What are some of the lessons learnt from in vitro studies of severe unpredictable drug reactions?
Authors: J.R. Sullivan, N.H. Shear
Journal: British Journal of Dermatology (2000)

We cannot, at this stage, diagnose an idiosyncratic drug reaction (IDR) solely by in vitro testing. In vitro tests for IDRs are not widely available and remain largely research tools. In individual cases they have improved diagnostic certainty, patient care and management options. Their greatest contribution has been to help identify pieces of the mechanistic puzzle underlying these reactions. Understanding the mechanisms by which drugs cause IDRs will aid development of safer drugs and help avoid the use of specific drugs in unusually susceptible individuals. IDRs, also called type B or ‘bizarre’ reactions, do not occur in most patients at any dose nor do they involve known pharmacologic properties of the drug. They are unpredictable and often serious in nature. In vitro tests provide clues to why some individuals are particularly susceptible. A large spectrum of IDRs occurs. Better clinical characterization of these drug–induced syndromes will aid application of laboratory studies. Clinical criteria are beginning to be prospectively studied. Diagnosis of drug–induced syndromes relies on a constellation of clinical features often supported by laboratory results. The drug–hypersensitivity syndrome (DHS), reported by Bedard et al. 1 in this issue, is a good example. Different authors detail varying criteria, acronyms and even names for diagnosis. 2-4 This multi-organ syndrome is frequently reported with a title and abstract highlighting only one organ such as hypersensitivity colitis or pneumonitis. We have no validated clinical gold standard in the DHS to compare results of laboratory studies. Despite the diversity of clinical features, many in vitro observations are consistently found throughout the spectrum of DHS reactions. The multiple mechanisms involved in IDRs are an obstacle for the development of diagnostic laboratory tests. Clinical features and more recently laboratory research provide evidence that drugs do indeed cause immune-mediated changes in the DHS. A large amount of circumstantial in vitro evidence also supports the role of reactive drug metabolites (RDM). 5 The interplay of these and other factors is probably a major reason for the unpredictability of the DHS. Increased levels of RDM, their impaired detoxification or decreased cellular defence against reactive drug products appear to be important initiating factors. 2,5,6 Association of slow acetylation with increased risk of DHS, including toxic epidermal necrolysis (TEN), highlights the importance of drug metabolism in causation. The role of RDM has mainly been studied in more severe IDRs such as the DHS including TEN and Stevens–Johnson syndrome, 7 halothane-hepatitis, and drug-induced cytopenias. 7 The lymphocyte toxicity assay (LTA) has been utilized to investigate the role of RDM and impaired drug detoxification mechanisms in vitro. The LTA is a measure of lymphocyte phenotypic detoxification systems. Patients’ lymphocytes are exposed to a drug’s metabolites, and markedly increased sensitivity to these RDM is used as evidence of impaired detoxification. 9,10 Studies of RDM have the ability to prospectively identify patients at increased risk of IDR to a specific drug. A relative of an index DHS case can be assessed for similarly impaired detoxification mechanisms. 10 LTAs can help enhance the accuracy of causality assessment of the likely agent and to screen for ‘cross-reacting’ drugs. 11,12 In the anticonvulsant-DHS the LTA may help in the choice of alternative safer therapies. 10 Impaired detoxification of several aromatic anticonvulsants is seen in 75% of anticonvulsant-DHS patients. This increased risk is confirmed by reports of DHS in the same patient to carbamazepine, phenytoin and/or phenobarbitone. 13 Unfortunately, the LTA is expensive and few laboratories utilize this test. A logical extension of this test is the determination of specific enzymatic genotype or phenotype involved in drug detoxification. This is not of confirmed value, probably because we don’t know all putative toxic metabolites and metabolic pathways involved in detoxification. Why does the DHS preferentially pick out different organs in patients on the same drug? What local factors determine why some predominantly suffer colitis or myocarditis vs. the more common occurrence of rash and hepatitis? A test solely of an individual’s lymphocytes is unlikely to provide answers to these questions. Oxidative RDM are found in organs and cells preferentially affected by IDRs. 5 Viral infection increases lymphocyte susceptibility to RDM 9confirming clinical suspicion that environmental factors are probably involved. Drugs can cause very selective organ toxicity as in halothane-hepatitis; others involve virtually every organ such as sulphonamide DHS. Immune events are less well characterized. Theories for the induction of immune mediated events to the drug, its metabolites, or changes caused by these include the hapten and ‘danger’ hypotheses. In the hapten hypothesis, sufficient reactive drug or metabolite binds to a carrier triggering specific activation of adaptive immune mechanisms. 