WO2002009732A2 - Verfahren und vorrichtung zur anreicherung und stabilisierung von konjugierten oestrogenen aus stutenharn - Google Patents
Verfahren und vorrichtung zur anreicherung und stabilisierung von konjugierten oestrogenen aus stutenharn Download PDFInfo
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- WO2002009732A2 WO2002009732A2 PCT/EP2001/008657 EP0108657W WO0209732A2 WO 2002009732 A2 WO2002009732 A2 WO 2002009732A2 EP 0108657 W EP0108657 W EP 0108657W WO 0209732 A2 WO0209732 A2 WO 0209732A2
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- urine
- cartridge
- adsorbent
- filter
- flow meter
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07J—STEROIDS
- C07J1/00—Normal steroids containing carbon, hydrogen, halogen or oxygen, not substituted in position 17 beta by a carbon atom, e.g. estrane, androstane
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07J—STEROIDS
- C07J75/00—Processes for the preparation of steroids in general
Definitions
- the present invention relates to a method and a device for the enrichment and stabilization of conjugated estrogens from the urine of pregnant mares on solid carriers in order to obtain suitable starting material for the production of pharmaceuticals which contain the natural mixture of these conjugated estrogens as active component.
- Estrogens are used in medicine for hormone replacement therapy.
- estrogen mixtures are used for the treatment and prophylaxis of the symptoms of menopause that occur in women after natural or artificial menopause.
- the solids content of the PMU contains phenolic constituents in amounts of approx. 2-5% by weight based on dry matter. These phenolic constituents include resole and the dihydro-3,4-bis [(3-hydroxyphenyl) methyl] -2 (3H) -furanone known as HPMF.
- the PMU contains a natural mixture of estrogens, which ches largely in conjugated form, for example as the sulfuric acid half-ester sodium salt (hereinafter abbreviated as "sulfate salt”).
- Extracts containing conjugated estrogens are usually obtained from the PMU by extraction with a polar organic solvent which is immiscible or only slightly miscible with water, such as, for example, ethyl acetate, n-butanol or cyclohexanol.
- a polar organic solvent which is immiscible or only slightly miscible with water, such as, for example, ethyl acetate, n-butanol or cyclohexanol.
- problems arise, such as strong foaming, sedimentation, emulsion formation and poor phase separation.
- Several extraction steps are generally required, which leads to losses and only a partial recovery of the estrogen content. To avoid these disadvantages, a number of solid phase extraction methods have therefore been proposed in the prior art.
- the steps upstream of the processing such as e.g. Ensuring an estrogen-friendly storage and handling of the collected urine at the collection point is of particular importance for the yield and quality of the raw material containing estrogen and the natural mixture of conjugated estrogens to be isolated therefrom.
- an optimization of the transport of the accumulated raw material containing estrogen from the collection point to the location of the actual processing and isolation of the conjugated estrogens should also be sought.
- PMU urine collection
- PMU urine collection
- Conjugated estrogens which are excreted in the same way as in pregnant mares' urine, are a complex mixture that contains sodium estrone sulfate, sodium equiline sulfate and other CE in particular.
- CE Conjugated estrogens
- the PMU is refurbished as quickly as possible.
- the urine begins to decompose rapidly.
- the urine turns dark, smells of ammonia and the CE content decreases very sharply.
- the rate of decomposition depends on the storage conditions and purity criteria.
- the collected urine (PMU) which is a large amount of liquid, has therefore had to be transported daily from the collection point to a central processing point in order to ensure the fastest possible processing.
- the method and the device are intended to enable a product-friendly, easy-to-use enrichment and stabilization of the conjugated estrogens from the PMU at the collection point of the urine, and to enable the estrogen-containing raw material provided in this way to be transported as efficiently as possible from the collection point to the site of the actual processing and isolation of the natural mixture of conjugated estrogens.
- the present invention therefore relates to a method for practical, decentralized, i.e. especially enrichment and / or pasture-related enrichment and stabilization of mixtures of conjugated estrogens (CE) from the urine of pregnant mares (PMU), which is characterized in that
- a predetermined maximum total amount of a collected, possibly previously sieved urine fluid from a storage vessel is pumped continuously or in discrete portions with a predetermined flow rate through an upright cartridge and the draining urine fluid is discarded, wherein
- the cartridge is filled with a (constantly) liquid-containing adsorbent suitable for adsorbing a predefined amount of the mixture of conjugated estrogens (CE) contained in the urine, and wherein
- the maximum total amount specified by the cartridge as the end point control for loading the adsorbent pumped urine is matched to the maximum loading capacity of the adsorbent for the conjugated estrogens (CE) contained in the urine.
- the term “decentralized” means here that the method at the respective location of the urine collection plant without special chemical-pharmaceutical facilities, i.e. , especially stable and / or pasture. Every type of closed column with lockable inlet and outlet and connection elements, e.g. Quick couplings, understood.
- the term “cartridge” thus also includes columns, such as these usually e.g. in the laboratory, in the technical center or in chemical operation, and which, as mentioned above, are equipped for use in accordance with the invention.
- a raw material or starting material is advantageously provided for the production of pharmaceuticals which contain the natural mixture of conjugated estrogens from the PMU as an active component, in which the natural estrogen mixture content of the PMU is practically complete
- Adsorbent carrier
- the cartridge can easily be replaced and replaced with a cartridge with an unloaded adsorbent.
- the cartridges with loaded adsorbent can still for some time on site at ambient temperature or, if desired, also with cooling down to about 4 ° C and under certain conditions also be stored deep frozen down to about - 20 ° C, e.g.
