Note: Descriptions are shown in the official language in which they were submitted.
CA 03113727 2021-03-17
WO 2020/058750 PCT/IB2018/057274
A SEMISYNTHETIC METHOD OF PREPARING NEOSAXITOXIN
TECHNICAL FIELD
The invention relates to the semisynthetic preparation of neosaxitoxin
(neoSTX) and the purification from extracts of cultures of selected isolates
of Alexandrium pacificum of the intermediate GTX1,4 used in its preparation.
BACKGROUND ART
As stated in the publication of Garcia-Altares (2017), marine microalgal
toxins constitute one of the most diverse and sophisticated groups of natural
products. Examples are paralytic shellfish toxins (PSTs) such as saxitoxin
(STX), its analogues and derivatives. Gonyautoxins (GTXs) are sulphated
analogues of STX and marine bacteria can transform GTXs into STX through
reductive eliminations. In marine environments the main producers of STX are
eukaryotic dinoflagellates.
STX is a monoterpenoid indole alkaloid containing a tricyclic 3,4-
system with 2 guanidinium moieties formed by the NH2
groups in the positions C2 and C8 of the reduced purine:
H2N,400
0
___________________________________________________ NH2
2 9
N3
12
OH
OH
STX blocks voltage-gated sodium channels (VGSCs), but also binds to calcium
and potassium channels. The nature of the substituents greatly influences the
overall toxicity of saxitoxin analogues. The hydroxylation of Ni, e.g. as in
neosaxitoxin (neoSTX) does not play a major role in binding affinity, but
seems to increase potency.
The prior art is replete with disclosures of the cosmetic and therapeutic
applications of PSTs, including their use as local anaesthetics and
analgesics. The publication of Mezher (2018) discloses that the US Food and
Drug Administration (FDA) plans to develop guidance documents to encourage
the development of extended-release local anaesthetics which could replace
the need for systemic oral opioids in certain situations. The expectations of
the US FDA are for the development of new non-opioid drugs to treat chronic
1
CA 03113727 2021-03-17
WO 2020/058750
PCT/IB2018/057274
pain that could provide a safer alternative for patients who require long-
term use of analgesic drugs. A limitation on the exploitation and widespread
adoption of these applications is the availability of the PSTs in sufficient
quantity and of sufficient purity to render their use in the manufacture of
pharmaceutical preparations commercially viable.
The following publications disclose the preparation of gonyautoxin 1 (GTX1),
gonyautoxin 4 (GTX4) or neosaxitoxin (neoSTX). Often the preparation is on an
analytical scale, or does not provide the quantity and purity required for
use of the preparation as an active pharmaceutical ingredient (API).
The publication of Hall et al (1984) discloses the confirmation by x-ray
crystallography of the position and identity of the three substituents which,
with the parent compound, form the array of twelve saxitoxins found in
protogonyaulax.
The publication of Daigo et al (1985) discloses the extraction and isolation
of neosaxitoxin (neoSTX) from specimens of crab. The dose-death time curve
obtained for the isolated neoSTX was clearly distinguishable from the curve
for saxitoxin (STX).
The publication of Laycock et al (1994) discloses methods for the
purification of some of the common paralytic shellfish poisoning (PSP) toxins
in quantities sufficient for use as analytical standards. The PSP toxins were
purified from the dinoflagellate Alexandrium excavatum, the giant sea scallop
(Plagopecten magellanicus) hepatopancreas and the cyanobacterium
Aphanizomenon flos-aquae.
The publication of Ravn et al (1995) discloses what are asserted to be
optimal conditions for extraction of paralytic shellfish toxins from a clone
of Alexandrium tamarense. The paralytic shellfish toxins are extracted with
acetic acid and hydrochloric acid in the concentration range 0.01 to 1.0 N.
Concentrations of hydrochloric acid in the range 0.03 to 1.0 N were observed
to cause the amount of Cl and C2 toxins to decrease sharply with a
concomitant increase in the amount of gonyautoxins 2 (GTX2) and 3 (GTX3).
The publication of Tsai et al (1997) discloses the detection of paralytic
toxicity by a tetrodotoxin bioassay in specimens of crab. Partial
purification and characterisation of the toxins demonstrated the main toxin
to be tetrodotoxin with minor amounts of gonyautoxins (GTXs) and neosaxitoxin
(neoSTX) .
The publication of Siu et al (1997) discloses the examination of the effects
of environmental and nutritional factors on population dynamics and toxin
production in Alexandrium catenella. Optimum conditions for the growth of
2
CA 03113727 2021-03-17
WO 2020/058750
PCT/IB2018/057274
this species of dinoflagellate are disclosed along with the toxin profile for
a species grown under these conditions. The toxin profile as detected by HPLC
was found to include in descending order GTX4, GTX3, GTX1, B2, neosaxitoxin
(neoSTX) and saxitoxin (STX).
The publication of Sato et al (2000) discloses the transformation of the 0-
sulfate group of GTX1 and GTX4 to methylene to form neosaxitoxin. The
transformation was achieved using thiols such as glutathione and
intermediates of the conversion were isolated.
The publication of Parker et al (2002) discloses an investigation of the
autotrophic growth of the toxic dinoflagellate, Alexandrium minutum, in three
different high biomass culture systems, assessing growth, productivity and
toxin production. The organism was grown in aerated and non-aerated two litre
Erlenmeyer flasks, 0.5 litre glass aerated tubes, and a four litre lab scale
alveolar panel photobioreactor. A marked increase in biomass and productivity
in response to aeration was observed. A maximum cell concentration of 3.3 x
10 cells/mL, a mean productivity of 0.4 x 10' cells/mL/day and toxin
production of approximately 20 pg/L/day with weekly harvesting was reported.
The publication of Baker et al (2003) discloses the production by bacterial
strains isolated from saxitoxin-producing dinoflagellates of compounds that
could easily be mistaken for gonyautoxin 4 (GTX4).
The publication of Miao et al (2004) discloses the isolation of gonyautoxins
(GTX1, GTX2, GTX3 and GTX4) from two strains of Alexandrium minutum Halim.
The strain of Alexandrium minutum Halim designated Amtk4 is asserted to be
suitable for the preparation of gonyautoxins.
The publication of Jiang and Jiang (2008) discloses the establishment of
optimal conditions for the extraction of paralytic shellfish poisoning toxins
from the gonad of Chlamys nobilis. The extraction uses acetic acid and
hydrochloric acid in the concentration range of 0.04 to 1.0 mol/L. The use of
hydrochloric acid in the concentration range of 0.25 to 1.0 mol/L was shown
to cause a significant decrease of the toxins Cl, C2 and GTX5 and the
concomitant increase in the toxins GTX2,3. The amount of the three unstable
toxins did not show any change when acetic acid was used in the extraction.
The publication of Liu et al (2010) discloses the culture of toxin producing
Alexandrium catenella in the laboratory. A maximum cell density of 0.4 x 10'
cells/mL was obtained within eight days of culture. Analysis by high
performance liquid chromatograph (HPLC) of a crude extraction showed the
major toxic components to be C1/2, GTX4, GTX5 and neoSTX at concentrations of
about 0.04550, 0.2526, 0.3392, 0.8275 and 0.1266 Timol/L, respectively.
3
CA 03113727 2021-03-17
WO 2020/058750
PCT/IB2018/057274
The publication of Foss et al (2012) discloses a comparison of extraction
methods for paralytic shellfish toxins (PSTs) from the filamentous
cyanobacterium Lyngbya wollei. In the absence of commercially available
standards for the unique toxins produced by this cyanobacterium it was not
possibly to quantify the toxins extracted.
