DE2260455A1 - Allergen-contg extracts prodn - by two-stage process using organic and aq. extractions for complete allergen recovery - Google Patents

Abstract

In the first stage extraction is with a lipophilic liq. and in the second with a conventional aq. extractant; the recovered extracts are then combined and opt. further processed in the usual manner. The pref. lipophilic liq. is a mixt. of THF, tetrahydropyran or dioxan with aq. NH3 esp. in ratio ether H2O:25% NH3 of 5:4:1. Pref. during at least one process stage irradiation with ultrasound (esp. 20-30 Hz) is applied esp. during the second extraction stage. The products are useful oth as diagnostic and therapeutic materials and may be converted to sustained release formulations by bonding to Al hydroxide. Two-stages gie complete allergen recovery without deactivation and ultrasonic treatment reduces process time.

Description

Process for the production of allergen-containing extracts and preparations The present invention relates to a new process for the production of allergen-containing Extracts and preparations used for the detection of sensitizing antigens and for specific desensitization of allergy sufferers are suitable.

It has long been known that numerous people come into contact with the contained materials show allergic reactions. Carrier of allergenic agents are e.g. pollen, seeds, epithelia, especially animal hair, dust, especially domestic stan, Insects and mushrooms.

Clinical experience has shown that allergic reactions are caused by subcutaneous injections of extracts from allergy-causing substances can be weakened. For the production of such allergenic extracts or preparations were previously generally, partly after previous degreasing of the allergen-containing Materials with ether, aqueous liquids such as physiological saline, weakly alkaline, possibly buffered saline solutions, or alcohol dilution in physiological saline solution used. Generally will the solutions obtained in this way are filtered sterile after removal of the solid constituents and injected into the patient in suitable dilutions for desensitization. the Use of these aqueous extracts requires a greater number of injections with slowly increasing amounts of allergens. Use is therefore advantageous of semi-depot preparations that significantly reduce the number required Injections are allowed and at the same time the risk of excessive reactions is reduced. Extraction processes are described in the literature for the production of such semi-depot preparations using aqueous solutions of heterocyclic tertiary amines, in particular Pyridine. The allergens are then separated off as aluminum hydroxide adsorbate by adding alum solution to the alkaline extract. The resulting the precipitate containing the active substances is removed after removal of the solvent and resuspended in a physiological solution after washing several times. These However, semi-depot preparations can only be used to treat patients but not too diag-.

nostic purposes.

Different allergists keep coming back to the fact pointed out that the commercially available allergen extracts from one and the same allergen carrier different reaction spectra result, so that already at. a diagnostic Debugging errors of up to 30% are possible.

For this reason there is considerable uncertainty after the diagnosis when choosing the appropriate therapeutic agent.

One tries to avoid this uncertainty by doing the appropriate 4af diagnostic and therapeutic allergen preparations from the same manufacturer are used, although it is known that these are obtained by completely schledenell methods and accordingly to show different tiWaktio; -; spectra.

The object of the present invention was to provide improved allergen-containing Manufacture extracts and preparations. In particular, it should be allowed out the same allergen extract as well as test solutions for diagnostic skin testing as well as aqueous treatment extracts. as well as semi-depot preparations for therapy to provide. This goal is achieved with the help of a special extraction process achieved. This new extraction process also takes into account the fact that the allergen carriers, such as pollen, dust, epithelia, fungi, insects, etc., im generally contain several 'different allergens', which also differ with regard to their solubility differ from each other.

The invention therefore relates to a method for production of allergen-containing extracts and possibly allergen preparations made from them with the help of an extraction of allergenic materials with aqueous extractants, that consists in carrying out the extraction using extraction agents carried out in two stages with different solution properties. In a lipophilic extraction liquid is used in the first stage and in the second Stage one of those commonly used for the extraction of allergenic materials aqueous extraction liquids are used. The two extracts thus obtained are then combined and, if necessary, in a manner known per se to form allergen preparations Developed. The lipophilic extraction liquids are preferably mixtures of water-soluble cyclic ethers with boiling points below 1059C (e.g. tetrahydrofuran, Tetrahydropyran, dioxane) with aqueous ammonia solutions.

