The decline in approval of new drugs during the past decade has led to a close analysis of the drug discovery process. One of the main reasons for attrition is preclinical toxicity, frequently attributed to the generation of protein-reactive drug metabolites. In this review, we present a critique of such reactive metabolites and evaluate the evidence linking them to observed toxic effects. Methodology for the characterization of reactive metabolites has advanced greatly in recent years, and is summarized first. Next, we consider the inhibition of key metabolic enzymes by electrophilic metabolites, as well as unfavorable drug-drug interactions that may ensue. One important class of protein-reactive metabolites, not linked conclusively to a toxic event, is acyl glucuronides. Their properties are discussed in light of the safety characteristics of carboxylic acid containing drugs. Many adverse drug reactions (ADRs) are known collectively as idiosyncratic events, that is, not predictable from knowledge of the pharmacology and pharmacokinetics of the parent compound. Observed ADRs may take various forms. Specific organ injury, particularly of the liver, is the most direct: we examine this in some detail. Moving to the cellular level, we also consider the upregulation of induced cellular processes. The related, but distinct, issue of hypersensitivity or allergic reactions to drugs and their metabolites, possibly via the immune system, is considered next. Finally, we discuss the impact of such data on the drug discovery process, both through early detection of reactive metabolites and informed synthetic design, which eliminates unfavorable functionality from drug candidates.
Recognition of drugs by immune cells is usually explained by the hapten model, which states that endogenous metabolites bind irreversibly to protein to stimulate immune cells. Synthetic metabolites interact directly with protein generating antigenic determinants for T-cells; however, experimental evidence relating intracellular metabolism in immune cells and the generation of physiologically relevant antigens to functional immune responses is lacking. The aim of this study was to develop an integrated approach using both animal and human experimental systems to characterize sulfamethoxazole (SMX) metabolism-derived antigenic protein adduct formation in immune cells and define the relationship between adduct formation, cell death, co-stimulatory signalling and stimulation of a T-cell response. Formation of SMX-derived adducts in antigen presenting cells was dose- and time-dependent, detectable at non-toxic concentrations and dependent on drug metabolizing enzyme activity. Adduct formation above a threshold induced necrotic cell death, dendritic cell co-stimulatory molecule expression and cytokine secretion. Antigen presenting cells cultured with SMX for 16h, the time needed for drug metabolism, stimulated T-cells from sensitized mice and lymphocytes and T-cell clones from allergic patients. Enzyme inhibition decreased SMX-derived protein adduct formation and the T-cell response. Dendritic cells cultured with SMX and adoptively transferred to recipient mice initiated an immune response; however, T-cells were stimulated with adducts derived from SMX metabolism in antigen presenting cells, not the parent drug. This study shows that antigen presenting cells metabolize SMX; subsequent protein binding generates a functional T-cell antigen. Adduct formation above a threshold stimulates cell death, which provides a maturation signal for dendritic cells.
The clustered regularly interspaced short palindromic repeat/CRISPR-associated protein 9 (CRISPR/Cas9) technology is a versatile and useful tool to perform genome editing in different organisms ranging from bacteria and yeast to plants and mammalian cells. For a couple of years, it was believed that the system was inefficient and toxic in the alga Chlamydomonas reinhardtii. However, recently the system has been successfully implemented in this model organism, albeit relying mostly on the electroporation of ribonucleoproteins (RNPs) into cell wall deficient strains. This requires a constant source of RNPs and limits the application of the technology to strains that are not necessarily the most relevant from a biotechnological point of view. Here, we show that transient expression of the Streptococcus pyogenes Cas9 gene and sgRNAs, targeted to the single-copy nuclear apt9 gene, encoding an adenine phosphoribosyl transferase (APT), results in efficient disruption at the expected locus. Introduction of indels to the apt9 locus results in cell insensitivity to the otherwise toxic compound 2-fluoroadenine (2-FA). We have used agitation with glass beads and particle bombardment to introduce the plasmids carrying the coding sequences for Cas9 and the sgRNAs in a cell-walled strain of C. reinhardtii (CC-125). Using sgRNAs targeting exons 1 and 3 of apt9, we obtained disruption efficiencies of 3 and 30% on preselected 2-FA resistant colonies, respectively. Our results show that transient expression of Cas9 and a sgRNA can be used for editing of the nuclear genome inexpensively and at high efficiency. Targeting of the APT gene could potentially be used as a pre-selection marker for multiplexed editing or disruption of genes of interest.
Exposure to sulfamethoxazole (SMX) is associated with T-cell-mediated hypersensitivity reactions in human patients. T-cells can be stimulated by the putative metabolite nitroso SMX, which binds irreversibly to protein. The hydroxylamine and nitroso derivatives of three arylamine benzenesulfonamides, namely, sulfamethozaxole, sulfadiazine, and sulfapyridine, were synthesized, and their T-cell stimulatory capacity in the mouse was explored. Nitroso derivatives were synthesized by a three-step procedure involving the formation of nitro and hydroxylamine sulfonamide intermediates. For immune activation, female Balb-c strain mice were administered nitroso sulfonamides four times weekly for 2 weeks. After 14 days, isolated splenocytes were incubated with the parent compounds, hydroxylamine metabolites, and nitroso derivatives to measure antigen-specific proliferation. To explore the requirement of irreversible protein binding for spleen cell activation, splenocytes were incubated with nitroso derivatives in the presence or absence of glutathione. Splenocytes from nitroso sulfonamide-sensitized mice proliferated and secreted interleukin (IL)-2, IL-4, IL-5, and granulocyte monocyte colony-stimulating factor following stimulation with nitroso derivatives but not the parent compounds. Splenocytes from sensitized mice were also stimulated to proliferate with hydroxylamine and nitroso derivatives of the structurally related sulfonamides. The addition of glutathione inhibited the nitroso-specific T-cell response. Hydroxylamine metabolites were unstable in aqueous solution: Spontaneous transformation yielded appreciable amounts of nitroso and azoxy compounds as well as the parent compounds within 0.1 h. T-cell cross-reactivity with nitroso sulfonamides provides a mechanistic explanation as to why structurally related arylamine benzenesulfonamides are contraindicated in hypersensitive patients.
Dysregulation in the expression of microRNAs (miRNAs), single-stranded RNAs which regulate gene expression, has been associated with diseases such as Stevens-Johnson syndrome (SJS)/toxic epidermal necrolysis (TEN), although their cellular origin has not been explored. Thus, the focus of this work was to study expression patterns of reported miRNAs involved in T-cell activation following drug-specific stimulation in peripheral blood mononuclear cells (PBMCs) and drug-specific CD4 T-cell clones (TCC) from patients with different cutaneous manifestations of delayed-type drug hypersensitivity reactions. CD4 T-cells from hypersensitive patients were stimulated to proliferate, secreted cytokines (IFN-γ and IL-22), cytolytic molecules (Granzyme B) and up-regulate miRNAs 24 to 48 h after drug exposure. Carbamazepine-specific CD4 T-cells that proliferated to the greatest extent and secreted the highest levels of IFN-γ showed an up-regulation of miR-18a and miR-155. In contrast, piperacillin-specific CD4 T-cells displaying high expression of miR-9 and miR-21 showed an association with the extent of proliferation, but not IFN-γ secretion. MiR-155 up-regulation was detected in PBMCs from all hypersensitive patients 24 h after drug treatment, while miR-18a and miR-21 expression was up-regulated after 48 h. These findings demonstrate that miRNAs are expressed during drug-specific CD4 T-cell activation and shows a new regulation path for drug hypersensitivity reactions.
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