HK1021884B - Method to obtain oestrogens from mare's urine - Google Patents

Description

Method for obtaining estrogen from mare's urine

The invention relates to a method for obtaining a natural mixture of conjugated estrogens from the urine of pregnant mares.

Estrogens are used medically in hormone replacement therapy. In particular, the estrogen mixtures are used for the treatment and prevention of climacteric complaints in women after natural or artificial menopause. Natural mixtures of conjugated estrogens, such as those present in the urine of pregnant mares, have particularly good therapeutic effects and good tolerability.

The content of solid matter dissolved in pregnant mare's urine (pregnant mare's urine, hereinafter abbreviated as "PMU") can naturally fluctuate over a wide range, generally in the range of 40-90g dry matter/liter. The phenolic components are contained in the solid matter of PMU in an amount of about 2-5 wt% of the dry matter, excluding urea and other common urine-containing substances. Among these phenolic components are cresol and dihydro-3, 4-bis [ (3-hydroxyphenyl) methyl ] -2(3H) -furanone, known as HPMF. They may be present in free or conjugated form. PMUs contain a natural mixture of estrogens, which may further be present in conjugated form, for example as the half-ester-sodium sulfate salt (hereinafter referred to as "sulfate"). The conjugated estrogens may be present in an amount of from 0.3 to 1% by weight of the dry product, calculated as estrogen sulfate.

Extracts containing conjugated estrogens are obtained from PMUs by extraction with a polar organic solvent such as ethyl acetate, n-butanol or cyclohexanol that is not miscible with water or only sparingly miscible with water. However, problems such as extensive foam formation, precipitate formation, emulsion formation and poor phase separation occur in such liquid-liquid extraction processes. Generally, multiple extraction steps are required to lose estrogen and only obtain a portion of the estrogen.

Bradlow suggested in 1968 (see steroid 11(1968), 265-) 272. for the extraction of estrogens from urine, macroreticular resin XAD-2 was used^RIt is a neutral, non-polar, hydrophobic, polyphenolic resin from Rohm and Haas. As stated, the adsorption capacity is very small. According to the method of Bradlow, the optionally diluted urine is passed through a column containing the resin at a relatively low flow rate. The estrogen is eluted with methanol or ethanol. But does not describe other substances contained in the estrogen-containing eluate.

It is an object of the present invention to develop an industrial process for obtaining natural conjugated estrogens from PMUs while avoiding the known disadvantages of the liquid-extraction processes heretofore commonly used, which provides a product that eliminates the phenolic urine content and is substantially free of cresols and HPMF.

A process has now been found which makes it possible, during the solid-phase extraction with a semipolar polymeric adsorbent resin of the non-ionic type, to obtain a mixture which, in the absence of phenolic urine inclusions, is free of cresols and HPMF, but retains almost completely the natural estrogen content of PMUs, and which can be used as a starting material for the preparation of a pharmaceutical containing an active substance of a mixture of natural conjugated estrogens derived from PMUs.

The process according to the invention for obtaining a mixture of natural conjugated estrogens of PMU origin that excludes phenolic urine inclusions is characterized in that a) a urine solution is obtained that is free of mucus substances and solid substances, a fraction of said urine solution

A concentrated solution or a concentrated urine retention solution obtained by filtering the urine through a membrane, and

a sufficient amount of semipolar to absorb the conjugated estrogens mixture contained in urine

Contacting polymeric adsorbent resin, and polymerizing the semi-polar polymer loaded with the conjugated estrogen mixture

Separating the adsorbent resin from the remaining urine, b) washing the urine with washing water adjusted to a pH of at least 12.0, in particular between 12.5 and 14

Washing the semipolar polymeric adsorbent resin loaded with the mixture of conjugated estrogens, c) contacting the washed adsorbent resin with the mixture of conjugated estrogens adsorbed thereon for desorption

Contacting with a sufficient amount of an eluent which is an organic solvent miscible with water

Agents derived from the combination of water-miscible ethers, lower alkanols and lower aliphatic ketones

Or the eluent is an organic solvent which can be mixed with water and water which is optionally adjusted to be alkaline

Then eluent containing natural conjugated estrogen mixture and adsorption resin

Isolation and optionally concentration.

