Note: Descriptions are shown in the official language in which they were submitted.
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Method to obtain oestrogens from mare's urine
Description
The present invention relates to obtaining a natural
mixture of conjugated oestrogens from the urine of
pregnant mares.
Oestrogens are used in medicine for hormone replacement
therapy. In particular, oestrogen mixtures are used
for the treatment and prophylaxis of the disorders of
the climacteric period which occur in women after
natural or artificial menopause. In this case, natural
mixtures of conjugated oestrogens such as are found in
the urine of pregnant mares have proved particularly
effective and readily compatible.
The dissolved solids content in the urine of pregnant
mares (= pregnant mares' urine, abbreviated hereafter
as "PMU") may naturally vary within wide ranges, and
may generally lie in a range of 40 - 90 g dry substance
per litre. In addition to urea and other usual urine
contents, phenolic constituents are contained in the
solids content of the PMU in quantities of about 2 - 5%
by weight relative to dry'substance. These phenolic
constituents include cresols and dihydro-3,4-bis[(3-
hydroxyphenyl)methyl]-2(3H)-furanone, known as HPMF.
These may be present in free or conjugated form. The
PMU contains a natural mixture of oestrogens which is
largely present in conjugated form, e.g. as sulphuric
acid semi-ester sodium salt (abbreviated hereafter as
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"sulphate salt"). The content of conjugated oestrogens
(calculated as oestrogen sulphate salt) may be between
0.3 and 1% by weight relative to dry substance.
Usually extracts containing conjugated oestrogens are
obtained from the PMU by extraction with a polar
organic solvent which is not miscible, or only slightly
miscible, with water, such as ethyl acetate, n-butanol
or cyclohexanol. In such liquid-liquid extractions,
however, a number of problems occur, such as severe
foaming, sedimentation, emulsification and poor phase
separation. Generally several extraction steps are
required, which results in losses and only partial
obtention of the oestrogen content.
In 1968 it was proposed by H. L. Bradlow (see Steroids
11 (1968), 265-272) to use Amberlite XAD-2 , a neutral,
non-polar hydrophobic polystyrene resin, manufactured
by Rohm und Haas, for the extraction of conjugated
oestrogens from urine. The adsorption capacity given
is low. According to Bradlow, an optionally diluted
urine is passed through a column containing the resin
at a low rate of flow. The oestrogens are eluted with
methanol or ethanol. However, no details are given of
the other substances contained in the oestrogen-
containing eluate.
It is an object of the present invention to develop an
industrial method for obtaining the natural mixture.of
conjugated oestrogens from the PMU, whilst avoiding the
disadvantages known from the liquid-liquid extractions
which have been usual hitherto, which method provides a
product which is largely cresol-free and HPMF-free and
which is depleted in phenolic urine contents.
A method has now been discovered with which a mixture
which is largely cresol-free and HPMF-free and which is
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depleted in phenolic urine contents, but contains the
natural oestrogen content of the PMU practically in'its
entirety can be obtained in a solid-phase.extraction on
a non-ionic, semi-polar polymeric adsorber resin, which
mixture can be used as a starting material for the
production of pharmaceuticals containing the natural
mixture of conjugated oestrogens from the PMU as active
constituent.
The method according to the invention for obtaining a
natural mixture, depleted in phenolic urine contents,
of conjugated oestrogens from PMU is characterised in
that
a) a urine, which represents the urine freed of
mucilaginous substances and solids, a reduced
concentrate of this urine or a reduced urine
retentate obtained by membrane filtration of this
urine, is contacted with a quantity of a non-ionic
semi-polar polymeric adsorber resin sufficient for
adsorption of the mixture of conjugated oestrogens
contained in the urine, and a non-ionic semi-polar
polymeric adsorber resin laden with the mixture of
conjugated oestrogens is separated off from the rest
of the urine,
b) the non-ionic semi-polar polymeric adsorber resin
laden with the mixture of conjugated oestrogens is
washed with a washing water set to a pH range of at
least 12.0, in particular of 12.5 to 14, and
C) the washed adsorber resin is contacted with a
quantity of an elution liquid sufficient for
desorption of the mixture of conjugated oestrogens
adsorbed thereon, which liquid represents a water-
miscible organic solvent from the group of water-
miscible ethers, lower alkanols and lower
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aliphatic ketones or a mixture of the water-
miscible organic solvent and water which has
optionally been rendered alkaline, and an eluate
containing the natural mixture of conjugated
oestrogens is separated off from the adsorber
resin and optionally reduced.
