AU722084B2 - Process to obtain oestrogens from mare's urine - Google Patents

Abstract

Recovery of a phenolics-depleted natural mixture of conjugated oestrogens from the urine of pregnant mares comprises (a) providing a liquid urine (which comprises a urine freed from mucous and solids, a concentrate of the latter or a urine retentate obtained by membrane filtration) and treating it with a water-soluble base to give a pH of at least 12 and subsequently treating with aqueous acid to adjust the pH to 5-8.5; (b) contacting the urine with a hydrophobised silica gel to adsorb the conjugated oestrogens and separating the gel from the mixture; (c) washing the gel with an aqueous buffer adjusted to pH 5-7; and (d) eluting the oestrogens from the gel using a mixture of water and a water-miscible solvent selected from ethers, lower alcohols and lower aliphatic ketones, and optionally concentrating the eluate;

Description

Process to obtain oestrogens from mares' 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 by weight relative to dry substance. These phenolic constituents include cresols and dihydro-3,4-bis[(3hydroxyphenyl)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.

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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. With 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.

A solid-phase extraction of oestrogens by means of a cartridge with silanised silica gel containing octadecylsilane radicals (Sep-Pak® C 1 8 cartridge, manufactured by Waters Ass. Inc. Milford, MA, USA) is proposed by Heikkinnen et al. (Clin. Chem. 27/7, (1981), 1186 1189) and by Shackleton et al. (Clinica Chimica Acta 107 (1980), 231-243) for the treatment of small quantities of urine and plasma liquids for analytical determination of oestrogens by means of gas chromatography. Therein, the oestrogens are eluted from the cartridge with methanol. 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 to obtain 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 HPMF-free and which is depleted in phenolic urine contents.

A method has now been discovered with which a mixture which is largely 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 hydrophobised silica gel, 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 to obtain a natural mixture, depleted in phenolic urine contents, of conjugated oestrogens from PMU is characterised in that a) a urine liquid, which represents the urine freed of mucilaginous substances and solids, a reduced concentrate of this urine or a reduced urine retentate obtained by ultrafiltration of this urine, is treated with a quantity of a watersoluble base sufficient for setting a pH value of at least 12 and then the pH value of the urine liquid treated with the base is set to a pH value in the range of 5 to 8.5 by the addition of a sufficient quantity of an aqueous acid solution, b) the urine liquid which was pre-treated with the base and set to pH 5 to 8.5 is contacted with a quantity of a hydrophobised silica gel sufficient for the adsorption of the mixture of conjugated oestrogens contained in the urine liquid, and a hydrophobised silica gel laden with the mixture of conjugated oestrogens is separated off from the rest of the urine liquid, c) the hydrophobised silica gel laden with the mixture of conjugated oestrogens is washed with an aqueous buffer solution set to a pH range of 5 to 7, in particular 5, and vAN 0 d) the washed hydrophobised silica gel is contacted with a quantity of an elution liquid sufficient for desorption of the mixture of conjugated oestrogens adsorbed thereon, which liquid represents a mixture of water and a water-miscible organic solvent from the group of water-miscible ethers, lower alkanols and lower aliphatic ketones, and an eluate containing the natural mixture of conjugated oestrogens is separated off from the hydrophobised silica gel 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 these are then separated off using 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.

If 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 virtually 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 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.

In method step a quantity of a water-soluble base sufficient for setting a pH value of at least 12, in particular 13 to 14, is added to the urine liquid.

Suitable water-soluble bases are inorganic or organic inert bases which are soluble in the urine liquid, which are strong enough to achieve a pH value of at least 12. For instance, alkali metal or alkaline-earth metal hydroxides, in particular sodium hydroxide, or alternatively organic bases such as quaternary loweralkylammonium hydroxides, are suitable. Preferably the base is added in the form of an aqueous solution having a base content of at least 20% by weight, preferably to 60% by weight. A 45-55% aqueous sodium hydroxide solution has proved particularly expedient. Upon this alkaline preliminary treatment, the pH value of the urine liquid is set to at least 12, for instance a range of 12 to 14, preferably approximately 13, and this alkaline pH value is maintained for a period sufficient for cleavage of lactone groups contained in urine contents, which may generally be 1/2 to 2 hours.