The publication of Li et al (2013) discloses a method for the rapid screening
and identification of paralytic shellfish poisoning (PSP) toxins in red tide
algae. The method utilises hydrophilic interaction chromatography-high
resolution mass spectrometry (HILIC-HR-MS) combined with an accurate mass
database. Limits of detection (LOD) of ten common PSP compounds were in the
range of 10 to 80 nmol/L. The developed method was asserted to be a useful
tool for the rapid screening and qualitative identification of common PSP
toxins in harmful algae.
The publication of Bernardi Bif et al (2013) discloses the sensitivity of sea
urchins to toxic cell extracts containing saxitoxins.
The publication of Poyer et al (2015) discloses the development of an
analytical method to characterise and differentiate saxitoxin analogs,
including sulfated (gonyautoxins) and non-sulfated analogs. Hydrophilic
interaction liquid chromatography (HILIC) was used to separate sulfated
analogs. Ion mobility mass spectrometry (IM-MS) was used as a new dimension
of separation based on ion gas phase confirmation to differentiate the
saxitoxin analogs. Positive and negative ionisation modes were used for
gonyautoxins. Positive ionisation mode was used for non-sulfated analogs. The
coupling of three complementary techniques, HILIC-IM-MS, permitted the
separation and identification of saxitoxin analogs, isomer differentiation
being achieved in the HILIC dimension with non-sulfated analogs separated in
the IM-MS dimension.
The publication of Rubio et al (2015) discloses a method to purify saxitoxin
using a liquid chromatography methodology based on ionic pairs. The saxitoxin
is extracted using hydrochloric acid and treated with ammonium sulfate
following a treatment with trichloroacetic acid and hexane/diethyl ether
(97/3). Samples were analysed by a semi-preparative HPLC in order to collect
pure fractions of saxitoxin and these fractions were eluted in solid-phase
cationic interchange STX extraction columns. The purified saxitoxin was
reported to be stable and homogenous and its identity confirmed by LC-MS-MS.
Analogs such as neosaxitoxin of a decarbamoyl saxitoxin were reported to be
absent from the purified saxitoxin.
The publication of Chen et al (2016) discloses the application of serial
coupling of reverse-phase liquid chromatography (RPLC) and hydrophilic
interaction chromatography (HILIC) combined with high resolution mass
4
CA 03113727 2021-03-17
WO 2020/058750
PCT/IB2018/057274
spectrometry (HR-MS) to the simultaneous screening and identification of
known lipophilic and hydrophilic toxins in the algae of harmful algal blooms
(HABs). Lipophilic and hydrophilic toxins were extracted simultaneously by
the use of ultrasound-assisted extraction (UAE). The publication demonstrated
that HPLC/HILIC-HR-MS combined with an accurate mass list of known marine
algal toxins may be used as a powerful tool for screening of different
classes of known toxins in marine harmful algae.
The publication of Cho et al (2016) discloses the analysis of crude extracts
of toxin-producing dinoflagellates by column switching and two-step gradient
elusion using hydrophilic-interaction chromatograph (HILIC) combined with
mass spectrometry. The publication states that the data obtained supports the
hypothesis that the early stages of the saxitoxin biosynthesis and shunt
pathways are the same in dinoflagellates and cyanobacteria.
The publication of Beach et al (2018) discloses the sensitive multiclass
analysis of paralytic shellfish toxins, tetrodotoxins and domoic acid in
seafood using a capillary electrophoresis (CE)-tandem mass spectrometry
(MS/MS) method. A novel, highly acidic background electrolyte comprising 5 M
formic acid was used to maximise protonation of analytes and is asserted to
be generally applicable to simultaneous analysis of other classes of small,
polar molecules with differing pKa values.
The publication of Kellmann and Neilan (2007) discloses the fermentative
production of neosaxitoxin and its analogs in recombinant Escherichia coli
strains.
The publications of Lagos Gonzales (2010, 2015a, 2015b and 2016) disclose the
purification of phycotoxins from cyanobacteria produced in a continuous
culture. The phycotoxins are isolated primarily from the bacteria, but can
also be isolated from the culture medium. In one embodiment of the process
disclosed only neosaxitoxin (neoSTX) and saxitoxin (STX)are produced. In
another embodiment of the process disclosed only gonyautoxin 2 (GTX2) and
gonyautoxin 3 (GTX3) are produced.
The publication of Wang et al (2010) discloses the preparation of a paralytic
shellfish poison (PSP) standard solution. The standard solution is prepared
by removing impurities from shellfish material, collecting shellfish meat,
adding distilled water and 0.1-0.3 mol/L hydrochloric acid solution,
regulating pH to 1.5 to 5.0, and homogenising to obtain homogenate,
precooling at -20 C for 30 minutes to 24 hours, and lyophilising to obtain a
core sample, grinding, and sieving, precooling at -20 C for 10 minutes to six
hours and lyophilising to obtain the standard sample. The method of
preparation is asserted to have the advantages of low raw material cost and a
simple preparation process.
5
CA 03113727 2021-03-17
WO 2020/058750
PCT/IB2018/057274
The publication of Xiong and Qiu (2009) discloses the application of
biguanido purine derivatives and their salts and esters for improving the
therapeutic effect and reducing the side effects of antitumor agents. The
biguanido purine derivates are saxitoxin analogs.
It is an object of the invention to provide a semisynthetic method of
producing neosaxitoxin in sufficient quantity and of sufficient purity to
enable its use in the manufacture of pharmaceutical preparations. It is a
further object of the invention to provide a method of producing the
intermediate for use in the semisynthetic method. These objects are to be
read in the alternative with the object to at least provide a useful choice.
SUMMARY OF INVENTION
In a first aspect the invention provides a method of preparing a volume of
concentrated aqueous extract for use in the preparation of a quantity of
GTX1,4 comprising the steps:
1. Culturing a selected isolate of a dinoflagellate in a vertical column of
aerated amended seawater for a period of time and at a temperature
sufficient to provide a culture having a predetermined cell density;
2. Harvesting the cells from the culture having the predetermined cell
density to provide a quantity of cellular biomass;
3. Resuspending the quantity of cellular biomass in an aqueous solution of
a
weak organic acid for a period of time and at a temperature sufficient to
provide a mixture of residual biomass and an extract in solution;
4. Separating the residual biomass from the extract in solution; and then
5. Reducing the volume of the extract in solution to provide the volume of
concentrated aqueous extract,
where the selected isolate has been selected to produce a ratio of GTX2,3 to
GTX1,4 of less than 0.1, the amended seawater is seawater amended with a
nutrient medium, and the predetermined cell density is in the range 7 x 104 to
105 cells/mL.
Preferably, the selected isolate is an isolate of the dinoflagellate
Alexandrium pacificum. More preferably, the isolate is an isolate of the
dinoflagellate Alexandrium pacificum that produces a ratio of GTX2,3 to
GTX1,4 of less than 0.01. Most preferably, the isolate is the isolate of the
dinoflagellate Alexandrium pacificum designated CAWD234.
Preferably, the nutrient medium comprises nitrates, phosphates, trace metals
and vitamins.
6
CA 03113727 2021-03-17
WO 2020/058750
PCT/IB2018/057274
Preferably, the aqueous solution of a weak organic acid is 0.25 to 0.75%
acetic acid.
Preferably, the volume of concentrated aqueous extract a density between 1.06
to 1.14 g/mL.