The PMU as such, a concentrate obtained therefrom by
reduction or a retentate obtained therefrom by membrane
filtration can be used for the method according to the
invention. The collected urine is first freed of
mucilaginous substances and solids in known manner.
Expediently, solids and mucilaginous substances are
allowed to settle and are then separated off according
to known separation methods, for instance decanting,
separation and/or filtration. Thus the PMU can for
instance be passed through a known separating
apparatus, e.g. a separator, a filtration unit or a
sedimenter. A sand bed, for example, may serve as a
separating apparatus, or commercially-available
separators, e.g. nozzle separators or chamber
separators, may be used. If desired, a microfiltration
installation or an ultrafiltration installation may
also be used, and if they are used it is possible to
obtain a largely germ-free and virus-free filtered PMU
at the same time.
If desired, preservatives, germicides, bactericides
and/or anthelmintics can be added to the urine.
I-f a concentrated PMU retentate is to be used instead
of the PMU, this may be obtained from the PMU by known
membrane filtration. The solids content of the
retentate and the composition thereof may vary
according to the PMU used and the membrane used for
membrane filtration, for instance the pore width
thereof, and the conditions of the filtration. For
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instance, when using a nanofiltration membrane, a loss-
free concentration of the oestrogen content in the PMU
retentate can be achieved with simultaneous removal of
up to 50% by weight of the lower-molecular PMU
5 contents. PMU retentates which have been concentrated
up to a ratio of approximately 1:10, for instance a
ratio of about 1:7, and the volume of which can thus be
reduced to approximately 1/10, for instance about 1/7,
of the original PMU volume, can be used for the method
according to the invention.
The semi-polar polymeric adsorber resins which can be
used in method step a) are porous organic non-ionic
polymers which, in contrast to non-polar hydrophobic
polymeric adsorber resins, have an intermediate
polarity (= e.g. with a dipole moment of the active
surface of the resin in the range of 1.0 to 3.0, in
particular 1.5 to 2.0, Debye) and a somewhat more
hydrophilic structure, for instance polycarboxylic acid
ester resins. Expediently, macroporous semi-polar
resins having a preferably macroreticular structure and
average pore diameters in the range of 50 to 150,
preferably 70 to 100, Angstrom, and a specific surface
area in the range of 300 to 900, preferably 400 to
500, m2/g are used. Macroporous cross-linked aliphatic
polycarboxylic acid ester resins, in particular cross-
linked polyacrylic ester resins such as Amberlite
XAD-7 , manufactured by Rohm und Haas, have proved
particularly suitable.
According to the invention, the adsorption of the
conjugated oestrogens on the semi-polar adsorber resin
can be effected by contacting the PMU or the retentate
thereof with the adsorber resin, in that the urine is
introduced into a reactor containing the adsorber resin
and is kept in contact with the adsorber resin therein
for a sufficient time for adsorption of the oestrogen
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content. Once adsorption of the conjugated oestrogens
on the semi-polar adsorber r.esin has taken place, the
adsorber resin.laderi with the mixture of conjugated
oestrogens can be separated from the rest of the urine
in known manner. Expediently, the urine can be passed
through a column containing the adsorber resin at such
a flow rate that the contact time is sufficient for
adsorption of the oestrogen content. Suitable flow
rates are for instance those which correspond to a
throughput of 3 to 10, preferably 5 to 7, parts by
volume PMU/1 part by volume adsorber resin/hour. The
adsorption is preferably effected at room temperature.
Expediently, the throughflow rate of the urine through
the reactor can be controlled by operating at a slight
excess pressure or under-pressure. The quantity of
semi-polar adsorber resin to be used may vary depending
on the type of adsorber resin used and the quantity of
the solids content in the urine. When using PMZT, for
instance one part by volume adsorber resin, e.g. cross-
linked aliphatic polycarboxylic acid ester adsorber
resin, can be loaded with up to 80 parts by volume
pretreated PMU, without perceptible quantities of
oestrogen being able to be detected in the urine
flowing out. When using a PMU concentrate or PMU
retentate, the loading capacity of the adsorber resin
is of course reduced to the extent at which it is
concentrated. For instance, 1 part by volume cross-
linked aliphatic polycarboxylic acid ester adsorber
resin may be laden with a quantity of urine
corresponding to 20 to 80, preferably 30 to 50, parts
by volume PMU.
