typically , the allergen is modified by treatment with a dialdehyde such as glutaraldehyde , in aqueous solution at a ph of between 5 and 10 , typically about 7 , and a temperature of between 0 ° c . and 100 ° c ., more usually between 4 ° c . and 37 ° c ., for up to 10 hours , for example about two hours at room temperature . the ratio of allergen to glutaraldehyde is typically in the range 50 : 1 to 2 : 1 , for example about 10 : 1 . the intermediate can be freeze dried or used in the next stage . a solution of the modified allergen , typically at ph 7 ± 1 , obtained either as the reaction mixture from the cross - linking process or from the solvation of a solid , is then mixed with a solution of tyrosine in a strong aqueous acid . the strong acid is usually an inorganic acid , preferably hydrochloric acid . the solution of allergen used in this step typically contains between 0 . 1 μg / ml and 1000 μg / ml allergen protein , for example about 400 μg / ml . the ratio of allergen : tyrosine in the mixture is typically in the range 1 : 4 × 105 to 1 : 1 × 10 2 w / w . the resulting mixture of solutions of allergen and tyrosine is neutralized . by neutralization is meant an adjustment of ph to a value within the range 4 . 0 to 7 . 5 . it is important that , at no time , or at least at no prolonged time , during the neutralization does the ph of the solution rise appreciably above 7 . 5 . this condition can be met by vigorous stirring of the solution and by the use only of the required amount of base , if desired . various buffering agents can usefully be added to the solutions of allergen to assist in ph control during the mixing and neutralizing stages . a particularly useful method of carrying out the neutralization is for separate streams of the solution of tyrosine in acid and the neutralizing base to be run into the solution of allergen . the rates of flow of the added solutions are controlled by ph - state , that is , by equipment which regulates the flow of one or both of the solutions so that the ph of the reaction mixture remains substantially constant at a predetermined level . we have found that optimum results are usually obtained by ph control within the range 6 . 5 to 7 . 5 though the precise ph may vary according to the nature of the allergen . the result of the neutralization is the immediate precipitation of the tyrosine , within and / or upon which the solution of allergen is occluded and / or adsorbed . after the precipitation the mixture is either washed immediately or allowed to stand for a period of from a few hours to a day or two prior to washing . the resulting precipitate may be removed from the solution by centrifugation or filtration and washed , e . g ., with phenol - saline , before being resuspended in a physiologically - acceptable carrier such as phenol - saline , or sterile water , to produce an injectable composition suitable for use in desensitization therapy in combination with 3 - dmpl . mpl which has been dissolved by the method described in preparation 3 below or by sonication can be diluted by various means prior to its addition to tyrosine adsorbates of allergens or modified allergens . the preparation of mpl is initially made at a concentration of typically between 0 . 5 mg / ml and 4 mg / ml , for example 1 mg per ml . it can then be diluted to a concentration of between 500 μg / ml and 20 μg / ml , preferably about 100 μg / ml . this dilution can be made in pure water , or in an aqueous glycerol solution containing between 1 % and 4 %, preferably 2 %, glycerol . such dilutions can then be added to a suspension of the tyrosine adsorbate prepared as described above . for convenience , the concentration of the mpl solution and the tyrosine adsorbate suspension , respectively , may be selected such that approximately equal volumes of each are admixed to obtain the final product for injection . a typical final product contains about 100 μg / ml of allergen and about 250 μg / ml of mpl . a neutral solution of approximately 0 . 5 mg / ml grass pollen extract which had been partially purified by dialysis or fractionation was chemically modified by the addition of an equal volume of 0 . 25 % w / v glutaraldehyde and the mixture stirred for approximately 2 hours at room temperature . to the above mixture was added phosphate buffer solution at a ph of 7 ± 1 . the allergen solution was co - precipitated with tyrosine by the simultaneous addition of one volume of l - tyrosine in hci ( prepared by dissolving 24 g l - tyrosine to 100 ml with 3 . 8m hci ) and one volume of 3 . 2m naoh , to four volumes of allergen solution , with vigorous agitation . the suspension so formed was centrifuged , washed repeatedly with buffered saline to remove contaminants and resuspended to the original volume in buffered saline ph 6 ± 1 . 3 - dmpl suitable for coadministration with the above formulation was prepared as described in preparation 3 below . eight mg of ovalbumin ( xoa ) were dissolved by mixing in 20 ml of evans solution . next , 6 . 