U.S. patent application number 17/116739 was filed with the patent office on 2021-03-25 for preparation of psilocybin, different polymorphic forms, intermediates, formulations and their use.
This patent application is currently assigned to COMPASS PATHFINDER LIMITED. The applicant listed for this patent is COMPASS PATHFINDER LIMITED. Invention is credited to Christopher BROWN, Derek John LONDESBROUGH, Gillian MOORE, David E. NICHOLS, Julian Scott NORTHEN, Hemant Kashinath PATIL.
Application Number | 20210087212 17/116739 |
Document ID | / |
Family ID | 1000005444572 |
Filed Date | 2021-03-25 |
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United States Patent
Application |
20210087212 |
Kind Code |
A1 |
LONDESBROUGH; Derek John ;
et al. |
March 25, 2021 |
PREPARATION OF PSILOCYBIN, DIFFERENT POLYMORPHIC FORMS,
INTERMEDIATES, FORMULATIONS AND THEIR USE
Abstract
This invention relates to the large-scale production of
psilocybin for use in medicine. More particularly, it relates to a
method of obtaining high purity crystalline psilocybin,
particularly, in the form of Polymorph A. It further relates to a
method for the manufacture of psilocybin and intermediates in the
production thereof and formulations containing psilocybin.
Inventors: |
LONDESBROUGH; Derek John;
(Hartlepool, GB) ; BROWN; Christopher; (Gateshead,
GB) ; NORTHEN; Julian Scott; (South Shields, GB)
; MOORE; Gillian; (Sedgefield, GB) ; PATIL; Hemant
Kashinath; (Sittingbourne, GB) ; NICHOLS; David
E.; (Chapel Hill, NC) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
COMPASS PATHFINDER LIMITED |
Altrincham |
|
GB |
|
|
Assignee: |
COMPASS PATHFINDER LIMITED
Altrincham
GB
|
Family ID: |
1000005444572 |
Appl. No.: |
17/116739 |
Filed: |
December 9, 2020 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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16920223 |
Jul 2, 2020 |
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17116739 |
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16679009 |
Nov 8, 2019 |
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16920223 |
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16155386 |
Oct 9, 2018 |
10519175 |
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16679009 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
C07D 209/16 20130101;
A61K 47/36 20130101; C07F 9/5728 20130101; C07B 2200/13 20130101;
A61K 47/38 20130101; A61K 9/0053 20130101; A61K 9/2054
20130101 |
International
Class: |
C07F 9/572 20060101
C07F009/572; C07D 209/16 20060101 C07D209/16; A61K 9/20 20060101
A61K009/20; A61K 9/00 20060101 A61K009/00; A61K 47/36 20060101
A61K047/36; A61K 47/38 20060101 A61K047/38 |
Foreign Application Data
Date |
Code |
Application Number |
Oct 9, 2017 |
GB |
1716505.1 |
Jun 28, 2018 |
GB |
1810588.2 |
Oct 9, 2018 |
GB |
1816438.4 |
Claims
1-30. (canceled)
31. A pharmaceutical composition, comprising crystalline Polymorph
A of psilocybin and a pharmaceutically acceptable excipient,
wherein the Polymorph A is characterized by X-ray powder
diffraction (XRPD) peaks at 11.5.+-.0.1, 12.0.+-.0.1, 14.5.+-.0.1,
17.5.+-.0.1 and 19.7.+-.0.1.degree.2.theta., wherein the psilocybin
has a chemical purity of greater than 97% and no single impurity of
greater than 1% as determined by HPLC analysis.
32. The pharmaceutical composition of claim 31, wherein the
composition is a capsule.
33. The pharmaceutical composition of claim 31, wherein the
composition is a tablet.
34. The pharmaceutical composition of claim 31, wherein the
Polymorph A is further characterized by at least one peak selected
from the group consisting of 20.4.+-.0.1, 22.2.+-.0.1, 24.3.+-.0.1,
and 25.7.+-.0.1.degree.2.theta..
35. The pharmaceutical composition of claim 31, wherein the
composition comprises about 5 mg of the crystalline Polymorph A of
psilocybin.
36. The pharmaceutical composition of claim 31, wherein the
composition comprises about 10 mg of the crystalline Polymorph A of
psilocybin.
37. The pharmaceutical composition of claim 31, wherein the
composition comprises about 25 mg of the crystalline Polymorph A of
psilocybin.
38. Crystalline Polymorph A of psilocybin, wherein the Polymorph A
is characterized by X-ray powder diffraction (XRPD) peaks at
11.5.+-.0.1, 12.0.+-.0.1, 14.5.+-.0.1, 17.5.+-.0.1 and
19.7.+-.0.1.degree.2.theta., wherein the psilocybin has a chemical
purity of greater than 97% and no single impurity of greater than
1% as determined by HPLC analysis.
39. The crystalline psilocybin of claim 38, wherein the Polymorph A
is further characterized by at least one peak selected from the
group consisting of 20.4.+-.0.1, 22.2.+-.0.1, 24.3.+-.0.1, and
25.7.+-.0.1.degree.2.theta..
40. The crystalline psilocybin of claim 38, wherein the Polymorph A
is characterized by a XRPD diffraction pattern that is
substantially the same as shown in FIG. 7a.
41. The crystalline psilocybin of claim 38, wherein the crystalline
psilocybin is further characterized by a water content of <0.5%
w/w.
42. The crystalline psilocybin of claim 38, wherein the crystalline
psilocybin is further characterized by a <0.5% w/w loss in the
TGA thermogram between 25.degree. C. and 200.degree. C.
43. The crystalline psilocybin of claim 38, wherein the crystalline
psilocybin is further characterized by an endothermic event in a
DSC thermogram having an onset temperature of between 205.degree.
C. and 220.degree. C.
44. The crystalline psilocybin of claim 38, wherein the crystalline
psilocybin is further characterized by an endothermic event in a
DSC thermogram having an onset temperature of between 145.degree.
C. and 155.degree. C.
45. The crystalline psilocybin of claim 38, wherein the crystalline
psilocybin is further characterized by one or more of the
following: a) a loss on drying of no more than 2% w/w; b) residue
on ignition of no more than 0.5% w/w; c) assay (on a dry basis) of
95-103% by weight as measured by HPLC; d) residual solvent content
of no more than 3000 ppm methanol; 5000 ppm ethanol, 720 ppm THF,
and 890 ppm toluene, as measured by high resolution gas
chromatography (HRGC); e) phosphoric acid content of no more than
1% w/w as measured by .sup.31P NMR; and f) Inductively Coupled
Plasma Mass Spectrometry (ICP-MS) elemental analysis of: i. no more
than 1.5 ppm Cd; ii. no more than 1.5 ppm Pb; iii. no more than 4.5
ppm As; iv. no more than 9.0 ppm Hg; v. no more than 15 ppm Co; vi.
no more than 30 ppm V; vii. no more than 60 ppm Ni; viii. no more
than 165 ppm Li; and ix. no more than 30 ppm Pd.
46. A method of treating major depressive disorder, the method
comprising: administering a therapeutically effective amount of
crystalline Polymorph A of psilocybin to a patient in need thereof,
wherein the Polymorph A is characterized by X-ray powder
diffraction (XRPD) peaks at 11.5.+-.0.1, 12.0.+-.0.1, 14.5.+-.0.1,
17.5.+-.0.1 and 19.7.+-.0.1.degree.2.theta., and wherein the
psilocybin has a chemical purity of greater than 97% and no single
impurity of greater than 1% as determined by HPLC analysis.
47. The method of claim 46, wherein about 5 mg of the crystalline
Polymorph A of psilocybin is administered.
48. The method of claim 46, wherein about 10 mg of the crystalline
Polymorph A of psilocybin is administered.
49. The method of claim 46, wherein about 25 mg of the crystalline
Polymorph A of psilocybin is administered.
50. The method of claim 46, wherein the crystalline Polymorph A of
psilocybin is orally administered.
51. The method of claim 50, wherein the crystalline Polymorph A of
psilocybin is administered in a capsule.
52. The method of claim 50, wherein the crystalline Polymorph A of
psilocybin is administered in a tablet.
53. The method of claim 46, wherein the Polymorph A is further
characterized by at least one peak selected from the group
consisting of 20.4.+-.0.1, 22.2.+-.0.1, 24.3.+-.0.1, and
25.7.+-.0.1.degree.2.theta..
Description
[0001] This application is a continuation of U.S. patent
application Ser. No. 16/920,223, filed Jul. 2, 2020; which is a
continuation of U.S. patent application Ser. No. 16/679,009, filed
Nov. 8, 2019; which is a continuation of U.S. patent application
Ser. No. 16/155,386, now U.S. Pat. No. 10,519,175, filed Oct. 9,
2018, which claims priority to United Kingdom Application No.
1816438.4, filed Oct. 9, 2018; United Kingdom Application No.
1810588.2, filed Jun. 28, 2018; and United Kingdom Application No.
1716505.1, filed Oct. 9, 2017. The disclosures of the above
applications are incorporated by reference herein in their
entireties.
[0002] This invention relates to the large-scale production of
psilocybin for use in medicine.
[0003] By large scale is meant producing batches of psilocybin with
a weight of greater than 10 g, more preferably greater than 100 g,
more preferably still greater than 250 g and up to, and above, Kg
levels.
[0004] It also relates to the production of intermediates,
including but not limited to psilocin, different polymorphic forms
of psilocybin, including isostructural variants, and their
formulation for use in medicine, particularly, but not exclusively
for the treatment of treatment resistant depression, as defined in
Diagnostic and Statistical Manual, 5.sup.th Edition, either alone
or in combination with psychological support which may be provided
digitally.
BACKGROUND
[0005] Psilocybin was first synthesised in 1958 by Sandoz, see
GB912714 and U.S. Pat. No. 3,075,992, and was widely available as a
research chemical until the mid-1960's.
[0006] A plant based psychedelic it has been used as an aide to
psychotherapy for the treatment of mood disorders and alcoholic
disorders and recently 3 clinical trials have reported its use for
depressive symptoms. [0007] Griffiths et al 2016; J Psychopharmacol
30 (12):1181-1197; [0008] Ross et al 2016; J Psychopharmacol 30
(12):1165-1180; and [0009] Carhart-Harris et al 2016, Lancet
Psychiatry 3(7): 619-627. [0010] Methods of manufacture of
psilocybin are limited and include: [0011] J Nat Prod 2003, 66,
pages 885-887; [0012] Helv Chim Acta 1959, 42, 2073-2103; [0013]
Experientia 1958, 15, 397-399; and [0014] Synthesis 1999,
935-938.
[0015] Based on this literature Applicant believed that the process
disclosed in J Nat Prod 2003, 66, pages 885-887 (hereafter JNP) was
the most suitable method for development into a commercial scaled
process.
[0016] The process disclosed therein produced quantities in the
order of 10 g and comprised 6 steps numbered (i) to (vi).
[0017] By analogy with the Applicants process, steps ii and iii are
hereafter discussed as a single step, (Step 2) and the JNP process
is reproduced as FIG. 1 herein.
[0018] Step 1 (i) comprised reacting 4-hydroxyindole ("3") with
acetic anhydride (Ac.sub.2O) in pyridine and anhydrous
dichloromethane (CH.sub.2Cl.sub.2) at 0.degree. C. Water was added,
the mixture evaporated, and the resulting concentrate was dissolved
in ethyl acetate, washed with water, and saturated sodium chloride,
and the organic phase dried over sodium sulphate and evaporated to
obtain 4-acetylindole ("4"), which was collected by filtration and
washed with water and ethyl acetate.
[0019] Step 2 (ii and iii), a two-step acylation (ii)--amidation
step (iii), comprised forming 3-Dimethylaminooxalyl-4-acetylindole
("6") by: (ii) reacting 4-acetylindole ("4") with oxalyl chloride
((COCl).sub.2) in anhydrous diethylether, stirring, adding n-hexane
and holding at -20.degree. C. to produce an intermediate
3-(2-chloro-2-oxoacetyl)-1H-indol-4-yl acetate ("5") which was
separated by filtration. The intermediate was dissolved in
anhydrous tetrahydrofuran (THF) and reacted with dimethylamine
((CH.sub.3).sub.2NH) in tetrahydrofuran and pyridine. Anhydrous
ether was added because of solidification, and the reaction product
separated by filtration and washed with n hexane, ethyl acetate,
and water to obtain 3-Dimethylaminooxalyl-4-acetylindole ("6").
[0020] Step 3 (iv) comprised the formation of psilocin ("1") by
reacting the 3-Dimethylaminooxalyl-4-acetylindole ("6") with
lithium aluminium hydride (LiAlH.sub.4) in anhydrous THF under an
argon atmosphere. After refluxing and cooling, anhydrous sodium
sulphate was added, followed by a solution of sodium sulphate, and
further anhydrous sodium sulphate. The reaction mixture was diluted
with ethyl acetate, quickly concentrated in vacuo, and the
resulting psilocin crystals briefly washed with methanol.
[0021] Step 4 (v) comprised the formation of benzyl
[2-(4-oxyindol-3-yl) ethyl]dimethylammonio-4-O-benzyl phosphate
("8") by reacting psilocin, dissolved in anhydrous THF, with
n-butyl lithium (n-BuLi) in n-hexane at -78.degree. C. and
tetrabenzylpyrophosphate [(BnO).sub.2PO].sub.2O, and the reaction
allowed to warm to 0.degree. C., and the production of intermediate
dibenzyl 3-[2-(dimethylamino)ethyl]-1H-indol-4-yl phosphate ("7")
monitored. On checking for its presence, aminopropyl silica gel was
added, the mixture diluted with ethyl acetate and filtered through
a Celite pad by suction, the filtrate concentrated in vacuo,
re-dissolved in CH.sub.2Cl.sub.2, and the precipitate collected by
filtration.
[0022] Step 5 (vi) comprised the formation of psilocybin ("2") by
reaction of ("8"), in methanol (MeOH), with hydrogen (H.sub.2)
using a palladium-activated carbon catalyst (Pd/C). Water was
added, because of product deposition, and ("8"), its mono
de-benzylated derivative were monitored along with the appearance
of psilocybin, the reaction solution was filtered through a Celite
pad. The product was collected by filtration and washed with
ethanol to provide a white needle crystalline form with a melting
point 190.degree. C.-198.degree. C.
[0023] In contrast to most processes, such as JNP, which use
non-aqueous solvents, such as methanol or ethanol, Experientia
1958, 15, 397-399 used a single re-crystalisation from water to
obtain psilocybin from a mushroom extraction. The teaching was to
use boiling water to dissolve the starting material, obtained at
small scale by chromatography, and the resulting high vacuum dried
material was stated to melt indistinctly between 185 and
195.degree. C., and showed a weight loss of 25.4%, suggesting it
clearly differs in purity and form to that obtained by
Applicant.
[0024] During the development of a synthesis to produce Psilocybin
the applicant conducted a number of hydrogenation reactions on a 5
g scale which resulted in different crystalline forms of Psilocybin
being obtained. The initial hydrogenation reaction yielded Hydrate
A (JCCA2157E) which exhibited a XRPD diffractogram as shown in FIG.
7d and DSC and TGA thermograms as shown in FIG. 8d. The DSC
exhibits an endotherm at .about.97.degree. C. which is coincidental
with a weight reduction in the TGA indicative of dehydration, and
an endothermic event with an onset temperature of
.about.216.degree. C. which was presumed to be the melt. Another
hydrogenation reaction yielded an ethanol solvate (JCCA2158 D)
which when analysed by XRPD (FIG. 7e), DSC (FIG. 8e), TGA (FIG. 8e)
and by .sup.1H NMR indicated 11% entrapped ethanol. The DSC
thermogram shows an endotherm having an onset of .about.154.degree.
C. that appeared to be a melt concurrent with the .about.13% weight
loss in the TGA. In another experiment performed during
development, the applicant performed a crystallisation of
psilocybin; rather than remain in solution in hot water allowing
for a polish filtration step, precipitation occurred at high
temperature (>90.degree. C.). The solids formed did not
re-dissolve upon further heating or addition of extra water. Upon
cooling and isolation of the solid (CB646E) XRPD was performed. The
XRPD diffractogram (FIG. 7f) suggested a mixed phase of Polymorph
A' (JCCA2160-F-D4) and Polymorph B (JCCA2160-F-TM2-C5). These
findings highlight the importance of developing a process which can
consistently produce the desired crystalline form so the Applicants
set about experiments to determine what these forms were in order
they could produce a chemically pure psilocybin, in a controlled
form suitable for use in medicine.
[0025] For clinical trials any New Active Substance (NAS) should be
capable of large scale production (typically 100 g plus, more
typically greater than 250 g, more preferably still greater than
500 g, to Kg plus batches), depending on the amount of active to be
dosed to a human subject. It should also be chemically pure, well
defined, and stable on storage.
[0026] Furthermore, any method of manufacture must be readily
reproducible, and provide batch to batch consistency.
[0027] It is a first object of this invention to provide
psilocybin, of consistent polymorphic form, for administration to
human subjects.
[0028] It is another object of this invention to provide chemically
pure psilocybin, of consistent polymorphic form, for administration
to human subjects.
[0029] It is yet a further object to provide chemically pure
psilocybin, in large scale batch quantities since for commercial
use, the pure psilocybin must be produced at scale.
[0030] It is yet a further object of the invention to provide a
method of crystallising psilocybin in a desired polymorphic
form.
[0031] It is yet a further object of the present invention to
provide a scalable method for manufacturing psilocybin, from
psilocin or 4 hydroxy-indole.
[0032] In developing suitable methodology Applicant experienced
numerous problems and difficulties which they had to be overcome,
and it is a separate, independent, object to overcome those
problems identified at each step, and use the inventions either
alone or in combination.
[0033] It is yet a further object of the invention to formulate the
psilocybin of the invention in a form suitable for administration
to human subjects and use it in medicine, particularly in the
treatment of central nervous system disorders (CNS), and more
particularly, but not exclusively, in the treatment of depression,
particularly, drug resistant depression either alone or in
combination with a digital health product or digital solution.
BRIEF SUMMARY OF THE DISCLOSURE
[0034] In accordance with a first aspect of the present inventions
there is provided crystalline psilocybin in the form Polymorph A or
Polymorph A', characterised by one or more of:
a. peaks in an XRPD diffractogram at 11.5, 12.0 and
14.5.degree.2.theta..+-.0.1.degree.2.theta.; b. peaks in an XRPD
diffractogram at 11.5, 12.0 and
14.5.degree.2.theta..+-.0.1.degree.2.theta., further characterised
by at least one further peak at 19.7, 20.4, 22.2, 24.3 or
25.7.degree.2.theta..+-.0.1.degree.2.theta.; c. an XRPD
diffractogram as substantially illustrated in FIG. 7a or 7b; or d.
an endothermic event in a DSC thermogram having an onset
temperature of between 205 and 220.degree. C. substantially as
illustrated in FIG. 8a or 8b.
Polymorph A
[0035] In accordance with a preferred embodiment of the present
invention there is provided crystalline psilocybin in the form
Polymorph A, characterised by one or more of: [0036] a. peaks in an
XRPD diffractogram at 11.5, 12.0, 14.5, and 17.5,
.degree.2.theta..+-.0.1.degree.2.theta.; [0037] b. peaks in an XRPD
diffractogram at 11.5, 12.0, 14.5 and 17.5,
.degree.2.theta..+-.0.1.degree.2.theta., further characterised by
at least one further peak at 19.7, 20.4, 22.2, 24.3 or
25.7.degree.2.theta..+-.0.1.degree.2.theta.; [0038] c. an XRPD
diffractogram as substantially illustrated in FIG. 7a; or [0039] d.
an endothermic event in a DSC thermogram having an onset
temperature of between 205 and 220.degree. C. substantially as
illustrated in FIG. 8a.
[0040] The peak at 17.5.degree.2.theta..+-.0.1.degree.2.theta. has
a relative intensity compared to the peak at
14.5.degree.2.theta..+-.0.1.degree.2.theta. of at least 5%,
preferably at least 6%, more preferably still at least 7%, through
8%, and 9% to at least 10%.
[0041] In one embodiment, psilocybin Polymorph A exhibits an XRPD
diffractogram characterised by the diffractogram summarised in
Table 1. In one embodiment, described herein, the crystalline
psilocybin Polymorph A comprises at least 3 peaks of
(.+-.0.1.degree.2.theta.) of Table 1. In a certain embodiment,
described herein, the crystalline psilocybin Polymorph A comprises
at least 4 peaks of (.+-.0.1.degree.2.theta.) of Table 1. In a
certain embodiment, described herein the crystalline psilocybin
Polymorph A comprises at least 5 peaks of (.+-.0.1.degree.2.theta.)
of Table 1. In a certain embodiment, described herein the
crystalline psilocybin Polymorph A comprises at least 6 peaks of
(.+-.0.1.degree.2.theta.) of Table 1. In a certain embodiment,
described herein the crystalline psilocybin Polymorph A comprises
at least 8 peaks of (.+-.0.1.degree.2.theta.) of Table 1. In a
certain embodiment, described herein the crystalline psilocybin
Polymorph A comprises at least 10 peaks of
(.+-.0.1.degree.2.theta.) of Table 1. In a certain embodiment,
described herein the crystalline psilocybin Polymorph A comprising
at least 15 peaks of (.+-.0.1.degree.2.theta.) of Table 1. A peak
at about 17.5, .degree.2.theta..+-.0.1.degree.2.theta.
distinguishes psilocybin Polymorph A from Polymorph A', in which
the peak is absent or substantially absent (i.e. has a relative
intensity compared to the peak at
14.5.degree.2.theta..+-.0.1.degree.2.theta. of less than 2%, more
preferably less than 1%).
TABLE-US-00001 TABLE 1 XRPD peak positions for Polymorph A Relative
Intensity Position [.degree.2 Th.] [%] 5.6 8.42 11.5 13.05 12.0
26.45 14.5 100 17.5 10.71 19.7 37.29 20.4 20.06 22.2 17.83 23.2
6.99 24.3 17.93 25.7 16.4 26.8 3.15 27.8 4.54 29.7 9.53 31.2 6.51
32.6 2.45 33.7 1.75
[0042] In one embodiment, crystalline psilocybin Polymorph A is
characterised by XRPD diffractogram peaks at 11.5, 12.0, 14.5, and
17.5.degree.2.theta..+-.0.1.degree.2.theta.. In another embodiment,
crystalline psilocybin Polymorph A is further characterised by at
least one additional peak appearing at 19.7, 20.4, 22.2, 24.3 or
25.7.degree.2.theta..+-.0.1.degree.2.theta.. In another embodiment,
crystalline psilocybin Polymorph A is further characterised by at
least two additional peaks appearing at 19.7, 20.4, 22.2, 24.3 or
25.7.degree.2.theta..+-.0.1.degree.2.theta.. In another embodiment,
crystalline psilocybin Polymorph A is further characterised by at
least three additional peaks appearing at 19.7, 20.4, 22.2, 24.3 or
25.7.degree.2.theta..+-.0.1.degree.2.theta.. In yet a further
embodiment, crystalline psilocybin Polymorph A exhibits an XRPD
diffractogram substantially the same as the XRPD diffractogram
shown in FIG. 7a.
[0043] In one embodiment, crystalline psilocybin Polymorph A is
characterised by XRPD diffractogram peaks at 14.5 and
17.5.degree.2.theta..+-.0.1.degree.2.theta. with the peak at
17.5.degree.2.theta. having an intensity which is at least 5% of
the intensity of the peak at 14.5.degree.2.theta., more preferably
still at least 6%, through at least 7%, at least 8%, at least 9%,
to at least 10%.
[0044] In one embodiment, crystalline psilocybin Polymorph A is
absent or substantially absent of an XRPD diffractogram peaks at
10.1. By substantially absent is meant than any XRPD diffractogram
peaks at 10.1 is less than 2% of the intensity of the peak at
14.5.degree.2.theta., such as less than 1%, or is not detectable in
the XRPD diffractogram,
[0045] In one embodiment, crystalline psilocybin Polymorph A is
characterised by an endothermic event in a DSC thermogram having an
onset temperature of between 205 and 220.degree. C., such as
between 210 and 220.degree. C., such as between 210 and 218.degree.
C., or such as between 210 and 216.degree. C. In another
embodiment, crystalline psilocybin Polymorph A is further
characterised by an endothermic event in the DSC thermogram having
an onset temperature of between 145 and 165.degree. C., such as
between 145 and 160.degree. C., or such as between 145 and
155.degree. C. In another embodiment, crystalline psilocybin
Polymorph A is characterised by an endothermic event having an
onset temperature of between 205 and 220.degree. C., such as
between 210 and 220.degree. C., such as between 210 and 218.degree.
C., or such as between 210 and 216.degree. C., and an endothermic
event having an onset temperature of between 145 and 165.degree.
C., such as between 145 and 160.degree. C., or such as between 145
and 155.degree. C., in a DSC thermogram. In yet another embodiment,
crystalline psilocybin Polymorph A exhibits a DSC thermogram
substantially the same as the DSC thermogram in FIG. 8a.
[0046] In another embodiment, crystalline psilocybin Polymorph A is
characterised by having a water content of <0.5% w/w, such as
<0.4% w/w, such as <0.3% w/w, such as <0.2% w/w, or such
as <0.1% w/w. The skilled person would know of methods to
determine the water content of a compound, for example Karl Fischer
Titration. In one embodiment, crystalline psilocybin Polymorph A is
characterised by having <0.5% w/w loss, such as <0.4% w/w,
such as <0.3% w/w, such as <0.2% w/w, such as <0.1% w/w,
in the TGA thermogram between ambient temperature, such as about
25.degree. C., and 200.degree. C. In one embodiment, crystalline
psilocybin Polymorph A loses less than 2% by weight in a loss on
drying test, such as less than 1% by weight, such as less than 0.5%
by weight. The loss on drying test is performed at 70.degree.
C.
[0047] In one embodiment, crystalline psilocybin Polymorph A is a
highly pure crystalline form of Polymorph A, for example,
psilocybin comprises at least 90% by weight, such as 95%, such as
99%, such as 99.5% of Polymorph A.