The semi-polar adsorber resin laden with the mixture of
conjugated oestrogens is washed in method step b) with
a washing water set to a pH range of at least 12.0, in
particular of 12.5 to 14, preferably about 12.5 to
13.5. Aqueous solutions of inert basic substances
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which are soluble in the urine and which are strong
enough to reach a pH value of at least 12.5 can be used
as washing water. Suitable water-soluble basic
substances which are inert to the semi-polar polymeric
adsorber resin are preferably water-soluble inorganic
bases such as alkali metal or alkaline-earth metal
hydroxides, in particular sodium hydroxide.
Expediently, the washing water only contains about the
quantity of basic substances which is required to
achieve the desired pH value, preferably approximately
pH 13. The quantity of washing water is selected such
that it is sufficient largely to remove phenolic urine
contents, without significant quantities of conjugated
oestrogens being washed out with them. For instance,
the use of 2 to 10, in particular 4 to 6, bed volumes
washing liquid per bed volume adsorber resin has proved
expedient. In this case, the washing water is
expediently passed through a reactor containing the
adsorber resin at a throughflow rate of 3 to 10,
preferably 5 to 7, parts by volume washing water/1 part
by volume adsorber resin/hour.
In method step c), the washed adsorber resin laden with
the mixture of conjugated oestrogens is then treated
with a quantity of an elution liquid sufficient for
elution of the mixture of conjugated oestrogens and an
eluate containing the natural mixture of conjugated
oestrogens of the PMU is obtained. The elution liquid
used according to the invention represents a water-
miscible organic solvent from the group of water-
miscible ethers, lower alkanols and lower aliphatic
ketones or a mixture of such a water-miscible organic
solvent and water which has optionally been rendered
alkaline. Suitable ether constituents of the elution
liquid are water-miscible cyclic ethers such as
tetrahydrofuran or dioxan, but also water-miscible
open-chain ethers such as ethylene glycol dimethyl
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ether (= monoglyme), diethylene glycol dimethyl ether
(= diglyme) or ethyloxyethyloxy ethanol (= Carbitol )
Suitable lower alkanols..are_water-miscible alkyl
alcohols with 1 to 4, preferably 1 to 3, carbon atoms,
in particular ethanol or isopropanol. Suitable lower
aliphatic ketones are water-miscible ketones with 3 to
5 carbon atoms, in particular acetone. Elution liquids
in which the organic solvent is ethanol have proved
particularly advantageous. Expediently, mixtures of
one of the afore-mentioned water-miscible organic
solvents and water which has optionally been rendered
alkaline are used as elution liquids. The pH value of
such water-containing eluents is in the neutral to
alkaline range up to pH 13 and may advantageously be
approximately 10 to 12. A solvent which is stable in
the pH range used is selected as the solvent component
in the water-containing elution liquid. In water-
containing alkaline elution liquids having pH values of
approximately 10 to 12, lower alkanols, preferably
ethanol, are suitable as solvent components. The
desired pH value of the water-containing eluent is set
by adding a corresponding quantity of a water-soluble
inert basic substance, preferably an inorganic base,
for instance an alkali metal or alkaline earth metal
hydroxide, in particular sodium hydroxide. In water-
containing elution liquids there may be a volume ratio
of water-miscible organic solvent to water in the range
of 40:60 to 20:80, preferably approximately 30:70. 'The
quantity of eluent used may be approximately 3 to 10,
in particular approximately 4 to 6, bed volumes per bed
volume adsorber resin. Expediently, the elution liquid
is passed through a reactor containing the adsorber
resin laden with the oestrogen mixture at such a flow
rate that the contact time is sufficient for complete
elution of the mixture of conjugated oestrogens. When
using a mixture of ethanol with water in a volume ratio
of 30:70, for instance flow rates of 3 to 10,
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preferably 5 to 7, parts by volume elution liquid per
1 part per volume adsorber resin per hour are suitable.