14 The ‘danger’ hypothesis proposes that presentation of antigen in the presence of cell necrosis or stress, a sign of ‘danger’, triggers an immune response. RDM, which are generally toxic to cells, and/or concurrent infection, may cause this stress or danger signal. A role for the innate immune system, whose cells are also modified by RDM, 8,15 or a combination of these and other factors, is possible. 5 Innate immune mechanisms lack specificity and memory and could partially explain the absence of recurrence on re-exposure of some drug-associated eruptions. IDRs occasionally follow the Gell and Coombs classification of hypersensitivity as in IgE-mediated (type I) urticaria accompanying anaphylaxis to penicillin or its metabolites. Classic mechanisms do not explain most IDRs 5,16,17 The DHS’s delayed-onset, rapid occurrence on re-exposure, and multiple organ involvement suggest adaptive immune mechanisms. Interplay of immune mechanisms may be important. Drug-hapten or carrier specific immunoglobulins can enhance or inhibit cell mediated hypersensitivity. 18 Some authors emphasize T cell-mediated changes in the DHS, 18 others including Bedard et al. in this issue have studied the antibody response. 20 Both arms of the adaptive and even innate systems could be involved. Limited knowledge of mechanisms of immune involvement in most IDRs complicate development of specific immunology-based diagnostic aids. Antibodies targeting self, RDM-altered native protein, and less commonly the drug, are detected in the DHS. 13,20 Their diagnostic or mechanistic value is unknown. The antibodies most often target subcellular structures, microsomes that are the site of drug bioactivation and binding site of RDM. 20 This supports a role for highly reactive, locally generated drug metabolites and the hapten and danger hypotheses. The “danger” hypothesis could explain why antibodies recognizing native proteins (not covalently modified by the causative drug) are more commonly found. The half-life of RDM/hapten-carrier complexes is such that they cannot account for the duration of DHS reactions. 21,22 Autoantibodies in DHS targeting microsomal antigens provide a clue to why DHS reactions are often remarkably persistent, as in the case detailed by Bedard et al. in this issue. Anti-microsomal antibodies in chronic autoimmune hepatitis are an interesting parallel. A humoral response to drugs can occur in the absence of clinical hypersensitivity. Thus, drug-specific antibodies alone do not necessarily lead to hypersensitivity, reducing diagnostic value of non-IgE drug-specific antibody tests. Molecular biology provides methods to study events during acute reactions. Profiles of changes could identify the mechanism operating in a specific DHS reaction. Differing cytokine profiles, such as those seen with Th1 and Th2 responses, may determine to what degree humoral or cellular adaptive systems are involved. In TEN elevated levels of TNF-α, IFN-γ and Fas ligand 23 are potentially synergistic causing widespread keratinocyte apoptosis, partially via the CD95 death receptor. These molecular biological changes carry implications for diagnosis and treatment. In summary, there are several issues that need to be addressed before in vitro evaluation of IDRs can be widely utilized for the routine assessment of IDRs. At present, their careful interpretation in the individual patient, taking into account their limitations, is critical. Controlled trials and refinements will hopefully lead to valuable future tests to improve diagnosis and prevent severe reactions prospectively. In vitro investigation contributes to our understanding of the fundamental biology underlying IDRs, providing clues to the aetiological and pathogenic puzzle. Supported by the Canadian Dermatology Foundation, Skin and Cancer Foundation of Australia, Florance Bequest of the Australasian College of Dermatologists, and Bernie Amos Travelling Fellowship, Westmead Hospital, Australia.