PMUs from which a concentrate is obtained by concentration or from which a retention fluid is obtained by membrane filtration can be used in the process according to the invention. The collected urine is first freed from mucus material and solid material in a manner known per se. It is appropriate to precipitate the solid and slime material and then separate them according to separation methods known per se, such as decantation, separation and/or filtration. This allows the PMU to be poured into, for example, a separator, a filtration device, or a settler. As the separation device, for example, a sand bath or a separator that is common on the market, for example, a jet separator or a chamber separator, can be used. If necessary, a microfiltration device or an ultrafiltration device can be applied, and the filtered PMU can be sterile and virus-free when the devices are applied.

If desired, storage agents, fungicides, (bactericides) and/or repellents can be added to the urine.

If instead of PMU a concentrated PMU-retention solution is applied, this can be obtained from PMU by membrane filtration methods known per se. The amount of solid material in the retention solution and its composition may vary depending on the PMU used, the membrane used in the membrane filtration, e.g., its pore size, and the filtration conditions. For example, in the case of nanofiltration membranes, estrogen concentrates can be obtained in PMU-retention with little loss, while removing up to 50 wt.% of the low molecular PMU-content. The PMU-retention used in the methods of the invention may be concentrated to a ratio of about 1: 10, for example about 1: 7, and its volume may be concentrated to about 1/10 of the original PMU-volume, for example about 1/7.

In method step a)Semi-polar polymeric adsorbent resins used are porous organic non-ionic polymers which, in contrast to non-polar hydrophobic polymeric adsorbent resins, have an intermediate polarity (e.g. the dipole moment of the active surface of the resin is in the range of 1.0 to 3.0 debye, especially 1.5 to 2.0 debye) and a hydrophilic structure, such as a polycarboxylate resin. It is suitable to use a specific surface in the range from 300 to 900, preferably from 400 to 500m, with a preferably large network structure, an average pore diameter in the range from 50 to 150, preferably from 70 to 100 angstroms²Macroporous semipolar resins in the range of/g. Especially suitable are macroporous, crosslinked aliphatic polycarboxylate resins, especially crosslinked polyacrylate resins, such as Amberlite XAD-7 from Rohm and Haas^R。

According to the present invention, the adsorption of conjugated estrogens onto semi-polar adsorption resins can be carried out by contacting PMU or its retention solution with the adsorption resin by injecting urine into a reactor containing the adsorption resin and therein contacting it with the adsorption resin for a time sufficient for the adsorption of estrogens. After the adsorption of the conjugated estrogens on the semipolar adsorption resin has been completed, the adsorption resin loaded with the conjugated estrogens mixture can be separated from the other urine in a manner known per se. Preferably, the urine is injected into a column containing an adsorbent resin, which is passed through the column at such a rate that the estrogen is sufficiently contacted to be adsorbed. For example, a suitable flow-through rate corresponds to a rate of 3 to 10, preferably 5 to 7 parts by volume PMU per part by volume adsorbent resin per hour. The adsorption is preferably carried out at room temperature. Preferably, the flow rate of urine through the reactor is regulated and controlled by operating at a slight overpressure or at a reduced pressure. The dosage of the semi-polar adsorbent resin used varies according to the type of adsorbent resin used and the solid content of urine. In the case of PMU, for example, one part of the adsorbent resin, e.g.a cross-linked aliphatic polycarboxylate-adsorbent resin, may be loaded with up to 80 parts by volume of the pretreated PMU, which proves to be free of significant estrogen doses in the urine effluent. In the application of PMU-concentrate or PMU-retention, the loading capacity of the adsorbent resins naturally decreases to the range where they are concentrated. Thus, for example, 1 part by volume of the crosslinked aliphatic polycarboxylate adsorbent resin can be loaded with urine equivalent to 20 to 80, preferably 30 to 50 parts by volume of PMU.

In process step b), the semipolar adsorption resin loaded with the conjugated estrogens mixture is washed with wash water having an adjusted pH of at least 12.0, especially between 12.5 and 14, preferably between about 12.5 and 13.5. As washing water, an aqueous solution of an inert alkaline substance soluble in urine and having an alkaline strength sufficient to bring the pH to at least 12.5 can be used. As inert water-soluble basic substance in contrast to the semipolar polymeric adsorbent resin, preference is given to water-soluble inorganic bases, such as alkali metal-or alkaline earth metal hydroxides, especially sodium hydroxide. Suitably, the amount of alkaline material contained in the wash water is only that amount which is required to achieve the desired pH-value, preferably about pH 13. The amount of wash water is selected to be sufficient to further reject the phenolic urine contents without concomitant washing of a significant dose of conjugated estrogens. It has proven to be suitable, for example, to use 2 to 10, in particular 4 to 6, cell volumes of washing liquid per unit cell volume of the adsorption resin. It is suitable that the flow rate of the washing water through the reactor containing the adsorbent resin is from 3 to 10, preferably from 5 to 7 parts by volume of washing water per 1 part by volume of adsorbent resin per hour.

