PL183484B1 - Composition of tyrosine and polymerised allergen - Google Patents

Abstract

A pharmaceutical composition comprising tyrosine and a polymerised allergen is prepared by (a) polymerising an allergen, (b) mixing an aqueous solution of the allergen with a solution of tyrosine in a strong aqueous acid, (c) neutralising the mixture of solutions, thereby co-precipitating tyrosine and polymerised allergen, and (d) optionally, mixing the product with a physiologically acceptable carrier.

Description

The present invention relates to novel compositions for use in the treatment or desensitization therapy of allergy sufferers, containing tyrosine and a polymerized allergen, and a process for the preparation of such compositions.

British patent specification GB-A-1492973 describes a method of producing tyrosine coprecipitates containing a dispersed modified allergen. The allergen was modified by treatment with an agent such as glutaraldehyde to cause intermolecular cross-linking and to reduce the allergenicity of the product compared to the unmodified allergen.

EP-A-0367306 and the publication Nakłady et al. (Allergy, 54 [1985] 437 et seq.) Describe methods for preparing polymerized allergens by prolonged treatment with a cross-linking agent to effect intermolecular cross-linking, followed by filtration or dialysis to remove unpolymerized product. Polymerized allergens are described as having reduced allergenicity.

The present invention relates to a pharmaceutical composition for the treatment or desensitization of allergy sufferers containing tyrosine and a polymerized allergen, characterized in that the polymerized allergen has a molecular weight of at least 100 kDalton. Preferably the allergen is coated and / or adsorbed onto tyrosine, e.g. by co-precipitation.

The allergen can be derived from any allergenic substance such as pollen (e.g., ragweed or birch pollen), food, insect venom, mold, animal dander, or house dust mites (D. farinae or D. pteronyssinus). In a preferred embodiment of the invention the allergen is derived from D. pteronyssinus. As used herein, the term "allergen" includes a mixture of allergens from a single source or from more than one source.

The invention also relates to a process for the preparation of the above-defined pharmaceutical composition, characterized in that it comprises the steps of (a) polymerizing the allergen by dialdehyde treatment, (b) removing by gel filtration or dialysis products with a molecular weight of less than 100 kDalton, (c) mixing the aqueous solution allergen with tyrosine solution in a free solution of strong acid, (d) neutralizing the mixture of solutions,

By co-precipitating tyrosine and the polymerized allergen, and (e) optionally, a mixture of the product with a physiologically acceptable carrier.

Suitable physiologically acceptable carriers include phenol-saline and sterile water.

The allergen is polymerized by the action of a dialdehyde such as glutaraldehyde in an aqueous solution at a pH of 3 to 10, typically 7 ± 1 and at a temperature between 0 and 100 ° C, typically between 4 and 37 ° C, for up to 10 hours, e.g. two hours at room temperature. The ratio of allergen to glutaraldehyde is usually in the range 1:25 to 1: 2, e.g. about 1: 4 by weight, although higher amounts of allergen can be used in combination with longer reaction times (see e.g. EP-A-0367306, using the ratio about 3: 1 and reaction time about 7 hours.

The low molecular weight product is then removed by gel filtration or dialysis, e.g. tangential flow dialysis, the product is freeze dried or used directly in the next step. The molecular weight cutoff is at least 100 kDa, e.g., at least 250 kDa, more preferably 300 kDa.

The polymerized allergen solution at pH 7 ± 1, obtained as a reaction mixture from the polymerization process or by dissolving the solid, is then mixed with a tyrosine solution in an aqueous strong acid solution. The strong acid is usually an inorganic acid, preferably hydrochloric acid. The polymerized allergen solution used in this step typically contains between 0.1 µg / ml and 100 µg / ml allergen protein. The ratio of allergen: tyrosine in the mixture is typically 1: 4x10 ⁵ to 1: 4x10 ² w / w.

The resulting mixture of allergen and tyrosine solutions is neutralized. By neutralization is meant the adjustment of the pH to a value in the range 4.0 to 7.5. It is important that the pH of the solution never rises well above 7.5 during neutralization, or at least for a long time. This condition is met by rapidly agitating the solution and by using only the required amount of base, if necessary. Various useful buffering agents can be added to the allergen solutions to facilitate pH control during the mixing and neutralization steps.

A particularly useful method of carrying out the neutralization is to introduce separate streams of the tyrosine in acid solution and the neutralizing base into the allergen solution. The flow rates of the added solutions are controlled by a pH stat, i.e. by a device regulating the flow of one or both of the solutions, such that the pH of the reaction mixture remains substantially constant at a given level. It has been found that the best results are usually obtained by adjusting the pH to the range 6.5 to 7.5, although the exact pH may vary depending on the nature of the allergen.

The neutralization result is the immediate precipitation of tyrosine on or in which the allergen solution is occluded and / or adsorbed. After precipitation, the mixture is washed immediately or left to stand for several hours to a day or two before washing. Preferably, a fine sediment is obtained, which is achieved by rapid neutralization of the solution combined with vigorous stirring at the same time.

The resulting precipitate may be removed from the solution by centrifugation or filtration and washing, e.g., with phenol-saline, then suspended in a physiologically acceptable carrier such as phenol-saline or sterile water, producing an injectable composition suitable for use in desensitisation therapy.

The following example illustrates the present invention:

Example. A neutral solution of about 2.5 mg / ml of D. pteronyssinus protein extract, partially purified by dialysis or fractionation, was polymerized by adding an equal volume of a 1% (w / w) glutaraldehyde solution and the mixture was stirred for about 2 hours at room temperature. The reaction was stopped by adding an equal volume of the 2% w / v glycine solution and the mixture was stirred for a further hour at room temperature. Low molecular weight material was removed by diafiltration through a membrane excluding 300 kDa molecular weight. The mixture was then sterile filtered and freeze dried.

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The polymerized allergen solution was prepared directly from the sterile filtered solution or by reconstitution of the freeze-dried solid. This solution contained 10 µg / ml in phosphate buffer pH 7 ± 1. The allergen solution was co-precipitated with tyrosine by simultaneously adding one volume of L-tyrosine in HCl (prepared by dissolving 24 g of L-tyrosine to 100 mL of 3.4M HCl) and one volume of 3.2M NaOH to 4 volumes of the allergen solution with vigorous stirring. The resulting suspension was centrifuged, washed again with buffered saline to remove impurities, and resuspended in solution to the original volume using buffered saline pH 6 ± 1.

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Claims (4)

Patent claims A pharmaceutical composition for the treatment or desensitisation of allergy sufferers, comprising tyrosine and a polymerized allergen, characterized in that the polymerized allergen has a molecular weight of at least 100,000. 2. A composition according to claim 1 5. A composition according to claim 1, characterized in that the allergen is coated and / or adsorbed on tyrosine. 3. A composition according to p. The composition of claim 1 or 2, characterized in that it comprises an allergen derived from D. pteronyssinus. 4. Process for the preparation of a pharmaceutical composition for the treatment or desensitization of allergy sufferers containing tyrosine and a polymerized allergen, characterized by the steps of (a) polymerizing the allergen by dialdehyde treatment, (b) removing products with a molecular weight of less than 100 by gel filtration or dialysis 000, (c) mixing the aqueous solution of the allergen with a solution of tyrosine in the aqueous solution of a strong acid, (d) neutralizing the mixture of solutions, co-precipitating tyrosine and the polymerized allergen, and (e) optionally mixing the product with a physiologically acceptable carrier.