ZA200402760B

ZA200402760B - Method for obtaining oestrogen from mare urine

Info

Publication number: ZA200402760B
Application number: ZA2004/02760A
Authority: ZA
Inventors: Helmer RASCHE Heinz; Rupp Olaf
Original assignee: Abbott Products Gmbh
Priority date: 2001-12-01
Filing date: 2004-04-08
Publication date: 2005-06-29

Description

HA0135 engl.

Solvay Pharmaceuticals GmbH 30173 Hannover }

Description

The present invention relates to obtaining a natural mixture of conjugated oestrogens from the urine of pregnant mares.

Oestrogens are used in medicine for hormone replacement therapy. In particular, oestrogen mixtures are used for the treatment and prophylaxis of the disorders of the climacteric period which occur in women after natural or artificial menopause. In these case, natural mixtures of conjugated oestrogens such as are found in the urine of pregnant mares have proved particularly effective and readily compatible. : The dissolved solids content in the urine of pregnant mares (= pregnant mares' urine, abbreviated hereafter as ~ "PMU") can naturally vary within wide ranges, and may generally lie in a range of 40 to 90 g dry matter per litre.

In addition to urea and other usual urine contents, phenolic constituents are contained in the solids content of the PMU in quantities of about 2 to 5% by weight relative to the dry matter. These phenolic constituents include cresols and dihydro-3,4-bis[(3-hydroxyphenyl) methyl] -2 (3H) -furanone, known as HPMF. These may be present in free or conjugated form. The PMU contains a natural mixture of oestrogens which ol is largely present in conjugated form, e.g. as sulphuric acid : semi-ester sodium salt (abbreviated hereafter as "sulphate . salt"). The content of conjugated oestrogens (calculated as

Co oestrogen sulphate salt) may be between 0.3 and 1% by weight, relative to the dry matter.

A suitable method is already known from WO 98/08526, . with which a largely cresol- and HPMF-free mixture which is depleted in phenolic urine contents and contains the natural : oestrogen content of the PMU practically completely can be’ obtained in a solid-phase extraction on a semipolar, in particular non-ionic semipolar, polymeric adsorption resin. oo This extract is suitable as a starting material for the . preparation of pharmaceuticals which contain the natural mixture of conjugated oestrogens as active substance } constituent . © In order to be able to be used as active substance constituent for pharmaceuticals, the natural mixture of" conjugated oestrogens which is obtained must however meet ‘ certain pharmaceutical specifications, e.g. the specification laid down in the USP (United States Pharmacopeia) or European

Pharmacopeia. Also, the content of free oestrogens relative to the dry matter should not exceed certain limit values.

Normally the pharmaceutical specification requirements set are met by the mixtures of conjugated oestrogens obtained from PMU in accordance with the method of WO 98/08526.

It has however turned out that the required pharmaceutical specifications, for example the above limit value of the maximum tolerable content of free oestrogens, can. often not be met when an "aged" urine, e.g. a PMU which has been stored or transported for relatively long periods and/or possibly at elevated temperatures, is used to obtain the natural mixture of conjugated oestrogens. This ageing of the PMU should probably be ascribed to the fact that depending on the storage and/or transport conditions the content of conjugated oestrogens can decrease over time or at elevated temperatures, while the content of undesirable free : oestrogens increases as a result. The original composition of the natural mixture of oestrogens can thus be adversely changed in aged urine compared with fresh urine. For the natural mixture of conjugated oestrogens obtained with the method of WO 98/08526, when the composition of an aged

Corrected sheet: 11 December 2007 starting product is possibly changed there is often the risk that the extract obtained can no longer meet the required pharmaceutical specifications, in particular the maximum tolerable content of free oestrogens. What is particularly disadvantageous about this is that batches obtained from aged

PMU with the active-substance extract, which is valuable in itself, of the mixture of conjugated oestrogens can then no longer be used for the production of a pharmaceutical preparation and therefore have to be discarded. The loss of a valuable natural pharmaceutical active substance, in view of the costly collection of PMU, which can only be carried out during certain periods of pregnancy, .and the logistical boundary conditions which this involves in practice and from economic points of view, is highly unsatisfactory.

