MXPA99001851A - Process to obtain oestrogens from mare's urine - Google Patents

Abstract

Disclosed is a process to obtain an extract containing a natural mixture of conjugated oestrogens from mare's urine by solid-phase extraction of the mixture of conjugated oestrogens from pregnant mare's urine on RP-silica gel.

Description

PROCEDURE FOR THE OBTAINING OF ESTROGENS FROM URINE OF YEGUAS DESCRIPTION OF THE INVENTION The present invention concerns the obtaining of a natural mixture of conjugated estrogens from the urine of pregnant mares. Estrogens are used in medicine for hormone replacement therapy. In particular, mixtures of estrogens are used for the treatment and prophylaxis of climacteric age disorders that manifest themselves in women after natural or artificial menopause. In this case, natural mixtures of conjugated estrogens, as they occur in the urine of pregnant mares, have been shown to be particularly effective and "well-compatible." The content of solid material dissolved in the urine of pregnant mares (abbreviated in the following) as "OYP") can vary by nature in wide ranges and can be, in general, in a range of 40-90 g of dry substance per liter, together with the urea and other common constituents of urine, in the content of solid material of the OYP are contained phenolic components in amounts of about 2-5% by weight, based on the dry substance. Among these phenolic components are cresols and dihydro-3,4-bis [(3-hydrofinyl) methyl] -2 (3H) -furanone, known as HPMF. These components can be present in free or conjugated form. The OYP contains a natural mixture of estrogens which is widely present in conjugated form, for example as sodium salt of sulfuric acid half ester (abbreviated below as "sulfate salt"). The content of conjugated estrogens (calculated as the sulfate salt of estrogen) can range between 0.3 and 1% by weight, based on the dry substance. Usually, extracts containing conjugated estrogens are obtained from the OYP by extraction with a polar organic solvent, immiscible or only slightly miscible with water, such as for example ethyl acetate, n-butanol or cyclohexanol. However, in the case of liquid-liquid extractions of this type, a plurality of problems are manifested, such as strong foaming, sediment formation, emulsion formation and poor phase separation. In general, several stages of extraction are required, which leads to losses and to a partial collection of the estrogen content. For the treatment of small amounts of urine and plasma fluids for the analytical determination of estrogens by gas chromatography, it is proposed by Heikkinen et al. (Clin Chem. 27/7, (1981), 1186-1189) and by Shackleton et al. (Chimica Clinic Acta 107 (1980) 231-243) a solid phase extraction of estrogens by means of a cartridge with silanized silica gel containing radicals or ct in accordance with 1 if 1 year (cartridge Cis Sep-PakR, manufacturer Waters Ass. Inc. Milford, MA, USA). In this case, the estrogens are eluted with methanol from the cartridge. However, no data are provided on the remaining substances contained in the eluted material containing estrogen. It is the object of the present invention to develop a technical process for obtaining the natural mixture of conjugated estrogens from the OYP, avoiding the known incontinence of the usual extractions of liquid, which provides a product depleted in urine constituents _phenolic and largely free of HPMF.

A process has now been found with which, in a solid-phase extraction in foamed hydrophilic silica gel - a mixture depleted in constituents of the phenolic urine, largely free of HPMF, but containing substantially all the natural estrogen content of the OYP, which can serve as a starting material for the preparation of pharmaceutical products containing the natural mixture of conjugated astrogens from the OYP as an active component. The method according to the invention for obtaining a natural mixture of conjugated estrogensIt is characterized by a) a urine liquid, which represents the urine released from mucosal substances, a concentrate of this urine or a retention of urine. Concentrated urine, obtained by ultrafiltration of this urine, is treated with an amount of a water-soluble base, sufficient for adjustment of a pH value of at least 12, and then the pH value of the treated urine fluid with the base is adjusted, by adding a quantity Sufficient of an aqueous solution of acids, at a pH value in the range of 5 to 8.5, b) the urine, previously treated with the base and adjusted to pH 5 to 8.5, is contacted with a quantity of a silica gel hi dr of ob izado, sufficient for the adsorption of the mixture of conjugated estrogens contained in the urine liquid, and a silica gel hi dr of ob izado, loaded with the mixture of conjugated estrogens, it is separated from the remaining urine fluid, c) the silica gel hi dr of ob izado, loaded with the mixture of conjugated estrogens, is washed with an aqueous buffer solution, adjusted to a pH range of 5 to 7 , in particular 5, d) the scrubbed and washed silica gel is contacted with an amount of an elution liquid, sufficient for the desorption of the conjugated estrogen mixture adsorbed to the silica gel, which represents a mixture based on water and an organic solvent miscible with water of the gru of the ethers, lower alkanols and lower aliphatic ketones miscible with water, and an eluted material containing the natural mixture of conjugated estrogens is separated from the hydrogenated silica gel and optionally concentrated.

