HK1091841B - Method for obtaining a natural mixture of conjugated equine estrogens - Google Patents

Description

method for obtaining natural mixture of conjugated equine estrogens

The invention relates to a process for obtaining natural mixtures of conjugated mare estrogens depleted in phenolic urine content and non-conjugated lipophilic compounds, including non-conjugated flavonoids, non-conjugated isoflavonoids, non-conjugated norisoprenoids, non-conjugated steroids, especially androstane-and pregnane-steroids, and similar non-conjugated compounds.

Estrogens are used in medicine for hormone replacement therapy. Especially, the estrogen mixture is used for treating and preventing climacteric symptoms of women after natural menopause or artificial menopause. Natural mixtures of conjugated estrogens, such as those present in the urine of pregnant mares, hereinafter referred to as natural mixtures of conjugated mares' estrogens, have proven to be particularly effective and well tolerated.

The amount of solids dissolved in pregnant mare's urine (hereinafter "PMU") naturally varies over a wide range, typically 40-90 grams dry matter per litre. The PMU has a solids content of phenolic components of about 2-5 wt% of dry matter, excluding urea and other common urine contents. Among these phenolic components are cresol and dihydro-3, 4-bis [ (3-hydroxyphenyl) methyl ] -2(3H) -furanone, known as HPMF. They are present in free or bound form. PMUs contain a natural mixture of estrogens, which are present to a large extent in bound form, for example as the half-ester-sodium sulfate salt (hereinafter referred to simply as "sulfate"). The content of conjugated estrogens (calculated as estrogen sulphate) is 0.1-0.5 wt.% of dry matter. In addition, other lipophilic compounds may be present in the solids content of the PMU, the content of which may vary within wide limits and is not foreseeable. These lipophilic compounds are mainly derived from plants ingested by pregnant mares as feeds and mainly include various flavonoids-, isoflavonoids-, norisoprenoid-derivatives and the like, such as 7-hydroxy-4' -methoxyisoflavone, genistein, daidzein, biochanin a, Equol (Equol) and coumestrol. These originally lipophilic compounds of plant origin may be present in the urine in bound or free (unbound) form. Among the lipophilic components present in the solid content of PMUs are non-conjugated steroid derivatives, in particular androstane-and pregnane steroids, also called non-conjugated estrogen derivatives.

The extract containing a natural mixture of conjugated estrogens is usually obtained by solid-phase extraction or by various liquid-extraction steps based on the use of organic solvents not mixed with water or only mixed in small amounts. It is generally appropriate that in order to obtain a natural mixture of conjugated estrogens which can be used as active ingredients in the manufacture of pharmaceuticals, it must meet specific pharmaceutical specifications, for example those established in the USP (united states pharmacopeia) or the european pharmacopeia. Thus, the content of conjugated estrogens relative to dry matter must be kept within a certain critical value.

U.S. patent nos. 2,551,205 and 2,429,398 describe a process for preparing a water-soluble estrogen preparation from PMUs in which an aqueous concentrate is obtained by first adsorption onto activated carbon or other suitable adsorbent material, elution with an organic solvent miscible with the aqueous phase, such as pyridine, and subsequent removal of the solvent, which contains a substantial portion of the water-soluble estrogen component of the originally used PMU. In us patent No.2,429,398 the concentrate is further purified by extraction with benzene and/or diethyl ether, whereas in us patent No.2,551,205 it is described that the concentrate is acidified to a pH-value of 2-6, preferably 4-5, and then rapidly extracted with an organic solvent which is hardly miscible with water, selected from aliphatic, aromatic or alicyclic hydrocarbons (e.g. hexane, benzene, toluene, cyclohexane) or chlorinated hydrocarbons (e.g. chloroform, dichloroethylene, trichloroethylene, carbon tetrachloride, chlorobenzene), so that undesired substances such as fats, oils, free phenolic components and unbound steroids are removed by transfer to the organic phase. Finally, the aqueous phase is stabilized by neutralization. U.S. Pat. No.2,551,205 recommends that the extract obtained be further purified by subsequent extraction steps and precipitation. In summary, after carrying out the process described in U.S. Pat. No.2,551,205, yields of only about 80% of the estrogenic component of the concentrate used are obtained.

U.S. patent No.2,565,115 describes extraction of conjugated estrogens from PMUs using acetone. But the purity of the obtained estrogen fraction is not stated.

U.S. Pat. No.2,696,265 describes a process in which the estrogen is first extracted with an aliphatic alcohol or ketone, such as hexanol, cyclohexanol or cyclohexanone. The estrogen is passed into the organic phase and subsequently further purified, and the aqueous phase containing the estrogen is brought to a pH of 4 with hydrochloric acid and extracted with ethylene dichloride.

U.S. patent No.2,834,712 discloses a process for preparing high purity low toxicity estrogen mixtures by employing multiple separate extraction steps with different solvents and different pH-value settings. Where large amounts of solvents such as hexane and benzene need to be used. For example, in a step the already purified concentrate is dissolved in water, adjusted to a pH of about 5.0 with hydrochloric acid, extracted with benzene and subsequently with diethyl ether to remove the phenolic constituents.

International patent application WO 01/27134 describes a relatively simple method for extracting conjugated estrogens from PMUs: after addition of a salt such as sodium chloride, PMU is extracted with at least equal parts by volume of an organic solvent such as ethyl acetate, as far as conjugated estrogens are introduced into the organic phase. The organic phase was separated and dried to obtain an extract. WO 01/27134 does not state the purity of the conjugated estrogens extract obtained.

However, many problems arise during the liquid-liquid extraction processes described above and known from the prior art, such as the generation of large amounts of foam, the generation of precipitates, the formation of emulsions and poor phase separation. Generally, many extraction steps are required, resulting in losses and only partial acquisition of estrogen content. These extraction steps also require a large fraction of solvents which are detrimental to health. Furthermore, in the patent documents cited above, neither the content of unbound lipophilic components (such as unbound flavonoids, isoflavonoids, norisoprenoid derivatives and similar unbound compounds or unbound steroids, in particular androstane and pregnane steroids) in the obtained product nor the removal of these components is stated. Said methods known from the prior art either do not provide satisfactory results in terms of yield or in terms of purity of the extract obtained (total amount of hormones obtained as measured relative to dry matter), or require numerous different process steps and the use of large amounts of organic and even partly unsuitable solvents from a toxicological point of view.

Furthermore, various solid phase extraction methods are known from the prior art for obtaining natural mixtures of conjugated mare's estrogen which are highly depleted in phenolic urine content.

International patent application WO 98/08526 thus describes a process which makes it possible to obtain a mixture depleted in phenolic urine contents, highly depleted in cresols and HPMF, containing almost entirely the natural estrogenic component of PMUs, by solid phase extraction on a semipolar, in particular non-ionic semipolar, polymeric adsorbent resin. International patent application WO 98/08526 describes a similar process using silica gel as the adsorbent material in the solid phase extraction. A similar process using polar adsorbent resins containing cyano groups is also described in chinese patent application CN 1308083. Further, U.S. patent application No. US 2002/0156303 describes a process in which PMU is first treated with a basic solvent (Solvenz) and pre-purified by filtration before being purified by polystyrene adsorbent resins. The extract thus obtained is suitable as a starting material for the preparation of a medicament containing a natural mixture of conjugated estrogens derived from PMUs as active ingredient.