It is therefore an object of the present invention to develop an industrially and economically optimum method for obtaining a natural mixture, depleted in phenolic urine contents, of conjugated oestrogens from PMU, in which even aged PMU, i.e. PMU which has been stored or transported for relatively long periods and/or at elevated temperatures, which may possibly have an elevated proportion of free oestrogens, can be used reliably as starting product without rejects, and in which in particular the natural mixture of conjugated oestrogens obtained from this PMU has good active- substance contents and meets the required pharmaceutical specification, in particular also with regard to the maximum tolerable content of free oestrogens.

A method has now been discovered with which in surprisingly simple manner a mixture of conjugated oestrogens can be obtained even from aged PMU, i.e. in particular from a

PMU possibly containing an increased proportion of free oestrogens, the mixture of conjugated oestrogens obtained having a high product quality and reliably meeting the requirements of pharmaceutical specification, in particular also with regard to the maximum tolerable content of free oestrogens.

The method according to the invention departs from the method described in WO 98/08526, which serves for obtaining a - natural mixture, depleted in phenolic urine contents, of conjugated oestrogens from the PMU. Accordingly, the ) invention relates to a method for obtaining a natural : mixture, depleted in phenolic urine contents, of conjugated ~ oestrogens from pregnant mares' urine, in which : a) a urine, which optionally represents a urine freed of mucilaginous substances and solids, a reduced concentrate of this urine or a reduced urine retentate obtained by nembrare filtration of this urine, is contacted with an amount of a semipolar polymeric adsorption resin sufficient for the adsorption of the mixture of conjugated oestrogens contained in the urine and a semipolar polymeric adsorption resin laden with the mixture of conjugated cestrogens is separated off from the rest of the urine, b) the semipolar polymeric adsorption resin laden with the mixture of conjugated oestrogens is washed with a washing water set to a pH range of at least 12.0, in particular of 12.5 to 14.0, c) the washed adsorption resin is contacted with an amount of an elution liquid, sufficient for the desorption of the mixture of conjugated oestrogens adsorbed thereon, which represents a water-miscible organic solvent from the group of water-miscible ethers, lower alkanols and lower aliphatic ketones or a mixture of the water-miscible organic solvent and water which has optionally been rendered alkaline, and an eluate containing the natural mixture of conjugated oestrogens is separated off from the adsorption resin and optionally reduced, the method according to the invention being distinguished with respect to the method of the prior art in that : between the process steps b) and c) an intermediate washing operation is carried out, in which the semipolar adsorption

Corrected sheet: 11 December 2007 resin laden with the mixture of conjugated oestrogens is washed with water.

The batch preparation, the known process steps a), Db) and c) and also the use of the eluate obtained in process step ¢), which contains a mixture of natural conjugated oestrogens, are already described generally in international patent application WO 98/08526 and are thus familiar to the person skilled in the art from this published patent application. The contents of this WO 98/08526 are also made the subject of the present application for the purposes of the disclosure. Further details on the general procedure and : materials which can be used are compiled in the examples section of the present application. For example, in accordance with WO 98/08526 semipolar, in particular non- ionic semipolar, adsorption resins may be used. Furthermore, according to the method of the present invention it is surprisingly also possible to use other adsorption resins together with the intermediate washing operation according to the invention, without the product quality or the pharmaceutical specification to be met being adversely affected. The adsorption resins usable within the scope of the present invention will be explained in greater detail further below in the description.

According to the invention, the semipolar adsorption resin laden with the mixture of conjugated oestrogens in process step a) is then washed with water in an intermediate washing operation following process step b). The amount of washing water is selected such that the eluate obtained in the subsequent process step c¢) has a mixture of conjugated oestrogens which meets the requirements of a maximum content of free oestrogens and thus can be used as active substance : constituent for pharmaceuticals. For example, the use of 2 to 8, preferably 3 to 5, bed volumes washing water per bed: volume adsorption resin has proved expedient. In this case, the washing water is expediently passed through a reactor containing the adsorption resin at a throughflow rate of 3 to

10, preferably 5 to 7, parts by volume washing water per 1 part by volume adsorption resin per hour.

