SK24499A3 - Process to obtain oestrogens from mare's urine - Google Patents

Abstract

A method for obtaining an extract from urine of pregnant mares containing a natural mixture of conjugated estrogens by solid-phase extraction of the mixture of conjugated estrogens from the urine of pregnant mares on RP silica gel.

Description

A process for obtaining an extract comprising a natural mixture of conjugated estrogens from marrow urine is carried out by extracting the solid phase of the mixture of conjugated estrogens from marrow urine on hydrophobized silica gel.

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-1 Method for obtaining estrogens from mares urine

A method for obtaining an extract comprising a natural mixture of conjugated estrogen from urine mares is described by solid phase extraction of a conjugated estrogen mixture from dung urine on RP-silica gel.

Technical field

The present invention relates to obtaining a natural mixture of conjugated estrogens from marrow urine.

BACKGROUND OF THE INVENTION

Estrogens are used in medicine for hormone replacement therapy (treatment). In particular, estrogen mixtures are used for the treatment and prevention of difficulties experienced in women at the time of transition (climacterium) after natural or artificial menopause. The natural mixtures of conjugated estrogens found in the urine of the mares have proved to be particularly effective and well tolerated.

The dissolved solids content of urine mares (= pregnant mares urine, hereinafter abbreviated as PMU) can fluctuate naturally over wide ranges and generally lies in the range of 40-90 g dry matter per liter. In addition to urea and other conventional urine-containing substances, the PMU solids content comprises phenolic components in amounts of about 2-5 wt. % based on dry matter. Among these phenolic components are cresols and known as HPMF dihydro-3,4-bis [3-hydroxyphenyl) methyl] -2 (3H) -furanone. These may be in free or conjugated form. The PMU contains a natural mixture of estrogens, which extensively exists in conjugated form, e.g. The content of conjugated estrogens (calculated as estrogen sulphate) may be between 0.3 and 1% by weight on dry weight basis.

Usually, PMU extracts containing conjugated estrogens are obtained by extraction with polar, water-immiscible or little

-2-miscible organic solvents such as e.g. ethyl acetate, n-butanol or cyclohexanol. However, such liquid-liquid extractions have a large number of problems, such as severe foam formation, sediment formation, emulsion formation and poor phase separation. In general, more extraction steps are required, resulting in losses and only partial recovery of estrogen content.

For the treatment of small amounts of urinary and plasma fluids for the analytical determination of estrogens by gas chromatography, Heikkinnen et al. (Clin. Chem. 27/7, (1981), 1186-1189) and Shackleton et al. (Clinica Chimica Acta 107 (1980), 31-243) proposed solid phase estrogen extraction using a silanized silica gel cartridge containing octadecylsilane residues (Sep-Pak ^R C-ig-cartridge, manufactured by Waters Ass. Inc. Milford, MA, USA) . The estrogens from the cartridge are eluted with methanol. However, no data are reported on other substances contained in the eluate containing estrogens.

SUMMARY OF THE INVENTION

SUMMARY OF THE INVENTION It is an object of the present invention to provide a technical process for obtaining a natural mixture of conjugated estrogens from PMU while avoiding the known drawbacks of hitherto conventional liquid-liquid extractions and delivering a product enriched with phenolic urine-containing substances.

We have now found a process whereby a single solid extraction on hydrophobized silica gel can obtain a mixture enriched with phenolic urine-containing substances, which is largely depleted of HPMF but practically completely contains the natural estrogen content of the PMU, which can serve as a starting material for pharmaceutical production. containing a natural mixture of conjugated estrogens from PMU as the active ingredient.

The process according to the invention for obtaining phenolic urine-containing substances enriched with natural mixtures of conjugated estrogens from PMU is characterized in that:

(a) urinary fluid, which is urine-free and solid-free urine, a concentrated concentrate of this urine, or a ultra-filtered urine retentate obtained by ultrafiltration of the urine. treated with an amount of water-soluble base sufficient to adjust the pH to a minimum of 12, and finally adjusting the pH with the base of the treated urine fluid by adding sufficient aqueous acid to a value in the range of 5 to 8.5,

b) the pretreated alkaline urine liquid, adjusted to a pH of 5 to 8.5, is contacted with an amount of hydrophobised silica gel sufficient to absorb the conjugated estrogen mixture contained in the urinary fluid and the hydrophobised silica loaded with the conjugated estrogen mixture is separated from the other urinary fluid;

c) washing the hydrophobised silica loaded with the conjugated estrogen mixture with an aqueous buffer solution adjusted to a pH of from 5 to 7, in particular 5,

d) contacting the washed hydrophobized silica gel with an amount of an elution liquid which is a mixture of water and a water-miscible organic solvent of the group of water-miscible ethers, lower alkanols and lower aliphatic ketones sufficient to desorb the adsorbed mixture of conjugated estrogens; oestrogens are separated and optionally tapered from hydrophobized silica gel.

