DE1953995A1 - Allergy desensitizers and process for their preparation - Google Patents

Description

DR. ELISABETH JUNG, DR. VOLKER VOSSIUS, DIPL.-ING. GERHARD COLDEWEY

Patent attorneys
MUNICH 23 ■ CLEMENSSTRASSE 30 - TELEPHONE 345067 · TELEGRAM ADDRESS: INVENT / MONCHEN · TELEX 5-29688

. 1953935

27.0KT.i959

uZ: E 814
A 307

BEECHAM GROUP LIMITED

Brentford, Middlesex, UK

"Allergy desensitizers and procedures too its manufacture "

Priority: October 25, 1968, Great Britain, Kr. 5C645 / 68

The invention relates to a desensitizing or immunizing agent for the treatment of hypersensitivity symptoms and allergies. The invention also relates to a process for the preparation of such desensitizers.

It is known that some people are “allergic or hypersensitive to allergens such as pollen, house dust, cat fur, grain and many other substances. These people can suffer from acute symptoms as a result of their allergic reactions, which manifest themselves in diseases such as asthma, hay fever, eczema, dermatitis and migraines. Much effort has therefore been and is being made to find suitable treatment methods which alleviate the suffering of allergic patients. 0 0 9818/1732

POST CHECK ACCOUNT) MÖNCHEN 60175 BANK ACCOUNT: DEUT8CHE BANK A.Q. MDNCHEN. LEOPOLDSTR. 71, KTO. NO. 80/36794

One method used in the past to treat allergic symptoms is so-called deseneibilization therapy. The patient to be treated is frequently administered repeated, gradually increasing doses of an extract of the allergen or allergens to which or to which he is sensitive. At the end of the treatment, the patient's natural resistance to the allergen has usually risen sharply, presumably as a result of the build-up of antibodies in the organism, which is stimulated by the administered extract.

However, this desensitization therapy has certain disadvantages. One such disadvantage is the possibility that a dangerously high dose of the allergen may be inadvertently administered, resulting in a general anaphylactic reaction in the patient. It has already been suggested that this difficulty can be overcome if it is possible to modify the allergen in such a way that the allergen effect is relatively reduced in relation to the desensitization properties and / or immunization properties. Thus, if the allergen containing material or this could be made harmless to the sensitive patients, or at least less harmful _t but its capacity could be maintained to stimulate the formation of antibodies simultaneously, one might be the desensitization therapy are off the 'main disadvantages.

According to the invention, a desensitizing agent against allergies is created which consists of an allergen that is at least partially intermolecular and / or intramolecular

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a halogenoformic acid ester of the general formula XOOOEi in which X is a fluorine, chlorine, bromine oäer iodato "- and R is an aliphatic, aromatic, araliphatic or heterocyclic optionally substituted group.

According to the invention, the deseneibilizing agent is prepared by that you can eliminate the allergen with the haloformic acid ester treated at a pH of 1 to 7 and the reaction product isolated.

"Allergen" is understood to mean all substances that have a allergic reaction in one sensitive to these substances Evoke patient. Examples of common allergens are pollen such as timothy grass (Phleum pratenae), Raigrae (Lolium perenne) and ice pen grass (Cocksfoot), house dust, mold spores, Animal hair or pels and fabrics. However, many other examples of allergens are also known.

According to a preferred embodiment of the invention, the Protein fraction of an allergen extract, usually in water, with the haloformic acid ester in the specified pH range, treated and then isolated.

A chloroformic acid ester, in particular a hydrocarbon chloroformic acid ester, such as an alkyl, aryl or aralkyl chloroformate, is preferably used as the haloformic acid ester.

Examples of suitable alkyl haloformates are esters having 1 to 8 carbon atoms in the alkyl chain, the alkyl chain being can be straight or branched, such as the methyl,

309818/1722 ""'"'"""'"'" ■ "'

Ethyl, η and isopropyl, η-butyl, zeobutyl and tert-butyl groups. The term alley groups should also include cycloalkyl groups skins. A suitable halogenoaeleneettureoycloalkyl ester is the cyclohexyl ester, in particular cyelohexyl chloroformate.