9 ml of phosphate buffer were added with mixing . the solution was placed in a 100 ml beaker containing a magnetic stir bar . while mixing using a magnetic stirrer , 6 . 9 ml of 3 . 2n naoh and 6 . 9 ml of 3 . 8n hcl containing 24 % w / v tyrosine were added simultaneously , dropwise , over a period of 5 min to form a precipitate . the mixture was allowed to stir for an additional 5 min and then transferred to a 50 ml centrifuge tube and centrifuged for 10 min at 2500 rpm . after centrifugation the supernatant was decanted and the pelleted precipitate resuspended in 40 ml of phosphate buffer . the mixture was centrifuged for 5 min at 2500 rpm . after centrifugation the supernatant was decanted and the precipitate resuspended in 40 ml of phosphate buffer . the mixture was centrifuged for 5 min at 2500 rpm . after centrifugation the supernatant was decanted and the pelleted precipitate resuspended in 40 ml of phosphate buffered saline , ph 7 . 2 , containing 0 . 4 % v / v glycerol and 0 . 0 1 % w / v thimerosal as a preservative . the final product contained approximately 40 mg / ml of tyrosine adsorbate . assuming 100 % binding of the xoa to the tyrosine adsorbate , the xao was at 200 μg / ml in the final product . the xoa - tyrosine adsorbate was stored at 4 ° c . until needed . a 4 mg / ml solution of 1 , 2 - dipalmitoyl - sn - glycero - 3 - phosphocholine (“ dppc ”) in absolute ethanol was prepared . for each 1 mg of mpl ®- tea salt to be solubilized , 27 μl of dppc was added to dissolve the mpl ®. the ethanol was removed by blowing a stream of n 2 gently into the vial . next 1 ml of pyrogen - free water for injection was added for each mg of mpl ® in the dried mpl ®/ dppc mixture . the solution was sonicated in a bath sonicator at 60 - 70 ° c . until clear . the mpl ®/ dppc solution was then filter sterilized by filtration through a sfca 290 - 4520 nalgene 0 . 2 μm filter . the mpl ®/ dppc solution was aseptically dispensed at 1 mg / ml into depyrogenated vials , labelled mpl ®- af , and stored at 4 ° c . th1 inducing activity in mice can be equated with the production of igg2a and igg2b antibodies and the th2 inducing activity with the production of igg 1 antibodies and ige antibodies . therefore , as an example , an experiment was carried out in mice to demonstrate the profiles of the allergen specific antibodies to an exemplar allergen ovalbumin ( xoa ), which is a well - known food allergen derived from chicken eggs . it was confirmed that a formulation consisting of mpl + xoa + tyrosine stimulated a more advantageous antibody profile than mpl + xoa , xoa + tyrosine , or xoa alone . groups of 8 balb / c female mice , 6 - 8 weeks of age , were injected subcutaneously in the inguinal area with 0 . 2 ml of one of the following vaccines : xoa + tyrosine : the xoa tyrosine adsorbate prepared in preparation 2 above was diluted with an equal volume of phosphate buffered saline within 30 min prior to injection . xoa + tyrosine + mpl : the xoa tyrosine adsorbate prepared in preparation 2 above was diluted with an equal volume of mpl ®- af at 500 μg / ml in phosphate buffered saline within 30 min prior to injection . xoa + mpl : xoa was dissolved in phosphate buffered saline at 200 μg / ml and diluted with an equal volume of mpl ®- af at 500 μg / ml in phosphate buffered saline within 30 min prior to injection . xoa alone : xoa was dissolved at 200 μg / ml in phosphate buffered saline and diluted with an equal volume of phosphate buffered saline . twenty - one days later the four groups of mice were boosted with 0 . 2 ml of freshly prepared vaccines . fourteen days following the booster the mice were bled and the sera separated and stored at − 70 ° c . until assay . the sera were assayed by conventional elisa technique using horseradish conjugated goat anti - mouse igg1 , igg2a , and igg2b antibodies purchased from southern biotechnology , inc . ( birmingham , ala .) and used according to the manufacturer &# 39 ; s instruction . the igg1 , igg2a , and igg2b titers represent the reciprocal serum dilution giving a reading of & gt ; 0 . 1 od units at a 490 . the serum ige levels were measured using an anti - ige capture elisa followed by the use of a biotinylated ovalbumin probe . binding was measured following the addition of a horseradish conjugated strepavidin preparation . the results are reported in table 1 as od units at a 490 . of particular important is the fact that the combination of allergen + tyrosine + mpl induces less allergen specific ige antibody than the other combinations . furthermore , the ratio of igg2a or igg2b to igg1 antibodies is greater and consistent with the highest levels of the two former antibody isotypes seen in the experiment in the mice given allergen + tyrosine + mpl than in any other group of mice . this is indicative of a better ratio of the th1 cell induction over the th2 cell induction in this group compared with that induced in the other groups of mice .
provided is a pharmaceutical composition comprising tyrosine , an optionally modified allergen , and 3 - dmpl , the composition is useful in the prevention and treatment of allergies .