It must be regarded as distinctly surprising that a supposedly simple washing of the adsorption resin with water performed between process steps b) and c), even when using aged PMU, in this way leads to the optimisation of the pharmaceutical specification of the natural mixtures of conjugated oestrogens obtained as active substance-extract, as was established according to the invention. In particular it is very surprising that the elevated proportion of free oestrogens possibly contained above all in aged PMU can be reduced reliably such that a mixture of natural conjugated oestrogens can be obtained as eluate in process step c) which meets the stringent requirements in terms of pharmaceutical specification, for example the requirements drawn up in accordance with the USP or the European Pharmacopeia, and in particular a high-quality and high-quantity active-substance extract containing natural conjugated oestrogen mixtures can be obtained even from aged PMU. These active-substance extracts reliably comply with the maximum tolerable maximum contents of free oestrogens independently of the age and origin of the PMU used, and thus prove an advantageous and valuable starting product for the preparation of pharmaceuticals. An undesirable and economically disadvantageous loss of valuable pharmaceutical raw materials, such as, in this case, the mixtures of natural conjugated oestrogens, can thus be avoided even under difficult conditions.

It has proved a further advantage in the method according to the invention that the eluate obtained in process step c¢), compared with the prior art, has, relative to the dry matter, an increased total hormone content. By means of this, a quality product is obtained which is distinctly improved e.g. in relation to the active-substance content.

: In an advantageous embodiment of the method according to the invention, the intermediate washing is carried out at temperatures below room temperature, particularly at temperatures between 0°C and 10°C, since it has been shown that losses of hormone or active substance possibly due to the additional intermediate washing operation can be considerably reduced. Usually the ambient temperature is : regarded as "room temperature", e.g. the term designates a temperature of between 20° and 30°C. It is very expedient to perform the method at temperatures of actually 0°C or approximately 0°C. In practice, it is therefore recommended

Lo operate at temperatures of close to but above 0°C and to ensure that the aforementioned temperature ranges are is maintained by suitable measures. Conventional measures for lowering the temperature may be used for this, e.g. the use of cooled reactors, cooled materials and/or cooled starting materials such as PMU. From practical points of view a - temperature range from 0°C to about 5°C, in particular of 0°C to about 3°C, can be considered as temperatures of 0°C or of approximately 06°C.

In order to keep any hormone losses during the intermediate washing as low as possible, according to this variant of the invention the washing water used in the intermediate washing operation and/or also the washing water which has been rendered alkaline used in process step b) will be precooled to temperatures below room temperature, in particular to temperatures between 0°C and 10°C. Further expedient or preferred temperature ranges are, as stated above, temperatures of 0°C to about 5°C, in particular of 0°C to about 3°C. Preferably operation is at temperatures of 0°C or of approximately 0°C, i.e. preferably the washing water used in the intermediate washing operation : and/or also the washing water which has been rendered alkaline used in process step b) is precooled to temperatures close to but above 0°C. By the use of cooled washing water which has been rendered alkaline in process step b), a type of precooling or maintaining of the cooling

Corrected sheet: 11 December 2007 ’ !

: of the adsorption resin which has already taken place is achieved, e.g. in order to prevent undesirable reheating of the water from taking place when using cooled washing water : for the intermediate washing. Preferably therefore the intermediate washing and the preceding process step b) are both carried out in the temperature range, e.g. at temperatures below room temperature, in particular at temperatures between 0°C and 10°C, or preferably in the same temperature ranges as stated above.

In the above variant of the invention, in which the method is carried out at temperatures below room temperature, it may be desirable to use all devices used, such as reactors for receiving the semipolar adsorption resin or reactors already containing same and/or the PMU used, precooled accordingly to temperatures below room temperature, in ” B particular to temperatures between 0°C and 10°C, or to the preferred temperature ranges given above.

