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Value of animal models for predicting hypersensitivity reactions to medicinal products
Toxicology 129 (1998) 27 – 35
Value of animal models for predicting hypersensitivity reactions to medicinal products G. Choquet-Kastylevsky *, J. Descotes Department of Pharmacology, Medical Toxicology and En6ironmental Medicine, et INSERM U98 -X, Faculte´ de Me´decine Lyon-RTH Lae¨nnec, 69008 Lyon, France
Abstract Although hypersensitivity reactions induced by medicinal products and chemicals are relatively common, few predictive models are available. A major difficulty is our currently limited understanding of the mechanisms involved, and efforts should be paid to better defining drug immunogenicity, hapten formation and immune effector mechanisms. A second difficulty is the multiplicity of clinical manifestations presumably due to varying mechanisms. Available models can only predict a few of these reactions. Anaphylaxis models in guinea-pigs can be only used for the safety assessment of macromolecules which are neither humanized or of human origin, whereas guinea-pig or mouse models can detect the majority of human contact sensitizers. In addition to the extensive validation of existing models, promising avenues of research are expected to be found in the use of novel animal models, particularly those using genetically modified animals, such as transgenic and knock-out mice. © 1998 Published by Elsevier Science Ireland Ltd. All rights reserved. Keywords: Allergy; Animal models; Hypersensitivity reactions; Immunotoxicity evaluation; Medicinal products
1. Introduction Hypersensitivity reactions are the most frequent adverse effects involving the immune system associated with medicinal products and chemical exposure. Even though reliable information is scarce on their actual incidence in humans, hypersensitivity reactions might account for approximately one third of recorded adverse drug reactions * Corresponding author.
based on the experience of post-marketing drug surveillance. Contact dermatitis and respiratory allergy are frequent complaints at the workplace, whereas allergy and chemical sensitivity are described in a significant fraction of the general population. Nevertheless, predictive animal models have not been considered a key priority until very recently. One explanation is that ‘allergic’ reactions in contrast to toxic effects have long been claimed to be unpredictable in animals. In addition, the search for understanding the mechanisms
0300-483X/98/$19.00 © 1998 Published by Elsevier Science Ireland Ltd. All rights reserved. PII S0300-483X(98)00060-2
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involved and the development of new diagnosis methods lagged behind recent advances in immunology as well as cellular/molecular biology. It is beyond doubt that hypersensitivity reactions induced by xenobiotics pose a major threat to human health. This review paper is an attempt to describe the current status of animal models predictive of hypersensitivity and to delineate possible avenues of research.
2. Immuno-allergy vs pseudo-allergy
2.1. Definitions Hypersensitivity reactions do not all involve the immune system, even though the generic term ‘allergy’ is commonly used. To avoid confusion, it is proposed to use the term ‘(immuno)-allergy’ only when highly specific mechanisms involving immunological memory and recognition are shown to be involved. The term ‘pseudo-allergic’ would describe those reactions which mimick immuno-allergy, but in which a specific immune mediated mechanism is not involved. The use of the term ‘pseudo-allergy’ as a synonym to ‘idiosyncrasy’, that is to say a reaction reflecting a particular susceptibility of individuals, is not recommended. When no indication of the mechanism involved is available, the general term ‘hypersensitivity’ is proposed to cover both possibly immuno-allergic and pseudo-allergic reactions.
2.2. Immuno-allergy One major feature of the immune system is the capacity to recognize foreign (‘non-self’) constituents and to memorize this encounter for subsequent accelerated and amplified immune responses. The mechanisms of immuno-allergic reactions to xenobiotics are ill-understood. In 1964, Gell and Coombs proposed a pathogenic classification of 4 types, namely immediate hypersensitivity or anaphylactic (type I, antibody-mediated (type II), immune complex-mediated (type III), and delayed hypersensitivity (type IV), reactions. Although this classification is still often referred to, it is too rigid and proves useless in
many instances, in particular when immuno-allergic reactions induced by xenobiotics are considered. In addition, two major limitations of this classification are the lack of updating to take into account recent advances in our understanding of the immune system, and the dissection of mechanisms into separate categories (types) excluding the likely simultaneous involvement of two or more mechanisms. Whatever the actual pathogenesis of immunoallergic reactions induced by xenobiotics, two features are absolutely essential: (i) the offending xenobiotic (or its metabolites) must be directly immunogenic or act as haptens, and (ii) the responding host must be sensitized prior to developing a patent immuno-allergic reaction. The immune response can be mounted against the drug or a reactive metabolite (haptenic epitope), the protein to which it is bound (auto-antigenic epitope), or a new epitope formed by the drugprotein complex. In the latter two situations, the response can result in a drug-induced systemic auto-immune reaction, such as the lupus syndrome or scleroderma-like disease, most often associated with the presence of antinuclear autoantibodies antibodies, or an organspecific autoimmune reaction, such as pemphigus or auto-immune hemolytic anemia. However, even though common immunological mechanisms are suggested to result in drug-induced hypersensitivity as well as autoimmune reactions (Griem et al., 1998), no clinical and biological similarities can be found between hydralazine-induced lupus and penicillin induced anaphylaxis, and auto-immune reactions will not be further considered.