In a second aspect the invention provides a method of fractionating a volume
of concentrated aqueous extract to provide a solution of partially purified
GXT1,4 comprising the steps:
1. Reducing the volume of the aqueous extract by ultrafiltration to
provide
a reduced volume;
2. Loading the reduced volume on a column of activated carbon sorbent to
provide a loaded column; and
3. Eluting the loaded column with a stepwise gradient of water followed by
aqueous acetic acid/acetonitrile to provide the solution of partially
purified GTX1,4,
where the volume of concentrated aqueous extract is an extract of a culture
of a dinoflagellate.
Preferably, the volume of concentrated aqueous extract is an extract of a
culture of a dinoflagellate prepared according to the method of the first
aspect of the invention.
In a third aspect the invention provides a method of preparing a quantity of
neoSTX comprising the steps of:
1. contacting in solution in a reaction solvent a quantity of purified
GTX1,4 and a quantity of dithiol for a period of time and at a
temperature sufficient to provide a reaction product in which greater
than 97.5% (w/w) of the GTX1,4 has been converted to neoSTX; and then
2. applying the reaction product to a silica based weak cation exchange
sorbent and eluting with an aqueous weak organic acid to separate the
neoSTX from the dithiol and provide the quantity of neoSTX,
where the pH of the solution is in the range 7.4 to 7.6.
Preferably, the reaction solvent is buffered aqueous acetic acid.
Preferably, the dithiol is selected from the group consisting of
dithiothreitol (DTT) and dithiobutylamine (DTBA). More preferably, the
dithiol is dithiothreitol (DTT).
7
CA 03113727 2021-03-17
WO 2020/058750
PCT/IB2018/057274
Preferably, the quantity of purified GTX1,4 is obtained from a solution of
purified GXT1,4 prepared according to the method of the second aspect of the
invention.
Preferably, the quantity of neoSTX is greater than 100 mg with a purity
greater than 99.5'8' (w/w).
The methods of the invention provide for the batch preparation of GTX1,4 and
neoSTX in quantities and of purities not previously obtainable (cf. Lagos
Gonzales (2010, 2015a, 2015b and 2016)).
In the description and claims of this specification the following
abbreviations, acronyms, phrases and terms have the meaning provided: "batch
preparation" means prepared discontinuously; "biosynthetic" means prepared
within living organisms or cells; "CAS RN" means Chemical Abstracts Service
(CAS, Columbus, Ohio) Registry Number; "comprising" means "including",
"containing" or "characterized by" and does not exclude any additional
element, ingredient or step; "consisting of" means excluding any element,
ingredient or step not specified except for impurities and other incidentals;
"consisting essentially of" means excluding any element, ingredient or step
that is a material limitation; "GTX" means gonyautoxin; "GTX1" mean
gonyautoxin 1 [CAS RN 60748-39-2]; "GTX4" means gonyautoxin 4 [CAS RN 64296-
26-0]; "GTX1,4" means an unresolved mixture (as solid or in solution)
comprising gonyautoxin 1 and gonyautoxin 4; "GTX2,3" means an unresolved
mixture (as solid or in solution) comprising gonyautoxin 2 and gonyautoxin 3;
"neoSTX" means (3aS,4R,10aS)-2-amino-4-[[(aminocarbonyl)oxy]methyl]-
3a,4,5,6,8,9-hexahydro-5-hydroxy-6-imino-1H,10H-pyrrolo[1,2-c]purine-10,10-
diol [CAS RN 64296-20-4]; "nutrient medium" means a medium comprising trace
metals and vitamins; "preparative scale" means prepared in batches of greater
than 100 mg; and "semi-synthetic" means prepared by chemical conversion of an
at least partially purified biosynthetic precursor. A paronym of any of the
defined terms has a corresponding meaning.
The terms "first", "second", "third", etc. used with reference to elements,
features or integers of the matter defined in the Summary of Invention and
Claims, or with reference to alternative embodiments of the invention, are
not intended to imply an order of preference. Where concentrations or ratios
of reagents are specified the concentration or ratio specified is the initial
concentration or ratio of the reagents. Where values are expressed to one or
more decimal places standard rounding applies. For example, 1.7 encompasses
the range 1.650 recurring to 1.749 recurring. Purity of the isolated neoSTX
is determined according to Method 3 [F. Analysis].
The invention will now be described with reference to embodiments or examples
8
CA 03113727 2021-03-17
WO 2020/058750
PCT/IB2018/057274
and the figures of the accompanying drawings pages.
BRIEF DESCRIPTION OF FIGURES
Figure 1. A plan view of a hanging bag (1) formed from a length of tubular
plastic for use in the bulk culture of isolates. Traversing single (3, 4, 5
and 6) and double solid lines (9 and 10) identify where two or more layers of
the tubular plastic are heat welded together. Traversing broken lines
identify where four (7 and 8) or two (12 and 13) layers of the tubular
plastic are cut to provide a hanging loop (2) and cone, respectively.
Figure 2. The profiles of toxins produced by individual isolates (Table 1)
when cultured in vertical columns of aerated amended sea water. The isolates
designated as CAWD12, CAWD20 and CAWD121 are identified as non-producers of
toxins under these culture conditions.
Figure 3. Amounts of toxins produced by individual isolates (Table 1) when
cultured in vertical columns of aerated amended sea water.
Figure 4. Plot of concentration of gonyautoxins (GTX 1 (*) and GTX 4 (M))
versus time.
DESCRIPTION
According to the present invention a quantity of GTX1,4 is purified on a
preparative scale, i.e. in batch quantities of greater than 100 mg, from a
concentrated extract of a culture of a dinoflagellate and converted in a
solution buffered to a pH around 7.5 to neoSTX by reductive desulfonation
using a dithiol as the preferred reducing agent.
Extracts of cultures of isolates of Alexandrium pacificum that produce
relatively low amounts of gonyautoxin 2 (GTX2) and gonyautoxin 3 (GTX3) have
been determined to be most suitable as a source for the purification of
GTX1,4 on a preparative scale. Extracts of cultures of dinoflagellates that
may be used for the purification of GTX1,4 in accordance with the method
described here are available from the Cawthron Institute, Nelson, New
Zealand.
The introduction of an ultrafiltration step prior to desalting has been found
to be particularly advantageous when purifying GTX1,4 from these aqueous
extracts on a preparative scale. Without wishing to be bound by theory it is
believed that ultrafiltration removes a substantial portion of the solutes
that might otherwise interfere with the desalting step on sorbents such as
activated carbon. The introduction of the ultrafiltration step thereby
reduces the quantity of sorbent that would otherwise be required.
9
CA 03113727 2021-03-17
WO 2020/058750
PCT/IB2018/057274
In solution, gonyautoxin 1 (GTX1) and gonyautoxin 4 (GTX4) exist as a pair of
epimers of which GTX1 is the thermodynamically most favoured. Epimerisation
is believed to occur under most conditions via keto-enol equilibration at C-
12. In the first step of the proposed 2-step reaction mechanism according to
SCHEME I a thiol group of the dithiol (R-SH) attacks the electrophilic C-12
of the keto form (I) to form a thiohemiketal (II). Conversion to a thioether
(IV) occurs via an episulfonium ion intermediate (III) when the leaving group
(0-sulfate) is oriented anti to the sulphur atom (as in the reactive epimer
GTX1). In the second step of the proposed reaction mechanism the thiol group
of the dithiol reacts with the sulphur of the thioether (IV) to form a
disulfide thereby yielding an enolate that readily hydrates to neoSTX (V).
