CA2263757C - Method to obtain oestrogens from mare's urine - Google Patents
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Abstract
A method for obtaining an extract containing the natural mixture of conjugated oestrogens from mares' urine by solid-phase extraction of the mixture of conjugated oestrogens from the urine of pregnant mares on non-ionic semi-polar polymeric adsorber resins is described.
Description
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 "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 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 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 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
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 "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 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 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 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 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 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 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, 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 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.
Zi 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 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 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)
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 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 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 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, 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 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.
Zi 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 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 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)
Claims (12)
1. A method for obtaining a natural mixture, depleted in phenolic urine contents, of conjugated oestrogens from the urine of pregnant mares, 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 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, 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 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.
2. A method according to claim 1, characterised in that in method step a) a macroporous polycarboxylic acid ester resin is used as the non-ionic semi-polar adsorber resin.
3. A method according to claim 2, characterised in that a cross-linked aliphatic polycarboxylic acid ester resin is used as the polycarboxylic acid ester resin.
4. A method according to anyone of claims 1 to 3, characterised in that in method step a) 1 part by volume non-ionic semi-polar adsorber resin is laden with a quantity of urine corresponding to 20 to 80 parts by volume urine.
5. A method according to anyone of claims 1 to 4, characterised in that in method step a) the urine is passed through a reactor containing the non-ionic semi-polar polymeric adsorber resin at a flow rate which corresponds to a throughput of 3 to 10 parts by volume urine/1 part by volume adsorber resin/hour.
6. A method according to anyone of claims 1 to 5, characterised in that the washing water used in method step b) is an aqueous sodium hydroxide solution set to approximately pH 12.5 to 13.5.
7. A method according to anyone of claims 1 to 6, characterised in that in method step c) a mixture of water and a water-miscible organic solvent having a volume ratio of organic solvent to water in the range of 20:80 to 40:60 is used as elution liquid.
8. A method according to anyone of claims 1 to 7, characterised in that in method step c) an ethanol-containing elution liquid is used.
9. A method according to claim 3, characterised in that the cross-linked aliphatic polycarboxylic acid ester resin is a cross-linked polyacrylic ester resin having a macroreticular structure.
10. A method according to claim 4, characterised in that in method step a) 1 part by volume semi-polar adsorber resin is laden with a quantity of urine corresponding to 30 to 50 parts by volume urine.
11. A method according to claim 5, characterised in that the urine is passed through the reactor at a flow rate which corresponds to 5 to 7 parts by volume urine/1 part by volume adsorber resin/hour.
12. A method according to claim 7, characterised in that a mixture of water and a water-miscible organic solvent having a volume ratio of organic solvent to water of 30:70 is used as elution liquid.
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| PCT/EP1996/003820 WO1998008526A1 (en) | 1996-08-30 | 1996-08-30 | Method to obtain oestrogens from mare's urine |
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| CA2263757A1 CA2263757A1 (en) | 1998-03-05 |
| CA2263757C true CA2263757C (en) | 2007-10-23 |
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| JP (1) | JP4285773B2 (en) |
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| AU (1) | AU722739B2 (en) |
| CA (1) | CA2263757C (en) |
| CZ (1) | CZ298761B6 (en) |
| DE (2) | DE19681621D2 (en) |
| DK (1) | DK0923381T3 (en) |
| ES (1) | ES2219694T3 (en) |
| HK (1) | HK1021884A1 (en) |
| HU (1) | HU228763B1 (en) |
| IL (1) | IL128670A (en) |
| NO (1) | NO318390B1 (en) |
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| PT (1) | PT923381E (en) |
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| US10143938B2 (en) | 2013-06-14 | 2018-12-04 | Dr. Reddy's Laboratories Ltd. | Process for purification and isolation of estrogens |
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| DE10037389A1 (en) | 2000-08-01 | 2002-02-14 | Solvay Pharm Gmbh | Method and device for the enrichment and stabilization of conjugated estrogens from mare's urine |
| DE10159161A1 (en) | 2001-12-01 | 2003-06-18 | Solvay Pharm Gmbh | Process for obtaining estrogens from mare's urine |
| TWI252111B (en) | 2001-12-14 | 2006-04-01 | Solvay Pharm Gmbh | Matrix film tablet with controlled release of a natural mixture of conjugated estrogens |
| TWI332400B (en) | 2001-12-14 | 2010-11-01 | Solvay Pharm Gmbh | Preformulation for the tableting of natural mixtures of conjugated estrogens |
| US20040072814A1 (en) | 2002-10-11 | 2004-04-15 | Solvay Pharmaceuticals Gmbh | Method for obtaining a natural mixture of conjugated equine estrogens depleted in non-conjugated lipophilic compounds |
| AR041121A1 (en) * | 2002-10-11 | 2005-05-04 | Solvay Pharm Gmbh | PROCEDURE FOR OBTAINING A NATURAL MIXTURE OF CONJUGATED STROGENS |
| CN1332973C (en) * | 2003-03-06 | 2007-08-22 | 深圳市海达克实业有限公司 | Extraction process of conjugated female hormone |
| RU2351607C2 (en) * | 2003-07-17 | 2009-04-10 | Зольвай Фармасьютиклз Гмбх | Method of separating natural mixture of conjugated horse estrogenes |
| CA2542515C (en) * | 2003-10-24 | 2012-08-14 | Thorgard Pharmaceuticals (Aust) Pty Ltd | Estrogen extraction from pregnant animal urine |
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| NL7111863A (en) * | 1970-10-13 | 1972-04-17 | ||
| CS224881B1 (en) * | 1981-10-21 | 1984-01-16 | Jiri Semenka | Means for isolation of 17-oxosteroids from biological material based on sorbents |
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- 1996-08-30 ES ES96930139T patent/ES2219694T3/en not_active Expired - Lifetime
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| US10143938B2 (en) | 2013-06-14 | 2018-12-04 | Dr. Reddy's Laboratories Ltd. | Process for purification and isolation of estrogens |
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|---|---|
| NO990952L (en) | 1999-02-26 |
| HUP9903776A2 (en) | 2000-03-28 |
| HUP9903776A3 (en) | 2000-04-28 |
| PL185919B1 (en) | 2003-08-29 |
| EP0923381A1 (en) | 1999-06-23 |
| PL331884A1 (en) | 1999-08-16 |
| DE59610985D1 (en) | 2004-05-19 |
| EP0923381B1 (en) | 2004-04-14 |
| HK1021884A1 (en) | 2000-07-14 |
| WO1998008526A1 (en) | 1998-03-05 |
| IL128670A0 (en) | 2000-01-31 |
| AU6930496A (en) | 1998-03-19 |
| SK24599A3 (en) | 1999-10-08 |
| ES2219694T3 (en) | 2004-12-01 |
| CZ59899A3 (en) | 1999-06-16 |
| NO990952D0 (en) | 1999-02-26 |
| HU228763B1 (en) | 2013-05-28 |
| CZ298761B6 (en) | 2008-01-23 |
| ATE264109T1 (en) | 2004-04-15 |
| DK0923381T3 (en) | 2004-05-10 |
| CA2263757A1 (en) | 1998-03-05 |
| NO318390B1 (en) | 2005-03-14 |
| JP2000516938A (en) | 2000-12-19 |
| IL128670A (en) | 2001-05-20 |
| AU722739B2 (en) | 2000-08-10 |
| JP4285773B2 (en) | 2009-06-24 |
| PT923381E (en) | 2004-09-30 |
| DE19681621D2 (en) | 1999-09-30 |
| SK281368B6 (en) | 2001-02-12 |
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