T4E The treatment conditions must be selected such that the conjugated oestrogens are not attacked. Thus the treatment preferably takes place at room temperature with a restriction of the addition of base to the quantity required to achieve the desired pH value in the range of 12 to 14. The quantity of base required to achieve a pH value in the range of 12 to 14 may vary depending on the composition of the PMU used. When using 50% sodium hydroxide solution, generally quantities of about 100 to 700 ml 50% sodium hydroxide solution relative to 10 1 PMU prove sufficient. Then the urine liquid is set to a pH value in the range of to 8.5, preferably 7 to 8.5, in particular 8 to 8.5, by addition of an aqueous acid solution. Suitable acids are inert inorganic or organic acids which are soluble in the urine liquid, such as hydrochloric acid, sulphuric acid, phosphoric acid or lower carboxylic acids such as acetic acid. For instance, a concentrated aqueous hydrochloric acid solution has proved expedient. The quantity of acid required to achieve the afore-mentioned pH range may vary according to the composition of the PMU used and the quantity of base used to render it alkaline. When using concentrated hydrochloric acid solution, the desired pH value can generally be achieved by the addition of approximately 25 to 100 g concentrated hydrochloric acid solution relative to 1 1 PMU.

The hydrophobised silica gels which can be used in method step b) are known reverse-phase silica gels (abbreviated to "RP silica gels"), that is to say, chemically modified silica gels which bear hydrophobic functional groups or ligands. For instance, silanised RP silica gels which contain n-octadecyldimethylsilyloxy, n-octyldimethylsilyloxy or dimethylhydroxysilyloxy radicals as hydrophobic functional groups are suitable. For instance, silanised silica gels having average grain sizes of 15 to 500 Am are suitable.

Silica gel containing dimethylhydroxylsilyloxy radicals and having an average grain size in the range of 0.05 0.3 mm have proved particularly expedient, for instance "Kieselgel 60/Dimethylsilanderivat", manufactured by Merck.

According to the invention, the adsorption of the conjugated oestrogens on the hydrophobised silica gel can be effected by contacting the optionally concentrated PMU or the retentate thereof with the hydrophobised silica gel, in that the urine liquid pretreated in method step a) is introduced into a reactor containing the silica gel and is kept in contact with the silica gel therein for a sufficient time for adsorption of the oestrogen content. Once adsorption of the conjugated oestrogens on the hydrophobised silica gel has taken place, the silica gel laden with the mixture of conjugated oestrogens can be separated from the rest of the urine liquid in known manner.

Expediently, the urine liquid can be passed through a column containing the silica gel 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 5 to 20 parts by volume PMU/1 part by volume silica gel/ hour. The adsorption is preferably effected at room temperature. Expediently, the flow rate of the urine liquid through the reactor can be controlled by operating at a slight excess pressure or underpressure. The quantity of hydrophobised silica gel to be used may vary depending on the type of silica gel used and the quantity of the solids content in the urine liquid pre-treated in method step When using pre-treated PMU, for instance one part by volume hydrophobised silica gel can be loaded with up to parts by volume pre-treated PMU, without perceptible quantities of oestrogen being detectable in the urine liquid flowing away. When using a pre-treated PMU concentrate or PMU retentate, the loading capacity of the hydrophobised silica gel is of course reduced to the extent at which it is concentrated. For instance, 1 part by volume hydrophobised silica gel may be laden with a quantity of urine liquid corresponding to 30 to preferably 40 to 50, parts by volume PMU.

The hydrophobised silica gel laden with the mixture of conjugated oestrogens is washed in method step c) with an aqueous buffer solution set to a pH range of 5 to 7, preferably about 5. Preferably an aqueous acetic acid/ acetate buffer solution set to about pH 5 is used as washing liquid. In particular 0.1 to 1-molar, preferably 0.1 to 0.2-molar, acetic acid/acetate buffer solutions are suitable. The quantity of washing liquid is selected such that it is sufficient largely to wash out phenolic urine contents, without significant quantities of conjugated oestrogens being washed out with them. For instance, the use of 8 to 15, in particular 9 to 10, bed volumes of washing liquid per bed volume of hydrophobised silica gel has proved expedient. In this case, the washing liquid is expediently passed through a reactor containing the hydrophobised silica gel at a flow rate of 5 to parts by volume washing liquid/i part by volume silica gel/hour.

In method step the washed hydrophobised silica gel 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 mixture of water and a water-miscible ether, lower alkanol and/or lower aliphatic ketone. Suitable ether constituents of the elution liquid are water-miscible cyclic ethers such as tetrahydrofuran or dioxan, but also watermiscible open-chain ethers such as ethylene glycol dimethyl ether monoglyme), diethylene glycol dimethyl ether diglyme) or ethyloxyethyloxy ethanol Carbitol). Suitable lower alkanols are watermiscible 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. In the elution liquid there may be a volume ratio of water-miscible organic solvent to water in the range of 60 to 20 80, preferably approximately 30 The quantity of eluent used may be approximately 3 to bed volumes per bed volume of hydrophobised silica gel.