The optimal pH for the conversion of GTX1,4 to neoSTX has been determined to
be around 7.5 and without wishing to be bound by theory is believed to ensure
both (i) an optimal rate of epimerisation between the gonyautoxin epimers and
(ii) optimal degrees of electrophilicity at C-12 and deprotonation of the
dithiol used as the reducing agent. The use of dithiols such as
dithiothreitol (DTT) and dithiobutylamine (DTBA) are preferred over the use
of monothiols such as glutathione (GSH) and mercaptoethanol (ME) (cf.
Sakamoto et al (2000) and Sato et al (2000)). Higher rates of conversion are
obtained when using the dithiols, rendering them more suitable for use in the
production of neoSTX on a preparative scale
The excess dithiol, sodium phosphate buffer and unreacted GTX1,4 has been
found to be most conveniently removed from the neoSTX containing conversion
product by the use of cation exchange chromatography. The silica based weak
cation exchange sorbent Sepram WCX has been determined to be a suitable
sorbent as it has been determined not to retain DTT. Trials of the polymeric
based weak cation exchange sorbent Strata_XTM CW (Phenomenex) determined this
sorbent to be unsuitable for purification of neoSTX from the conversion
product on a preparative scale. The excess dithiol is retained by both an ion
exchange and a reverse phase mechanism when using this sorbent. Although a
portion of the excess DTT is eluted with organic solvents such as
acetonitrile/water a further portion is eluted with 1 M acetic acid
frustrating the purification of the neoSTX when using this sorbent.
CA 03113727 2021-03-17
WO 2020/058750 PCT/IB2018/057274
=,, '.....,
= tt.
=====. w=:..
=
,..4
k,
,../
H N 0.;.t-t.)IN;M: 0
604.
H
/ H '..7. ¨ H
P--- "'
to.
/ \
0 0 A
0
s:
1.0
tv
r 0:
, =vr vt
----..... 1
*"....
t4
v N
cv ,A1
,
H
).....w, ., X.:
,...
,
Pi icel N
to
al
r
or:
0;
..---- 1
a r-
z
I
or
X:
Itox =,,,,= C.,
...........
H
A,
\ *
SF2...
i 0
0 r....
'v..'
Z3
0
M
N
CO
0
C.,
0
0
CA 03113727 2021-03-17
WO 2020/058750
PCT/IB2018/057274
EXAMPLE
A. Materials
mM acetic acid (0.6 g/L in deionised water); amended seawater (7 mL/L Li
nutrient medium in seawater); deionised water (Milli-Qm, Merck-Millipore); Li
5 nutrient medium (1.25 g/L EDTA, 0.91 g/L FeC13.6H20, 0.29 mL/L trace
metal
stock solution, 21.6 g/L NaNO3, 1.44 g/L NaH2PO4 and 14.4 mL/L vitamin stock
solution); Mobile Phase A (2.2 g/L sodium heptane sulphonic acid (sodium
salt) and 0.31 g/L 85% phosphoric acid adjusted to pH 7.1 with 25% ammonium
hydroxide); Mobile Phase B (0.1% acid in acetonitrile); Mobile Phase C (0.1%
10 acetic acid in deionised water); roll of tubular (230 mm x 200 m x 250
pm)
low density poly(ethylene) (LDPE) plastic (Amcor Limited); seawater (30 to 37
ppt salinity); trace metal stock solution (2.5 g/L CuSO4.5H20, 20 g/L
Na2Mo04.2H20, 23 g/L ZnSO4.7H20, 11.9 g/L CoC12.6H20, 178 g/L MnC12.4H20, 1.3
g/L
H2Se03, 2.6 g/L NiSO4.6H20, 1.8 g/L Na3VO4 and 1.9 g/L K2Cr04); vitamin stock
solution (0.01 g/L biotin, 2 g/L thiamine and 0.01 g/L vitamin B12).
Vitamin stock solutions are filter sterilised and aseptically added through a
0.22 pm syringe filter during preparation of amended seawater following the
autoclaving of other ingredients.
B.Inocula
Isolates are obtained from naturally occurring algal blooms in coastal
waters. Species responsible for harmful algal blooms include Alexandrium
minutum, Alexandrium pacificum (formerly referred to as Alexandrium
catenella), Alexandrium tamarense and Gymnodium catenatum.
The dinoflagellate Alexandrium pacificum is an armoured, marine, planktonic
dinoflagellate. The species was originally identified as Alexandrium
catenella using the morphological characteristics described in the
publication of Balech (1985). A more recent description of the
characteristics of species of the genus is provided in the publication of
MacKenzie et al (2004).
A chain-forming species, Alexandrium pacificum typically occurs in
characteristic short chains of 2, 4 or 8 cells. Single cells are round,
slightly wider than long, and are anterio-posteriorly compressed. A small to
medium sized species, it has a rounded apex and a slightly concave antapex.
The thecal plates are thin and sparsely porulated. Cells range in size
between 20 to 48 pm in length and 18 to 32 pm in width.
12
CA 03113727 2021-03-17
WO 2020/058750
PCT/IB2018/057274
Designation Original identification Provenance
CAWD11 Alexandrium minutum Anakohe Bay, New Zealand
CAWD12 Alexandrium minutum Anakohe Bay, New Zealand
CAWD20 Alexandrium tamarense
Tamar East, Plymouth, United Kingdom
CAWD44 Alexandrium catanella Tauranga, New Zealand
CAWD45 Alexandrium catanella Tauranga, New Zealand
CAWD46 Alexandrium catanella Tauranga, New Zealand
CAWD47 Alexandrium catanella Tauranga, New Zealand
CAWD49 Alexandrium catanella Te Kaha, New Zealand
CAWD50 Alexandrium catanella Te Kaha, New Zealand
CAWD101 Gymnodinium catenatum Kaitaia Spat, New Zealand
CAWD102 Gymnodinium catenatum Kaitaia Spat, New Zealand
CAWD121 Alexandrium tamarense Marsden Point, New Zealand
CAWD234 Alexandrium catanella Opua Bay, New Zealand
CAWD235 Alexandrium catanella Opua Bay, New Zealand
Table 1. Isolates evaluated for their production of toxins and isolated from
New Zealand coastal waters prior to October 2015.
As stated in the publication of MacKenzie (2014), Alexandrium pacificum is
the cause of most paralytic shellfish toxin contamination in New Zealand.
Individual isolates (Table 1), including isolates of the species Alexandrium
pacificum, have been evaluated for their production of toxins when cultured
in bulk according to the following protocols. Representations of the
structures of the toxins referred to in Figure 2 and Figure 3 are provided
below. The representations are of the structures of the toxins in their
neutral (uncharged) form.
A. Culture
Individual isolates are cultured in aerated vertical columns of amended
seawater in a plurality of hanging bags. The bags are hung in an incubation
room maintained at a temperature of 16 to 20 C. The bags are illuminated
according to a 16-hour/8-hour light/dark cycle. Automated carbon dioxide (CO2)
dosing is used to maintain the pH in the range 8 to 9 during the light phase
of the light/dark cycle. Standard personal protection equipment is worn by
operators to minimise the risk of exposure to toxins.
Reference is made to Figure 1 of the accompanying drawings pages where a plan
view of a cut and welded hanging bag (1) for use in the bulk culture of
isolates is provided. The bags are formed from a roll of tubular plastic by
cutting and heat welding according the following protocol.