In process step c), the washed adsorption resin loaded with the mixture of conjugated estrogens is then treated with a sufficient amount of eluent to elute the mixture of conjugated estrogens, after which an eluent containing the mixture of PMU's natural conjugated estrogens is obtained. The eluent used in the present invention is a water-miscible organic solvent derived from a water-miscible compound group of ether, lower alkanol and lower aliphatic ketone, or a mixture of a water-miscible organic solvent and optionally water adjusted to be alkaline. As the ether component of the eluent, suitable are water-miscible cyclic ethers such as tetrahydrofuran or dioxane, and also suitable are water-miscible open-chain ethers such as ethylene glycol dimethyl ether (═ monoglyme), diethylene glycol dimethyl ether (═ diglyme) or ethoxyethoxyethanol (═ diethylene glycol monoethyl ether). As lower alkanols, suitable are water-miscible alkyl alcohols having 1 to 4, preferably 1 to 3, carbon atoms, in particular ethanol or isopropanol. As lower aliphatic ketones, suitable are ketones which can be mixed with water and have 3 to 5 carbon atoms, in particular acetone. Particularly suitable eluents are those in which the organic solvent is ethanol. It is suitable to use as eluent a mixture of the above-mentioned water-miscible organic solvent and optionally water adjusted to be alkaline. The pH of the aqueous eluent is in the range from neutral to basic at pH below 13, advantageously between about 10 and 12. The solvent component of the aqueous eluent is preferably selected to be stable over the pH range of use. In aqueous alkaline eluents having a pH-value of between about 10 and 12, suitable solvent components are lower alkanols, preferably ethanol. The desired pH-value of the aqueous eluate can be adjusted by adding corresponding amounts of water-soluble inert basic substances, preferably inorganic bases such as alkali metal hydroxides or alkaline earth metal hydroxides, in particular sodium hydroxide. The volume ratio of water-miscible organic solvent to water in the aqueous eluent may be in the range 40: 60 to 20: 80, preferably about 30: 70. The eluent dosage applied may be about 3 to 10, especially about 4 to 6 cell volumes per cell volume of adsorbent resin. Suitably, the eluate is passed through a reactor containing an adsorption resin loaded with a mixture of oestrogens, at a rate such that the oestrogen is contacted sufficiently to be adsorbed. In the case of using a mixture of ethanol and water in a volume ratio of 30: 70, a suitable flow rate is, for example, 3 to 10, preferably 5 to 7 parts by volume of eluent per 1 part by volume of adsorbent resin per hour. Suitably, the elution is carried out at a temperature of between room temperature and about 60 c, preferably between about 40 and 50 c. The passage speed is adjusted, if necessary, by working the treatment at slightly elevated pressure, for example at an excess pressure of up to 0.2 bar, and the eluate is received in a plurality of fractions. The contents of conjugated estrogens and phenolic urinary contents such as cresol and HPMF in the individual eluates can be determined in a manner known per se by High Performance Liquid Chromatography (HPLC).

During elution, a lightly colored or even colorless, estrogen-free precursor fraction is first obtained, typically in a dose corresponding to about one cell volume. The amount of conjugated estrogens present in the bulk of the dose, e.g., between 80-99% of the starter-PMU, is present in the following dark yellowish-brown main eluate fraction, which is typically dosed at about 2 to 4 cell volumes. The latter subsequent fractions generally contain only trace amounts of their conjugated estrogens. The subsequent fractions also contain more than 10% by weight of conjugated estrogens and less than 0.6% by weight of cresols and HPMF as dry substances, and if these subsequent fractions are retained, they can be combined with the estrogen-rich main eluate for further processing.

The main eluent separated from the adsorbent resin according to the method described above contains the natural conjugated estrogens mixture present in PMU, in addition to traces of phenolic urine content originally present in PMU. This eluate can be used as a starting material for the preparation of a medicament containing a mixture of natural conjugated estrogens. This eluate may, if desired, be further concentrated in a manner known per se in order to obtain a concentrate free of organic solvents suitable for further processing in galens. Mixtures of solid substances without eluent can also be prepared by spray drying if desired. If a mixture of natural conjugated estrogens is used to prepare the solid pharmaceutical, it is appropriate to add the solid carrier substance to the eluate containing the conjugated estrogens before concentrating or spray drying, in such a way that a solid mixture containing the conjugated estrogens and the carrier substance is obtained. The eluate containing the estrogen mixture, and the concentrate or spray-dried solid product prepared therefrom, can be prepared in a known manner in solid or liquid galenical form, for example as tablets, dragees, capsules or emulsions. These galenic preparations can be prepared in a manner known per se using customary solid or liquid carrier substances, for example starch, cellulose, lactose or talc, or liquid paraffin, and/or using customary pharmaceutical adjuvants, for example tablet disintegrants, solubilizers or storage agents. The product containing conjugated estrogens can thus be mixed together with pharmaceutical carrier substances or adjuvants in a manner known per se, and the mixture can then be converted into a suitable dosage form.