- the transport can take place at ambient temperatures or, as described above for storage, with cooling or deep-freezing.
- the actual work-up and isolation of the natural mixture of the conjugated estrogens (CE) can then take place in the central work-up point in the usual manner for the respective adsorbent, for example by washing and eluting the CE from the adsorbent and further processing of the eluate in the usual way.
- the eluate can be further concentrated in a manner known per se in order to obtain a concentrate which is largely or completely freed from organic solvent and is suitable for further galenical processing.
- an eluant-free solid mixture can also be produced by other suitable drying methods, such as, for example, spray drying, fluidized-bed drying, freeze-drying or vacuum drying. Both the eluate containing the estrogen mixture and a concentrate produced therefrom or spray-dried solid product can be incorporated into solid or liquid pharmaceutical preparations such as tablets, dragees, capsules or emulsions in a manner known per se.
- the invention also relates to a device which can be used in this method and which takes account of the simple conditions at the place of use in an expedient manner and ensures that the method is carried out safely and in a manner which is gentle on the product.
- This device according to the invention is suitable for practical, decentralized, ie in particular stable or pasture-rich, enrichment and stabilization.
- some further process parameters that are important for carrying out the process are explained in general.
- special constructive or apparatus configurations of devices which are suitable for carrying out the method according to the invention. rens are suitable, reference is made to the detailed description of the device according to the invention below, which, if appropriate, is to be used in addition to the following general description of the implementation of the method.
- the PMU collected can be used as such and the amount of PMU collected daily is initially kept in a storage container for this purpose. If necessary, the prior separation of coarse mechanical contaminants such as straw, hay or the like, e.g. by means of a simple coarse mesh sieve when filling the storage container with the freshly collected PMU. If desired, preservatives, germicides, bactericides and / or anthelmintics can be added to the urine collected in the storage container to reduce the number of germs and viruses.
- the PMU is regularly pumped from the storage container, depending on the amount of urine accumulated, for example once or twice a day, to the cartridge containing adsorbent, for example by means of a peristaltic pump.
- This cartridge usually has dimensions and weights that allow manual handling by normal strong people.
- it is advisable to dimension the internal dimensions of the cartridge for the adsorbent bed to be accommodated in such a way that it can hold about 30 to 50 liters of adsorbent and the PMU has a distance of about 80 to 120 cm from the inlet to the outlet , preferably must cover.
- the conjugated estrogens are adsorbed on the adsorbent by contacting the PMU with the adsorbent, by introducing the urine into an upright cartridge containing the adsorbent, optionally, for example, on the head or foot side, and therein during the flow for an adsorption of the estrogen. sufficient content is kept in contact with the adsorbent until the residual urine finally leaves the cartridge on the opposite side.
- the PMU is doing this so, for example on the head or foot side, introduced into the cartridge to ensure that the adsorbent is constantly surrounded by liquid in order to prevent the cartridge from running dry if possible.
- suitable loading speeds are e.g. 4.5 to 5.5 adsorbent bed volume / h.
- the maximum total amount of urine that can be passed through the cartridge depends on the adsorption capacity of the respective adsorbent and should be in the range of 20 to 60 adsorbent bed volumes, preferably 30 to 40 adsorbent bed volumes, for adsorbents suitable for the process. Usually, the maximum total amount of urine that can be passed over the cartridge for loading should therefore be in the range of 900 to 2,000 liters depending on the adsorbent and cartridge size.
- the respective final value of the flow rate is predefined for the operating personnel at the collection point for the respective cartridge type.
- a simple flow meter preferably e.g. a water meter. After the maximum load has been reached, a switch can optionally be made to a parallel second cartridge and the adsorption process can be continued there.
- the cartridge with the adsorbed CE is, if desired still washed, for example, with water and / or another suitable aqueous washing solution before the liquid supply is interrupted. Then the liquid outlet and the liquid inlet are closed and then the connection, for example a simple quick coupling, of the liquid inlet to the cartridge is released. The loaded cartridge can then be removed and stored in a suitable location until it is transported to the central processing point, and a fresh, unloaded cartridge can be connected to the liquid inlet.
- the cartridge can be loaded with PMU, e.g. before replacing a cartridge fully loaded with PMU or even before reaching the total loading capacity after any individual partial loading steps, if necessary with water and / or another suitable aqueous washing solution, e.g. a basic washing solution such as e.g. dilute aqueous sodium hydroxide solution (e.g. aqueous 0.5-2 N NaOH) to remove residual urine from the cartridge.
- a basic washing solution such as e.g. dilute aqueous sodium hydroxide solution (e.g. aqueous 0.5-2 N NaOH) to remove residual urine from the cartridge.
- the amount of washing solution is not critical and is preferably chosen so that it is sufficient to displace the residual urine from the cartridge without having to wash out significant amounts of conjugated estrogens.
- the wash water or the wash solution is expediently passed through the cartridge containing the adsorbent at a flow rate of 3 to 10, preferably 5 to 7, parts by volume of wash water per 1 part by volume of adsorbent / hour.
- the adsorbent loaded with CE in the cartridge can serve as raw or starting material for the isolation of pure CE for the production of medicaments containing the natural mixture of conjugated estrogens.
- the cartridge can be used for further storage after washing with water and / or washing solution.
- bilization of the adsorbed CE finally be rinsed with a preserving solution, for example a solution containing preservatives, a pH-adjusted aqueous solution and / or an aqueous salt solution, before the cartridge is released from the device and replaced by a fresh one.