For the method according to the invention, to the OYP as. such a concentrate obtained by concentration or a retentate obtained by membrane filtration can be added. The collected urine is first released in a manner known per se from solid to solid substances. Conveniently, the solid and mucilage substances are allowed to settle and then separated according to known separation methods, for example decanting, separation and / or filtration. Thus, the OYP can be passed, for example, by means of a separation device known per se, for example a separator, a filtration plant or a sediment. As a separating device, for example, a sand bed can be used or customary separators can be used, for example nozzle or chamber separators. If desired, a microfiltration facility or an ultrafiltration facility can also be used, with the use of which at the same time a broad lack of germs and viruses of the filtered OYP can be achieved. If desired, preservatives, germicides, bactericides and / or anthelmintics may be added to the urine.

If a OYP concentrate retentate is to be used instead of the OYP, it can be obtained from the OYP by filtration through a membrane known per se. The content of solid materials of the retentate and its composition can vary, depending on the OYP used and the membrane used for filtering through the membrane, for example in its pore width, as well as in the conditions of the filtration. For example, in the case of using a nanofiltration membrane, a concentration of the estrogen content free of losses in the OYP retentate can be achieved with simultaneous elimination of up to 50% by weight of the constituents of the low molecular weight OYP. . For the process according to the invention, OYP retentates can be used which were concentrated to a ratio of about 1:10, for example a ratio of about 1: 7, and whose volume can therefore be reduced to approximately 1/10. , for example approximately 1/7 of the original volume of the OYP. In step a) of the procedure, an amount of a water soluble base is added to the urine liquid, sufficient for the adjustment of a pH value of at least 12, in particular 13 to 14. Inorganic or organic bases, inert and soluble in the urine liquid, which are sufficiently strong to reach a pH value of at least 12, are suitable as water-soluble bases. Thus, hydroxides of alkali metals or alkaline earth metals, in particular sodium hydroxide, are suitable, or also organic bases, such as alkyl hydroxides in fe rior-quaternary ammonium. Preferably, the base is added in the form of an aqueous solution with a base content of at least 20% by weight, preferably 40 to 60% by weight. Particularly suitable is a lye of aqueous soda at 45 to 55%. In this alkaline pretreatment the pH value of the urine liquid is adjusted to at least 12, for example in a range of 12 to 14, preferably approximately 13, and this alkaline pH value is maintained for a sufficient period of time for the separation of lactone clusters contained in substances constituting the urine which, in general, can be from 1/2 to 2 'hours. The treatment conditions have to be chosen so that in this case the estrogen-conjugates are not attacked. Thus, the treatment preferably takes place at room temperature with limitation of the addition of bases to the amount required to reach the desired pH value in the range of 12 to 14. The amount of base required to reach a pH value in the range of 12 to 14 may vary depending on the of the composition of the OYP used. In the case of using 50% sodium hydroxide solution, quantities of approximately 100 to 700 ml of 50% sodium hydroxide solution, based on 10 1 OYP, are generally sufficient. Then, the urine liquid is adjusted, by the addition of an aqueous solution of acids, to a pH value in the range of 5 to 8.5, preferably 7 to 8.5, in particular 8 to 8.5. . Suitable acids are inert inorganic or organic acids soluble in the urine liquid, such as, for example, hydrochloric acid, sulfuric acid, phosphoric acid or lower carboxylic acids, such as acetic acid. A concentrated aqueous solution of hydrochloric acid is shown to be convenient, for example. The amount of acid needed to reach the aforementioned pH range can vary depending on the composition of the OYP used and the amount of base used for the alkaline adjustment. In the case of using a solution of concentrated hydrochloric acid, the desired pH value can be achieved, in general, by the addition of about 25 to 100 g of concentrated hydrochloric acid solution, based on 1 l of OYP. The hi-fied silica gels, employable in step b) of