ALLERGENIC EXTRACTS

Abstract: This invention relates to a preparation of allergenic (antigenic) extracts useful in a systematic approach to diagnosis, immunotherapy and the monitoring thereof of allergic patients, and also to be used in diagnosis of other immunological disorders. The method comprises of an initial quick extraction step, wherein most of the immunologically irrelevant material is removed in the solute and discarded. The allergenic residue is recovered and is repeatedly extracted while retaining the solute, resulting in a sequence of allergenic extracts that are immunologically potent.

Description: Allergenic extracts The present invention relates to allergenic (antigenic) extracts to be used in diagnostics of immunological disorders preferably allergic diseases, the immunotherapy and the monitoring of the immunotherapy in allergic patients and to a method for the preparation of the same. People who suffer from allergy have allergic (reaginic) antibodies, called IgE antibodies. These antibodies combine with the offending substances, called allergens (antigens) to manifest an allergic reaction. It has been a practice to extract these allergens from their source to be used for diagnostic and therapeutic purposes. The prior art methods used in preparation of an allergenic extract especially for inhalation allergens such as pollens, mites, insects, molds, allergens from foods and other sources are to extract the source material in water or various solutions from two hours to overnight or more. This extract is then dialyzed to remove low molecular compounds and then lyophilized. In vivo and in vitro diagnostic test systems based on this so called crude extract show manifestation of allergic reaction and presence of IgE antibodies but fails to give more information which the clinician requires for the proper management of the patient. The extract so obtained contains about 90% irrelevant waste material consisting of carbohydrates, lipids, enzymes, toxic materials, irritants and number of other molecules which have no significance in immune response. This makes the test systems less sensitive and inaccurate. When used for the purpose of immunotherapy, the crude extract has not delivered any significant results and the presence of the waste materials causes mild to serious side effects during immunotherapy. Moreover this crude extract is not suitable in immunoassays to monitor the levels of IgG antibodies, the so called "blocking antibodies" during immunotherapy, because of the interference and high background level caused by irrelevant nonimmunological materials in the crude extract. Among the irrelevant waste materials, the crude extract contains only about 10% of number of different allergenic molecules. Attempts are also made to obtain purified allergens from these crude preparations through techniques such as gel filtration, ion exchange chromatography, monoclonal antibodies, DNA technology, synthetic peptides etc. Until now the use of these purified allergens have not led to any significant improvement in diagnosis, immunotherapy or monitoring the effects of immunotherapy. Because the purified allergens become denatured as a result of the purification processes and the immune system fails to recognize these denatured purified allergens. Extensive literature is available on the subject of methods for the preparation of allergenic extracts. The U.S. patents 3.591.677; 3.798.319; 4.031.199; 4.774.226 and 4.963.356 describe various methods and processes to prepare allergenic extract. In all of these methods the allergenic source material is initially extracted during 16 hours to as long as 5 days. Contrary to the conventional methods, the U.S. patent 4.364.938 employs a method wherein the source material is repeatedly extracted. The extracted solutes containing most of the allergenic activity is discarded and the residue is recovered. This residue either directly or after further treatment is suspended in 0.9% saline and administered to the patient by way of injections. The allergen release from the source material has also been studied by a number of investigators among them Marsh et al. J. Allergy Clin. Immunol. 1981:67.206-16 and Baraniuk et al. J. Allergy Clin.Immunol 1988: 81.1126- 34. To summarize problems in allergy, the crude extract contains too much irrelevant material and the purified allergens add to the complexity of the problem because most patients have IgE antibodies to more than one allergen in that particular source material. The method of the present invention overcomes these problems and it has neither been disclosed nor suggested in the prior art. It is the object of this invention to provide a novel process for the preparation of allergenic (antigenic) extracts which contain almost none of the immunologically irrelevant waste materials. It is the object of this invention to provide a systematic approach to diagnosis of patients with immunological disorders preferably allergies, a systematic approach to immunotherapy of an allergic patient and a systematic approach to monitoring the allergic patients who are undergoing immunotherapy. I have unexpectedly found that after the extraction of the source material according to the conventional methods, the residue which is left behind and discarded contains more immunological activity than the extract itself. After further experimentation it became quite clear that most of this i munogenic activity was in fact of allergenic nature and it depended upon the rate of release of allergenic molecules from the source material. The present invention provides a method to prepare an allergenic residue obtainable by extracting the allergenic source in an aqueous solvent whereby this extraction does not last longer than about five minutes. The solute containing most of the immunologically irrelevant material is discarded and the allergenic residue is recovered. Optionally this procedure may be repeated depending upon the allergenic source material. As soon as the extraction medium comes in contact with the source material, most of the irrelevant material immediately enters into the solution whereas there is a slight delay of the allergenic molecules to enter into the solution. This time of delay of the entry of the allergenic molecules into the solution is variable in each particular allergenic source material. Therefore outermost care should be taken to minimize the duration of this extraction step. The purpose of this step is the removal of most of the CO It was then extracted with 3 times 0.1 M NaOH as in the last step. The filtrates were gently neutralized with acetic acid and mixed to obtain Extract D Basic. All the extracts so obtained and concentrations or dilutions thereof are to be used in vivo and vitro diagnostic methods. These extracts are also to be used for immunotherapy and for the monitoring thereof. EXAMPLE 2 Repeating the procedure of example 1 , but replacing the pollen Dactylus glomerata with english rye grasspollen (Lolium perrene). In this case step 3 was repeated only one time ( different from two times in example 1 ) . EXAMPLE 3 Repeating the procedure of example 1 , but replacing the pollen Dactylus glomerata with timothy pollen (Phelum pratense). EXAMPLE 4 Repeating the procedure of example 1 , but replacing the pollen Dactylus glomerata with birch tree pollen (Betula verrucosa). In this case step 3 was repeated only one time ( different from two times in example 1 ) . EXAMPLE 5 Repeating the procedure of example 1 , but replacing the pollen Dactylus glomerata with house dust mite (Dermatophagoides pteronyssinus) . In this case step 3 was repeated four times ( different from only two times in example 1 ) .

Claims: Claims 1. A process for preparing an allergenic residue from an allergenic source material comprising: subjecting the said allergenic source material to at least one extraction step, said extraction step lasts not longer than five minutes, said extraction step resulting in removal of most of the immunologically irrelevant material in the extracted solute and minimum or no loss of allergenic activity from the recovered allergenic residue. 2. An allergenic residue obtainable according to the process of claim 1. 3. A process for preparing at least one allergenic extract comprising: subjecting the residue of claim 2 to at least one subsequent extraction step (a), the total extraction time of said subsequent extraction (a) being shorter than four hours and said extraction step (a) resulting in the release of rapidly releasing allergenic activity in the extracted solute. 4. A process according to claim 3 wherein the total extraction time of extraction step (a) is shorter than two hours. 5. A process according to claim 3 or 4 wherein said extraction (a) comprises at least two sequential extractions. 6. A process according to claim 5 wherein the subsequent extractions of extraction (a) each lasts less than an hour. 7. A process to prepare at least one allergenic extract wherein the extracts obtainable according to any of claims 3 to 6 are combined in a pool. 8. A process for preparing at least one allergenic extract comprising: subjecting the residue obtainable from any of the subsequent extraction steps (a) according to any of claims 3 to 6 to at least one further extraction step (b), said further extraction step (b) lasting longer than four hours and said extraction step (b) resulting in release of most of the slowly releasing allergenic activity in the extracted solute. 9. A process according to claim 8 wherein said further extraction (b) comprises at least two sequential extractions. 10. A process to prepare at least one allergenic extract wherein the extracts obtainable according to any of claim 8 or 9 are combined in a pool. 11. A process for preparing an acidic allergenic extract wherein the allergenic residue obtainable from any of the extraction steps (b) of claims 8 or 9 is subjected to at least one additional extraction step (c) in an acidic solution and said extraction step (c) resulting in release of most of the immunogenic activity in the extracted solute. 12. A process for preparing an acidic allergenic extract according to claim 11 wherein the extraction step (c) lasts not longer than thirty minutes. 13. A process for preparing a series of acidic allergenic extracts according to claim 11 or 12 comprising carrying out repeated sequential extractions each lasting not longer than thirty minutes. 14. A process to prepare at least one allergenic extract wherein the extracts obtainable according to any of claims 11 to 13 are combined in a pool. 15. A process for preparing an alkaline allergenic extract wherein allergenic residue obtainable from any of the extraction steps (b) of claims 8 or 9, or any of the extraction steps (c) of any of claims 11 to 13 is subjected to at least one additional extraction step (d) in an alkaline solution and said extraction step (d) resulting in release of most of the immunogenic activity in the extracted solute. 16. A process for preparing an alkaline allergenic extract according to claim 15 wherein the extraction step (d) lasts not longer than thirty minutes. 17. A process for preparing a series of alkaline allergenic extracts according to claim 15 or 16 comprising carrying out repeated sequential extractions each lasting 14 not longer than thirty minutes. 18. A process to prepare at least one allergenic extract wherein the extracts obtainable according to any of claims 15 to 17 are combined in a pool. 19. The allergenic extract obtainable according to the process of any one of claims 3 to 18. 20. The use of at least one allergenic extract according to claim 19 in diagnostics for immunological disorders, preferably to detect allergic disease. 21. The use of at least one allergenic extract according to claim 19 in therapeutic treatment of patients, preferably in immunotherapy. 22. The use of at least one allergenic extract according to claim 19 in monitoring the patients during immunotherapy.

Inventors: KOSHTE VINAY LUCAS, KOSHTE, VINAY, LUCAS
Applicants: KOSHTE VINAY LUCAS, KOSHTE, VINAY, LUCAS
Application year: 1993
Main classification: A61K39
IPC codes: A61K35, A61K36, A61K39, A61P37, C07G99, C07K1, C07K14