By "alkyl groups * ^1" both unsaturated and saturated

Alkyl groups fall. An example of a useful unsaturated haloformic acid ester is the compound of the formula CH _{2 * = CHO.CO.Cl.}

Examples of suitable haloformic acid eryl esters are haloformic acid phenyl esters and naphthyl esters.

Examples of suitable lialogenemic acid aralkyl ethers are those esters which have 1 to 8 carbon atoms in the alkyl chain have, in particular Halogenformiseneäurebenzyleeter and particularly preferably chloroform acid benzyl ether.

A compilation of most of those known from literature Chloroformic acid ester can be found in "Chemical Reviews", 1964 » Page 64.

As a rule, bind allergen proteins are quite resistant to denaturation. You can therefore usually click on relative high temperatures can be heated without denaturing. The process for the preparation of the desensitizers according to the invention can therefore be carried out over a wide temperature range, although in practice it is not generally necessary or desirable, a temperature above

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T953-9.S5

10O ⁰ C to use. Practically, it is preferable to perform the tree Ümeetaung at temperature, ie at about 20 ⁰ C.

When carrying out the process , the pH is 1 to 7. The process is preferably carried out at a pH of 4 to 6, preferably around 5 »

At a pH below about 4, the reaction evidently proceeds only with great difficulty , while at a pH above 6 it only proceeds completely satisfactorily if the pH drops to below 6 soon after the onset of the reaction.

It is preferred to treat the protein fraction of an aqueous allergen extract with the haloformic acid ester in order to obtain the desired desensitizing agent, that is to say the modified allergen. The protein fraction of the allergen extract is usually obtained by ex- the allergen- containing substance with a suitable solvent in a known manner. tracted, for which water is usually used, but oily extraction agents can also be used in some cases, and then the non-protein material is removed, for example by dialysis, precipitation or gel filtration. Such methods are described in "Journal of Allergy", Volume 13, 1942, pages 177 - 203 describes, in particular page 187, in more detail. Usually, the extract is thoroughly purified, for example by exhaustive dialysis.

When an aqueous allergen extract is used, the haloformic acid ester usually becomes immediately aqueous

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Extract added. The reaction mixture becomes violent for some time stirred / usually about 15 minutes. Then you leave that Stand the reaction mixture while the insoluble precipitate of the modified allergen settles »The modified allergen can then centrifuged off. It is preferred to wash the precipitate to remove impurities. this can by washing with a urea solution and then done with a sodium chloride solution.

The modified allergens produced in accordance with the invention are almost always insoluble if the starting material has been sufficiently purified to remove solubilizing impurities. Although the invention is not limited to any particular theoretical explanation of the implementation «; it is assumed that haloformic acid esters form crosslinking points or bridges between reactive groups on separate molecules of the allergen protein and also between reactive groups on the same molecule, thus causing both intermolecular and intramolecular crosslinking. The fact that the allergens modified according to the invention are mainly insoluble can be related to the formation of such cross-links, with the extent to which the allergen protein is purified before the reaction may play a role. Soluble "modified allergens " can be produced under special conditions. However, it is preferred to use the insoluble desensitizing agents for the treatment of allergies.

The desensitizing agent according to the invention is known in one Execution forms of the invention are used as medicaments,

0038Ag /

that an allergen modified according to the invention together mil; contains a parenterally safe carrier. Suitable carrier substances are isotonic salt solutions or buffer solutions, oily carriers and other substances known per se. The modified allergen is preferably water-insoluble here.

The desensitizing agent according to the invention or modified Allergen is generally administered by subcutaneous injection. The dosages vary depending on the allergic conditions of the Patient. .

When the modified allergen is administered parenterally According to the invention in animals it has been found that significant amounts of antibodies are formed in the bloodstream that are responsible for the modified allergen are specific. The modified allergens studied caused a high percentage of the test animals showed no anaphylactic reaction.