For the process according to the invention, PMU can be used as such, the concentrate obtained therefrom or the retentate obtained by membrane filtration. The collected urine is first freed of mucilaginous and solid substances in a known manner. The deposition of solid and mucilaginous substances proves expedient and is then separated by known separation methods, e.g. decanting, separating and / or filtering. Thus, the PMU may e.g. guided through a known separating device, e.g. separator, filter device or sedimentation device. As a separating device, e.g. sand bed, or microfiltration or ultrafiltration devices can also be used, where the use of far-reaching PMU-filtered germs and vortexes can be achieved simultaneously.

If desired, preservatives, disinfectants (germicides), bactericides and / or anthelmintics may be added to the urine.

If PMU retentate is to be used instead of PMU, it can be obtained from PMU by known membrane filtration. The solids content of the retentate and its composition may vary according to the PMU used and the membrane used for membrane filtration, e.g. according to its pore size or filtration conditions. E.g. when using a nanofiltration membrane, almost lossless concentration can be achieved

-4 estrogen content in the PMU-retentate while removing up to 50% by weight of the low molecular weight substances contained in the PMU. PMU retentates which have been concentrated up to about 1:10, preferably about 1: 7, and whose volume can be reduced to about 1/10, preferably 1/7, of the original volume of PMU.

In process step a), a water-soluble base is added to the urine liquid in an amount sufficient to adjust the pH to a minimum of 12, especially 13 to 14. Water-soluble bases are inorganic or organic, inert, urine-soluble bases which are strong enough to Thus, alkali metal or alkaline earth metal hydroxides, especially sodium hydroxide, or organic bases such as quaternary ammonium hydroxides with lower alkyls 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 40 to 60% by weight. In particular, a 45% to 55% aqueous sodium hydroxide solution is found to be suitable. In this alkaline pretreatment, the pH of the urinary fluid is adjusted to a minimum of 12, for example in the range of 12 to 14, preferably about 13, and this alkaline pH will be maintained for a period sufficient to cleave the lactone moieties contained in the urine containing substances. the period may generally be 1/2 to 2 hours. The processing conditions must be chosen so that conjugated estrogens are not attacked. Thus, the treatment is preferably carried out at room temperature while limiting the addition of the base to an amount necessary to achieve a desired pH in the range of 12 to 14. The amount of base required to achieve a pH in the range of 12 to 14 may vary according to the composition of the PMU used. When using 50% sodium hydroxide solution, generally sufficient amounts of from about 100 to 700 ml of 50% sodium hydroxide solution, based on 10 L of PMU, are shown to be sufficient. Finally, the urine fluid is adjusted to a pH in the range of 5 to 8.5, preferably 7 to 8.5, especially 8 to 8.5, by adding an aqueous acid solution. Suitable acids are inert inorganic or organic acids soluble in the urine liquid, such as e.g. hydrochloric acid, sulfuric acid, phosphoric acid or lower carbonic acids such as acetic acid. A concentrated aqueous hydrochloric acid solution, for example, has proved suitable. The amount of acid required to achieve the aforementioned pH range may vary depending on the composition used

-5PMU and the amount of base used in the initial alkaline adjustment. When using a concentrated hydrochloric acid solution, the desired pH value can generally be achieved by adding about 25 to 100 g of concentrated hydrochloric acid solution based on 1 L of PMU.

Hydrophobized silica gels usable in process step b) are known inverse phase (= Reverse-Phase-Kieselgele) abbreviated RP-silica gels. chemically modified silica gels carrying hydrophobic functional groups; ligands. Thus, e.g. silanized RP-silica gels containing n-octadecyldimethylsilyoxy, n-octyldimethylsilyoxy- or dimethylhydroxysilyoxy groups as hydrophobic functional groups. Suitable are e.g. silanized silica gel with a mean grain size of from 15 to 500 µm. Silica gel containing dimethylhydroxysilyloxy residues having an average grain size in the range of 0.05-0.3 mm, e.g. a silica silane derivative of Kieselgel 60 / Dimethylsilanderivat from Merck.