After discarding the first two metres of a new roll of tubular plastic a 20
cm section is cut and the open ends of the excised section each sealed by a
heat weld. The inner surfaces of the sealed section of tubular plastic roll
are subjected to microbiological evaluation before the remainder of the new
roll of tubular plastic is used for the fabrication of hanging bags.
13
CA 03113727 2021-03-17
WO 2020/058750 PCT/IB2018/057274
HAT o H2N 0 H2N 0
0 0 0
E E ...H
E E
N .....11-4>_ N
HON EN EN
NH
EN)N N ¨NE
EN40N __ N NH
N
H N H
OH OH OH
H HN
OH OH OH
H H HO3SO
OSO3H OSO3H H
Gonyautoxin 1 (GTX1) Gonyautoxin 2 (GTX2) Gonyautoxin 3 (GTX3)
H2N,I00
0/eNHS031-1 0/eNHSO3E
0
0 0
H
H H
N H H
...--1
HON N> __ NH N
HNN N ¨NH
).-INN N HN
> __________________________________________________________________ NH
H
HN N40N HN
OH
OH OH
OH
HO SO OH OH
H
Gonyautoxin 4 (GTX4) Gonyautoxin 5 (GTX5(B1)) Gonyautoxin 6 (GTX6(B2))
0,eNASO3H 01/eNHSO3H Oy NHSO3H
0 0 0
H H H
H H H
N N N
HN HN HON
HN4 N ;>---NH ;>===NH
HN40LN > __ NH
" N
HN4OLN
H H H
OH OH OH
OH OH OH
H HO3S0 H
OSO3H H OSO3H
Cl C2 C3
0/eNHSO3H
H2N,400 H2Nr
0
0 0
H
H E.::
N
HON E:::5:
HN HON
N NH
HN4"N
HN401N HN N N
H H
OH
OH OH OH
HO3S0 OH OH
H
C4 Saxitoxin (STX) Neosaxitoxin (neoSTX)
14
CA 03113727 2021-03-17
WO 2020/058750
PCT/IB2018/057274
A two-metre length (A+G) is dispensed from the tubular plastic roll and a
hanging loop (2) formed at the first end. The hanging loop (2) is formed by
folding back a 10 cm section (A) of the tubular plastic and heat welding
together the four layers of plastic to provide a first heat weld (3) proximal
to the cut first end. A second heat weld (4) is formed within the folded back
section (A) parallel and spaced apart from the first heat weld (3) by about 2
cm followed by diagonal heat welds (5,6) traversing the region between the
horizontal first and second heat welds (3,4). The corners of the folded back
section distal from the welds are then cut (7,8) to provide a hanging loop
(2) of a width (B) capable of supporting the length of tubular plastic when
filled with a volume of amended seawater.
The second end of the length of tubular plastic is sealed by double diagonal
heat welds (9,10) converging to a point (11) proximal to the centre of the
second end. The integrity of each double diagonal heat weld (9,10) is
inspected visually before each triangle of plastic outside the conical sealed
end is cut (12,13) away. The conical portion of the sealed end has a depth
(C) of around 20 cm. Each hanging bag (1) is capable of containing a culture
volume of approximately 24 L
Prior to filling with inoculum and amended seawater a hanging bag (1) is hung
in position and the top outside corner surface sterilised by wiping with
isopropanol. A downward pointing first hole is formed in the surface
sterilised region using a sterile pick. The tip of a sterilised air vent
inserted into the hole and taped to the outside of the bag using PVC
insulation tape (50 mm width). A region of the outer surface of the conical
sealed end is also surface sterilised by wiping with isopropanol. A second
hole is formed in the surface sterilised region using a sterile pick. The
downward pointing tip of a sterile inoculation line is inserted into the hole
and the line taped to the outside of the bag using PVC insulation tape (50 mm
width).
Inoculum is fed into the hanging bag from a parent culture via the
inoculation line. The line from the parent culture and the inoculation line
to the hanging bag are each connected to a manifold. Air is purged from the
lines by pumping amended seawater into both. The parent culture is then
allowed to flow into the hanging bag. Pressurised air is introduced into the
hanging bag containing the parent culture via its sterilised air vent. Equal
volumes of the parent culture are transferred to multiple hanging bags. The
inoculum containing hanging bags are then filled with the amended seawater to
a fill line (D). Once the hanging bags are filled, the inoculation lines are
disconnected from the manifold and connected to an air line via a sterilised
air filter. The culture volume is aerated via the air line.
CA 03113727 2021-03-17
WO 2020/058750
PCT/IB2018/057274
To monitor the pH of the medium during the culture the surface of a pH probe,
including the glass bulb, is washed with deionised water and 70% (w/w)
ethanol. A region of the outer surface of the hanging bag around 4 cm above
the fill line is surface sterilised by wiping with isopropanol and a hole
made in the bag within this region using a sterilised 10 mL pipette tip. The
pH probe is inserted via the hole and into the culture volume and held flat
against the inner side wall of the bag.
Under these incubation conditions the isolates have a doubling time of about
two days and are harvested when the cell density has reached 7 x 10 to 10'
cells/mL as determined by microscopic image cytometry.
B. Harvest
With aeration maintained the pH probe is removed and a volume of about 9 mL
glacial acetic acid introduced into the culture volume. After about 10 to 20
minutes a volume of about 100 mL of a suspension of hydrated bentonite clay
is added to the culture to provide a dosage of about 4 mL/L of culture. After
a further 5 to 10 minutes the inoculation line is clamped and disconnected
from the air filter. Settling of cells occurs over a period of time of at
least 2 hours. The settled cells are drained into a centrifuge bottle and the
collected volume (300 to 400 mL/bag) centrifuged in balanced bottles at a
force of 1,500 x g for a period of time of 5 minutes. The supernatant is
discarded, and the harvested cells weighed before storing frozen at -20 C.
C. Extraction
Extracts for (i) determining toxin profiles and monitoring toxin production,
or (ii) preparation of GTX1,4 are prepared according to the following
methods.
For monitoring toxin production, 10 mL of the culture volume is transferred
to a polypropylene centrifuge tube and the cells pelleted by centrifugation
at a force of 1,500 x g for a period of time of 5 minutes. The supernatant is
discarded, and the pellet resuspended in a volume of 250 pL 1mM acetic acid.
The volume is sonicated for a period of time of 2 minutes and heated to 80 C
for a period of time of 10 minutes. The cellular material is pelleted by
centrifugation at a force of 3,220 x g for a period of time of 5 minutes and
10-fold and 20-fold dilutions of the supernatant then prepared in 80% (v/v)
acetonitrile/0.25% (v/v) acetic acid.
For preparation of GTX1,4, the frozen pellet of harvested cells is defrosted
before resuspending in an equal volume of 0.5% acetic acid and leaving at
room temperature for a period of time of 30 to 60 minutes. The suspension in
acetic acid is then heated and maintained at a temperature of 85 2 C in a
16
CA 03113727 2021-03-17
WO 2020/058750
PCT/IB2018/057274
water bath for a period of time of 10 to 15 minutes. The heated suspension is
then cooled in an ice slurry before centrifugation at a force of 3,990 x g
for a period of time of 2 minutes and the first supernatant decanted to a
collection vessel. An equal weight of 0.5% acetic acid is added to the
pelleted cellular material, mixed well, and centrifuged at a force of 3,990 x
g for a period of time of 5 minutes. The second supernatant is decanted to
the collection vessel and the total volume of collected supernatants reduced
by rotary evaporation under vacuum (less than 15 mBar) at a temperature of 30
C. (For example, portions of a total volume of 10 litres of supernatant are
transferred from the collection vessel to a weighed 5 litre round bottom
flask and the volume reduced by rotary evaporation until an extract having a
weight of 800 50 g and a density between 1.08 to 1.12 g/mL is obtained.) The
concentrated extract is stored frozen at -20 C.