Suitable adsorbents for use for the method according to the invention are polymeric adsorption resins. Particularly preferred adsorption resins are semipolar, in particular non- " ionic semipolar, polymeric adsorption resins. The semipolar polymeric adsorption resins usable as adsorbent in the method according to the invention are preferably porous organic non- ionic polymers, which in contrast to non-polar hydrophobic polymeric adsorption resins have an intermediate polarity (= e.g. with a dipole moment of the active surface of the resin in the range of 1.0 to 3.0, in particular 1.5 to 2.0, Debye) and a somewhat more hydrophilic structure, for example polycarboxylic acid ester resins. Expediently, macroporous semipolar resins with preferably macroreticular structure and with average pore diameters in the range of 50 to 150, preferably 70 to 100, Angstrom and a specific surface area in the range of 300 to 900, preferably 400 to 500, m?/g are used. Macroporous cross-linked aliphatic polycarboxylic acid ester resins, in particular cross-linked polyacrylate resins such as e.g. Amberlite XAD-7R from Rohm und Haas, which

Tepresent non-ionic semipolar adsorption resins, have proved : particularly suitable.

In addition to the adsorbents named as preferable, other ‘adsorption resins may also be used. Non-polar, semipolar and also polar adsorption resins are all suitable as adsorption resins. The amount of urine which can be pumped through the adsorber should in this case be determined beforehand by means of the respective adsorber capacity. Examples of : adsorption resins which can be used are commercially available types such as polymeric Amberlite adsorbents with styrene divinylbenzene parent structures (e.g. types XAD- 1180, XaD-2, XAD-4, XAD-16), with acrylic ester parent structures (e.g. XAD-7) or those with highly polar parent structures containing nitrogen and oxygen (e.g. XAD-12). - Other adsorption resins are Dowex resins (copolymers of styrene and divinylbenzene), such as Dowex 112, Dowex ‘Optipore, Dowex Optipore V 493; Lewatits (cross-linked polystyrenes), e.g. Lewatit OC 1064, Lewatit OC 1066 or

Lewatit OC 1163, polyamine anion-exchanger resins, e.g. Dowex resins. Advantageous adsorption resins are in particular ]

XAD-7, XAD-16 (Type HP), XAD 118 and Dowex Optipore, preferably as Dowex Optipore V 493, and Lewatits OC 1064, . OC 1066 and OC 1163.

The method according to the invention, as already described above in detail, has a number of advantages and improvements compared with the prior art. Thus the invention permits the use even of aged PMU, which may also have an elevated proportion of free cestrogens, without jeopardising pharmaceutical specifications which have to be met. The method according to the invention therefore also has economic advantages, since the risk of losing valuable active substances if the pharmaceutical specification is not met, e.g. in the case of contents of free oestrogens which are no longer tolerable, is considerably reduced. The method } according to the invention provides an improved-quality

Corrected sheet: 11 December 2007 active substance constituent with an increased hormone content relative to the dry-matter content. This active substance constituent is excellently suitable for the preparation of pharmaceuticals which contain a mixture of natural conjugated oestrogens as active substance.

The following examples are intended to explain the invention further, but without limiting its scope.

Examples

In the following examples there are given general operating procedures for obtaining active-substance extracts from PMU which contain the natural mixture of the conjugated oestrogens contained in the PMU and are largely depleted in phenolic urine contents. It is demonstrated how a quality extract with high active-substance contents can be obtained according to the invention even from aged PMU, which may have an elevated proportion of free oestrogens. "Method of WO 98/08526

For the method according to the invention, just as in accordance with WO 98/08526, the PMU as such, a concentrate obtained therefrom by reduction or a retentate obtained therefrom by membrane filtration can be used. The collected urine is first freed in known manner from mucilaginous substances and solids. Expediently, solids and mucilaginous substances are allowed to settle and are then separated off using known separation methods, for example decanting, separation and/or filtering. Thus the PMU may be passed for example through a known separating means, e.g. a separator, a filtration unit or a sedimenter. A sand bed for example may serve as separating means, or commercially available separators may be used, e.g. nozzle or chamber separators. If desired, a microfiltration unit or an ultrafiltration unit may also be used, and if these are used it is possible to achieve a largely bacteria-free and virus-free filtered PMU at the same time.