[0048] In one embodiment, crystalline psilocybin Polymorph A is a
white to off white solid.
[0049] In another embodiment, crystalline psilocybin Polymorph A is
chemically pure, for example the psilocybin has a chemical purity
of greater than 97%, such as greater than 98%, or such as greater
than 99% by HPLC. In one embodiment, crystalline psilocybin
Polymorph A has no single impurity of greater than 1%, more
preferably less than 0.5%, including phosphoric acid as measured by
.sup.31P NMR, and psilocin as measured by HPLC. In one embodiment,
crystalline psilocybin Polymorph A has a chemical purity of greater
than 97 area %, more preferably still greater than 98 area %, and
most preferably greater than 99 area % by HPLC. In one embodiment,
crystalline psilocybin Polymorph A has no single impurity greater
than 1 area %, more preferably less than 0.5 area % as measured by
HPLC. In one embodiment, crystalline psilocybin Polymorph A does
not contain psilocin at a level greater than 1 area %, more
preferably less than 0.5 area % as measured by HPLC. In one
embodiment, crystalline psilocybin Polymorph A does not contain
phosphoric acid at a level greater than 1 weight %, more preferably
less than 0.5 weight % as measured by .sup.31P NMR. In one
embodiment, crystalline psilocybin Polymorph A has a chemical assay
of at least 95 weight %, such as at least 96 weight %, or such as
at least 98 weight %.
Polymorph A'
[0050] In accordance with another embodiment of the invention,
there is provided crystalline psilocybin Polymorph A' characterised
by one or more of: [0051] a. peaks in an XRPD diffractogram at
11.5, 12.0 and 14.5.degree.2.theta..+-.0.1.degree.2.theta., but
absent or substantially absent of a peak at
17.5.degree.2.theta..+-.0.1.degree.2.theta.; [0052] b. peaks in an
XRPD diffractogram at 11.5, 12.0 and
14.5.degree.2.theta..+-.0.1.degree.2.theta., but absent or
substantially absent of a peak at
17.5.degree.2.theta..+-.0.1.degree.2.theta., further characterised
by at least one further peak at 19.7, 20.4, 22.2, 24.3 or
25.7.degree.2.theta..+-.0.1.degree.2.theta.; [0053] c. an XRPD
diffractogram as substantially illustrated in FIG. 7b; or [0054] d.
an endothermic event in a DSC thermogram having an onset
temperature of between 205 and 220.degree. C. substantially as
illustrated in FIG. 8b.
[0055] By substantially absent of a peak at
17.5.degree.2.theta..+-.0.1.degree.2.theta. is meant, if present,
the peak has a relative intensity, compared to a peak at
14.5.degree.2.theta..+-.0.1.degree.2.theta., of less than 5%, more
preferably less than 4%, through less than 3%, to 2%, 1% or
less.
[0056] In one embodiment, psilocybin Polymorph A' exhibits an XRPD
diffractogram characterised by the diffractogram summarised in
Table 2. In one embodiment, described herein the crystalline
psilocybin Polymorph A' comprises at least 3 peaks of
(.+-.0.1.degree.2.theta.) of Table 2 but absent or substantially
absent of a peak at 17.5.degree.2.theta..+-.0.1.degree.2.theta.. In
a certain embodiment, described herein the crystalline psilocybin
Polymorph A' comprising at least 4 peaks of
(.+-.0.1.degree.2.theta.) of Table 2 but absent or substantially
absent of a peak at 17.5.degree.2.theta..+-.0.1.degree.2.theta.. In
a certain embodiment, described herein the crystalline psilocybin
Polymorph A' comprises at least 5 peaks of
(.+-.0.1.degree.2.theta.) of Table 2 but absent or substantially
absent of a peak at 17.5.degree.2.theta..+-.0.1.degree.2.theta.. In
a certain embodiment, described herein the crystalline psilocybin
Polymorph A' comprises at least 6 peaks of
(.+-.0.1.degree.2.theta.) of Table 2 but absent or substantially
absent of a peak at 17.5.degree.2.theta..+-.0.1.degree.2.theta.. In
a certain embodiment, described herein the crystalline psilocybin
Polymorph A' comprises at least 8 peaks of
(.+-.0.1.degree.2.theta.) of Table 2 but absent or substantially
absent of a peak at 17.5.degree.2.theta..+-.0.1.degree.2.theta.. In
a certain embodiment, described herein the crystalline psilocybin
Polymorph A' comprises at least 10 peaks of
(.+-.0.1.degree.2.theta.) of Table 2 but absent or substantially
absent of a peak at 17.5.degree.2.theta..+-.0.1.degree.2.theta.. In
a certain embodiment, described herein the crystalline psilocybin
Polymorph A' comprises at least 15 peaks of
(.+-.0.1.degree.2.theta.) of Table 2 but absent or substantially
absent of a peak at 17.5.degree.2.theta..+-.0.1.degree.2.theta.. In
a certain embodiment, described herein the crystalline psilocybin
Polymorph A' comprises at least 20 peaks of
(.+-.0.1.degree.2.theta.) of Table 2 but absent or substantially
absent of a peak at 17.5.degree.2.theta..+-.0.1.degree.2.theta.. In
a certain embodiment, described herein the crystalline psilocybin
Polymorph A' comprises at least 25 peaks of
(.+-.0.1.degree.2.theta.) of Table 2 but absent or substantially
absent of a peak at
17.5.degree.2.theta..+-.0.1.degree.2.theta..
TABLE-US-00002 TABLE 2 XRPD peak positions for Polymorph A`
Position [.degree.2 Th.] Relative Intensity [%] 5.5 4.89 10.1 4.09
11.5 22.05 12.0 22.77 14.5 100 14.9 11.29 17.5 1.08 18.7 2.44 19.4
23.02 19.6 33.7 20.3 17.01 21.1 12.08 21.6 8.51 22.2 15.54 22.6
8.78 23.1 10.11 24.3 21.83 25.1 4.36 25.8 15.4 26.3 4.28 26.8 2.86
27.8 5.96 28.6 1.91 29.7 10.56 31.1 7.35 32.6 3.72 33.8 1.54
[0057] In one embodiment, crystalline psilocybin Polymorph A' is
characterised by XRPD diffractogram peaks at 11.5, 12.0, and
14.5.degree.2.theta..+-.0.1.degree.2.theta. but substantially
absent of a peak at 17.5.degree.2.theta..+-.0.1.degree.2.theta.. In
another embodiment, crystalline psilocybin Polymorph A' is further
characterised by at least one additional peak appearing at 19.7,
20.4, 22.2, 24.3, or 25.7.degree.2.theta..+-.0.1.degree.2.theta..
In another embodiment, crystalline psilocybin Polymorph A' is
further characterised by at least two additional peaks appearing at
19.7, 20.4, 22.2, 24.3, or
25.7.degree.2.theta..+-.0.1.degree.2.theta.. In another embodiment,
crystalline psilocybin Polymorph A' is further characterised, and
distinguished from Polymorph A by the presence of a peak appearing
at 10.1.degree.2.theta..+-.0.1.degree.2.theta.. In yet a further
embodiment, crystalline psilocybin Polymorph A' exhibits an XRPD
diffractogram substantially the same as the XRPD diffractogram
shown in FIG. 7b.
[0058] In one embodiment, crystalline psilocybin Polymorph A' is
characterised by XRPD diffractogram peaks at 14.5 and
17.5.degree.2.theta..+-.0.1.degree.2.theta. wherein the intensity
of the peak at 17.5.degree.2.theta. is less than 5% of the
intensity of the peak at 14.5.degree.2.theta., such as less than
4%, such as less than 3%, such as at less than 2%, such as less
than 1%, or such as about 1%.
[0059] In one embodiment, crystalline psilocybin Polymorph A' is
characterised by XRPD diffractogram peaks at 10.1 and
14.5.degree.2.theta..+-.0.1.degree.2.theta. wherein the intensity
of the peak at 10.1.degree.2.theta. is at least 1% of the intensity
of the peak at 14.5.degree.2.theta., such as at least than 2%, such
as at least than 3%, or such as about 4%.
[0060] In one embodiment, crystalline psilocybin Polymorph A' is
characterised by an endothermic event in a DSC thermogram having an
onset temperature of between 205 and 220.degree. C., such as
between 210 and 220.degree. C., such as between 210 and 218.degree.
C., or such as between 210 and 216.degree. C. In another
embodiment, crystalline psilocybin Polymorph A' is further
characterised by an endothermic event in the DSC thermogram having
an onset temperature of between 145 and 165.degree. C., such as
between 145 and 160.degree. C., or such as between 145 and
155.degree. C. In another embodiment, crystalline psilocybin
Polymorph A' is characterised by an endothermic event having an
onset temperature of between 205 and 220.degree. C., such as
between 210 and 220.degree. C., such as between 210 and 218.degree.
C., or such as between 210 and 216.degree. C., and an endothermic
event having an onset temperature of between 145 and 165.degree.
C., such as between 145 and 160.degree. C., or such as between 145
and 155.degree. C., in a DSC thermogram. In yet another embodiment,
crystalline psilocybin Polymorph A' exhibits a DSC thermogram
substantially the same as the DSC thermogram in FIG. 8b.
[0061] In another embodiment, crystalline psilocybin Polymorph A'
is characterised by having a water content of <0.5% w/w, such as
<0.4% w/w, such as <0.3% w/w, such as <0.2% w/w, or such
as <0.1% w/w. The skilled person would know of methods to
determine the water content of a compound, for example Karl Fischer
Titration. In one embodiment, crystalline psilocybin Polymorph A'
is characterised by having <0.5% w/w loss, such as <0.4% w/w,
such as <0.3% w/w, such as <0.2% w/w, such as <0.1% w/w,
in the TGA thermogram between ambient temperature, such as
25.degree. C., and 200.degree. C. In one embodiment, crystalline
psilocybin Polymorph A' loses less than 2% by weight in a loss on
drying test, such as less than 1% by weight, such as less than 0.5%
by weight. The loss on drying test is performed at 70.degree.
C.
[0062] In one embodiment, crystalline psilocybin Polymorph A' is a
highly pure crystalline form of Polymorph A', for example,
psilocybin comprises at least 90% by weight, such as 95%, such as
99%, such as 99.5% of Polymorph A'.
[0063] In one embodiment, crystalline psilocybin Polymorph A's is a
white to off white solid.
[0064] In another embodiment, crystalline psilocybin Polymorph A'
is chemically pure, for example the psilocybin has a chemical
purity of greater than 97%, more preferably still greater than 98%,
and most preferably greater than 99% by HPLC. In one embodiment,
crystalline psilocybin Polymorph A' has no single impurity of
greater than 1%, more preferably less than 0.5%, including
phosphoric acid as measured by .sup.31P NMR, and psilocin as
measured by HPLC. In one embodiment, crystalline psilocybin
Polymorph A' has a chemical purity of greater than 97 area %, more
preferably still greater than 98 area %, and most preferably
greater than 99 area % by HPLC. In one embodiment, crystalline
psilocybin Polymorph A' has no single impurity greater than 1 area
%, more preferably less than 0.5 area % as measured by HPLC. In one
embodiment, crystalline psilocybin Polymorph A' does not contain
psilocin at a level greater than 1 area %, more preferably less
than 0.5 area % as measured by HPLC. In one embodiment, crystalline
psilocybin Polymorph A' does not contain phosphoric acid at a level
greater than 1 weight %, more preferably less than 0.5 weight % as
measured by .sup.31P NMR. In one embodiment, crystalline psilocybin
Polymorph A' has a chemical assay of at least 95 weight %, such as
at least 96 weight %, or such as at least 98 weight %.
[0065] XRPD diffractograms and XRPD peak positions are acquired
using Cu K.alpha. radiation.
[0066] DSC and TGA thermograms are acquired using a heating rate of
20.degree. C./min.
[0067] In one embodiment, there is provided high purity crystalline
psilocybin, Polymorph A or Polymorph A' (12 A or 12A'), exhibiting
an XRPD diffractogram as substantially illustrated in FIG. 7a or 7b
and a DSC thermograph as substantially illustrated in FIG. 8a or 8b
or a mixture thereof.
[0068] Preferably the crystalline psilocybin Polymorph A (12A)
exhibits an XRPD diffractogram as illustrated in FIG. 7a and a DSC
thermograph as illustrated in FIG. 8a.
[0069] Preferably the crystalline psilocybin Polymorph A' (12A')
exhibits an XRPD diffractogram as substantially illustrated in FIG.
7b and a DSC thermograph as substantially illustrated in FIG.
8b.
[0070] Preferably the high purity crystalline psilocybin Polymorph
A (12A) is characterised by a XRPD diffractogram as substantially
illustrated in FIG. 7a and a DSC thermograph as substantially
illustrated in FIG. 8a.
[0071] Preferably the high purity crystalline psilocybin Polymorph
A (12A') is characterised by a XRPD diffractogram as illustrated in
FIG. 7b and a DSC thermograph as illustrated in FIG. 8b.
[0072] Polymorph A (including its isostructural variant Polymorph
A') (FIGS. 7a and 7b) differs from Polymorph B (FIG. 7c), the
Hydrate A (FIG. 7d) and the ethanol solvate (FIG. 7e: Solvate A),
and the relationship between some of the different forms is
illustrated in FIG. 9.
[0073] The crystalline psilocybin Polymorph A or Polymorph A', is a
white to off white solid, and/or has a chemical purity of greater
than 97%, more preferably still greater than 98%, and most
preferably greater than 99% by HPLC, and has no single impurity of
greater than 1%, more preferably less than 0.5%, including
phosphoric acid as measured by .sup.31P NMR, and psilocin as
measured by HPLC. In one embodiment, there is provided high purity
crystalline psilocybin, Polymorph A or Polymorph A'. In one
embodiment, crystalline psilocybin, Polymorph A or Polymorph A',
has a chemical purity of greater than 97 area %, more preferably
still greater than 98 area %, and most preferably greater than 99
area % by HPLC. In one embodiment, crystalline psilocybin,
Polymorph A or Polymorph A', has no single impurity greater than 1
area %, more preferably less than 0.5 area % as measured by HPLC.
In one embodiment, crystalline psilocybin, Polymorph A or Polymorph
A', does not contain psilocin at a level greater than 1 area %,
more preferably less than 0.5 area % as measured by HPLC. In one
embodiment, crystalline psilocybin, Polymorph A or Polymorph A',
does not contain phosphoric acid at a level greater than 1 weight
%, more preferably less than 0.5 weight % as measured by .sup.31P
NMR. In one embodiment, crystalline psilocybin, Polymorph A or
Polymorph A', has a chemical assay of at least 95 weight %, such as
at least 96 weight %, or such as at least 98 weight %.
[0074] The heating of Polymorph A or A' results in an endothermic
event having an onset temperature of circa 150.degree. C.
corresponding to solid-solid transition of Polymorph A or Polymorph
A' to Polymorph B. Continued heating of the resulting solid, i.e.,
Polymorph B, results in a second endothermic event corresponding to
a melting point having an onset temperature of between 205 and
220.degree. C. (see FIGS. 8a and 8b).
[0075] In accordance with another independent aspect of the present
invention there is provided a crystalline form of psilocybin,
Hydrate A, characterised by one or more of: [0076] a. peaks in an
XRPD diffractogram at 8.9, 12.6 and
13.8.degree.2.theta..+-.0.1.degree.2.theta.; [0077] b. peaks in an
XRPD diffractogram at 8.9, 12.6 and
13.8.degree.2.theta..+-.0.1.degree.2.theta., further characterised
by at least one further peak at 6.5, 12.2, 19.4, 20.4 or
20.8.degree.2.theta..+-.0.1.degree.2.theta.; [0078] c. an XRPD
diffractogram as substantially illustrated in FIG. 7d; or [0079] d.
an endothermic event in a DSC thermogram having an onset
temperature of between 205 and 220.degree. C. substantially as
illustrated in FIG. 8d.
[0080] In one embodiment, psilocybin Hydrate A exhibits an XRPD
diffractogram characterised by the diffractogram summarised in
Table 3. In one embodiment, described herein the crystalline
psilocybin Hydrate A comprises at least 3 peaks of
(.+-.0.1.degree.2.theta.) of Table 3. In a certain embodiment,
described herein the crystalline psilocybin Hydrate A comprises at
least 4 peaks of (.+-.0.1.degree.2.theta.) of Table 3. In a certain
embodiment, described herein the crystalline psilocybin Hydrate A
comprises at least 5 peaks of (.+-.0.1.degree.2.theta.) of Table 3.
In a certain embodiment, described herein the crystalline
psilocybin Hydrate A comprises at least 8 peaks of
(.+-.0.1.degree.2.theta.) of Table 3. In a certain embodiment,
described herein the crystalline psilocybin Hydrate A comprises at
least 10 peaks of (.+-.0.1.degree.2.theta.) of Table 3.
TABLE-US-00003 TABLE 3 XRPD peak positions for Hydrate A Position
[.degree.2 Th.] Relative Intensity [%] 5.6 14.4 6.5 18.84 8.9 100
12.2 11.51 12.6 18.65 13.8 44.22 16.2 21.22 18.9 6.62 19.4 38.68
20.4 21.32 20.8 19.73 21.5 20.75 22.3 12.8 22.5 19.38 23.1 47.53
23.5 25.79 24.3 5.62 24.8 14.62 25.4 5.27 26.9 6.53 27.9 7.82 28.4
5.78 29.0 5.09 29.7 4.83 32.1 8.27 32.8 4.81 33.4 3.74 34.2
5.96
[0081] In one embodiment, crystalline psilocybin Hydrate A is
characterised by XRPD diffractogram peaks at 8.9, 12.6 and
13.8.degree.2.theta..+-.0.1.degree.2.theta.. In another embodiment,
crystalline psilocybin Hydrate A is further characterised by at
least one peak appearing at 6.5, 12.2, 19.4, 20.4 or
20.8.degree.2.theta..+-.0.1.degree.2.theta.. In another embodiment,
crystalline psilocybin Hydrate A is further characterised by at
least two peaks appearing at 6.5, 12.2, 19.4, 20.4 or
20.8.degree.2.theta..+-.0.1.degree.2.theta.. In yet a further
embodiment, crystalline psilocybin Hydrate A exhibits an XRPD
diffractogram substantially the same as the XRPD diffractogram
shown in FIG. 7d.
[0082] In one embodiment, crystalline psilocybin Hydrate A is
characterised by an endothermic event in a DSC thermogram having an
onset temperature of between 205 and 220.degree. C., such as
between 210 and 220.degree. C., such as between 210 and 218.degree.
C., or such as between 210 and 216.degree. C. In another
embodiment, crystalline psilocybin Hydrate A is further
characterised by an endothermic event in the DSC thermogram having
an onset temperature of between 85 and 105.degree. C., or such as
between 90 and 100.degree. C. In another embodiment, crystalline
psilocybin Hydrate A is characterised by an endothermic event
having an onset temperature of between 205 and 220.degree. C., such
as between 210 and 220.degree. C., such as between 210 and
218.degree. C., or such as between 210 and 216.degree. C., and an
endothermic event having an onset temperature of between 85 and
105.degree. C., or such as between 90 and 100.degree. C., in a DSC
thermogram. In yet another embodiment, crystalline psilocybin
Hydrate A exhibits a DSC thermogram substantially the same as the
DSC thermogram in FIG. 8d.
[0083] In another embodiment, crystalline psilocybin Hydrate A is
characterised by having a water content of between 10 and 18%, such
as between 12 and 16%, or such as about 13%. The skilled person
would know of methods to determine the water content of a compound,
for example Karl Fischer Titration. In one embodiment, crystalline
psilocybin Hydrate A is characterised by having a weight loss in
the TGA thermogram of between 10 and 18%, such as between 12 and
16%, or such as about 13%, between ambient temperature, such as
about 25.degree. C., and 120.degree. C.
[0084] In one embodiment, crystalline psilocybin Hydrate A is a
highly pure crystalline form of Hydrate A, for example, psilocybin
comprises at least 90% by weight, such as 95%, such as 99%, such as
99.5% of Hydrate A.
[0085] In accordance with another independent aspect of the present
invention there is provided a crystalline form of psilocybin,
Polymorph B, characterised by one or more of: [0086] a. peaks in an
XRPD diffractogram at 11.1, 11.8 and
14.3.degree.2.theta..+-.0.1.degree.2.theta.; [0087] b. peaks in an
XRPD diffractogram at 11.1, 11.8 and
14.3.degree.2.theta..+-.0.1.degree.2.theta., further characterised
by at least one further peak at 14.9, 15.4, 19.3, 20.0 or
20.6.degree.2.theta..+-.0.1.degree.2.theta.; [0088] c. an XRPD
diffractogram as substantially illustrated in FIG. 7c; or [0089] d.
an endothermic event in a DSC thermogram having an onset
temperature of between 205 and 220.degree. C. substantially as
illustrated in FIG. 8c.
[0090] In one embodiment, psilocybin Polymorph B exhibits an XRPD
diffractogram characterised by the diffractogram summarised in
Table 4. In one embodiment, described herein the crystalline
psilocybin Polymorph B comprises at least 3 peaks of
(.+-.0.1.degree.2.theta.) of Table 4. In a certain embodiment,
described herein the crystalline psilocybin Polymorph B comprises
at least 4 peaks of (.+-.0.1.degree.2.theta.) of Table 4. In a
certain embodiment, described herein the crystalline psilocybin
Polymorph B comprises at least 5 peaks of (.+-.0.1.degree.2.theta.)
of Table 4. In a certain embodiment, described herein the
crystalline psilocybin Polymorph B comprising at least 8 peaks of
(.+-.0.1.degree.2.theta.) of Table 4. In a certain embodiment,
described herein the crystalline psilocybin Polymorph B comprises
at least 10 peaks of (.+-.0.1.degree.2.theta.) of Table 4.
TABLE-US-00004 TABLE 4 XRPD peak positions for Polymorph B Position
Relative Intensity [.degree.2Th.] [%] 5.5 21.33 11.1 36.91 11.8
100.00 12.5 12.73 14.3 70.23 14.9 50.01 15.4 23.67 17.1 51.58 17.4
91.25 18.0 12.61 19.3 39.33 20.0 76.61 20.6 50.26 21.5 20.77 22.3
40.19 23.9 13.32 24.3 16.03 25.3 32.94 28.3 7.60 28.9 17.89 29.3
8.96 31.3 6.57 32.2 6.90 33.8 2.37
[0091] In one embodiment, crystalline psilocybin Polymorph B is
characterised by XRPD diffractogram peaks at 11.1, 11.8 and
14.3.degree.2.theta..+-.0.1.degree.2.theta.. In another embodiment,
crystalline psilocybin Polymorph B is further characterised by at
least one peak appearing at 14.9, 15.4, 19.3, 20.0 or
20.6.degree.2.theta..+-.0.1.degree.2.theta.. In another embodiment,
crystalline psilocybin Polymorph B is further characterised by at
least two peaks appearing at 14.9, 15.4, 19.3, 20.0 or
20.6.degree.2.theta..+-.0.1.degree.2.theta.. In yet a further
embodiment, crystalline psilocybin Polymorph B exhibits an XRPD
diffractogram substantially the same as the XRPD diffractogram
shown in FIG. 7c.
[0092] In one embodiment, crystalline psilocybin Polymorph B is
characterised by an endothermic event in a DSC thermogram having an
onset temperature of between 205 and 220.degree. C., such as
between 210 and 220.degree. C., such as between 210 and 218.degree.
C., or such as between 210 and 216.degree. C. In yet another
embodiment, crystalline psilocybin Polymorph B exhibits a DSC
thermogram substantially the same as the DSC thermogram in FIG.
8c.
[0093] In another embodiment, crystalline psilocybin Polymorph B is
characterised by having a water content of <0.5% w/w, such as
<0.4% w/w, such as <0.3% w/w, such as <0.2% w/w, or such
as <0.1% w/w. The skilled person would know of methods to
determine the water content of a compound, for example Karl Fischer
Titration. In one embodiment, crystalline psilocybin Polymorph B is
characterised by having <0.5% w/w loss, such as <0.4% w/w,
such as <0.3% w/w, such as <0.2% w/w, such as <0.1% w/w,
in the TGA thermogram between ambient temperature, such as about
25.degree. C., and 200.degree. C. In one embodiment, crystalline
psilocybin Polymorph B loses less than 2% by weight in a loss on
drying test, such as less than 1% by weight, such as less than 0.5%
by weight. The loss on drying test is performed at 70.degree.
C.
[0094] In one embodiment, crystalline psilocybin Polymorph B is a
highly pure crystalline form of Polymorph B, for example,
psilocybin comprises at least 90% by weight, such as 95%, such as
99%, such as 99.5% of Polymorph B.
[0095] In another embodiment, crystalline psilocybin Polymorph B is
chemically pure, for example the psilocybin has a chemical purity
of greater than 97%, such as greater than 98%, or such as greater
than 99% by HPLC. In one embodiment, crystalline psilocybin
Polymorph B has no single impurity of greater than 1%, more
preferably less than 0.5%, including phosphoric acid as measured by
.sup.31P NMR, and psilocin as measured by HPLC. In one embodiment,
crystalline psilocybin Polymorph B has a chemical purity of greater
than 97 area %, more preferably still greater than 98 area %, and
most preferably greater than 99 area % by HPLC. In one embodiment,
crystalline psilocybin Polymorph B has no single impurity greater
than 1 area %, more preferably less than 0.5 area % as measured by
HPLC. In one embodiment, crystalline psilocybin Polymorph B does
not contain psilocin at a level greater than 1 area %, more
preferably less than 0.5 area % as measured by HPLC. In one
embodiment, crystalline psilocybin Polymorph B does not contain
phosphoric acid at a level greater than 1 weight %, more preferably
less than 0.5 weight % as measured by .sup.31P NMR. In one
embodiment, crystalline psilocybin Polymorph B has a chemical assay
of at least 95 weight %, such as at least 96 weight %, or such as
at least 98 weight %.