- a preserving solution for example a solution containing preservatives, a pH-adjusted aqueous solution and / or an aqueous salt solution, before the cartridge is released from the device and replaced by a fresh one.
- Conventional preservatives and, for example, germicides, bactericides and / or anthelmintics can be used to prevent the column from becoming contaminated, in particular during storage or transport without cooling.
- a pH adjustment of the wash water will depend on the adsorbent used.
- salt solutions Variously concentrated aqueous solutions of inorganic salts can be used as salt solutions, examples of which are given in Table III.1.
- the use of salt solutions is particularly recommended when the cartridges loaded with CE are to be refrigerated or, in particular, stored deep-frozen.
- Preferred salt solutions are saline solutions with salt concentrations of about 10 to 35% by weight, preferably 25 to 33% by weight.
- the collected urine is preferably freed of slime and finely divided solids before it is pumped from the storage container to the cartridge. It is therefore expedient if the urine is first passed out of the storage container through one or more pre-filters before the urine is pumped over the cartridge.
- the PMU can be routed via at least one separation device known per se, for example via a filtration system with at least one depth filter or precoat filter.
- a depth filter with a sand bed or a commercially available filter cartridge can serve as the separation device, or commercially available precoat filters or plate filters, candle filters, filter bags or also filtration tubes can be used.
- the filters can be used individually or in any combination with one another, for example in series and / or in parallel become.
- filtration aids can be added to the urinary fluid (PMU) before filtration. Suitable filtration aids are, for example, those that bind calcium carbonate and / or muccine and thus improve urine filtration.
- a particle separator can also be connected downstream of the filter, for example in the case of a sand bed as a filter, a downstream sand separator.
- the principles or the design of filtration are known per se to the person skilled in the art. With regard to the object to be achieved by the invention, the usual principles of clarification filtration are therefore suitable for the use according to the invention.
- the clarifying filtration aims at the purification of the liquid phase, so that for them the purity of the filtrate is an essential evaluation parameter for the achievement of the process goal.
- the separation process is basically realized through the interaction between the filter medium and the suspension. This must be taken into account both when selecting the filter media, the process modeling and the process engineering design of filter devices. In most cases, the separated solid significantly influences the process. With a sufficient proportion of solid and its ability to form bridges (the particles are supported above the filter medium without clogging it), the filter cake that forms and grows continuously takes on the function of the filter medium.
- the structure of the filter cake determines the course of the process, while the actual filter medium only has a supporting function after an initial phase.
- the location of the solids separation is another essential feature for the differentiation of filtration processes.
- the solid is deposited on the surface of the filter medium by means of a sieve action, ie on account of the size relationships between solid particles and pores of the filter medium. In contrast, the solid particles into the filter medium and are deposited inside it.
- the adsorbents that can be used in the cartridge can in themselves be any inorganic or organic adsorbents that have sufficient adsorption capacity for the natural mixtures conjugated estrogens contained in the PMU.
- Polymeric adsorber resins, silica gel, RP silica gel or preferably semipolar polymeric adsorber resins are therefore suitable as adsorbents for use in the cartridge.
- RP silica gels reverse-phase silica gels
- silanized RP silica gels which contain n-octadecyldimethylsilyloxy, n-octyldimethylsilyloxy or dimethylhydroxysilyloxy radicals as hydrophobic functional groups are suitable.
- silanized silica gels with average grain sizes of 15 to 500 ⁇ m are suitable.
- Silica gel containing dimethylhydroxylsilyloxy radicals and having an average grain size in the range of 0.05-0.3 mm for example the "Silica gel 60 / dimethylsilane derivative" from Merck, has proven to be particularly useful.
- the urine fluid can expediently be passed through the cartridge containing the silica gel at such a throughput speed that the contact time is sufficient for the adsorption of the estrogen content.
- Throughput speeds are suitable, for example, which correspond to a throughput of 5 to 20 parts by volume of PMU per 1 part by volume of silica gel / hour.
- the adsorption is preferably carried out at room temperature.
- the flow rate of the urine liquid through the reactor can expediently be controlled by working at a slight excess pressure (regulated by the power of the pump).
- the amount of hydrophobized silica gel to be used can vary depending on the type of silica gel used and the amount of solids in the urine collected.
- prefiltered PMU that is, from which mucus and solids have been removed
- up to 80 parts by volume of pretreated PMU can be loaded onto a volume part of hydrophobized silica gel, for example, without noticeable amounts of estrogen being detectable in the urine fluid flowing off.
- Macroporous semipolar resins with a preferably acreticular structure and with average pore diameters in the range from 50 to 150, preferably 70 to 100 angstroms and a specific surface area in the range from 300 to 900, preferably 400 to 500, m / g are expediently used.
- Macroporous cross-linked aliphatic polycarboxylic acid ester resins in particular cross-linked polyacrylic ester resins such as Amberlite XAD-7 R from Rohm and Haas, have proven particularly suitable.
- the urine liquid can expediently be passed through the cartridge containing the adsorber resin at such a throughput speed that the contact time is sufficient to adsorb the estrogen content.
- Throughput speeds are suitable, for example, which correspond to a throughput of 3 to 10, preferably 5 to 7, parts by volume of PMU per 1 part by volume of adsorber resin / hour.
- the adsorption is preferably carried out at room temperature.
- the flow rate of the urine liquid through the reactor can expediently be achieved by working at a slight excess pressure (through Pump output regulated) can be controlled.