It has been shown that this new extraction process is quite significant Has advantages. For example, the denaturation of allergens during extraction largely prevented. The gentle one according to the invention is essential for this Extraction method using the proven aqueous extraction solutions with a pretreatment with inert organic, water-miscible solvents with good solution properties. On the organic solvents can normally not be dispensed with, as allergens that are sparingly soluble in water are as complete as possible should be extracted. The organic solvents previously used for extraction However, (e.g. pyridine) cannot be used without the use of higher temperatures remove from the extracts. This and the nature of the solvent can but always a change or denaturation of the allergens are caused. In addition, the pyridine is usually very solid, e.g. T. also complex, bound, so that a complete removal of this extractant presents considerable difficulties bumps.

When performing the two-stage extraction, be on very gentle way allergens with different dissolving properties are obtained, and the various components of the allergenic material are recorded.

Those to be used for the pretreatment according to the present invention cyclic ethers have excellent solubility properties. You are in everyone Ratio miscible with water and lose their lipophilic properties even in Do not use 50% aqueous dilution. They are also chemically inert and leave easily evaporation due to their relatively low boiling point the end removed the extracts. For this reason, tetrahydrofuran is also still opposite the solvents tetrahydropyran and dioxane, which are also suitable, are preferred.

Due to their miscibility with water, these solvents extract also by watery ones. Barriers masked lipids and lipophilic allergens and prepare the allergen-containing starting material optimally to the aqueous extraction in the second Extraction step.

So far, a pretreatment of allergenic materials, especially for degreasing, ether decayed. However, this is not the case an extraction, but only the removal of unwanted lipids and lipids, although this pretreatment certainly also includes allergens Active ingredients are extracted. The ether used so far is not in the Able to dissolve lipoids masked by water barriers.

The lipophilic extractant to be used in the first stage preferably contains the cyclic ethers with a boiling point below 105 ° C. in a mixture with aqueous ammonia. Here are mixtures of organic solvents: water in a ratio of 5: 1 to 1: 2, preferably about 1: 1, preferred. The watery one Component advantageously contains about 1 to 10, preferably about 5% by weight, NH3. It has been found to be particularly useful, the cyclic ether, water and concentrated Use ammonia (25 dig) in a ratio of 5: 4: 1.

In many cases, however, the amount of ammonia can also be used can be significantly reduced, so that the extraction also with one as defined above Mixture in the ratio 10: 8: 1 can be carried out. The pH of the lipophilic The extraction agent is generally conveniently between 11 and 12.

The extraction itself takes place in a conventional manner.

The allergen-containing, preferably previously comminuted or ground Extracting agent is added to the material and several hours (e.g. 5 to 60 hours) left to stand or shaken in a shaker. Will be expedient the extraction is carried out at room temperature, although in principle also at lower temperatures Temperatures can be worked. Then the solid ingredients separated from the liquid. The latter is concentrated under gentle conditions or evaporated to dryness. This is done, for example, conveniently on a rotary evaporator with water bath temperatures that do not exceed about 400C.

The residue obtained in this way is the end product of the first extraction stage.

Those prepared by extraction with the lipophilic solvent and partially extracted allergen carriers are then extracted in the second extraction step subjected to a customary extraction with the tried and tested aqueous extractants. Summaries of the extraction fluids used can be found E.g. in "KON5TITUTIONJ ALLERGISCHE KJNKE9RITEN} DISEASES OF THE BONES, JOINTS UND MUSKELN1 ?, edited by F. Curtius and others, Springer Verlag 1954, pages 389-392 and in the Journal of Allergy, Volume 30, 1959, pages G6-82. The sediment the extract obtained after the first stage is used in one of the known aqueous extraction liquids and suspended according to methods known from the literature extracted.

All aqueous extractants can be used here, which also previously commonly used for the extraction of allergenic materials have been. In particular, the solutions known from the literature come into play, such as physiological saline solution, saline buffer solutions, 12 figes ethanol or Cocas solution, in question (as is known, 100 ml of Cocas solution contains: 0.5 g NaCl, 0.25 g NaHCO 3 and 0.4 g phenol).

In this way, all of the water-soluble, allergen-containing ones Materials contained effective allergens obtained. Again, that will be the case Sediment separated from the liquid, preferably by centrifugation. It is makes sense to carry out the extraction in this second stage so that the resulting Extract is obtained as a physiologically adjusted salt solution.