Expediently, the elution is performed in a temperature
range from room temperature to approximately 60 C,
preferably at approximately 40 to 50 C. If desired,
the flow rate is regulated by operating at slightly
elevated pressure, e.g. at an excess pressure of up to
0.2 bar, and the eluate is collected in several
fractions. The contents of conjugated oestrogens and
phenolic urine contents such as cresols and HPMF in the
individual eluate fractions may be determined in known
manner by high-performance liquid chromatography
(abbreviated "HPLC").
Upon elution, first of all a slightly-coloured to
colourless, practically oestrogen-free preliminary
fraction is obtained, the quantity of which corresponds
generally to approximately one bed volume. The bulk of
the conjugated oestrogens, for instance between 80 and
99% of the conjugated oestrogens present in the
starting PMU, is in the subsequent dark-yeilow-brown
coloured main eluate fractions, the quantity of which
is generally 2 to 4 bed volumes. Generally only traces
of conjugated oestrogens are contained in the
subsequent last fractions. If succeeding fractions are
obtained which still have a content of conjugated
oestrogens of above 10% by weight relative to dry
substance and less than 0.6% by weight relative to dry
substance of cresols and HPMF, these may be combined
with the oestrogen-rich main eluate for further
processing.
The main eluate separated from the adsorber resin in
the manner previously described contains the natural
mixture of conjugated oestrogens occurring in the PMU
in addition to only a small proportion of the content
of phenolic urine contents originally present in the
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PMU. This eluate may be used as a starting material
for the production of inedicaments containing the
natural mixture of conjugated oestrogens. If desired,
the eluate may be further reduced in known manner, in
5 order to obtain a concentrate largely freed of organic
solvent which is suitable for further galenic
processing. If desired, an eluent-free solids mixture
can also be produced by spray-drying. If the natural
mixture of conjugated oestrogens is to be used for the
10 production of solid medicaments, it may be expedient to
admix a solid carrier substance to the eluate
containing the conjugated oestrogens already before
concentration or spray-drying, in order to obtain in
this manner a solids mixture containing the conjugated
oestrogens and carrier substances. Both the eluate
containing the oestrogen mixture and a concentrate
produced therefrom or spray-dried solids product may be
processed in known manner into solid or liquid galenic
preparations such as tablets, dragees, capsules or
emulsions. These galenic preparations can be produced
according to known methods using conventional solid or
liquid carrier substances such as starch, cellulose,
lactose or talcum, or liquid paraffins and/or using
conventional pharmaceutical auxiliaries, for instance
tablet disintegrating agents, solubilisers or
preservatives. For instance, the product dontaining
the conjugated oestrogens may be mixed with the
pharmaceutical carrier substances and auxiliaries in
known manner and the mixture converted into a suitable
dosing form.
The following examples are intended to explain the
invention further, but without restricting its scope.
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Examples. 1 - 3
General operating.directions for obtaining an.extract
from PMU which is largely depleted in phenolic urine
contents and contains the natural mixture of the
conjugated oestrogens contained in the PMU.
A) Adsorption of the oestrogen content of the PMU on
a semi-polar polyacrylic ester adsorber resin.
A column of a height of 30 cm and a diameter of
2.4 cm was filled with 65 ml of a semi-polar
.)10 polyacrylic ester adsorber resin (= Amberlite
XAD-7, manufactured by Rohm und Haas, grain size
0.3 to 1.2 mm, dipole moment 1.8 Debye, average
pore diameter 80 Angstrom, specific surface area
approximately 450 m2/g dry) swollen in water. 2 1
of a PMU (for dry substance content (= DS) and
also contents of conjugated oestrogens (calculated
as oestrone sulphate salt), cresol and HPMF
determined by means of HPLC see following table of
examples) filtered.through a microfiltration unit
or purified by passing through a separator were
passed through the column at room temperature at a
flow rate of 6 ml/min. (= approximately 5.5 bed
volumes per hour). The oestrogen content of the
PMU was fully adsorbed on the semi-polar adsorber
resin column thus laden. The urine running off
was investigated for its content of conjugated
oestrogens (calculated as oestrone sulphate salt)
by means of HPLC and"proved to be practically
oestrogen-free. The bottom product was discarded.
B) Washing of the laden adsorber resin column.
