DE2352724A1 - Allergenic extracts prepn - by treating allergen contg materials with an aq lipophilic agent before extraction of the allergens - Google Patents

Abstract

Extracts contg. allergens, and opt. allergenic prepns. obtd. from these extracts, are prepd. by trating materials contg. allergens with an aq. lipophilic extracting agent before extraction of the allergens with an aq. extracting agent. The lipophilic extracting agents are pref. mixts. of hydrosol. ethers having b. pts. 105 degrees C and water or aq. ammoniacal solns. Pref. ethers are THF, tetrahydropyran and dioxane, and the ratio of ether to water or aq. NH3 is 1:2-5:1, pref. 1:1. The material contg. allergens may be pollen, seeds, skins, esp. animal fur, dusts, esp. house dust, insects,fungi, etc. The allergenic extracts are opt. formulated as slow-release prepns.

Description

Merck Patent Society
with limited liability

Darmstadt October 15, 1973

Process for the production of allergenic extracts and preparations

Addition to DBP (registration P 22 '60 455)

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

The object of the present invention was to produce improved allergen-containing extracts and preparations. It in particular should be made possible from the same allergen extract both test solutions for diagnostic skin testing and aqueous treatment extracts as well To make semi-depot preparations available for therapy. 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 .. generally contain several different allergens, which differ from one another in terms of their solubility differentiate.

According to the main patent, this problem is solved by removing the allergen-containing materials before the extraction with aqueous extractants with a lipophilic Treated extraction liquid.

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The two extracts thus obtained are then combined and processed genpräparaten if appropriate in the manner known per se to Aller. The lipophilic extracting liquids are preferably mixtures of water-soluble cyclic ethers having boiling points below 105 ⁰ C (for example, tetrahydrofuran, tetrahydropyran, dioxane) with aqueous ammonia solutions.

It has now been found that this extraction is also successful when the lipophilic extraction liquid is used a mixture of water and a water-soluble cyclic ether is used.

The subject of the invention is thus a process for the production of allergen-containing extracts and possibly allergen preparations made therefrom by extraction of allergenic materials with aqueous extractants, the allergen-containing materials being treated with a lipophilic extraction liquid prior to extraction according to DBP (now patent application P 22 60 455), which consists in that there is used as lipophilic extraction liquid mixtures of water-soluble cyclic ethers having boiling points below 105 ⁰ C with water.

It has been shown that this new extraction process has very considerable advantages. So z. B. the Most allergens are denatured during extraction be prevented. The gentle extraction method according to the invention using is essential for this the. proven aqueous extraction solutions with a pretreatment by inert organic, with water miscible solvents with good dissolving properties. On the organic solvents can normally do not do without v / grounding, as allergens that are sparingly soluble in water are extracted as completely as possible

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the should. The organic solvents previously used for extraction (e.g. fyridine) can, however, be used do not remove from the extracts without applying higher temperatures. Because of that and because of nature however, the solvent can always cause a change or denaturation of the allergens. aside from that the pyridine is usually very solid, e.g. T. also complex, bound, so that a complete removal this extractant encounters considerable difficulties.

When the extraction according to the invention is carried out, allergens with different dissolving properties are obtained 3xx. ± very gently, and the various components of the allergen-containing material are recorded. '

The cyclic ethers to be used for the pretreatment according to the present invention are excellent Solution properties. They can be mixed with water in any ratio and lose their lipophilic properties not even in strong aqueous dilution. They are also chemically inert and, because of their relatively low boiling point can easily be removed from the extracts by evaporation. For this reason is also Tetrahydrofuran is still preferred over the also well-suited solvents tetrahydropyran and dioxane. Due to their miscibility with water, these solvents extract even those masked by aqueous barriers Lipoid and lipophilic allergens and prepare the allergen-containing The starting material is optimally prepared for the aqueous extraction in the second extraction step.

So far, ether has been used to pretreat allergenic materials, especially for degreasing. However, this is not an ex-

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traction, but only to remove undesirable Lipoids and lipids, although with this pretreatment it is certain that allergenic agents are inevitably also present extracted. However, the aether used up to now is not able to pass through water barriers Bringing masked lipoids into solution.

The lipophilic extraction agent to be used in the first stage 'contains the cyclic ethers with a boiling point below 105 ° C mixed with water, mixtures in a ratio of 5: 1 to 1: 2, preferably about 1 1, are preferred.