Expediently, the elution liquid is passed through a reactor containing the hydrophobised silica gel 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 tetrahydrofuran and/or ethanol with water in a volume ratio of 30 70, for instance flow rates of to 20 parts by volume elution liquid per 1 part per volume silica gel per hour are suitable. Expediently, 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-yellow-brown coloured main eluate fractions, the quantity of which is generally 1 to 2 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 silica gel 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 medicaments containing the natural mixture of conjugated oestrogens. If desired, the eluate may be further reduced in known manner, in order to obtain a concentrate largely freed of organic solvent which is suitable for further pharmaceutical 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 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 incorporated in known manner into solid or liquid pharmaceutical preparations such as tablets, dragees, capsules or emulsions. These pharmaceutical 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 containing 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.

Examples 1 General operating procedure to obtain 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) Preliminary treatment of the PMU.

10 1 of a PMU filtered through a sand bed 5 cm high or through a microfiltration installation (for dry substance content DS) and also contents of oestrone sulphate salt, cresol and HPMF determined by means of HPLC see following table of examples) were set to pH 13 with vigorous stirring by the addition of a 50% aqueous sodium hydroxide solution (density at 20'C 1.52 g/ml) ii7CZN (for quantity of NaOH solution added see table of examples) and stirred for one hour at room temperature. Then the alkaline urine liquid was set to a pH value of 8 to 8.5 by the addition of a concentrated aqueous hydrochloric acid solution (approximately B) Adsorption of the oestrogen content of the PMU on RP silica gel.

A column of a height of 50 cm and a diameter of cm was filled with 100 g (A a volume of approximately 193 ml 1 bed volume) RP silica gel "Kieselgel manufactured by Merck, Order No. 7719, grain size 63 to 200 Am). The column was washed first with 1 water, then with 0.25 1 methanol and once again with 0.5 1 water.

The urine liquid pre-treated under A) was passed through the column at an excess pressure of 0.2 bar and at a flow rate of 60 ml/minute. The oestrogen content of the PMU was fully adsorbed on the RP silica gel column thus laden. The urine liquid running off was investigated for its content of oestrone sulphate salt by means of HPLC and proved to be practically oestrogen-free. The bottom product was discarded.

C) Washing of the laden RP silica gel column.

The laden RP silica gel column was washed with 2 1 (A approximately 10 bed volumes) of a 0.15-molar acetic acid/sodium acetate buffer solution. The buffer solution was produced by adding solid sodium acetate (approximately 107 g) to a 0.15molar aqueous acetic acid solution until pH 5 was reached. The washing liquid running off was ainvestigated in terms of its content of oestrone sulphate salt, cresol and HPMF by means of HPLC, and had less than 5% of the oestrogen content of the starting PMU.

D) Desorption of the conjugated oestrogens from the washed RP silica gel column and separation of an oestrogen-rich eluate fraction.

1 1 of the elution liquid (water/solvent mixture, for composition see following table of examples) were passed through the column at an excess pressure of 0.2 bar and at a flow rate of approximately 60 ml/min. The eluate running off was collected in several fractions (volume each approximately 200 ml approximately 1 bed volume). The individual fractions were investigated in terms of their content of 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. The volume of this fraction was 150 to 200 ml. This fraction contained only traces of oestrogen sulphate salt.

As soon as a colour change in the eluate to an intensive dark-yellow to brown colouring took place, the next fraction of approximately 200 ml was collected. This fraction contained approximately 80 to 98% of the total quantity of conjugated oestrogens laden on the column. The remaining fractions contained only small quantities of oestrogen sulphate salt. If desired they can be returned to method step B) once the solvent content has been distilled off.

The DS content in by weight and the contents of oestrone sulphate salt, cresol and HPMF determined by HPLC in each case are given in the following 0 A table of examples for the second fraction COlAn 14 containing the majority of the conjugated oestrogens. This fraction represents an extract suitable for further pharmaceutical processing.

E) Regeneration of the RP silica gel column.

For regeneration, the column was first washed with 2 1 water, then with 0.5 1 methanol and again with 2 1 water. Then the column can be used again for the process. The column can be regenerated and re-used many times, for instance up to ten times.

"Comprises/comprising" when used in this specification is taken to specify the presence of stated features, integers, steps or components but does not preclude the presence or addition of one or more other features, integers, steps, components or groups thereof.