2.3. Pseudo-allergy Many hypersensitivity reactions do not involve immune-mediated mechanisms. Because preformed (mainly histamine) and neoformed (e.g. arachidonic acid metabolites) mediators can be released by nonspecific immunological mechanisms, the clinical symptoms of pseudo-allergic reactions often mimick those of immuno-allergic reactions. The best recognized pseudo-allergic mechanisms (Dejarnatt and Grant, 1992) include:
G. Choquet-Kastyle6sky, J. Descotes / Toxicology 129 (1998) 27–35
activation of the complement system resulting in the release of biologically active peptidic by-products, such as the anaphylatoxins C3a, C4a and C5a. The pharmaceutical solvent Cremophor El° is an illustrative example of this mechanism (Benoit et al., 1983); direct histamine release from mast cells and basophils presumably involving histamine-releasing factors, cytotoxicity or hyperosmolarity. Morphine and other opiate derivatives are amongst the most potent histamine-releasing drugs (Rosow et al., 1982) alterations in the synthesis and release pathways of arachidonic acid metabolites resulting in intolerance to nonsteroidal anti-inflammatory drugs (Manning and Stevenson, 1991) The role of other putative mechanisms, involving cytokines and kinins for instance, remains to be fully ascertained. At the present time, no standardized and validated animal models are available to predict pseudoallergic reactions.
3. Immunogenicity A prior contact with the antigen is a prerequisite to the development of immuno-allergic reactions, even though it is generally impossible to demonstrate whether one given contact was actually sensitizing in a given individual. After a subsequent contact with the antigen and not necessarily the next contact, sensitization will become patent with the development of a clinical reaction. Two features are required for a xenobiotic to be a sensitizer: it must be foreign (‘non-self’), as are the vast majority of drugs and chemicals, and the molecular weight must be large enough (at least \1000 and more probably \5000). Foreign macromolecules, such as proteins, polypeptides, microbial extracts, can thus be directly immunogenic. Because the molecular weight of most xenobiotics, particularly medicinal products, except for a few of them, such as heparin and insulin, is too small ( B 500), they cannot be direct immunogens, but can act as haptens when strongly bound to carrier macromolecules. For hapten-carrier immunogenic complexes to be formed in vivo, xeno-
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biotics must be chemically reactive (Park et al., 1987). In contrast to most industrial chemicals, medicinal products are chemically inert to avoid toxicity. It is therefore likely that metabolites instead of the parent molecules are actually involved in hapten formation. As many biotransformation processes can lead to reactive, intermediate metabolites, it is indeed tempting to assume they act as haptens. This mechanism was conclusively shown to be involved in penicillin allergy, but there is limited direct and conclusive evidence that this mechanism is involved in all cases. Should what is still a postulate be accepted, then the use of appropriate probes, namely metabolites instead of the parent molecules, would have to be recommended for in vitro diagnosis testing in order to limit false negative responses, as the immune system can discriminate between very closely related chemical structures. Whatever the immunogenic potential of xenobiotics, not all exposed individuals become sensitized, so that the role of predisposing factors, such as age, gender, the route of exposure (e.g. topical vs oral administration), modalities of exposure (e.g. intermittent vs continuous exposure), and individual (genetic) traits, has to be considered. Although the influence of these factors was demonstrated in a number of experimental models, it remains often speculative in human beings.
4. Pathogenesis and symptoms of immuno-allergic reactions to xenobiotics
4.1. Anaphylaxis Anaphylaxis is an immediate-type hypersensitivity reaction involving specific IgE antibodies (Atkinson and Kaliner, 1992). Following a sensitizing contact, IgE bind to high affinity receptors on mast cells and basophils. After a subsequent contact, the reaction between a divalent antigen and bound IgE results in the degranulation of target cells with the immediate release of stored vaso-active mediators (histamine) and the synthesis of arachidonic acid derivatives (e.g. prostaglandins and leucotrienes). These mediators exert a wide array of biological effects which
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G. Choquet-Kastyle6sky, J. Descotes / Toxicology 129 (1998) 27–35
account for the clinical symptoms of anaphylaxis, such as urticaria, angioedema, bronchospasm, and shock. Drug-induced anaphylaxis is a relatively rare, but life-threatening event (Van der Klauw et al., 1996). It as been reported with protein derived drugs (i.e. heparin, insulin...), penicillin, curares and miscellaneous drugs.
sensitivity, immune complex response, complement activation and cell mediated response (Bernstein and Bernstein, 1994). The pathogenesis of immune-mediated skin (Merk and Hertl, 1996) or liver (Losser and payen, 1996) drug damage is still less clearly understood.
5. Predictive animal models
4.2. Contact dermatitis 5.1. Anaphylaxis Allergic contact dermatitis (Krasteva, 1993) is a delayed-type hypersensitivity reaction characterized by the infiltration of T-lymphocytes into the dermis and epidermis. It is caused by skin contact with a chemical which triggers an immunological response leading to inflammatory skin lesions. A hapten for contact hypersensitivity is usually a low-molecular weight, lipid-soluble substance which binds or complexes with cell surface or structural proteins on various cells, including Langerhans cells and keratinocytes. Langerhans cells process and present the antigen to Tlymphocytes, which leads to the clonal proliferation of sensitized lymphocytes and to a clinically patent inflammatory reaction. Acute allergic contact dermatitis usually develops as an erythematous, vesicular, oedematous eruption at the sites of skin contact with the sensitizing substance. Following repeated exposure, chronic contact dermatitis will develop with erythematous, scalling and thickened skin lesions. Occupational exposure and drug treatments are major causes of contact dermatitis (Storrs, 1991; Stewart, 1992).