[0096] The psilocybin of the invention in the form Polymorph A or
A' has the general properties illustrated in Table 5 below:
TABLE-US-00005 TABLE 5 Appearance: White to off white solid Major
endothermic event in 210-215.degree. C. DSC (onset temperature)
(corresponding to a melt): Hygroscopicity: Psilocybin forms Hydrate
A at high humidity and when added to water but the water of
hydration is lost rapidly on drying. The anhydrous form is
therefore being developed. Crystalline form: Anhydrous Polymorph A
and/ or A` pKa (calculated): 1.74, 6.71, 9.75 Solubility approx. 15
mg/ml in Water
[0097] The psilocybin conforms to the spectra as set out in Table 6
below and illustrated in the spectra of FIGS. 10-13.
TABLE-US-00006 TABLE 6 Technique Conclusions Proton (.sup.1H) and
Assignment of the proton (FIG. 10) and Carbon carbon spectra (FIG.
11) are concordant with (.sup.13C) NMR Psilocybin. FT-Infrared
Assignment of the FT-IR spectrum (FIG. 12) Spectroscopy is
concordant with Psilocybin. (FT-IR) Mass Spectroscopy Assignment of
the mass spectrum (FIG. 13) (MS) is concordant with Psilocybin.
[0098] The high purity is attained by careful control of reaction
conditions to ensure that potential organic impurities are
significantly reduced.
[0099] Known and potential impurities in Psilocybin are shown in
Table 7 below:
TABLE-US-00007 TABLE 7 Relative Retention Time Impurity (RRT)
Structure Origin Psilocin 1.65 ##STR00001## Starting material
(stage 3). Also generated by hydrolysis of Psilocybin. Only
significant impurity observed in psilocybin batches. Stage 4A
##STR00002## Initial product formed in the stage 4 reaction.
Converts to stage 4 on stirring in THF. Converts to Psilocybin in
stage 5. Stage 4 2.74 ##STR00003## Intermediate N-Denzylated stage
4 ##STR00004## Identified by MS in Stage 4. Converts to Psilocybin
in stage 5. Stage 4 Anhydride Impurity ##STR00005## Identified by
MS in Stage 4. Converts to Stage 5 Pyrophosphoric acid impurity in
stage 5. Stage 5 Pyrophosphoric acid impurity ##STR00006##
Identified by MS Formed from the stage 4 anhydride. Removed in the
stage 6 re- crystallisation by a combination of hydrolysis to
Psilocybin and increased solubility due to the extra phosphate
group. Stage 5 (intermediates) 1.89 and 2.45 ##STR00007## 2
Intermediates are formed during the hydrogenation. These
subsequently convert to product (structures based on chemistry).
Monitored and controlled in stage 5 reaction. ##STR00008##
[0100] Similarly, the careful processing ensures solvent levels are
kept to below levels as indicated in Table 8.
TABLE-US-00008 TABLE 8 Chemical Stage Solvent Controlled to solvent
is used in Methanol 3000 ppm Stage 5 Ethanol 5000 ppm Stage 5 THF
720 ppm Stage 4 Toluene 890 ppm Generated as a by-product in stage
5
[0101] Through careful selection of operating methodology the
psilocybin drug substance of the invention meets the acceptance
criteria set out in Table 9 below:
TABLE-US-00009 TABLE 9 Quality attribute Acceptance criteria Test
method 1. Appearance For information only. Visual 2. Identity by
.sup.1H NMR Compares well with refe- .sup.1H NMR rence. FIG. 10 3.
Identity by .sup.13C NMR Compares well with refe- .sup.13C NMR
rence. FIG. 11 4. Identity by MS Compares well with refe- MS rence.
FIG. 12 5. Identity by FT-IR Compares well with refe- FT-IR rence.
FIG. 13 6. Loss on drying NMT 2% w/w European Pharmacopoeia 2.2.32
7. Residue on Ignition NMT 0.5% w/w US Pharmaco- poeia <281>
8. Chemical purity NLT 97 area % HPLC 9. Drug Related No single
impurity NMT HPLC Impurities 1.0 area % 10. Assay (on a dry basis)
95-103 weight % HPLC 11. Residual Solvent Methanol NMT 3000 ppm
HRGC Content Ethanol NMT 5000ppm THF NMT 720 ppm Toluene NMT 890
ppm 12. Phosphoric acid NMT 1% w/w .sup.31P NMR content 13.
Elemental analysis Cd NMT 1.5 ppm US Pharmaco- by ICP-MS Pb NMT
1.5ppm poeia <233> As NMT 4.5 ppm Hg NMT 9.0 ppm Co NMT 15
ppm V NMT 30 ppm Ni NMT 60 ppm Li NMT 165 ppm Pd NMT 30 ppm 14.
Polymorphism Conforms to reference XRPD FIG. 7a 15. Melting Poin t
Report result DSC FIG. 8a Abbreviations used in table: NMT = not
more than, NLT = not less than.
[0102] The methodology used to verify the purity is provided in the
detailed description.
[0103] In fact, the criteria 6-13 are far exceeded in practice, as
noted in Table 10 below:
TABLE-US-00010 TABLE 10 Acceptance Quality attribute criteria
Typically Test method 1. Loss on drying Typically less than 1% w/w
European Pharmaco- poeia 2.2.32 2. Residue on Ignition Typically
less than 0.2% w/w US Pharmaco- poeia <281> 3. Chemical
purity Typically NLT 99% HPLC 4. Drug Related Impurity No single
RRT 1.49: 0.06% HPLC impurity NMT RRT 1.69 (Psilocin): 1.0% 0.39%
RRT 1.70: 0.05% Others LT 0.05%: 0.22% 5. Assay (on a dry basis)
95-103 98.65% HPLC 6. Residual Solvent Methanol NMT NMT 5 ppm HRGC
Content 3000 ppm NMT 10 ppm Ethanol NMT NMT 5 ppm 5000 ppm NMT 5
ppm THF NMT 720 ppm Toluene NMT 890 ppm 7. Phosphoric acid NMT 1%
w/w 0.2% .sup.31P NMR content Absence of phosphoric acid
(H.sub.3PO.sub.4) which comes at approx. 0 ppm 8. Elemental
analysis Cd NMT LT 0.5 ppm US Pharmaco- by ICP-MS 1.5 ppm LT 0.5
ppm poeia <233> Pb NMT LT 1 ppm 1.5 ppm LT 1 ppm As NMT LT 5
ppm 4.5 ppm LT 20 ppm Hg NMT LT 10 ppm 9.0 ppm LT 20 ppm Co NMT LT
5 ppm 15 ppm V NMT 30 ppm Ni NMT 60 ppm Li NMT 165 ppm Pd NMT 30
ppm Abbreviation used in table: NMT = not more than, LT = less
than.
[0104] Thus, crystalline psilocybin, in the form Polymorph A or
Polymorph A', has spectra that conform with Proton (.sup.1H) and
Carbon (.sup.13C) NMR, FT-Infrared Spectroscopy (FT-IR), and Mass
Spectroscopy (MS)--FIGS. 10-13.
[0105] It also conforms to any of the criteria specified in Table 9
or Table 10.
[0106] In accordance with a second aspect of the present invention
there is provided a batch of crystalline psilocybin, in the form
Polymorph A or Polymorph A' according to the first aspect of the
present invention. In one embodiment, there is provided a batch of
crystalline psilocybin, Polymorph A or Polymorph A', comprising at
least 10 g, more preferably at least 100 g, and most preferably at
least 250 g. In one embodiment, there is provided a batch of
crystalline psilocybin, Polymorph A or Polymorph A', comprising at
least 10 g, more preferably at least 100 g, and most preferably at
least 250 g. In one embodiment, there is provided a batch of high
purity psilocybin comprising at least 10 g, more preferably at
least 100 g, and most preferably at least 250 g. In one embodiment,
there is provided a batch of high purity psilocybin Polymorph A
comprising at least 10 g, more preferably at least 100 g, and most
preferably at least 250 g. In one embodiment, there is provided a
batch of high purity psilocybin Polymorph A' comprising at least 10
g, more preferably at least 100 g, and most preferably at least 250
g.
[0107] Alternatively, and independently, the crystalline psilocybin
may take the form of Hydrate A or Polymorph B.
[0108] In accordance with a third aspect of the present invention
there is provided a pharmaceutical formulation comprising
crystalline psilocybin and one or more excipients.
[0109] In one embodiment, there is provided a pharmaceutical
formulation comprising high purity psilocybin and one or more
excipients. In another embodiment, there is provided a
pharmaceutical formulation comprising crystalline psilocybin
Polymorph A and one or more excipients. In another embodiment,
there is provided a pharmaceutical formulation comprising
crystalline psilocybin Polymorph A' and one or more excipients. In
another embodiment, there is provided a pharmaceutical formulation
comprising high purity crystalline psilocybin, Polymorph A or
Polymorph A', and one or more excipients. In another embodiment,
there is provided a pharmaceutical formulation comprising high
purity crystalline psilocybin Polymorph A and one or more
excipients. In another embodiment, there is provided a
pharmaceutical formulation comprising high purity crystalline
psilocybin Polymorph A' and one or more excipients.
[0110] Alternatively, and independently, the crystalline psilocybin
in the formulation may take the form of Hydrate A or Polymorph
B.
[0111] Preferred pharmaceutical excipients for an oral formulation
include: diluents, such as, microcrystalline cellulose, starch,
mannitol, calcium hydrogen phosphate anhydrous or co-mixtures of
silicon dioxide, calcium carbonate, microcrystalline cellulose and
talc; disintegrants, such as, sodium starch glycolate or
croscarmellose sodium; binders, such as, povidone, co-povidone or
hydroxyl propyl cellulose; lubricants, such as, magnesium stearate
or sodium stearyl fumurate; glidants, such as, colloidal silicon
dioxide; and film coats, such as, Opadry II white or PVA based
brown Opadry II.
[0112] Psilocybin is a difficult active to formulate for a number
of reasons. Firstly it has poor flow characteristics, and secondly
it is used in relatively low doses which combination makes it
challenging to ensure content uniformity in tabletting.
[0113] A good blend will have an Acceptance Value, AV value of less
than 15, and more preferably less than 10.
[0114] It will also have a % Label claim of greater than 90% more
preferably greater than 94%.
[0115] Between them these parameters indicate consistent dosing of
the psilocybin between tablets.
[0116] For most pharmaceutical tablets, standard excipients,
particularly fillers, can be used. However, in the course of
formulating psilocybin tablets, applicant found that in order to
achieve a satisfactory product, a non-standard filler was
preferred.
[0117] In this regard a functional filler was selected. The
functional filler was a silicified filler, preferably a silicified
microcrystalline cellulose. The preferred forms comprises high
compactability grades with a particle size range of from about 45
to 150 microns.
[0118] In fact a mixture of two functional fillers having different
particle size ranges may be used with the wt percentages of the two
favouring the larger sized particles.
[0119] In one embodiment the silicified microcrystalline filler may
comprise a first filler, having a particle size range of from about
45 to 80 microns in an amount of up to 30%, more preferably up to
20%, more preferably still up to 15% or less and a second filler,
having a particle size range of from about 90 to 150 microns, in an
amount of up to 70%, more preferably up to 80, and more preferably
still up to 85% or more, by weight.
[0120] The formulation may further comprise or consist of a
disintegrant, preferably sodium starch glycolate, a glidant,
preferably colloidal silicon dioxide and a lubricant, preferably
sodium stearyl fumarate.
[0121] Further details of formulation development are given in
Example 12.
[0122] It should be noted that the formulations may comprise
psilocybin in any form, not only the preferred polymorphic forms
disclosed.
[0123] Studerus et al (2011) J Psychopharmacol 25(11) 1434-1452
classified oral doses of psilocybin as follows: Very low doses at
0.045 mgkg; low doses between 0.115-0.125 mg/kg, medium doses
between 0.115-0.260 mg/kg, and high doses at 0.315 mg/kg.
[0124] The psilocybin would typically be present in a formulated
dose in an amount of from 0.01 mg/kg to 1 mg/kg. A typical human
dose (for an adult weighing 60-80 kg) would equate to a dose of
somewhere between 0.60 mg and 80 mg. In one embodiment, between 2
and 50 mg of crystalline psilocybin, most preferably Polymorph A or
Polymorph A', is present in a formulated dose, such as between 2
and 40 mg, such as between 2 and 10 mg, such as 5 mg, such as
between 5 and 30 mg, such as between 5 and 15 mg, such as 10 mg,
such as between 20 and 30 mg, or such as 25 mg. In one embodiment,
between 2 and 50 mg of crystalline psilocybin, particularly
Polymorph A, is present in a formulated dose, such as between 2 and
40 mg, such as between 2 and 10 mg, such as 5 mg, such as between 5
and 30 mg, such as between 5 and 15 mg, such as 10 mg, such as
between 20 and 30 mg, or such as 25 mg. In one embodiment, between
2 and 50 mg of crystalline psilocybin, particularly Polymorph A' is
present in a formulated dose, such as between 2 and 40 mg, such as
between 2 and 10 mg, such as 5 mg, such as between 5 and 30 mg,
such as between 5 and 15 mg, such as 10 mg, such as between 20 and
30 mg, or such as 25 mg.
[0125] Favoured adult oral doses are likely to be in the range 1 mg
to 40 mg, preferably 2 to 30 mg, more preferably 15 to 30 mg, for
example 5 mg, 10 mg or 25 mg. Micro-dosing, typically at about a
tenth of these doses, is also possible with micro dose formulations
typically lying within the range 0.05 mg to 2.5 mg.
[0126] A preferred pharmaceutical formulation is an oral dosage
form.
[0127] The oral dosage form may be a tablet or a capsule.
[0128] For a tablet it is necessary to be able to accurately
disperse the active. This is challenging due to the low doses and
the hygroscopic and sticky nature of the active which limits its
flowability.
[0129] The psilocybin will be present together with one or more
excipients. Preferred excipients include microcrystalline cellulose
and starch, more particularly still a silicified microcrystalline
cellulose.
[0130] In accordance with a fourth aspect of the present invention
there is provided the crystalline psilocybin in the form Polymorph
A or Polymorph A' according to the first aspect of the present
invention for use in medicine. In one embodiment, there is provided
crystalline psilocybin Polymorph A for use in medicine. In one
embodiment, there is provided crystalline psilocybin Polymorph A'
for use in medicine. In one embodiment, there is provided a high
purity crystalline psilocybin Polymorph A for use in medicine. In
one embodiment, there is provided a high purity crystalline
psilocybin Polymorph A' for use in medicine.
[0131] Alternatively, and independently, the crystalline psilocybin
may take the form of Hydrate A or Polymorph B.
[0132] In accordance with a fifth aspect of the present invention
there is provided crystalline psilocybin in the form Polymorph A or
Polymorph A' of the first aspect of the present invention for use
in treating central nervous disorders.
[0133] Alternatively, and independently, the crystalline psilocybin
may take the form of Hydrate A or Polymorph B.
[0134] In one embodiment, there is provided crystalline psilocybin,
Polymorph A or Polymorph A', for use in treating depression. In one
embodiment, there is provided crystalline psilocybin, Polymorph A
or Polymorph A', for use in treating drug resistant depression. In
one embodiment, there is provided crystalline psilocybin Polymorph
A for use in treating drug resistant depression. In one embodiment,
there is provided crystalline psilocybin Polymorph A' for use in
treating drug resistant depression. In one embodiment, there is
provided a high purity crystalline psilocybin Polymorph A for use
in treating drug resistant depression. In one embodiment, there is
provided a high purity crystalline psilocybin Polymorph A' for use
in treating drug resistant depression.
[0135] Other conditions that may be treated include: anxiety
disorders, including anxiety in advanced stage illness e.g. cancer
as well as Generalized Anxiety Disorder, Depression including Major
Depressive Disorder, Cluster Headaches, Obsessive Compulsive
Disorder, Personality Disorders including Conduct Disorder, Drug
Disorders including: alcohol dependence, nicotine dependence,
opioid dependence, cocaine dependence and other addictions
including Gambling Disorder, Eating Disorder and Body Dysmorphic
Disorder. A still further condition is the treatment of pain.
[0136] In accordance with a sixth aspect of the present invention
there is provided a method of treating central nervous disorders
comprising administering to a subject in need thereof an effective
dose of crystalline psilocybin in the form Polymorph A or Polymorph
A' according to the first aspect of the present invention.
[0137] In one embodiment, there is provided a method of treating
depression comprising administering to a subject in need thereof an
effective dose of crystalline psilocybin in the form of Polymorph A
or Polymorph A'. In one embodiment, there is provided a method of
treating drug resistant depression comprising administering to a
subject in need thereof an effective dose of crystalline psilocybin
in the form Polymorph A or Polymorph A'. In one embodiment, there
is provided a method of treating drug resistant depression
comprising administering to a subject in need thereof an effective
dose of psilocybin Polymorph A. In one embodiment, there is
provided a method of treating drug resistant depression comprising
administering to a subject in need thereof an effective dose of
psilocybin Polymorph A'. In one embodiment, there is provided a
method of treating drug resistant depression comprising
administering to a subject in need thereof an effective dose of a
high purity crystalline psilocybin Polymorph A. In one embodiment,
there is provided a method of treating drug resistant depression
comprising administering to a subject in need thereof an effective
dose of a high purity crystalline psilocybin Polymorph A'.
[0138] Alternatively, and independently, the crystalline psilocybin
may take the form of Hydrate A or Polymorph B.
[0139] To produce the psilocybin of the invention the psilocybin
was crystallised from water in a controlled manner.
[0140] According to a seventh aspect of the present invention there
is provided a method for large scale manufacture of psilocybin
characterised in that the method comprises subjecting psilocybin to
a water crystallization step, with controlled drying, to produce
crystalline psilocybin Polymorph A according to the first aspect of
the present invention.
[0141] In one embodiment, there is provided a method for large
scale manufacture of psilocybin characterised in that the method
comprises subjecting psilocybin to a water crystallization step,
with controlled drying, to produced crystalline psilocybin
Polymorph A with an XRPD diffractogram as illustrated in FIG. 7a
and a DSC and TGA thermograph as illustrated in FIG. 8a. In one
embodiment, there is provided a method for large scale manufacture
of psilocybin characterised in that the method comprises subjecting
psilocybin to a water crystallization step, with controlled drying,
to produce a high purity crystalline psilocybin--Polymorph A with
an XRPD diffractogram as illustrated in FIG. 7a and a DSC
thermograph as illustrated in FIG. 8a.
[0142] Preferably Polymorph A is an isostructural variant with an
XRPD diffractogram as illustrated in FIG. 7a and a DSC thermograph
as illustrated in FIG. 8a.
[0143] More preferably the psilocybin is recrystallized in
typically about 10-20 volumes of water, heated with agitation to a
temperature of at least 70.degree. C., polish filtered with a
suitable cut off (typically, below 5 .mu.m), seeded at a
temperature of about 70.degree. C., and cooled in a controlled
manner to about 5.degree. C. over a period of more than 2
hours.
[0144] More preferably the method comprises controlled cooling
which drops the temperature by about 5.degree. C.-15.degree. C. an
hour, more preferably about 10.degree. C. an hour.
[0145] Preferably the polish filter step is done through an
appropriately sized filter such as a 1.2 .mu.m in line filter.
[0146] Preferably the agitation is by stirring at about 400-500
rpm, typically about 450 rpm.
[0147] Preferably the seed is psilocybin Hydrate A. In one
embodiment, 0.1% weight or less of seed is added to the
process.
[0148] Preferably the crystalline psilocybin is isolated by vacuum
filtration.
[0149] In one embodiment, the isolated crystals are dried in vacuo
at a temperature of at least 30.degree. C., such as between 30 and
50.degree. C., or such as between 40 and 50.degree. C. In one
embodiment, the isolated crystals are dried in vacuo for at least
10 hours, such as between 12 and 18 hours, or such as about 30
hours. In one embodiment, the isolated crystals are dried in vacuo
at a temperature of at least 30.degree. C., such as between 30 and
50.degree. C., or such as between 40 and 50.degree. C., for at
least 10 hours, such as between 12 and 18 hours, or such as about
30 hours. In one embodiment, the isolated crystals are dried until
the isolated crystals lose less than 2% weight in a loss on drying
test, such as less than 0.5% weight.
[0150] Preferably the isolated crystals are washed, several times,
in water and dried in vacuo at about 50.degree. C. for at least 12
hours.
[0151] The crystals obtained are typically relatively large (range
50 to 200 microns) and uniform when viewed under the
microscope.times.10, as illustrated in FIG. 16a.
[0152] This differs from crystals obtained without controlled
cooling which are much smaller in size (typically 5 to 50 microns)
when viewed under the microscope.times.10, as illustrated in FIG.
16b.
[0153] In accordance with an eighth aspect of the present invention
there is provided Psilocybin according to the first aspect of the
present invention obtained by the method of crystallisation of the
invention.
[0154] In accordance with a ninth aspect of the present invention
there is provided a pharmaceutical formulation comprising
psilocybin according to the first aspect of the present invention
obtained by the method of crystallisation of the invention.
[0155] The psilocybin manufactured prior to crystallisation may be
produced using any method: synthetic or biological, e.g. by
fermentation or obtained by extraction from mushrooms.
[0156] Preferred manufacturing methods use psilocin, or 4
hydroxy-indole, as a starting material.
[0157] In accordance with a tenth aspect of the present invention
there is provided a method for large scale manufacture of
psilocybin from psilocin comprising the steps of:
i) Stage 4--Reacting psilocin with tetrabenzylpyrophosphate to form
benzyl 3-[2-(benzyldimethylazaniumyl)ethyl]-1H-indol-4-yl
phosphate; and ii) Stage 5--Reacting benzyl
3-[2-(benzyldimethylazaniumyl) ethyl]-1H-indol-4-yl phosphate with
hydrogen to form psilocybin.
[0158] In accordance with an eleventh aspect of the present
invention there is provided a method for large scale manufacture of
psilocybin from 4-hydroxyindole comprising the steps of:
i) Stage 1--Reacting 4-hydroxyindole with acetic anhydride to form
1H-indol-4-yl acetate; ii) Stage 2--Reacting 1H-indol-4-yl acetate
with oxalyl chloride and dimethylamine to form
3[(dimethylcarbamoyl)carbonyl]-1H-indol-4yl-acetate; iii) Stage
3--Reacting 3[(dimethylcarbamoyl)carbonyl]-1H-indol-4yl-acetate
with lithium aluminium hydride to form psilocin; iv) Stage
4--Reacting psilocin with tetrabenzylpyrophosphate to form benzyl
3-[2-(benzyldimethylazaniumyl)ethyl]-1H-indol-4-yl phosphate; and
v) Stage 5--Reacting benzyl 3-[2-(benzyldimethylazaniumyl)
ethyl]-1H-indol-4-yl phosphate with hydrogen to form
psilocybin.
[0159] In accordance with a twelfth aspect of the present invention
there is provided a method for large scale manufacture of
psilocybin as per the tenth or eleventh aspect of the present
invention further comprising:
vi) Stage 6--a water crystallization step, with controlled drying,
to produce crystalline psilocybin Polymorph A according to the
first aspect of the present invention.
[0160] In one embodiment, there is provided a method for large
scale manufacture of psilocybin as per the tenth or eleventh aspect
of the present invention further comprising:
vi) Stage 6--a water crystallization step, with controlled drying,
to produce crystalline psilocybin--Polymorph A with an XRPD
diffractogram as substantially illustrated in FIG. 7a and a DSC
thermograph as substantially illustrated in FIG. 8a.
[0161] In one embodiment, there is provided a method for large
scale manufacture of psilocybin as per the tenth or eleventh aspect
of the present invention further comprising:
vi) Stage 6--a water crystallization step, with controlled drying,
to produce a high purity crystalline psilocybin--Polymorph A with
an XRPD diffractogram as illustrated in FIG. 7a and a DSC
thermograph as illustrated in FIG. 8a.
[0162] Preferably the crystalline psilocybin is Polymorph A.
[0163] In developing methodology for the large scale production of
psilocin or psilocybin the Applicant overcame one or more
significant problems at each of Stages 1 to 5, and whilst these
problems are considered in the context of the large scale
production of psilocin or psilocybin each step, or rather the way
each problem was overcome, are considered separate and independent
inventions as they have application in the manufacture of other
actives be they intermediates to psilocin, psilocybin, or other
derivatives, salts, esters or the like which provide a prodrug.
[0164] Preferably the Stage 4 (i) reaction comprises the use of
sodium hexamethyldisilazide (NaHMDS).
[0165] This has the benefits over the use of Butyl lithium in that:
i) it is easier to handle, and ii) it does not introduce lithium
into the reaction which causes issues in downstream processing.
[0166] Preferably the reaction uses the solvent THF.
[0167] This has the benefit that resulting product is obtained in
significantly higher purity.
[0168] Preferably in (i) the reaction is initiated below
-50.degree. C.
[0169] This has the benefit of reducing the levels of impurities
(m/z 295.2 observed by LCMS) that will subsequently effect purity
downstream.
[0170] More preferably still the Stage 4 (ii) step uses THF as the
solvent.
[0171] This has the benefit of ensuring thickening is avoided and
facilitates a simple stir out process for obtaining the
product.
[0172] Preferably the Stage 4 (ii) step comprises a stir out
process to obtain benzyl 3-[2-(benzyldimethylazaniumyl)
ethyl]-1H-indol-4-yl phosphate.
[0173] A stir out process has the advantage that the process is
simplified and yields are improved.
[0174] To ensure the Stage 4 (ii) reaction is run to completion,
levels of Intermediate 4A are monitored, and on completion, the
benzyl 3-[2-(benzyldimethylazaniumyl) ethyl]-1H-indol-4-yl
phosphate is filtered and oven dried.
[0175] This has the advantage that impurities are minimised and a
purer product is obtained
[0176] Preferably the Stage 5 reaction is monitored for levels of
intermediates by HPLC, using relative retention times (RRT) and
completion is determined by the intermediates being present at less
than 0.2%.
[0177] Psilocybin crude (Stage 5 product, (12)) has main stage 5
impurities whose relative retention times (RRT) in the HPLC method
are about 1.89 and 2.45 respectively, and psilocin (RRT 1.66).
