HLA-DQ allele-restricted activation of nitroso sulfamethoxazole-specific CD4-positive T lymphocytes from patients with cystic fibrosis.

BACKGROUND: For certain HLA allele-associated drug hypersensitivity reactions, the parent drug has been shown to associate directly with the risk allele. In other forms of hypersensitivity, HLA risk alleles have not been identified and T cells are activated in an allele unrestricted manner. Chemically reactive drug metabolites bind to multiple proteins; thus, it is assumed that the derived peptide antigens interact with a number of HLA molecules to activate T cells; however, HLA restriction of the drug metabolite-specific T-cell response has not been studied. OBJECTIVE: To utilize T cells from sulfamethoxazole (SMX) hypersensitive patients with cystic fibrosis to examine the HLA molecules that interact with nitroso SMX (SMX-NO)-derived antigens. METHODS: T-cell clones were generated from 4 hypersensitive patients. Drug-specific proliferative responses and cytokine secretion were measured. Anti-human class I and class II antibodies were used to analyse HLA restriction. Antigen-presenting cells expressing different HLA molecules were used to determine the alleles involved in the presentation of SMX-NO-derived antigens to T cells. RESULTS: A total of 976 clones were tested for SMX-NO reactivity. Thirty-nine CD4+ clones were activated with SMX-NO and found to proliferate and secrete cytokines. The SMX-NO-specific response was blocked with an antibody against HLA-DQ. SMX-NO-specific responses were detected with antigen-presenting cells expressing HLA-DQB1*05:01 (patient 1) and HLA-DQB1*02:01 (patient 2), but not other HLA-DQB1 alleles. CONCLUSION AND CLINICAL RELEVANCE: HLA-DQ plays an important role in the activation of SMX-NO-specific CD4+ T cells. Detection of HLA-DQ allele-restricted responses suggests that T cells are activated by a limited repertoire of SMX-NO-modified peptides.

Current status of GV1001 and other telomerase vaccination strategies in the treatment of cancer.

GV1001 is a telomerase-specific, promiscuous class II peptide vaccine which is currently in an advanced stage of clinical development. This article reviews the biological rationale underpinning the design of ongoing studies with the vaccine as well as its immunogenicity and clinical activity. It places GV1001 in the context of other immunotherapeutic approaches targeting telomerase and assesses the chances of the vaccine becoming a future standard of care in the treatment of cancer.

"Danger" conditions increase sulfamethoxazole-protein adduct formation in human antigen-presenting cells.

Antigen-presenting cells (APC) are thought to play an important role in the pathogenesis of drug-induced immune reactions. Various pathological factors can activate APC and therefore influence the immune equilibrium. It is interesting that several diseases have been associated with an increased rate of drug allergy. The aim of this project was to evaluate the impact of such "danger signals" on sulfamethoxazole (SMX) metabolism in human APC (peripheral blood mononuclear cells, Epstein-Barr virus-modified B lymphocytes, monocyte-derived dendritic cells, and two cell lines). APC were incubated with SMX (100 microM-2 mM; 5 min-24 h), in the presence of pathological factors: bacterial endotoxins (lipopolysaccharide and staphylococcal enterotoxin B), flu viral proteins, cytokines [interleukin (IL)-1beta, IL-6, IL-10; tumor necrosis factor-alpha; interferon-gamma; and transforming growth factor-beta], inflammatory molecules (prostaglandin E2, human serum complement, and activated protein C), oxidants (buthionine sulfoximine and H(2)O(2)), and hyperthermia (37.5-39.5 degrees C). Adduct formation was evaluated by enzyme-linked immunosorbent assay and confocal microscopy. SMX-protein adduct formation was time- and concentration-dependent for each cell type tested, in both physiological and danger conditions. A danger environment significantly increased the formation of SMX-protein adducts and significantly shortened the delay for their detection. An additive effect was observed with a combination of danger signals. Dimedone (chemical selectively binding cysteine sulfenic acid) and antioxidants decreased both baseline and danger-enhanced SMX-adduct formation. Various enzyme inhibitors were associated with a significant decrease in SMX-adduct levels, with a pattern varying depending on the cell type and the culture conditions. These results illustrate that danger signals enhance the formation of intracellular SMX-protein adducts in human APC. These findings might be relevant to the increased frequency of drug allergy in certain disease states.

Are chemically reactive metabolites responsible for adverse reactions to drugs?

Low molecular weight organic chemicals can be transformed by normal drug-metabolising systems into short-lived metabolites that are inherently reactive towards cellular macromolecules. There is direct evidence that the formation of such chemically reactive metabolites may lead to mutagenesis, carcinogenicity, apoptosis and necrosis in both cell and animal models. A number of drugs associated with non-pharmacological drug toxicities in man have been shown to undergo bioactivation either in vivo or in vitro. We have therefore examined the evidence for the role of reactive metabolites in the three most common drug-induced toxicities: hepatotoxicity, skin reactions and blood dyscrasias.

Influence of reduced glutathione on the proliferative response of sulfamethoxazole-specific and sulfamethoxazole-metabolite-specific human CD4+ T-cells.

1. Hypersensitivity to the drug sulfamethoxazole (SMX) is thought to be a consequence of bioactivation to the hydroxylamine metabolite (SMX-NHOH) and further oxidation to the ultimate reactive metabolite, nitroso-sulfamethoxazole (SMX-NO). SMX-NO covalently modifies self proteins which in turn might be recognized as neo-antigens by T-cells. The antioxidant glutathione (GSH) is known to protect cells from reactive metabolites by conjugation and subsequent dissociation to SMX-NHOH and/or SMX. 2. To study the reactivity of T-cells to SMX metabolites and their respective role in the generation of drug-specific T-cells, we analysed the effect of GSH on the response of PBMC to SMX and its metabolites SMX-NHOH and SMX-NO. Furthermore, we monitored the proliferative response of drug-specific T-cell clones in the presence or absence of GSH. 3. We found that addition of GSH to peripheral blood mononuclear cells had no effect on the SMX-specific response but enhanced the proliferation to SMX-metabolites. The response of SMX-NO-specific T-cell clones was abrogated when GSH was present during the covalent haptenation of antigen presenting cells (APC). Conversely, SMX-specific T-cell clones gained reactivity through the conversion of SMX-NO to the parent drug by GSH. While GSH had no effect on the initial activation of T-cell clones, it prevented covalent binding to APCs, reduced toxicity and thereby led to proliferation of drug-specific T-cells to non-reactive drug metabolites. 4. Our data support the concept that in allergic individuals T-cells recognize the non-covalently bound parent drug rather than APC covalently modified by SMX-NO.

Metabolism of lamotrigine to a reactive arene oxide intermediate.

Lamotrigine [3,5-diamino-6-(2,3-dichlorophenyl)-1,2,4-triazine] is an antiepileptic drug associated with hypersensitivity reactions which are thought to be an immunological response to metabolically generated drug-protein adducts. The o-dichlorophenyl moiety is a potential site for bioactivation of the drug to an arene oxide. The metabolites of [(14)C]lamotrigine (78 micromol/kg, iv) in adult male Wistar rats were characterized with particular reference to thioether derivatives of an epoxide intermediate. Biliary recovery of radioactivity from anesthetized and cannulated animals was 7.3 +/- 3.0% (mean +/- SD, n = 4) of the dose over 4 h; 5.5 +/- 0.5% was recovered in bladder urine after 4 h. Bile contained [(14)C]lamotrigine (1.4 +/- 0.3%), a glutathione adduct of [(14)C]dihydrohydroxylamotrigine (1.8 +/- 0.3%), i.e., an adduct of an arene oxide, and the glutathione (1.5 +/- 0.7%), cysteinylglycine (1.9 +/- 0.5%), and N-acetylcysteine (0.4 +/- 0.2%) adducts of [(14)C]lamotrigine. Formation of the thioether metabolites was partially blocked by the cytochrome P450 inhibitor, ketoconazole. Urine contained [(14)C]lamotrigine (4.5 +/- 0.5%) and [(14)C]lamotrigine N-oxide (0.9 +/- 0.2%). The radiolabeled material in skin (15.6 +/- 1.4%) was almost entirely [(14)C]lamotrigine. Isolated rat hepatocytes achieved a low rate of turnover to the glutathione adduct and N-oxide. However, neither rat nor human liver microsomes catalyzed NADPH-dependent irreversible binding. Lamotrigine can be bioactivated to an arene oxide by rat hepatocytes in the absence of a major competing pathway such as N-glucuronidation. Inhibition of N-glucuronidation has been associated with an increased risk of skin reactions in patients.

Induction of metabolism-dependent and -independent neutrophil apoptosis by clozapine.

Clozapine, an atypical antipsychotic used in the treatment of refractory schizophrenia, causes neutropenia and agranulocytosis in 3 and 0.8% of patients, respectively. Clozapine undergoes bioactivation to a chemically reactive nitrenium ion, which has been shown to cause neutrophil cytotoxicity. To define further the mechanism of cell death, we have investigated the toxicity of clozapine, its stable metabolites, and its chemically reactive nitrenium ion to neutrophils and lymphocytes. Clozapine was able to induce neutrophil apoptosis at therapeutic concentrations (1-3 microM) only when it was bioactivated to the nitrenium ion. The parent drug caused apoptosis at supratherapeutic concentrations (100-300 microM) only. Neutrophil apoptosis induced by the nitrenium ion, but not by the parent drug itself, was inhibited by antioxidants and genistein and was accompanied by cell surface haptenation (assessed by flow cytometry) and glutathione depletion. Dual-color flow cytometry showed that neutrophils that were haptenated were the same cells that underwent apoptosis. No apoptosis of lymphocytes was evident with the nitrenium ion or the parent drug, despite the fact that the former caused cell surface haptenation, glutathione depletion, and loss of membrane integrity. Demethylclozapine, the major stable metabolite in vivo, showed a profile that was similar to, although less marked than that observed with clozapine. N-oxidation of clozapine or replacement of the nitrogen (at position 5) by sulfur produced compounds that were entirely nontoxic to neutrophils. In conclusion, the findings of the study expand on potential mechanisms of clozapine-induced cytotoxicity, which may be of relevance to the major forms of toxicity encountered in patients taking this drug.

Plasma cysteine deficiency and decreased reduction of nitrososulfamethoxazole with HIV infection.

The aim of these studies was to determine whether HIV-infected patients have a plasma thiol deficiency and whether this is associated with decreased detoxification of the toxic metabolites of sulfamethoxazole. Reduced, oxidized, protein-bound, and total thiol levels were measured in 33 HIV-positive patients and 33 control subjects by an HPLC method utilizing the fluorescent probe bromobimane. The reduction of sulfamethoxazole hydroxylamine and nitrososulfamethoxazole by plasma and the plasma redox balance in the presence of nitrososulphamethoxazole were also determined by HPLC. Reduced plasma cysteine was significantly (p<0.0001) lower in HIV-positive patients (13.0+/-3.0 microM) when compared with control subjects (16.9+/-3.0 microM). Although there was no difference in oxidized, protein-bound, and total cysteine, the thiol/disulfide ratios were lower in HIV-positive patients. Reduced homocysteine was elevated in patients. Plasma from HIV-positive patients was less able to detoxify nitrososulfamethoxazole than control plasma. These findings show that the disturbance in redox balance in HIV-positive patients may alter metabolic detoxification capacity, and thereby predispose to sulfamethoxazole hypersensitivity.

Cellular disposition of sulphamethoxazole and its metabolites: implications for hypersensitivity.

1. 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. 2. 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). 3. 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. 4. Fluorescence HPLC showed that SMX-NHOH and SMX-NO depleted CYS and GSH in buffer, and to a lesser extent, in cells and plasma. 5. 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. 6. Partitioning of SMX-NHOH into red blood cells was significantly (P<0.05) lower than with the hydroxylamine of dapsone. 7. 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.

Metabolism-dependent neutrophil cytotoxicity of amodiaquine: A comparison with pyronaridine and related antimalarial drugs.

Life-threatening agranulocytosis and hepatotoxicity during prophylactic administration of amodiaquine have led to its withdrawal. Agranulocytosis is thought to involve bioactivation to a protein-reactive quinoneimine metabolite. The toxicity of amodiaquine and the lack of cheap drugs have prompted a search for alternative antimalarial agents. The aim of this study was to determine the metabolism and neutrophil toxicity of amodiaquine, pyronaridine, and other related antimalarial agents. Horseradish peroxidase and hydrogen peroxide were used to activate drugs to their respective quinoneimine metabolites. Metabolites were trapped as stable glutathione conjugates, prior to analysis by LC/MS. Amodiaquine was metabolized to a polar metabolite (m/z 661), identified as a glutathione adduct. Tebuquine was converted to two polar metabolites. The principal metabolite (m/z 686) was derived from glutathione conjugation and side chain elimination, while the minor metabolite gave a protonated molecule (m/z 496). Only parent ions were identified when chloroquine, cycloquine, or pyronaridine was incubated with the activating system and glutathione. Calculation of the heat of formation of the drugs, however, demonstrated that amodiaquine, tebuquine, cycloquine, and pyronaridine readily undergo oxidation to their quinoneimine. None of the antimalarial compounds depleted the level of intracellular glutathione (1-300 microM) when incubated with neutrophils alone. Additionally, with the exception of tebuquine, no cytotoxicity below 100 microM was observed. In the presence of the full activating system, however, all compounds except chloroquine resulted in depletion of the level of glutathione and were cytotoxic. Pretreating the cells with glutathione and other antioxidants inhibited metabolism-dependent cytotoxicity. In summary, our data show that amodiaquine and related antimalarials containing a p-aminophenol moiety undergo bioactivation in vitro to chemically reactive and cytotoxic intermediates. In particular, pyronaridine, which is currently being investigated in humans, was metabolized to a compound which was toxic to neutrophils. Thus, the possibility that it will cause agranulocytosis in clinical practice cannot be excluded, and will require careful monitoring.

Disposition of amodiaquine and related antimalarial agents in human neutrophils: implications for drug design.

The development and clinical use of 4-aminoquinoline antimalarial agents such as amodiaquine have been limited by toxicity to neutrophils. We have investigated the chemical basis of amodiaquine-induced toxicity and compared the findings with those for established antimalarial drugs proposed for human use. Amodiaquine, like chloroquine, mefloquine and halofantrine, was lysosomotropic and accumulated in human neutrophils. Amodiaquine did not lead to impairment of either cellular function or cell viability at therapeutic levels. In contrast to other antimalarial agents, amodiaquine (because it contains a 4-aminophenol function) depleted glutathione in activated neutrophils, by formation of an electrophilic quinoneimine metabolite. Bioactivation was accompanied by the expression of a drug-related antigen on the cell surface, which was recognized by drug-specific antibodies, suggesting that a type II hypersensitivity reaction is responsible for the observed toxicity. Similar bioactivation and accumulation were observed for the structurally related amopyroquine. The effects of chemical modifications at the 3'- and 5'-positions, which are known to enhance antimalarial activity, were also investigated. The introduction of a lipophilic 5'-chlorophenyl group and 3'-t-butyl group blocked bioactivation but enhanced cellular accumulation, with resultant impairment of function and neutrophil viability, whereas introduction of a second cationic dialkylamino group (bis-mannich compounds) blocked bioactivation and reduced cellular accumulation, without producing noticeable effects on cellular function and viability. These data provide a chemical rationale for the idiosyncratic agranulocytosis observed with amodiaquine, and they suggest that similar toxicity might be anticipated for amopyroquine but is less likely with bis-mannich antimalarial agents such as pyronaridine.

Neutrophil cytotoxicity of the chemically reactive metabolite(s) of clozapine: possible role in agranulocytosis.

Clozapine is associated with a 0.8% incidence of agranulocytosis. Bioactivation to an unstable protein-reactive metabolite, identified as a nitrenium intermediate, has been implicated in the toxicity. In this study, we investigated whether the reactive metabolite is cytotoxic toward polymorphonuclear leukocytes and mononuclear leukocytes using horseradish peroxidase and H2O2 to generate the metabolite in situ. In the absence of a full metabolizing system (i. e., lack of horseradish peroxidase and/or H2O2), clozapine (0-100 microM) and its stable metabolites were not cytotoxic. With a full metabolizing system, both clozapine (30 microM) and demethylclozapine exhibited cytotoxicity toward polymorphonuclear leukocytes (50.7 +/- 7.7% and 17.6 +/- 1.2% cell death, respectively) and mononuclear leukocytes (36.6 +/- 2.1% and 24.6 +/- 4.1%, respectively), whereas clozapine N-oxide was not cytotoxic. Exogenous glutathione (GSH), N-acetylcysteine and ascorbic acid all protected the cells. Bioactivation of clozapine and demethylclozapine, but not the N-oxide, was accompanied by depletion of intracellular GSH. [14C]Clozapine was metabolized to the previously identified C6 and C9 glutathionyl conjugates; GSH conjugates were also detected when demethylclozapine and clozapine N-oxide were bioactivated by horseradish peroxidase and H2O2. In conclusion, using a novel in vitro assay, we have shown that clozapine and its stable metabolites are not cytotoxic per se but are bioactivated to cytotoxic metabolites. The cytotoxic metabolite of clozapine is identical to the protein-reactive metabolite that has been characterized previously. These cytotoxic metabolites may play an important role in the pathogenesis of clozapine agranulocytosis; the mechanism by which this occurs is currently being investigated.

Manipulation of the N-alkyl substituent in amodiaquine to overcome the verapamil-sensitive chloroquine resistance component.

Aminoquinoline resistance correlates with lipid solubility at pH 7.2. Consequently, the in vivo dealkylation of amodiaquine, to the less lipid-soluble desethylamodiaquine, is a likely contributor to therapeutic failure in vivo. Therefore, 4-aminoquinoline drugs with lipid solubilities similar to that of amodiaquine, but which are not subject to side chain modification in vivo, should be superior antimalarial agents. In this study, we have identified amopyroquine and N-tertbutylamodiaquine as two such compounds. The values for the logarithms of the partition coefficients for amopyroquine and N-tertbutylamodiaquine are between those for amodiaquine and its dealkylated metabolite, desethylamodiaquine. Both amopyroquine and N-tertbutylamodiaquine possess levels of antimalarial activity greater than that of desethylamodiaquine and significantly reduced cross-resistance patterns; i.e., the former two compounds are not subject to the verapamil-sensitive resistance mechanism. Simple in vitro markers of direct toxicity and potential reactive metabolite formation suggest that these two compounds are no more toxic than amodiaquine and desethylamodiaquine.