The following examples further illustrate the invention but are not intended to be limiting thereof.Examples 1 to 3

General procedures for obtaining extracts from PMUs that have eliminated the phenolic urine content and contain a mixture of conjugated estrogens contained in natural PMUs. A)The estrogen component of PMU is absorbed on the semipolar polyacrylate-adsorption resin

Semi-polar polyacrylate-adsorption resin swollen with 65ml in water (═ Rohm and

amberlite XAD-7 from Haas corporation^RParticle size of about 0.3-1.2mm and dipole moment of 1.8

Debye, average pore diameter of 80 angstroms, specific surface of about 450m²In dry state) is filled to 30cm high and straight

A column with a diameter of 2.4 cm. 2 liters are filtered through a microfiltration device or by separation

Purified PMU (dry matter content (TS), and female measured by HPLC

Contents of hormones sulfate, cresol and HPMF, see table of examples below), at room temperature

The column was next injected at a flow rate of 6 ml/min (═ about 5.5 cell volumes per hour). PMU contains

Some of the estrogen was completely desorbed on the thus-loaded semipolar adsorption resin-column. By means of

PHLC examination of the conjugated Estrogen content (as estrogen sulfate) in urine voided

Calculated), it was confirmed that it contains almost no conjugated estrogens. Is abandonedAnd (4) discharging liquid. B)Washing loaded adsorbent resin column

The loaded adsorption resin column was loaded with 300ml of a solution having a pH value of the value given in the example table,

And (4) washing with a water-soluble sodium hydroxide solution. For this purpose, the alkaline washing water is used at 6 ml/min (about 5.5)

Bath volume per hour) was passed through the column. Determination of the eluting wash by means of HPLC

The contents of estrogen sulfate, cresol and HPMF in the composition. The examination result showed that the detergent was washed

During which less than 5% of the total estrogen loading on the column was washed out. C)Desorption of conjugated estrogens through washed adsorption resin column

At a flow rate of about 6 ml/min, at the elution temperature given in the example table 315

ml of eluent (water/solvent-mixed solution adjusted to be alkaline by adding sodium hydroxide, composition

And pH see table of examples below) was passed through the preheated column. Separating the effluent eluent into

Received in 6 fractions. The first distillate was about 65ml (about 1 cell volume), the remainder was

The distillate was about 50ml each. Detection of Estrogen sulphuric acid in the respective fractions by means of HPLC

Content of salts, cresols and HPMF. Collecting the first distillate until the eluate is colorless

It turned pale yellow. The distillate contained only trace amounts of estrogen sulfate.

When the first bath volume of eluent is run out, the color of the eluent turns dark

Dark brown. Containing conjugated estrogens adsorbed on the column in subsequent fractions 2 to 4

About 80-98% of the total dose, the remaining fraction containing only trace amounts of estrogen sulfate

And (3) salt. This can also be seen by the reduction in color depth. After removal of the solvent by distillation

Optionally, the remaining fraction is again subjected to process step A).

The following example tables illustrate major amounts of conjugated estrogen-containing fractions, respectively

And TS-content in weight%, and estrogen sulfate measured by HPLC,

Cresol and HPMF content. These fractions are suitable for further galenic treatment

Extracting the liquid. D)Regeneration of adsorbent resin column

For regeneration, first 100ml of ethanol solution adjusted to pH12 containing 50% ethanol

Alcohol/water mixture, then 150ml of 10% aqueous sodium citrate solution, and then

The column was washed once with 150ml of ethanol/water mixture and finally with 100ml of distilled water. All of

The regeneration process of (a) is carried out at a temperature of 45 ℃. The column may be used a plurality of times, for example up to 40 times

The ground is loaded and then regenerated again.