- the amount of semipolar adsorber resin to be used can vary depending on the type of adsorber resin used and the amount of solids in the collected liquid.
- a volume part of adsorber resin for example cross-linked aliphatic polycarboxylic acid ester adsorber resin, can be loaded with up to 80 volume parts of pretreated PMU without noticeable amounts of estrogen being detectable in the urine fluid flowing off.
- adsorber resins or types of silica gel can also be used. Both non-polar, semipolar and polar adsorber resins are suitable as adsorber resins.
- the amount of urine that can be pumped through the adsorber must be determined beforehand based on the respective adsorber capacity.
- usable adsorber resins are commercially available types such as polymeric Amberlite adsorbents with styrene-divinylbenzene backbones (for example types XAD-1180, XAD-2, XAD-4, XAD-16), with acrylic ester backbones (for example XAD-7) or those with highly polar backbones containing nitrogen and oxygen (eg XAD-12).
- Adorber resins are Dowex resins (copolymers of styrene and divinylbenzene), e.g. Dowex 112, Dowex Optipore, Dowex Optipore V 493; Lewatite (cross-linked polystyrenes), e.g. Lewatit OC 1064, Lewatit OC 1066 or Lewatit OC 1163, polyamine anion exchange resins, e.g. Dowex resins.
- Advantageous adsorber resins are in particular XAD-7, XAD-16 (type HP), XAD 118 and Dowex Optipore, preferably as Dowex Optipore V 493, and Lewatite OC 1064, OC 1066 and OC 1163.
- the invention also relates to a device which can be used in this method and which takes account of the simple conditions at the place of use in an expedient manner and ensures that the method is carried out safely and in a manner which is gentle on the product.
- An upright cartridge which is filled with an (always) liquid-surrounded adsorbent suitable for adsorbing a predefined amount of the mixture of conjugated estrogens (CE) contained in pregnant urine, and the cartridge either a) has a liquid inlet arranged on the foot side and has a liquid outlet arranged at the top or b) a liquid inlet arranged at the top and a liquid outlet arranged at the foot,
- the device according to the invention is intended for carrying out the above-described method for practical, decentralized enrichment and stabilization of conjugated estrogens (CE) contained in the PMU.
- CE conjugated estrogens
- the device can also contain two cartridges connected in parallel, which can be operated one after the other or alternately.
- the cartridge used in the device according to the invention can be dimensioned in a wide range in terms of height and diameter.
- the size and weight of the cartridge should be such that it can be loaded by a normal person, for example when replacing a loaded one Cartridges against fresh, empty cartridges, can still be handled and carried manually.
- the cartridge should therefore be dimensioned such that the cartridge has internal dimensions for the adsorbent bed which allow 30 to 50 l of adsorbent to be taken up, the internal height in particular being approximately in the range from 80 to 120 cm.
- the inside diameter of the cartridge will generally be 10 to 25 cm. The person skilled in the art will be able to dimension the cartridge precisely in order to ensure optimal flow conditions during operation.
- the cartridge used in the device according to the invention expediently consists of robust, hard-wearing and chemical-resistant materials as are usually used for the production of devices for the chemical industry, e.g. impact-resistant laboratory glass, plastic and / or metal such as Sheet steel.
- any simple and robust pumps can be used in the device according to the invention, e.g. Monopumps, peristaltic pumps or diaphragm pumps.
- a peristaltic pump has proven to be useful as a pump for the device according to the invention.
- any simple and robust flow meter can be used in the device according to the invention, e.g. Rotameter, rotary vane flow meter or inductive flow meter.
- a rotameter with a float has proven to be useful as a flow meter for the device according to the invention.
- any simple and robust flow meter such as water meters or inductive flow meters, can be used to measure the amount of urine fluid pumped through the cartridge.
- a convenient flow meter for the invented device according to the invention has proven, for example, a water meter.
- At least one or more prefilters are additionally interposed between the pump and the flow meter.
- the filters can be, for example, a depth filter with a sand bed or a commercially available filter cartridge, or a commercially available precoat filter or plate filter, candle filter, filter bag or also filtration tubes.
- a particle separator can optionally be connected downstream of the prefilter, for example in the case of a sand bed as a prefilter, a downstream sand separator.
- the tALters can be connected individually or in any combination with one another, for example in series and / or in parallel.
- the pre-filter is a module of two parallel pre-filters, which are operated individually in alternation and can also be exchanged for a fresh pre-filter while the device is in operation.
- the filter system consists of a module of a prefiltration unit connected in series, consisting of a prefilter (e.g. filter bag) and one or two deep bed filters and then two absolute filters (e.g. membrane filter) connected in parallel, the Absolute filters can be operated alternately and can be exchanged for a fresh filter even while the device is running.
- FIG. 1 shows a schematic device for stabilizing and enriching CE from PMU, serves to further explain the device according to the invention in a preferred embodiment.
- FIG. 2 A schematic representation of the procedure for a variant of the filtration of crude urine is shown in FIG. 2.
- the method and the device according to the invention have a number of advantages, of which the important ones are briefly mentioned below.
- the enrichment of the CE i.e. the adsorber columns can be loaded directly at the collection site. This eliminates the daily transport of large amounts of urine to a central processing point.
- the loading can take place continuously over a longer period of time up to several weeks until the adsorber column is saturated; e.g. in the adsorber resin XAD-7, the amount of urine adsorbed is approximately 35 bed volumes.
- the residual urine exempt from CE remains in place, where there are already suitable facilities for disposal.