The extraction itself takes place in a conventional manner. The allergen-containing material, preferably previously comminuted or ground, is mixed with the extractant and left to stand for several hours (eg 5 to 60 hours) or shaken in a shaking machine. The extraction is expediently carried out at room temperature, although in principle it is also possible to work at lower temperatures. The solid components are then separated from the liquid. The latter is concentrated under gentle conditions or evaporated to dryness. This can be accomplished conveniently on a rotary evaporator with the water bath temperature not exceeding about 40 ⁰ C. The residue obtained in this way is the end product of the first extraction stage. . .

The prepared by the extraction with the lipophilic solvent and partially extracted allergen carriers are then in the second step of a conventional extract ions Ex · -. ■ traction with the proven aqueous extractants subjected. Summaries about 'the extraction liquids used can be found in "CONSTITUTION, ALLERGIC DISEASES, DISEASES OF THE BONES, JOINTS' -. '

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UND MUSKELIi ", edited by F. Curtius and others, Springer Verlag 1954, pages 389-392, and in the Journal of Allergy, Volume 30, 1959, pages 66-82. The sediment of the extract obtained after the first stage is in a -the known aqueous extraction liquids ■ suspended and extracted by methods known from the literature. All aqueous extractants can be used that have been used previously for the extraction of allergenic materials. In particular, the solutions known from the literature, such as physiological saline, Salty buffer solutions, 12% ethanol or Cocas solution, in question (as is well 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. Here, too, the sediment is removed from the liquid separated, preferably by centrifugation. It makes sense to carry out the extraction in this second stage in such a way that that the resulting extract is obtained as a physiologically adjusted salt solution. ·.

It has proven to be very advantageous to then combine the two extracts obtained in this way. In this way an extract is obtained that contains all the effective principles of the allergenic material. It is particularly advantageous to free the extract obtained in the first stage from the solvents and to combine the residue obtained in this way with the extract obtained in the second stage. In this way, a strain extract with high allergenic activity and free from organic solvents is obtained. This extract is then treated further in the usual way, so z. B. i'iltrisrt clear and then sterile via membrane filter. In the usual way, about 0.4 %

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Phenol can be added. Such an extract is then equally suitable for the production of diagnostic as well as therapeutically applicable preparations. The extract obtained in this way, which contains the combined allergens with different dissolving properties, can now be used in a manner which is conventional per se, namely like the allergen extracts known up to now. So körinen z. B. by VeT-. dilution, for example with physiological saline solution or with Cocas solution, extracts suitable for testing can be obtained. It can also be used to prepare provocation test solutions in a known manner. Injection solutions suitable for therapy can also be prepared without difficulty from the same stock extract according to standard procedures in the desired dilutions adapted to the respective treatment goal.

This extract is also suitable for the production of so-called Seini depot preparations, which are produced by methods known from the literature with the aid of aluminum hydroxide. The extract is mixed with an aluminum salt solution, for example alum, and. aluminum hydroxide is precipitated by changing the pH. The precipitate obtained settles out. The supernatant liquid is clear and only slightly colored, while the aluminum hydrOxide adsorbate shows a strong gray-green color. The supernatant clear solution is filtered off with suction, and the suspension is washed very thoroughly with a solution which usually consists of physiological saline solution with 0.4 % phenol. A preparation suitable for therapy is then obtained by diluting it with sterile physiological saline solution to the desired PNU content.

It goes without saying that all work with sterile solutions and in sterile devices is carried out in one closed place System.

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~ 7 -

It has also proven to be very beneficial during the manufacturing process of the allergen-containing Let the extracts and preparations work with ultrasound. Ultrasound treatment can be applied take place at any stage of the manufacturing process, but an action in the is preferred second extraction stage. This has the particular advantage that tedious extraction can be avoided can. With the extraction methods customary up to now, relatively long extraction times have to be accepted e.g. usually 1 to 10 days. It can of course never be ruled out that chemically or microbiologically triggered changes in the allergens and allergen carriers.

Extraction with simultaneous sonication can reduce the extraction times considerably. According to the preferred embodiment according to the invention, it is sufficient to use the material together with the Sonicate the extraction liquid for a few minutes in order to achieve a good extraction. In many cases it is advisable to divide larger amounts of liquid into smaller portions for sonication. As a rule, it is sufficient to carry out the sonication for about 0.5 to 5 minutes. Lower frequency is preferred Ultrasound is used, for example in a frequency range of about 20 to 30, preferably about up to 25 kHz. It is advisable to cool during the sonication, e.g. with ice water, so that the sonication no heating of the solution occurs. In addition to the drastic shortening of the extraction time, the Targeted substantial increase in the yield of allergen-active material of particular advantage. Furthermore in this way becomes a bacterial and fungal contamination of the allergen-containing starting material is inhibited.