These impurities are illustrated in Table 7. Typically, psilocybin
crude (Stage 5 product (12)) has 0.24 area % of the RRT 1.89
impurity, 0.03 area % of the RRT 2.45 impurity and 1.86 area % of
psilocin. In addition, the pyrophosphoric acid impurity (RRT 0.31)
is present in psilocybin crude, for example at a level of about 2-6
area % by HPLC.
[0178] At this level subsequent crystallisation processes can be
conducted to provide substantially pure psilocybin, for example
psilocybin having a purity of at least 95 area % by HPLC, such as
at least 98 area %, or such as at least 99 area %. In one
embodiment, the pyrophosphoric acid impurity (RRT 0.31) is present
in the substantially pure psilocybin at a level of less than 0.3
area % by HPLC, such as less than 0.2 area %, or such as less than
0.1 area %.
[0179] In addition, during this stage water is added to the
reaction to maintain the psilocybin in solution.
[0180] Preferably the catalyst is recovered by filtration.
[0181] Preferably in Stage 1 the reaction is conducted in DCM and
pyridine.
[0182] This has the advantage that flammable solvents are
avoided.
[0183] Preferably the reaction mixture is washed with citric acid,
to give a pH of about 2-3, to remove excess pyridine, and the acid
phase is separated from the DCM phase.
[0184] This has the advantage that the Intermediate 2A can be
isolated, allowing purification away from excess oxalyl
chloride.
[0185] More preferably the DCM phase is further washed with sodium
bicarbonate at about pH 8.
[0186] This has the advantage of purer processing.
[0187] Preferably the 1H-indol-4-yl acetate is precipitated in
heptane.
[0188] This aids precipitation and overcomes partial solubility
issues.
[0189] Preferably magnesium sulphate is used as a drying agent.
[0190] Preferably the solvents tert butyl methyl ether (TBME) and
tetrahydrofuran (THF) are used.
[0191] Preferably the reaction with oxalyl chloride is conducted at
about 30.degree. C.-40.degree. C.
[0192] This has the advantage that a high reaction rate is ensured
giving improved levels of completion.
[0193] Preferably Intermediate 2A is isolated by filtration.
[0194] This has the advantage that the intermediate is purified
away from excess oxalyl chloride.
[0195] Preferably in Stage 2, step i the Intermediate 2A is also
washed to remove excess oxalyl chloride.
[0196] Preferably the Intermediate 2A is washed with TBME.
[0197] Preferably a heptane addition is made to precipitate out
further Intermediate 2A.
[0198] Preferably in Stage 2, step ii, dimethyl amine is used in
excess.
[0199] This has the advantage that a much improved impurity profile
and yield is obtained.
[0200] Preferably the pH is maintained at about or above pH 7.
[0201] Preferably the reaction is carried out in TBME.
[0202] Preferably this stage further comprises a purification step
that removes dimethyl amine salts.
[0203] This has the advantage that purity is improved.
[0204] Preferably this stage comprises a slurry and filtration
step.
[0205] This has the advantage that handling and purity is
improved.
[0206] More preferably it comprises slurrying with water and/or
IPA, filtering, and drying the isolated
3[(dimethylcarbamoyl)carbonyl]-1H-indol-4yl-acetate.
[0207] This has the advantage that purity and yields are improved
and hydrolysis reduced.
[0208] Preferably in Stage 3 the reaction is conducted in the
solvent THF.
[0209] This has the advantage that a suspension/emulsion is formed
without thickening.
[0210] Preferably the
3[(dimethylcarbamoyl)carbonyl]-1H-indol-4yl-acetate is added to a
solution of LiAlH.sub.4 in THF.
[0211] Preferably the reaction is quenched with acetone, followed
by citric acid ensuring the mixture remains strongly basic (pH11 or
above).
[0212] This has the advantage that high yields are obtained.
[0213] Preferably the psilocin is filtered and washed in THF and
slurried in PrOAc:TBME, filtered, washed in TBME, and dried.
[0214] This has the advantage that a high purity product is
obtained, for example, at least 95% pure by HPLC, such as at least
98% pure by HPLC, or such as at least 99% pure by HPLC.
[0215] The favoured production method comprises each of Stages 1 to
6 but it will be appreciated that each of the features of each
stage can stand alone or be used in combination with any other
feature from the same or a different step of the reaction.
[0216] Psilocybin of a given form, Polymorph A or Polymorph A', and
psilocybin of such high purity has not previously been obtained,
and to Applicants knowledge their production of Polymorph A and
Polymorph A' particularly (as illustrated in FIGS. 7a & 7b and
8a & 8b) is novel. Indeed, the production of large batch
quantities of Polymorph A, is new. A consequence of the
crystallisation methodology of the invention and, in part, the
manufacturing process enable such high chemical purity of
crystalline Psilocybin to be obtained.
[0217] Furthermore, given the unstable nature of the compound they
have obtained a crystalline form which they have shown to be
stable, under accelerated conditions, (described later) for at
least 12 months.
[0218] Polymorph A and A' (FIGS. 7a and 7b) differs from Polymorph
B (FIG. 7c), a Hydrate A (FIG. 7d) and ethanol solvate (FIG. 7e)
and mixture (FIG. 7f (upper)) as will be apparent from their XRPD
diffractograms and DSC thermographs--as described hereafter.
[0219] The relationship between the different polymorphs is shown
in FIG. 9.
[0220] Indeed, the size and shape of the crystals are determined by
the crystallisation methodology, and these in turn can affect
stability and the ability to formulate the product.
[0221] In a particularly preferred embodiment, the psilocybin is
manufactured through a 6-stage process as outlined below:
[0222] In accordance with another aspect of the present invention
there is provided a method for manufacture of crystalline
psilocybin according to the first aspect of the present invention,
characterised in that the method comprises subjecting psilocybin to
a water crystallization step, with controlled drying, to produce
crystalline psilocybin Polymorph A or Polymorph A' according to the
first aspect of the present invention. In one embodiment, there is
provided a method for manufacture of crystalline psilocybin
according to the first aspect of the present invention,
characterised in that the method comprises a water crystallization
step, with controlled drying, to produce crystalline
psilocybin--Polymorph A or Polymorph A' with an XRPD diffractogram
as substantially illustrated in FIG. 7a or FIG. 7b and a DSC
thermograph as substantially illustrated in FIG. 8a or 8b. In one
embodiment, there is provided a method for manufacture of
psilocybin according to the first aspect of the present invention
characterised in that the method comprises a water crystallization
step, with controlled drying, to produce a high purity crystalline
psilocybin--Polymorph A or Polymorph A' with an XRPD diffractogram
as illustrated in FIG. 7a or FIG. 7b and a DSC thermograph as
illustrated in FIG. 8a or FIG. 8b.
[0223] Preferably Polymorph A and Polymorph A' are isostructural
variants with XRPD diffractograms as substantially illustrated in
FIG. 7a and FIG. 7b and DSC thermographs as substantially
illustrated in FIG. 8a and FIG. 8b.
[0224] More preferably the psilocybin is recrystallized in about
10-20 volumes of water, heated with agitation to a temperature of
at least 70.degree. C., polish filtered with a suitable cut off
(typically, below 5 .mu.m), seeded at a temperature of about
70.degree. C., and cooled in a controlled manner to about 5.degree.
C. over a period of more than 2 hours.
[0225] More preferably the method comprises controlled cooling
which drops the temperature by about 5.degree. C.-15.degree. C. an
hour, more preferably about 10.degree. C. an hour.
[0226] Preferably the polish filter step is done through an
appropriately sized filter such as a 1.2 .mu.m or a 0.45 .mu.m in
line filter.
[0227] Preferably the agitation is by stirring at about 400-500
rpm, typically about 450 rpm.
[0228] Preferably the seed is psilocybin Hydrate A. In one
embodiment, 0.1% weight or less of seed is added to the
process.
[0229] Preferably the crystalline psilocybin is isolated by vacuum
filtration.
[0230] In one embodiment, the isolated crystals are dried in vacuo
at a temperature of at least 30.degree. C., such as between 30 and
50.degree. C., or such as between 40 and 50.degree. C. In one
embodiment, the isolated crystals are dried in vacuo for at least
10 hours, such as between 12 and 18 hours, or such as about 30
hours. In one embodiment, the isolated crystals are dried in vacuo
at a temperature of at least 30.degree. C., such as between 30 and
50.degree. C., or such as between 40 and 50.degree. C., for at
least 10 hours, such as between 12 and 18 hours, or such as about
30 hours. In one embodiment, the isolated crystals are dried until
the isolated crystals lose less than 2% weight in a loss on drying
test, such as less than 0.5% weight.
[0231] Preferably the isolated crystals are washed, several times,
in water and dried in vacuo at about 50.degree. C. for at least 12
hours.
[0232] The crystals obtained are typically relatively large (range
50 to 200 microns) and uniform when viewed under the
microscope.times.10, as illustrated in FIG. 16a.
[0233] This differs from crystals obtained without controlled
cooling which are much smaller in size (typically 5 to 50 microns)
when viewed under the microscope.times.10, as illustrated in FIG.
16b.
Stage 1: Synthesis of 1H-indol-4-yl acetate (3)
[0234] The core reaction is the reaction of 4-hydroxyindole (1)
with acetic anhydride (2) to form 1H-indol-4-yl acetate (3); (FIG.
2)
[0235] Most preferably stage 1 is as follows:
[0236] 4-hydroxyindole (1), DCM (12), and pyridine (13) are added
to a vessel and cooled to about 0-5.degree. C. Acetic anhydride (2)
is added dropwise, and the mixture warmed to about 20-25.degree. C.
and stirred until complete by HPLC. The reactants are washed with
aqueous citric acid solution (14) and aqueous NaHCO.sub.3 (15),
dried over MgSO.sub.4 (16) filtered and evaporated to approximately
half volume. Heptane (17) is added, and distillation continued to
remove the majority of the DCM. The mixture is cooled to about
5-25.degree. C., filtered, washed with heptane and dried in a
vacuum oven overnight to isolate 1H-indol-4-yl acetate (3) as a
solid suitable for use in the following stage.
Stage 2: Synthesis of
3[(dimethylcarbamoyl)carbonyl]-1H-indol-4yl-acetate (6)
[0237] The core reaction is the reaction of 1H-indol-4-yl acetate
(3) with Oxalyl chloride (4) and dimethylamine (5) to form
3[(dimethylcarbamoyl)carbonyl]-1H-indol-4yl-acetate (6); (FIG.
3)
[0238] Most preferably stage 2 is as follows:
[0239] 1H-indol-4-yl acetate (3) is dissolved in a mixture of THF
(19) and TBME (18) at room temperature. Oxalyl chloride (4) was
added dropwise allowing the reaction to exotherm at about
35-40.degree. C. The temperature range is maintained throughout the
remainder of the addition. The reaction is then stirred at about
40.degree. C. until complete by HPLC. The reaction is cooled to
room temperature and heptane (17) added resulting in precipitation
of further solids. The slurry is stirred, then allowed to settle,
followed by removal of the majority of the solvent (18/19) by
decanting. The solid was washed in the vessel twice with heptane
(17). TBME (18) is added to give a yellow slurry and the mixture
cooled to about -20.degree. C. Dimethylamine solution (5) is added
maintaining the temperature at -20.degree. C. to -10.degree. C. The
reaction was then warmed to room temperature and stirred until
complete, adding extra dimethylamine if necessary. The reaction was
filtered, washed with heptane (17) and dried in a vacuum oven. The
crude 3[(dimethylcarbamoyl)carbonyl]-1H-indol-4yl-acetate (6) was
further purified by a slurry in water (20), then IPA (21) and then
dried in a vacuum oven to yield (6) as a solid suitable for use in
the following stage.
Stage 3: Synthesis of 3-(2-(dimethylamino) ethyl)-1H-indol-4-ol
(Psilocin) (8)
[0240] The core reaction is the reaction of
3[(dimethylcarbamoyl)carbonyl]-1H-indol-4yl-acetate (6) with
lithium aluminium hydride (7) to form psilocin (8); (FIG. 4)
[0241] Most preferably stage 3 is as follows:
[0242] The 3[(dimethylcarbamoyl)carbonyl]-1H-indol-4yl-acetate (6)
was slurried in THF (19) and cooled to about 0.degree. C. A THF
solution of LiAlH.sub.4 (7) was added dropwise maintaining the
temperature at about 0-20.degree. C. The reaction was then refluxed
until complete by HPLC. The reaction was cooled to 0.degree. C. and
the excess LiAlH.sub.4 quenched by addition of acetone (22)
followed by aqueous citric acid solution (14). The batch was
filtered to remove Lithium and Aluminium salts. The filtrate was
dried over MgSO.sub.4 (16), filtered and concentrated and loaded
onto a silica pad (23). The pad was eluted with THF (19) and the
product containing fractions evaporated. The resulting solid was
slurried in iPrOAc:TBME (24/18) mixture, filtered and washed with
TBME. The solid was dried in the oven to yield high purity psilocin
(8) as an off white solid.
Stage 4: Synthesis of benzyl
3-[2-(benzyldimethylazaniumyl)ethyl]-1H-indol-4-yl phosphate
(10)
[0243] The core reaction is the reaction of psilocin (8) with
tetrabenzylpyrophosphate (9) to form benzyl
3-[2-(benzyldimethylazaniumyl)ethyl]-1H-indol-4-yl phosphate (10),
(FIG. 5)
[0244] Most preferably stage 4 is as follows:
[0245] Charge psilocin (8) to a vessel followed by THF (19). The
reaction was cooled to -50.degree. C. to -70.degree. C. and NaHMDS
(25) was added dropwise at about -45.degree. C. to -70.degree. C.
The temperature was adjusted to about -45.degree. C. to -60.degree.
C. and tetrabenzylpyrophosphate in THF was added. The batch was
allowed to warm to 0.degree. C. after which the solid by products
were removed by filtration and the filtrate concentrated in vacuo.
The concentrated mixture was then heated to about 40.degree. C. and
stirred until the intermediate had converted to the stage 4 product
(10)--controlled by monitoring and the use of HPLC. The batch was
cooled to about 0-5.degree. C. and the resulting solid isolated by
filtration and dried in vacuo to provide benzyl
3-[2-(benzyldimethylazaniumyl)ethyl]-1H-indol-4-yl phosphate (10)
as a solid.
Stage 5: Synthesis of Intermediate Grade 3-[2-(dimethylazaniumyl)
ethyl]-1H-indol-4-yl hydrogen phosphate (Psilocybin Crude) (12)
[0246] The core reaction comprises reacting benzyl
3-[2-(benzyldimethylazaniumyl) ethyl]-1H-indol-4-yl phosphate (10)
with hydrogen (11) to form psilocybin (12), (FIG. 6).
[0247] Most preferably stage 5 is as follows:
[0248] To a vessel was charged Pd/C (26), methanol (24) and
3-[2-(benzyldimethylazaniumyl)ethyl]-1H-indol-4-yl phosphate (10)
and the resulting mixture sparged with hydrogen (11) until complete
by HPLC. Purified water (20) is added during this process to retain
the product in solution. The mixture was heated to about 35.degree.
C.-45.degree. C. and then filtered through a bed of Celite (27)
washing with methanol (24) and purified water (20). The filtrate
was evaporated in vacuo, azeotroping with ethanol (28) to obtain
intermediate grade psilocybin (12).
Stage 6: Synthesis of 3-[2-(dimethylazaniumyl) ethyl]-1H-indol-4-yl
hydrogen phosphate (Psilocybin)
[0249] The core purifying/polymorph determining step is a water
crystallization step, followed by a controlled cooling and drying
step, to produce high purity crystalline psilocybin, Polymorph A or
Polymorph A'.
[0250] Most preferably stage 6 is as follows:
[0251] The intermediate grade Psilocybin (12) (stage 5) was charged
to a vessel with purified water (20) and the mixture heated until
the psilocybin (12) dissolved. The resulting bulk solution was then
polish filtered into a pre-warmed vessel. The temperature was
adjusted to, preferably, about 68.degree. C.-70.degree. C., and a
Psilocybin hydrate seed (i.e., Hydrate A) was added to the
reaction. The batch was then cooled in a controlled manner to about
0-10.degree. C. and stirred, and the solids were collected by
filtration and washed with purified water. The isolated solids were
then dried in vacuo to yield high purity crystalline Psilocybin,
Polymorph A or A', as an off white solid.
BRIEF DESCRIPTION OF THE DRAWINGS
[0252] Embodiments of the invention are further described
hereinafter with reference to the accompanying drawings, in
which:
[0253] FIG. 1 is a schematic of the reaction taught in JNP;
[0254] FIG. 2 is a schematic of the Stage 1 reaction of one aspect
of the present invention;
[0255] FIG. 3 is a schematic of the Stage 2 reaction of one aspect
of the present invention;
[0256] FIG. 4 is a schematic of the Stage 3 reaction of one aspect
of the present invention;
[0257] FIG. 5 is a schematic of the Stage 4 reaction of one aspect
of the present invention;
[0258] FIG. 6 is a schematic of the Stage 5 reaction of one aspect
of the present invention;
[0259] FIG. 7a is a XRPD diffractogram of Polymorph A (GM764B);
[0260] FIG. 7b is a XRPD diffractogram of Polymorph A'
(JCCA2160F);
[0261] FIG. 7c is a XRPD diffractogram of Polymorph B;
(JCCA2160-F-TM2);
[0262] FIG. 7d is a XRPD diffractogram of a Hydrate A
(JCCA2157E);
[0263] FIG. 7e is a XRPD diffractogram of an ethanol solvate
(JCCA2158D);
[0264] FIG. 7f is a XRPD diffractogram of product obtained during
development of the process (CB646-E) (top)--compared to the
diffractograms Polymorph A' (JCCA2160F) (middle) and Polymorph B
(JCCA2160-TM2) (bottom);
[0265] FIG. 8a is a DSC and TGA thermograph of Polymorph A
(GM764B);
[0266] FIG. 8b is a DSC and TGA thermograph of Polymorph A'
(JCCA2160F);
[0267] FIG. 8c is a DSC thermograph of Polymorph B (GM748A);
[0268] FIG. 8d is a DSC and TGA thermograph of Hydrate A
(JCCA2157E);
[0269] FIG. 8e is a DSC and TGA thermograph of ethanol solvate
(JCCA2158D);
[0270] FIG. 9 is a form phase diagram showing the
inter-relationship of form in water-based systems;
[0271] FIG. 10 is a 1H NMR spectrum of Psilocybin; (Read alongside
assignment Example 7);
[0272] FIG. 11 is a .sup.13C NMR spectrum of Psilocybin; (Read
alongside assignment Example 7);
[0273] FIG. 12 is a FT-IR Spectrum of Psilocybin;
[0274] FIG. 13 is a Mass Spectrum of Psilocybin;
[0275] FIG. 14 is a numbered structural formula of Psilocybin;
[0276] FIG. 15 is a temperature solubility curve for Psilocybin in
water;
[0277] FIG. 16a is a micrograph showing crystals obtained by
controlled cooling;
[0278] FIG. 16b is a micrograph showing crystals obtained by
uncontrolled cooling drying;
[0279] FIG. 17 is a form phase diagram showing the
inter-relationship of forms in different solvent systems;
[0280] FIG. 18 is XRPD diffractogram--Pattern C for solids isolated
at 25 and 50.degree. C.;
[0281] FIG. 19 is XRPD diffractograms--Patterns D, E and F for
solids isolated at 25 and 50.degree. C.;
[0282] FIG. 20 is a comparison of the XRPD diffractograms acquired
for the solids isolated from the equilibration of amorphous
Psilocybin in solvents A to H;
[0283] FIG. 21 is a comparison of the XRPD diffractograms acquired
for the solids isolated from the equilibration of amorphous
Psilocybin in solvents I to P; and
[0284] FIG. 22 is a comparison of the XRPD diffractograms acquired
for the solids isolated from the equilibration of amorphous
Psilocybin in solvents R to Y.
DETAILED DESCRIPTION
[0285] In contrast to the prior art, the present invention sought
to produce psilocybin at a commercial large scale, in amounts or
batches of at least 100 g, and more preferably at least 250 g,
levels 1 log or 2 logs higher than the levels described in JNP,
which describes a "large" scale method to producing gram quantities
on a 10 g scale.
To demonstrate the many significant development steps from JNP, the
description below sets out details of experiments and
investigations undertaken at each of the process stages, which
illustrate the selections made to overcome the numerous technical
problems faced, in producing psilocybin (7) to GMP at a large scale
(including the various intermediates (2-6)) starting from
4-hydroxyindole (1).
[0286] Reference to a particular numerical value includes at least
that particular value, unless the context clearly dictates
otherwise. When a range of values is expressed, another embodiment
includes from the one particular value and/or to the other
particular value. Further, reference to values stated in ranges
include each and every value within that range. All ranges are
inclusive and combinable.
[0287] When values are expressed as approximations, by use of the
antecedent "about," it will be understood that the particular value
forms another embodiment.
[0288] As used herein, the singular forms "a," "an," and "the"
include the plural.
[0289] The term "about" when used in reference to numerical ranges,
cut-offs, or specific values is used to indicate that the recited
values may vary by up to as much as 10% from the listed value. As
many of the numerical values used herein are experimentally
determined, it should be understood by those skilled in the art
that such determinations can, and often times will, vary among
different experiments. The values used herein should not be
considered unduly limiting by virtue of this inherent variation.
Thus, the term "about" is used to encompass variations of .+-.10%
or less, variations of .+-.5% or less, variations of .+-.1% or
less, variations of .+-.0.5% or less, or variations of .+-.0.1% or
less from the specified value.
[0290] As used herein, "treating" and like terms refer to reducing
the severity and/or frequency of symptoms, eliminating symptoms
and/or the underlying cause of said symptoms, reducing the
frequency or likelihood of symptoms and/or their underlying cause,
delaying, preventing and/or slowing the progression of diseases
and/or disorders and improving or remediating damage caused,
directly or indirectly, by the diseases and/or disorders.
[0291] The following abbreviations have been used herein:
DSC--Differential Scanning Calorimetry
[0292] RT--room temperature TBME--methyl tert-butyl ether
TGA--Thermogravimetric Analysis
[0293] THF--tetrahydrofuran wrt--with respect to
XRPD--X-Ray Powder Diffraction
Example 1
Stage 6 Crystalisation Process and Resulting Polymorphs
Experimental to Produce Form A':
[0294] 1.0 g of crude Psilocybin was charged to a 25 mL flask.
Water (12.8 mL/16 volumes based on activity of input material) was
added. The mixture was agitated and heated to 80.degree. C. A dark
brown solution with visible undissolved solids was obtained. The
mixture was polish filtered through a warmed 0.45 .mu.m filter into
a hot 25 mL flask. The undissolved solids were removed to give a
dark brown solution. The solution was re-equilibrated at 75.degree.
C. and then cooled slowly (10.degree. C./hour) to ambient
temperature. The resulting pale brown solution was equilibrated at
ambient temperature for 16 hours. The suspension was cooled to
5.degree. C. prior to isolation of the solid by vacuum filtration.
The filter cake was washed with water (0.8 mL/1 volume) and dried
in vacuo at 50.degree. C. for 16 hours. Yield of 75%, chemical
purity 99%, NMR assay >98%.
[0295] The procedure above was repeated with 14 volumes (11.2 mL)
of water. Yield of 69%, chemical purity 99%, NMR assay >98%.
[0296] In both cases, dissolution of crude Psilocybin was achieved
at ca. 75.degree. C. On gradual cooling, precipitation was observed
at ca. 60.degree. C.
[0297] In both cases, psilocybin Polymorph A' was produced,
confirmed by XRPD (diffractogram consistent with FIG. 7b) and DSC
(thermogram consistent with FIG. 8b).
Experimental to Produce Form A:
[0298] 94 g of crude Psilocybin obtained from the Stage 5 process
(about 93% pure by HPLC with about 4% pyrophosphate impurity) was
subject to an aqueous re-crystallisation as set out below:
[0299] The protocol used sufficient water (12 volumes), a rapid
agitation rate (450 rpm) and a controlled cooling profile
(10.degree. C./hr).
[0300] Psilocybin (94.0 g) (CB650E) was charged to a 2 L flask.
Water (902 ml, 12 volumes based upon activity of input material)
was added. The mixture was agitated and heated to about 78.degree.
C. A dark brown solution with visible undissolved solids was
obtained. The mixture was polish filtered through a 1.2 .mu.m
in-line filter into a hot 5 L flask fitted with an overhead stirrer
(450 rpm). The undissolved solids were removed to give a clarified
dark brown solution. The solution was re-equilibrated at about
75.degree. C. for 15 minutes and then cooled slowly (10.degree.
C./hour) to ambient temperature. The solution was seeded with
Psilocybin Hydrate A (GM758A-XRPD diffractogram consistent with
FIG. 7d) following maturation in water) at 68.degree. C.-70.degree.
C. The resulting pale brown suspension was equilibrated at ambient
temperature for about 16 hours. The suspension was cooled to
5.degree. C. for one hour prior to isolation of the solid by vacuum
filtration. The filter cake was washed with water (282 mL, 3
volumes) and dried in vacuo at about 50.degree. C. for 30
hours.
[0301] The process was completed successfully with a yield of 75%
achieved. Chemical purity of the solid was confirmed as 99.3%.
Analysis of the solid by XRPD post drying for 30 hours showed
Polymorph A (FIG. 7a). A characteristic perturbation was observed
at ca .about.17.degree.2.theta., such as 17.5.degree.2.theta., and
was pronounced in the bulk material.
Solid State Characterisation of Polymorph A and Polymorph A'
[0302] The DSC and TGA thermograms (FIG. 8a) obtained for Polymorph
A were comparable to the DSC and TGA thermograms obtained for
Polymorph A' (FIG. 8b). The TGA thermograms (FIG. 8a) obtained for
Polymorph A and Polymorph A' show no weight loss prior to
decomposition. This suggested that the difference between the XRPDs
obtained for Polymorph A (FIG. 7a; perturbation present at ca.