the process, are reversed-phase silica gels (abbreviated, "silica gels RP") known per se, which are chemically modified silica gels which they carry functional hydrophobic groups or ligands. Thus, for example, silanized RP silica gels are suitable which, as functional hydrophobic groups, contain n-oct radicals in addition to C1 1 if 1 to 1 x i, n-octyl-dime ti ls to y i xoximeime ti lhidr oxy if 1 i 1 oxy. Suitable are, for example, silanized silica gels with average particle sizes of 15 to 500 μm. A silica gel having a content of radicals d ime ti lhidr oxy si loxi with an average grain size in the range of 0.05-0.3 mm, for example the "gel si si 1", has proved to be particularly suitable. i ce / de ri vado de dime ti 1 si 1 ano "of the Merck company name. According to the invention, the absorption of the conjugated estrogens to the silica gel hydr it can be effected by contacting the partially concentrated OYP or its retentate with the hydrous silica gel, and introducing the urine liquid, previously treated in step a) of the process, into a reactor containing the gel. silica and keeping it there in contact with the silica gel for a sufficient time for the adsorption of the estrogen content. After the adsorption of the conjugated estrogens to the hi-fied silica gel, the silica gel loaded with the conjugated estrogen mixture can be separated, in a manner known per se, from the remaining urine fluid. Conveniently, the urine liquid can be passed through a column containing the silica gel at a flow rate such that the contact time for the adsorption of the estrogen content is sufficient. For example, flow rates corresponding to a flow rate of 5 to 20 parts by volume of OYP / 1 part by volume of silica gel / hour are suitable. The adsorption is preferably carried out at room temperature. Advantageously, the flow rate of the urine liquid through the reactor can be controlled by operating at a slight overpressure or depression. The amount of silica gel hydr or f obi zed to be used it can vary depending on the type of silica gel used and the amount of solid content of the urine content previously treated in step a) of the process. In the case of using previously treated OYP, for example, one part by volume of silica gel can be loaded or fired with up to 80 parts by volume of OYP previously treated., without noticeable quantities of e s t r o_g e no_ can be detected in the effluent urine fluid. In the case of using an OYP concentrate or a previously treated OYP retentate, the loading capacity of the hydrogenated silica gel is naturally reduced to the extent that these are concentrated. Thus, for example, 1 part by volume of silica gel hydrated or filled with a quantity of urine fluid corresponding to 30 to 80, preferably 40 to 50, parts by volume of OYP can be charged. The silica gel loaded with the mixture of conjugated estrogens is washed in step c) of the process with a buffer solution adjusted to a pH range of 5 to 7, preferably approximately 5. As a washing liquid preferably an aqueous solution of acetic acid co / t and acetate is used, adjusted to about pH 5. Acetic acid solutions / acetate buffer 0.1 to 1 molar, preferably 0.1 to 0.2 molar are particularly suitable. The amount of washing liquid is chosen so that it is sufficient to wash out phenolic constituents of the urine widely by washing, without there being washed together amounts of conjugated estrogens worthy of mention. For example, the use of 8 to 15, in particular 9 to 10, bed volumes of washing liquid per volume of silica gel bed is desirable. In this case, the washing liquid is suitably passed through a reactor containing hi-silica silica gel with a flow rate of 5 to 20 parts by volume of washing liquid / 1 part by volume of water. silica gel / hour. In step d) of the process, the washed silica gel washed with the mixture of conjugated estrogens is then treated with an amount of an elution liquid sufficient for the elution of the mixture of the conjugated estrogens , and an eluate is obtained that contains the natural mixture of the conjugated estrogens of the OYP. The elution liquid used according to the invention represents a mixture based on water and an ether, lower alkanol and / or lower aliphatic ketone miscible with water. Suitable ether components of the eluting liquid are cyclic ethers which are miscible with water, such as tetrahydrofuran or dioxane, but also open-chain ethers which are miscible with water, such as, for example, eti 1 engl i coldime ti 1 é ter (= monoglyme), dieti 1 eng 1 ico 1 di eti 1 é ter (= diglima) or etiloxi-e ti 1 or ie tano 1 (= ca rb it ol). Suitable lower alkanols are alkyl alcohols miscible with water having 1-4, preferably 1-3 carbon atoms, in particular ethanol or isopropanol. Suitable lower aliphatic ketones are ketones miscible with water having 3-5 carbon atoms, in particular acetone. Particularly favorable are elution liquids in which the organic solvent is ethanol. A volume ratio of organic solvent miscible with water to water may be present in the elution liquid in the range of 40:60 to 20:80, preferably about 30:70. The amount of eluent employed can amount to about 3 to 5 bed volumes per volume of silica gel bed f. Conveniently, the liquid The elution is passed through a reactor containing the hydrogenated silica gel charged with the estrogen mixture at a flow rate such that the contact time is sufficient for the complete elution of the conjugated estrogen mixture. When using a mixture of tetrahydrofuran and / or ethanol with water in the volume ratio 30:70, flow rates of 5 to 20 parts by volume of elution liquid per 1 part by volume of silica gel are suitable, for example. per hour. Conveniently, the flow rate is regulated by working at a slightly elevated pressure, for example at an overpressure of up to 0.2 bar, and the eluate is collected in several fractions. The contents of the various fractions of material eluted in conjugated estrogens and phenolic urine constituents, such as cresols and HPMF, can be determined in a manner known per se by high performance liquid chromatography (abbreviated as "HPLC"). During the elution, a head fraction is first obtained from weakly colored to colorless, practically free of estrogens, the amount of which generally corresponds to approximately one bed volume. The main quantity of conjugated estrogens, for example between 80 and 99% of the conjugated estrogens present in the starting OYP, is found in the following fractions of eluted main material of yellow color curo-pardo, whose quantity generally covers 1 to 2 bed volumes . In the following fractions of tail, in general, only traces of conjugated estrogens are already contained. If tail fractions are obtained which still contain a conjugated estrogen content of more than 10% by weight, based on the dry substance, and less than 0.6% by weight, based on the dry substance, in cresols and HPMF, these fractions they can be brought together for further treatment with the main estrogen-rich eluted material. The primary eluted material separated in the manner described above from the silica gel contains the natural conjugated estrogen mixture which is manifested in the OYP together with only a small proportion of the content of phenolic urine constituents, originally present in the OYP. This eluate can serve as a starting material for the preparation of medicaments containing the natural mixture of conjugated estrogens. If desired, the material The eluate can be further concentrated, in a manner known per se, in order to obtain a concentrate that is largely freed from organic solvent and suitable for further galenic treatment. If desired, a mixture of solid materials free of eluting agents can also be prepared by spray drying. If the natural mixture of conjugated estrogens is to be used for the preparation of solid medicaments, it may be convenient to add a solid support material already before a concentration or spray drying by mixing the eluted material containing the conjugated estrogens with the purpose of to thereby obtain a mixture of solid materials containing the conjugated estrogens and the support materials. Both the eluted material containing the estrogen mixture, as well as a concentrate prepared from the previous one or a solid spray-dried material product, can be incorporated, in a manner known per se, into solid or liquid pharmaceutical preparations, such as for example tablets, dragees, capsules or emulsions. These galenic preparations can be produced according to methods known per se using conventional solid or liquid support substances, such as, for example, starch, cellulose, lactose or talc, or liquid paraffins and / or with the use of customary pharmaceutical auxiliaries, for example tablet disintegrating agents, dissolving agents or preservatives. Thus, the product containing the conjugated estrogens can be mixed in a manner known per se with the pharmaceutical auxiliary and support substances and the mixture can be converted into a suitable dosage form. The following examples have to explain the invention in more detail, but without limiting its scope.