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Surprisingly, the intensity of action of the allergen extracts produced in this way is significantly increased. The yield of protein-bound nitrogen (PNU) in the extracts, which is regarded as a measure of the allergen content, is increased by up to 100 % through this additional treatment with ultrasound.

Even if you use the extraction method according to the invention resulting extracts or solutions prepared therefrom by customary dilution treated with ultrasound, the intensity of the action is significantly increased. The sound effects generally one compared to the unsound extract Increase in allergen potential by about ten times. Here, too, it is expedient to work with cooling Ice water. As already mentioned, the point in time of the sonication can also be selected here as desired; he however, it is expedient for the extracts to be filled into ampoules, which may be provided.

The new process thus makes the production particularly possible highly effective allergen extracts and preparations that are clearly superior to those known so far. the Extracts and preparations are used in the same way as for diagnosis and therapy in allergic diseases the previously available extracts and preparations are used.

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example 1

9 g of Festuca elatior pollen were used for 16 hours ground in a ball mill. The ground pollen material was in a 1 liter Erlenmeyer flask with 300 ml of a mixture of tetrahydrofuran and water (1: 1) are added and the mixture is carried out for 16 hours at room temperature shaken in a shaking machine. By centrifugation at 4500 rpm, the solid components were separated from the supernatant. This is called extract I.

The sediment was filled with 200 ml of a 1: \ 1 suspended with water diluted Cocas solution (Cocas solution contains in 100 ml solution: 0.5 g NaCl, 0.25 g NaHCO ^ and 0.4 g phenol). The suspension was made in 3 servings divided up. Each part was subjected to ultrasonic ice water cooling for 3 minutes at a frequency of 22 kHz and a maximum power of 150 watts ". The three servings were combined again and that Sediment was separated off by centrifugation. This ultrasound-treated extract is extract II.

Extract I was in a rotary evaporator at a water bath temperature of 36 ° C and at about 10 torr to. evaporated to dryness; the residue was dissolved in 10 ml · 1 η NaOH, and the solution was neutralized with 10 ml 1 η HCl and this solution was again brought to dryness in the rotary evaporator. *

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 and then sterile filtered with a membrane filter.

There were 270 ml of a green, cloudy stock extract with received a content of 31,650 PNU / ml, corresponding to a

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Total PNU yield of 950,000 PIiU / g pollen material. . ·.

A portion of the strain obtained extract was diluted with unver- · dilute Cocas solution directly to a form suitable for skin testing extract wird- said default set to 100 PNU / ml Before use, DIE '· sev _t extract as needed diluted further, in usually by ■ at least two powers of ten.

Another part of the starfish extract was used for production a provocation test solution diluted with undiluted Cocas solution to a PNU content of 5000 PNU / ml. '

To produce a semi-depot preparation, a third part of the strain extract was demineralized, sterile Water brought to 20,000 PNU / ml. The following procedure was used to manufacture the SemiDepot preparation:

The adsorptive binding of the allergens to an active aluminum hydroxide took place by precipitation with an aluminum salt solution. To this end, 200 ml of a 2 follow potassium aluminum alum solution in 0.25 η HpSO / were added to 180 ml of the extract solution, which was concentrated with 20 ml. (25 aqueous ammonia solution had been added. The mixture was then vigorously stirred. It was then dialyzed for 24 hours against tap water and twice for 1 hour against demineralized water. The aluminum hydroxide adsorbate showed a very strong gray-green color. The supernatant clear solution became suctioned off, and the suspension v / urde washed very often with a solution containing 0.9 % sodium chloride and 0.4 % phenol.

All work was carried out with sterile solutions and sterile devices ■ in a closed system. 300 ml of adsorbate suspension with a content of 7,590 PNU / ml were obtained, which corresponds to a yield of 80 %, based on the PNU units used. The suspension was brought to a content of 10,000 PNU / ml by dilution with sterile physiological saline solution. A sterile control carried out afterwards showed sterility.