D. Analysis
A sample of extract is prepared for analysis using activated carbon solid
phase extraction (SPE). A Supelcleanm ENVI-Carbm SPE tube (bed wt. 250 mg,
volume 6 mL) is conditioned with a volume of 3 mL of 20% acetonitrile/1%
acetic acid followed by a volume of 3 mL of 0.025% ammonia. Following elution
to the top frit the tube is loaded with a total volume of 400 pL consisting
of 10 to 400 pL of extract and quantum sufficit deionised water. The
cartridge is eluted to the top frit using a vacuum of -15 to -20 kPa and
washed with a volume of 3 mL of deionised water before elution with a volume
of 5 mL of 20% acetonitrile/1% acetic acid. The eluate is collected in a
polypropylene tube and a volume of 10 pL diluted 4-fold with the addition of
acetonitrile in a polypropylene autosampler vial. The contents of the vial
are mixed on a vortex mixture with further dilution as required.
Method 1
Aqueous extracts and fractions enriched for the presence of GTX1,4 are
analysed by high pressure liquid chromatography (HPLC) (Shimadzu Prominence)
with ultraviolet (UV) detection according to this Method 1.
Samples are diluted with 10 mM acetic acid to provide a nominal concentration
of 200 pg/mL. A volume of 40 pL of diluted sample is injected onto a column
(4.6 x 150 mm) of 3.5 pm Zorbax Bonus RP eluted with Mobile Phase A at a flow
rate of 1 mL/min for a period of time of 30 minutes while being maintained at
a temperature of 20 C. The absorbance of the eluate is monitored at
wavelengths of 210 nm (purity) and 245 nm (quantity) using a photodiode array
detector (PAD). The purity of samples is calculated based on the percentage
area at 210 nm and retention time with reference to a standard at a
concentration of approximately 200 pg/mL GTX1,4. Samples are analysed in
17
CA 03113727 2021-03-17
WO 2020/058750
PCT/IB2018/057274
triplicate with 10 mM acetic acid used as a blank and the chromatogram
obtained subtracted from that obtained for all samples.
Method 2
Extracts and fractions enriched for the presence of GTX1,4 are analysed and
quantified by liquid chromatography-mass spectrometry (LC-MS)(Shimadzu 8050)
according to this Method 2.
Samples are diluted to an appropriate concentration using 80$
acetonitrile/0.25% acetic acid. A mixed standard containing a number of
reference paralytic shellfish toxins (PSTs) is used. A maximum volume of 2 pL
of diluted sample is injected by means of an auto sampler maintained at 4 C
onto a column (2.1 x 100 mm) of 1.7 pM Waters Acquity UPLC BEH amide eluted
at a flow rate of 0.4 mL/min while being maintained at a temperature of 20 C.
The column is eluted stepwise with 75% Mobile Phase B/25% Mobile Phase C for
a period of time of 5 minutes following injection, followed by 55% Mobile
Phase B/45% Mobile Phase C for a period of time of 0.50 min before reverting
to 75% Mobile Phase B/25% Mobile Phase C. The eluate is monitored by mass
spectrometry using selective ion monitoring in ESI- and ESI+ ionisation
modes.
Method 3
Purified products of extraction, fractionation and conversion are analysed
for quantity and purity according to this Method 3.
Isolated products (GTX1,4 or neoSTX) are diluted to a concentration of 200
pg/mL in 10 mM acetic acid. The diluted sample is then further diluted 100-
fold in 8% acetonitrile/0.25% acetic acid to provide a solution of product at
a concentration of 20 mg/mL for quantitative analysis. A mixed standard
containing a number of reference paralytic shellfish toxins (PSTs) is also
prepared in the same solvent. A solution of 2 pL of the diluted product (20
ng/mL) is injected by means of an autosampler maintained at a temperature of
40 C onto a column (2.1 x 100 mm) of 1.7 pm Waters Acquity UPLC BEH amide
eluted at a flow rate of 0.6 mL/min while being maintained at a temperature
of 60 C. The column is eluted stepwise with 80% Mobile Phase B/20% Mobile
Phase C for a period of time of 6 minutes following injection, followed by
55% Mobile Phase B/45% Mobile Phase C for a period of time of 0.50 minutes
before reverting to 80$ Mobile Phase B/20% Mobile Phase C. The eluate is
monitored by mass spectrometry monitoring in ESI- and ESI+ ionisation modes.
E. Purification
The concentrated extract is thawed at room temperature. The extract is
divided between two balanced 500 mL conical centrifuge bottles and
18
CA 03113727 2021-03-17
WO 2020/058750
PCT/IB2018/057274
centrifuged at a force of 4000 x g for a period of time of 10 minutes. The
supernatant is decanted and filtered under reduced pressure through a series
of 110 mm diameter hardened ashless and glass microfiber filter papers
(Whatmanm grade 540, Whatmanm grade 542 and Whatmanm grade GF/A).
The filtered extract is then subjected to crossflow ultrafiltration by
recirculation through two filters (VivaFlow 200) connected in parallel with
an outlet pressure no greater than 2.5 bar until the volume of the extract
has been reduced to a volume of 10 to 20 mL of retentate.
The retentate is transferred to a 100 mL bottle and made up to a total volume
of 100 mL with deionised water. The diluted retentate is similarly subjected
to rounds of crossflow ultrafiltration reducing the volume of retentate to 10
to 20 mL before making up to a total volume of 100 mL with deionised water.
A 32 mm 5pm syringe filter (Pall Corp.) is installed on the inlet of a 50 g
Sepabeadsm SP-270 (Supelco) SPE cartridge conditioned with 500 mL of
deionised water at a flow rate of 30 mL/minute. The washed retentate is
passed through the conditioned SPE cartridge at a flow rate of 30 mL/minute
and the effluent collected. The SPE cartridge is then eluted with 200 mL of
deionised water at a flowrate of 30 mL/minute and the effluent collected.
The volume of the combined effluents is reduced by rotary evaporation under
vacuum (less than 15 mBar) at a temperature of 30 2 C. The volume is reduced
to provide a weight of 500 50 g, the density determined gravimetrically, and
the total reduced volume calculated on this basis.
Prior to purification of the GTX1,4 from the reduced volume on a preparative
scale a sample of the reduced volume is prepared and analysed as described
(F. Analysis). A 10,000-fold dilution of the sample should provide a
concentration of GTX1,4 in the range 20 to 50 ng/mL. The calculated yield of
the GTX1,4 should exceed 90% (w/w).
The total reduced volume is desalted by loading on a 100 g 25 x 450 mm carbon
column (Enviro-Cleanm Graphitized Carbon Non-Porous, UCT) conditioned with 1L
of deionised water. The reduced volume is loaded at a flow rate of 30 mL/min
using a Mini-Flash Pump (Sorbent Technologies, Inc.) and eluted at a flow
rate of 15 mL/min with a stepwise gradient of deionised water for a period of
time of 40 minutes followed by 0.2% (v/v) acetic acid/30% (v/v) acetonitrile
for a period of time of 40 minutes.