Examples 1-5 General working prescription for obtaining an extract based on OYP widely impoverished in constituents of the phenolic urine and containing the natural mixture of the conjugated estrogens contained in the OYP. A) Previous treatment of the OYP 10 1 of an OYP filtered through a sand bed of 5 cm in height or through a microfiltration facility (dry substance content (= SS) and also contained in Conjugated estrogens (calculated as estrone sulfate salt) determined by HPLC, see the following table of examples) are adjusted to pH 13 under intense agitation by the addition of a 50% aqueous sodium hydroxide solution (density at 20 ° C = 1.52 g / ml) (amount added of NaOH, see Table of examples) and stirred for one hour at room temperature. Next, the alkaline urine liquid is adjusted to a pH value of 8 to 8.5 by the addition of an aqueous solution of concentrated hydrochloric acid (approximately 35%).

B) Adsorption of the estrogen content of the OYP to silica gel RP A 50 cm high column with a diameter of 5 cm is filled with 100 g (a volume of approximately 193 ml = 1 bed per volume) of gel silica RP (= "silica gel 1 60 / dimethylsilane derivative" from Merck, trade name 7719, grain size 63-200 μm). The column is washed first with 0.5 1 of water, then with 0.25 1 of methanol and again with 0.5 1 of water.

The urine content previously treated in section A) is passed through the column with a 0.2 bar pre squeeder and a flow rate of 60 ml / minute. The estrogen content of OYP is completely adsorbed to the RP silica gel column thus loaded. The evacuated urine fluid is examined by HPLC for its content of conjugated estrone (calculated as sulfate salt of estrogen) and manifests as practically free of estrogen. The evacuated material is discarded.

C) Flushing the loaded RP silica gel column The loaded RP silica gel column is washed with 2 1 (^ _ about 10 beds in volume) of a 0.15 molar solution of acetic acid co / t sodium acetate ampoule. The buffer solution is prepared by adding 0.15 molar solid sodium acetate to an aqueous solution of acetic acid. (approximately 107 g) until reaching a pH of 5.

The washing liquid that is evacuated is examined by HPLC for its content of conjugated estrogens (calculated as estrone sulfate salt), cresol and HPMF, and contains less than 5% of the estrogen content of the starting OYP.

D) Desorption of the conjugated estrogens from the column of washed silica gel RP and separation of a fraction of material eluted rich in estrogen 1 1 of the elution liquid (water / solvent mixture, composition see the following Table of examples) is made pass through the column with an overpressure of 0.2 bar and a flow rate of approximately 60 ml / min. The eluate that is evacuated is collected in several fractions (volume in each case approximately 200 ml d approximately 1 bed in volume). The individual fractions are examined by HPLC for their conjugated estrogen content (calculated as estrone sulfate salt), cresol and HPMF. The first fraction is collected until the eluate appears colorless to pale yellow. The volume of this fraction is 150 to 200 ml. This fraction contains only traces of estrone sulfate salt. As soon as a color change of the material eluted in a deep dark yellow to brown color manifests, the next fraction of approximately 200 ml is collected. In this fraction approximately 80 to 98% of the total amount of conjugated estrogens loaded in the column is contained. The remaining fractions already contain only small amounts of estrone sulfate salt. If desired, they can be added to step B) of the process after distillation of the solvent content. For the 2nd fraction containing the main amount of the conjugated estrogens, the content of SS in% by weight and the contents in conjugated estrogens (calculated as sulfate sulfate salt) determined by HPLC are indicated in the following Table of examples in each case. cresol and HPMF. This fraction represents an adequate extract for a subsequent galenic treatment.

E) Regeneration of the silica gel column RP For regeneration, the column is washed first with 2 1 of water, then with 0.5 1 of methanol and again with 2, 1 of water. Then, the column can be used again for the procedure. The column can be regenerated several times, for example up to ten times, and used again.