The laden adsorber resin column was washed with
300 ml of an aqueous sodium hydroxide solution
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having the pH value given in the table of
examples. To this end, the alkaline washing water
was.passed through the column..at a flow rate of
6 ml/min. (= approximately 5.5. bed volumes per
hour). The washing liquid running off was
investigated in terms of its content of conjugated
oestrogens (calculated as oestrone sulphate salt),
cresol and HPMF by means of HPLC. The
investigation showed that during the washing phase
less than 5% of the total oestrogens charged on to
the column was washed out.
C) Desorption of the conjugated oestrogens from the
.washed adsorber resin column.
315 mi of the elution liquid (water/solvent
mixture rendered alkaline by the addition of
sodium hydroxide, for composition and pH see
following table of examples) were passed through
the column, which had been preheated to the
elution temperature given in the table of
examples, at a flow rate of approximately
6 ml/min. The eluate running off was collected in
6 fractions. The first fraction was about 65 ml
(= approximately 1 bed volume), and the remaining
fractions were each about 50 ml. The individual
fractions were investigated in terms of their
content of conjugated oestrogens (calculated as
oestrone sulphate salt) cresol and HPMF by means
of HPLC. The first fraction was collected for as
long as the eluate appeared colourless to slightly
yellowish in colour. This fraction contained only
traces of oestrogen sulphate salt.
Once the first bed volume of eluate had run off, a
colour change in the eluate to an intensive dark-
brown colouring took place. Then approximately 80
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to 98% of the total quantity of conjugated
oestrogens adsorbed on the column were contained
in the subsequent fractions 2 to 4. The remaining
fractions contained only a small quantity of
oestrogen sulphate salt. This can also clearly be
seen in the decrease in colour intensity.
Optionally the remaining fractions can be returned
to method step A) after the solvent content has
been distilled off.
The DS content in % by weight and the contents of
conjugated oestrogens (calculated as oestrone
sulphate salt), cresol and HPMF determined by HPLC
in each case are given in the following table of
examples for the fractions containing the majority
of the conjugated oestrogens. These fractions
represent extracts suitable for further galenic
processing.
D) Regeneration of the adsorber resin column.
For regeneration, the column was first washed with
100 ml of an ethanol/water mixture containing 50%
ethanol and set to pH 12, then with 150 ml 10%
aqueous sodium citrate solution and again with
150 ml of the ethanol/water mixture, and finally
with 100 ml distilled water. The entire
regeneration took place at a temperature of 45 C.
The column can be laden and regenerated many
times, for instance up to 40 times.
Example No. 1 2 3
7,3 6, 5 7, 1
Starting PMU % by weight DS
Content oestrone sulphate salt mg/1 (% wt. 110 (0,15) 125 (0,19) 124 (0,17)
Content cresol mg/1 (% wt. DS) DS) 232 (0,32) 263 (0,40) 269 (0,38)
74 (0,10) 86 (0,13) 88 (0,12)
Content HPMF mg/1 (% wt. DS)
pH 12,5 pH 13,0 pH 13,5
Washing water = a ueous a
Elution liquid Ethanol/water Ethanol/water Ethanol/water
30 ; 70 pH 12 30 s 70 pH 12 30 : 70 pH 12
Elution temperature 45 C 45 C 45 C ~
N
Eluate fraction 2
% wt. DS 1,0 1, 5 3, 9
(22,41) 2217 (14,78) 1461 (3,75)
Content oestrone sulphate salt mg/1 (% wt 2214121 (1,12) 82 (0,55) 161 (0,41)
o
Content cresol mg/l (% wt. DS) DS)
~
0 (0,00) 0 (0,00) 0 (0,00)
Content HPMF mg/1 (% wt. DS) o
Eluate fraction 3 % wt.. DS 1,6 0,7 1,2
Content oestrone sulphate salt mg/1 (% wt. 1625 (10,16) 1731 (24,73) 2201
(18,34)
Content cresol mg/1 (% wt. DS) DS) 62 (0,39) 0 (0,00) 59 (0,49)
Content HPMF mg/l (% wt. DS) 2 (0,01) 0 (0,00) 0 (0,00)
Eluate fraction 4 % wt. DS 0,1 0,1 0,4
Content oestrone sulphate salt mg/i (% wt. 240 (24,00) 285 (28,50) 652 (16,30)
Content cresol mg/l (% wt. DS) DS) 16 (1,60) 9 (0,90) 9 (0,22)
Content HPMF m/1 (% wt. DS) 3 (0,30) 0 (0,00) 2 (0,05)