4.3. Other immune-mediated hypersensiti6ity reactions Respiratory sensitivity and blood dyscrasias are other examples of immune-mediated adverse effects due to xenobiotics. However, different mechanisms can lead to similar reactions: for instance, drug-induced immune haemolytic anaemias can result from an immune complex, a hapten or an autoimmune-mediated mechanism (Claas, 1996), whereas the immunological mechanisms of asthma include IgE-mediated immediate hyper-
Guinea-pig models have been used for years, even though limited efforts have been paid to standardizing and validating these models. Using a panel of 12 microbial vaccines and medicinal macromolecules, systemic anaphylaxis in guineapigs was suggested to be a valuable model, even though the predictive value was found to be limited to non humanized or non human substances (Brutzkus et al., 1997). The relevance of this model for low-molecular-weight molecules remains to be carefully assessed: using different panels of prototypic medicinal compounds, Chazal et al. (1994), in contrast to Nagami et al. (1995), could not induce systemic anaphylaxis. Passive cutaneous anaphylaxis is another typical guinea-pig model, but it did not seem to be more sensitive than systemic anaphylaxis (Chazal et al., 1994). Another approach for predicting the anaphylaxis-inducing potential of xenobiotics is the mouse IgE test which was suggested to help identify respiratory sensitizers (Hilton et al., 1996). Respiratory sensitization resulting in IgE-mediated immediate hypersensitivity reactions can also be investigated in guinea-pigs following exposure to atmospheres of free and protein-bound allergens and subsequent inhalation challenge with the same form of the allergen, or following either topical, subcutaneous or intradermal exposure to the free chemical, and inhalation challenge with the free or protein-bound chemical (Sarlo and Karol, 1994). Overall, these models indicate that possibilities do exist to predict the sensitizing potential of xenobiotics resulting in anaphylaxis, but their value is limited to certain chemicals, such as non
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human macromolecules or highly reactive chemicals. Therefore, they are of very limited, if any, value as regards most medicinal products.
5.2. Contact sensitization Contact sensitizers can be best identified using guinea-pig models, such as the Buehler test and the Magnusson and Kligman (maximisation) test (Maurer et al., 1994). Recently, mouse models, including the Mouse Ear Swelling Test or MEST (Gad, 1994), and the Local Lymph Node Assay or LLNA (Kimber et al., 1994) were developed, and the latter proved to be similarly predictive to the guinea-pig maximisation test (Basketter and Scholes, 1992). Despite some limitations and varying results depending on the model used, these models can reasonably identify the majority of human contact sensitizers.
5.3. Immunogenicity and systemic sensitization The best approach to induce a specific immune response against low molecular-weight substances is the use of hapten-carrier conjugates. However, these conjugates (complexes) are likely to be different from those actually formed in vivo and resulting in clinical adverse reactions. However, the use of such conjugates can be helpful to assess the potential for cross-allergenicity between compounds of closely related structures, such as the b-lactam antibiotics (Saxon et al. 1985). Animals given repeated injections of a substance can develop specific IgG and IgM antibodies in their sera. This approach is particularly used for the safety assessment of biotechnology-derived products. However, as antibodies detected in the sera of treated patients are inconsistently associated with overt adverse consequences (Vial and Descotes, 1997), it is unsure whether the observed immunogenicity, even in primates, is clinically relevant. Despite known or suspected differences in the sensitizing potential of chemicals according to the route of exposure, the general mechanisms involved in sensitization are similar. This was exemplified by the finding that results of contact
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sensitization assays in guinea-pigs and the clinical experience regarding immune-allergic reactions paralleled for 59 of 70 medicinal products and chemicals (Vial and Descotes, 1994). Regarding the estimated incidence of human allergic reactions, the guinea pig assays had a sensitivity of 85% and a specificity of 83%. Examples of medicinal products and chemicals are presented in Table 1. Because of discrepancies in the results of 11 products, caution should be exercised when extrapolating positive as well as animal data to man. However, several findings are particularly worth of consideration: (1) few reactions have been reported in man with the six negative products in guinea-pigs; (2) reactive metabolites can be involved; (3) the lack of known allergic reactions in man with five positive products in guinea-pigs does not necessarily mean that no reactions have ever developed. In addition, the inhibition of macrophage migration in guinea-pigs sensitized with a panel of medicinal products using the maximisation test, paralleled their potential for inducing immune-allergic reactions in humans (Laschi-Loquerie et al., 1987). Obviously, these results cannot lead to definite conclusions, but they suggest that correlation’s, although empirically obtained, can be used for risk assessment, at least when no other data are available.