The invention is illustrated in more detail by the following examples.

example 1

Complete pollen from timothy grass was extracted with 10 times the volume of 0.001 M ammonium bicarbonate solution at pH 8 for 2 days, starting from a temperature of 4 ^° C. The filtered

Extract was exhaustively dialyo against the same solvent

^ siert. The retained solution was freeze-dried. A

«^ Part of 300 mg of the substance obtained was in 10 ml of a

* "v 0.2 M sodium acetate buffer solution dissolved at pH 7.0. The

The pH was adjusted to 7.0 by adding sodium hydroxide solution held. Somewhat insoluble substances were centrifuged off. It

BA *

0.3 ml of ethyl chloroformate were added. The mixture was stirred vigorously for 15 minutes at room temperature and then allowed to stand overnight. The precipitate formed was centrifuged off, washed four times in 15 ml of an 8 in urea solution and three times in 15 ml of a 0.9% sodium chloride solution and suspended in a sodium chloride solution. Estimated yield (from the dry weight of a sample) 100 mg.

When injected into guinea pigs, the product did not release any antigens, as can be seen from the following experiment.

Guinea pigs were passively sensitized by specific Rabbit antiserum of timothy grass pollen in dilutions in the Skin has been injected, as is done with a passive skin test for anaphylaxis.

After an 18 hour latency period, doses of the treated timothy grass pollen product were injected subcutaneously. Intravenously a blue dye was injected, causing the reaction of the grass antigen with the antibody would be visible in the skin as blue welts. The results obtained are in Table I listed.

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subcutaneous in
injected Guinea pig
Allergen 2.5 mg Thinning of the kenxneh
Antieerums and Strisa-
sea in am
1:10 1:50 1: 250 Is1250 modified 1.25 mg Allergen auE 250
125 mg
mg Timothy grass 32 mg negative 10 mg 500 mg Timothy grass 100 mg > 30> 30> 30 28 50 mg > 30> 30 28 25. Allergen 10 mg 18 + - 18 -

It follows that with the pollen preparation according to the invention injected guinea pigs gave no reaction, while the guinea pigs given only 10 ug of untreated timothy pollen allergen injected gave a positive reaction. The product was able to induce the formation of serum antibodies, * which react against a complete Lyschgrass pollen tract, as emerged from the following experiment.

6 guinea pigs were given 2.5 mg of the Lieechgraspollenpoly » kidneys injected subcutaneously. After three weeks the following tests were carried out to show that this was treating Foil product the formation of antibodies in the guinea pigs Chen had stimulated and that the antibodies were specific for timothy grass. Dilutions of timothy grass pollen allergen were intradermally into the skin on the crouching of the guinea pigs injected. Immediately thereafter, oin blue dye was intravenous

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- ιό - '■■■ -.

administered. It has been found that injection of as little as 0.016 µg of Lyme grass pollen xlergen causes a striatum remission in the Skin of 12 mm in diameter appeared. The fact that through the ' Polymers that raised antibody imaging in guinea pigs.

Example 2

Production of insoluble m All erge nderivat with Chlorameiaen-Bäureäthyle ater

50 mg of partially purified bluegrass pollen extract were dissolved in 5 ml of a 0.2 M sodium acetate buffer solution at pH 5.3. 5 ml of a 20% ethyl chloroformate solution in chloroform were added. The mixture was stirred at room temperature for 2 hours. The 33 precipitate was centrifuged off, washed three times in 15 μl of absolute alcohol and three times in 15 ml of 0.5% borate buffer solution at pH 8.85 and finally suspended in a phenol saline solution.

Allergieve rs uoh

The suspension of insolubilized pollen was in a Concentration of 1 mg / ml in the skin of those sensitive to grass rubbed into allergic patients. At the same time it was the starting material and the phenol saline separately into others Rubbed in areas of skin from the same patient. You welt areas were measured after 10 minutes. You are in the following

Table II given in mm. It follows that the SiOH _r insoluble gemeoJbte allergen preparation at 20 times the concentration of the Äusgengsmaterials less than 1 # has on its allergen activity. The retained allergy is therefore negligibly small,

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- n - ■ ■

compared to that produced by the parent material.