According to the invention, adsorption of conjugated estrogens on hydrophobized silica gel can be effected by contacting the optionally concentrated PMU or retentate thereof with hydrophobized silica gel, wherein in step a) the pretreated urine fluid is introduced into the silica-containing reactor and maintained in contact with the silica gel for sufficient adsorption. estrogen content. After adsorption of the conjugated estrogens onto the hydrophobized silica gel is carried out, the silica gel coupled with the conjugated estrogen mixture can be separated from the other urinary fluid in a known manner. More preferably, the urine liquid may be passed through a column containing silica gel at a flow rate such that the contact time is sufficient to adsorb the estrogen content. Suitable are e.g. flow rates corresponding to a flow rate of 5 to 20 parts by volume PMU / 1 part by volume silica gel / hour. The adsorption is preferably carried out at room temperature. It is expedient to control the urinary fluid flow rate through the reactor by operating at low or negative pressure. The amount of hydrophobized silica gel used may vary depending on the type of silica gel used and the amount of solids content in the urine liquid pretreated in process step a). When using pre-treated PMU, for example, 1 part by volume of hydrophobized silica gel can be attached up to 80 parts by volume

- 6 pre-treated PMU, without visible levels of estrogen in the urinary effluent. When using pre-treated PMU concentrate or PMU retentate, the bonding capacity of the hydrophobized silica gel is naturally reduced to the extent that they are concentrated. Thus, for example, 1 part by volume on hydrophobized silica gel may be fed with an amount of urine liquid corresponding to 30 to 80, preferably 40 to 50, parts by volume of PMU.

The hydrophobised silica gel coupled with the conjugated estrogen mixture is washed in step c) with an aqueous buffer solution adjusted to a pH range of 5 to 7, preferably about 5. Preferably, an aqueous buffer solution composed of acetic acid / acetate adjusted to a pH of about 5 is used. Particularly suitable are 0.1 to 1 molar, preferably 0.1 to 0.2 molar acetic acid / acetate buffer solutions. The amount of washing liquid is selected so as to be sufficient for the far-reaching elimination of phenolic urine-containing substances, without mentioning the amount of conjugated estrogens mentioned. By way of example, the use of 8 to 15, in particular 9 to 10, bed volumes of washing liquid per bed volume of hydrophobized silica gel has proven suitable. More preferably, the washing liquid is passed through a reactor containing hydrophobized silica gel at a flow rate of 5 to 20 parts by volume of washing liquid / 1 part by volume of silica gel / hour.

In process step d), the washed hydrophobised silica gel is finally treated with a mixture of conjugated estrogens with an amount of elution liquid sufficient to elute the mixture of conjugated estrogens PMU. The elution liquid used in the invention is a mixture of water and a water-miscible ether, a lower alkanol or a lower aliphatic ketone. Suitable etheric components of the elution liquid are water-miscible cyclic ethers such as tetrahydrofuran or dioxane, but also water-miscible open-chain ethers such as e.g. ethylene glycol dimethyl ether (= Monoglyme), diethylene glycol dimethyl ether (= Diglyme) or ethyloxyethyloxyethanol (= carbitol). Suitable lower alcohols are water-miscible alkyl alcohols having 1-4, preferably 1-3, carbon atoms, in particular ethanol or isopropanol. Suitable lower aliphatic ketones are water-miscible ketones having 3-5 carbon atoms, especially acetone. Elution has proven to be particularly favorable

-Ί liquids in which the organic solvent is ethanol. In the elution liquid, the volume ratio of the water miscible organic solvent to the water may range from 40:60 to 20:80, preferably about 30:70. The amount of eluent used may be about 3-5 bed volumes per bed of hydrophobized silica gel. More preferably, the elution liquid is passed through a reactor containing hydrophobized silica gel coupled with an estrogen mixture at a flow rate such that the contact time is sufficient to completely elute the conjugated estrogen mixture. When using a 30:70 mixture of tetrahydrofuran and / or ethanol with water, flow rates of from 5 to 20 parts by volume of elution liquid per 1 part by volume of silica gel per hour are suitable, for example. Suitably, the flow rate is controlled by operating at a slightly elevated pressure, e.g. at overpressure up to 0.2 bar. and the eluate is collected in several fractions. The contents of the individual eluate fractions of conjugated estrogens and phenolic urine-containing substances, such as cresols and HPMF, can be determined in a known manner by high performance liquid chromatography (abbreviated as HPLC).