6. Perspectives From the data reviewed above, it appears that most available models are of limited value to predict the risk of hypersensitivity reactions due to xenobiotics, particularly medicinal products, so that new avenues of research are needed. Significant efforts should be paid to gaining a better understanding of the fundamental mechanisms involved in order to design more reliable tests. Immuno-allergic mechanisms are presumably not fundamentally different from other immune responses. The development of new animal models, e.g. transgenic and knock-out mice, is expected to help better understand the mechanisms of allergic responses (Lamers and Yu, 1995), even though their utility to predict the sensitizing potential of xenobiotics remains to es-
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Table 1 Examples of drugs, drug additives and chemicals with their sensitivity potentials in guinea-pig, and reported human allergic reactions (adapted from Vial and Descotes, 1994) Drugs, additives and chemicals
Sensitivity in guinea-pig models
Estimated incidence of human allergic reactions
Althesin® Cloxacillin Penicillin G Quinine Chloramine T Diisocyanate (TDI) Trimellitic anhydre
+ +++ +++ +++ +++ +++ ++
++ + +++ ++ +++ +++ ++
Salicylic acid Miconazole Naftidrofuryl Acetone Acroleine
0 0 0 0 0
0 0 0 0 0
Gentamicin Kanamycin Phenazone Phenobarbital Propylene glycol Copper salts
0 0 0 0 0 0
+ + + + + +
Clioquinol Propyl gallate Acrylonitrile Amyl acetate Aniline
+ +++ +++ + +++
0 0 0 0 0
tablish. CD4+ and CD8+ T-lymphocytes are pivotal and their role in these reactions can be analyzed using MHC class I or class II molecules knock-out mice deficient in CD8+ and CD4+ T cells, respectively (Bour et al., 1995). The balanced function of T-lymphocytes, including the different profiles of released cytokines by so-called Thl and Th2 CD4 cells, is a critical aspect of allergic sensitization to chemicals (Kimber, 1994). However, it is not known why a chemical substance can preferentially trigger a Thl (e.g. contact dermatitis) instead of a Th2 (e.g. respiratory allergy) response (Dearman et al., 1996). Expert systems are under development to determine the qualitative and/or quantitative structural-activity relationships in the field of chemical sensitivity. Examples of such computer-aided systems include contact (Barratt et al., 1994) and respiratory sensitizers (Karol et al., 1996). Unfortunately, the question why a chemical substance is
a sensitizer remains largely unanswered (Baldo and Pham, 1994; Basketter et al., 1995). Existing models, such as the LLNA or the mouse IgE test, should be more extensively validated in order to assess to what extent they can be useful as regards medicinal products. Previous validation efforts mainly focused on non medicinal compounds (Kimber et al., 1995), so that it is largely unknown whether these results can be extrapolated to medicinal products with far lesser chemical reactivity. Novel animal models are needed to better predict the sensitizing potential of medicinal products and chemicals. As Thl cytokines (IL-2 and IFN-g) are preferentially involved in delayed hypersensitivity reactions, whereas Th2 cytokines (IL-4, IL5) are involved in antibody-dependent reactions, such as anaphylaxis and respiratory allergies, the effects of known sensitizers could be investigated in mice with different cytokine profiles, such as
G. Choquet-Kastyle6sky, J. Descotes / Toxicology 129 (1998) 27–35
C57BL/10 (Thl) and Balb/c (Th2) mice (Sun et al., 1997). Similarly, IL-4 transgenic mice are expected to react more strongly and/or more readily to chemical sensitizers, as IgE production is under the control of IL-4 (Tepper et al., 1990). IL-5 transgenic mice develop hypereosinophilia and could help predict medicinal products with the potential to induce hypereosinophilia-associated hypersensitivity syndromes (Van Oosterhout and Ladenius, 1993). On the opposite, the use of animals with a preferential Thl response, such as IFN-g transgenic mice (Saulnier et al., 1995), could be useful to investigate cell-mediated reactions, as might be animals with an increased number of mast cells or basophils and supposedly an increased susceptibility to nonspecific degranulation, to identify pseudo-allergic drug reactions. SCID mice because of altered VDJ recombination have no B- and T- lymphocytes (Løvik, 1997). They can be reconstituted with human cells (SCID-hu mice) from the peripheral blood, the bone marrow or fetal liver. They can be considered as closer to the human situation. Reconstitution with peripheral blood cells obtained from patients who developed an immune-allergic reactions can also be considered. Many immune-mediated reactions caused by medicinal products, such as epidermal necrolysis and fixed pigmented erythema, supposedly involve cytotoxic pathways. The use of animals with a genetic defect in such pathways, such as Fas, Fas ligand and perforin knockout mice, is likely to open new avenues of research. Extra-immunological mechanisms are also likely to be involved in hypersensitivity reactions, particularly metabolic and detoxification pathways. The study of cell sensitivity to toxic metabolites in relatives of patients with a history of drug-induced hypersensitivity reaction (Wolkenstein et al., 1995; Shapiro and Shear, 1996) is illustrative of the role of pharmacogenetic predisposition. The greater incidence of drug-induced hypersensitivity reactions in HIV patients was suggested to be associated with a defect in acetylation or glutathione related pathways (Koopmans et al., 1995). Genetic defects in cytochrome P450 isoenzymes, such as CYP2-D-6, were suggested to be associated with more fre-
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quent sensitization to drugs (Larrey and Pageaux, 1997). The use of animals with a genetic defect in glutathione-dependent pathways or with a slow acetylator phenotype could therefore be considered. Finally, the use of animals with combined modifications in genes involving the immune system and biotransformation pathways could be useful. In conclusion, predicting hypersensitivity reactions to xenobiotics, particularly medicinal products, is often impossible today. More information is required on the mechanisms involved and more adequate models are needed. Research efforts in the academia and the industry are urgently needed. Regulatory agencies should also play a positive role in this process by avoiding to promote tests, such as the Japanese guidelines on antigenicity, which are based on ill-validated data (Udaka, 1992) or to abruptly deny any value to tests, such as systemic anaphylactic models (ICH Steering Committee, 1997).