- the loaded column is transported, i.e. only approx. 1/35 of the original weight of the urinary fluid. The transport can take place in larger time intervals.
- the transport can also take a long time, e.g. can take up to a few weeks. Due to the good stability of the CE on the adsorber column, no decomposition is to be expected. There is no need to treat large amounts of urine by means of a separator and ultrafiltration system, since the separation of any mucilages and sediments that may be present in the original urine via robust and maintenance-free precoat or depth filters (e.g. sand filters, filter cartridges, precoat filters, plate filters, candle filters, filter bags or filtration tubes) takes place, which can be easily replaced if necessary.
- precoat or depth filters e.g. sand filters, filter cartridges, precoat filters, plate filters, candle filters, filter bags or filtration tubes
- the filter system consists of a module of pre-filtration unit connected in series, consisting of pre-filter (e.g. filter bag) and one or two deep bed filters and then two absolute filters connected in parallel (e.g. membrane filter).
- pre-filter e.g. filter bag
- pre-filter e.g. filter bag
- absolute filters e.g. membrane filter
- the collected PMU is filled into a storage vessel via a funnel equipped with a sieve.
- the sieve has a coarse mesh size and is only used to remove coarse mechanical impurities such as straw, hay, etc.
- the method is explained below using an adsorber column (as a cartridge) with a semi-polar adsorber resin. If cartridges with other adsorbents are used, the handling and, in particular, the refurbishment of the loaded cartridge may depend on the type of the respective adsorbent.
- the loading process and the final washing of the cartridge at the urine collection point are essentially maintained as described under A) and B) or, if necessary, only slightly adapted to any special conditions on site or the respective adsorbent.
- the adsorption of the CE content of the PMU is carried out in an adsorption system as outlined in FIG. 1 by way of example.
- a total amount of PMU based on the cartridge or adsorber column capacity, is pumped at ambient temperature, if necessary after prior screening and if necessary filtration, in total or in parts of a predetermined flow rate via a column or cartridge filled with an adsorbent.
- the loaded adsorber column or cartridge is optionally washed with water or another aqueous washing solution, e.g. a basic washing solution such as in particular dilute sodium hydroxide solution (e.g. 0.5-2 N NaOH).
- a basic washing solution such as in particular dilute sodium hydroxide solution (e.g. 0.5-2 N NaOH).
- the wash water or the wash solution at ambient temperature with a predetermined flow rate of e.g. 5.5 bed volumes per hour also pumped through the column or cartridge.
- the washing liquid running off is discarded.
- the optional washing for example with or with another aqueous, optionally basic washing solution, such as in particular dilute sodium hydroxide solution, for example 0.5- 2 N NaOH
- Storage can take place at ambient temperatures, e.g. 15 to 30 ° C in a normal warehouse or in a cold room down to about 4 ° C. If the liquid in the adsorber column is protected against freezing, even at lower temperatures, e.g. be deep-frozen down to around 20 ° C.
- the loaded columns or cartridges are transported at regular intervals from a few days to a few weeks from the collection and storage location to a central processing point, where the chemical processing and isolation of the conjugated estrogens is guaranteed in a professional manner.
- the loaded columns or cartridges are worked up in a separator for the respective adsorbent of accompanying substances and isolation from CE usual ways.
- a semi-polar adsorber resin of the Amberlite type, eg XAD 7 the processing can be carried out as in the international patent application WO 98/08526.
- the elution liquid is passed through the column or cartridge preheated to 45 ° C. at a suitable flow rate.
- the eluate is collected in fractions.
- the individual fractions are examined for their content of oestrone sulfate salt, cresol and HPMF by means of HPLC.
- the first fraction is collected as long as the eluate appears colorless to pale yellow. This fraction regularly contains only traces of estrogen sulfate salt.
- the color of the eluate changes to an intense dark brown color.
- the subsequent 2 to 4 fractions then regularly contain approximately 80 to 98% of the total amount of conjugated estrogens adsorbed on the column.
- the remaining fractions contain only a small amount of estrogen sulfate salt. This is also clearly visible in the decrease in color intensity. If necessary, the residual fractions can also be further processed after the solvent content has been distilled off and further CEs can be isolated.
- TS dry matter, determined by HPLC
- the loaded columns or cartridges are regenerated in a manner customary for the respective adsorbent.
- a semipolar Amberlite type adsorbent resin e.g. XAD 7
- the regeneration can be carried out as in international patent application WO 98/08526.
- the column or cartridge is e.g. first washed with an ethanol / water mixture containing 50% by weight of ethanol adjusted to pH 12, then with 10% by weight aqueous sodium citrate solution and again with an ethanol / water mixture and finally with distilled water.
- the entire regeneration takes place for the exemplary adsorber resin at a temperature of 45 ° C, and here, too, the regeneration and washing solutions can be passed over the column or cartridge at the foot or head end.
- the column or cartridge can be loaded and regenerated several times, for example up to 40 times.
- the absorption was in each case in a semipolar XAD 7 adsorber resin (semipolar polyacrylic ester adsorber resin, ie Amberlite XAD-7 from Rohm and Haas, particle size 0.3 to 1.2 mm, dipole moment 1.8 Debye, average pore diameter 80 Angstrom, specific surface area approx. 450 m 2 / g dry) in a known 31-column.
- the estrogen content of the PMU was completely adsorbed on the semi-polar adsorber resin column loaded in this way. After loading the column with the mare's urine was washed with 3 l of water and the loaded resin was discharged from the column and divided into 10 samples of 300 ml and transferred to smaller columns. A sample was discarded.