The mixture of conjugated estrogens obtained from PMUs according to the method of WO 98/08526 or WO 98/08525 generally meets the requirements of the pharmaceutical specifications specified, for example, in terms of conjugated estrogen content relative to dry matter, critical values to be observed. In particular, highly cresol-and HPMF-depleted products of phenolic urine content can be obtained by means of the process disclosed therein. However, it has been shown that dry matter obtained may contain, in addition to the desired conjugated oestrogen component, non-conjugated lipophilic compounds. These non-conjugated lipophilic compounds include, for example, various non-conjugated flavonoids, isoflavonoids, norisoprenoid derivatives and similar non-conjugated compounds, such as 7-hydroxy-4' -methoxyisoflavone, genistein, daidzein, biochanin A, equol and coumestrol, and also non-conjugated steroids, especially androstane-and pregnane-steroids, and non-conjugated estrogens, to name but a few possibilities. The presence of these non-conjugated lipophilic compounds in mixtures of conjugated estrogens obtained from PMUs cannot be standardized, while the content and composition of free and conjugated lipophilic compounds vary, for example, according to the feed ingested by pregnant mares.

Although the composition of natural mixtures of conjugated mare's estrogen is not altered by the additional presence of non-conjugated lipophilic compounds, the content of conjugated mare's estrogen in relation to dry matter is thus reduced. By targeted removal of unconjugated lipophilic components, higher concentrations of active substance (i.e. conjugated mare's estrogen) can be achieved in the obtained extract. Furthermore, the separation of the unconjugated lipophilic compounds ensures a uniform composition of the individual batches of extract, since the unconjugated lipophilic components are removed, the content and composition of which in PMUs varies in relation to seasonal variations in the type of feed ingested by pregnant mares, and the extract thus obtained has an approximate content of conjugated mares' oestrogens relative to dry matter. Furthermore, the isolation of non-conjugated lipophilic compounds is also advantageous for achieving a uniform spectrum of physiological activity. It may be of interest, for example, to remove possibly present unconjugated lipophilic compounds from natural mixtures of conjugated mare's estrogen, since they may have intrinsic physiological effects in themselves.

One possibility for removing undesired non-conjugated lipophilic compounds is to subject natural mixtures of conjugated mare's estrogen, for example obtained by means of the known solid-phase extraction methods described above, separately to liquid-liquid extraction with suitable organic solvents, which pertinently extracts the undesired non-conjugated lipophilic compounds without leading to a loss of conjugated mare's estrogen. Such a process is generally described in the pending international patent application PCT/EP 03/50703.

The object of the present invention is to develop a technically and economically optimized process for obtaining natural mixtures of conjugated equine estrogens, which are highly depleted in both phenolic urine content and in non-conjugated lipophilic compounds. The non-conjugated lipophilic compound is especially non-conjugated flavonoid, non-conjugated isoflavonoid, non-conjugated norisoprenoid derivative. In particular, it is an object of the present invention to develop a process which does not require additional process steps with respect to the known processes for obtaining natural mixtures of conjugated equine estrogens. The aim of the invention is to develop a method for achieving depletion of both phenolic urine contents and unconjugated lipophilic compounds in a solid phase extraction process. A method was further developed which required only a few method steps to be able to provide an extract of conjugated mare's estrogen with a higher content of conjugated estrogens relative to dry matter. It should furthermore be possible to obtain in a simple manner a natural mixture of conjugated estrogens from the urine of pregnant mares even if the urine contains varying and possibly elevated amounts of unconjugated lipophilic compounds. The task of the present invention is to develop an optimized process for solid phase extraction so that natural mixtures of conjugated mare's estrogen are obtained with excellent active substance content and meet the required pharmaceutical specifications, especially in view of the fact that the content of conjugated estrogens relative to dry matter should comply with the required critical values.

It has now been found that a process is possible in a surprisingly simple manner to obtain from PMUs a highly cresol-and HPMF-depleted mixture of conjugated mare's estrogen which is depleted in phenolic urine content and which is at the same time highly depleted in unconjugated lipophilic compounds, in particular unconjugated flavonoids, unconjugated isoflavonoids, unconjugated norisoprenoid derivatives, even if the PMU contains a varying and possibly elevated amount of unconjugated lipophilic compounds.

The basic steps of the method according to the invention are based on the method described in WO 98/08526 for obtaining a natural mixture of conjugated estrogens depleted in phenolic urine content from PMUs. The method according to the invention is furthermore based on the method described in international patent application WO 03/048183, which also makes it possible to obtain natural mixtures of conjugated estrogens that meet the required pharmaceutical specifications using an aged PMU that may contain an elevated share of free estrogens.

The invention accordingly relates to a process for obtaining natural mixtures of conjugated estrogens from the urine of pregnant mares, which comprises

a) Urine, optionally urine depleted of mucus and solid material, a concentrated urine retentate obtained by membrane filtration of said urine, is contacted with a polymeric adsorbent resin in an amount sufficient to adsorb the mixture of conjugated estrogens contained in the urine and the polymeric adsorbent resin loaded with the mixture of conjugated estrogens is separated from the remaining urine, and

b) the polymeric adsorbent resin loaded with the mixture of conjugated estrogens is washed with a washing water adjusted to a pH of at least 12.0, in particular 12.5 to 14.0, and

c) optionally performing an intermediate washing step in which the polymeric adsorbent resin loaded with the mixture of conjugated estrogens is washed with water, and

d) contacting the washed adsorbent resin with an eluent in an amount sufficient to dissociate the adsorbed mixture of conjugated estrogens, and

e) the eluate of the natural mixture containing conjugated estrogens is separated from the adsorption resin and optionally concentrated,

wherein the method according to the invention is characterized in relation to prior art methods,

the obtained natural mixture of conjugated mare's estrogen is depleted in phenolic urine content and non-conjugated lipophilic compounds including non-conjugated flavonoids, non-conjugated isoflavonoids, non-conjugated norisoprenoids, non-conjugated steroids, especially androstane-and pregnane-steroids, and similar non-conjugated compounds, and

in process step (d), a single-phase or two-phase mixture is used as eluent, which contains

(i) Water, optionally adjusted to a pH in the alkaline range,

and

(ii) at least one organic solvent immiscible or nearly immiscible with water suitable for eluting the non-binding lipophilic compound selected from the list above,

and optionally

(iii) At least one water-miscible organic solvent selected from the group consisting of water-miscible ethers, lower alkanols and lower aliphatic ketones, and mixtures of the aforementioned solvents,

and is

If the eluate obtained in process step e), which is optionally concentrated, is a biphasic mixture, the aqueous phase of the eluate obtained and composed of the two phases is separated and the aqueous phase of the natural mixture containing conjugated estrogens is obtained and optionally concentrated.

The preparation of the individual batches, i.e. the process steps a), b), d) and e) known per se and the use of the eluate obtained in process step e) containing the natural mixture of conjugated estrogens, has been described generally as process steps a), b) and c) in international patent application WO 98/08526, and the patent applications derived from these publications are therefore familiar to the person skilled in the art. The process step c) of the invention, which is known per se, is generally described in international patent application WO 03/048183 as an "intermediate wash", and the patent applications derived from these publications are thus familiar to the person skilled in the art. The contents of said WO 98/08526 and WO 03/048183 are also disclosed for the present invention as content of the present application. Further details of the general method and the materials that can be used are summarized in the examples section of this application.

Semipolar, in particular nonionic semipolar, adsorption resins can be used, for example, according to WO 98/08526. Furthermore, it is surprisingly also possible to use other adsorbent resins with the process according to the invention without affecting the product quality or the pharmaceutical specifications to be observed. Polymeric adsorbent resins are thus within the scope of the present invention suitable as adsorbent materials for the process of the present invention. Polymeric adsorbent resins that can be used within the scope of the present invention are further illustrated in the examples section of the specification.

The method according to the invention is used to obtain natural mixtures of conjugated mare's oestrogens, which are depleted in phenolic urine content such as cresol and HPMF, or in non-conjugated lipophilic compounds including non-conjugated flavonoids, non-conjugated isoflavonoids, non-conjugated norisoprenoids, non-conjugated steroids (especially androstane-and pregnane-steroids) and similar non-conjugated compounds.