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ft o ftft f Example No. 1 2 3 Starting PMU wt. DS 6,0 7,3 7,3 Content oestrone sulphate salt in mg wt. DS) 860,0 (0,14) 1040 (0,14) 1090 (0,15) Content cresol in mg wt. DS) 10170,0 (1,70) 2220 (0,30) 2410 (0,33) Content HPMF in mg wt. DS) 680,0 (0,11) 840 (0,12) 830 (0,11) Preliminary treatment Addition g 50% NaOH (pH) 629 (13) 250 (13) 252 (13) Addition g cone. HC1 (pH) 767 284 263 Elution liquid Tetrahydrofuran/- Ethanol/ Isopropanol/ water 30 70 water 30 70 water 30 Eluate fraction 2 wt. DS 3,1 1,8 Content oestrone sulphate salt in mg wt. DS) 858,2 (13,84) 852 (23,7) 894,2 (21,3) Content cresol in mg wt. DS) 18,0 (0,29) 7,4 (0,21) 10,9 (0,26) Content HPMF in mg wt. DS) 6,4 (0,10) 9,2 (0,26) 10,7 (0,25) R\ Example No. 4 Starting PMU wt. DS 7,7 7,7 Content oestrone sulphate salt in mg wt. DS) 1250 (0,16) 1250 (0,16) Content cresol in mg DS) 2940 (0,38) 2940 (0,38) Content HPMF in mg DS) 890 (0,12) 890 (0,12) Addition g 50% NaOH (pH) 291 (13) 305 (13) Addition g conc. HCl (pH) 334 347 Elution liquid Monoglyme/ Acetone/ water 30 70 water 30 Eluate fractions 2 and 3 Content oestrone sulphate salt in mg wt. DS) 1045,1 (19,97) 971,9 (20,9) Content cresol in mg wt. DS) 33,7 (0,46) 14,7 (0,32) Content HPMF in mg wt. DS 1 7,3 14) 0 (0,0)

Claims (7)

1. A method to obtain a natural mixture, depleted in phenolic urine contents, of conjugated oestrogens from the urine of pregnant mares, characterised in that a) a urine liquid, 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 treated with a quantity of a water- soluble base sufficient for setting a pH value of at least 12 and then the pH value of the urine liquid treated with the base is set to a pH value in the range of 5 to 8.5 by the addition of a sufficient quantity of an aqueous acid solution, b) the urine liquid which was pre-treated with base and set to pH 5 to 8.5 is contacted with a quantity of a hydrophobised silica gel sufficient for the adsorption of the mixture of conjugated oestrogens contained in the urine liquid, and a hydrophobised silica gel laden with the mixture of conjugated oestrogens is separated off from the rest of the urine liquid, c) the hydrophobised silica gel laden with the mixture of conjugated oestrogens is washed with an aqueous buffer solution set to a pH range of 5 to 7, and d) the washed hydrophobised silica gel is contacted with a quantity of an elution liquid sufficient for desorption of the mixture of conjugated oestrogens adsorbed thereon, which liquid represents a mixture of water and a water-miscible 18 organic solvent from the group of water-miscible ethers, lower alkanols and lower aliphatic ketones, and an eluate containing the natural mixture of conjugated oestrogens is separated off from the hydrophobised silica gel and optionally reduced.

2. A method according to Claim 1, characterised in that in method step a) the urine liquid is mixed with the base, preferably with an aqueous solution of the base, and is kept at the pH value of at least 12 for a period sufficient for cleavage of lactone groups contained in urine contents.

3. A method according to one of the preceding claims, characterised in that a silanised silica gel bearing dimethylhydroxysilyloxy radicals is used as the hydrophobised silica gel.

4. A method according to one of the preceding claims, characterised in that in method step b) 1 part by volume hydrophobised silica gel is laden with a quantity of urine liquid corresponding to 30 to preferably 40 to 50, parts by volume urine.

A method according to one of the preceding claims, characterised in that in method step b) the urine liquid is passed through a reactor containing the hydrophobised silica gel at a flow rate which corresponds to a throughput of 5 to 20 parts by volume urine/I part by volume hydrophobised silica gel/hour.

6. A method according to one of the preceding claims, characterised in that the aqueous buffer solution used as washing liquid in method step c) is an aqueous acetic acid/acetate buffer solution set to approximately pH 19

7. A method according to one of the preceding claims, characterised in that in method step d) 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, in particular 30 70, is used as elution liquid.