- the elution was carried out in a manner known per se by first washing the loaded adsorber resin with an aqueous sodium hydroxide solution. After each washing step, the respective washing liquid was determined using HPLC examined for their content of estrone (as sulfate salt), equiline, cresol and HPMF.
- the water / ethanol mixture (30% by weight, pH 12) made alkaline by adding sodium hydroxide was used as the elution liquid and the resin was eluted at an elution temperature of 45 ° C.
- the eluate was collected in fractions and the individual fractions were again examined for their content of estrone, cresol and HPMF by means of HPLC.
- the first fraction was collected as long as the eluate appeared colorless to pale yellow. This fraction contains only traces of estrone (estrogen sulfate salt). After the first bed volume of eluate has run out, the color of the eluate changes to an intense dark brown color.
- the subsequent 2 to 4 fractions then contain approximately 80 to 98% of the total amount of conjugated estrogens adsorbed on the column.
- the remaining fractions for the regeneration of the resin (at 45 ° C. in the order given with 50% by weight ethanol with pH 12, 10% by weight sodium citrate and water) only contain a small amount of estrogen sulfate salt. This is also clearly visible in the decrease in color intensity.
- Tables 1.2 to 1.9 show the results for the eluation after one, two, five or eight weeks of storage.
- the adsorbed hormones CE
- the adsorbed hormones are usually desorbed quantitatively.
- quantitative desorption is only determined after 50% ethanol regeneration, although this is only the desorption of residual amounts of the CE and the majority has already been isolated in the actual desorption could be.
- a dependence of the desorption on storage time and temperature does not seem to exist or at best only to a limited extent, for example if optimal flow conditions in the small columns may not play a major role.
- the eluate is largely free of cresol and HPMF.
- care should be taken to ensure optimum column flow regardless of the storage time.
- the columns or cartridges should therefore always be under liquid, i.e. Avoid running dry.
- the HPMF and cresol contents in the eluate are very low in both cases.
- Table 1.10 shows all experiments in a clear context. The summation e.g. The estrone content of the eluate and regenerate is in all cases within approx. 1055 mg + 4% and thus corresponds to the expected accounting.
- resin After application to the column, resin is discharged and washed with 3 liters of water.
- the resin is divided into equal 300 ml portions and subjected to a column stability test.
- One resin package is eluted immediately, the others are stored at room temperature and in the refrigerator.
- the results of the bacterial count determinations are shown in Table II.
- the total bacterial count for a storage temperature of 4 ° C can be described as low.
- the column wash water has 1.2 to 4.6 • 10 6 germs, 80 to 170 germs were found in the eluate.
- the stored column adsorbents had not been washed or preserved beforehand, and were therefore exposed to an increased risk of nucleation from the outset.
- thorough washing of the loaded columns is therefore recommended at least with water, but advantageously also with additional stabilizing or preserving washing solutions, for example saline.
- temperatures below freezing can occur when columns or cartridges are loaded during storage or transport, with the undesirable consequence that the aqueous content of the columns or cartridges solidifies.
- Corresponding experiments suggest that the freezing process appears to have a negative effect on the elution of the thawed column or cartridge. If the column cannot be tempered, or if it is stored at low temperatures, to prevent freezing, it is recommended to carry out the last wash instead of with water using suitable cold mixtures.
- a saline solution was selected as an example for further experiments from the large number of possible cold mixtures, some of which are given by way of example in Table III .1. With the addition of 23 g NaCl per 100 g water, the freezing point is -21 ° C.
- the column loading was carried out in the usual way. After loading with urine, 3 bed volumes of water were rinsed, followed by 2 bed volumes of a 33% by weight NaCl solution. The column was then stored in a freezer at -19 ° C. for 9 days.
- Fresh urine of sufficient quality was collected in a stud farm. With three horses and 15 to 20 l urine / day, a total of about 150 to 200 were obtained over 10 days. A 5 1 adsorber column was therefore chosen for adsorption. Daily urine loading, except on weekends, was carried out in the experiment according to the following schedule:
- the column is moved from the bottom up.
- Fresh urine was placed on a 5 l adsorber column daily for a test period of 10 days.
- the total urine volume was 140 l with an average estrone or equiline content of 123 mg / l or 74 mg / l.
- the partly stirred urine of pregnant mares collected in barrels was pumped out of the barrels by means of a peristaltic pump over a 5 ⁇ bag (prefilter) into a container.
- a sediment of about 0.5% by weight remained in the container.
- FIG. 2 A schematic representation of the procedure for the filtration of raw urine is shown in FIG. 2.