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

If a concentrated PMU retentate is to be used instead of the PMU, this can be obtained from the PMU by known membrane filtration. The solids content of the retentate and the composition thereof may vary depending on the PMU used and the membrane used for the membrane filtration, for example the pore width thereof, and also the conditions of filtration. For example, when using a nanofiltration membrane virtually loss-free concentration of the oestrogen content in the PMU retentate can be achieved while simultaneously removing up to 50% by weight of the low-molecular PMU contents. PMU retentates which have been concentrated up to a ratio of approximately 1:10, for example a ratio of approximately 1:7, and the volume of which can thus be reduced to approximately 1/10, for example approximately 1/7, of the original PMU volume can be used for the method according to the invention.

The semipolar polymeric adsorption resins usable in process step a) are porous organic non-ionic polymers, which in contrast to non-polar hydrophobic polymeric adsorption resins have an intermediate polarity (= e.g. with a dipole moment of the active surface of the resin in the range of 1.0 " to 3.0, in particular 1.5 to 2.0, Debye) and a somewhat more hydrophilic structure, for example polycarboxylic acid ester resins. Expediently, macroporous semipolar resins with preferably macroreticular structure and with average pore diameters in the range of 50 to 150, preferably 70 to 100,

Angstrom and a specific surface area in the range of 300 to 900, preferably 400 to 500, m?/g are used. Macroporous Cross- linked aliphatic polycarboxylic acid ester resins, in particular cross-linked polyacrylate resins such as e.g.

Amberlite XAD-7% from Rohm und Haas, have proved particularly suitable.

The adsorption of the conjugated oestrogens on the semipolar adsorption resin can be effected in accordance with

WO 98/08526 and also in the present method according to the invention by contacting the PMU or the retentate thereof with the adsorption resin, by introducing the urine into a reactor containing the adsorption resin and keeping it in contact ‘with the adsorption resin therein for a sufficient time for adsorption of the oestrogen content. Once adsorption of the conjugated oestrogens on the semipolar adsorption resin has taken place, the adsorption resin laden with the mixture of conjugated oestrogens can be separated off from the rest of the urine in known manner. Expediently, the urine can be passed through a column containing the adsorption resin at such a throughflow rate that the contact time is sufficient for adsorption of the oestrogen content. Suitable examples are throughflow rates which correspond to a throughflow of 3 to 10, preferably 5 to 7, parts by volume PMU/1 part by volume adsorption resin/hour. Expediently, the throughflow rate of the urine through the reactor can be controlled by operating at a slight excess pressure or partial vacuum. The amount of semipolar adsorption resin to be used may vary according to the type of adsorption resin used and the amount of the solids content in the urine. When using PMU, for example one part by volume adsorption resin, e.g. cross- linked aliphatic polycarboxylic acid ester adsorption resin, may be laden with up to 80 parts by volume pretreated PMU, without perceptible amounts of oestrogen being detectable in the urine flowing out. When using a PMU concentrate or PMU retentate, the loading capacity of the adsorption resin is of course reduced to the extent by which they are concentrated.

Thus for example 1 part by volume of cross-linked aliphatic polycarboxylic acid ester adsorption resin may be laden with an amount of urine corresponding to 20 to 80, preferably 30 to 50, parts by volume PMU.

The semipolar adsorption resin laden with the mixture of conjugated oestrogens is washed in process step b) with a washing water set to a pH range of at least 12.0, in particular of 12.5 to 14, preferably approximately 12.5 to 13.5. Washing waters which can be used are aqueous solutions of inert basic substances soluble in the urine, which are strong enough to achieve a pH value of at least 12.5.

Suitable water-soluble basic substances which are inert with respect to the semipolar polymeric adsorption resin are preferably water-soluble inorganic bases such as alkali metal or alkaline-earth metal hydroxides, in particular sodium hydroxide. Expediently the washing water contains only about the amount of basic substances which is required to achieve the desired pH value, preferably approximately pH 13. The amount of washing water is selected such that it is sufficient largely to remove phenolic urine contents without significant amounts of conjugated oestrogens being washed out with them. For example, the use of 2 to 10, in particular 4 to 6, bed volumes washing liquid per bed volume adsorption resin has proved expedient. In this case, the washing water is expediently passed through a cartridge containing the adsorption resin at a throughflow rate of 3 to 10, preferably to 7, parts by volume washing water/1l part by volume adsorbent /hour.