The eluate is monitored at 205 nm using an inline UV detector and sequential
volumes of 10 mL of eluate collected as fractions. Volumes of 5 pL of
selected fractions are diluted to a total volume of 10 mL with 0.25% (v/v)
acetic acid/80% (v/v) acetonitrile and submitted to LC-MS analysis as
described (F. Analysis). The GTX1,4 containing fractions are combined.
19
CA 03113727 2021-03-17
WO 2020/058750
PCT/IB2018/057274
The pH of the desalted combined fractions collected from the carbon column is
adjusted to 7.8 using concentrated ammonium hydroxide at a rate of
approximately 2 11L/mL. The pH adjusted volume is then loaded on a 35 x 460 mm
column of 280 g Sepram WCX-NH4" conditioned using a volume of 1 L of 50 mM
ammonium bicarbonate at a flow rate of 30 mL/min. The column is eluted with a
gradient of 10 to 80$ 0.5 M ammonium bicarbonate over a period of time of 100
minutes. The eluate is monitored at wavelengths of 205 nm and 245 nm using an
inline UV detector. Sequential volumes of 25 ml are collected as fractions
and combined according to the UV monitoring and LC-MS analysis where required
(F. Analysis). Fractions containing greater than 2% of the total amount of
GTX1,4 are combined and the pH of the combined fractions reduced to 6.5 by
the dropwise addition of glacial acetic acid. A 5,000-fold dilution of the
acidified volume is subjected to LC-MS analysis as described (F. Analysis)
and the total quantity and yield of GTX1,4 and the ratio to GTX5 calculated.
The yield should be greater than 90$ and the ratio of GTX5 to GTX 1,4 should
be less than 1%.
The combined fractions from the weak cation exchange chromatography are
loaded onto a 35 x 480 mm column of 175 g Sepram ZT-WCX-H form conditioned
using 1 L of deionised water at a flowrate of 30 mL/min. The loaded column is
eluted with a continuous gradient of 0 to 100% 1M acetic acid in water over a
period of time of 100 minutes and sequential volumes of 25 mL of eluate
collected as fractions while monitoring at a wavelength of 205 nm using an
inline UV detector.
The GTX1,4 containing fractions are collected from baseline to baseline and
the total volume reduced to a volume of 10 to 20 mL by rotary evaporation at
C under reduced pressure of less than 15 mBar. A 500,000-fold dilution of
the reduced volume is subjected to LC-MS analysis as described (F. Analysis)
and the total quantity and yield of GTX1,4 calculated on this basis.
The GTX1,4 containing reduced volume is loaded on a 50 x 500 mm column of P2
30 gel conditioned with 2 L of 100 mM acetic acid at a flowrate of 50
mL/min and
eluted isocratically with the same conditioning mobile phase. The eluate is
monitored at a wavelength of 205 nm for a period of time of 200 minutes using
an inline UV detector and sequential volumes of 10 mL of eluate collected as
fractions. The GTX1,4 containing fractions are collected from baseline to
baseline and the total volume reduced by rotary evaporation at a temperature
of 30 C under reduced pressure of less than 15 mBar.
The reduced volume of combined fractions obtained by gel filtration is
transferred to a pre-weighed 100 mL round bottom flask and evaporated to
dryness by rotary evaporation at a temperature of 30 C under a reduced
pressure of less than 15 mbar followed by drying in a freeze dryer at a shelf
CA 03113727 2021-03-17
WO 2020/058750
PCT/IB2018/057274
temperature of 10 C and pressure of 0.05 mbar for 24 hours. The open mouth of
the round bottom flask is securely covered with an air permeable, lint free
tissue before placing in the freeze dryer. The yield of purified GTX1,4 is
determined gravimetrically. The purified GTX1,4 is dissolved in a known
volume of deionised water to provide a solution containing 70 to 100 mg/mL of
GTX 1,4.
A 500-fold dilution of the solution in 10 mM acetic acid is prepared and
analysed as described (F. Analysis). The final volume required to provide a
concentration of 40 to 45 mg/mL of GTX 1,4 is calculated and the solution
transferred via a filter to a pre-weighed 10 mL amber glass vial, rinsing
with deionised water to provide a transferred volume having the target
concentration of 40 to 45 mg/mL. A 200-fold dilution of the transferred
solution is prepared in 10 mM acetic acid and analysed as described (F.
Analysis). The dilution is diluted a further 10-fold in 10 mM acetic acid and
analysed for purity as described (F. Analysis).
F. Conversion
A quantity of 183 mg (as the free base) of GTX1,4 purified according to the
preceding steps from an extract of a culture of the isolate of Alexandrium
pacificum designated CAWD234 is dissolved in a total volume of 5 mL of dilute
acetic acid and mixed with a volume of 45 mL of 0.2 M phosphate buffer at a
pH of 7.5 in a 100 mL round bottom flask. The mixture is placed on ice and
the pH adjusted from 6.8 to 7.5 with the addition whilst stirring of solid
sodium carbonate. A quantity of 1.5 g of dithiothreitol (DTT) is added to the
pH adjusted mixture and its dissolution promoted by placing the reaction
mixture containing round bottom flask in an ultrasonic bath before
transferring to a water bath maintained at a temperature of 50 C. Aliquots of
a volume of 10 pL are removed from the reaction mixture and transferred to
the water bath (T=0) and periodically (every 15 minutes) thereafter. Aliquots
are diluted 50-fold by the addition of a volume of 490 pL 80% acetonitrile
0.25% acetic acid immediately following removal from the reaction mixture and
analysed by LC-MS as described (F. Analysis) to monitor the progress of the
reaction in near real time (Figure 4). After incubation for 45 minutes at
50 C the reaction mixture is chilled by transferring the round bottom flask
to an ice slurry. Under these conditions near quantitative conversion of
GTX1,4 to neoSTX is observed with close to 100% yield.
G. Isolation
The conversion product is loaded onto a quantity of 39 g Sepram WCX packed in
an empty flash cartridge (Grace) and preconditioned with a volume of 250 mL
of 50% (w/w) acetonitrile followed by a volume of 250 mL of deionised water.
21
CA 03113727 2021-03-17
WO 2020/058750
PCT/IB2018/057274
The conversion product is loaded onto the packed cartridge with rinses of
deionised water with collection of the effluent (about 200 mL). The
dissolution of any crystals formed during storage of the conversion product
at 4 C is achieved by the addition of a minimal amount of deionised water.
The loaded packed cartridge is then eluted at a rate of 50 mL/min with a
total volume of 1.5 L, followed by elution with a continuous gradient to 1 M
acetic acid over 20 minutes and the collection of sequential volumes of 10 mL
of eluate as fractions while monitoring UV absorbance at 205 nm and 254 nm.
A volume of 5 pL of fractions demonstrating UV absorbance at 205 nm is
diluted 100,000-fold in 813$' acetonitrile 0.25$ acetic acid and analysed by
LC-MS. Fractions confirmed to comprise neoSTX are combined, frozen at -70 C
and lyophilised. The dried neoSTX is dissolved in a small volume of 10 mM and
transferred to a pre-weighed 10 mL glass vial and a volume of 10 pL analysed.
The purity and quantity of a batch (CNC00063) of neoSTX prepared according to
the foregoing method is provided in Table 2.
Component Quantity (mg) % (w/w)
neoSTX 118 99.58
L-arginine 0.444 0.37
STX 0.0546 0.05
DTT <0.005 <0.004
TOTAL 119 100
Table 2. Specification for a batch (CNC00063) of neoSTX prepared according to
the semisynthetic method described.