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Example 2 "

9 g of Dactylis glomerata pollen was ground for 16 hours in a ball mill with porcelain balls. The extraction of the pollen material was carried out as in Example 1, but in contrast to there with a solvent mixture of dioxane and HpO in a ratio of 1: 1. In the second extraction step, instead of the Cocas solution diluted with water, the Evans solution diluted in the same ratio was used
(Evans solution: 5.0 g NaCl

0.36 g KH2PO4 4.0 g phenol

distilled in 1000 ml

Water.

7.0 g Na ₂ HPO ₄

The treatment with ultrasound was carried out in the same way, in that the extraction solution was sonicated in three portions for 3 minutes. The combined stock extract obtained had a total volume of 240 ml and was brown-green in color. The yield was 25,800 PNU / ml, corresponding to ^N corresponding to a total PNU content of 688,000 PNU / g of pollen material used. The precipitation or binding to aluminum hydroxide was carried out as described in Example 1. 300 ml of an aluminum hydroxide adsorbate suspension with a content of 4,500 Pwü / ml were obtained, which corresponded to a PNU yield of 65 % .

Example 3

Analogously to Example 1, 9 g of pollen were obtained from. Seeale cereale extracted. Instead of tetrahydrofuran, however, the same volume fraction of tetrahydropyran was used. As a result, an aluminum hydroxide adsorbate having a specific content of 7,800 PNU / ml of suspension was obtained. 300 ml of this suspension thus corresponded to a total content of 3.99 million PNU (68 % yield).

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Corresponding results are obtained with the following allergenic materials: House dust, Mucor mocedo (mold), Bed feathers (consisting mainly of goose feathers) and honey bees killed at low temperatures.

Example 4

House dust collected with the vacuum cleaner was sieved through a series of test sieves. Only the faction that one A sieve with a mesh size of 0.3 mm was used for extraction in order to retain fullness and hair .

70 g of house dust were sieved in a 1 liter Erlenmeyer flask with 300 ml of a mixture of tetrahydrofuran and water (volume ratio 1: 1) and for 16 hours shaken in a shaker. The solids were removed from the supernatant by centrifugation at 4500 rpm (Extract I) separately. The sediment was suspended with 300 ml of a Cocas solution diluted 1: 1 with water and alternating 3 times for 8 hours at 45 ° C and then for 8 hours at room temperature extracted with occasional shaking. By centrifugation at 4500 rpm the sediment was separated from the supernatant (Extract II).

I extract was evaporated in a rotary evaporator at a bath temperature of Wasser-- ■ 36 ⁰ C and about 10 torr to dryness; the residue was dissolved in 10 ml of NaOH, the solution was neutralized with 10 ml of 1N HCl and brought to dryness again in a rotary evaporator. The dry residue obtained in this way was dissolved in extract II. The solution was successively filtered clear and then sterile through membrane filters with different pore sizes in a pressure filtration device.

There were 270 ml of a clear, dark brown stock extract with 39,000 PNCJ / ml, corresponding to a total PNU yield of 150,000 PNU / g of house dust used.

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Claims (9)

Claims Process for the production of allergen-containing extracts and, if appropriate, produced therefrom Allergen preparations by extraction, allergenic materials with aqueous Extraction agents, the allergenic materials with a lipophilic extraction liquid are treated according to DBP (now patent application P 22 60 455), characterized in that the lipophilic extraction liquid is mixtures of water-soluble, cyclic ethers with boiling points below 105 C with water. 2. The method according to claim 1, characterized in that the cyclic ether is tetrahydrofuran, Tetrahydropyran or dioxane is used. 3. The method according to claim 1, characterized in that the cyclic ether and water in Ratio 1: 2 to 5: 1, preferably 1: 1, is used. 4. Process according to claims 1 to 3, characterized in that at least one of any Stage during the manufacturing process allows ultrasound to act. 5. The method according to claim 4, characterized in that one ultrasound with a frequency of 20 to 30 kHz used. 5 0 9 8 17/1138 JH 6. The method according to claims 4 and 5, characterized in that the treatment with ultrasound is carried out during the second extraction stage. 7. Process according to Claims 1 to 6, characterized in that the combined extracts converted into a semi-depot preparation by binding to aluminum hydroxide. 8. Use of the prepared according to claims 1 to 7 Extracts for the production of diagnostic and therapeutic allergen preparations. 9. Allergen-containing extracts and preparations according to claims 1 to 7. 509817/1138