It has been found to be very beneficial by the two different ones Extracts obtained in the extraction stages to then combine. In this way one obtains an extract that contains all the effective principles of the allergenic material contains. It is particularly advantageous to use the extract obtained in the first stage to free from the solvents and the residue thus obtained with the in the to combine the extract obtained in the second stage.

This gives a strain extract with high allergenic activity, the is free from organic solvents. This extract is then used in usual In a manner that is further treated, e.g. clear through a membrane filter and then sterile filtered.

For preservation, about 0.4% phenol can be added in the usual way Such an extract is then equally suitable for both production of diagnostic as well as therapeutically applicable preparations. The thus obtained Extract containing the combined allergens with different solution properties contains, can now in a conventional manner, namely like the previously known allergen extracts, can be used. B. by dilution, e.g. with physiological saline solution or with Cocas solution, extracts suitable for testing can be obtained. It can provocation test solutions can also be prepared therefrom in a known manner. Even Injection solutions suitable for therapy can be prepared according to standard procedures in the generalized and adapted to the respective treatment goal. Dilutions can easily be made from the same strain extract.

This extract is also suitable for the production of so-called Semi-depot preparations made according to methods known from the literature with the aid of aluminum hydroxide getting produced. The extract is mixed with an aluminum salt solution, e.g. Alum, added and by changing the pH value, aluminum hydroxide is precipitated. The precipitate obtained settles out. The supernatant liquid is clear and only slightly colored, while the aluminum hydroxide adsorbate is a strong one shows gray-green color. The supernatant clear solution is suctioned off, and the suspension is washed very thoroughly with a solution, mostly from physiological Saline solution with 0.4% phenol. It is then obtained by diluting with sterile physiological saline solution to the desired PNU content for therapy suitable preparation.

Of course, all work with sterile solutions and in sterile devices in a closed system.

It has also been found to be very beneficial during the manufacturing process Let the allergen-containing extracts and preparations act ultrasound. the Ultrasound treatment can be used at any stage of the manufacturing process take place, however, an action in the second extraction stage is preferred. This has the particular advantage that tedious extraction can be avoided can. The extraction methods customary up to now require relatively long extraction times be accepted, e.g. usually 1 to 10 days. Of course, you can never do this it is ruled out that chemically or microbiologically triggered changes occur on the allergens and allergen carriers.

By extraction with simultaneous sonication, the Significantly reduce extraction times. According to the invention, preferred Embodiment, it is sufficient to put the material together with the extraction liquid Sonicate a few minutes to achieve a good extraction. In many cases it is advisable to use larger amounts of liquid for sonication in smaller portions to subdivide.

As a rule, it is sufficient to provide the sound for about 0.5 to 5 minutes. Preferably, low frequency ultrasound is used, e.g. in a frequency range of about 20 to 30, preferably about 20 to 25 kHz. Appropriate is cooled during the sonication, e.g. with ice water, so that the sonication no heating of the <solution occurs. In addition to the drastic reduction in the extraction time is the considerable increase in the yield of all allergen-active material that can be achieved as a result of particular advantage. In addition, this way a bacterial and mycotic contamination of the allergen-containing starting material is inhibited.

Surprisingly, the intensity of the effect is the same as that shown Allergen extracts increased significantly. Also the yield of protein-bound nitrogen (PNU) in the extracts, which is regarded as a measure of the allergen content this additional treatment with ultrasound increased up to 100%.

Even if you have the resulting in the extraction process according to the invention Extracts or solutions made from them by customary dilution can still be done later treated with ultrasound, the intensity of the action is significantly increased. the Sonication generally brings about an increase compared to the unsound extract the allergen potential by about ten times.

Here, too, it is expedient to work with cooling with ice water. The timing act of the sound reinforcement can, as already mentioned, too can be chosen at will here; however, it is expediently before the eventual intended filling of the extracts in ampoules.

The new process thus enables production to be particularly highly effective Allergen extracts and preparations that are clearly superior to those previously known. The extracts and preparations are used for diagnosis and in allergic diseases Therapy in the same way as the previously available extracts and preparations used.