.about.17.degree.2.theta.) and for Polymorph A' which was obtained
at small scale (FIG. 7b; perturbation not present) was not due to
excess hydration.
[0303] Microscopy of the solid (FIG. 16a) shows rod shaped crystals
with good uniformity with a size range of between 50 and 200
micron.
[0304] The XRPD diffractogram obtained for Polymorph A' does not
demonstrate a perturbation at ca. .about.17.degree.2.theta. to the
same extent as Polymorph A. The perturbation in the XRPD
diffractogram at ca. .about.17.degree.2.theta. is more pronounced
for psilocybin produced at large scale (compared to that obtained
at small scale) and was unexpected. Applicant has demonstrated that
the Hydrate A is the only polymorphic form that exists across a
range of temperatures with no diffraction peak in the 17.degree.2
theta region (see FIG. 7d). This strongly suggests a collapse of
Hydrate A upon dehydration to yield Polymorph A or A' that varies
with scale and that Polymorph A is the true form with Polymorph A'
formed at a small scale being atypical.
[0305] To test the robustness of this theory and to demonstrate a
return to Polymorph A, a small portion of the bulk was re-dried
following another soak in water (to reproduce Hydrate A). A small
sample (250 mg--psilocybin Polymorph A) was equilibrated in water
(10 vols) for one hour. The suspension was filtered and analysis of
the damp solid confirmed that Hydrate A had been generated (FIG.
7d), no perturbation at 17.degree. 2 theta. The material was dried
in vacuo for 16 hours and the solid reassessed by XRPD. Polymorph
A' material was confirmed by XRPD (FIG. 7b) with the reduction in
the XRPD perturbation noted. Additional drying of the original bulk
solid and ageing at ambient temperature did not change the XRPD
diffractogram of the solid. The two solid versions obtained, the
XRPD diffractograms for Polymorph A and Polymorph A' are virtually
identical other than the .about.17.5.degree. 2 theta peak. The
thermal properties are also identical. The distinction between the
XRPD diffractograms for Polymorph A versus Polymorph A' is subtle
and both polymorphs kinetically convert to the hydrated state very
rapidly.
[0306] Additional experiments were performed to ascertain if the
differences in the XRPD diffractograms for Polymorph A and
Polymorph A' were due to the larger scale crystallisation process
delivering solids of a larger particle size that subsequently did
not dry as effectively and caused the change, or whether the habit
and size difference of the crystalline solid was the cause.
Psilocybin Polymorph A (polymorphic form confirmed prior to
experiment) was ground via a mortar and pestle and assessed by
XRPD. No change in polymorph was observed. Another portion (51 mg)
was charged with water (<1 mL) and assessed damp to confirm that
the hydrate was formed. Both lots were dried in vacuo at 50.degree.
C. for ca. 18 hours and re-assessed by XRPD. The ground sample
remained as Polymorph A. The hydrated sample after dehydration was
shown to be Polymorph A' (i.e., no reflection at
.about.17.5.degree.2.theta.). This suggested that size/habit alone
were not the sole reason for the original reflection peaks.
[0307] TGA assessment revealed that the input lot demonstrated a
small mass loss (0.139% weight) by ca. 70.degree. C. The particle
size reduced and subsequently dried solid demonstrated a greater
mass loss of 0.343 wt % by ca. 75.degree. C. whereas the hydrated
and dried solid demonstrated the smallest mass loss of 0.069 wt %
by ca. 80.degree. C. The particle size reduced and subsequently
dried solid was held at 80.degree. C. for 10 minutes (past the
point of mass loss by TGA) but assessment by XRPD revealed no
change from the input, meaning that low levels of hydration and
partial swelling of the crystalline lattice were not the cause of
the variation
[0308] It is possible to generate Polymorph A' via the hydration of
Polymorph A and subsequent drying of the isolated solid on a small
scale.
[0309] Psilocybin Polymorph A and Polymorph A', ca. 60 mg each,
were charged with water, 0.2 ml, to deliver Hydrate A from both
lots. Half of each Hydrate A was dried in vacuo at 25.degree. C.
for ca. 171/4 hours and the remainder of each Hydrate A was dried
at ambient temperature under a stream of N.sub.2 for ca. 171/4
hours. The solids were isolated following drying and assessed by
XRPD. XRPD assessment of the solids isolated from the Polymorph A
input confirmed that Hydrate A was successfully generated and that
the solids dried to give Polymorph A' from both drying methods.
XRPD assessment of the solids isolated from the Polymorph A' input
confirmed that Hydrate A was successfully generated and that the
solids dried to remain as Polymorph A' from both drying
methods.
[0310] On the small scale investigated, Polymorph A and Polymorph
A' will dry to give Polymorph A' via conversion to Hydrate A.
[0311] Psilocybin Polymorph A (100 mg) was particle size reduced
via mortar and pestle grinding. The ground lot was subject to two
different drying regimes in order to assess whether reducing the
particle size affected the dehydration of the sample. The first
sample was held at 80.degree. C. for 10 minutes and the second
sample held at 110.degree. C. for 10 minutes. Both solids were
assessed by XRPD which revealed that Polymorph A was retained. It
was considered whether the ground lot in the prior isothermal
stresses were not held at 110.degree. C. for long enough to impact
the form and so a portion of the ground lot was dried in vacuo at
110.degree. C. for ca. 24 hours. Assessment by XRPD revealed a
subtle change in form with the Polymorph A reflections at ca. 17
still present but at a slightly reduced intensity.
[0312] It was concluded that Polymorph A would not readily convert
to Polymorph A' via particle size reduction and/or drying at high
temperature.
Methodology
[0313] Stability assessments of Psilocybin, not containing the
pyrophosphate impurity, indicated that at temperatures in excess of
80.degree. C., the level of the Stage 3 intermediate impurity
(psilocin) generated by hydrolysis of Psilocybin is of concern. For
example, when a 83 mg/mL Psilocybin aqueous solution is heated to
90.degree. C. and analysed by HPLC at 1, 2 and 4 hours, the level
of the Stage 3 impurity were determined as 0.28, 1.82 and 7.79 area
% respectively. In comparison, when 50 mg of psilocybin is
dissolved in water (1.2-1.8 ml; volume sufficient to maintain a
solution) and heated to 70, 75 and 80.degree. C. for 4 hours, the
level of the Stage 3 impurity was determined, by HPLC, as 0.53,
0.74 and 2.30 area % respectively. The recrystallization heats the
crude Psilocybin to between 75.degree. C. and 80.degree. C. in
order to achieve dissolution, and polish filtration. The immediate
cooling of the solution limits the level of Psilocybin hydrolysis
by reducing the residency time of the material to excessive
temperature.
[0314] Further trial re-crystallisation's of Psilocybin were
conducted introducing the following variations:
[0315] Varying the volumes of water used:
[0316] Varying agitation;
[0317] Having a controlled cooling profile;
[0318] Having a rapid (uncontrolled) cooling profile.
[0319] Using smaller volumes of water (as little as 12 volumes) did
not hinder the re-crystallisation process and dissolution of
Psilocybin was achieved at a temperature to enable a polish
filtration step. Different cooling rates were shown to result in
different crystal size distributions; a slow controlled cool at ca
10.degree. C. per hour produced a relatively larger and more even
average crystal size (FIG. 16a) whereas a rapid cooling profile
delivered smaller crystals (FIG. 16b). A controlled cooling profile
is preferred and this was reflected in an improved purity for the
controlled cool.
[0320] Using the process resulted in Psilocybin of 99.3% chemical
purity, with a 75% yield. Thermal characteristics of the solid
corresponded with those desired. Differences in the XRPD
diffractogram of the dry solid have suggested that the drying
profile may be important in determining how the Hydrate A collapses
to give the preferred solid form. Polymorph A has been demonstrated
to be stable under accelerated stability testing conditions for 12
months.
[0321] Experimental
[0322] Stage 5 was charged to vessel under N.sub.2 followed by
water (approx. 12-15 vol based on active Stage 5). The mixture was
heated to about 80.degree. C. to achieve dissolution and polish
filtered through a 1.2 .mu.m in-line filter into a clean new flask
heated to 80.degree. C. The agitation rate was set to a high level
(450 rpm) and the solution equilibrated at 70-75.degree. C. The
solution was cooled to ambient temperature, at approx. 10.degree.
C./hour, seeding with Psilocybin Hydrate A (0.001.times.stage 5
charge) at 68-70.degree. C. The suspension was held at ambient
temperature overnight then cooled to approx. 5.degree. C. and held
for 1 hour. The suspension was filtered, washing with water (2-3
volumes based upon active charge of Stage 5). The pure Psilocybin
was dried in vacuo at 50.degree. C. Crystalline material psilocybin
(Polymorph A or Polymorph A' dependent on scale) was obtained, for
example using 94 g input of psilocybin yielded Polymorph A and
using 1 g input of psilocybin yielded Polymorph A'. Typically,
batch sizes of greater than 5 g deliver Polymorph A, while batch
sizes less than 5 g deliver Polymorph A'.
[0323] The differences from JNP and the benefits can be summarised
as follows:
i) This additional crystallisation step gives rise to a defined
crystalline form--Polymorph A (or A'). ii) Heating to about
80.degree. C., for a short period, has the advantage that
solubility is maximised (and hydrolysis avoided), which ensures
good yields. iii) At about 70-80.degree. C. polish filtration can
be used to remove insoluble impurities. This is best achieved using
an in-line filter--typically about 1.2 .mu.m. This ensures good
chemical purity. iv) Using a high agitation rate (typically about
450 rpm) ensures speedy dissolution allowing the time at which the
solution is kept at 80.degree. C. to be minimised, thus avoiding
increased levels of the Stage 3 intermediate impurity formed by
hydrolysis of Psilocybin. v) The provision of controlled cooling,
typically cooling at about 10.degree. C. per hour, delivers a more
uniform crystal size and maintains form as crystalline Hydrate A.
vi) Seeding the solution at about 70.degree. C. with Psilocybin
Hydrate A facilitates crystallisation as the Hydrate A. vii) The
crystals are washed in water and dried at about 50.degree. C. to
maximise purity and deliver Polymorph A or A' depending on
scale.
Examples 2 to 6
Stages 1 to 5 Production of Psilocybin
[0324] The following Examples illustrate significant developments
from the process described in JNP, and illustrated in FIG. 1, as
described herein before.
Example 2
Stage 1 (FIG. 2)
[0325] The stage 1 conditions in JNP used 1.1 eq Ac.sub.2O, and 1.2
eq Pyridine in the solvent DCM. The reaction was found to be
complete (99% product, 0% SM) after stirring overnight. The
reaction mixture was washed with water and concentrated in vacuo
giving a brown oil. In the literature, the oil was taken up in
EtOAc and concentrated by evaporation, giving precipitation of
solids at low volume.
Investigation
[0326] However, in the Applicants hands precipitation of solids
from EtOAc was not observed. Precipitation of solids was encouraged
by trituration with heptane, however this would not form a scalable
process. The solids were collected giving high purity stage 1
product (75% yield, >95% pure by NMR).
[0327] While the reaction worked well, the isolation procedure
required further development in order that an easy to handle solid
could be obtained. It was hoped that isolation of the solids by
filtration would then also offer a means of purification.
[0328] The reaction was first trialled in EtOAc to see if
precipitation of solids could be encouraged allowing isolation
directly from the reaction mixture. However, the reaction profile
in EtOAc was found to be less favourable than in DCM and therefore
the reaction was abandoned.
[0329] Applicant washed out the pyridine from the DCM reaction
mixture, as it was believed this may be preventing
re-crystallisation of the product. The reaction was repeated
(completion of 0.4% SM, 98.7% product by HPLC) and the reaction
mixture washed with 20% citric acid to achieve pH 2/3, removing
pyridine and then saturated NaHCO.sub.3 (aq) to avoid low pH in the
evaporation steps. The organics were dried and a solvent swap to
heptane was carried out giving precipitation of stage 1. The solids
were collected by filtration yielding pure stage 1 after drying in
vacuo (87% yield, >95% purity by NMR).
[0330] Stability trials were carried out that confirmed the
reaction mixture was stable overnight when stirred with 20% citric
acid, and also saturated NaHCO.sub.3. The product was found to be
stable when oven dried at 40.degree. C. and 60.degree. C.
Scale Up
[0331] The stage 1 reaction was successfully scaled up processing
>100 g of 4-hydroxyindole. The reaction progressed as expected
and was worked up to give stage 1 product (93% yield, .about.98%
NMR purity).
GMP Raw Material Synthesis
[0332] A large scale stage 1 reaction was carried out to supply GMP
starting material (processing >500 g of 4-hydroxyindole). The
reaction proceeded as expected giving consumption of SM by HPLC
(99.2% product, <0.1% SM). The reaction was worked up using the
established procedure to give stage 1 product after drying (94%
yield, 99.1% by HPLC, 99% NMR assay).
[0333] It was also noted in development that the stage 1 procedure
was effective in removing minor impurities present in some batches
of 4-hydroxyindole. The low level impurities present in
4-hydroxyindole were completely removed after the stage 1 reaction
providing clean material in high yield (89%) and purity (99% by
HPLC, 99% by NMR assay).
[0334] Experimental
[0335] 4-hydroxyindole (1 eq. limiting reagent) was charged to a
vessel under N.sub.2 followed by DCM (dichloromethane; 6 vol based
on 4-hydroxyindole charge). The reaction was cooled to 0-5.degree.
C. and pyridine added (1.2 eq) dropwise at 0-5.degree. C. Acetic
anhydride (1.1 eq) was added dropwise at 0-5.degree. C. and the
reaction warmed to 20-25.degree. C. for 1-1.5 hrs and stirred at
20-25.degree. C. for a further 3 hours. The reaction was sampled
and analysed for completion. The reaction was then washed three
times with 20% aqueous citric acid solution (3.times.3 vol based on
4-hydroxyindole charge) and once with sat. NaHCO.sub.3 (3 vol based
on 4-hydroxyindole charge). The DCM solution was dried over
MgSO.sub.4 and filtered and the DCM layer concentrated to half
volume by distillation. Heptane (6 vol based on 4-hydroxyindole
charge) was added and further DCM was removed by distillation until
full precipitation of the Stage 1 had occurred. The reaction was
cooled to 15-25.degree. C. and the solids collected by filtration,
washing with heptane (1 vol based on 4-hydroxyindole charge) dried
under vacuum overnight at 60.degree. C.
[0336] The differences from JNP and the benefits can be summarised
as follows:
i) Applicant washed out the pyridine using citric acid at a pH of
about 2-3. This facilitated improved isolation and crystallisation.
In practice the DCM phase is separated and the aqueous citric acid
phase discarded. ii) An additional wash in sodium bicarbonate
resulted in further improvement. iii) A solvent swap to heptane
improved solid precipitation maximising yield and resulting in
reproducible high purity Stage 1.
Example 3
Stage 2 (FIG. 3)
[0337] Step i--Acid Chloride Formation
[0338] Formation of reactive Intermediate 2A by reaction of the
stage 1 product with oxalyl chloride (1.5 eq) was initially
trialled in a mixture of TBME and THF (6 vol/1 vol) to determine if
it was a viable alternative to volatile and highly flammable
Et.sub.2O as used in the literature. The reaction gave completion
after .about.18 hours with a similar solubility profile to
Et.sub.2O (stage 1 in solution, precipitation of stage 2A).
[0339] As the acid chloride intermediate is prone to hydrolysis,
leading to variable analytical results a more robust sample make up
and analysis was developed in which the reaction was quenched into
THF/NMe.sub.2 (to give stage 2) and then analysed by HPLC.
[0340] The ratio of TBME and THF was optimised to give the highest
purity and yield of intermediate with a preferred ratio of TBME:THF
of 6:1 chosen for scale up. Other ratios of TBME:THF may be
used.
[0341] A scale up reaction was carried out using the preferred
solvent mixture (1 vol THF, 6 vol TBME) but with the oxalyl
chloride addition carried out at 30-35.degree. C. The resulting
solution was then heated at 40.degree. C. for 2.5 hrs giving a
completion with .about.1% stage 1 product remaining. Carrying out
the addition hot to maintain a solution ensures a high reaction
rate and gave an improved level of completion with a much shorter
reaction time (2.5 hrs vs overnight). The product was still seen to
precipitate at temperature after .about.15 min and no detrimental
effect on the reaction profile was observed by HPLC.
[0342] As the stability of Intermediate 2A was not known,
telescoping of material through to stage 2 was attempted rather
than isolating the intermediate and risking degradation
(hydrolysis). The reaction profile was complex with multiple
components present at a low level. TBME was added and the
precipitate collected. However, this was also found to be a complex
mixture by HPLC/NMR.
[0343] Due to the poor reaction profile it was deemed necessary to
isolate Intermediate 2A to allow for purification away from excess
oxalyl chloride. The reaction was repeated and the yellow
precipitate collected by filtration and washed with TBME to remove
the excess oxalyl chloride (80% yield). NMR analysis confirmed the
product to be of sufficient purity (.about.95% by NMR). However
despite storing under nitrogen, some decomposition was noted over
the following days giving partial hydrolysis including
de-protection of the acetate group.
[0344] In order to try and reduce the potential for hydrolysis of
the intermediate acid chloride during isolation, further
investigations into a telescoped procedure were carried out. It was
found that by allowing the reaction mixture to settle the TBME
liquors could easily be decanted and then the residual solids
washed with further portions of TBME in a similar manner. This
allowed purification of Intermediate 2A away from excess oxalyl
chloride whilst minimising exposure to moisture.
[0345] It was felt that some reaction yield may be lost due to
partial solubility of the intermediate in the THF/TBME mixture.
This was confirmed by adding heptane to the decantation liquors
which gave precipitation of further solids. To limit this
solubility, a heptane (8 vol) addition was made prior to
decantation. Rather than washing the solids with TBME, heptane was
also used for the washes (3.times.6 vol) which maximised the yield
while maintaining the high purity of the intermediate. This
methodology was successfully scaled up and is the preferred
process.
Step ii--Reaction with Dimethylamine
[0346] The literature (Synthesis, 1999, 6, 935-938; D. E. Nichols)
suggested HNMe.sub.2 gas was effective for this transformation.
However to simplify large scale processing this was substituted for
either solid HNMe.sub.2.HCl, with an additional excess of base, or
a solution of HNMe.sub.2 in THF. JNP uses HNMe.sub.2 in the
presence of excess base (pyridine).
[0347] Initially isolated Intermediate 2A was used to optimise the
reaction with dimethylamine via a series of trial reactions (see
Table 11).
TABLE-US-00011 TABLE 11 Stage 2 reaction optimisation trials
Dimethylamine HPLC # Source Base Solvent completion Isolated solids
1 2M Pyridine THF/TBME 80% product, 64% yield, ~70% pure by NMR,
HNMe.sub.2/THF 1.3 eq 1.2 eq 2 HNMe.sub.2.HCl K.sub.2CO.sub.3
THF/H.sub.2O 70% product 40% yield ~98% by NMR. 1.5eq 3 2M Pyridine
Et.sub.2O 81% product 63% yield, ~90% by NMR. HNMe.sub.2/THF 3.6 eq
1.33 eq 4 2M N/A Et.sub.2O 93% product 72% yield, ~98% by NMR.
HNMe.sub.2/THF 2.9 eq
[0348] The literature supplied conditions with pyridine (#1) were
trialled along with a similar reaction in Et.sub.2O (#3), a
biphasic reaction using Me.sub.2NH.HCl and aqueous K.sub.2CO.sub.3
(#2) and a reaction with excess 2M Me.sub.2NH in THF (#4). The
major component by HPLC was the desired product in all cases with
the conditions using aqueous base and excess Me.sub.2NH being
generally much cleaner than those with pyridine. While significant
hydrolysis product was seen in all cases this was thought to be the
result of unreacted Intermediate 2A which was quenched during
sample makeup for HPLC analysis. The reactions were worked up by
addition of water and then the organic solvent was removed in vacuo
giving precipitation of solids.
[0349] The reaction with excess amine showed a much improved
impurity profile which translated into a higher yield (72% vs 63%)
and purity (98% vs 90%). This approach limited the water content of
the reaction and therefore minimised the opportunity for hydrolysis
to occur. Purification was also expected to be more facile due to
the absence of pyridine in the isolated solids. For these reasons
the conditions with excess HNMe.sub.2 as base were chosen for scale
up.
[0350] The reaction in Et.sub.2O gave a clean (#4) profile.
However, to facilitate large scale processing it proved
advantageous to switch to a less volatile solvent, such as TBME.
This would facilitate telescoping the acid chloride into this
reaction. For these reasons it was chosen to carry out the reaction
in TBME using excess 2M Me.sub.2NH in THF.
[0351] It was believed that the addition of water would aid the
workup by solubilising the HNMe.sub.2.HCl salts that were present
and resulted in a very thick mixture and slow filtrations. This was
trialled. However, when water was added to the reactions in TBME
and THF a poor recovery was obtained with analysis of the liquors
showing additional impurities and extensive acetate de-protection
(phenol product). Further development of the purification was
therefore required.
Purification Development
[0352] It was desirable to develop a purification strategy that
would remove the hydrolysis product and other impurities observed.
It was also desirable to include water in the crystallisation to
reduce the salt content of the isolated material (assumed
HNMe.sub.2.HCl). To this end a series of 15 solvents and solvent
mixtures were screened (100 mg scale, 10 vol solvent, heat cycling
to 60.degree. C.).
TABLE-US-00012 TABLE 12 Stage 2 purification trials Purity (hydro-
lysis imp of Solvent Observations Recovery Intermediate 2A) TBME
Slurry 47 mg* 96.4% (2.8%) DCM Slurry 60 mg 99.5% (0.5%) Toluene
Slurry 30 mg* 95.8% (3.4%) EtOAc Slurry 66 mg* 97.8% (1.6%) iPrOAc
Slurry 40 mg* 97.3% (2.0%) IPA Slurry 65 mg 99.4% (0.4%)
EtOH/H.sub.2O, 1:1 Slurry 62 mg 99.4% (0.5%) MeCN/H.sub.2O, 1:1
Partial solution at RT 30 mg 99.0% (0.7%) Solution at 60.degree. C.
Acetone/H.sub.2O, 1:1 Slurry at RT 51 mg 99.4% (0.5%) Solution at
60.degree. C. THF/H.sub.2O, 1:1 Partial solution at RT No n/a
Solution at 60.degree. C. precipitate Heptane Slurry 34 mg* 95.0%
(3.6%) MIBK Slurry 65 mg 97.9% (1.4%) MEK Slurry 60 mg 99.6% (0.3%)
Cyclohexane Slurry 41 mg* 94.4% (4%) Xylenes Slurry 56 mg* 96.1%
(3%) *Recovery not representative due to thick suspensions and
solids adhering to the glass vial.
[0353] From the solvents screened acetone/water gave a
re-crystallisation with little observed solubility at room
temperature. Since this was an aqueous system it had the advantage
of helping to purge Me.sub.2NH.HCl from the solids.
[0354] The acetone/water re-crystallisation was scaled up. A
solution was obtained at temperature (5 vol acetone, 1 vol water)
prior to addition of further water (4 vol) and the mixture cooled
to RT giving crystallisation (62% recovery, >99% HPLC purity).
This process was subsequently scaled up further with addition of
more water to aid the recovery (in total 5 vol acetone, 10 vol
water, 78% recovery).
[0355] The process was scaled up further (30 g) and the crude
solids taken through the re-crystallisation procedure. While
product purity was high, there was a drop in yield (56% yield, 99%
by NMR assay, 99.4% by HPLC).
[0356] In order to improve the recovery, the amount of water added
was further increased from 10 vol to 15 vol. This maintained
product purity at greater than 99% and gave a higher recovery on a
small scale (90% recovery, 56-70% previously observed). However,
scale up of this amended procedure again gave a low recovery (58%
yield). Therefore, due to the issues encountered when scaling up
the re-crystallisation, an alternative means of purification was
sought based on the original slurry screen that was carried out
(Table 12 above).
[0357] Redevelopment of the purification strategy took place using
material isolated from a large scale, stage 2, reaction. The
reaction progressed as expected to give crude product after oven
drying (70% by NMR assay, 79% active yield). To remove the
significant salt component (presumed to be HNMe.sub.2.HCl) a
portion was water slurried at RT. After drying this gave 75%
recovery (95% by NMR assay) showing this to be an effective means
of reducing the salt content. HPLC purity remained unchanged at
.about.93%. A method was then sought to increase the chemical
purity of the solids.
[0358] From the initial screen both EtOH:H.sub.2O, IPA and
acetone:H.sub.2O appeared to give high purity product with a good
recovery and so these solvent systems were chosen for further
investigation. Input purity was 92.7% with the main impurities at
levels of 1.4%, 1.0% and 0.8%.
TABLE-US-00013 TABLE 13 Further purification development (slurry at
40.degree. C. 1 hr, filtration at RT, 1 vol wash) HPLC purity (main
impurity levels) Solvent system Recovery Product Imp 1 Imp 2 Imp 3
Acetone/Water 67% 98.8% 0.7% 0.2% 0.1% 6/16 vol 1:1 EtOH:H.sub.2O
79% 98.0% 0.9% 0.5% 0.2% 10 vol IPA 90% 97.5% 0.8% 0.8% 0.3% 10 vol
IPA 92% 97.2% 1.0% 0.7% 0.3% 5 vol 4.5:2.5 IPA:H.sub.2O * 79% 98.7%
0.6% 0.4% 0.1% 7 vol 6:1 IPA:H.sub.2O 82% ** 98.0% 0.9% 0.6% 0.2% 7
vol * The reaction was initially run in 1:1 IPA:H.sub.2O at 5 vol.
However it became too thick to stir and so a further 2 vol of IPA
was added. ** The mixture was thick and the solids present were
very fine making filtration difficult with some solids beating the
filter.
[0359] The results of these trials suggested that good recoveries
were possible from these systems, particularly those based on IPA.