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Value of animal models for predicting hypersensitivity reactions to medicinal products G. Choquet-Kastylevsky *, J. Descotes Department of Pharmacology, Medical Toxicology and En6ironmental Medicine, et INSERM U98 -X, Faculte´ de Me´decine Lyon-RTH Lae¨nnec, 69008 Lyon, France
Abstract Although hypersensitivity reactions induced by medicinal products and chemicals are relatively common, few predictive models are available. A major difficulty is our currently limited understanding of the mechanisms involved, and efforts should be paid to better defining drug immunogenicity, hapten formation and immune effector mechanisms. A second difficulty is the multiplicity of clinical manifestations presumably due to varying mechanisms. Available models can only predict a few of these reactions. Anaphylaxis models in guinea-pigs can be only used for the safety assessment of macromolecules which are neither humanized or of human origin, whereas guinea-pig or mouse models can detect the majority of human contact sensitizers. In addition to the extensive validation of existing models, promising avenues of research are expected to be found in the use of novel animal models, particularly those using genetically modified animals, such as transgenic and knock-out mice. © 1998 Published by Elsevier Science Ireland Ltd. All rights reserved. Keywords: Allergy; Animal models; Hypersensitivity reactions; Immunotoxicity evaluation; Medicinal products
1. Introduction Hypersensitivity reactions are the most frequent adverse effects involving the immune system associated with medicinal products and chemical exposure. Even though reliable information is scarce on their actual incidence in humans, hypersensitivity reactions might account for approximately one third of recorded adverse drug reactions * Corresponding author.
based on the experience of post-marketing drug surveillance. Contact dermatitis and respiratory allergy are frequent complaints at the workplace, whereas allergy and chemical sensitivity are described in a significant fraction of the general population. Nevertheless, predictive animal models have not been considered a key priority until very recently. One explanation is that ‘allergic’ reactions in contrast to toxic effects have long been claimed to be unpredictable in animals. In addition, the search for understanding the mechanisms
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involved and the development of new diagnosis methods lagged behind recent advances in immunology as well as cellular/molecular biology. It is beyond doubt that hypersensitivity reactions induced by xenobiotics pose a major threat to human health. This review paper is an attempt to describe the current status of animal models predictive of hypersensitivity and to delineate possible avenues of research.
2. Immuno-allergy vs pseudo-allergy
2.1. Definitions Hypersensitivity reactions do not all involve the immune system, even though the generic term ‘allergy’ is commonly used. To avoid confusion, it is proposed to use the term ‘(immuno)-allergy’ only when highly specific mechanisms involving immunological memory and recognition are shown to be involved. The term ‘pseudo-allergic’ would describe those reactions which mimick immuno-allergy, but in which a specific immune mediated mechanism is not involved. The use of the term ‘pseudo-allergy’ as a synonym to ‘idiosyncrasy’, that is to say a reaction reflecting a particular susceptibility of individuals, is not recommended. When no indication of the mechanism involved is available, the general term ‘hypersensitivity’ is proposed to cover both possibly immuno-allergic and pseudo-allergic reactions.
2.2. Immuno-allergy One major feature of the immune system is the capacity to recognize foreign (‘non-self’) constituents and to memorize this encounter for subsequent accelerated and amplified immune responses. The mechanisms of immuno-allergic reactions to xenobiotics are ill-understood. In 1964, Gell and Coombs proposed a pathogenic classification of 4 types, namely immediate hypersensitivity or anaphylactic (type I, antibody-mediated (type II), immune complex-mediated (type III), and delayed hypersensitivity (type IV), reactions. Although this classification is still often referred to, it is too rigid and proves useless in
many instances, in particular when immuno-allergic reactions induced by xenobiotics are considered. In addition, two major limitations of this classification are the lack of updating to take into account recent advances in our understanding of the immune system, and the dissection of mechanisms into separate categories (types) excluding the likely simultaneous involvement of two or more mechanisms. Whatever the actual pathogenesis of immunoallergic reactions induced by xenobiotics, two features are absolutely essential: (i) the offending xenobiotic (or its metabolites) must be directly immunogenic or act as haptens, and (ii) the responding host must be sensitized prior to developing a patent immuno-allergic reaction. The immune response can be mounted against the drug or a reactive metabolite (haptenic epitope), the protein to which it is bound (auto-antigenic epitope), or a new epitope formed by the drugprotein complex. In the latter two situations, the response can result in a drug-induced systemic auto-immune reaction, such as the lupus syndrome or scleroderma-like disease, most often associated with the presence of antinuclear autoantibodies antibodies, or an organspecific autoimmune reaction, such as pemphigus or auto-immune hemolytic anemia. However, even though common immunological mechanisms are suggested to result in drug-induced hypersensitivity as well as autoimmune reactions (Griem et al., 1998), no clinical and biological similarities can be found between hydralazine-induced lupus and penicillin induced anaphylaxis, and auto-immune reactions will not be further considered.