Although the invention has been described with reference to embodiments or
examples it should be appreciated that variations and modifications may be
made to these embodiments or examples without departing from the scope of the
invention. Where known equivalents exist to specific elements, features or
integers, such equivalents are incorporated as if specifically referred to in
this specification. Variations and modifications to the embodiments or
examples that include elements, features or integers disclosed in and
selected from the referenced publications are within the scope of the
invention unless specifically disclaimed. The advantages provided by the
invention and discussed in the description may be provided in the alternative
or in combination in these different embodiments of the invention.
REFERENCED PUBLICATIONS
Baker et al (2003) GTX4 imposters: characterization of fluorescent compounds
synthesized by Pseudomonas stutzeri SF/PS and Pseudomonas/Alteromonas PTB-1,
symbionts of saxitoxin-producing Alexandrium spp. Toxicon, 41(3), 339-347.
Balech (1995) The genus Alexandrium Halim (Dinoflagellata) Sherkin Island
Marine Station, Sherkin Island, Ireland 151 pp.
22
CA 03113727 2021-03-17
WO 2020/058750
PCT/IB2018/057274
Beach et al (2018) Capillary electrophoresis-tandem mass spectrometry for
multiclass analysis of polar marine toxins Analytical and Bioanalytical
Chemistry, 410(22), 5405-5420.
Bernardi Bif et al (2013) Evaluation of mysids and sea urchins exposed to
saxitoxins Environmental Toxicology and Pharmacology, 36(3), 819-825.
Chen et al (2016) Simultaneous screening for lipophilic and hydrophilic
toxins in marine harmful algae using a serially coupled reversed-phase and
hydrophilic interaction liquid chromatography separation system with high-
resolution mass spectrometry Analytica Chimica Acta, 914, 117-126.
Cho et al (2016) Column switching combined with hydrophilic interaction
chromatography-tandem mass spectrometry for the analysis of saxitoxin
analogues, and their biosynthetic intermediates in dinoflagellates Journal of
Chromatography A, 1474, 109-120.
Daigo et al (1985) Isolation and some properties of neosaxitoxin from a
xanthid crab Zosimus aeneus Nippon Suisan Gakkaishi, 51(2), 309-13.
Foss et al (2012) Investigation of extraction and analysis techniques for
Lyngbya wollei derived Paralytic Shellfish Toxins Toxicon, 60(6), 1148-1158.
Garcia-Altares (2017) Structural diversity of microalgal marine toxins
Comprehensive Analytical Chemistry, 78, 35-75.
Hall et al (1984) Dinoflagellate neurotoxins related to saxitoxin: structures
of toxins C3 and C4, and confirmation of the structure of neosaxitoxin
Tetrahedron Letters, 25(33), 3537-8.
Jiang and Jiang (2008) Investigation of extraction method for paralytic
shellfish poisoning toxins in shellfish Fenxi Huaxue, 36(11), 1460-1464.
Kellmann and Neilan (2007) Fermentative production of neosaxitoxin and its
analogs in recombinant Escherichia coli strains International application no.
PCT/EP2017/053077 [publ. no. WO 2017/137606 Al].
Lagos Gonzales (2015a) Methods for producing phycotoxins United States patent
no. 8,957,207 B2.
Lagos Gonzales (2015b) Methods for purifying phycotoxins, pharmaceutical
compositions containing purified phycotoxins and methods of use thereof
United States patent no. 8,952,152 B2.
Lagos Gonzales (2016) Methods for purifying phycotoxins, pharmaceutical
compositions containing purified phycotoxins and methods of use thereof
United States patent no. 9,249,150 B2.
Lagos Gonzales (2010) Methods for purifying phycotoxins, pharmaceutical
compositions containing purified phycotoxins, and methods of use thereof
International application nos. PCT/IB2010/051187 [publ. no. WO 2010/109386
23
CA 03113727 2021-03-17
WO 2020/058750
PCT/IB2018/057274
Al] and PCT/IB2010/051188 [publ. no. WO 2010/109387 Al].
Laycock et al (1994) Isolation and purification procedures for the
preparation of paralytic shellfish poisoning toxin standards Natural Toxins,
2(4), 175-83.
Li et al (2013) Rapid screening, identification of paralytic shellfish
poisoning toxins in red tide algae using hydrophilic interaction
chromatography-high resolution mass spectrometry with an accurate-mass
database Fenxi Huaxue, 41(7), 979-985.
Liu et al (2010) Cultivation of Alexandrium catenella and extraction and
detection of paralytic shellfish poisoning toxins Shuichan Xuebao, 34(11),
1783-1788.
MacKenzie (2014) The risk to New Zealand shellfish aquaculture from paralytic
shellfish poisoning (PSP) toxins: A review New Zealand Journal of Marine and
Freshwater Research, 48(3), 430-465.
MacKenzie et al (2004) The dinoflagellate genus Alexandrium (Halim) in New
Zealand coastal waters: comparative morphology, toxicity and molecular
genetics Harmful Algae, 3, 71-92.
Mezher (2018) FDA to replace analgesic drug development guidance with new
documents Regulatory Focus News Article (www.raps.org/news-and-articles/news-
articles/2018/8/fda-2-replace-analgesic-drug-development-guidance).
Miao et al (2004) Isolation and purification of gonyautoxins from two strain
of Alexandrium minutum Halim Journal of Chinese Pharmaceutical Sciences,
13(2), 103-105.
Parker et al (2002) Growth of the toxic dinoflagellate Alexandrium minutum
(Dinophyceae) using high biomass culture systems Journal of Applied
Phycology, 14(5), 313-324.
Poyer et al (2015) Identification and separation of saxitoxins using
hydrophilic interaction liquid chromatography coupled to traveling wave ion
mobility-mass spectrometry Journal of Mass Spectrometry, 50(1), 175-181.
Ravn et al (1995) Standardized extraction method for paralytic shellfish
toxins in phytoplankton Journal of Applied Phycology, 7(6), 589-94.
Rubio et al (2015) Purification and characterization of saxitoxin from
Mytilus chilensis of southern Chile Toxicon, 108, 147-153.
Rutman et al (2011) Treatment of loss of sense of touch with saxitoxin
derivatives International application no. PCT/EP2011/051992 [Publ. no. WO
2011/098539 Al].
Rutman et al (2013) Paralytic shellfish poison International application no.
PCT/EP2013/055448 [Publ. no. WO 2013/135884 Al].
24
CA 03113727 2021-03-17
WO 2020/058750
PCT/IB2018/057274
Sakamoto et al (2000) Formation of intermediate conjugates in the reductive
transformation of gonyautoxins to saxitoxins by thiol compounds Fisheries
Science, 66, 136-141.
Sato et al (2000) Identification of thioether intermediates in the reductive
transformation of gonyautoxins into saxitoxins by thiols Bioorganic and
Medicinal Chemistry Letters 10, 1787-1789.
Siu et al (1997) Environmental and nutritional factors which regulate
population dynamics and toxin production in the dinoflagellate Alexandrium
catenella Hydrobiologia, 352, 117-140.
Tsai et al (1997) Toxicity and toxic components of two xanthid crabs,
Atergatis floridus and Demania reynaudi, of Taiwan Toxicon, 35(8), 1327-1335.
Wang et al (2010) Paralytic shellfish poison or PSP standard substances and
preparations for monitoring sea food industries Chinese patent application
no. 2010-10179534 [publ. no. CN 101858833 A].
Xiong and Qui (2009) Application of biguanido purine derivatives for
improving therapeutic effect and reducing side effect of antitumor agents
Chinese patent application no. 2008-10007215 [publ. no CN101513408].