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- Chemical & Material Sciences (AREA)
- Organic Chemistry (AREA)
- Health & Medical Sciences (AREA)
- General Health & Medical Sciences (AREA)
- Investigating Or Analysing Biological Materials (AREA)
- Medicines Containing Material From Animals Or Micro-Organisms (AREA)
- Steroid Compounds (AREA)
- Compounds Of Unknown Constitution (AREA)
- Organic Low-Molecular-Weight Compounds And Preparation Thereof (AREA)
- Treatment Of Liquids With Adsorbents In General (AREA)
- Solid-Sorbent Or Filter-Aiding Compositions (AREA)
- Peptides Or Proteins (AREA)
- Treatment Of Sludge (AREA)
- Medicines Containing Plant Substances (AREA)
- Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)
Abstract
Description
Claims
Priority Applications (18)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
SI200130560T SI1307210T1 (sl) | 2000-08-01 | 2001-07-26 | Postopek in naprava za obogatitev in stabilizacijo konjugiranega estrogena iz kobiljega urina |
HU0301283A HU228396B1 (en) | 2000-08-01 | 2001-07-26 | Method and device for concentrating and stabilising conjugated oestrogens from the urine of mares |
BRPI0107062-2A BR0107062B1 (pt) | 2000-08-01 | 2001-07-26 | Processo e dispositivo para o enriquecimento e estabilização de estrogênios conjugados de urina de égua |
MXPA02002835A MXPA02002835A (es) | 2000-08-01 | 2001-07-26 | Metodo y dispositivo para concentrar y estabilizar estrogenos de la orina de las yeguas. |
AU77553/01A AU785438B2 (en) | 2000-08-01 | 2001-07-26 | Method and device for concentrating and stabilising conjugated oestrogens from the urine of mares |
IL14887801A IL148878A0 (en) | 2000-08-01 | 2001-07-26 | Method and device for concentrating and stabilising conjugated oestrogens from the urine of mares |
CA2410654A CA2410654C (en) | 2000-08-01 | 2001-07-26 | Method and device for concentrating and stabilizing conjugated estrogens from mares' urine |
PL354451A PL202238B1 (pl) | 2000-08-01 | 2001-07-26 | Sposób i urządzenie do zatężania i stabilizacji koniugowanych estrogenów z moczu klaczy |
NZ518645A NZ518645A (en) | 2000-08-01 | 2001-07-26 | Method and device for concentrating and stabilising conjugated oestrogens from the urine of pregnant mares onto solid supports |
EEP200200170A EE04541B1 (et) | 2000-08-01 | 2001-07-26 | Meetod ja seade tiinete mrade uriinist konjugeeritud strogeenide kontsentreerimiseksja stabiliseerimiseks |
UA2002043558A UA79067C2 (en) | 2000-08-01 | 2001-07-26 | Method and device for concentrating and stabilising conjugated oestrogen mixtures from the urine of pregnant mares |
SK442-2002A SK287663B6 (sk) | 2000-08-01 | 2001-07-26 | Spôsob a zariadenie na obohacovanie a stabilizáciu konjugovaných estrogénov z moču kobýl |
EP01955372A EP1307210B1 (de) | 2000-08-01 | 2001-07-26 | Verfahren und vorrichtung zur anreicherung und stabilisierung von konjugierten oestrogenen aus stutenharn |
DE50110057T DE50110057D1 (de) | 2000-08-01 | 2001-07-26 | Verfahren und vorrichtung zur anreicherung und stabilisierung von konjugierten oestrogenen aus stutenharn |
HR20020248A HRP20020248B1 (en) | 2000-08-01 | 2002-03-22 | Method and device for concentrating and stabilising conjugated oestrogens from the urine of mares |
US10/112,040 US7081451B2 (en) | 2000-08-01 | 2002-04-01 | Method and apparatus for concentrating and stabilizing conjugated estrogens from mare urine |
US11/141,390 US7074325B2 (en) | 2000-08-01 | 2005-06-01 | Device for concentrating and stabilizing conjugated estrogens from mare urine |
US11/451,472 US7524833B2 (en) | 2000-08-01 | 2006-06-13 | Method and apparatus for concentrating and stabilizing conjugated estrogens from mare urine |
Applications Claiming Priority (2)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
DE10037389.5 | 2000-08-01 | ||
DE10037389A DE10037389A1 (de) | 2000-08-01 | 2000-08-01 | Verfahren und Vorrichtung zur Anreicherung und Stabilisierung von konjugierten Oestrogenen aus Stutenharn |
Related Child Applications (1)
Application Number | Title | Priority Date | Filing Date |
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US10/112,040 Continuation US7081451B2 (en) | 2000-08-01 | 2002-04-01 | Method and apparatus for concentrating and stabilizing conjugated estrogens from mare urine |
Publications (2)
Publication Number | Publication Date |
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WO2002009732A2 true WO2002009732A2 (de) | 2002-02-07 |
WO2002009732A3 WO2002009732A3 (de) | 2003-03-13 |
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ID=7650908
Family Applications (1)
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PCT/EP2001/008657 WO2002009732A2 (de) | 2000-08-01 | 2001-07-26 | Verfahren und vorrichtung zur anreicherung und stabilisierung von konjugierten oestrogenen aus stutenharn |
Country Status (25)
Country | Link |
---|---|
US (3) | US7081451B2 (de) |
EP (1) | EP1307210B1 (de) |
CN (1) | CN100482239C (de) |
AT (1) | ATE328600T1 (de) |
AU (1) | AU785438B2 (de) |
BG (1) | BG65554B1 (de) |
BR (1) | BR0107062B1 (de) |
CA (1) | CA2410654C (de) |