2.3. Pseudo-allergy Many hypersensitivity reactions do not involve immune-mediated mechanisms. Because preformed (mainly histamine) and neoformed (e.g. arachidonic acid metabolites) mediators can be released by nonspecific immunological mechanisms, the clinical symptoms of pseudo-allergic reactions often mimick those of immuno-allergic reactions. The best recognized pseudo-allergic mechanisms (Dejarnatt and Grant, 1992) include:
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activation of the complement system resulting in the release of biologically active peptidic by-products, such as the anaphylatoxins C3a, C4a and C5a. The pharmaceutical solvent Cremophor El° is an illustrative example of this mechanism (Benoit et al., 1983); direct histamine release from mast cells and basophils presumably involving histamine-releasing factors, cytotoxicity or hyperosmolarity. Morphine and other opiate derivatives are amongst the most potent histamine-releasing drugs (Rosow et al., 1982) alterations in the synthesis and release pathways of arachidonic acid metabolites resulting in intolerance to nonsteroidal anti-inflammatory drugs (Manning and Stevenson, 1991) The role of other putative mechanisms, involving cytokines and kinins for instance, remains to be fully ascertained. At the present time, no standardized and validated animal models are available to predict pseudoallergic reactions.
3. Immunogenicity A prior contact with the antigen is a prerequisite to the development of immuno-allergic reactions, even though it is generally impossible to demonstrate whether one given contact was actually sensitizing in a given individual. After a subsequent contact with the antigen and not necessarily the next contact, sensitization will become patent with the development of a clinical reaction. Two features are required for a xenobiotic to be a sensitizer: it must be foreign (‘non-self’), as are the vast majority of drugs and chemicals, and the molecular weight must be large enough (at least \1000 and more probably \5000). Foreign macromolecules, such as proteins, polypeptides, microbial extracts, can thus be directly immunogenic. Because the molecular weight of most xenobiotics, particularly medicinal products, except for a few of them, such as heparin and insulin, is too small ( B 500), they cannot be direct immunogens, but can act as haptens when strongly bound to carrier macromolecules. For hapten-carrier immunogenic complexes to be formed in vivo, xeno-
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biotics must be chemically reactive (Park et al., 1987). In contrast to most industrial chemicals, medicinal products are chemically inert to avoid toxicity. It is therefore likely that metabolites instead of the parent molecules are actually involved in hapten formation. As many biotransformation processes can lead to reactive, intermediate metabolites, it is indeed tempting to assume they act as haptens. This mechanism was conclusively shown to be involved in penicillin allergy, but there is limited direct and conclusive evidence that this mechanism is involved in all cases. Should what is still a postulate be accepted, then the use of appropriate probes, namely metabolites instead of the parent molecules, would have to be recommended for in vitro diagnosis testing in order to limit false negative responses, as the immune system can discriminate between very closely related chemical structures. Whatever the immunogenic potential of xenobiotics, not all exposed individuals become sensitized, so that the role of predisposing factors, such as age, gender, the route of exposure (e.g. topical vs oral administration), modalities of exposure (e.g. intermittent vs continuous exposure), and individual (genetic) traits, has to be considered. Although the influence of these factors was demonstrated in a number of experimental models, it remains often speculative in human beings.
4. Pathogenesis and symptoms of immuno-allergic reactions to xenobiotics
4.1. Anaphylaxis Anaphylaxis is an immediate-type hypersensitivity reaction involving specific IgE antibodies (Atkinson and Kaliner, 1992). Following a sensitizing contact, IgE bind to high affinity receptors on mast cells and basophils. After a subsequent contact, the reaction between a divalent antigen and bound IgE results in the degranulation of target cells with the immediate release of stored vaso-active mediators (histamine) and the synthesis of arachidonic acid derivatives (e.g. prostaglandins and leucotrienes). These mediators exert a wide array of biological effects which
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account for the clinical symptoms of anaphylaxis, such as urticaria, angioedema, bronchospasm, and shock. Drug-induced anaphylaxis is a relatively rare, but life-threatening event (Van der Klauw et al., 1996). It as been reported with protein derived drugs (i.e. heparin, insulin...), penicillin, curares and miscellaneous drugs.
sensitivity, immune complex response, complement activation and cell mediated response (Bernstein and Bernstein, 1994). The pathogenesis of immune-mediated skin (Merk and Hertl, 1996) or liver (Losser and payen, 1996) drug damage is still less clearly understood.
5. Predictive animal models
4.2. Contact dermatitis 5.1. Anaphylaxis Allergic contact dermatitis (Krasteva, 1993) is a delayed-type hypersensitivity reaction characterized by the infiltration of T-lymphocytes into the dermis and epidermis. It is caused by skin contact with a chemical which triggers an immunological response leading to inflammatory skin lesions. A hapten for contact hypersensitivity is usually a low-molecular weight, lipid-soluble substance which binds or complexes with cell surface or structural proteins on various cells, including Langerhans cells and keratinocytes. Langerhans cells process and present the antigen to Tlymphocytes, which leads to the clonal proliferation of sensitized lymphocytes and to a clinically patent inflammatory reaction. Acute allergic contact dermatitis usually develops as an erythematous, vesicular, oedematous eruption at the sites of skin contact with the sensitizing substance. Following repeated exposure, chronic contact dermatitis will develop with erythematous, scalling and thickened skin lesions. Occupational exposure and drug treatments are major causes of contact dermatitis (Storrs, 1991; Stewart, 1992).