Example No. 2	1	2	3
				Start-PMU weight% TS Estrogen sulfate content mg/l (weight% TS) cresol content mg/l (weight% TS) HPMF content mg/l (weight% TS)	7.3110 (0.15)232 (0.32)74 (0.10)	6.5125 (0.19)263 (0.40)86 (0.13)	7.1124 (0.17)269 (0.38)88 (0.12)
Water-soluble NaOH solution	pH12.5	pH13.0	pH13.5
				Eluent	Ethanol/water 30: 70pH12	Ethanol/water 30: 70pH12	Ethanol/water 30: 70pH12
Elution temperature	45℃	45℃	45℃
				Eluent 2 wt% TS Estrogen sulfate content mg/l (wt% TS) Cresol content mg/l (wt% TS) HPMF content mg/l (wt% TS) eluent 3 wt% TS Estrogen sulfate content mg/l (wt% TS) Cresol content mg/l (wt% TS) HPMF content mg/l (wt% TS) eluent 4 wt% TS Estrogen sulfate content mg/l (wt% TS) Cresol content mg/l (wt% TS) HPMF content mg/l (wt% TS)	1.02241(22.41)112 (1.120)0 (0.00)1.61625(10.16)62 (0.39)2 (0.01)0.1240 (24.00)16 (1.60)3 (0.30)	1.52217(14.78)82 (0.55)0 (0.00)0.71731(24.73)0 (0.00)0 (0.00)0.1285 (28.50)9 (0.90)0 (0.00)	3.91461(3.75)161 (0.41)0 (0.00)1.22201 (18.34)59 (0.49)0 (0.00)0.4652 (16.30)9 (0.22)2 (0.05)

Claims (11)

1. A process for obtaining a mixture of natural conjugated estrogens from the urine of pregnant mares, which abandons the content of phenolic urine, characterized in that,

a) contacting a urine solution which is a urine solution free from mucus substances and solid substances, a concentrated solution of the urine solution or a concentrated urine retention solution obtained by filtering the urine solution through a membrane with a nonionic semipolar polymeric adsorbent resin in an amount sufficient to absorb a mixture of conjugated estrogens contained in the urine solution, the nonionic semipolar polymeric adsorbent resin being a macroporous polycarboxylate resin, and then separating the semipolar polymeric adsorbent resin loaded with the mixture of conjugated estrogens from the remaining urine solution,

b) washing the non-ionic semi-polar polymeric adsorbent resin loaded with the conjugated estrogen mixture with washing water adjusted to a pH of at least 12.0,

c) contacting the washed adsorbent resin with a sufficient amount of an eluent, which is a water-miscible organic solvent derived from water-miscible compounds such as ethers, lower alkanols and lower aliphatic ketones or a mixture of water-miscible organic solvents and optionally water adjusted to alkaline in a volume ratio of organic solvent to water in the range of 20: 80 to 40: 60, required for desorbing the conjugated estrogens mixture therefrom, and separating and optionally concentrating the eluent containing the natural conjugated estrogens mixture from the adsorbent resin.

2. A process according to claim 1, characterized in that as polycarboxylate resin a cross-linked aliphatic polycarboxylate resin is used.

3. A method according to claim 2, characterized in that as polycarboxylate resin a cross-linked polyacrylate resin is used.

4. A process according to claim 3, wherein the polycarboxylate resin is a crosslinked polyacrylate resin having a macroreticular structure.

5. The process according to claim 1 or 2, characterized in that in process step a) 1 part by volume of the non-ionic, semi-polar adsorption resin is loaded with urine-containing liquid corresponding to 20 to 80 parts by volume of urine.

6. The process according to claim 5, wherein 1 part by volume of the non-ionic, semi-polar adsorption resin is loaded with urine-containing liquid corresponding to 30 to 50 parts by volume of urine in process step a).

7. The process according to claim 1 or 2, characterized in that in process step a) the urine is passed through a reactor containing a non-ionic, semi-polar polymeric adsorbent resin at a flow rate corresponding to 3-10 parts by volume of urine per l parts by volume of adsorbent resin per hour.

8. A method according to claim 7, characterized in that the flow-through rate corresponds to 5-7 parts by volume of urine per l parts by volume of adsorbent resin per hour.

9. The process according to claim 1 or 2, characterized in that the wash water used in process step b) is an aqueous sodium hydroxide solution adjusted to a pH value of 12.5 to 13.5.

10. The process according to claim 1 or 2, characterized in that the eluent used in process step c) is a mixture of water and a water-miscible organic solvent in a volume ratio of water to organic solvent in the range of 30: 70.

11. The process according to claim 1 or 2, characterized in that an alcoholic eluent is used in process step c).