CZ (1) | CZ303159B6 (de) |
DE (2) | DE10037389A1 (de) |
EE (1) | EE04541B1 (de) |
ES (1) | ES2266221T3 (de) |
HR (1) | HRP20020248B1 (de) |
HU (1) | HU228396B1 (de) |
IL (1) | IL148878A0 (de) |
MX (1) | MXPA02002835A (de) |
NZ (1) | NZ518645A (de) |
PL (1) | PL202238B1 (de) |
PT (1) | PT1307210E (de) |
RS (1) | RS50238B (de) |
RU (1) | RU2277241C2 (de) |
SI (1) | SI1307210T1 (de) |
SK (1) | SK287663B6 (de) |
UA (1) | UA79067C2 (de) |
WO (1) | WO2002009732A2 (de) |
Families Citing this family (12)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
DE10159161A1 (de) * | 2001-12-01 | 2003-06-18 | Solvay Pharm Gmbh | Verfahren zur Gewinnung von Oestrogenen aus Stutenharn |
US8349819B2 (en) * | 2002-10-09 | 2013-01-08 | Dr. Reddy's Laboratories New York, Inc. | Steroid extraction process from urine sources |
EP1648916B1 (de) * | 2003-07-17 | 2008-10-08 | Solvay Pharmaceuticals GmbH | Verfahren zur gewinnung eines natürlichen gemisches konjugierter equin esrtrogene |
EP1675867A4 (de) * | 2003-10-24 | 2013-04-10 | Thorgard Pharmaceuticals Asut Pty Ltd | Extraktion von östrogen aus dem urin schwangerer tiere |
ITMI20052516A1 (it) | 2005-12-29 | 2007-06-30 | Evultis Sa | PROCESSO PER L'ISOLAMENTO DI PèRINCIPI FARMACOLOGICAMENTE ATTIVI DI ORIGINE VEGETALE ED ANIMALE |
US20070212751A1 (en) * | 2006-01-18 | 2007-09-13 | Solvay Pharmaceuticals Gmbh | Microbial method for the 11beta hydroxylation of 9beta, 10alpha-steriods |
CA2761844C (en) | 2009-05-14 | 2017-11-28 | University Health Network | Quantitative endoscopy |
US10391419B1 (en) | 2015-02-12 | 2019-08-27 | Nostrum Pharmaceuticals. Llc. | Isolation and purification of conjugated estrogens |
WO2017192668A1 (en) * | 2016-05-05 | 2017-11-09 | Indiana University Research & Technology Corporation | Quantitative profiling of progesterone metabolites for the prediction of spontaneous preterm delivery |
RU180071U1 (ru) * | 2016-05-23 | 2018-05-31 | Федеральное государственное бюджетное учреждение науки "Федеральный исследовательский центр "Казанский научный центр Российской академии наук" | Линия получения лекарственного средства Na-, Fe-, Ca-полигалактуронат |
US11014025B1 (en) * | 2020-03-06 | 2021-05-25 | Kevin-Steven Creagh Buford | Pressurized filtration system and device for rapid extraction and recycling of medication from body fluid |
CN116920459B (zh) * | 2023-09-15 | 2023-11-24 | 东营平明医药科技有限公司 | 一种2-巯基苯并噻唑粗品的提纯装置 |
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US2834712A (en) * | 1953-05-27 | 1958-05-13 | American Home Prod | Urinary estrogen compositions and methods for preparing them |
WO1998008525A1 (de) * | 1996-08-30 | 1998-03-05 | Solvay Deutschland Gmbh | Prozess zur gewinnung von oestrogenen aus stutenharn |
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US2519743A (en) * | 1947-12-29 | 1950-08-22 | Ivy H Dameron | Adjustable protector cap |
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HU9500323D0 (en) * | 1994-02-08 | 1995-03-28 | Solvay Deutschland | Process for separating estrogenes from urine of mares with foal |
HU228763B1 (hu) | 1996-08-30 | 2013-05-28 | Abbott Products Gmbh | Eljárás ösztrogének kinyerésére kancavizeletbõl |
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2000
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2001
- 2001-07-26 AU AU77553/01A patent/AU785438B2/en not_active Ceased
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- 2001-07-26 CZ CZ20021409A patent/CZ303159B6/cs not_active IP Right Cessation
- 2001-07-26 PL PL354451A patent/PL202238B1/pl unknown
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- 2001-07-26 SI SI200130560T patent/SI1307210T1/sl unknown
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- 2001-07-26 MX MXPA02002835A patent/MXPA02002835A/es active IP Right Grant
- 2001-07-26 WO PCT/EP2001/008657 patent/WO2002009732A2/de active IP Right Grant
- 2001-07-26 PT PT01955372T patent/PT1307210E/pt unknown
- 2001-07-26 ES ES01955372T patent/ES2266221T3/es not_active Expired - Lifetime
- 2001-07-26 CN CNB018022588A patent/CN100482239C/zh not_active Expired - Fee Related
- 2001-07-26 RS YU24102A patent/RS50238B/sr unknown
- 2001-07-26 EE EEP200200170A patent/EE04541B1/xx not_active IP Right Cessation
- 2001-07-26 DE DE50110057T patent/DE50110057D1/de not_active Expired - Lifetime
- 2001-07-26 NZ NZ518645A patent/NZ518645A/en not_active IP Right Cessation
- 2001-07-26 IL IL14887801A patent/IL148878A0/xx unknown
- 2001-07-26 EP EP01955372A patent/EP1307210B1/de not_active Expired - Lifetime
-
2002
- 2002-03-22 HR HR20020248A patent/HRP20020248B1/xx not_active IP Right Cessation
- 2002-04-01 US US10/112,040 patent/US7081451B2/en not_active Expired - Fee Related
- 2002-04-25 BG BG106649A patent/BG65554B1/bg unknown
-
2005
- 2005-06-01 US US11/141,390 patent/US7074325B2/en not_active Expired - Fee Related
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WO1998008525A1 (de) * | 1996-08-30 | 1998-03-05 | Solvay Deutschland Gmbh | Prozess zur gewinnung von oestrogenen aus stutenharn |
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