4.3. Other immune-mediated hypersensiti6ity reactions Respiratory sensitivity and blood dyscrasias are other examples of immune-mediated adverse effects due to xenobiotics. However, different mechanisms can lead to similar reactions: for instance, drug-induced immune haemolytic anaemias can result from an immune complex, a hapten or an autoimmune-mediated mechanism (Claas, 1996), whereas the immunological mechanisms of asthma include IgE-mediated immediate hyper-
Guinea-pig models have been used for years, even though limited efforts have been paid to standardizing and validating these models. Using a panel of 12 microbial vaccines and medicinal macromolecules, systemic anaphylaxis in guineapigs was suggested to be a valuable model, even though the predictive value was found to be limited to non humanized or non human substances (Brutzkus et al., 1997). The relevance of this model for low-molecular-weight molecules remains to be carefully assessed: using different panels of prototypic medicinal compounds, Chazal et al. (1994), in contrast to Nagami et al. (1995), could not induce systemic anaphylaxis. Passive cutaneous anaphylaxis is another typical guinea-pig model, but it did not seem to be more sensitive than systemic anaphylaxis (Chazal et al., 1994). Another approach for predicting the anaphylaxis-inducing potential of xenobiotics is the mouse IgE test which was suggested to help identify respiratory sensitizers (Hilton et al., 1996). Respiratory sensitization resulting in IgE-mediated immediate hypersensitivity reactions can also be investigated in guinea-pigs following exposure to atmospheres of free and protein-bound allergens and subsequent inhalation challenge with the same form of the allergen, or following either topical, subcutaneous or intradermal exposure to the free chemical, and inhalation challenge with the free or protein-bound chemical (Sarlo and Karol, 1994). Overall, these models indicate that possibilities do exist to predict the sensitizing potential of xenobiotics resulting in anaphylaxis, but their value is limited to certain chemicals, such as non
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human macromolecules or highly reactive chemicals. Therefore, they are of very limited, if any, value as regards most medicinal products.
5.2. Contact sensitization Contact sensitizers can be best identified using guinea-pig models, such as the Buehler test and the Magnusson and Kligman (maximisation) test (Maurer et al., 1994). Recently, mouse models, including the Mouse Ear Swelling Test or MEST (Gad, 1994), and the Local Lymph Node Assay or LLNA (Kimber et al., 1994) were developed, and the latter proved to be similarly predictive to the guinea-pig maximisation test (Basketter and Scholes, 1992). Despite some limitations and varying results depending on the model used, these models can reasonably identify the majority of human contact sensitizers.
5.3. Immunogenicity and systemic sensitization The best approach to induce a specific immune response against low molecular-weight substances is the use of hapten-carrier conjugates. However, these conjugates (complexes) are likely to be different from those actually formed in vivo and resulting in clinical adverse reactions. However, the use of such conjugates can be helpful to assess the potential for cross-allergenicity between compounds of closely related structures, such as the b-lactam antibiotics (Saxon et al. 1985). Animals given repeated injections of a substance can develop specific IgG and IgM antibodies in their sera. This approach is particularly used for the safety assessment of biotechnology-derived products. However, as antibodies detected in the sera of treated patients are inconsistently associated with overt adverse consequences (Vial and Descotes, 1997), it is unsure whether the observed immunogenicity, even in primates, is clinically relevant. Despite known or suspected differences in the sensitizing potential of chemicals according to the route of exposure, the general mechanisms involved in sensitization are similar. This was exemplified by the finding that results of contact
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sensitization assays in guinea-pigs and the clinical experience regarding immune-allergic reactions paralleled for 59 of 70 medicinal products and chemicals (Vial and Descotes, 1994). Regarding the estimated incidence of human allergic reactions, the guinea pig assays had a sensitivity of 85% and a specificity of 83%. Examples of medicinal products and chemicals are presented in Table 1. Because of discrepancies in the results of 11 products, caution should be exercised when extrapolating positive as well as animal data to man. However, several findings are particularly worth of consideration: (1) few reactions have been reported in man with the six negative products in guinea-pigs; (2) reactive metabolites can be involved; (3) the lack of known allergic reactions in man with five positive products in guinea-pigs does not necessarily mean that no reactions have ever developed. In addition, the inhibition of macrophage migration in guinea-pigs sensitized with a panel of medicinal products using the maximisation test, paralleled their potential for inducing immune-allergic reactions in humans (Laschi-Loquerie et al., 1987). Obviously, these results cannot lead to definite conclusions, but they suggest that correlation’s, although empirically obtained, can be used for risk assessment, at least when no other data are available.
