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1.
J Viral Hepat ; 28(2): 420-430, 2021 02.
Artigo em Inglês | MEDLINE | ID: mdl-33073452

RESUMO

The first clinical case of persistent HEV infection in England was reported in 2009. We describe the demography, virology and outcomes of patients identified with persistent HEV infection in England and Wales between 2009 and 2017. A series of 94 patients with persistent HEV infection, defined by HEV viraemia of more than 12 weeks, was identified through routine reference laboratory testing. Virology, serology and clinical data were recorded through an approved PHE Enhanced Surveillance System. Sixty-six cases (70.2%) were transplant recipients, 16 (17.0%) had an underlying haematological malignancy without stem cell transplantation, six (6.4%) had advanced HIV infection, five (5.3%) were otherwise immunosuppressed, and one patient (1.1%) had no identified immunosuppression. Retrospective analysis of 46 patients demonstrated a median 38 weeks of viraemia before diagnostic HEV testing. At initial diagnosis, 16 patients (17.0%) had no detectable anti-HEV serological response. Of 65 patients treated with ribavirin monotherapy, 11 (16.9%) suffered virological relapse despite undetectable RNA in plasma or stool at treatment cessation. Persistent HEV infection remains a rare diagnosis, but we demonstrate that a broad range of immunocompromised patients are susceptible. Both lack of awareness and the pauci-symptomatic nature of persistent HEV infection likely contribute to significant delays in diagnosis. Diagnosis should rely on molecular testing since anti-HEV serology is insufficient to exclude persistent HEV infection. Finally, despite treatment with ribavirin, relapses occur even after cessation of detectable faecal shedding of HEV RNA, further emphasising the requirement to demonstrate sustained virological responses to treatment.


Assuntos
Infecções por HIV , Vírus da Hepatite E , Hepatite E , Demografia , Hepatite E/diagnóstico , Hepatite E/epidemiologia , Vírus da Hepatite E/genética , Humanos , Hospedeiro Imunocomprometido , Recidiva Local de Neoplasia , RNA Viral , Estudos Retrospectivos , País de Gales/epidemiologia
2.
Br J Clin Pharmacol ; 87(6): 2572-2588, 2021 06.
Artigo em Inglês | MEDLINE | ID: mdl-33217013

RESUMO

AIMS: Carbamazepine can cause hypersensitivity reactions in ~10% of patients. An immunogenic effect can be produced by the electrophilic 10,11-epoxide metabolite but not by carbamazepine. Hypothetically, certain single nucleotide polymorphisms might increase the formation of immunogenic metabolites, leading ultimately to hypersensitivity reactions. This study explores the role of clinical and genetic factors in the pharmacokinetics (PK) of carbamazepine and 3 metabolites known to be chemically reactive or formed through reactive intermediates. METHODS: A combination of rich and sparse PK samples were collected from healthy volunteers and epilepsy patients. All subjects were genotyped for 20 single nucleotide polymorphisms in 11 genes known to be involved in the metabolism or transport of carbamazepine and carbamazepine 10,11-epoxide. Nonlinear mixed effects modelling was used to build a population-PK model. RESULTS: In total, 248 observations were collected from 80 subjects. A 1-compartment PK model with first-order absorption and elimination best described the parent carbamazepine data, with a total clearance of 1.96 L/h, central distribution volume of 164 L and absorption rate constant of 0.45 h-1 . Total daily dose and coadministration of phenytoin were significant covariates for total clearance of carbamazepine. EPHX1-416G/G genotype was a significant covariate for the clearance of carbamazepine 10,11-epoxide. CONCLUSION: Our data indicate that carbamazepine clearance was affected by total dose and phenytoin coadministration, but not by genetic factors, while carbamazepine 10,11-epoxide clearance was affected by a variant in the microsomal epoxide hydrolase gene. A much larger sample size would be required to fully evaluate the role of genetic variation in carbamazepine pharmacokinetics, and thereby predisposition to carbamazepine hypersensitivity.


Assuntos
Anticonvulsivantes , Carbamazepina , Epilepsia , Anticonvulsivantes/farmacocinética , Anticonvulsivantes/uso terapêutico , Carbamazepina/farmacocinética , Carbamazepina/uso terapêutico , Epilepsia/tratamento farmacológico , Epilepsia/genética , Epóxido Hidrolases/genética , Humanos , Fenitoína/uso terapêutico
3.
Chem Res Toxicol ; 32(10): 2095-2106, 2019 10 21.
Artigo em Inglês | MEDLINE | ID: mdl-31468968

RESUMO

Hypersensitivity reactions occur frequently in patients upon treatment with sulfamethoxazole (SMX). These adverse effects have been attributed to nitroso sulfamethoxazole (SMX-NO), the reactive product formed from auto-oxidation of the metabolite SMX hydroxylamine. The ability of SMX-NO to prime naïve T-cells in vitro and also activate T-cells derived from hypersensitive patients has illustrated that T-cell activation may occur through the binding of SMX-NO to proteins or through the direct modification of MHC-bound peptides. SMX-NO has been shown to modify cysteine residues in glutathione, designer peptides, and proteins in vitro; however, the presence of these adducts have not yet been characterized in vivo. In this study a parallel in vitro and in vivo analysis of SMX-NO adducts was conducted using mass spectrometry. In addition to the known cysteine adducts, multiple SMX-NO-derived haptenic structures were found on lysine and tyrosine residues of human serum albumin (HSA) in vitro. On lysine residues two haptenic structures were identified including an arylazoalkane adduct and a Schiff base adduct. Interestingly, these adducts are labile to heat and susceptible to hydrolysis as shown by the presence of allysine. Furthermore, SMX-modified HSA adducts were detected in patients on long-term SMX therapy illustrated by the presence of an arylazoalkane adduct derived from a proposed carboxylic acid metabolite of SMX-NO. The presence of these adducts could provide an explanation for the immunogenicity of SMX and the strong responses to SMX-NO observed in T-cell culture assays. Also, the degradation of these adducts to allysine could lead to a stress-related innate immune response required for T-cell activation.


Assuntos
Haptenos/imunologia , Compostos Nitrosos/química , Sulfametoxazol/química , Linfócitos T/efeitos dos fármacos , Linfócitos T/imunologia , Células Cultivadas , Estudos de Coortes , Haptenos/química , Humanos , Espectrometria de Massas , Modelos Moleculares , Estrutura Molecular , Compostos Nitrosos/imunologia , Albumina Sérica Humana/química , Albumina Sérica Humana/isolamento & purificação , Sulfametoxazol/imunologia
4.
Arch Toxicol ; 93(2): 385-399, 2019 02.
Artigo em Inglês | MEDLINE | ID: mdl-30426165

RESUMO

The transcription factor NRF2, governed by its repressor KEAP1, protects cells against oxidative stress. There is interest in modelling the NRF2 response to improve the prediction of clinical toxicities such as drug-induced liver injury (DILI). However, very little is known about the makeup of the NRF2 transcriptional network and its response to chemical perturbation in primary human hepatocytes (PHH), which are often used as a translational model for investigating DILI. Here, microarray analysis identified 108 transcripts (including several putative novel NRF2-regulated genes) that were both downregulated by siRNA targeting NRF2 and upregulated by siRNA targeting KEAP1 in PHH. Applying weighted gene co-expression network analysis (WGCNA) to transcriptomic data from the Open TG-GATES toxicogenomics repository (representing PHH exposed to 158 compounds) revealed four co-expressed gene sets or 'modules' enriched for these and other NRF2-associated genes. By classifying the 158 TG-GATES compounds based on published evidence, and employing the four modules as network perturbation metrics, we found that the activation of NRF2 is a very good indicator of the intrinsic biochemical reactivity of a compound (i.e. its propensity to cause direct chemical stress), with relatively high sensitivity, specificity, accuracy and positive/negative predictive values. We also found that NRF2 activation has lower sensitivity for the prediction of clinical DILI risk, although relatively high specificity and positive predictive values indicate that false positive detection rates are likely to be low in this setting. Underpinned by our comprehensive analysis, activation of the NRF2 network is one of several mechanism-based components that can be incorporated into holistic systems toxicology models to improve mechanistic understanding and preclinical prediction of DILI in man.


Assuntos
Doença Hepática Induzida por Substâncias e Drogas/genética , Redes Reguladoras de Genes/efeitos dos fármacos , Hepatócitos/efeitos dos fármacos , Fator 2 Relacionado a NF-E2/genética , Células Cultivadas , Doença Hepática Induzida por Substâncias e Drogas/patologia , Regulação da Expressão Gênica/efeitos dos fármacos , Hepatócitos/patologia , Humanos , Isotiocianatos/efeitos adversos , Proteína 1 Associada a ECH Semelhante a Kelch/genética , Análise de Sequência com Séries de Oligonucleotídeos , Estresse Oxidativo/efeitos dos fármacos , Estresse Oxidativo/genética , RNA Interferente Pequeno , Sulfóxidos
6.
Chem Res Toxicol ; 30(12): 2174-2186, 2017 12 18.
Artigo em Inglês | MEDLINE | ID: mdl-29045131

RESUMO

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 N-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 in vitro 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.


Assuntos
Dapsona/farmacologia , Ativação Linfocitária/efeitos dos fármacos , Compostos Nitrosos/farmacologia , Linfócitos T/citologia , Linfócitos T/efeitos dos fármacos , Animais , Proliferação de Células/efeitos dos fármacos , Células Cultivadas , Cromatografia Líquida de Alta Pressão , Dapsona/administração & dosagem , Dapsona/química , Voluntários Saudáveis , Humanos , Espectrometria de Massas , Camundongos , Estrutura Molecular , Compostos Nitrosos/administração & dosagem , Compostos Nitrosos/química , Albumina Sérica/química , Baço/citologia , Baço/efeitos dos fármacos , Linfócitos T/imunologia
7.
Chem Res Toxicol ; 30(7): 1419-1435, 2017 07 17.
Artigo em Inglês | MEDLINE | ID: mdl-28562019

RESUMO

Carbamazepine (CBZ) is an effective antiepileptic drug that has been associated with hypersensitivity reactions. The pathogenesis of those reactions is incompletely understood but is postulated to involve a complex interplay between the drug's metabolism, genetic variation in human leukocyte antigens, and adverse activation of the immune system. Multiple T-cell activation mechanisms have been hypothesized including activation by drug-peptide conjugates derived from proteins haptenated by reactive metabolites. However, definitive evidence of the drug-protein adducts in patients has been lacking. In this study, mass spectrometry was used to characterize protein modifications by microsomally-generated metabolites of CBZ and in patients taking CBZ therapy. CBZ 10,11-epoxide (CBZE), a major electrophilic plasma metabolite of CBZ, formed adducts with glutathione-S-transferase pi (GSTP; Cys47) and human serum albumin (HSA; His146 and His338, but not Cys34) in vitro via notably divergent side-chain selectivity. Both proteins were adducted at the same residues by undefined monoxygenated metabolites ([O]CBZ) when they were incubated with human liver microsomes, NADPH and CBZ. There was also evidence for formation of a CBZ adduct at His146 and His338 of HSA derived via dehydration from an intermediate arene oxide adduct. Glutathione trapping of reactive metabolites confirmed microsomal production of CBZE and indicated simultaneous production of arene oxides. In 15 patients prescribed CBZ therapy, [O]CBZ-modified HSA (His146) was detected in all subjects. The relative amount of adduct was moderately positively correlated with plasma concentrations of CBZ (r2 = 0.44, p = 0.002) and CBZE (r2 = 0.35, p = 0.006). Our results have provided the first chemical evidence for microsomal production of [O]CBZ species that are able to escape the microsomal domain to react covalently with soluble proteins. This study has also demonstrated the presence of circulating [O]CBZ-modified HSA in patients without hypersensitivity reactions who were receiving standard CBZ therapy. The implications of those circulating adducts for susceptibility to CBZ hypersensitivity merit further immunological investigation in hypersensitive patients.


Assuntos
Carbamazepina/sangue , Compostos de Epóxi/sangue , Glutationa S-Transferase pi/sangue , Albumina Sérica/análise , Carbamazepina/química , Carbamazepina/metabolismo , Compostos de Epóxi/metabolismo , Glutationa S-Transferase pi/metabolismo , Humanos , Espectrometria de Massas , Estrutura Molecular , Albumina Sérica/metabolismo
8.
Hepatology ; 64(6): 1934-1950, 2016 12.
Artigo em Inglês | MEDLINE | ID: mdl-27631819

RESUMO

The interplay between host antiviral immunity and immunopathology during hepatitis E virus (HEV) infection determines important clinical outcomes. We characterized the specificity, functionality, and durability of host T-cell responses against the full-length HEV virus and assessed a novel "Quantiferon" assay for the rapid diagnosis of HEV infection. Eighty-nine volunteers were recruited from Oxford, Truro (UK), and Toulouse (France), including 44 immune-competent patients with acute HEV infection, 18 HEV-exposed immunosuppressed organ-transplant recipients (8 with chronic HEV), and 27 healthy volunteers. A genotype 3a peptide library (616 overlapping peptides spanning open reading frames [ORFs] 1-3) was used in interferon-gamma (IFN-γ) T-cell ELISpot assays. CD4+ /CD8+ T-cell subsets and polyfunctionality were defined using ICCS and SPICE analysis. Quantification of IFN-γ used whole-blood stimulation with recombinant HEV-capsid protein in the QuantiFERON kit. HEV-specific T-cell responses were detected in 41/44 immune-competent HEV exposed volunteers (median magnitude: 397 spot-forming units/106 peripheral blood mononuclear cells), most frequently targeting ORF2. High-magnitude, polyfunctional CD4 and CD8+ T cells were detected during acute disease and maintained to 12 years, but these declined over time, with CD8+ responses becoming more monofunctional. Low-level responses were detectable in immunosuppressed patients. Twenty-three novel HEV CD4+ and CD8+ T-cell targets were mapped predominantly to conserved genomic regions. QuantiFERON testing demonstrated an inverse correlation between IFN-γ production and the time from clinical presentation, providing 100% specificity, and 71% sensitivity (area under the receiver operator characteristic curve of 0.86) for HEV exposure at 0.3 IU/mL. CONCLUSION: Robust HEV-specific T-cell responses generated during acute disease predominantly target ORF2, but decline in magnitude and polyfunctionality over time. Defining HEV T-cell targets will be important for the investigation of HEV-associated autoimmune disease. (Hepatology 2016;64:1934-1950).


Assuntos
Vírus da Hepatite E/imunologia , Hepatite E/imunologia , Hepatite E/virologia , Especificidade do Receptor de Antígeno de Linfócitos T , Linfócitos T/imunologia , Linfócitos T/virologia , Adulto , Idoso , Idoso de 80 Anos ou mais , Feminino , Hepatite E/sangue , Hepatite E/diagnóstico , Humanos , Interferon gama/sangue , Masculino , Pessoa de Meia-Idade , Fatores de Tempo , Adulto Jovem
9.
Lancet Gastroenterol Hepatol ; 1(3): 217-225, 2016 11.
Artigo em Inglês | MEDLINE | ID: mdl-28404094

RESUMO

BACKGROUND: Early, accurate prediction of survival is central to management of patients with paracetamol-induced acute liver failure to identify those needing emergency liver transplantation. Current prognostic tools are confounded by recent improvements in outcome independent of emergency liver transplantation, and constrained by static binary outcome prediction. We aimed to develop a simple prognostic tool to reflect current outcomes and generate a dynamic updated estimation of risk of death. METHODS: Patients with paracetamol-induced acute liver failure managed at intensive care units in the UK (London, Birmingham, and Edinburgh) and Denmark (Copenhagen) were studied. We developed prognostic models, excluding patients who underwent transplantation, using Cox proportional hazards in a derivation dataset, and tested in initial and recent external validation datasets. Mortality was estimated in patients who had emergency liver transplantation. Model discrimination was assessed using area under receiver operating characteristic curve (AUROC) and calibration by root mean square error (RMSE). Admission (day 1) variables of age, Glasgow coma scale, arterial pH and lactate, creatinine, international normalised ratio (INR), and cardiovascular failure were used to derive an initial predictive model, with a second (day 2) model including additional changes in INR and lactate. FINDINGS: We developed and validated new high-performance statistical models to support decision making in patients with paracetamol-induced acute liver failure. Applied to the derivation dataset (n=350), the AUROC for 30-day survival was 0·92 (95% CI 0·88-0·96) using the day 1 model and 0·93 (0·88-0·97) using the day 2 model. In the initial validation dataset (n=150), the AUROC for 30-day survival was 0·89 (0·84-0·95) using the day 1 model and 0·90 (0·85-0·95) using the day 2 model. Assessment of calibration using RMSE in prediction of 30-day survival gave values of 0·1642 for the day 1 model and 0·0626 for the day 2 model. In the external validation dataset (n=412), the AUROC for 30-day survival was 0·91 (0·87-0·94) using the day 1 model and 0·91 (0·88-0·95) using the day 2 model, and assessment of calibration using RMSE gave values of 0·079 for the day 1 model and 0·107 for the day 2 model. Applied to patients who underwent emergency liver transplantation (n=116), median predicted 30-day survival was 51% (95% CI 33-85). INTERPRETATION: The models developed here show very good discrimination and calibration, confirmed in independent datasets, and suggest that many patients undergoing transplantation based on existing criteria might have survived with medical management alone. The role and indications for emergency liver transplantation in paracetamol-induced acute liver failure require re-evaluation. FUNDING: Foundation for Liver Research.


Assuntos
Acetaminofen/efeitos adversos , Analgésicos não Narcóticos/efeitos adversos , Doença Hepática Induzida por Substâncias e Drogas/diagnóstico , Tomada de Decisão Clínica/métodos , Técnicas de Apoio para a Decisão , Transplante de Fígado/mortalidade , Modelos Estatísticos , Adulto , Área Sob a Curva , Doença Hepática Induzida por Substâncias e Drogas/etiologia , Doença Hepática Induzida por Substâncias e Drogas/mortalidade , Doença Hepática Induzida por Substâncias e Drogas/cirurgia , Estudos de Coortes , Emergências , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Prognóstico , Modelos de Riscos Proporcionais , Curva ROC
10.
Chem Res Toxicol ; 28(1): 51-8, 2015 Jan 20.
Artigo em Inglês | MEDLINE | ID: mdl-25489718

RESUMO

Isoniazid (INH), a widely used antituberculosis drug, has been associated with serious drug-induced liver injury (DILI). INH-modified proteins have been proposed to play important roles in INH DILI; however, it remains to be determined whether INH or reactive metabolites bind irreversibly to proteins. In this study, mass spectrometry was used to define protein modifications by INH in vitro and in patients taking INH therapy. When INH was incubated with N-acetyl lysine (NAL), the same isonicotinic-NAL (IN-NAL) adducts were detected irrespective of the presence or absence of any oxidative enzymes, indicating auto-oxidation may have been involved. In addition, we found that INH could also bind to human serum albumin (HSA) via an auto-oxidation pathway, forming isonicotinic amide adducts with lysine residues in HSA. Similar adducts were detected in plasma samples isolated from patients taking INH therapy. Our results show that INH forms protein adducts in the absence of metabolism.


Assuntos
Isoniazida/metabolismo , Lisina/metabolismo , Albumina Sérica/metabolismo , Adulto , Idoso de 80 Anos ou mais , Feminino , Humanos , Isoniazida/efeitos adversos , Isoniazida/sangue , Masculino , Microssomos Hepáticos/metabolismo , Pessoa de Meia-Idade , NADP/metabolismo , Oxirredução , Ligação Proteica
11.
Chem Res Toxicol ; 28(1): 144-54, 2015 Jan 20.
Artigo em Inglês | MEDLINE | ID: mdl-25531135

RESUMO

Drug hypersensitivity remains a major concern, as it causes high morbidity and mortality. Understanding the mechanistic basis of drug hypersensitivity is complicated by the multiple risk factors implicated. This study utilized sulfamethoxazole (SMX) as a model drug to (1) relate SMX metabolism in antigen presenting cells (APCs) to the activation of T-cells and (2) characterize covalent adducts of SMX and myeloperoxidase, which might represent antigenic determinants for T-cells. The SMX metabolite nitroso-SMX (SMX-NO) was found to bind irreversibly to APCs. Time- and concentration-dependent drug-protein adducts were also detected when APCs were cultured with SMX. Metabolic activation of SMX was significantly reduced by the oxygenase/peroxidase inhibitor methimazole. Similarly, SMX-NO-specific T-cells were activated by APCs pulsed with SMX, and the response was inhibited by pretreatment with methimazole or glutaraldehyde, which blocks antigen processing. Western blotting, real-time polymerase chain reaction (RT-PCR), and mass spectrometry analyses suggested the presence of low concentrations of myeloperoxidase in APCs. RT-PCR revealed mRNA expression for flavin-containing monooxygenases (FMO1-5), thyroid peroxidase, and lactoperoxidase, but the corresponding proteins were not detected. Mass spectrometric characterization of SMX-NO-modified myeloperoxidase revealed the formation of N-hydroxysulfinamide adducts on Cys309 and Cys398. These data show that SMX's metabolism in APCs generates antigenic determinants for T-cells. Peptides derived from SMX-NO-modified myeloperoxidase may represent one form of functional antigen.


Assuntos
Células Apresentadoras de Antígenos/metabolismo , Peroxidases/metabolismo , Sulfametoxazol/metabolismo , Linfócitos B/metabolismo , Células HL-60 , Humanos , