Dean J. Naisbitt

Managing the challenge of chemically reactive metabolites in drug development

The normal metabolism of drugs can generate metabolites that have intrinsic chemical reactivity towards cellular molecules, and therefore have the potential to alter biological function and initiate serious adverse drug reactions. Here, we present an assessment of the current approaches used for the evaluation of chemically reactive metabolites. We also describe how these approaches are being used within the pharmaceutical industry to assess and minimize the potential of drug candidates to cause toxicity. At early stages of drug discovery, iteration between medicinal chemistry and drug metabolism can eliminate perceived reactive metabolite-mediated chemical liabilities without compromising pharmacological activity or the need for extensive safety evaluation beyond standard practices. In the future, reactive metabolite evaluation may also be useful during clinical development for improving clinical risk assessment and risk management. Currently, there remains a huge gap in our understanding of the basic mechanisms that underlie chemical stress-mediated adverse reactions in humans. This Review summarizes our views on this complex topic, and includes insights into practices considered by the pharmaceutical industry.

Recognition of Sulfamethoxazole and Its Reactive Metabolites by Drug-Specific CD4+ T Cells from Allergic Individuals

The recognition of the antibiotic sulfamethoxazole (SMX) by T cells is usually explained with the hapten-carrier model. However, recent investigations have revealed a MHC-restricted but processing- and metabolism-independent pathway of drug presentation. This suggested a labile, low-affinity binding of SMX to MHC-peptide complexes on APC. To study the role of covalent vs noncovalent drug presentation in SMX allergy, we analyzed the proliferative response of PBMC and T cell clones from patients with SMX allergy to SMX and its reactive oxidative metabolites SMX-hydroxylamine and nitroso-SMX. Although the great majority of T cell clones were specific for noncovalently bound SMX, PBMC and a small fraction of clones responded to nitroso-SMX-modified cells or were cross-reactive. Rapid down-regulation of TCR expression in T cell clones upon stimulation indicated a processing-independent activation irrespective of specificity for covalently or noncovalently presented Ag. In conclusion, our data show that recognition of SMX presented in covalent and noncovalent bound form is possible by the same TCR but that the former is the exception rather than the rule. The scarcity of cross-reactivity between covalently and noncovalently bound SMX suggests that the primary stimulation may be directed to the noncovalently bound SMX.

Idiosyncratic Adverse Drug Reactions: Current Concepts

Idiosyncratic drug reactions are a significant cause of morbidity and mortality for patients; they also markedly increase the uncertainty of drug development. The major targets are skin, liver, and bone marrow. Clinical characteristics suggest that IDRs are immune mediated, and there is substantive evidence that most, but not all, IDRs are caused by chemically reactive species. However, rigorous mechanistic studies are very difficult to perform, especially in the absence of valid animal models. Models to explain how drugs or reactive metabolites interact with the MHC/T-cell receptor complex include the hapten and P-I models, and most recently it was found that abacavir can interact reversibly with MHC to alter the endogenous peptides that are presented to T cells. The discovery of HLA molecules as important risk factors for some IDRs has also significantly contributed to our understanding of these adverse reactions, but it is not yet clear what fraction of IDRs have a strong HLA dependence. In addition, with the exception of abacavir, most patients who have the HLA that confers a higher IDR risk with a specific drug will not have an IDR when treated with that drug. Interindividual differences in T-cell receptors and other factors also presumably play a role in determining which patients will have an IDR. The immune response represents a delicate balance, and immune tolerance may be the dominant response to a drug that can cause IDRs.

Immunological principles of adverse drug reactions: the initiation and propagation of immune responses elicited by drug treatment.

Adverse drug reactions account for between 2 to 5% of all hospital admissions and can prevent the administration of an otherwise effective therapeutic agent. Hypersensitivity or immune-mediated reactions, although less common, tend to be proportionately more serious. There is convincing evidence to implicate the immune system in the pathogenesis of hypersensitivity reactions. Our understanding of the way in which the immune system recognises drugs is based on the hapten hypothesis; the onset of hypersensitivity involves drug bioactivation, covalent binding to proteins, followed by uptake, antigen processing and T cell proliferation. Central to this hypothesis is the critical role of drug metabolism, with the balance between metabolic bioactivation and detoxification being one important component of individual susceptibility.The purpose of this review is to classify drug hypersensitivity reactions in terms of their clinical presentation, and also to consider recent advances in our understanding of the chemical, biochemical and, in particular, cellular immunological mechanisms of hypersensitivity. The following topics are reviewed: (i) drug disposition and cellular metabolism; (ii) mechanisms of antigen processing and presentation; (iii) the role of cytokines and co-stimulatory molecules in the induction and maintenance of a polarised immune response; and (iv) the application of the hapten hypothesis, danger hypothesis and serial triggering model to drug hypersensitivity. Agreater understanding of the mechanism(s) of hypersensitivity may identify novel therapeutic strategies and help to combat one of the more severe forms of adverse reactions to drugs.

Gemcitabine and capecitabine with or without telomerase peptide vaccine GV1001 in patients with locally advanced or metastatic pancreatic cancer (TeloVac): an open-label, randomised, phase 3 trial

BACKGROUND We aimed to assess the efficacy and safety of sequential or simultaneous telomerase vaccination (GV1001) in combination with chemotherapy in patients with locally advanced or metastatic pancreatic cancer. METHODS TeloVac was a three-group, open-label, randomised phase 3 trial. We recruited patients from 51 UK hospitals. Eligible patients were treatment naive, aged older than 18 years, with locally advanced or metastatic pancreatic ductal adenocarcinoma, and Eastern Cooperative Oncology Group performance status of 0-2. Patients were randomly assigned (1:1:1) to receive either chemotherapy alone, chemotherapy with sequential GV1001 (sequential chemoimmunotherapy), or chemotherapy with concurrent GV1001 (concurrent chemoimmunotherapy). Treatments were allocated with equal probability by means of computer-generated random permuted blocks of sizes 3 and 6 in equal proportion. Chemotherapy included six cycles of gemcitabine (1000 mg/m(2), 30 min intravenous infusion, at days 1, 8, and 15) and capecitabine (830 mg/m(2) orally twice daily for 21 days, repeated every 28 days). Sequential chemoimmunotherapy included two cycles of combination chemotherapy, then an intradermal lower abdominal injection of granulocyte-macrophage colony-stimulating factor (GM-CSF; 75 μg) and GV1001 (0·56 mg; days 1, 3, and 5, once on weeks 2-4, and six monthly thereafter). Concurrent chemoimmunotherapy included giving GV1001 from the start of chemotherapy with GM-CSF as an adjuvant. The primary endpoint was overall survival; analysis was by intention to treat. This study is registered as an International Standard Randomised Controlled Trial, number ISRCTN4382138. FINDINGS The first patient was randomly assigned to treatment on March 29, 2007, and the trial was terminated on March 27, 2011. Of 1572 patients screened, 1062 were randomly assigned to treatment (358 patients were allocated to the chemotherapy group, 350 to the sequential chemoimmunotherapy group, and 354 to the concurrent chemoimmunotherapy group). We recorded 772 deaths; the 290 patients still alive were followed up for a median of 6·0 months (IQR 2·4-12·2). Median overall survival was not significantly different in the chemotherapy group than in the sequential chemoimmunotherapy group (7·9 months [95% CI 7·1-8·8] vs 6·9 months [6·4-7·6]; hazard ratio [HR] 1·19, 98·25% CI 0·97-1·48, p=0·05), or in the concurrent chemoimmunotherapy group (8·4 months [95% CI 7·3-9·7], HR 1·05, 98·25% CI 0·85-1·29, p=0·64; overall log-rank of χ(2)2df=4·3; p=0·11). The commonest grade 3-4 toxic effects were neutropenia (68 [19%] patients in the chemotherapy group, 58 [17%] patients in the sequential chemoimmunotherapy group, and 79 [22%] patients in the concurrent chemoimmunotherapy group; fatigue (27 [8%] in the chemotherapy group, 35 [10%] in the sequential chemoimmunotherapy group, and 44 [12%] in the concurrent chemoimmunotherapy group); and pain (34 [9%] patients in the chemotherapy group, 39 [11%] in the sequential chemoimmunotherapy group, and 41 [12%] in the concurrent chemoimmunotherapy group). INTERPRETATION Adding GV1001 vaccination to chemotherapy did not improve overall survival. New strategies to enhance the immune response effect of telomerase vaccination during chemotherapy are required for clinical efficacy. FUNDING Cancer Research UK and KAEL-GemVax.

Antigenicity and immunogenicity of sulphamethoxazole: demonstration of metabolism-dependent haptenation and T-cell proliferation in vivo

Sulphamethoxazole has been associated with the occurrence of hypersensitivity reactions. There is controversy as to whether the immune response is metabolism‐dependent or ‐independent. We have therefore investigated the site of antigen formation and the nature of the drug signal presented to the immune system in vivo. Male Wistar rats were dosed with sulphamethoxazole, sulphamethoxazole hydroxylamine or nitroso sulphamethoxazole. Antigen formation on cell surfaces was determined by flow cytometry using a specific anti‐sulphamethoxazole antibody. Immunogenicity was determined by assessment of ex vivo T‐cell proliferation. Administration of nitroso sulphamethoxazole, but not sulphamethoxazole or sulphamethoxazole hydroxylamine, resulted in antigen formation on the surface of lymphocytes, splenocytes and epidermal keratinocytes, and a strong proliferative response of splenocytes on re‐stimulation with nitroso sulphamethoxazole. Rats dosed with sulphamethoxazole or sulphamethoxazole hydroxylamine did not respond to any of the test compounds. CD4+ or CD8+ depleted cells responded equally to nitroso sulphamethoxazole. The proliferative response to nitroso sulphamethoxazole was seen even after pulsing for only 5 min, and was not inhibited by glutathione. Responding cells produced IFN‐γ, but not IL‐4. Haptenation of cells by sulphamethoxazole hydroxylamine was seen after depletion of glutathione by pre‐treating the rats with diethyl maleate. Splenocytes from the glutathione‐depleted sulphamethoxazole hydroxylamine‐treated rats responded weakly to nitroso sulphamethoxazole, but not to sulphamethoxazole or sulphamethoxazole hydroxylamine. Dosing of rats with sulphamethoxazole produced a cellular response to nitroso sulphamethoxazole (but not to sulphamethoxazole or its hydroxylamine) when the animals were primed with complete Freunds adjuvant. These studies demonstrate the antigenicity of nitroso sulphamethoxazole in vivo and provide evidence for the role of drug metabolism and cell surface haptenation in the induction of a cellular immune response in the rat.

Cellular and molecular pathophysiology of cutaneous drug reactions.

Hypersensitivity reactions to drugs can cause a variety of skin diseases like maculopapular, bullous and pustular eruptions. In recent years increasing evidence indicates the important role of T cells in these drug-induced skin diseases. Analysis of such drug-specific T cell clones has revealed that drugs can be recognized by αβ-T cell receptors, not only if bound covalently to peptides, but also if the drug binds in a rather labile way to the presenting major histocompatibility complex (MHC)-peptide. This presentation is sufficient to stimulate T cells.In maculopapular exanthema (MPE), histopathological analysis typically shows a dominant T cell infiltration together with a vacuolar interface dermatitis. Immunohistochemical studies demonstrate the presence of cytotoxic CD4+ and to a lesser degree of CD8+ T cells, which contain perforin and granzyme B. They are close to keratinocytes that show signs of cell destruction. Expression of Fas ligand is barely detectable, suggesting that cytotoxic granule exocytosis may be the dominant pathway leading to keratinocyte cell damage. While in MPE, the killing of cells seems to be predominately mediated by CD4+ T cells, patients with bullous skin disease show a strong CD8+ T cell migration to the epidermis. This is probably due to a preferential presentation of the drug by MHC class I molecules, and a more extensive killing of cells that present drugs on MHC class I molecules. This might lead to bullous skin diseases.In addition to the presence of cytotoxic T cells, drug-specific T cells also orchestrate the inflammatory skin reaction through the release and induction of various cytokines [i.e. interleukin (IL)-5, IL-6, tumor necrosis factor-α and interferon-γ] and chemokines (RANTES, eotaxin or IL-8). The increased expression of these mediators seems to contribute to the generation of tissue and blood eosinophilia, a hallmark of many drug-induced allergic reactions. However, in acute generalized exanthematous pustulosis (a peculiar form of drug allergy), neutrophils represent the predominant cell type within pustules, probably due to their recruitment by IL-8 secreting drug specific T cells and keratinocytes.

Cellular disposition of sulphamethoxazole and its metabolites: implications for hypersensitivity

Bioactivation of sulphamethoxazole (SMX) to chemically‐reactive metabolites and subsequent protein conjugation is thought to be involved in SMX hypersensitivity. We have therefore examined the cellular metabolism, disposition and conjugation of SMX and its metabolites in vitro. Flow cytometry revealed binding of N‐hydroxy (SMX‐NHOH) and nitroso (SMX‐NO) metabolites of SMX, but not of SMX itself, to the surface of viable white blood cells. Cellular haptenation by SMX‐NO was reduced by exogenous glutathione (GSH). SMX‐NHOH and SMX‐NO were rapidly reduced back to the parent compound by cysteine (CYS), GSH, human peripheral blood cells and plasma, suggesting that this is an important and ubiquitous bioinactivation mechanism. Fluorescence HPLC showed that SMX‐NHOH and SMX‐NO depleted CYS and GSH in buffer, and to a lesser extent, in cells and plasma. Neutrophil apoptosis and inhibition of neutrophil function were induced at lower concentrations of SMX‐NHOH and SMX‐NO than those inducing loss of membrane viability, with SMX having no effect. Lymphocytes were significantly (P<0.05) more sensitive to the direct cytotoxic effects of SMX‐NO than neutrophils. Partitioning of SMX‐NHOH into red blood cells was significantly (P<0.05) lower than with the hydroxylamine of dapsone. Our results suggest that the balance between oxidation of SMX to its toxic metabolites and their reduction is an important protective cellular mechanism. If an imbalance exists, haptenation of the toxic metabolites to bodily proteins including the surface of viable cells can occur, and may result in drug hypersensitivity.

Human leukocyte antigen (HLA)‐B*57:01‐restricted activation of drug‐specific T cells provides the immunological basis for flucloxacillin‐induced liver injury

The role of the adaptive immune system in adverse drug reactions that target the liver has not been defined. For flucloxacillin, a delay in the reaction onset and identification of human leukocyte antigen (HLA)‐B*57:01 as a susceptibility factor are indicative of an immune pathogenesis. Thus, we characterize flucloxacillin‐responsive CD4+ and CD8+ T cells from patients with liver injury and show that naive CD45RA+CD8+ T cells from volunteers expressing HLA‐B*57:01 are activated with flucloxacillin when dendritic cells present the drug antigen. T‐cell clones expressing CCR4 and CCR9 migrated toward CCL17 and CCL 25, and secreted interferon‐gamma (IFN‐γ), T helper (Th)2 cytokines, perforin, granzyme B, and FasL following drug stimulation. Flucloxacillin bound covalently to selective lysine residues on albumin in a time‐dependent manner and the level of binding correlated directly with the stimulation of clones. Activation of CD8+ clones with flucloxacillin was processing‐dependent and restricted by HLA‐B*57:01 and the closely related HLA‐B*58:01. Clones displayed additional reactivity against β‐lactam antibiotics including oxacillin, cloxacillin, and dicloxacillin, but not abacavir or nitroso sulfamethoxazole. Conclusion: This work defines the immune basis for flucloxacillin‐induced liver injury and links the genetic association to the iatrogenic disease. (HEPATOLOGY 2013;)

Metabolic activation in drug allergies

Drug allergies are a major problem in the clinic and during drug development. At the present time, it is not possible to predict the potential of a new chemical entity to produce an allergic reaction (hypersensitivity) in patients in preclinical development. Such adverse reactions, because of their idiosyncratic nature, only become apparent once the drug has been licensed. Our present chemical understanding of drug hypersensitivity is based on the hapten hypothesis, in which covalent binding of the drug (metabolite) plays a central role in drug immunogenicity and antigenicity. If this theory is correct, then it should be possible to develop in vitro systems to assess the potential of drugs to bind to critical proteins, either directly or indirectly after metabolic activation to protein-reactive metabolites (bioactivation) and initiate hypersensitivity. The purpose of this review is to assess critically the evidence to support the hapten mechanism, and also to consider alternative mechanisms by which drugs cause idiosyncratic toxicity.