Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving antigen-antibody binding – specific binding protein...
Reissue Patent
2002-04-04
2004-02-10
Mertz, Prema (Department: 1647)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving antigen-antibody binding, specific binding protein...
C435S007200, C435S007210, C435S325000, C435S326000, C435S372000, C435S372300, C435S373000, C435S375000, C530S351000
Reissue Patent
active
RE038423
ABSTRACT:
CROSS-REFERENCE TO RELATED APPLICATIONS
This application is a 371 of WO97/16732.
The present invention concerns a process for in vitro analysis of toxic and allergenic substances. Substances intended for use as pharmaceuticals, food additives, cosmetic or hygienic products, industrial chemicals and other sub-stances are analysed for adverse reactions. Such reactions may be allergic reactions, skin irritating effects and toxic effects.
Today such tests are performed in vivo on animals. Animals are expensive to raise and keep. Further, some tests may involve suffering of the animals.
The present invention provides a process for in vitro analysis of the adverse effects of substances. The analysis of the substances is performed on blood cells from warm blooded animals. No animals are involved since the substances are tested on human cells i.e. cells from the same species as they are intended for. Some substances may not have the same effect on different species, and tests performed on animals may give false results. Therefore the test is preferably performed with human blood.
Thus, the method will be offered as an alternative to animal tests for food additives, cosmetic or hygienic products, industrial chemical, drugs and other substances where adverse reactions are to be avoided.
In vitro evaluation of toxic effects on the immune system has been tested on splenocytes from mice (“In vitro evaluation of drug-induced toxic effects on the immune system as assessed by proliferative assays and cytokine production”, M. Pallardy et al. Eur. Cytokine Net., Vol. 2 No. 3, May-June 1991, pp. 201-206). The method was poorly effective detecting molecules inducing autoimmunity and hypersensitivity.
It has now turned out that according to the present invention it is possible to analyse adverse tissue reactions to foreign substances in vitro by cultivating them with human whole blood, prevented from coagulating and analysing the cytokine produced by the human cells.
Before a substance has been in use and before the target group has been exposed the present method offers the possibility to predict whether a risk for adverse reactions of inflammatory or allergic type will occur.
The invention provides an interpretation key for evaluating the adverse tissue reactions to foreign substances.
SUMMARY OF THE INVENTION
The method is defined in claim 1 and is based on cultivation of blood cells in vitro. The substance to be evaluated is added to the blood culture. If the reactive cells of the blood recognises the test material as foreign or damaging a production of inflammatory signal substances, cytokines occurs. The blood contains different white blood cells with different functions in the response to foreign substances. The different types of inflammatory cells secrete different repertoirs of cytokines. The patterns, (profiles) of the secreted cytokines indicate what compartment of the immune system that has been activated. Thereby is it also possible to predict what kind of adverse reaction to the expected from a substance if administered to humans or experimental animals. Some substances will induce a typical cytokine profile driving the immune system towards IgE mediated allergy, this indicating a potential of those substances to evoke asthma, hay fever and urticaria. Other substances will induce a cytokine pattern driving the immune system to cellular immunity thus having the potential to result in contact excema as an adverse reaction. Finally, a chemical or toxic effect will result in a cytokine pattern typical of alarm reactions seen in tissue damage and operations. Thus, the concept that the present method is based on is to use an interpretation of the cytokine pattern that a substance can induce in blood in vitro cells to predict whether the substance has the potential to cause allergy of different types or is generally hazardous to induce tissue damaging or toxic effects.
We classify adverse tissue reactions to foreign substances as follows.
Class 0: Tissue damage. Unspecific chemical, mechanical or physical damage.
Class I: Allergic immune reaction type I. This is also named immediate type hypersensitivity and is mediated by IgE antibodies produced specifically to the foreign substance. An acute inflammatory reaction is produced, histamine is often produced and examples of symptoms are asthma hay fever, urticaria and rhinitis. This type requires that the substance can induce a fully mature immune response where all components of the immune system participates. This is also considered as a final step immune reaction.
Class IV: Inflammatory immune reaction type IV. This is also named delayed type hypersensitivity and is mediated by sensitised T lymphoccytes type TH-I, considered as a first step type immune reaction. Allergic contact dermatitis is an example of this type of reaction.
Class 0 is our own classification of unspecific damage to the immune system. For Class I and Class IV see “Immunology” by Ivan Roitt, Jonathan Brostoff and David Male, Gower Medical Publishing London—New York, 1989, page 1.9-19.20 and 22.1-22.10, which is incorporated as a reference.
Three main types of cytokine profiles have been identified in the present work.
Class 0 type: Secretion of alarm cytokines indicating damage to connective tissue, fibroblasts, endothelial cells, epithelial cells and unspecific inflammatory white blood cells. Members of this group are IL-1, IL-6, IL-12 and TNF.
Class I type: Secretion of cytokines of an immune response type from lymphocytes and inflammatory cells. These includes cytokines from Class 0 and in addition IFN-gamma, Neopterin, IL-2 and IL-10. Theoretically as known from animal in vivo studies also IL-4 and IL-5 should be produced here, but these substances are notoriously difficult to determine and are therefore not routinely included in our test protocols.
Class IV type: Secretion of the cytokines of Class 0 unspecific type and in addition IL-2, IL-10 and IFN-gamma.
A finding that could not predicted was that the complete final type immune response Class I drives to Neopterin production whereas the Class IV primary type immune response does not stimulate the immune system as long as to Neopterin production.
In the three groups of cytokines listed above only preferably selected representative members are listed. The groups may contain other cytokines to be used and the patent is claimed for the use of analysis of all substances listed in The Cytokine Facts Book, Eds Callard R. E. and Gearing, A. J. H., Academic Press 1994 hereby incorporated as a reference and future updated issues to predict the grade of maturity of the response that a substance has the potential to evoke. At present 50 substances.
Interleukins
IL-1, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-11, IL-12, IL-13, IL-14, IL-15
Other Cytokines (in alphabetical order)
BDNF, CNTF, EGF, Epo, FGF, G-CSF, GM-CSF, I-309/TCA-3, &ggr;IP-10, INF&agr;, IFN&bgr;, IFN&ggr;, LIF, LT(TNF&bgr;), MCP-1,2 and 3, M-CSF, MIF, MIP-1&agr;, MIP-I1&bgr;, MIP-2, NGF, NT-3, NT-4, OSM, PBP, PBSF, PDGF, PG-4, RANTES, SCF, TGF&agr;, TGF&bgr;, TNF&agr;, Tpo, VEGF
According to the invention the presence of inflammatory immune reaction type IV is preferably analyses by using IL-8. It has turned out that IL-8 is developed and is present in raised levels during type IV reactions.
Neopterin being present in higher amounts when type I reaction is at hand, IL-8 and/or neopterin can especially be used to distinguish between the inflammatory immune reaction type IV and the allergic immune reaction type I.
Based on the outcome of the in vitro test, the cytokine pattern induced by a the substance under investigation, a prediction will be made regarding the potential of the substance to cause adverse reaction if humans or animals are exposed to it. Whole blood, preferably venous blood is used. The blood is prevented from coagulating. This can be done by the use of endotoxin free heparin tubes. Other methods may also be used such as addition of EDTA or citrate. Alternatively the blood can be shaken with glass beads. The leukocyte counts
Biovator Technologies AB
Hamud Fozia
Mertz Prema
Young & Thompson
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