Evidence for the involvement of carbon-centred radicals in the induction of apoptotic cell death by artemisinin compounds

Artemisinin and its derivatives are currently recommended as first-line antimalarials in regions where Plasmodium falciparum is resistant to traditional drugs. The cytotoxic activity of these compounds towards rapidly dividing human carcinoma cells and cell lines has been reported and it is hypothesised that activation of the endoperoxide bridge, by an iron (II) species, to form C-centred radicals, is essential for cytotoxicity. The studies described here have utilised artemisinin derivatives ; dihydroartemisinin, 10β-(p-bromophenoxy) dihydroartemisinin and 10β-(p-fluorophenoxy) dihydroartemisinin to determine the chemistry of endoperoxide bridge activation to reactive intermediates responsible for initiating cell death, and to elucidate the molecular mechanism of cell death. In vitro studies have demonstrated the selective cytotoxic activity of the endoperoxides toward leukaemia cell lines (HL-60 and Jurkat) over quiescent peripheral blood mononuclear cells (PBMC). Deoxy-10β-(p-fluorophenoxy) dihydroartemisinin, which lacks the peroxide bridge, was 50- to 130-fold less active in the same cells confirming the importance of this functional group for cytotoxicity. We have shown that chemical activation is responsible for cytotoxicity using LC-MS analysis to monitor endoperoxide bridge activation by measurement of a stable rearrangement product of endoperoxide-derived radicals, which was formed in sensitive HL-60 cells but not insensitive PBMC. In HL-60 cells the endoperoxides induce caspase-dependent apoptotic cell death; characterized by concentration- and time-dependent mitochondrial membrane depolarisation, activation of caspases-3 and –7, sub-G0/G1 DNA formation and attenuation by z-VAD.fmk, a caspase inhibitor. Overall, these results indicate that endoperoxide-induced cell death is a consequence of metabolic activation of the peroxide bridge to radical species, which trigger caspase-dependent apoptosis

Detection of Drug Bioactivation in Vivo: Mechanism of Nevirapine–Albumin Conjugate Formation in Patients

The non-nucleoside reverse transcriptase inhibitor nevirapine (NVP) is widely used for the treatment of human immunodeficiency virus type 1 (HIV-1), particularly in developing countries. Despite its therapeutic benefits, NVP has been associated with skin and liver injury in exposed patients. Although the mechanism of the tissue injury is not yet clear, it has been suggested that reactive metabolites of NVP may be involved. The detection of NVP mercapturate in the urine of patients undergoing standard antiretroviral chemotherapy indicates that NVP undergoes bioactivation in vivo. However, covalent binding of drug to protein in patients remains to be determined. In this study, we investigate the chemical basis of NVP protein adduct formation by using human serum albumin (HSA) and glutathione <i>S</i>-transferase pi (GSTP) as model proteins in vitro. In addition, HSA was isolated from serum samples of HIV-1 patients undergoing NVP therapy to measure NVP haptenation. Mass spectrometric analysis of 12-sulfoxyl-NVP-treated HSA revealed that the drug bound selectively to histidine (His146, His242, and His338) and a cysteine residue (Cys34). The reaction proceeds most likely by a concerted elimination–addition mechanism. This pathway was further confirmed by the observation of NVP-modified Cys47 in GSTP. Importantly, the same adduct (His146) was detected in HSA isolated from the blood of patients receiving NVP, providing direct evidence that NVP modifies protein in vivo, via the formation of a reactive metabolite

Dapsone and Nitroso Dapsone Activation of Naı̈ve T‑Cells from Healthy Donors

Dapsone (DDS) causes hypersensitivity reactions in 0.5–3.6% of patients. Although clinical diagnosis is indicative of a hypersensitivity reaction, studies have not been performed to define whether dapsone or a metabolite activates specific T-cells. Thus, the aims of this study were to explore the immunogenicity DDS and nitroso DDS (DDS-NO) using peripheral blood mononuclear cells from healthy donors and splenocytes from mice and generate human T-cell clones to characterize mechanisms of T-cell activation. DDS-NO was synthesized from DDS-hydroxylamine and shown to bind to the thiol group of glutathione and human and mouse albumin through sulfonamide and <i>N</i>-hydroxyl sulphonamide adducts. Naïve T-cell priming to DDS and DDS-NO was successful in three human donors. DDS-specific CD4+ T-cell clones were stimulated to proliferate in response to drug via a MHC class II restricted direct binding interaction. Cross reactivity with DDS-NO, DDS-analogues, and sulfonamides was not observed. DDS-NO clones were CD4+ and CD8+, MHC class II and I restricted, respectively, and activated via a pathway dependent on covalent binding and antigen processing. DDS and DDS-NO-specific clones secreted a mixture of Th1 and Th2 cytokines, but not granzyme-B. Splenocytes from mice immunized with DDS-NO were stimulated to proliferate <i>in vitro</i> with the nitroso metabolite, but not DDS. In contrast, immunization with DDS did not activate T-cells. These data show that DDS- and DDS-NO-specific T-cell responses are readily detectable

Longitudinal biomarker analysis of infants treated with multiple courses of gentamicin without a change in serum creatinine concentration.