Example 1 9 g of Festuca elatior pollen were in ground in a ball mill. The ground pollen material was placed in a 1 liter Erlenmeyer flask with 300 ml of a mixture of tetrahydrofuran: water: conc. Ammonia (25%) as 5: 4: 1 added and for 16 hours at room temperature in a shaker shaken.

The solid components were removed by centrifugation at 4500 rpm Separate supernatant. This is called extract I.

The sediment was with 200 ml of a 1: 1 with water diluted Cocas solution suspended (Cocas solution contains in 100 ml of solution: 0.5 g NaCl; 0.25 g NatIC03 and 0.4 g phenol). The suspension was divided into 3 portions. Each part was ultrasonically scanned for 3 minutes while cooling with ice water Treated frequency of 22 kHz and a maximum power of 150 watts. The three Portions were recombined and the sediment was separated by centrifugation. This ultrasound-treated extract is extract II.

Extract I was made on a rotary evaporator at a water bath temperature from 360C and up to about 10 Torr.

evaporated to dryness; the residue was dissolved in 10 ml of 1N NaOH, the solution was neutralized with 10 ml of 1N HCl and this solution was again on a rotary evaporator brought to dryness.

The dry residue thus obtained was dissolved in extract II. the Solution was successively through membrane filters with different pore sizes in one Pressure filtration device and then sterile filtered with a membrane filter.

There were 270 ml of a green, cloudy stock extract with a content of 51900 PNU / ml obtained, corresponding to one Total PNU yield of 1,557,000 PNU / g pollen material.

Part of the obtained stock extract was mixed with neat Cocas solution directly diluted to an extract suitable for skin testing, with standard is set to 100 PNU / ml. Before using, this extract is made as needed further diluted, usually by at least two powers of ten.

Another part of the stock extract was used to make a provocation test solution diluted with undiluted Cocas solution to a PNU content of 5000 PNU / ml.

A third part was used to manufacture a semi-depot preparation of the stock extract brought to 20,000 PNU / ml with demineralized, sterile water. The procedure for producing the semi-depot preparation was as follows: The adsorptive The allergens were bound to an active aluminum hydroxide by precipitation with an aluminum salt solution. For this purpose, 200 ml of a 2 point potassium aluminum alum solution were added in 0.25 N H2S04 added to 180 ml of the extract solution, which is concentrated with 20 ml. (25 iger) aqueous ammonia solution had been added. The mixture then became vigorous touched. Then there was 24 hours against tap water and 2 times 1 hour against demineralized water Dialyzed water. The aluminum hydroxide adsorbate showed a very strong gray-green Colour. The supernatant clear solution was suctioned off and the suspension became very washed frequently with a solution containing 0.9% sodium chloride and 0.4% phenol.

All work was done with sterile solutions and sterile equipment in carried out in a closed system. There were 300 ml of adsorbate suspension with a content of 14 <400 PNU / ml, which corresponds to a yield of 80 %, based on the PNU units used.

The suspension became physiological by diluting it with sterile Saline solution brought to a content of 10,000 PNU / ml. A subsequently carried out Sterile control, enex «anb, germ freedom.

Example 2 9 g of pollen from Dactylis glomerata were collected for 16 hours a ball mill. ground with porcelain balls. The extraction of the pollen material was carried out analogously to Example 1, but in contrast to there with a solvent mixture from dioxane: H20: conc. Ammonia (25%) as 5: 4: 1. In the second extraction step instead of the Cocas solution diluted with water, they were also used in the same Ratio of dilute solution according to Evans used (Evans solution: 5.0 g NaCl) 0.36 g KH2PO4) in 1000 ml distilled 4.0 g phenol) water.

7.0 6 Na2HPO4) The treatment with ultrasound was carried out in the same way Way by sonicating the extraction solution in three portions for 3 minutes. The resulting combined stock extract had a total volume of 240 ml and showed a brownish-white color. The yield was 41,500 PNU / ml, corresponding to a total PNU content of 1,106,700 PNU / g of pollen material used. The precipitation or binding to aluminum hydroxide took place as described in Example 1.

There were 300 ml of an aluminum hydroxide adsorbate suspension with a Content of 12,700 PNU / ml received what a PNU-.