EtOH:H.sub.2O gave a marginally better impurity profile than IPA
alone; however the recovery was not as good (79 vs 90%). The
impurity profile with IPA was greatly enhanced by the presence of
water (98.7% vs .about.97.5%) however this led to a lower recovery
(79 vs 90%). This suggested a certain level of water solubility for
the compound. A final trial in IPA and EtOH:Water was conducted
with reduced water volumes to see if a balance could be found that
provided high purity and a recovery of .about.90%. While this
system improved the yield the filtration was slow and therefore
further solvent mixtures were also evaluated.
TABLE-US-00014 TABLE 14 Examination of further solvent mixtures
(500 mg scale, slurry at 40.degree. C. 1 hr, filtration at RT, 1
vol wash) HPLC purity (impurity levels) Solvent system Recovery
Product Imp 1 Imp 2 Imp 3 IPA 460 mg (92%) 97.6% 1.1% 0.7% 0.3% 10
vol 1:3 EtOH:H2O 451 mg (90%) 96.2% 1.4% 0.8% 0.5% 5 vol 1:2
EtOH:H2O 440 mg (88%) 97.5% 1.3% 0.7% 0.4% 5 vol 1:1 EtOH:H2O 375
mg (75%) 97.9% 1.0% 0.7% 0.3% 5 vol 2:1 EtOH:H2O 354 mg (71%) 97.6%
1.0% 0.6% 0.4% 5 vol 3:1 EtOH:H2O 369 mg (74%) 97.9% 0.9% 0.5% 0.3%
5 vol
[0360] The 5 vol EtOH/Water slurries were very thick and not easily
handled. Since the purity of the solids was comparable from all the
trials (slight variations are likely due to the quality of the
filtration and wash), the 100% IPA conditions were scaled up as
they offered a high recovery and the resulting suspension was
easily handled.
[0361] An initial scale up of the preferred slurry gave (92%
recovery) with HPLC purity of 96.4% (Impurity levels of 1.2%, 0.7%,
0.4%). Liquors analysis showed them to be enriched in all of the
main impurities--72% (8.6%, 3.8%, 3.4%) by HPLC. This was deemed a
suitable purification method offering a high recovery and the
material was use tested in the following stage to ensure tracking
and removal of the impurities was achieved downstream (>99% at
stage 3, no single impurity >0.5%).
[0362] The slurry proved scalable when remaining crude stage 2
material (70% assay) was water slurried to remove inorganics, and
then slurried in IPA to give material of improved purity (97% by
NMR assay, 76% yield for stage 2, 96.8% by HPLC, impurities at
1.1%, 0.8%, 0.4%).
GMP Raw Material Synthesis
[0363] A large scale, stage 2 reaction was carried out to supply
the GMP campaign. The reaction progressed as expected to provide
crude product that was water slurried and filtered to provide stage
2 that was 93% pure by HPLC. This was further slurried in 8 vol IPA
and filtered to give stage 2 product (93.7% by HPLC, 92% assay, 66%
active yield). Since the purity obtained was lower than that
observed during the development campaign, a use test was conducted
which confirmed that high purity stage 3 obtained was suitable for
onward processing (GMP raw material).
[0364] A second batch was carried out under identical conditions to
give crude product which after a water slurry was 90% pure by HPLC.
This material was subsequently water slurried and purified by IPA
slurry to give 384 g of stage 2 product (93.0% by HPLC, 91% NMR
assay, 60% active yield).
[0365] A third batch was carried out to resupply the GMP synthesis.
The crude product was successfully purified by a water then IPA
slurry to deliver stage 2 (79% yield) with an increased purity when
compared to previous batches (97.3% by HPLC, 96% NMR assay).
[0366] Experimental
[0367] Stage 1 (1 eq. limiting reagent) was charged to a vessel
under N.sub.2 followed by THF (1 vol wrt stage 1 charge) and TBME
(6 vol wrt stage 1 charge). Oxalyl chloride was then added dropwise
to the vessel (1.5 eq,) allowing the exotherm to initially raise
the temperature to 35-40.degree. C. and then applying cooling as
required to maintain 35-40.degree. C. Immediately following the
addition the reaction was heated to 40.degree. C. and stirred for
2-6 hours. The reaction was sampled and analysed for completion,
then cooled to RT and heptane (8 vol wrt stage 1 charge) added
giving precipitation of further solids. The reaction was stirred
for 10 min and then the solids were allowed to settle. The majority
of the solvent was decanted from the solid which was then washed
twice with heptane (2.times.6 vol wrt stage 1 charge), decanting in
a similar manner after each wash. The solids were then sampled and
analysed. TBME was charged to the vessel (4 vol wrt stage 1 charge)
to give a yellow slurry which was cooled to -20.degree. C. using a
dry ice/acetone bath. A 2M solution of Me.sub.2NH in THF (2 eq,)
was added dropwise to the vessel over .about.15 min maintaining the
temperature at -20.degree. C. to -10.degree. C. The reaction
mixture was allowed to warm slowly to RT and stirred overnight.
Further Me.sub.2NH can be added at this point if required. The
reaction was sampled and analysed for completion. The reaction was
filtered, washing with heptane (2.times.2 vol wrt stage 1 charge)
and the isolated solids dried at 60.degree. C. under vacuum. The
crude stage 2 was slurried in water (8 vol wrt stage 1 charge) for
2-18 hours and then filtered, washing with water (2 vol wrt stage 1
charge). The solids were dried at 60.degree. C. under vacuum to
obtain crude stage 2 with <2% w/w water (determined by Karl
Fischer titration (KF)). The crude stage 2 was slurried in IPA (10
vol) for 2-18 hrs and then filtered, washed with IPA (1 vol wrt
mass of crude Stage 2) and oven dried under vacuum at 60.degree.
C.
[0368] The differences from JNP and the benefits can be summarised
as follows:
Step 1
[0369] i) Firstly, the use of a THF/TBME solvent system in place of
diethyl ether was less volatile and flammable. ii) Secondly, the
addition of oxalyl chloride was conducted at an elevated
temperature, heated to 40.degree. C., giving rise to improved
solubility and preventing entrapment of stage 1 product in the
precipitate. It also provided a high reaction rate, improved levels
of completion and shorter reaction times. iii) Thirdly, the
Intermediate 2A was isolated to allow for purification away from
excess oxalyl chloride. iv) Fourthly, heptane was added to help
precipitate Intermediate 2A.
Step 2
[0370] v) By ensuring the amine was used in excess much improved
purity and yields were obtained, due to minimal water being
present, and hence reduced hydrolysis. vi) Finally, the use of
water and IPA slurries provided good purity of the Stage 2
product.
Example 4
Stage 3
[0371] An initial stage 3 trial reaction was carried out using
purified stage 2 material (>99% by HPLC) and the supplied
reaction conditions. The stage 2 input material was found to be
largely insoluble in THF, and so rather than adding a solution of
stage 2 to LiAlH.sub.4 the reverse addition was carried out. 4 eq
of LiAlH.sub.4 was used as a 1M solution in THF with the addition
made at 20-25.degree. C., over .about.2 hrs. At this point 10%
product was observed with several intermediate species present. The
reaction was heated at reflux for .about.7 hrs to give 93% product
conversion (by HPLC). The reaction was worked up to give crude
stage 3 product (.about.90% by HPLC, .about.90% by NMR, .about.87%
corrected yield).
[0372] A trial reaction was carried out in which the LiAlH.sub.4
charge employed was successfully reduced (3 eq vs 4 eq). It was
hoped this would benefit the work up by reducing the quantity of Li
and Al salts generated. After prolonged heating at reflux (10-18
hrs) the reaction intermediate was largely consumed (2-3%
remaining) with .about.95% product by HPLC.
Workup Development
[0373] Although the first trial reaction was successfully worked up
using Rochelles salt, the volumes employed were very high
(.about.100 vol) and this procedure would not form a viable process
for scale up. A variety of alternative workup procedures were
examined in order to try and reduce the volumes required and aid
removal of Li/Al salts.
[0374] A reduced volume quench was trialled with EtOAc and then
Rochelles salt. Grey solids were present as a thick paste which
settled to the bottom of the flask. While filtration failed, the
liquors could be decanted and the solids re-slurried in THF/EtOAc
to extract the product. An aqueous workup was then carried out and
the product isolated by concentration. This yielded product of good
purity (90-95% by NMR) in good yield (94% uncorrected for purity).
However the process was not readily amenable to scale up.
[0375] A reaction was quenched by addition of EtOAc and then sat.
Na.sub.2SO.sub.4 in the presence of anhydrous Na.sub.2SO.sub.4 to
act as a binding agent. The reaction gave granular solids which
could be readily filtered. An aqueous workup was then carried out
and the product isolated by concentration. A good yield was
obtained (.about.94% uncorrected for purity) but the product
contained higher levels of the main impurity by NMR (10% vs 2-4%
usually observed).
[0376] A reaction was quenched with 20% AcOH at 0.degree. C.
leading to the formation of a gel which could not be filtered. The
reaction was abandoned.
[0377] A reaction was quenched with EtOAc and then 20% citric acid
to give solids which could be separated by filtration. The liquors
were concentrated to obtain the product. While this procedure was
slightly lower yielding (.about.77% uncorrected for purity), the
product was of very high purity (>95%).
[0378] A further reaction was quenched by the addition of EtOAc and
then water (3 mL per g of LiAlH.sub.4 in THF). A gel formed which
could not be readily filtered and the reaction was abandoned.
[0379] Finally, a reaction was quenched by the Fieser method. An
addition of water (1 mL per g of LiAlH.sub.4) was made then 15%
NaOH (1 mL per g of LiAlH.sub.4) and finally water (3 mL per g of
LiAlH.sub.4). This gave solids which could be filtered from the
reaction mixture. The liquors were then partitioned and
concentrated in vacuo (87% yield, 90-95% by NMR).
[0380] These Experiments are summarised in Table 15 below:
TABLE-US-00015 TABLE 15 Summary of alternative workup conditions
Workup # procedure Yield Purity 3.1 EtOAc 2.8 g (94% uncorrected)
90-95% by NMR quench Rochelles salt (reduced vol.) 3.2 EtOAc 2.8 g
(94% uncorrected) ~80-85% by NMR, quench in 10% imp. presence of
Na.sub.2SO.sub.4 3.3 20% AcOH Emulsion (reaction to waste) quench
3.4 EtOAc i) 529 mg (71% uncorrected) >95% purity quench ii) 2.3
g (77% uncorrected) by NMR 20% citric acid 3.5 Water Emulsion
(reaction to waste) quench 3.6 Water/NaOH i) 615 mg (83%
uncorrected) 90-95% by NMR quench ii) 2.6 g (87% uncorrected)
[0381] Both quenches with citric acid and NaOH gave solids that
could be readily filtered from the reaction mixture and required
minimal solvent volumes. While the conditions with NaOH were higher
yielding (.about.10%), the product obtained from this procedure was
less pure and would likely require further purification before use
in the next stage. The lower yield with citric acid was likely due
to some precipitation of the product citrate salt. This had a
purifying effect with clean product obtained directly after
concentration. These conditions were chosen for scale up and it was
hoped that further optimisation of the citric acid charge would
enable clean product to be isolated in high yield from this
process.
[0382] The reaction was repeated with a slightly reduced citric
acid charge in order to try and maximise the recovery. This
reaction yielded product in 57% yield with a further 20% yield
obtained by re-slurry of the filter cake in THF (both samples 97.7%
by HPLC).
[0383] The reaction was scaled up. However during the EtOAc quench,
where the reaction was previously seen to thicken, the reaction
gummed in the flask to form a thick mass which restricted mixing.
While the addition of citric acid then led to the usual slurry/gel
this did not represent a viable process. This reaction was worked
up with the filter cake re-slurried in THF to maximise the recovery
giving 76% active yield, 95.0% by HPLC.
[0384] The reaction was repeated in order to develop a better
quench and avoid the gum formation seen with EtOAc. A portion was
quenched by addition of acetone which led to a readily stirred
suspension/emulsion with no sign of thickening. The citric acid
treatment was then carried out to give a filterable mixture. This
quench was successfully carried out on the remainder of the
reaction and worked up to provide crude product in good yield (71%
assay, 82% corrected yield, 98.0% by HPLC).
[0385] After the quench the reaction mixture was generally found to
be pH 8/9. As part of the workup optimisation process different pHs
were investigated. A reaction was split for workup with half
receiving a slightly reduced citric acid charge (to obtain pH 11/12
after quench) and the other half taken to pH 7 by addition of
further citric acid. The pH 11 reaction was worked up to give
material of 85% NMR assay (73% yield) with the pH 7 reaction giving
60% NMR assay (62% yield). It was clear from this result that
obtaining the correct pH after quench was critical in order to give
a >70% yield. By reducing citric acid charge only slightly
(still approx. 2 vol of 20% citric acid) an approx. 8% increase in
yield was obtained. With this information in hand the pH of future
reactions was monitored during the quench in order to ensure the
mixture remained strongly basic.
Purification Development
[0386] A purification screen was carried out using 100 mg portions
of crude Psilocin product which were slurried in 10 vol of solvent
with heat cycling to 60.degree. C. The slurries were cooled to RT
over the weekend and then any solids collected by filtration.
Stability to acid and base was also tested with the view to
carrying out an acid/base workup. The results of the screen are
presented in Table 16 below:
TABLE-US-00016 TABLE 16 Psilocin purification screen. (Input purity
and 3 main impurity levels: 90.2%, 3.8%, 0.9%, 0%). Recovery HPLC
Purity (and main Solvent Observations (approx.) impurity levels)
MeOH Solution at RT n/a n/a EtOH Solution at 60.degree. C. 35 mg
97.2%, 0.4%, 1.1%, 0.8% Precipitate at RT IPA Slurry at 60.degree.
C. 51 mg 97.6%, 0.5%, 0.5%, 1.1% MeCN Solution at 60.degree. C. 46
mg 96.8%, 0.6%, 0.4%, 1.6% Precipitate at RT EtOAc Slurry at
60.degree. C. 58 mg 97.1%, 0.9%, 0.2%, 1.3% .sup.iPrOAc Slurry at
60.degree. C. 58 mg 98.2%, 0.8%, 0.2%, 0.4% Toluene Slurry at
60.degree. C. 70 mg 93.3%, 3.6%, 0.2%, 0.8% Heptane Slurry at
60.degree. C. 77 mg 91.3%, 3.8%, 0.2%, 0.7% Acetone Solution at
60.degree. C. 30 mg 97.7%, 0.4%, 0.3%, 0.9% Precipitate at RT MEK
Solution at 60.degree. C. 24 mg 97.3%, 0.5%, 0.5%, 1.2% Precipitate
at RT MIBK Slurry at 60.degree. C. 49 mg 97.4%, 0.6%, 0.2%, 1.3%
THF Solution at RT n/a n/a TBME Slurry at 60.degree. C. 67 mg
95.5%, 2.0%, 0.1%, 1.5% DCM Solution at RT n/a n/a 1M HCl Solution
at RT n/a 83.7%, new imp 8% 1M KOH Slurry at RT (black) n/a 89.1%,
5.4%, 0.9%, 2.2%
[0387] The first of the three highlighted impurities corresponded
with the most stable reaction intermediate that is observed at
.about.70%, when the LiAlH.sub.4 addition is complete (requiring
refluxing to convert to product). The third impurity was not
present in the input and appeared to be generated during the slurry
procedure. Of the solvents that remained as a slurry, .sup.iPrOAc
gave the highest purity. Several re-crystallisations were found
with MeCN having the potential to remove impurities during the
crystallisation and having a recovery which had the potential to
improve during development. Some degradation was observed in both
acid and base with the KOH sample rapidly turning black.
[0388] Purification of the crude stage 3 material was scaled up
using the two most promising solvents (MeCN and .sup.iPrOAc). The
solvent volumes were reduced to a minimum in order to improve the
recovery. The results of these trials are presented in Table 17
below.
TABLE-US-00017 TABLE 17 Further development of MeCN/.sup.iPrOAc
purification Recovery HPLC Purity (and main Solvent Observations
(%) impurity levels) MeCN (5 vol) Recryst in 5 vol 512 mg 97.6%,
0.7%, 0.8%, 0% (51%) .sup.iPrOAc (3 vol) Slurry in 3 vol 706 mg
95.8%, 1.3%, 0.6%, 0% (71%)
[0389] A re-crystallisation was obtained from MeCN in 5 vol and a
hot slurry was achieved in .sup.iPrOAc at 3 vol (both at 75.degree.
C.). The recovery from MeCN was again poor despite reduced volumes,
however the product was of very high purity (>>95% by NMR).
The recovery from .sup.iPrOAc was better with a large increase in
product purity when analysed by NMR (.about.95%).
[0390] Although HPLC and NMR purity of material from the
.sup.iPrOAc slurry was high, a low assay value (85% by NMR assay)
was observed. In order to improve the assay value of the material,
as well as remove colour, (all materials obtained so far were
strongly purple, green or brown) purification by silica pad was
investigated.
[0391] Crude Psilocin (71% assay, 98.0% by HPLC) was passed through
4 eq of silica eluting with THF. An 80% recovery of an off white
solid with slightly improved HPLC purity (98.4%) and assay value
(.about.82% assay) was obtained. This proved to be an effective
means of increasing the product assay value and was therefore
included as part of the reaction workup.
[0392] A series of .sup.iPrOAc/anti-solvent slurries (Table 18)
were then performed using the silica treated input (100 mg per
slurry) to try and improve the recovery, whist maintaining chemical
purity (input purity 98.4%).
TABLE-US-00018 TABLE 18 Results of .sup.iPrOAc/anti-solvent
additions. Solvent system Recovery HPLC purity .sup.iPrOAc 78%
99.7% 5 vol 1:1 .sup.iPrOAc:Heptane 70% 99.6% 5 vol 1:1
.sup.iPrOAc:TBME 83% 99.7% 5 vol 1:1 .sup.iPrOAc:Toluene 84% 99.4%
5 vol .sup.iBuOAc 80% 99.6% 5 vol
[0393] Since all purity values were comparable, two solvent systems
were chosen for scale up based on the highest recoveries obtained.
The two favoured slurries (TBME and Toluene as anti-solvent) were
scaled up (1.0 g per slurry) to better assess the recovery.
TABLE-US-00019 TABLE 19 Scale up of favoured purification methods
Solvent system Recovery HPLC purity 1:1 .sup.iPrOAc:TBME 79.9%
99.1% 5 vol 1:1 .sup.iPrOAc:Toluene 79.4% 99.6% 5 vol
[0394] Both of these options provided material of >99% HPLC
purity at .about.80% recovery and, combined with a silica pad,
appear to provide an effective means of purification for the
Psilocin product. Further colour was removed into the liquors
during the slurry giving Psilocin as a white solid. All impurities
were effectively removed to below 0.5%. The .sup.iPrOAc:TBME slurry
was chosen for scale up as this used non-toxic ICH class 3
solvents.
Scale Up
[0395] The developed stage 3 conditions were scaled up and the
reaction progressed to give a completion of 94.4% product with 2.9%
of the reaction intermediate present by HPLC after overnight at
reflux (typical of the process). After a silica pad Psilocin was
obtained in 83% purity by NMR assay, 66% active yield, 97.0% by
HPLC. This material was further purified by slurry in iPrOAc/TBME
to give material 100% by NMR assay in 62% yield and 99.7% by
HPLC.
[0396] Due to the crude yield from the reaction being lower than
expected (66% vs .about.75%) the filter cake and silica pad were
reinvestigated in order to try and recover additional material.
However, this was unsuccessful.
[0397] The lower than expected yield may have been due to
decomposition of the product during workup, although previous
stress tests had indicated the material to be stable under the
conditions used. To investigate this further the reaction was
repeated. The crude product was isolated before the silica pad and
additional stress test samples taken to confirm degradation of the
product was not occurring during workup.
[0398] The reaction progressed as expected to give completion
(93.7% product, 2.9% intermediate) and was concentrated yielding
crude material (77% NMR assay, 66% active yield). The filter cake
was re-slurried in THF/MeOH but no significant Psilocin was
isolated. In order to try and displace any product that was
coordinated to the aluminium salts, further citric acid was added
to take the pH to 4 (from pH 8) and the cake re-slurried in THF,
but again no significant Psilocin was isolated. Mass balance was
not obtained from the reaction with the 66% active yield closely
matching what was previously obtained. This batch was purified by
silica pad and slurry in .sup.iPrOAc/TBME to give a 62% yield of
high purity material (99.8% by HPLC).
[0399] Despite the solvent volumes employed being relatively high
and a silica pad being required for removal of aluminium and
lithium species, the process was still well suited to the required
scale.
[0400] The stage 3 reaction was further scaled up to process. The
reaction proceeded as expected to give completion after 18 hours
(.about.91% product, .about.3% reaction intermediate remaining).
Workup by silica pad and slurry gave a 57% yield of high purity
Psilocin (>99% by HPLC, 99% NMR assay, 0.35% w/w water Karl
Fischer).
[0401] Experimental
[0402] Stage 2 (1 eq. limiting reagent) was charged to the vessel
followed by THF (5 vol wrt stage 2 charge). The mixture was cooled
to 0.degree. C. and a 1M THF solution of LiAlH.sub.4 (3 eq,) added
dropwise over 30-45 min maintaining the temperature at 0-20.degree.
C. Following the addition, the reaction was stirred for 30 min at
10-20.degree. C. and then heated to reflux and stirred for
.about.16 hrs. The reaction was sampled and analysed for
completion, cooled to 0.degree. C. and quenched by dropwise
addition of acetone (9.3 eq.) at 0-30.degree. C. followed by a 20%
aq citric acid soln (1.9 vol wrt stage 2 charge) at 0-30.degree. C.
The pH of the addition was monitored to ensure that it remains at
pH>11 and the addition was stopped early if required. The
resulting suspension was stirred for 1 hr and filtered, washing
with THF (2 vol wrt stage 2 charge) to remove Li and Al salts. The
filter cake was slurried in THF (12.5 vol wrt stage 2 charge) for
.about.1 hr and filtered, washing with THF (5 vol wrt stage 2
charge) to recover product from the Li and Al salts. The combined
organics were dried over MgSO.sub.4 and filtered. The filtrate was
evaporated in vacuo until approximately 10 volumes remained (wrt
stage 2 charge) and this solution was applied to a silica pad (3 eq
wrt stage 2 charge). The silica pad was eluted with THF and the
product fractions were combined and evaporated to dryness in vacuo.
The crude stage 3 (Psilocin) was slurried in 1:1 iPrOAc:TBME (5 vol
wrt mass at step 18) for 2-18 hrs, filtered, washing with TBME (2.5
vol wrt mass at step 18) and dried in vacuo at 40.degree. C. to
isolate pure Psilocin.
[0403] The differences from JNP and the benefits can be summarised
as follows:
i) Firstly, Applicant, whilst using THF as a solvent, quenched the
reaching using acetone. This lead to a suspension/emulsion without
thickening. ii) Secondly, Applicant quenched with citric acid
maintaining a basic pH, typically about 11. The pH control ensured
high yields were obtained. iii) Thirdly, following purification by
silica pad, to remove residual Li/Al salts, eluting with THF, a
iPrOAc:TBME slurry provides a highly purified product which was
then dried.
Example 5
Stage 4
[0404] Initially the literature conditions were used to process a
2.58 g sample giving .about.88% conversion to Intermediate 4A when
analysed by HPLC. The product was purified by the addition of
aminopropyl silica and filtration through Celite. The resulting
green oil (5.5 g) was slurried in DCM giving benzyl transfer and
precipitation of the zwitterionic stage 4 (4.1 g, 70% yield,
.about.95% by NMR).
Step i
[0405] Initial development at this stage was focussed on finding an
alternative to .sup.nBuLi that was easier to handle and ideally did
not introduce further lithium into the synthesis. An initial screen
of alternative conditions was carried out including the following
bases: Li.sup.tBuO, K.sup.tBuO, NaH, NaHMDS, and NaNH.sub.2. All of
the reactions gave product with NaHMDS performing as well as
.sup.nBuLi. All of the reactions became very thick with gelling
observed and overhead stirring was recommended for efficient
stirring.
[0406] The initial screen suggested NaHMDS would be a suitable
alternative to .sup.nBuLi (81% conversion to product/Intermediate
4A). These conditions were scaled up to 1.5 g alongside a reference
reaction with .sup.nBuLi. Overhead stirring was used in both
cases.
TABLE-US-00020 TABLE 20 Comparison of .sup.nBuLi and NaHMDS
Timepoint HPLC-.sup.nBuLi HPLC-NaHMDS 1 hr, -30.degree. C. 6.6% St
3, 78% <1% St 3, 78% Int 4A, 1% St 4 Int 4A, 4% St 4 2 hr,
0.degree. C. 6.5% St 3, 77% <1% St 3, 76% Int 4A, 1% St 4 Int
4A, 4% St 4 Crude 4.89 g 4.38 g product <1% St 3, 2% <1% St
3, 5% Int 4A, 60% St 4 Int 4A, 68% St 4 Abbreviations used in the
table: St 3 = Stage 3, Int 4A = Intermediate 4A, St 4 = Stage 4
[0407] The reaction profile obtained in both cases was very similar
with the NaHMDS reaction giving consumption of stage 3. Both
reactions were filtered on Celite to remove a white precipitate and
concentrated. By NMR excess benzyl protons were present in both
cases (especially in the example with .sup.nBuLi) and the isolated
yield was >100%. The NaHMDS conditions proved successful giving
a favourable reaction profile and were chosen for further scale up.
However, workup and purification development was required.
Step ii
[0408] HPLC data indicated the material isolated from the trials
above using NaHMDS and .sup.nBuLi had rearranged to give
zwitterionic stage 4 upon concentration. Purification of this
material away from the benzylphosphoric acid by-products and other
impurities was attempted by slurry in a number of solvents.