The elution initially yields a slightly colored to colorless, practically estrogen-free pre-drip fraction, the amount of which generally corresponds to approximately one bed volume. A major amount of conjugated estrogens, for example between 80 and 99% of the conjugated estrogens existing in the starting PMU, are found in the following dark yellow to brown colored eluate fractions, the amount generally comprising 1-2 bed volumes. In general, only traces of conjugated estrogens are generally contained in the subsequent post-run fractions. If back fractions are obtained, which still contain conjugated estrogens above 10 wt% on dry weight basis and less than 0.6 wt% cresols and HPMF on dry weight basis, these fractions may be combined with the main estrogen rich eluate for further processing

The main eluate separated from the silica gel as described above contains a naturally occurring mixture of conjugated estrogens present in the PMU besides only a small fraction of the phenolic urine constituents initially present in the PMU. This eluate can serve as a starting material for the manufacture of medicaments containing a natural mixture of conjugated estrogens. If desired, the eluate can be further narrowed in a known manner so as to obtain a largely free organic solvent concentrate which is suitable for further galenic processing. In any case

The eluent composition can also be produced by spray drying. If the natural mixture of conjugated estrogens is to be used for the manufacture of solid medicaments, it may be appropriate to admix the solid carrier conjugate to the eluate containing the conjugated estrogens prior to concentrating or spray drying to obtain a solids mixture containing the conjugated estrogens and carriers. Both the estrogen-containing eluate and the concentrate produced therefrom or the spray-dried solid product can be incorporated into solid or liquid galenic formulations, such as e.g. tablets, dragees, capsules or emulsions. These galenic formulations can be manufactured according to known methods using conventional solid or liquid carriers such as e.g. starch, cellulose, milk sugar (lactose) or talc or liquid paraffins and / or using conventional pharmaceutical excipients, e.g. tablets, diluents or preservatives. Thus, the conjugated estrogen-containing product may be admixed in a known manner with pharmaceutical carriers and excipients, and the mixture may be made into a suitable dosage form.

The following examples are intended to illustrate the invention in more detail, but without limiting its scope.

DETAILED DESCRIPTION OF THE INVENTION

Examples 1-3

General Working Instructions for Obtaining a PMU Extract Containing a Natural Mixture of PMU Contained Conjugated Oestrogens That Are Widely Enriched with Phenolic Substances Contained in the Urine.

A) Pre-treatment of PMU

Even the PMU filtered through a 5 cm high sand bed or microfiltration device (dry matter content (= TS) as well as estrone sulfate salt, cresol and HPMF determined by HPLC (see the following table of examples)) is adjusted by adding 50% aqueous hydroxide solution under vigorous stirring. Sodium (density at 20 ° C = 1.52 g / ml) to pH 13 (amount of solution added)

NaOH (see example table) and stirred at room temperature for 1 hour. Finally, the alkaline urine liquid is adjusted to a pH of from 8 to 8.5 by addition of a concentrated aqueous hydrochloric acid solution (about 35%).

B) Adsorption of estrogen content PMU on RP-silica gel

A 50 cm column with a diameter of 5 cm is filled with 100 g (approx. 193 ml = 1 bed volume) of RP-silica gel (silica gel 60 / dimethylsilanderivative from Merck, order No. 7719, grain size 63 to 200 µm) . The column is washed first with 0.5 L of water, then with 0.25 L of methanol and then with 0.5 L of water.

The urinary liquid pretreated according to A) is passed through the column at an overpressure of 0.2 bar and at a flow rate of 60 ml / minute. The estrogen content of the PMU is completely adsorbed on the RP-silica gel column thus connected. The spent urine fluid is analyzed by HPLC for estrone sulfate salt content and appears to be practically estrogen free. The effluent is discarded.

C) Wash the connected RP-silica gel column

The loaded RP-silica gel column is washed with 2 L (ca. 10 bed volumes) of a 0.1 molar acetic acid / sodium acetate buffer solution. Buffer solution is prepared when solid sodium acetate (ca. 107 g) is added to a 0.15 molar aqueous acetic acid solution to reach pH 5. The overflowing wash liquid is analyzed by HPLC for estrone sulfate, cresol and HPMF content and contains less than 5% estrogen content of the starting PMU.

D) Desorption of conjugated estrogens from washed RP-silica gel column and separation of estrogen rich eluate fraction

I of the elution liquid (water-solvent mixture, composition see table below) is conducted with an overpressure of 0.2 bar and a flow rate of about 60 ml / min through the column. The eluted eluate is collected in several fractions (approximately 200 ml volume to approximately 1 bed volume). The individual fractions are assayed by HPLC for estrone sulfate salt, cresol and HPMF.

The first fraction was collected when the eluate showed colorless to pale yellow. The volume of this fraction is 150 to 200 ml. This fraction contains only traces of estrone sulfate salt.