Corresponding to a yield of 70.5%.

Example 3 Analogously to Example 1, 9 g of pollen from Secale cereale extracted. Instead of tetrahydrofuran, however, the same volume fraction was used Tetrahydropyran used in the 1st extraction stage. As a result, it became an aluminum hydroxide adsorbate get with. a specific content of ~ -13-.500 ~ PNU / ml suspension. 300 ml this suspension therefore corresponded to a total content of 3.99 million PNU (73.9 O / o yield).

Corresponding results are obtained with the following allergenic materials preserved: house dust, mucor mucedo (mold), bed feathers (consisting mainly of from goose feathers) and honey bees killed at low temperatures.

Example 4 House dust collected with the vacuum cleaner was removed by a Sieved series of test sieves. Only the fraction that has a sieve with a mesh size 0.3 mm passed, was used for extraction to retain wool and hair.

70 g of house dust were sieved in a 1 l Erlenmeyer flask with 300 ml of a mixture of tetrahydrofuran: water: conc. Ammonia (25%) = 5: 4: 1 and shaken for 16 hours in a shaker. By Centrifugation at 4500 rpm, the solid components were removed from the supernatant (extract I) separated. The sediment was diluted with 300 ml of a 1: 1 ratio of water Cocas solution suspended and alternating 3 times for 8 hours at 450C and then Extracted for 8 hours at room temperature with occasional shaking. By Centrifugation at 4500 rpm, the sediment was separated from the supernatant (extract II).

Extract I was made on a rotary evaporator at a water bath temperature evaporated to dryness from 360C and about 10 torr; the residue was in 10 ml of NaOH dissolved, the solution neutralized with 10 ml of 1 N HCl and again in a rotary evaporator brought to dryness. The dry residue obtained in this way was dissolved in extract II. The solution was successively passed through membrane filters with different pore sizes in a pressure filtration device clear and then filtered sterile.

There were 270 ml of a clear, dark brown stock extract with 60 000 PNU / ml obtained, corresponding to a total PNU yield of 240,000 PNU / g used House dust.

Claims (14)

Claims 1. Process for the production of allergen-containing extracts as well allergen preparations possibly produced therefrom by extraction of allergenic substances Materials with aqueous extractants, characterized in that the Extraction using extraction agents with different dissolving properties carried out in two stages, with a lipophilic extraction liquid in the first stage and in the second stage one usually for the extraction of allergens Materials used aqueous extraction liquid is used and that the two extracts obtained in this way are combined and, if appropriate, known per se Processed into allergen preparations. 2. The method according to claim 1, characterized in that as lipophilic extraction liquid mixtures of water-soluble, cyclic ethers used with boiling points below 105 ° C with aqueous ammonia solutions. 3. The method according to claim 2, characterized in that as cyclic ethers tetrahydrofuran, tetrahydropyran or dioxane are used. 4. The method according to claim '2, characterized in that the cyclic ether and the aqueous ammonia solutions in a ratio of 1: 2 to 5: 1, preferably 1: 1, is used. 5. The method according to claim 4, characterized in that one aqueous ammonia solution used, which about 1 - 10 wt .-%, preferably about 5 Wt .-%, contains NH3. 6. The method according to claim 5, characterized in that the cyclic ether, water and ammonia (25 Sig) in a ratio of 5: 4: 1. 7. Process according to claims 1 to 6, characterized in that the extract obtained after the first stage at temperatures below 400C of freed from the solvents. 8. The method according to claim 7, characterized in that the residue thus obtained with the extraction liquid obtained in the second stage united. 9. The method according to claims 1 to 8, characterized in that at least at one stage during the manufacturing process Let ultrasound act. 10. The method according to claim 9, characterized in that one ultrasound used at a frequency of 20 to 70 kHz. 11. The method according to claims 9 and 10, characterized in that that the treatment with ultrasound is carried out during the second extraction stage. 12. Vei'iahren according to claims 1 to 11, characterized in that that the combined extracts can be converted into a semi-depot preparation by acidifying aluminum hydroxide convicted. 13. Use of the extracts prepared according to responses 1 to 12 for the production of diagnostic and irotherapeutic allergen preparations. 14. Allergen-containing extracts and preparations, produced according to the claims 1 to 12.