TABLE-US-00021 TABLE 21 Trial purification of crude stage 4 product
Solvent Mass recovered HPLC Purity DCM White solid 84% St 4 EtOH
Gum -- EtOAc Gum -- IPA White solid 88% St 4 Toluene Gum -- TBME
White solid 62% St 4 MIBK Gum -- MeCN Gum -- Acetone White solid
86% St 4 Abbreviations used in table: St 4 = Stage 4 * filtration
poor
[0409] White solids were obtained from several solvents however the
solids obtained from DCM and TBME turned to a pale purple gum when
stored over the weekend. Those obtained from IPA and acetone
remained as free flowing white solids on storage suggesting that
the stability of these solids was likely to be higher and that they
would allow for easier handling.
[0410] The slurries in IPA and acetone were scaled up to 1 g.
However, gumming was noticed immediately on addition of the
solvent. The gum was slowly dispersed by vigorous stirring and
eventually showed signs of crystallisation, with a white slurry
forming after an overnight stir. However, this process was not
suitable for scale up. Solids were isolated in good yield with IPA
providing the highest purity.
[0411] THF was also investigated as this had advantages in that it
was also the reaction solvent. However, when this was trialled
initial gum formation was again observed (isolated .about.80%
yield, .about.92% by HPLC). In order to try and avoid the gum
formation and give a more controlled crystallisation the crude
stage 4 was first solubilised in a low volume of DMSO (2 vol). THF
was then added to this (10 vol) and the solution stirred over the
weekend. This slowly gave precipitation of the product which was
collected by filtration and washed with THF to yield stage 4 (86%
yield) with 96% HPLC purity (>95% by NMR).
[0412] As the THF crystallisation was successful and it was
previously noted that complete conversion to zwitterionic stage 4
occurred during concentration of the reaction liquors (THF/EtOAc)
at 40.degree. C., it was hoped that the changes of solvent could be
avoided and the product crystallised directly by stirring out the
reaction mixture at 40.degree. C.
[0413] Two 4 g NaHMDS reactions were carried out with both
reactions reaching completion with .about.80% conversion to
Intermediate 4A. One reaction was diluted with EtOAc and the other
with THF and both were filtered to remove phosphate by-products. In
order to further reduce the phosphate impurity levels a brine wash
was carried out and the organics dried and concentrated to 10 vol.
These solutions were stirred overnight at 40.degree. C. to give
conversion to, and precipitation of, stage 4 (.about.1% stage 3,
.about.0.2% Intermediate 4A, .about.82% stage 4). The solids were
collected by filtration giving 8.03 g (88% yield) from EtOAc/THF
and 5.65 g (62% yield) from THF. The brown/grey solids obtained
from EtOAc/THF were of lower purity (.about.90% by HPLC, 78% assay)
when compared to the white solids obtained from THF (97% by HPLC,
88% assay). Analysis of the aqueous layer from the THF reaction
showed product to be present and additional losses were incurred to
the final THF filtrate.
[0414] Due to the higher purity obtained from THF, this solvent was
investigated further in order to optimise the recovery. The brine
wash was omitted due to product losses to the aqueous layer and the
reaction mixture was further concentrated after the reaction to
minimise losses during the final filtration step. This new
procedure was trialled on a 75 g scale with portions of the
reaction mixture concentrated to 8 vol and 6 vol. Upon filtration
no difference in yield was noted between the two portions with an
overall yield of 140.4 g (90% by NMR assay, 74% active yield, 90%
by HPLC).
Impurity Tracking
[0415] Three main impurities were observed in the isolated product,
with identities for two of these species proposed based on MS
data.
[0416] The debenzylated impurity (typically .about.2-5% by HPLC)
was shown to give psilocybin during the following hydrogenation and
could therefore be tolerated at a higher level. The main observed
impurity in the isolated stage 4 (typically .about.5-8% by HPLC)
was the anhydride impurity. This was tracked through the subsequent
hydrogenation and shown to be readily removed by re-crystallisation
from water as the highly soluble pyrophosphorate impurity that
results from debenzylation. The other main observed impurity (m/z
295.2 observed by LCMS) was found to be controlled to less than 2%
by limiting the reaction temperature (below -50.degree. C.) and was
not observed in Psilocybin after hydrogenation.
[0417] The impurity profile of the 140 g batch produced above
showed 90.0% stage 4, 6.4% anhydride impurity, 0.2% N-debenzylated
impurity and 1.2% of the m/z 295.2 impurity.
GMP Synthesis
[0418] The first large scale stage 3 batch (544 g input) was
completed using the established procedure to give 213.5 g (53%
yield, 99% by HPLC). A second batch (628.2 g input) was also
processed successfully to give 295.2 g (66% yield, 99% by
HPLC).
[0419] Some variability in yield at this stage was noted over 3
large scale batches (57%, 53% and 66%). This is probably a
consequence of minor differences in the workup and quench
procedure.
[0420] Experimental
[0421] Stage 3 was charged to a vessel followed by THF (15 vol wrt
stage 3 charge) and cooled to .ltoreq.-50.degree. C. using a dry
ice/acetone bath. 1M NaHMDS solution in THF (1.13 eq) was charged
maintaining a temperature of .ltoreq.-45.degree. C., target
<-50.degree. C. The reaction was stirred for 30 minutes at -60
to -50.degree. C. Tetrabenzylpyrophosphate (2.26 eq) was charged to
the reaction in a single portion followed by additional THF (20
vol) while maintaining the reaction temperature <-30.degree. C.
The reaction was warmed to 0.degree. C., over 1.5-2 hours and
sampled for completion. The reaction was filtered to remove
phosphate salts washing with THF (8 vol). The filtrate was
concentrated until 6-8 vol remains and stirred overnight at
40.degree. C. to convert Intermediate 4A to stage 4 product. The
reaction was sampled for completion and then filtered and the solid
washed with THF (2 vol). The stage 4 product was dried in a vacuum
oven at 40.degree. C.
[0422] The differences from JNP and the benefits can be summarised
as follows:
Step i
[0423] i) Firstly, sodium hexamethyldisilazide was introduced to
support deprotonation. This proved an effective alternative to
Butyl Lithium, which was easier to handle, and did not introduce
further lithium into the reaction. ii) Secondly, by diluting the
reaction with THF, a much higher purity Intermediate 4A was
obtained. iii) Thirdly, by controlling the reaction temperature at
below -50.degree. C., undesirable mz 295.2 observed by LCMS was
controlled to levels of less than 2%.
Step ii
[0424] iv) Fourthly, by monitoring levels of stage 4A impurities,
particularly the N-debenzylated Stage 4 (Table 7) and anhydride
Stage 4 (Table 7), a pure product can be produced reproducibly. v)
The intermediate stage 4A to stage 4 conversion can be carried out
in the reaction solvent, avoiding the need for time consuming
solvent swaps. vi) Finally, the obtained solid is washed with THF
and oven dried to obtain stage 4.
Example 6
Stage 5
[0425] Catalyst poisoning was noted during development of this
stage and a charcolation step can be included in the process when
required to prevent incomplete hydrogenation. However, charcolation
is not routinely required.
[0426] After sparging with hydrogen for 3 hours typical reactions
showed high levels of completion (>90% product, 3-5% SM
remaining). A small amount of water was added to aid solubility and
after sparging with hydrogen for a further 1 hour, consumption of
stage 4 was achieved.
[0427] A successful reaction was worked up by filtration, followed
by evaporation to remove methanol, leaving the product as a thick
suspension in water. Ethanol was added and the solid filtered to
give Psilocybin in 69% yield. .sup.1H NMR confirmed the identity of
the product but indicated a minor related impurity was present.
LCMS analysis indicated a purity of 95.2% with the major impurity
(4.1%) being identified as the pyrophosphoric acid impurity. (Table
7) deriving from the anhydride impurity at stage 4. It was later
shown that this impurity was effectively purged during the final
product re-crystallisation (Stage 6).
[0428] A further reaction was then carried out using stage 4
material from the finalised THF workup which was 88.0% pure by HPLC
and contained 7.3% N-debenzylated stage 4 (converted to product),
with none of the anhydride impurity. Again completion was noted and
the reaction worked up as previously to give Psilocybin in 46%
yield. The low yield was believed to result from precipitation of
the product during the catalyst filtration step. .sup.1H NMR
confirmed the identity of the product and HPLC indicated a purity
of 98.9%.
[0429] Further development of the reaction conditions was carried
out to optimise the water volume employed and minimise product
losses during the filtration step. After the reaction, a solution
was obtained by addition of 10 volumes of water with heating to
40.degree. C. This allowed for removal of the catalyst by
filtration without incurring product losses on the filter.
[0430] Some stage 3 was generated by hydrolysis during the reaction
and workup with levels of approx. 1-2.5% appearing to be typical of
the process. A reduction in the stage 3 level was demonstrated
during the final product re-crystallisation.
Scale Up
[0431] The large scale stage 4 batch (non-GMP) was processed as a
single batch (148 g active input). Consumption of stage 4 was
achieved with 88% product and 0.9% stage 3 resulting from
hydrolysis. The anhydride impurity (6.4%) was completely converted
to the corresponding pyrophosphoric acid impurity (5.2%).
[0432] The large scale hydrogenation was filtered and concentrated
to yield 109 g of crude product after stripping back from ethanol
to reduce the water content (.about.71% by NMR assay, 86%
yield).
Experimental
[0433] 10% Pd/C (.about.50% water wet, type 87L, 0.1.times.stage 4
charge) was charged to a vessel under N.sub.2 followed by Methanol
(20 vol wrt stage 4) and Stage 4. The N.sub.2 was replaced with
H.sub.2 and the reaction was stirred under H.sub.2 (atmospheric
pressure) for 1-2 hours. The reaction was sampled for completion
and then water was added (10 vol wrt stage 4) maintaining a
temperature of <25.degree. C. The mixture stirred for a further
1-2 hours under H.sub.2 (atmospheric pressure). The reaction was
sampled and checked for completion.
[0434] If the reaction was incomplete, H.sub.2 was recharged and
the reaction continued for a further 1-12 hr until completion was
observed. The reaction was then placed under N.sub.2 and warmed to
40.degree. C. and held for 15-45 minutes. The reaction was filtered
through celite to remove catalyst, washing with methanol (13.3 vol
wrt stage 4 charge) and water (6.7 vol wrt stage 4 charge). The
filtrate was concentrated in vacuo, azeotroping with ethanol to
remove water until a solid was obtained. The differences from JNP
and the benefits can be summarised as follows:
[0435] Primarily, the reaction is monitored for levels of
intermediates by HPLC, using relative retention times (RRT) and
completion controlled with intermediates being present at less than
0.2%. The stage 5 pyrophosphoric acid impurity is also carefully
monitored to confirm that it can be controlled in the final
re-crystallisation.
[0436] The final Stage 6 process is as described in Example 1.
Example 7
Testing Methodology and Protocols
[0437] To test for purity etc the following methodology/protocols
were employed. 7.1 NMR
[0438] .sup.1H and .sup.13C NMR spectra of Psilocybin in D.sub.2O
were obtained using 400 MHz spectrometer. Chemical shifts are
reported in ppm relative to D.sub.2O in the .sup.1H NMR
(.quadrature.=4.75 ppm) and relative to MeOH (.quadrature.=49.5
ppm), which was added as a reference, in the .sup.13C NMR the
spectrum. Literature values for Psilocybin are reported in JNP.
Analysis of Psilocybin by NMR gave data that was consistent with
the structure and consistent with that reported in the literature
with only minor variations in chemical shifts for protons near the
ionisable groups which is expected as the zwitterionic nature of
the compound makes the material very sensitive to small changes in
pH.
[0439] The .sup.1H NMR and .sup.13C NMR data are outlined below and
the spectra are shown in FIGS. 10 and 11.
[0440] .sup.1H NMR Data (400 MHz, D.sub.2O): 2.79 (s, 3H), 3.18 (t,
J=7.4 Hz, 2H), 3.31 (t, J=7.4 Hz, 2H), 6.97 (d, J=8.0 Hz, 1H), 7.08
(s, 1H), 7.10 (t, J=8.0 Hz, 1H), 7.19 (d, 8.2 Hz, 1H).
[0441] .sup.13C NMR Data (400 MHz, D.sub.2O (+ trace MeOH): 22.3
(1.times.CH.sub.2), 43.4 (2.times.CH.sub.3), 59.6
(1.times..times.CH.sub.2), 108.4 (1.times.CH), 108.6 (1.times.C),
109.5 (1.times.CH), 119.1 (d, .sup.3J.sub.P-H=6.7 Hz, 1.times.C),
123.3 (1.times.CH2), 124.8 (1.times.CH), 139.3 (1.times.C), 146.3
(d, .sup.2J.sub.P-H=6.7 Hz, 1.times.C)
7.2 FT-IR
[0442] Data was collected on a Perkin Elmer Spectrum Two.TM.
Spectrometer with UATR Two accessory. Analysis of Psilocybin
(Batch: AR755) by FT-IR spectroscopy gave a spectrum (FIG. 12) that
is consistent with the proposed structure. The broad peak at 3244
cm.sup.-1 is typical of an amine salt. The remainder of the peaks
are in the fingerprint region and therefore can't be assigned
individually.
7.3. Mass Spectrometry
[0443] The mass spectrum of Psilocybin (AR755) was obtained on a
Bruker Esquire 3000 plus Ion Trap Mass Spectrometer and was
concordant with the structure. The mass spectrum (FIG. 13) showed a
main peak at m/z=284.8 and 568.1 that corresponded to (M+H).sup.+
and (2M+H).sup.+ of Psilocybin. This implied the molecular ion has
m/z 284 corresponding to the molecular formula of Psilocybin
(C.sub.12H.sub.17N.sub.2O.sub.4P) (FIG. 14).
7.4 Residue on Ignition
[0444] The residue on ignition method follows the pharmacopeia
method with one adjustment. Inconsistent results were obtained when
the crucible was heated to 500.degree. C. and it is believed this
is due to low volatility of the phosphate residues that are
generated. The temperature was therefore increased to 800.degree.
C. for Psilocybin and consistent and accurate results were
obtained.
7.5 HPLC--Assay and Purity Determination
[0445] The HPLC method used for assay, chemical purity and
quantifying the impurities of Psilocybin is a gradient HPLC-UV
method and the conditions are outlined in Table 22. External
standards are used for quantification. Approximately 1 mg/mL of
Psilocybin was dissolved in Purified Water:MeOH (95:5). Sonicate to
fully dissolve.
[0446] Purity by HPLC is calculated in the basis of area % and is
correlated against a known retention time standard.
[0447] Assay by HPLC is calculated on an anhydrous basis, based on
weight % versus a standard of known purity and composition.
TABLE-US-00022 TABLE 22 Typical HPLC Conditions for Identification,
Purity and Assay Parameter Conditions System Agilent 1100 series
liquid chromatograph or equivalent Column XBridge C18, 4.6
.times.150 .mu.m; 3.5 .mu.m (Ex; waters PN: 186003034) Flow Rate
1.0 ml.min.sup.-1 Injection Volume 5 .mu.l Detection UV @ 267 nm
Column Temperature 30.degree. C. Mobile Phase A-Purified
Water:Methanol:TFA (95:5:0.1) B-Methanol:Purified Water:TFA
(95:5:0.1) Gradient Time (mins) % A % B 0 100 0 2 100 0 15 0 100 20
0 100 22 100 0
7.6 Residual Solvent Content by HRGC
[0448] The HRGC method for quantifying residual solvents is a
headspace method and is described in Table 23 below:
TABLE-US-00023 TABLE 23 Typical Residual Solvent GC Method
Parameter Conditions System Agilent 6890/7890 HRGC or similar
Column DB-624 60 m .times. 0.32 mm, 1.80 .mu.m film thickness (or
equivalent) Oven Program 40.degree. C. (hold for 15 min) then ramp
(20.degree. C. min.sup.-1) to 200.degree. C. (hold 5 min) Headspace
Parameters Oven Temp 125.degree. C. Loop Temp 140.degree. C.
Transfer Line Temp 150.degree. C. Split Ratio 10:1 Injector
temperature 200.degree. C. Detector temperature 250.degree. C., FID
Head pressure 15 psi, constant pressure Carrier gas Nitrogen Column
flow 2.0 ml min.sup.-1 @ 40.degree. C. Internal Standard
1,2-Difluorobenzene Levels of the following solvents and reagents
are determined: Methanol, Ethanol, THF and Toluene.
7.7 Melting Point by DSC
[0449] DSC data was collected on a PerkinElmer Pyris 6000 DSC (or
similar). The instrument was verified for energy and temperature
calibration using certified indium. The sample was weighed
(typically 0.5 to 3.0 mg) into a pin-holed aluminium sample pan.
The pan was crimped with an aluminium pan cover. The pan was heated
at 20.degree. C./min from 30 to 300.degree. C. with a purge of dry
nitrogen at 20 mL/min. During the melting point procedure, each
batch of Psilocybin Polymorph A or A' exhibited two endothermic
events the latter; the first of which was attributed to solid-solid
transition of Polymorph A or A' to Polymorph B, and the second of
which was attributed to melting of Polymorph B.
7.8 Polymorphism by XRPD
[0450] The solid state form of Psilocybin is determined by XRPD.
XRPD diffractograms were collected on a diffractometer (such as a
PANalytical X'Pert PRO or equivalent) using Cu K.alpha. radiation
(45 kV, 40 mA), .theta.-.theta. goniometer, focusing mirror,
divergence slit (1/2''), soller slits at both incident and
divergent beam (4 mm) under ambient conditions. The data collection
range was 3-35.degree.2.theta. with a continuous scan speed of 0.2
s.sup.-1. The resulting diffractogram is compared to that of a
reference diffractogram of Polymorph A or A' to ensure that it is
concordant (FIG. 7a or 7b respectively).
7.9 Thermo-Gravimetric Analysis (TGA)
[0451] TGA data was collected on a PerkinElmer Pyris 1 TGA (or
similar). The instrument was calibrated using a certified weight
and certified Alumel and Perkalloy for temperature. A predefined
amount of sample (typically ca. 5 mg) was loaded into an aluminium
crucible, and was heated at 20.degree. C./min from ambient
temperature to 400.degree. C. A nitrogen purge at 20 mL/min was
maintained over the sample.
7.10 Loss on Drying
[0452] Determine in duplicate the loss on drying of the sample
using a 1 g portion, accurately weighed, dried at 70.degree. C.,
under vacuum to constant weight.
[0453] Calculation:
% Loss on Drying = ( W I N T I A L - W F I NAL ) W S A M P L E
.times. 1 0 0 ##EQU00001##
Where:
[0454] W.sub.INITIAL=Initial weight of dish and sample prior to
drying (g) W.sub.FINAL=Final weight of dish and dried sample (g)
W.sub.SAMPLE=Weight of sample (g)
Example 8
Forced Degradation Studies
[0455] Psilocybin drug substance was stressed under various
conditions in solution and in the solid state to provide samples to
evaluate analytical method selectivity.
[0456] The forced degradation study was performed on Psilocybin;
based on the requirements of ICH Q1A(R2). Testing under stressed
conditions has provided information on potential degradation
pathways and the intrinsic stability of Psilocybin. The optimised
analytical method employed demonstrated specificity to Psilocybin;
it was shown to be suitable and changes to identity, purity and
potency of the product can be detected using this method. The
method used has also been shown to be free from interferences from
likely impurities and degradation products in accordance with ICH
Q2(R1) (Validation of Analytical Procedures) with reference to
specificity. Therefore, the HPLC method is deemed suitable for
determining purity of Psilocybin and related impurities.
[0457] The control sample of Psilocybin was stable in solution over
the study period (study period was 7 days for non-photostability
samples). Psilocybin degraded slowly when heated in solution
producing psilocin as the major impurity. Psilocybin was also
stable under acid conditions at room temperature. However, at
60.degree. C. a slow and steady degradation was observed producing
psilocin as the main impurity. Psilocybin was slightly unstable at
room temperature in the presence of base with slow degradation to a
range of impurities over the study period. Only very low levels of
impurities were formed under the peroxide conditions with the
overall purity dropping by .about.0.5%. In the solid state, slow
chemical degradation was noted (3 days at 150.degree. C.)
predominantly producing psilocin (stage 3) as an impurity.
Psilocybin was stable under photostability conditions both as a
solid and when in solution.
Stability Studies
[0458] Stability studies were undertaken with two batches of
Psilocybin as shown in Table 24.
TABLE-US-00024 TABLE 24 Study number/ Drug Intended time Study
Substance Site of Lot Storage points/Study start Lot No. Packaging
Manufacture Use Condition Status ON- GM764B Double Onyx Ref.
2-8.degree. C. 1, 3, 6 months YXSTAB0138 food Scientific Std.
25.degree. C./60% ongoing grade RH 1, 3, 6 months Polythene
40.degree. C./75% ongoing bags. RH 1, 3, 6 months Outer ongoing
Polythene container ON- 170231 Double Onyx Clinical 2-8.degree. C.
1, 3, 6, 9, 12, YXSTAB0139 food Scientific 25.degree. C./60% 18,
24, 36 grade RH months ongoing Polythene 40.degree. C./75% 1, 3, 6,
9, 12, bags. RH 18, 24, 36 Outer months ongoing Polythene 1, 3, 6
months container ongoing
[0459] Samples were double bagged in food grade polythene bags and
sealed in an outer polythene container and placed on storage at
2-8.degree. C., 25.degree. C./60% RH and 45.degree. C./75% RH, a
desiccant bag is included between the inner polythene bags to
prevent moisture uptake. Tests for appearance, water content,
purity and assay were carried out.
[0460] The protocols for the two studies are shown in Table 25 and
Table 26. The one month and three months stability data for batch
GM764B are detailed in Table 27 and Table 28 below. The one, three,
six, nine and twelve month stability data for GMP batch 170231 are
provided in Table 29, Table 30, Table 31, Table 32 and Table 33
respectively below.
TABLE-US-00025 TABLE 25 Onyx Stability Trial Protocol Sheet
Product: Psilocybin Onyx trial number: ONYXSTAB0138 Batch number:
GM764B Trial due start date: 10 Mar. 2017 Test method: N/A Date of
manufacture: 6 Feb. 2017 Additional information: 1200 mg of
material required in each container. Packaging components: Double
polythene bagged lined contained within 300 ml HDPE container (food
grade). Insert a desiccant bag between the two polythene bags. Test
parameters Routine tests Appearance Assay (Anhydrous basis) by
.sup.1H-NMR Water Content by loss on drying Chemical
Purity/Impurities by HPLC Months 1 3 6 Spares Total 2.degree.
C.-8.degree. C. X X X 2 5 25.degree. C./60% RH X X X 2 5 40.degree.
C./75% RH X X X 0 3 Date due off 10 Apr. 10 Jun. 10 Sep. 13 17 17
17
TABLE-US-00026 TABLE 26 Onyx Stability Trial Protocol Sheet
Product: Psilocybin Onyx trial number: ONYXSTAB0139 Batch number:
170231 Trial due start date: 31 Mar. 2017 Test method:
SS/PSILOCYBIN/ Date of manufacture: 27 Feb. 2017 Additional
information: 2200 mg of material required in each container.
Packaging components: Double polythene bagged lined contained
within 300 ml HDPE container (food grade). Insert a desiccant bag
between the two polythene bags. Test parameters Routine tests
Appearance Assay (on a dry basis) by HPLC Water Content by loss on
drying Chemical Purity/Impurities by HPLC Timepoint 1 3 6 9 12 18
24 36 month months months months months months months months Spares
Total 2-8.degree. C. X X X X X X X X 2 10 25.degree. C./60% RH X X
X X X X X X 2 10 40.degree. C./75% RH X X X 1 4 Date due off 31
Apr. 30 Jun. 30 Sep. 31 Dec. 31 Mar. 30 Sep. 31 Mar. 31 Mar. 24 17
17 17 17 18 18 19 20
TABLE-US-00027 TABLE 27 One Month Stability Data for Batch GM764B
Test Specification Limit T = 0 T = 1 month T = 1 month T = 1 month
Condition N/A N/A 2.degree. C.-8.degree. C. 25.degree. C./60%RH
40.degree. C./75%RH Appearance For information only. An off white
solid. An off white solid. An off white solid. An off white solid.
Free from visible Free from visible Free from visible Free from
visible contamination contamination contamination contamination
Assay by .sup.1H-NMR For information only. 97% w/w 99% w/w 98% w/w
96% w/w Water content by loss For information only. 0.86% w/w 0.35%
w/w 0.20% w/w 0.14% w/w on drying Chemical Purity By For
information only. 99.24% 99.24% 99.22% 99.23% HPLC Impurities by
HPLC: For Information only. (Quote all GT 0.05%) RRT 0.86 0.05%
0.05% 0.05% 0.05% RRT 1.46 0.05% 0.09% 0.10% 0.10% RRT 1.59
(Psilocin) 0.37% 0.35% 0.34% 0.34% Total Impurities 0.76% 0.76%
0.78% 0.77%
TABLE-US-00028 TABLE 28 Three Month Stability Data for Batch GM764B
Test Specification Limit T = 0 T = 3 month T = 3 month T = 3 month
Condition N/A N/A 2.degree. C.-8.degree. C. 25.degree. C.-60%RH
40.degree. C.-75%RH Appearance For information only. An off white
solid. An off white solid. An off white solid. An off white solid.
Free from visible Free from visible Free from visible Free from
visible contamination contamination contamination contamination
Assay by .sup.1H-NMR For information only. 97% w/w 97% w/w 99% w/w
97% w/w Water content by loss For information only. 0.86% w/w 0.26%
w/w 0.08% w/w 0.14% w/w on drying Chemical Purity By For
information only. 99.24% 99.31% 99.27% 99.26% HPLC Impurities by
HPLC: For Information only. (Quote all GT 0.05%) RRT 0.86 0.05% LT
0.05% LT 0.05% LT 0.05% RRT 1.46 0.05% 0.10% 0.09% 0.10% RRT 1.59
(Psilocin) 0.37% 0.37% 0.36% 0.37% Total Impurities 0.76% 0.69%
0.73% 0.74%
TABLE-US-00029 TABLE 29 One Month Stability Data for Batch 170231
Test Specification Limit T = 0 T = 1 month T = 1 month T = 1 month
Condition N/A N/A 2.degree. C.-8.degree. C. 25.degree. C./60%RH
40.degree. C./75%RH Appearance For information only. An off white
solid. An off white solid. An off white solid. An off white solid.