<p>Representative figures demonstrating the longitudinal quantification of the biomarkers KIM-1 (blue; ng/mg. uCr), NGAL (green; ng/mg. uCr), NAG (yellow; IU/mg. uCr) and serum creatinine (red; µmol/L) for three infants treated with gentamicin (A–C). Gentamicin treatment episode and length of treatment (days) are indicated by the black horizontal bar on each figure for that individual patient.</p

Haloarene Derivatives of Carbamazepine with Reduced Bioactivation Liabilities: 2‑Monohalo and 2,8-Dihalo Derivatives

The anticonvulsant carbamazepine <b>1</b> is associated with adverse drug reactions (ADRs), including hepatotoxicity; oxidative metabolism of <b>1</b> has been implicated in the pathogenesis of the ADRs. We report the synthesis and evaluation of 2-monohalo and 2,8-dihalo analogues of <b>1</b> that were intended to minimize reactive metabolite formation via arene oxidation and 10,11-epoxidation. Halo analogues were obtained either by rearrangement of halogenated <i>N</i>-arylindoles or from specifically halogenated iminodibenzyl derivatives. In rat hepatocytes, none of the analogues underwent oxidative dehalogenation or glutathione adduction. Some formation of the 10,11-epoxide still occurred, but aromatic hydroxylation was not seen with the exception of 2-fluoro, which allowed minor monohydroxylation. Complete inhibition of aromatic hydroxylation required at least monochlorination or difluorination of <b>1</b>. In human liver microsomes, difluoro analogue <b>5b</b> underwent 10,11-epoxidation but gave no arene oxidation

Baseline characteristics and clinical signs of neonates treated with gentamicin.

<p>Patients are subdivided according to gestational age. Mean biomarker values presented include samples collected both on and off gentamicin treatment over the whole time course of inclusion in the study.</p

HLA Restriction of Carbamazepine-Specific T‑Cell Clones from an HLA-A*31:01-Positive Hypersensitive Patient

HLA-A*31:01 is associated with carbamazepine (CBZ) hypersensitivity in Caucasian and Japanese populations. Herein, we show that <i>HLA-A*31:01+</i> restricted the activation of carbamazepine-specific CD8⁺ T-cells, which provides an immunological basis for the genetic association. Furthermore, CD4⁺ T-cells were activated with carbamazepine in a <i>HLA-DRB1*04:04</i>-restricted manner, indicating that a common HLA haplotype may contribute to the multiclonal T-cell response seen in European patients with CBZ hypersensitivity

Oxidative Bioactivation of Abacavir in Subcellular Fractions of Human Antigen Presenting Cells

Human exposure to abacavir, a primary alcohol antiretroviral, is associated with the development of immunological drug reactions in individuals carrying the HLA risk allele B*57:01. Interaction of abacavir with antigen presenting cells results in cell activation through an Hsp70-mediated Toll-like receptor pathway and the provision of T-cell antigenic determinants. Abacavir’s electrophilic aldehyde metabolites are potential precursors of neoantigens. Herein, we have used mass spectrometry to study the oxidative metabolism of abacavir in EBV-transformed human B-cells. RNA and protein were isolated from the cells and subjected to transcriptomic and mass spectrometric analyses to identify the redox enzymes expressed. Low levels of isomeric abacavir carboxylic acids were detected in subcellular fractions of EBV-transformed human B-cells incubated with abacavir. Metabolite formation was time-dependent but was not reduced by an inhibitor of Class I alcohol dehydrogenases. Relatively high levels of mRNA were detected for several redox enzymes, including alcohol dehydrogenase 5 (Class III), aldehyde dehydrogenases (ALDH3A2, ALDH6A1, and ALDH9A1), CYP1B1, CYP2R1, CYP7B1, and hydroxysteroid dehydrogenase 10. Over 2600 proteins were identified by mass spectrometry. More than 1000 of these proteins exhibited catalytic activity, and 80 were oxido-reductases. This is the first proteomic inventory of enzymes in antigen presenting cells. However, neither of the hepatic alcohol dehydrogenases of Class I which metabolize abacavir <i>in vitro</i> was expressed at the protein level. Nevertheless the metabolic production of abacavir carboxylic acids by B-cell fractions implies abacavir-treated immune cells might be exposed to the drug’s protein-reactive aldehyde metabolites <i>in vivo</i>

Longitudinal biomarker analysis of infants treated with multiple courses of gentamicin with a change in serum creatinine concentration (AKI).

<p>Representative figures demonstrating the longitudinal quantification of the biomarkers KIM-1 (blue; ng/mg. uCr), NGAL (green; ng/mg. uCr), NAG (yellow; IU/mg. uCr) and serum creatinine (red; µmol/L) for three infants treated with gentamicin (A–C). Gentamicin treatment episode and length of treatment (days) are indicated by the black horizontal bar on each figure for that individual patient.</p

Association between gentamicin treatment and the change in biomarker values.

<p>The mean baseline biomarker values in the absence of any gentamicin treatment were 1.91 ng/mg uCr (95% CI 1.07, 2.76) for KIM-1, 0.13 IU/mg uCr (0.07, 0.19) for NAG, 425.4 ng/mg uCr (162.6, 688.3) for NGAL, and 62.39 µmol/l (53.1, 71.69) for creatinine.</p