After carrying out process steps (a) to (c) known per se, the washed adsorption resin loaded with the mixture of conjugated estrogens is treated for this purpose in process step (d) with a sufficient amount of eluant for eluting the mixture of conjugated estrogens. According to the invention, a single-phase or two-phase mixture is used as eluent in method step (d) and contains

(i) Water, optionally adjusted to a pH in the alkaline range,

and

(ii) at least one water-immiscible or almost-immiscible organic solvent suitable for eluting unbound lipophilic compounds including unbound flavonoids, unbound isoflavonoids, unbound norisoprenoids, unbound steroids, especially androstane-and pregnane-steroids, and similar unbound compounds,

and optionally

(iii) At least one water-miscible organic solvent selected from the group consisting of water-miscible ethers, lower alkanols and lower aliphatic ketones, and mixtures of the aforementioned solvents,

in a preferred embodiment of the invention, a single-phase or two-phase mixture is used as eluent in process step (d), which contains

(i) Water, optionally adjusted to a pH in the alkaline range,

and

(ii) at least one water-immiscible or almost-immiscible organic solvent suitable for eluting unbound lipophilic compounds including unbound flavonoids, unbound isoflavonoids, unbound norisoprenoids, unbound steroids, especially androstane-and pregnane-steroids, and similar unbound compounds,

and

(iii) at least one water-miscible organic solvent selected from the group consisting of water-miscible ethers, lower alkanols and lower aliphatic ketones, and mixtures of the aforementioned solvents,

the single-phase or two-phase mixture used as eluent in process step (d) contains water (i), optionally adjusted to a pH value in the alkaline range.

If the eluent used in process step (d) is a single-phase mixture, the pH of this aqueous eluent is in the alkaline range from neutral to up to pH 13, advantageously between pH 7 and 10. The solvent component in the aqueous eluate is selected from organic solvents which are stable in the pH range used. The desired pH value of the aqueous, single-phase eluate is 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.

If the eluent used in process step (d) is a two-phase mixture, the pH of the aqueous phase of this eluent after thorough mixing of the two phases can advantageously be in the alkaline range from neutral to pH 13, between pH 7 and 10. If the aqueous phase of the biphasic eluent contains an organic solvent component, the organic solvent component is selected from organic solvents which are stable in the pH-range used. The desired pH value of the aqueous phase of the biphasic eluate is 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.

In addition to the optionally basic water (i), the single-phase or two-phase mixture used as eluent in process step (d) also contains at least one organic solvent (ii) for eluting the unbound hydrophilic speciesLipid compounds, including unbound flavonoids, unbound isoflavonoids, unbound norisoprenoids, unbound steroids, especially androstane-and pregnane-steroids, and similar unbound compounds. Furthermore, such suitable organic solvents (ii) should be immiscible or hardly miscible with water, by hardly miscible here is meant that at most 6% by volume of dissolved organic solvent is present in the aqueous phase. Organic solvents (ii) suitable for eluting unconjugated lipophilic compounds within the scope of the present invention may be, for example, the following organic solvents having from 1 to 10 carbon atoms in a linear, branched or cyclic arrangement: c₄-C₁₀Alcohols (e.g. butanol, hexanol, cyclohexanol and pentanol), C₁-C₁₀Esterified acids such as ethyl acetate, methyl acetate, propyl acetate, isopropyl acetate, butyl acetate, pentyl acetate, ethylmethylmalonate, dimethylphosphonate), C₃-C₁₀Aldehydes and C₄-C₁₀Ketones (e.g. butanone, pentanone, hexane-2, 4-dione, hexanedial, heptanal, butane-1, 2, 4-trimethylaldehyde, methylphenylketone and the like), or all C₃-C₁₀Alkoxy-compounds, C₃-C₁₀Ethers (diethyl ether, methyl tert-butyl ether), C₃-C₆Nitriles and C₁-C₃Halogenated alkanes (dichloromethane), and mixtures of the foregoing solvents. In particular, acetic acid-C can be used within the scope of the invention₁-C₄-alkyl esters, butanol, cyclohexanol, hexanol, diethyl ether, dichloromethane, methyl tert-butyl ether and mixtures of said solvents as water-immiscible or almost immiscible organic solvents (ii) suitable for eluting unconjugated lipophilic compounds. Within this optional range, acetic acid-C₁-C₄The alkyl esters, especially ethyl acetate and/or isopropyl acetate, are preferably the organic solvents (ii) of the invention used which are immiscible or barely miscible with water and which are suitable for eluting unconjugated lipophilic compounds.

Furthermore, the single-phase or two-phase mixture used as eluent in step (d) of the process according to the invention optionally also contains at least one water-miscible organic solvent (iii) selected from the group consisting of water-miscible ethers, lower alkanols and lower aliphatic ketones, and mixtures of the aforementioned solvents. The ether component of the eluent is suitably a water-miscible cyclic ether such as tetrahydrofuran or dioxane, but may also be a water-miscible open-chain ether such as ethylene glycol dimethyl ether (Monoglyme), diethylene glycol dimethyl ether (Diglyme) or ethoxyethoxyethanol (Carbitol). Suitable lower alkanols are water-miscible alkyl alcohols having 1 to 4, preferably 1 to 3, carbon atoms, in particular ethanol or isopropanol. Suitable lower aliphatic ketones are water-miscible ketones having 3 to 5 carbon atoms, in particular acetone. Furthermore, it has been shown to be particularly advantageous for the eluent to be one in which the water-miscible organic solvent (iii) is acetone, ethanol or a mixture of acetone and ethanol.

In a preferred embodiment of the invention, the single-phase or two-phase mixture used as eluent in process step (d) contains, in addition to the optionally basic water (i), acetic acid-C as an organic solvent (ii) which is suitable for eluting the non-conjugated lipophilic compound and is immiscible or barely miscible with water₁-C₄-an alkyl ester, preferably ethyl acetate and/or isopropyl acetate, and acetone and/or ethanol as water-miscible organic solvent (iii).

In a further preferred embodiment of the present invention, the single-phase or two-phase mixture used as eluent in process step (d) contains, in addition to water (i), optionally adjusted to basic, ethyl acetate as organic solvent (ii) which is suitable for eluting the non-conjugated lipophilic compound and is immiscible or barely miscible with water, and acetone as organic solvent (iii) which is miscible with water.

The volume ratio of water (i) and the water-immiscible or virtually immiscible organic solvent (ii) in the single-phase or two-phase mixture used as eluent in process step (d) can be in the range from 5: 1 to 1: 5, in particular from 2: 1 to 1: 2, is regarded as suitable.

Furthermore, the volume ratio of water (i) and water-miscible organic solvent (iii) in the single-phase or two-phase mixture used as eluent in process step (d) may be in the range from 4: 1 to 1: 5, preferably from 2: 1 to 1: 3, is regarded as suitable.

In particular, the volume ratio of the total volume of water (i) and the water-immiscible or virtually immiscible organic solvent (ii) to the volume ratio of the water-miscible organic solvent (iii) in the single-phase or two-phase mixture used as eluent in process step (d) can be in the range from 5: 1 to 1: 5, preferably in the range from 2: 1 to 1: 2.

In a particularly preferred embodiment of the invention, the single-phase or two-phase mixture used as eluent in process step (d) is prepared from (i) water, (ii) acetic acid-C₁-C₄(ii) -an alkyl ester, preferably ethyl acetate and/or isopropyl acetate as organic solvent which is immiscible or nearly immiscible with water, and (iii) acetone and/or ethanol as organic solvent which is miscible with water, in a volume ratio in the range from 1: 1 to 1: 2. In particular, in process step (d), a single-phase mixture is used as eluent, which consists of (i) water, (ii) ethyl acetate as organic solvent which is immiscible or barely miscible with water, and (iii) acetone as organic solvent which is miscible with water in a volume ratio of 1: 1.4.