6. Perspectives From the data reviewed above, it appears that most available models are of limited value to predict the risk of hypersensitivity reactions due to xenobiotics, particularly medicinal products, so that new avenues of research are needed. Significant efforts should be paid to gaining a better understanding of the fundamental mechanisms involved in order to design more reliable tests. Immuno-allergic mechanisms are presumably not fundamentally different from other immune responses. The development of new animal models, e.g. transgenic and knock-out mice, is expected to help better understand the mechanisms of allergic responses (Lamers and Yu, 1995), even though their utility to predict the sensitizing potential of xenobiotics remains to es-
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Table 1 Examples of drugs, drug additives and chemicals with their sensitivity potentials in guinea-pig, and reported human allergic reactions (adapted from Vial and Descotes, 1994) Drugs, additives and chemicals
Sensitivity in guinea-pig models
Estimated incidence of human allergic reactions
Althesin® Cloxacillin Penicillin G Quinine Chloramine T Diisocyanate (TDI) Trimellitic anhydre
+ +++ +++ +++ +++ +++ ++
++ + +++ ++ +++ +++ ++
Salicylic acid Miconazole Naftidrofuryl Acetone Acroleine
0 0 0 0 0
0 0 0 0 0
Gentamicin Kanamycin Phenazone Phenobarbital Propylene glycol Copper salts
0 0 0 0 0 0
+ + + + + +
Clioquinol Propyl gallate Acrylonitrile Amyl acetate Aniline
+ +++ +++ + +++
0 0 0 0 0
tablish. CD4+ and CD8+ T-lymphocytes are pivotal and their role in these reactions can be analyzed using MHC class I or class II molecules knock-out mice deficient in CD8+ and CD4+ T cells, respectively (Bour et al., 1995). The balanced function of T-lymphocytes, including the different profiles of released cytokines by so-called Thl and Th2 CD4 cells, is a critical aspect of allergic sensitization to chemicals (Kimber, 1994). However, it is not known why a chemical substance can preferentially trigger a Thl (e.g. contact dermatitis) instead of a Th2 (e.g. respiratory allergy) response (Dearman et al., 1996). Expert systems are under development to determine the qualitative and/or quantitative structural-activity relationships in the field of chemical sensitivity. Examples of such computer-aided systems include contact (Barratt et al., 1994) and respiratory sensitizers (Karol et al., 1996). Unfortunately, the question why a chemical substance is
a sensitizer remains largely unanswered (Baldo and Pham, 1994; Basketter et al., 1995). Existing models, such as the LLNA or the mouse IgE test, should be more extensively validated in order to assess to what extent they can be useful as regards medicinal products. Previous validation efforts mainly focused on non medicinal compounds (Kimber et al., 1995), so that it is largely unknown whether these results can be extrapolated to medicinal products with far lesser chemical reactivity. Novel animal models are needed to better predict the sensitizing potential of medicinal products and chemicals. As Thl cytokines (IL-2 and IFN-g) are preferentially involved in delayed hypersensitivity reactions, whereas Th2 cytokines (IL-4, IL5) are involved in antibody-dependent reactions, such as anaphylaxis and respiratory allergies, the effects of known sensitizers could be investigated in mice with different cytokine profiles, such as
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C57BL/10 (Thl) and Balb/c (Th2) mice (Sun et al., 1997). Similarly, IL-4 transgenic mice are expected to react more strongly and/or more readily to chemical sensitizers, as IgE production is under the control of IL-4 (Tepper et al., 1990). IL-5 transgenic mice develop hypereosinophilia and could help predict medicinal products with the potential to induce hypereosinophilia-associated hypersensitivity syndromes (Van Oosterhout and Ladenius, 1993). On the opposite, the use of animals with a preferential Thl response, such as IFN-g transgenic mice (Saulnier et al., 1995), could be useful to investigate cell-mediated reactions, as might be animals with an increased number of mast cells or basophils and supposedly an increased susceptibility to nonspecific degranulation, to identify pseudo-allergic drug reactions. SCID mice because of altered VDJ recombination have no B- and T- lymphocytes (Løvik, 1997). They can be reconstituted with human cells (SCID-hu mice) from the peripheral blood, the bone marrow or fetal liver. They can be considered as closer to the human situation. Reconstitution with peripheral blood cells obtained from patients who developed an immune-allergic reactions can also be considered. Many immune-mediated reactions caused by medicinal products, such as epidermal necrolysis and fixed pigmented erythema, supposedly involve cytotoxic pathways. The use of animals with a genetic defect in such pathways, such as Fas, Fas ligand and perforin knockout mice, is likely to open new avenues of research. Extra-immunological mechanisms are also likely to be involved in hypersensitivity reactions, particularly metabolic and detoxification pathways. The study of cell sensitivity to toxic metabolites in relatives of patients with a history of drug-induced hypersensitivity reaction (Wolkenstein et al., 1995; Shapiro and Shear, 1996) is illustrative of the role of pharmacogenetic predisposition. The greater incidence of drug-induced hypersensitivity reactions in HIV patients was suggested to be associated with a defect in acetylation or glutathione related pathways (Koopmans et al., 1995). Genetic defects in cytochrome P450 isoenzymes, such as CYP2-D-6, were suggested to be associated with more fre-
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quent sensitization to drugs (Larrey and Pageaux, 1997). The use of animals with a genetic defect in glutathione-dependent pathways or with a slow acetylator phenotype could therefore be considered. Finally, the use of animals with combined modifications in genes involving the immune system and biotransformation pathways could be useful. In conclusion, predicting hypersensitivity reactions to xenobiotics, particularly medicinal products, is often impossible today. More information is required on the mechanisms involved and more adequate models are needed. Research efforts in the academia and the industry are urgently needed. Regulatory agencies should also play a positive role in this process by avoiding to promote tests, such as the Japanese guidelines on antigenicity, which are based on ill-validated data (Udaka, 1992) or to abruptly deny any value to tests, such as systemic anaphylactic models (ICH Steering Committee, 1997).
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