Ing quni ta attempt

The insolubilized allergen and the starting material were each in a complete auxiliary solution according to Freud in a Emulsified concentration of 1 mg / ml. Groups of guinea pigs were placed subcutaneously with one and the other emulsion (0.5 ml). After 22 days, the animals were shaved off the planks. In the bald spots there was a number of intradernal injections of 0.5 ml each from a dilution series made from purified bluegrass pollen extract. A five pound Pontamine sky blue solution (0.4 ml) was immediately injected intravenously. After 20 minutes, the Strieae saturation eater was measured. They are given in Table III. It turns out that the pollen allergen treated with ethyl chloroformate Forms antibodies that react with the starting material.

Table Il

Patient 'treated extract of purified bluegrass phenol

No. of bluegrass pollen pollen extract cooking

1 mg / ml ~ 50 pg / ml sale

1
2

total

20th

46

42 115

0 0 1 2

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100 10 1 ο, ι 0.01 7th 0.001 - Ηβ pe m W. 6th W. - 14th 12th 10 8th • τ 8th - 15th 14th 12th 10 - 7th ie 12th 11th 10 7th - - 14th 12th 10 10 - - 14th 12th 9 7th 14th 12th 8th - 14th 12th 10 8th 14th 12th 9 «.

- 12 -

Table III

Guinea Pig Immuni Strlesiendurchnieeaer (en} for intrachen Numbering dermal extracts from Etspen fabric (0.5 mg) ffraapollen

1 purified

2 panicle grass pollen

1 extrajct

more insoluble

treated

Eispengrae-

pollen

extract

Example 3

Production of an insoluble allergen preparation with ChlorameisenaäureiBetliyleter 50 mg of a partially purified Eispengraapollen extract were dissolved in 5 ml of a 0.2 M sodium acetate buffer solution of pH 5-3. 5 ml of a 20 follow chloroformate · metnylesterlösung added. The mixture was stirred at room temperature for 2 hours. The precipitate was centrifuged off, washed three times in 15 ml of absolute alcohol and three times in 15 ml of a 0 _v 5% borate buffer solution of pH 8.85 and finally suspended in a phenol saline solution.

Allergy trial

The suspension of the insolubilized pollen preparation at a concentration of 1 mg / ml was in the skin of grass-sensitive rubbed into allergic patients. At the same time, in other skin areas of the same patients, the starting point

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material and the phenol saline solution rubbed in separately. The welt areas were measured after 10 minutes. They are given in the following Table XV in mm ^2, it is found that the insolubilized Allergenpräparai has less than $ 1 of the allergy effect in the 20-faohen concentration of the starting material. The lasting allergy effect is therefore negligibly small compared to that of the original material.

TableJEV

Patient insoluble, cleaned panicle phenolic

Kr, delter bluegrass extract, table salt

pollen extract 50 µg / ml 1 mg / ml

1 4 14 0

2 3 0 ^v 31

3 1 12 1

4 1 12 1 total 9 69

I ^ iun j. t ^ tsyersuch

The TmmunitSitsversuph was carried out according to Example 2, however, the treated insolubilized allergen preparation and the starting material each in a concentration of 10 mg / ml were extracted.

The results are shown in Table V below. It shows that the insolubilized allergen preparation retains the immunization specificity of the starting material.

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Table V

Heerschwein- Imrnuni- StriemendurefaBteeser (am) for intrachen Kr, yearing venous extract & close from fiispest- ' substance (5 mg) grass pollen

100 10 1 0.1 0.01 O ₁ OOl

Uä ryp ujt Ujt VX

1 cleaned 14 13 10

"Eispengras- _{17 ll5 12} "

* pollen- ^{x / X5 i2 1} ^

3 extract 16 15 12 12

4 Χβ 15 10 10

5 insoluble 14 12 8

c behsndslter _ΛΑ ,, _Q

^ö bluegrass ¹⁴ - ^{11 3}

7 pollen- 12 11 8

8 extract _{12 10?}

Manufacture of an insoluble allergy preparation with chloroformic acid secondary ester.