In process step c), the washed adsorption resin laden with the mixture of conjugated oestrogens is then treated with an amount of an elution liquid sufficient for elution of the mixture of conjugated oestrogens and an eluate containing the natural mixture of the conjugated oestrogens of the PMU

Claims

1. A method for obtaining a natural mixture, depleted in phenolic urine contents, of conjugated oestrogens from pregnant mares' urine, in which a) a urine,. which optionally represents a urine freed of mucilaginous substances and solids, a reduced concentrate of this urine or a reduced urine retentate obtained by membrane filtration of this urine, is contacted with an amount of a polymeric adsorption resin sufficient for the adsorption of the mixture of conjugated oestrogens contained in the urine and a polymeric adsorption resin laden with the mixture of conjugated oestrogens is separated off from the rest of the urine, b) the polymeric adsorption resin laden with the mixture of conjugated oestrogens is washed with a washing water which has been set to a pH range of at least 12.0, and c) the washed adsorption resin is contacted with an amount of an elution liquid, sufficient for the desorption of the mixture of conjugated oestrogens adsorbed thereon, which represents a water-miscible organic solvent from the group of water-miscible ethers, lower alkanols and lower aliphatic ketones or a mixture of the water- miscible organic solvent and water which has optionally been rendered alkaline, and an eluate containing the natural mixture of conjugated oestrogens is separated off from the adsorption resin and optionally reduced, characterised in that between the process steps b) and c¢) an intermediate washing operation is carried out, in which the polymeric adsorption resin laden with the mixture of conjugated oestrogens is washed with water.

2. A method according to Claim 1, characterised in that the intermediate washing is carried out at temperatures below room ; temperature.

. 3. A method according to Claim 1, characterised in that the intermediate washing is carried out at temperatures between 0°C and 10°C.

4. A method according to Claim 1, characterised in that the intermediate washing is carried out at temperatures close to but above 0°C.

5. A method according to one of the preceding claims, characterised in that the washing water used for the intermediate washing operation is precooled to temperatures below room temperature.

6. A method according to Claim 5, characterised in that the washing water used for the intermediate washing operation is precooled to temperatures between 0°C and 10°C.

7. A method according to Claim 5, characterised in that the washing water used for the intermediate washing operation is precooled to temperatures close to but above 0°C.

8. A method according to Claim 1, characterised in that the washing water which has been rendered alkaline which is used in process step b) is precooled to temperatures below room temperature.

9. A method according to Claim 8, characterised in that the washing water which has been rendered alkaline which is used in process step b) is precooled to temperatures between 0°C and 10°C. Bmended Sheet - 27-06-2005

10. A method according to Claim 8, characterised in that the washing water which has been rendered alkaline which is used in process step b) 1s precooled to temperatures close to but above 0°C.

11. A method according to one of the preceding claims, characterised in that the intermediate washing and the preceding process step b) are carried out at temperatures below room temperature.

12. A method according to Claim 11, characterised in that the intermediate washing and the preceding process step b) are carried out at temperatures between 0°C and 10°C.

13. A method according to Claim 11, characterised in that the intermediate washing and the preceding process step b) are carried out at temperatures close to but above 0°C.

14. A method according to one of the preceding claims, characterised in that the devices used and/or the PMU used are pre-cooled accordingly to temperatures below room temperature.

15. A method according to Claim 14, characterised in that the devices are reactors for holding the polymeric adsorption resin or reactors already containing such.

16. A method according to Claim 14 or Claim 15, characterised in that the devices and/or the PMU used are pre-cooled to temperatures between 0°C and 10°C.

17. A method according to Claim 14 or Claim 15, characterised in that the devices and/or the PMU used are pre-cooled to temperatures close to but above 0°C. Amended Sheet - 27-06-2005

18. A method according to one of the preceding claims, characterised in that semipolar adsorption resins are used as polymeric adsorption resins.

19. A method according to Claim 18, characterised in that the semipolar adsorption resins are non-ionic semipolar adsorption resins.

20. A method according to Claim 1, substantially as herein described with reference to any one of Examples 2 to 4. Amended Sheet - 27-06-2005