Free from visible Free from visible Free from visible Free from
visible contamination contamination contamination contamination
Chemical Purity By For information only. 99.28% 99.20% 99.16%
99.17% HPLC Impurities by HPLC: (Quote all GT 0.05%) RRT 1.49 For
Information only. 0.06% 0.05% 0.05% 0.06% RRT 1.62 (Psilocin) 0.39%
0.36% 0.37% 0.36% RRT 1.70 0.05% LT 0.05% LT 0.05% LT 0.05%
Impurity at RRT 1.89 LT 0.05% LT 0.05% LT 0.05% LT 0.05% Impurity
at RRT 2.45 LT 0.05% LT 0.05% LT 0.05% LT 0.05% Impurities LT 0.05%
0.22% 0.39% 0.42% 0.41% Total Impurities 0.72% 0.80% 0.84% 0.83%
Assay by HPLC For information only 98.65% w/w 98.76% w/w 97.98% w/w
98.52% w/w (on a dry basis) Water content by loss For information
only. 0.32% w/w 0.27% w/w 0.17% w/w 0.19% w/w on drying
TABLE-US-00030 TABLE 30 Three Month Stability Data for Batch 170231
Test Specification Limit T = 0 T = 3 months T = 3 month T = 3 month
Condition N/A N/A 2.degree. C.-8.degree. C. 25.degree. C./60%RH
40.degree. C./75%RH Appearance For information only. An off white
solid. An off white solid. An off white solid. An off white solid.
Free from visible Free from visible Free from visible Free from
visible contamination contamination contamination contamination
Chemical Purity By For information only. 99.28% 99.30% 99.31%
99.17% HPLC Impurities by HPLC: (Quote all GT 0.05%) RRT 0.69 For
Information only. LT 0.05% 0.05% LT 0.05% LT 0.05% RRT 1.49 0.06%
0.05% 0.05% 0.06% RRT 1.62 (Psilocin) 0.39% 0.37% 0.36% 0.39% RRT
1.70 0.05% LT 0.05% LT 0.05% LT 0.05% Impurity at RRT 1.89 LT 0.05%
LT 0.05% LT 0.05% LT 0.05% Impurity at RRT 2.45 LT 0.05% LT 0.05%
LT 0.05% LT 0.05% Impurities LT 0.05% 0.22% 0.22% 0.27% 0.34% Total
Impurities 0.72% 0.70% 0.69% 0.79% Assay by HPLC For information
only 98.65% w/w 98.45% w/w 99.46% w/w 98.64% w/w (on a dry basis)
Water content by loss For information only. 0.32% w/w 0.17% w/w
0.01% w/w 0.19% w/w on drying
TABLE-US-00031 TABLE 31 Six Month Stability Data for Batch 170231
Test Specification Limit T = 0 T = 6 months T = 6 months T = 6
months Condition N/A N/A 2.degree. C.-8.degree. C. 25.degree.
C.-60%RH 40.degree. C.-75%RH Appearance For information only. An
off white solid. An off white solid. An off white solid. An off
white solid. Free from visible Free from visible Free from visible
Free from visible contamination contamination contamination
contamination Chemical Purity By For information only. 99.28%
99.20% 99.19% 99.12% HPLC Impurities by HPLC: (Quote all GT 0.05%)
RRT 0.69 For Information only. LT 0.05% 0.06% 0.06% 0.06% RRT 1.49
0.06% 0.07% 0.07% 0.08% RRT 1.62 (Psilocin) 0.39% LT 0.05% LT 0.05%
LT 0.05% RRT 1.70 0.05% 0.35% 0.34% 0.38% Impurity at RRT 1.89 LT
0.05% LT 0.05% LT 0.05% LT 0.05% Impurity at RRT 2.45 LT 0.05% LT
0.05% LT 0.05% LT 0.05% Impurities LT 0.05% 0.22% LT 0.05% LT 0.05%
LT 0.05% 0.32% 0.34% 0.36% Total Impurities 0.72% 0.80% 0.81% 0.88%
Assay by HPLC For information only 98.65% w/w 97.97% w/w 98.04% w/w
100.10% w/w (on a dry basis) Water content by loss For information
only. 0.32% w/w 0.06% w/w 0.32% w/w 2.26% w/w on drying
TABLE-US-00032 TABLE 32 Nine Month Stability Data for Batch 170231
Test Specification Limit T = 0 T = 9 months T = 9 months Condition
N/A N/A 2.degree. C.-8.degree. C. 25.degree. C.-60%RH Appearance
For information only. An off white solid. An off white solid. An
off white solid. Free from visible Free from visible Free from
visible contamination contamination contamination Chemical Purity
By For information only. 99.28% 99.16% 99.16% HPLC Impurities by
HPLC: (Quote all GT 0.05%) RRT 0.69 For Information only. LT 0.05%
LT 0.05% LT 0.05% RRT 1.49 0.06% LT 0.05% LT 0.05% RRT 1.62
(Psilocin) 0.39% 0.07% 0.05% RRT 1.70 0.05% 0.06% 0.06% Impurity at
RRT 1.89 LT 0.05% 0.37% 0.37% Impurity at RRT 2.45 LT 0.05% LT
0.05% LT 0.05% Impurities LT 0.05% 0.22% LT 0.05% LT 0.05% LT 0.05%
LT 0.05% 0.34% 0.35% Total Impurities 0.72% 0.84% 0.84% Assay by
HPLC For information only 98.65% w/w 97.53% w/w 98.12% w/w (on a
dry basis) Water content by loss For information only. 0.32% w/w
0.21% w/w 0.10% w/w on drying
TABLE-US-00033 TABLE 33 Twelve Month Stability Data for Batch
170231 Test Specification Limit T = 0 T = 12 months T = 12 months
Condition N/A N/A 2.degree. C.-8.degree. C. 25.degree. C./60%RH
Appearance For information only. An off white solid. An off white
solid. An off white solid. Free from visible Free from visible Free
from visible contamination contamination contamination Chemical
Purity By For information only. 99.28% 99.25% 99.25% HPLC
Impurities by HPLC: (Quote all GT 0.05%) RRT 0.69 For Information
only. LT 0.05% LT 0.05% LT 0.05% RRT 1.49 0.06% LT 0.05% LT 0.05%
RRT 1.62 (Psilocin) 0.39% 0.37% 0.37% RRT 1.70 0.05% LT 0.05% LT
0.05% Impurity at RRT 1.89 LT 0.05% LT 0.05% ND Impurity at RRT
2.45 LT 0.05% LT 0.05% LT 0.05% Impurities LT 0.05% 0.22% 0.38%
0.38% Total Impurities 0.72% 0.75% 0.75% Assay by HPLC For
information only 98.65% w/w 99.63% w/w 98.97% w/w (on a dry basis)
Water content by loss For information only. 0.32% w/w 0.49% w/w
0.61% w/w on drying
[0461] Over the first 12 months of the ICH stability study
Psilocybin has proven to be chemically stable under low temperature
(2-8.degree. C.), ambient (25.degree. C./60% RH) and accelerated
(40.degree. C./75% RH) conditions. There has been no change in the
appearance and HPLC analysis has also remained consistent. The
water content has varied in all samples, due to the initial impact
and then aging of the desiccant bags used in the study.
Example 9--Experimental to Form Hydrate A
[0462] Psilocybin (200 mg) was charged to a crystallisation tube
followed by deionised water (4 ml). The mixture was equilibrated at
25.degree. C. for 2 hours before the solid was isolated by vacuum
filtration. The material was split into two equal portions. One
portion was not subjected to further drying to give Hydrate A, lot
GK2, by XRPD and DSC (diffractogram and thermogram consistent with
FIG. 7d and FIG. 8d respectively).
Example 10--Experimental to Form Polymorph B
[0463] Psilocybin Polymorph A (250 mg) was charged to a round
bottom flask, heated to 173.degree. C. using an oil bath and held
at temperature for 5 minutes. The solid was cooled to ambient
temperature and isolated to give lot GK3 with a recovery of 93%.
Analysis by XRPD and DSC revealed lot GK3 to be Polymorph B
(diffractogram and thermogram consistent with FIG. 7c and FIG. 8c
respectively).
Example 11--Solid State Investigations
[0464] A number of polymorphism investigations were completed. A
summary of the solid forms found is shown in FIG. 17. The majority
of the forms found were derived from solvent perturbation; in some
cases stoichiometric solvates were isolated and in other cases
non-stoichiometric solvates.
Slurries of Polymorph A
[0465] Solvent mediated equilibrations of Psilocybin Pattern A were
conducted as a primary route into modification of the solid form
and to visually assess the solubility of the material in a range of
24 solvents between 25 and 50.degree. C.
[0466] Psilocybin Pattern A (40 mg) was dosed into tubes at room
temperature and solvents as listed in Table 34 were added in
aliquots of 0.4 ml (10 vol.) to a total volume of 1.2 ml (30 vol.)
and observations noted. The mixtures were agitated constantly. Heat
cycling was conducted as follows: 50.degree. C. for 18 hours, cool
over 2 hours to 20.degree. C., mature for 4 hours, heat to
50.degree. C. for 4 hours, cool to 20.degree. C. over 2 hours,
mature for 18 hours. A repeat 50.degree. C.-20.degree. C. cycle
over 24 hours was conducted and the following applied:
[0467] Isolation post heating to 50.degree. C. where solids were
sufficient=A series
[0468] Isolation post cooling to 20.degree. C. where solids were
sufficient=B series
All isolated solids were dried in vacuo at 50.degree. C. for 24
hours and analysed by XRPD. The observations are provided in Table
34.
[0469] The API was largely insoluble in the solvents and solvent
mixtures tested in 30 volumes at 50.degree. C. resulting in heavy
suspensions. Water did solubilise Psilocybin at 50.degree. C.
TABLE-US-00034 TABLE 34 Tabulated observations for heat cycling
slurry maturations and using Pattern A blend as input Obs.
20.degree. C., Obs. 20.degree. C., Obs. 20.degree. C., Obs. XRPD
XRPD Entry Solvent 0.4 ml 0.8 ml 1.2 ml 50.degree. C. A series B
series 1 Cyclohexane Susp. Susp. Susp. Susp. A A 2 Chlorobenzene
Susp. Susp. Susp. Susp. A A 3 2-Chlorobutane Susp. Susp. Susp.
Susp. A A 4 Benzotrifluoride Susp. Susp. Susp. Susp. A A 5 Anisole
Susp. Susp. Susp. Susp. A A 6 Nitromethane Susp. Susp. Susp. Susp.
C C 7 CPME Susp. Susp. Susp. Susp. A A 8 Heptane Susp. Susp. Susp.
Susp. A A 9 TBME Susp. Susp. Susp. Susp. C A 10 MIBK Susp. Susp.
Susp. Susp. A A 11 MEK Susp. Susp. Susp. Susp. A A 12 iPrOAc Susp.
Susp. Susp. Susp. C C 13 EtOAc Susp. Susp. Susp. Susp. A A 14
Toluene Susp. Susp. Susp. Susp. A A 15 THF Susp. Susp. Susp. Susp.
A A 16 CHCl3 Susp. Susp. Susp. Susp. A A 17 MeOH Susp. Susp. Susp.
Susp. D D 18 EtOH Susp. Susp. Susp. Susp. E E 19 IPA Susp. Susp.
Susp. Susp. F F 20 MeCN Susp. Susp. Susp. Susp. C A 21 Water Susp.
Susp. Susp. Solution n/a A 22 4:1 EtOH/water Susp. Susp. Susp.
Susp. A A 23 4:1 THF/water Susp. Susp. Susp. Susp. A Hydrate A 24
4:1 IPA/water Susp. Susp. Susp. Susp. A C
[0470] Results:
[0471] In the figures (FIG. 18 and FIG. 19), "25 C" denotes
isolation of the solid at 25.degree. C. and "50 C" denotes
isolation of the solid at 50.degree. C. For example, GM832-20_50_A9
represents GM832 entry 20 (MeCN) isolated at 50.degree. C.
[0472] 50.degree. C. Slurries
[0473] Entries 1, 2, 3, 4, 5, 7, 8, 10, 11, 13, 14, 15, 16, 22, 23,
24: XRPD diffractogram broadly consistent with Polymorph A, but
with an additional peak of varying intensity at
18.degree.2.theta..
[0474] Entries 6, 9, 12, 20: XRPD diffractogram acquired for the
isolated solids were broadly consistent (see FIG. 18) with
additional peaks at 10.degree.2.theta. and 13.2.degree.2.theta.
observed for some samples. This XRPD diffractogram was designated
Pattern C. There is no chemotype correlation between the solvents
(CH.sub.3NO.sub.2, TBME, iPrOAc and CH.sub.3CN).
[0475] Entry 17: XRPD diffractogram acquired had multiple
diffraction peaks (FIG. 19). The XRPD diffractogram was designated
Pattern D.
[0476] Entry 18: XRPD diffractogram acquired had multiple
diffraction peaks (FIG. 19). The XRPD diffractogram was designated
Pattern E.
[0477] Entry 19: XRPD diffractogram acquired had multiple
diffraction peaks (FIG. 19). The XRPD diffractogram was designated
Pattern F.
[0478] 25.degree. C. slurries:
[0479] Entries 1, 2, 3, 4, 5, 7, 8, 9, 10, 11, 13, 14, 15, 16, 20,
21, 22: XRPD diffractograms are all similar to that acquired for
Polymorph A.
[0480] Entries 6, 12, 24: XRPD diffractograms acquired for the
isolated solids were broadly consistent (see FIG. 18) with Pattern
C.
[0481] Entry 23: XRPD analysis showed Hydrate A had formed.
[0482] Entry 17: XRPD diffractogram acquired had multiple
diffraction peaks (FIG. 19). The XRPD diffractogram was designated
Pattern D.
[0483] Entry 18: XRPD diffractogram acquired had multiple
diffraction peaks (FIG. 19). The XRPD diffractogram was designated
Pattern E.
[0484] Entry 19: XRPD diffractogram acquired had multiple
diffraction peaks (FIG. 19). The XRPD diffractogram was designated
Pattern F.
[0485] Analysis of Results:
[0486] The XRPD diffractograms for the solids isolated at
25.degree. C. are broadly the same as for the XRPD diffractograms
acquired for the solids isolated at 50.degree. C.
[0487] Patterns D, E and F were derived from alcohols (MeOH, EtOH
and IPA). Solvated states were postulated considering an Ethanol
Solvate was previously isolated during development. The XRPD
diffractograms for the Ethanol Solvate are not identical, however,
given that exact solvent level variation may deliver varying states
of order within the lattice, the comparison between these XRPD
diffractograms provides a strong hypothesis that these more
significant phase variations are invoked by solvent
entrainment.
[0488] XRPD patterns D, E and F (FIG. 19) are all dissimilar to the
XRPD diffractogram for Hydrate (FIG. 7d).
[0489] Direct comparison of the XRPD diffractograms acquired for
the MeOH, EtOH and IPA derived solids (Patterns D, E and F--FIG.
19) isolated at the two temperatures shows conforming
diffractograms; the two MeOH diffractograms are similar while the
EtOH and IPA are directly comparable.
[0490] DSC analysis was performed on the isolated solids, and where
sufficient sample was available, TGA. The solids that delivered
Patterns D, E and F all features endotherms at ca. 170-180.degree.
C. but otherwise proffered distinct thermal profiles. TGA analysis
for the MeOH slurry isolated solid showed one protracted mass loss
from ca. 25-190.degree. C. (3.1%). A stochiometric methanol solvate
would require 10.3% weight solvent. TGA analysis of the EtOH slurry
isolated solid showed two distinct mass loss steps. The first one
occurred before 100.degree. C. (0.3% weight) is considered to be
due to water, and the second larger loss (11.5% weight) due to
solvent. A stoichiometric ethanol solvate requires 13.9% weight
solvent. TGA analysis of the IPA slurry isolated solid featured two
distinct mass loss steps. The first mass loss before 100.degree. C.
(0.4% weight) is considered to be due to water, while the second
larger mass loss (13.9% weight) is considered to be due to residual
solvent. A stoichiometric IPA solvate requires 17.5% weight
solvent.
[0491] Slurries of Amorphous Psilocybin
[0492] In order to generate amorphous material a small sample of
Psilocybin (0.5 g) was dissolved in water (0.5 L, 1000 vol.),
polish filtered and lyophilised. Psilocybin was recovered as an off
white fibrous material (lot MC1368A; 412 mg, 82%, XRPD
amorphous).
[0493] To assess visually solubility of the amorphous API and to
induce form modification, a series of slurry maturations were
performed as follows:
Psilocybin (15 mg) was charged to tubes. Solvent was then added at
ambient temperature (20.degree. C., 0.3 ml, 20 vol.) and the
suspensions agitated. Observations were made. After 1 hour of
stirring, samples were heated to 45.degree. C. for 18 hours and
observations made. Samples were heated to 50.degree. C. for 8 hours
and observations were made. The samples were agitated for 72 hours
at 25.degree. C. and subject to a final heat cycle, prior to
isolation. Observations are shown in Table 35.
TABLE-US-00035 TABLE 35 Observations of amorphous Psilocybin during
heat cycling slurry maturation and form fate Obs. at Obs. at Obs.
at Entry Solvent 20.degree. C. 45.degree. C. 50.degree. C. XRPD
Data A Cyclohexane Susp. Susp. Susp. Semi-crystalline B
Chlorobenzene Susp. Susp. Susp. Semi-crystalline C Chlorobutane
Susp. Susp. Susp. Pattern B D Benzotrifluoride Susp. Susp. Susp.
Semi-crystalline E Anisole Susp. Susp. Susp. Semi-crystalline F
Nitromethane Susp. Susp. Susp. Pattern B G CPME Susp. Susp. Susp.
Semi-crystalline H Heptane Susp. Susp. Susp. Semi-crystalline I
TBME Susp Susp. Susp. Semi-crystalline J MIBK Susp. Susp. Susp.
Semi-crystalline K MEK Susp. Susp. Susp. Semi-crystalline L iPrOAc
Susp. Susp. Susp. Semi-crystalline M EtOAc Susp. Susp. Susp.
Semi-crystalline N Toluene Susp. Susp. Susp. Similar to Solvate A O
THF Susp. Susp. Susp. Semi-crystalline P Chloroform Susp. Susp.
Susp. Similar to Pattern E R MeOH Susp. Susp. Susp.
Semi-crystalline S EtOH Susp. Susp. Susp. Pattern D T IPA Susp.
Susp. Susp. Pattern B U Acetonitrile Susp. Susp. Susp. Amorphous V
Water Susp. Susp. Susp. Similar to Pattern A W 4:1 EtOH/water Susp.
Susp. Susp. Similar to Pattern D X 4:1 THF/water Susp. Susp. Susp.
Similar to Pattern A Y 4:1 IPA/water Susp. Susp. Susp. Similar to
Pattern A
[0494] Results
[0495] The majority of solvents returned a solid that was
considered to be semi-crystalline (predominantly amorphous with a
notable reflection at ca. 18.degree.2.theta.).
[0496] Truly amorphous was returned from equilibration in MeCN.
[0497] Polymorph B was returned from chlorobutane, nitromethane and
IPA (FIGS. 20 and 22).
[0498] Pattern D, which was isolated from MeOH in the Polymorph A
slurry experiments discussed above, was returned from the EtOH
equilibration whereas MeOH in this study returned a
semi-crystalline solid.
[0499] Solids similar to Pattern A were recovered from water,
THF:Water and IPA:Water (4:1).
[0500] A solid similar to Pattern D was recovered from EtOH:Water
(4:1), supporting the finding of the isolation of Pattern D from
EtOH alone.
[0501] A solid similar to Pattern E was recovered from
Chloroform.
[0502] From none of the solvents investigated was true Polymorph A
or A' isolated following extended equilibration and thermal
maturation of amorphous Psilocybin.
Example 12--Formulation Development
[0503] An initial series of experiments were conducted using
formulations as set out in Table 36 below. The objective was to
identify suitable single filler or combination fillers for large
scale formulation.
TABLE-US-00036 TABLE 36 Batch No (% w/w) APL-117- APL-117- APL-117-
Material Name 6085-01 6085-02 6085-03 Psilocybin 1.0 1.0 1.0
Microcrystalline 91.5 49.5 81.5 cellulose Ph 102 Pregelatinised
Starch -- 45.0 -- (Starch 1500) Compact Cel MAB -- -- 10
Hydroxpropyl 3.0 3.0 3.0 Cellulose (Klucel EXF) Sodium Starch 3.0
3.0 3.0 Glycolate Colloidal silicon 0.5 0.5 0.5 Dioxide Magnesium
Stearate 1.0 1.0 1.0 (Vegetable Derived) Sodium Stearyl -- -- --
Fumarate TOTAL 100.0 100.0 100.0
[0504] The outcome, in terms of key physiochemical
properties--Material flow, Blend Uniformity, and Content Uniformity
are set out in Table 37 below:
TABLE-US-00037 TABLE 37 Strength Material flow Blend Content Batch
No (mg) (Carrs Index) Uniformity uniformity APL-117- 1.0 19.1 TOP =
127.9 % Label 6085-01 Middle = 106.4 claim = 92.4 Bottom = 104.5 AV
= 7.9 Mean = 112.9 % RSD = 10.9 APL-117- 1.0 19.1 TOP = 115.9 %
Label 6085-02 Middle = 106.6 claim = 95.2 Bottom = 106.1 AV = 5.9
Mean = 109.6 % RSD = 4.9 APL-117- 1.0 22.4 TOP = 105.0 % Label
6085-03 Middle = 101.4 claim = 96.3 Bottom = 98.7 AV = 4.6 Mean =
101.7 % RSD = 3.8
[0505] Whilst batch (APL-117-6085-03) showed good blend uniformity
across different sample analysed (TOP, MIDDLE and BOTTOM) and very
good content uniformity its flow property (based on Carr's index)
were towards the high end and it was predicted that the formulation
would not accommodate higher drug loads.
[0506] For this reason, a number of alternative formulations were
trialled. The objective was to consider other filler combinations
with the aim of improving the powder flow as well as achieving good
blend uniformity and content uniformity.
[0507] Formulations containing less Compactcel MAB and higher
amount of glidant compared to Batch 3 (APL-117-6085-03) were also
trialed
[0508] These further formulations are set out in Table 38
below.
TABLE-US-00038 TABLE 38 Batch No (% w/w) APL-117- APL-117- APL-117-
Material Name 6085-05 6085-06 6085-07 Psilocybin 1.0 1.0 5.0
Microcrystalline cellulose Ph 102 -- 89.0 85.0 Pregelatinised
Starch (Starch 45.0 -- -- 1500) Compact Cel MAB -- 5.0 5.0
Microcrystaline Cellulose 49.5 -- -- CEOLUS UF 702 Sodium Starch
Glycolate 3.0 3.0 3.0 Colloidal silicon Dioxide 0.5 1.0 1.0 Sodium
Stearyl Fumarate 1.0 1.0 1.0 TOTAL 100.0 100.0 100.0
[0509] The results for these Batches are shown in Table 39
below:
TABLE-US-00039 TABLE 39 Strength Material flow Blend Content Batch
No (mg) (Carrs Index) Uniformity uniformity APL-117- 1.0 20.9 TOP =
130.0 % Label 6085-05 Middle = 105.4 claim = 88.3 Bottom = 107.2 AV
= 16.5 Mean = 114.2 % RSD = 12.6% APL-117- 1.0 20.0 TOP = 107.0 %
Label 6085-06 Middle = 96.2 claim = 96.2 Bottom = 95.5 AV = 10.5
Mean = 99.6 % RSD = 6.5 APL-117- 5.0 24.3 TOP = 91.5 % Label
6085-07 Middle = 94.2 claim = 96.0 Bottom = 94.8 AV = 11.9 Mean =
93.5 % RSD = 7.0
[0510] APL-117-6085-05 failed to achieve good blend uniformity, and
also failed on content uniformity criteria.
[0511] APL-117-6085-06 and APL-117-6085-07 both exhibited improved
powder flow, but the blend uniformity for both formulations was
poorer than APL-117-6085-03.
[0512] As a consequence, Applicant looked at modified excipients
and more particularly silicified fillers with different particle
sizes. These formulations are set out in Table 40 below:
TABLE-US-00040 TABLE 40 Batch No (% w/w) Material Name
APL-117-6085-11 APL-117-6085-12 Psilocybin 5.0 1.0 Prosolv 50 10.5
15.5 Prosolv 90 80.0 79.0 Sodium Starch Glycolate 3.0 3.0 Colloidal
silicon Dioxide 0.5 0.5 Sodium Stearyl Fumarate 1.0 1.0 TOTAL 100.0
100.0
[0513] Prosolv is a silicified microcrystalline cellulose, and the
two variants were selected to determine if particle size affected
outcome. Compared to standard microcrystalline cellulose (typical
size range, depending on sieving, is 80-350 microns) the Prosolv
has a finer particle size distribution, and that gives an increased
surface area. The increased surface area it was hypothesised might
provide superior flow and increased compaction together with
improved content uniformity and stability in the formulation. The
ratio of Prosolv 50 and Prosolv 90 was to produce a particle size
distribution across both finer and coarser particles.
[0514] The results are set out in Table 41 below
TABLE-US-00041 TABLE 41 Strength Material flow Blend Content Batch
No (mg) (Carrs Index) Uniformity uniformity APL-117- 5.0 24.3 TOP =
103.4 % Label 6085-11 Middle = 100.4 claim = 94.1 Bottom = 100.2 AV
= 6.0 Mean = 101.5 % RSD = 2.0 APL-117- 1.0 21.1 TOP = 101.9 %
Label 6085-12 Middle = 98.4 claim = 100.5 Bottom = 99.9 AV = 5.8
Mean = 100.1 % RSD = 3.8%
[0515] It can be seen that the key parameters of content uniformity
(greater than 90%, and in fact greater than 94%) and AV (less than
10, and in fact less than 7) are excellent as is the consistency in
blend uniformity (greater than 95% allowing for error).
* * * * *