Once the color of the eluate has changed to an intense dark yellow to brown color, another fraction of about 200 ml is collected. About 80 to 98% of the total adsorbed amount of conjugated estrogens is contained in this fraction. The remaining fractions contain only a small amount of the estrogen sulfate salt. If desired, these fractions can be added to process step B) after distilling off the solvent. For the 2 nd fraction containing the major amount of conjugated estrogens, the following table each contains the dry matter (TS) content by weight and the estrone sulfate, cresol and HPMF content determined by HPLC. This fraction is an extract suitable for further galenic processing.

E) Regeneration of RP-silica gel column

For regeneration, the column is washed first with 2 L of water, then with 0.5 L of methanol and then with 2 L of water. The column can then be reused for that column. The column may be multiple times, e.g. up to ten times, regenerated and reused.

η 7.3 1090 (0.15) 2410 '(0.33) 830 (0.11) 252 (12) 263 (8.0) water 30: 70 894.2 (21.3) 10.9 (0.25) 10.7 (0.25) CN 7.3 1040 (0.14) 2220 (0.30) 840 (0.12) 250 (12) 284 (8.5) water 30: 70 1.8 852 (23.7) 7.4 (0.23) 9.2 (0.26) - 6.0 860.0 (0.14) 10170.0 (1.70) 680.0 (0.10) 629 (12) 767 (8.5) water 30: 70 3.1 858.2 (13.84) 18.0 (0.29) 6.4 (0.10) Example # Default PMU Weight-% dry matter (TS) Content of estrone sulphate salt in mg (wt-% TS) Cresol content in mg (wt-% TS) HPMF content in mg (wt-% TS) Pre - processing Add g 50% NaOH (pH) Add g horse. HCl (pH) Elution fluid Eluate fraction 2 Mass-% dry matter (TS) Content of estrone sulphate salt in mg (wt-% TS) Cresol content in mg (wt-% TS) HPMF content in mg (wt-% TS)

Claims (7)

A process for obtaining a phenolic, urine-containing substance enriched in a natural mixture of conjugated estrogens from urine mares, comprising: (a) the urinary fluid, which is urine-free and solid-free urine, the concentrated urine concentrate or the membrane-filtered urine retentate obtained, treats with an amount of water-soluble base sufficient to adjust pH 12 and finally adjusts the pH of the urinary liquid treated with the base by adding an amount of an aqueous acid solution to a value in the range of 5 to 8.5, (b) the pretreated alkaline urine fluid, adjusted to a pH of 5 to 8.5, is contacted with an amount of hydrophobized silica gel sufficient to adsorb the urinary liquid contained in the conjugated estrogen mixture and separates the hydrophobised silica gel conjugated estrogen mixture from the other urinary liquid; (c) washing the hydrophobised silica gel coupled with the conjugated estrogen mixture with an aqueous buffer solution adjusted to a pH of from 5 to 7, and (d) contacting the washed hydrophobized silica gel with an amount of eluting liquid sufficient to desorption the adsorbed mixture of conjugated estrogens, the liquid being a mixture of water and a water miscible organic solvent of the water miscible ethers group, lower alkanols and lower aliphatic ketones; the eluate containing the natural mixture of conjugated estrogens separates and optionally narrows from hydrophobized silica gel. Process according to claim 1, characterized in that in a step of said process (a) the urinary fluid is mixed with a base, preferably an aqueous solution of the base, and maintained at a pH of at least 12 for a period sufficient to break down the lactone moieties contained in the urine. Process according to one of the preceding claims, characterized in that silanized silica gel bearing dimethylhydroxysilyloxy residues is used as hydrophobized silica gel. Process according to one of the preceding claims, characterized in that in process step b) 1 part by volume of hydrophobized silica gel is connected with an amount of urine liquid corresponding to 30 to 80, preferably 40 to 50 parts by volume of urine. Process according to one of the preceding claims, characterized in that, in process step b), the urine liquid is passed through a reactor containing hydrophobized silica gel at a flow rate corresponding to a flow rate of from 5 to 20 parts by volume of urine / 1 part by volume of hydrophobized silica / hour. Process according to one of the preceding claims, characterized in that B in process step c) the aqueous buffer solution used as the washing liquid is an aqueous acetic acid / acetate buffer solution which is adjusted to a pH of about 5. Process according to one of the preceding claims, characterized in that in process step d) a mixture of water and a water-miscible organic solvent with a volume ratio of organic solvent to water in the range 20:80 to 40:60 is used as elution liquid, in particular 30:70.