50 mg of a partially purified hispengrass pollen extract were dissolved in 5 ml of a 0.2 M sodium acetate buffer solution of pH 5.3. 5 ml of a 20% benzyl chloroformate solution in chloroform were added. The mixture was stirred at room temperature for 2 hours. The precipitate was centrifuged off and washed three times in 15 ml of absolute alcohol and three times in 15 ml of a 0.5% borate buffer solution with a pH of 0.85 and finally suspended in one.

The suspension of the solubilized filler in a concentration rm% 1 mg / mX ^ n ^ us in the skin of a grass

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rubbed into sensitive allergic patients. Equilateral became raw material and the phenolic koohsalm solution in others rubbed in separate areas of skin of the same patient. The welt areas were measured after 10 minutes. they are

2
given in mm in Table VI below. It shows that the insoluble pollen preparation at 10 times the concentration of the starting material has less than 15% of its allergy. The lasting allergy effect is therefore negligibly small compared to that of the exposure material.

* Table VI

Patient insoluble, cleaned panicle phenolic

No. delter bluegrass extract table salt

pollen extract 100 µg / ml

1 mg / ml - _ ^ ·

1 2 '30 2

2 0 20 θ '

3 0 21 0

4 .0 23 0 total 2 94 '.

Iamunitätverreu ch

The experiment was carried out according to Example 3. Results are shown in Table VII below. It turns out that the insolubilized allergen preparation retains the immunization specificity of the starting material.

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Table VII

Guinea pig immune welts (mm) for intrachial amounts of venous extract aue Kispen fabric (9 ag) grass pollen

purified Eispengrasll

extrcött

100

14 14 12

0.1

μ «

0.01 0.001

μβ

ίο ίο 7 9

9 6 7

insoluble 13 10 10 8 treated

Icepengrass »~ ~

pollen extract 11 10 7 -

- patent claims -

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Claims (1)

Fatentan s d ruche 1. Allergy desensitizers from one Allergen, at least partially intermolecular and / or intramolecular to a haloformic acid ester of the general formula XCOOR, in which X is a fluorine, chlorine, bromine or iodine atom and R is an aliphatic ©, aromatic, araliphatic or heterocyclic, optionally substituted group is bonded. 2. Desensitization agent according to claim 1, daduroh It is labeled as an allergen, a protein fraction from an aqueous extract of an allergenic substance. 3. Desensitizer according to claim 1 or 2, characterized gekennzeichne t that there is an alkyl, aryl or aralkyl chloroformate as the haloformate contains. 4. Desensitizer according to claim 1 to 3, characterized Marked that there is also a parenteral contains harmless carrier. 5: Desensitizer according to claim 4, characterized in that it contains an isotonic salt solution or a buffer solution or an oil as a carrier. 5. Process for the preparation of the broom sensitizer also claims 1 to 3, characterized in that 009818/1732 I · i M - 18 - that Ban is an allargen with a Halogenaaeisaneäureeater of the general formula XCOOR, in which X is a fluorine, chlorine, broa or iodine atom and H an aliphatic, aromatic, araliphatic or heterocyclic optionally substituted group, at a pH of 1 to 7 treated and the modified Allergan obtained isolated. 7 * The method according to claim 6, characterized in that that the protein fraction of an aqueous extract of an allergen-containing substance is used as the allergen. 8. The method according to claim 6 to 7, characterized in that one is used as a haloformic acid ester Chloroformic acid ester used. 9. The method according to claim 6 to 8, characterized in that one is used as a haloformic acid ester Alkyl, aryl or aralkyl esters are used. 10. The method according to claim 9, characterized in that that one as halogen formic acid ester chloroformic acid methyl, ethyl, propyl, butyl, cyclohexyl, phenyl or -benzyl ester used. 11. The method according to claim 6 to 10, characterized in that the reaction is carried out at a pH of 4 to S _9, in particular of about 5 _t . 12. The method according to claim 6 to 11, characterized in that the reaction is carried out at a temperature of about 2O ⁰ C. 008818/1732