Ejeaplo »* 1 2 3 O? P starting 6, 7, 3, 7.3 I in weight ie SS Cog (emitted in the sulphate ie estonate ea i ((- I e weight of SS J • 860.0 (0.14) 1040 (0.14) 1090 (0.15) Coaleaiio in cresol in a ((- I by weight ie SS] 10170,0 (1,70) 2220 (0.30) 2410 (0,33) Copteiido tu H tnij iea pesóle SS) 680.0 (0.11) 840 (0.12) 830 (0.11) Triliary price ilicift en j ie KiOl il SOI (p8 | 629 (13) 250 (13) 252 (13) Addiction in J ie SCI coac. (pl) 767 (8.5) 284 (8.5) 263 (8.0) Elution liquid Telnaidrofaran / Etiaol / Isopropanol / ai »30:70 api 10:70 api 30:70 Flaccida 2 ie uterial elaide 3.1 e weight ie SS l ,? Content in const syllable and i. { (- I SS weight) 858.2 (13.84) 852 (23.7) 894.2 (21.3) Coateniio in cresol ea t, (-Jen esodeSS) 18.0 (0.29) 7.4 (0.21) 10.9 (0.26) IW in a | (i I by weight of SS.) 6.4 (0.10) 9.2 (0.26) 10.7 (0.25)

Claims (7)

1. Procedure for obtaining a natural mixture of conjugated estrogens, impoverished in constituents of phenolic urine from the urea of pregnant mares, characterized by a) a urine liguid, which represents the urine released from muci lagi substances. sasy solids, a concentrate of this urine or a concentrated urine retentate, obtained by filtration through this urine membrane, is treated with an amount of a water soluble base, sufficient for the adjustment of a pH value of minus 12, and then the pH value of the urine-treated base is adjusted, by the addition of a sufficient amount of an aqueous solution of acids, to a pH value in the range of 5 to 8.5, b) the urine liguid, previously treated with the base and adjusted to pH 5 to 8.5, is contacted with an amount of a silica gel hi dr of obi zed, sufficient for the adsorption of the estrogen mixture. conjugates contained in the urine liguid, and a silica gel h i dr or f ob i zado, loaded with the mixture of conjugated estrogens, it is separated from the remaining urine fluid, c) the silica gel hi dr of ob izado, loaded with the mixture of conjugated estrogens, is washed with an aqueous buffer solution, adjusted to a range of pH from 5 to 7, and d) the hydrophobic and washed silica gel is contacted with an amount of an elution liquid, sufficient for the desorption of the conjugated estrogen mixture adsorbed to the silica gel, which represents a water-based mixture and an organic solvent miscible with water of the group of ethers, lower alkanols and lower aliphatic ketones miscible with water, and an eluate containing the natural mixture of conjugated estrogens is separated from the hydrophobic silica gel and is concentrate

2. Process according to the rei indication 1, characterized in that in step a) of the process the urine liquid is mixed with the base, preferably with an aqueous solution of the base- and kept at a pH value of at least 12 during a space enough time for the dissociation of lactone groupings contained in the substances constituting the urine.

3. Process according to one of the preceding claims, characterized in that a silanized silica gel carrying dimethylhydroxysilyloxy radicals is used as the hydrophobic silica gel.

4. Process according to one of the preceding claims, characterized in that in stage b) of the process 1 part by volume of hydrophobic silica gel is charged with a quantity of urine liquid corresponding to 30 to 80, preferably 40 to 50 parts by volume of urine .

5. Method according to one of the preceding claims, characterized in that in step b) of the process the urine liquid is passed through a reactor containing the hydrophobic silica gel, with a flow rate corresponding to a flow rate of 5 to 20 parts by volume of urea / 1 part by volume of silica gel hi dr of obi z ado / hor a.

6. A process according to one of the preceding claims, characterized in that the aqueous buffer solution used as the washing guide in step c) of the process is a solution of aqueous acetate adjusted to approximately pH 5.

7. A process according to one of the preceding claims, characterized in that a water-based mixture and an organic solvent miscible with water with a volume ratio of organic solvent to water in the range of water are used as the eluting liquid. 20:80 to 40:60, in particular 30:70.