If a two-phase mixture is used as eluent in process step (d), it is advisable to mix the two phases thoroughly directly before loading the column.

The volume of eluent used may be about 3 to 10 (especially about 4 to 6 column volumes) per column volume of polymeric adsorbent resin. Suitably, the elution is carried out at an elution rate by passing the eluate through a reactor containing an adsorption resin loaded with a mixture of estrogens, for a contact time sufficient for adequate elution of the mixture of conjugated estrogens. Suitably, the elution rate is, for example, from 3 to 10 (preferably from 5 to 7) parts by volume of eluent per part by volume of adsorbent resin per hour using a single phase mixture of water, ethyl acetate and acetone in a volume ratio of 10: 14. Suitably, elution is carried out at a temperature in the range of between about 20 ℃ and 80 ℃, preferably between about 30 ℃ and about 50 ℃. Ideally, the elution rate is adjusted and the eluate is collected in multiple fractions by operating at slightly elevated pressure (e.g. at an overpressure of up to 0.2 bar).

The content of conjugated estrogens, phenolic urine contents such as cresols and HPMF, and non-conjugated lipophilic compounds in the individual eluate fractions can be determined in a manner known per se by high performance liquid chromatography ("HPLC") or by gas chromatography ("GC" for short).

During the elution, a light to colorless, virtually estrogen-free front elution fraction (Vorlauffrakation) is first obtained in method step e), the volume of which corresponds approximately to 1 column volume. The main volume of conjugated estrogens, e.g. 80-99% of the conjugated estrogens present in the initial PMU, is typically 2 to 4 column volumes in the subsequent dark yellow-brown main elution fraction. Only traces of conjugated estrogens are generally contained in the later late eluting fractions. If the obtained post-elution fraction also contains more than 10 wt.% conjugated estrogens relative to dry matter and less than 0.6 wt.% cresol and HPMF relative to dry matter, it can be combined with the estrogen-rich main eluate for further processing.

The eluate obtained in process step (e) may be a single-phase or a two-phase mixture. Even the use of a single-phase mixture as the eluent results in an eluate consisting of two phases through the discharge of the column for the water present in the column of the adsorption resin before elution and the consequent change in the volume ratio of the aqueous phase to the organic phase. It may be desirable to further concentrate the single-phase or two-phase eluate obtained in process step (e) in a manner known per se. In particular, if the eluate obtained in process step (e) is single-phase, the eluate is concentrated in order to preferably remove the water-miscible organic solvent (iii) until the concentrated eluate is a two-phase mixture.

If the eluate obtained in process step (e), which is optionally concentrated, is a biphasic mixture, the aqueous phase of the eluate obtained from the two phases is separated in process step (f) according to the invention to obtain an aqueous phase containing the natural mixture of conjugated estrogens. For this purpose the two-phase eluate obtained is left to stand in order to achieve phase separation. The phase separation may take a time of 10 minutes to 24 hours, respectively, depending on the volume obtained, preferably the phase is left to stand for 5 to 10 hours. When the aqueous phase and the organic phase have been separated from each other, the aqueous phase is separated and used for further applications, and the organic phase is discarded.

After separation of the organic and aqueous phases, an aqueous phase containing a natural mixture of conjugated estrogens is obtained in step (f). The aqueous phase contains the natural mixture of conjugated estrogens present in PMU, with only a small fraction of urine phenolic constituents present in PMU, such as cresol and HPMF, and a very small fraction of lipophilic non-conjugated constituents present in PMU. The aqueous phase can optionally be further concentrated in a manner known per se to give a concentrate which is highly freed from organic solvents and is suitable for further processing. It is thus possible, for example, to distill off the organic solvent residue still present from the aqueous phase obtained. The dry matter content of the aqueous extract phase can also be adjusted to a specific value by distillation, preferably to a dry matter content in the range between 5 and 15%, particularly preferably to a dry matter content of 9%. It is then also possible to adjust the pH of the aqueous extract to a basic range, preferably to a range between 8 and 13, more preferably to a value range between 9 and 12, in order to stabilize the natural mixture of conjugated mare's estrogen obtained. Adjusting the pH value with a base usually suitable for adjusting the pH value, e.g. 1N NaOH or Na₂CO₃。

The aqueous phase obtained in process step (f) according to the invention, optionally further treated or concentrated, can be used as starting material for the preparation of medicaments containing natural mixtures of conjugated mare's estrogen. The solid mixture without eluent can be prepared by a suitable drying procedure, such as spray drying or fluidized bed drying, if desired. If a natural mixture of conjugated estrogens is used for the preparation of solid drugs, it is appropriate to mix a solid carrier substance into the aqueous phase containing the conjugated estrogens before concentrating and drying, in such a way that a solid mixture containing the conjugated estrogens and the carrier substance is obtained. For example, the aqueous phase containing the conjugated estrogens may be sprayed in a fluidized bed onto a solid carrier material, such as cellulose. Whether an aqueous phase containing the estrogen mixture or a concentrate or a dried solid product prepared therefrom, can be processed in a manner known per se to form solid or liquid galenic preparations, such as tablets, dragees, capsules or emulsions. These galenical preparations can be prepared in a manner known per se using conventional solid or liquid carrier substances, such as starch, cellulose, lactose or talc or liquid paraffin, and/or using conventional pharmaceutical adjuvants, such as tablet disintegrants, solubilizers or preservatives. The product containing conjugated estrogens can thus be mixed with pharmaceutical carrier substances and adjuvants in a manner known per se, and the mixture can then be converted into a suitable dosage form.

According to the solid phase-extraction method described in the present invention, a natural mixture of conjugated mare's estrogen is obtained from urine, optionally urine freed from mucous substances and solid matter, a concentrated concentrate of the urine or a concentrated urine retentate of the urine obtained by membrane filtration, wherein the mixture is highly depleted in the mixture of both phenolic urine contents (such as in particular cresol and HPMF) and also of non-conjugated lipophilic compounds in a simple manner. The unbound lipophilic compounds removed are in particular unbound flavonoids, unbound isoflavonoids, unbound norisoprenoids and unbound steroids, in this case in particular unbound androstane and unbound pregnane derivatives.

The solid phase-extraction method according to the invention provides a natural mixture of conjugated mare's estrogen which is highly depleted in lipophilic compounds, whether phenolic urine content or unconjugated, compared to known solid phase-extraction methods, wherein the method according to the invention does not present additional process steps, since the depletion of lipophilic compounds, even unconjugated, has already been accomplished in the solid phase extraction method.

In the process known from WO 98/08526, a part of the unbound lipophilic compounds, for example isoflavone-massol, is washed out in process step b), while the other unbound lipophilic compounds, for example 7-hydroxy-4' -methoxyisoflavone, are taken along into the eluate and, if appropriate, have to be separated off by subsequent liquid-extraction. The same is true for the unconjugated, i.e. free, steroid hormones, which are likewise detected in certain fractions in the eluate. It is therefore extremely surprising that for quantitative elution, a single-phase or even two-phase mixture of water, an organic solvent which is immiscible or barely miscible with water and optionally a water-miscible organic solvent such as lower ethers, alkanols or ketones can be used for the conjugated estrogens adsorbed on polymeric adsorbent resins, and the resulting, optionally concentrated eluate is divided into two phases, wherein the quantitatively eluted conjugated estrogens are almost exclusively rediscovered in the aqueous phase and the undesired, unconjugated lipophilic compounds are present in the organic phase and can be removed in a simple manner.

The method of the invention further provides an extract of conjugated mare's estrogen which has a relatively high content of conjugated estrogens relative to dry matter. Thus with the method of the invention, a natural mixture of conjugated estrogens can be obtained in a simple manner from the urine of pregnant mares, even when the urine contains varying and possibly increasing amounts of non-conjugated lipophilic compounds, without the need for further purification steps after the solid phase extraction process.

The natural mixtures of conjugated mare's estrogen, which are depleted in unconjugated lipophilic constituents and phenolic urine constituents, obtained as active substance-extracts by the process according to the invention exhibit a significantly optimized pharmaceutical specification, as established according to the invention, compared with active substance-extracts obtained by known solid-phase extraction methods.

It must be stated as extremely surprising that the known solid-phase extraction processes, with the variations according to the invention, can lead to such an improvement in the quality of the active substance extract obtained even if pregnant mares' urine, which provides a natural mixture of conjugated estrogens, contains varying and varying amounts of unconjugated lipophilic components. It is particularly surprising that the share of unconjugated lipophilic compounds, which is relevant to the PMUs used, whether in content or in composition, can be reduced so assuredly by the process of the invention that a mixture of natural conjugated mare estrogens meeting the high requirements of pharmaceutical specifications, for example according to the requirements set forth in the USP or European pharmacopoeia, can be obtained as the aqueous phase in process step f).

As described in detail above, the method according to the invention has a series of advantages and improvements with respect to the prior art. The invention therefore also makes it possible to use PMUs with a modified content of unbound lipophilic constituents, which may for example have a relatively high content of free flavonoids, free isoflavonoids, free norisoprenoids or free steroid derivatives, in particular free androstane or free pregnane steroids, without affecting the pharmaceutical specifications to be adhered to. In which the process according to the invention is based on known solid phase extraction processes, surprisingly no further extraction steps are required. The method according to the invention thus ensures a uniform composition of the individual batches of extract, since unconjugated lipophilic components, the content and composition of which vary in PMUs depending on the type of feed ingested by pregnant mares, can always be removed, and the extract thus obtained has an approximate content of conjugated mare's estrogen relative to dry matter. Furthermore, by using the method according to the invention not only the targeted removal of phenolic urine contents but also the specific removal of non-conjugated lipophilic components in a single operation step, higher concentrations of active substance, i.e. conjugated mare's estrogen, are obtained in the obtained extract. The method according to the invention also has economic advantages, since the risk of giving up valuable active substances in situations where pharmaceutical specifications cannot be complied with, for example, insufficient conjugated estrogens relative to dry matter, is significantly reduced. Furthermore, the method according to the invention allows the content of active substance in the obtained extract to be substantially accurately and repeatedly adjusted. The active substance components are outstandingly suitable for the preparation of medicaments which contain natural mixtures of conjugated mare's estrogen as active substance.

The following examples are intended to further illustrate the invention without limiting its scope.

Examples

The following example will describe the general working rules for obtaining an extract of active substances from a PMU, said extract containing a natural mixture of conjugated estrogens contained in the PMU and in which not only the phenolic urinary contents (e.g. cresol or HPMF) but also the non-conjugated lipophilic compounds (e.g. non-conjugated flavonoids, non-conjugated isoflavonoids, non-conjugated norisoprenoids, non-conjugated steroids, in particular androstane-and pregnane-steroids and similar non-conjugated compounds) are highly depleted. It shows how a high-quality extract with a high active substance content can be obtained from a PMU according to the invention with a shifted or increased fraction of unconjugated lipophilic compounds.

Urine:

for the method of the invention, the PMU that can be used as urine is, as described in WO 98/08526, a PMU obtained by concentrating a PMU or a PMU concentrate that has been previously purified by filtration or the like. The collected urine (PMU) is first freed from mucus and solid matter in a manner known per se. It is expedient to deposit the solid matter and the mucous substance and then to separate them in accordance with separation methods known per se, for example decanting, partitioning and/or filtration. The PMUs can be led, for example, via a separation device known per se, for example a separator, a filter or a precipitator. As a separating device, for example, a sand bed can be used, or commercially available separators, such as a nozzle separator (Dusen separator) or a chamber separator (Kammersescaroren), can be used. If desired, a microfiltration or ultrafiltration apparatus may also be used, which in its application simultaneously results in a high degree of mold and virus removal from the filtered PMU.

Optionally preservatives, microbicides, bactericides and/or anthelmintics may be added to the urine or urine concentrate.

It is also possible to use as pre-purified urine concentrate a concentrated PMU retentate obtained from PMU by membrane filtration methods known per se. The solids content in the retentate and its composition may vary depending on the PMU used and the membrane used for membrane filtration, e.g. its pore size, and the filtration conditions, respectively. For example, it is possible to achieve almost no loss of the concentration of estrogen content in the PMU retentate using nanofiltration membranes, and at the same time remove up to 50 wt.% of the low molecular weight PMU content. PMU retentate that is concentrated in a ratio of up to about 1: 10, for example in a ratio of about 1: 7, may be used for the process of the present invention, whereby its volume may be concentrated to about 1/10, for example about 1/7, of the original PMU volume.

Adsorption resin:

the polymeric adsorbent resins which can be used in process step a) are semipolar, in particular nonionic semipolar, polymeric adsorbent resins according to WO 98/08526. The polymeric adsorbent resins which can be used as adsorbent material in the process according to the invention are preferably porous organic nonionic polymers which, in contrast to the nonpolar hydrophobic polymeric adsorbent resins, have a moderate polarity (═ e.g. the dipole moment of the active surface of the resin in the range from 1.0 to 3.0, in particular from 1.5 to 2.0 debye) and have a slightly hydrophilic structure, for example polycarbonate resins. It is suitable to use a macroporous semipolar resin, preferably having a macroreticular structure, with an average pore diameter of from 50 to 150 angstroms, preferably from 70 to 150 angstromsIn the range of 100 angstroms and has a specific surface area of 300 to 900m²G, preferably from 400 to 500m²In the range of/g. Particularly suitable are indicated to be macroporous, crosslinked, aliphatic polycarbonate resins, in particular crosslinked polyacrylate resins such as Amberlite XAD-7(HP type) from Rohm and Haas, which are nonionic, semi-polar adsorption resins.

Other adsorbent resins than the preferred adsorbent materials may be used. Suitable as the adsorbent resin herein are not only nonpolar, semipolar, but also polar adsorbent resins. The amount of urine that can be pumped into the adsorption material is first determined by the respective adsorption capacity of the adsorption resin. Examples of adsorbent resins which may be used are commercially available types, such as polymeric Amberlite adsorbent materials having basic building blocks of styrene divinylbenzene (e.g. XAD-1180, XAD-2, XAD-16), polymeric Amberlite adsorbent materials having basic building blocks of acrylates (e.g. XAD-7) or those having nitrogen and oxygen containing highly polar basic structures (e.g. XAD-12). Other adsorbent resins are Dowex-resins (copolymers of styrene and divinylbenzene), such as Dowex 112, Dowex Optipore V493; lewatit (cross-linked polystyrene), such as Lewatit OC 1064, Lewatit OC 1066 or Lewatit OC 1163, polyamine anion exchange resins, such as Dowex-resins. Advantageous adsorption resins are in particular XAD-7(HP type), XAD-16(HP type), XAD 118 and Dowex Optipore, preferably Dowex Optipore V493 and Lewatit OC 1064, OC 1066 and OC 1163.

Method step a):

adsorption of conjugated estrogens on polymeric adsorbent resins can be carried out according to WO 98/08526, also in the process according to the invention, by bringing the optionally pre-processed PMU or its retentate into contact with the adsorbent resin by introducing urine into a reactor containing the adsorbent resin and maintaining contact with the adsorbent resin therein for a time sufficient for the estrogenic component to be adsorbed. The adsorption resin loaded with the mixture of conjugated estrogens can be separated from the remaining urine after successful adsorption of the conjugated estrogens on the polymeric adsorption resin in a manner known per se. It is desirable that the urine is passed through a column containing an adsorbent resin at such a flow rate that the contact time is sufficient for adsorption of the estrogenic component. It is suitable, for example, for the flow-through rate to correspond to a throughput of 3 to 10 (preferably 5 to 7) parts by volume of PMU per 1 part by volume of adsorption resin per hour. It is desirable that the rate of passage of urine through the reactor can be controlled by processing at a slight overpressure or underpressure. The amount of polymeric adsorbent resin applied may vary depending on the type of adsorbent resin used and the solids content in the urine, respectively. In the case of PMUs, it is possible, for example, to load a volume of adsorbent resin, for example a cross-linked aliphatic polycarbonate-adsorbent resin, with up to 80, preferably 30 to 50, volume parts of the preconditioned PMU, without a significant amount of estrogen being detected in the urine effluent. In the case of PMU-concentrate or PMU-retentate, the loading capacity of the adsorbent resin decreases, of course, with the degree of concentration. The crosslinked aliphatic polycarbonate-adsorbing resin can thus be loaded, for example, with 1 part by volume of urine in an amount corresponding to 20 to 80, preferably 30 to 50 parts by volume of PMU.

Method step b):

the polymeric adsorbent resin loaded with the mixture of conjugated estrogens is washed in process step b) with washing water adjusted to a pH-range of at least 12.0, in particular from 12.5 to 14, preferably from about 12.5 to 13.5. As washing water, an aqueous solution of an inert alkaline substance dissolved in urine can be used, which is sufficient to bring the pH to at least 12.5. As the water-soluble basic substance inert to the polymeric adsorbent resin, a water-soluble inorganic base such as an alkali metal hydroxide or an alkaline earth metal hydroxide, particularly sodium hydroxide is preferable. Suitably, the wash water contains only about the amount of alkaline material required to achieve the desired pH-value, preferably about pH 13. The amount of wash water is selected such that it is sufficiently high to remove phenolic urine contents without this being accompanied by washing off valuable amounts of conjugated estrogens. It has been shown to be suitable, for example, to use from 2 to 10, in particular from 4 to 6, column volumes of washing liquid per column volume of adsorbent resin. The washing water is here suitably passed through the reactor containing the adsorption resin at a flow rate of from 3 to 10, preferably from 5 to 7, parts by volume of washing water per 1 part by volume of adsorption resin per hour.

Method step c) -intermediate washing steps:

the polymeric adsorbent resin loaded with the mixture of conjugated estrogens in process step a) is washed with water in an intermediate washing step after process step b). The amount of wash water is selected such that the eluate obtained in the last process step e) contains a mixture of conjugated estrogens which meets the requirements for the highest content of free estrogens and can thus be used in the pharmaceutical industry as active substance component. It has been shown to be appropriate to use, for example, from 1 to 8, preferably from 1 to 4, column volumes of wash water per column volume of adsorbent resin. The washing water is passed through a reactor containing the adsorption resin, expediently at a flow rate of from 3 to 10, preferably from 5 to 7, parts by volume of washing water per 1 part by volume of adsorption resin per hour.

In an advantageous embodiment of the process according to the invention, the intermediate washing step is carried out at a temperature below room temperature, in particular at a temperature between 0 ℃ and 10 ℃, since it was found that the loss of hormones or active substances which may be accompanied by conditions by additional intermediate washing steps can be significantly reduced. Room temperature is what is generally considered ambient temperature, for example temperatures between 20 ℃ and 30 ℃ are described. It is highly suitable that the process is carried out at a temperature of practically 0 ℃ or approximately 0 ℃. In practice, it is therefore advisable to operate at temperatures slightly above 0 ℃ and to maintain the stated temperature range by corresponding measures. For this purpose, conventional measures can be used for reducing the temperature, for example the use of cooled reactors, cooled materials and/or cooled feedstocks such as PMUs. Temperatures of 0 ℃ or close to 0 ℃ are understood from the practical standpoint to be from about 0 ℃ to about 5 ℃, in particular from about 0 ℃ to about 3 ℃.

In order to maintain as little possible hormone loss in the intermediate washing step as possible, the alkalinized washing water used in the intermediate washing step and/or also in process step b) is pre-cooled to a temperature below room temperature, in particular between 0 ℃ and 10 ℃, according to this variant. Further suitable or preferred temperature ranges are temperatures from about 0 ℃ to about 5 ℃, in particular from about 0 ℃ to about 3 ℃ as listed before. Preference is given to operating at a temperature of 0 ℃ or close to 0 ℃, i.e. preferably the basified wash water used in the intermediate washing step and/or also in process step b) is pre-cooled to a temperature slightly above 0 ℃. The already completed cooling of the adsorption resin is precooled or maintained by the use of cooled, alkalinized washing water in method step b), for example in order to avoid an undesired renewed heating up when cooled washing water for the intermediate washing step is added. It is therefore preferred that both the intermediate washing step and the preceding process step b) are carried out in the same temperature range, for example at a temperature below room temperature, in particular between 0 ℃ and 10 ℃ or preferably in the temperature range as given above.

In the aforementioned variants of the invention which are carried out at temperatures below room temperature, it is desirable that all the devices used, such as the reactor for carrying the polymeric adsorbent resin or the reactor already filled with polymeric adsorbent resin and/or the PMU used, can be precooled to temperatures below room temperature, in particular to temperatures between 0 ℃ and 10 ℃ or within the above-listed preferred maintained temperature ranges.

Method steps d), e) and f) according to the invention:

the washed adsorption resin loaded with the mixture of conjugated estrogens is then treated with an amount of eluent sufficient for eluting the mixture of conjugated estrogens, and in process step e) an eluate of the natural mixture of conjugated estrogens containing PMU is obtained and optionally concentrated, according to the description of process step d) of the present invention. If the eluate obtained, optionally concentrated, is a biphasic mixture, the aqueous phase of the eluate is separated in process step f) in order to obtain an aqueous phase containing a natural mixture of conjugated estrogens from the PMU, which may optionally be further concentrated.

Regeneration of adsorbent resin column

For the regeneration of the column, first 1 to 4, in particular 2 to 3 column volumes of an ethanol/water mixture containing 50% ethanol, adjusted to a pH of 13, are used per column volume of the adsorption resin, then the corresponding volume of 10% aqueous sodium citrate solution and finally the corresponding volume of distilled water are used. The entire regeneration process is carried out at a temperature of 40 ℃ to 45 ℃. The column may be loaded and regenerated repeatedly a number of times, for example up to 40 times.

Example 1:

comparative examples corresponding to the processes of WO 98/08526 and WO 03/048183

Examples 2a, 2b and 3 (method according to the invention)

Example 2 a: elution with 50% by volume ethyl acetate at room temperature

Example 2 b: eluting with 50 vol.% ethyl acetate at 45 deg.C

Example 3: eluting with a single phase ethyl acetate-containing eluent mixture at 40 deg.C

a) Adsorption of the estrogenic component of PMU on a semipolar polyacrylate-adsorbent resin (used in all examples)

A column height of 330 mm with a diameter of 40 mm was prepared using 200 ml of a semipolar polyacrylate-adsorbent resin swollen in water (Amberlite XAD-7(HP type) from Rohm and Haas, particle size 0.3 to 1.2 mm, dipole moment 1.8 Debye, average pore diameter 80 angstroms, specific surface area about 450m²Dry,/g) loading. 7 l (═ 35 column volume, abbreviated hereinafter as BV) of PMU filtered through an ultrafiltration device were passed through the column at room temperature with a flow rate of on average 16.7 ml/min (═ 5BV/h) in example 1, 40 ml/min (═ 12BV/h) in example 2a and 24.4 ml/min (═ 7.3BV/h) in examples 2b and 3, the conjugated estrogen content of the PMU (calculated as the sum of estrogen sulfate, equilenin sulfate and 17- α -dihydro-equilenin) and the cresol content being determined by means of HPLC (see table I for values).The estrogenic component of PMU will be completely adsorbed on the semipolar adsorption resin column thus loaded. The urine effluent was tested for conjugated estrogen content by HPLC and indicated virtually no estrogen. Discarding the urine flow.

b) Washing of loaded adsorbent resin column (for all examples)

The loaded adsorption resin column was washed with 1.0 liter (═ 5BV) of aqueous sodium hydroxide solution having pH-13. For this purpose, the alkaline wash water was passed through the column with a flow rate on average of 16.7 ml/min (═ 5 BV/h). The effluent washing liquid was tested for its content of conjugated estrogens and cresols by means of HPLC. This test showed that less than 5% of all estrogen loaded on the column was washed out during the wash.

c) Intermediate washing step (for all examples)

The loaded adsorption resin column was washed with 600 ml of water (═ 3 BV). Here neutral wash water was passed through the column at a flow rate of 16.7 ml/min on average (═ 5 BV/h). The effluent washing liquid was tested for its content of conjugated estrogens and cresols by means of HPLC. This test shows that only a very small fraction (at most about a few percent) of the total estrogen loaded on the column is washed out during the washing.

d) And e) dissociating the conjugated estrogens from the washed adsorbent resin column

Example 1 comparative example

1.1 l (═ 5.5BV) of the eluent (ethanol/water 30: 70) was passed through the column preheated to 45 ℃ using an average flow rate of 8.2 ml/min (═ 2.4 BV/h). The eluate which flows out is collected in 6 fractions E1 to E5. The fractions were each 200 ml (═ 1 column volume) or 100 ml (═ 0.5 column volume for E6) and the content of conjugated estrogens and cresols therein was determined by means of HPLC (see table I for values).

The first four fractions contained about 80 to 98% of the total bound estrogen adsorbed by the column. The subsequent fractions contain only small amounts of conjugated estrogens. The fraction containing the major amount of conjugated estrogens is an extract suitable for further processing in galens.

Example 2:

elution with 50 vol.% ethyl acetate at room temperature (example 2a)

Elution with 50 vol.% ethyl acetate at 45 deg.C (example 2b)

900 ml (═ 4.5 column volumes) of biphasic eluent (50: 50 ethyl acetate-water mixture) was passed through the column at the elution temperature described above, using an average flow rate of 16.7 ml/min (═ 5.0 BV/h). The eluate was collected in 9 fractions E1 to E9. The fractions were each 100 ml (═ 0.5BV), and the eluate obtained starting from the fourth fraction was a two-phase mixture which, for the analysis of the respective extracts, was partitioned into an aqueous phase and an organic phase (E4 to E9, respectively denoted solvent by LM and aqueous phase by W). All fractions will be tested for their conjugated estrogens and cresol content by HPLC (see table I for values). The content of unbound lipophilic compounds, such as 7-hydroxy-4' -methoxyisoflavone, will also be determined by means of GC for certain fractions (see Table II for values).

The first three poles contained only trace amounts of estrogen. The aqueous phase of the subsequent fractions 4 to 8 contains about 80 to 98% of the total amount of conjugated estrogens adsorbed by the column. The subsequent fractions contained only very small amounts of estrogen. The fraction containing the major amount of conjugated estrogens is an extract suitable for further processing in galens.

In order to control the thoroughness of the elution according to the invention with the ethyl acetate-water mixture, a known extraction with a single-phase ethanol-water mixture (30: 70) was then connected after the extraction.

By subsequent ethanol elution, a significant amount (equivalent to about 6.8% of the total loading) of conjugated estrogens still eluted in example 2 a. The amount of conjugated estrogens remaining on the column that can only be eluted by ethanol can be reduced to less than 1% by increasing the elution temperature to 45 ℃.

Example 3:

eluting with a single phase eluent mixture containing ethyl acetate at 40 deg.C

1.0 l (═ 5BV) of single phase eluent (a 1: 1.4 volume ethyl acetate-water-acetone mixture) was passed through the column at a temperature of 40 ℃ with an average flow rate of 16.7 ml/min (═ 5.0 BV/h). The eluate was collected in 13 different amounts of fractions. The first fraction was 100 ml (═ 0.5BV), the next 10 fractions were 50 ml each (═ 0.25BV), and the last two fractions were 200 ml each. The wash water after removal from the column surprisingly already separated into two phases in the eluate (starting from fraction 4). All fractions were checked for their conjugated estrogens and cresol content by HPLC (see table II for values).

The first fraction contained only trace amounts of estrogen. The subsequent fractions 2 to 7 contained about 80 to 98% of the total bound estrogen adsorbed on the column. The final fraction contained only very small amounts of estrogen. The fraction containing the major amount of conjugated estrogens is an extract suitable for further processing in galens.

The eluates E7 to E13 did not separate into two phases until after about 24 hours. The eluates E1 to E7 were combined and processed as follows: the eluates E1 to E7 were combined with a 500 ml separatory funnel to obtain about 350 ml of solution. No phase separation occurred immediately, but a small amount of about 10 ml of organic phase was visible at the upper end of the separatory funnel after standing for one weekend. The phases were tested for their content of conjugated estrogens and cresols by means of HPLC. After phase separation, the aqueous phase is concentrated in a rotary evaporator at 70 ℃ until the TS is 8-10% (here 8.16% TS, see Table I for values)

Regeneration of the adsorbent resin column (for all examples)

For regeneration, the column was first washed with 400 ml of an ethanol/water mixture containing 50% ethanol adjusted to pH 13, then with 400 ml of a 10% aqueous solution of sodium citrate and finally with 400 ml of distilled water. The entire regeneration process is carried out at a temperature in the range of 40 ℃ to 45 ℃. The column may be loaded and regenerated repeatedly a number of times, for example up to 40-times.

The TS content in weight% and the content of conjugated estrogens and cresol as determined by HPLC (CE calculated as the sum of the estrogen sodium sulfate, the equestrone sodium sulfate and 17- α -dihydro-equestrone) are shown in the table below for the fractions obtained in the elution containing the major amount of conjugated estrogens, respectively. The yields of extraction are also listed in numerical form.

Table I: comparing the methods according to the composition of the fraction containing the major amount of conjugated estrogens

Determined value of content by calculation

For example, during the experiment of example 1, i.e. in the case of ethanol elution, a portion of the isoflavonoid, e.g. horse alcohol, is washed out with an alkaline wash, and a portion of the isoflavonoid, e.g. 7-hydroxy-4' -methoxyisoflavone, also enters the eluate and can only be isolated by subsequent extraction. This is also similar for other non-conjugated lipophilic compounds, such as free steroid hormones. In contrast, the elution method according to the invention enables the isoflavonoid and free steroid still present in the column to enter in major portions into the water-immiscible solvent phase of the optionally concentrated eluate, while up to about 98% of the conjugated estrogens is located in the aqueous phase of the eluate which requires further processing. The aqueous phase is free of unbound steroid hormones and is highly depleted of isoflavonoids and similar unbound lipophilic compounds. This distribution can be shown in the following table by GC-analysis of the aqueous and organic phases of the eluate 4 from example 2 a.

Table II: distribution of conjugated estrogens, free steroid hormone and 7-hydroxy-4' -methoxyisoflavone (as an example of isoflavonoids) in the aqueous and organic phases of the eluent of example 2a, 4, calculated by GC-analysis (the aqueous phase is approximately concentrated 1.8 times before the test):

Claims (24)

1. A process for obtaining natural mixtures of conjugated estrogens from the urine of pregnant mares, which comprises

a) Urine which is urine from which mucus material and solid matter have not been removed or removed, a concentrated urine retentate obtained by membrane filtration of said urine, is contacted with a polymeric adsorbent resin in an amount sufficient to adsorb a mixture of conjugated estrogens contained in the urine, and the polymeric adsorbent resin loaded with the mixture of conjugated estrogens is separated from the remaining urine, and

b) the polymeric adsorbent resin loaded with the mixture of conjugated estrogens is washed with a wash water having a pH adjusted to at least 12.0, and

c) optionally performing an intermediate washing step in which the polymeric adsorbent resin loaded with the mixture of conjugated estrogens is washed with water, and

d) contacting the washed adsorbent resin with an eluent in an amount sufficient to dissociate the adsorbed mixture of conjugated estrogens, and

e) separating the eluate of the natural mixture containing conjugated estrogens from the adsorption resin and optionally concentrating,

it is characterized in that the preparation method is characterized in that,

the obtained natural mixture of conjugated mare's estrogen is depleted in phenolic urine content and non-conjugated lipophilic compounds including non-conjugated flavonoids, non-conjugated isoflavonoids, non-conjugated norisoprenoids, non-conjugated steroids, and in process step (d) a single-phase or two-phase mixture is used as eluent, which contains

(i) Water, optionally adjusted to a pH in the alkaline range,

and

(ii) at least one organic solvent which is immiscible or nearly immiscible with water and is selected from among the linear, branched or cyclic C's, suitable for eluting the non-conjugated lipophilic compounds selected from the list cited above₄-C₁₀Alcohol, C₁-C₁₀Esterified acid, C₃-C₁₀Aldehydes, C₄-C₁₀Ketones, C₃-C₁₀Ethers, C₃-C₆Nitriles and C₁-C₃Halogenated alkanes, as well as mixtures of these solvents,

and optionally

(iii) At least one water-miscible organic solvent selected from the group consisting of water-miscible ethers, lower alkanols and lower aliphatic ketones, and mixtures of these solvents,

and is

If the eluate obtained in process step e), which is optionally concentrated, is a two-phase mixture, process step e) is carried out

f) The aqueous phase of the eluate obtained, consisting of two phases, is separated and the aqueous phase of the natural mixture containing conjugated estrogens is obtained and optionally concentrated.

2. The process according to claim 1, wherein a single-phase or two-phase mixture containing

(i) Water, optionally adjusted to a pH in the alkaline range, and

(ii) at least one organic solvent immiscible or practically immiscible with water suitable for eluting the non-conjugated lipophilic compounds including non-conjugated flavonoids, non-conjugated isoflavonoids, non-conjugated norisoprenoids, non-conjugated steroids, said organic solvent being chosen from C, in linear, branched or cyclic form, and C₄-C₁₀Alcohol, C₁-C₁₀Esterified acid, C₃-C₁₀Aldehydes, C₄-C₁₀Ketones, C₃-C₁₀Ethers, C₃-C₆Nitriles and C₁-C₃Halogenated alkanes, and mixtures of these solvents, and

(iii) at least one water-miscible organic solvent selected from the group consisting of water-miscible ethers, lower alkanols and lower aliphatic ketones, and mixtures of these solvents.

3. The process according to claim 1 or 2, wherein the water-immiscible or almost immiscible organic solvent (ii) suitable for eluting the non-conjugated lipophilic compound is selected from acetic acid-C₁-C₄Alkyl esters, butanol, cyclohexanol, hexanol, diethyl ether, dichloromethane, methyl tert-butyl ether and mixtures of these solvents.

4. A process according to claim 3, wherein the process is adapted for elution of unbound lipophilic substancesThe water-immiscible or virtually immiscible organic solvent (ii) of the compound is selected from acetic acid-C₁-C₄-an alkyl ester.

5. The process according to claim 4, wherein acetic acid-C₁-C₄-the alkyl ester is selected from the group consisting of: ethyl acetate, isopropyl acetate, or a mixture thereof.

6. The process according to claim 1 or 2, wherein the water-miscible organic solvent (iii) is selected from acetone, ethanol and tetrahydrofuran and mixtures of these solvents.

7. The process according to claim 6, wherein the water-miscible organic solvent (iii) is acetone and/or ethanol.

8. The process according to claim 7, wherein the water-miscible organic solvent (iii) is acetone.

9. The process according to claim 1 or 2, wherein the water-immiscible or almost immiscible organic solvent (ii) suitable for eluting the non-conjugated lipophilic compound is acetic acid-C₁-C₄-alkyl esters, and the water-miscible organic solvent (iii) is acetone and/or ethanol.

10. The process according to claim 9, wherein acetic acid-C₁-C₄-the alkyl ester is selected from the group consisting of: ethyl acetate, isopropyl acetate, or a mixture thereof.

11. The method according to claim 1 or 2, wherein the water-immiscible or almost immiscible organic solvent (ii) suitable for eluting the non-conjugated lipophilic compound is ethyl acetate and the water-miscible organic solvent (iii) is acetone.

12. The process according to claim 1 or 2, wherein the volume ratio of the single-phase or two-phase mixture of water (i) and the water-immiscible or virtually-immiscible organic solvent (ii) used as eluent in process step (d) is in the range from 5: 1 to 1: 5.

13. The process according to claim 12, wherein the volume ratio of the single-phase or two-phase mixture of water (i) and the water-immiscible or virtually immiscible organic solvent (ii) used as eluent in process step (d) is in the range from 2: 1 to 1: 2.

14. The process according to claim 1 or 2, wherein the volume ratio of the single-phase or two-phase mixture of water (i) and water-miscible organic solvent (iii) used as eluent in process step (d) is in the range from 4: 1 to 1: 5.

15. The process according to claim 14, wherein the volume ratio of water (i) and water-miscible organic solvent (iii) of the single-phase or two-phase mixture used as eluent in process step (d) is in the range from 2: 1 to 1: 3.

16. The process according to claim 1 or 2, wherein the volume ratio of the single-phase or two-phase mixture of water (i) and the water-immiscible or virtually-immiscible organic solvent (ii) to the water-miscible organic solvent (iii) used as eluent in process step (d) is in the range from 5: 1 to 1: 5.

17. The process according to claim 16, wherein the volume ratio of the single-phase or two-phase mixture of water (i) and the water-immiscible or virtually-immiscible organic solvent (ii) together relative to the water-miscible organic solvent (iii) used as eluent in process step (d) is in the range from 2: 1 to 1: 2.

18. The process according to claim 9, wherein the water of the single-phase or two-phase mixture used as eluent in process step (d) (i) is compared with the acetic acid-C used as organic solvent which is immiscible or barely miscible with water (ii)₁-C₄-alkyl esters and a volume ratio relative to the acetone and/or ethanol used as water-miscible organic solvent (iii) in the range of 1: 1 to 1: 2.

19. The process according to claim 18, wherein the water of the single-phase or two-phase mixture used as eluent in process step (d) (i) is compared with the acetic acid-C used as organic solvent which is immiscible or barely miscible with water (ii)₁-C₄-alkyl esters and a volume ratio of 1: 1.4 with respect to acetone and/or ethanol used as water-miscible organic solvent (iii).

20. The process according to claim 18 or 19, wherein acetic acid-C₁-C₄-the alkyl ester is selected from the group consisting of: ethyl acetate, isopropyl acetate, or a mixture thereof.

21. The process according to claim 1 or 2, wherein a semipolar adsorption resin is used as the polymeric adsorption resin.

22. The process according to claim 21, wherein a non-ionic semi-polar adsorption resin is used as the polymeric adsorption resin.

23. A process according to claim 1 or 2, wherein elution of the natural mixture of conjugated estrogens from the washed adsorption resin is carried out in process step d) at a temperature in the range between 20 ℃ and 80 ℃.

24. A process according to claim 23, wherein elution of the natural mixture of conjugated estrogens from the washed adsorption resin is carried out in process step d) at a temperature in the range between 30 ℃ and 50 ℃.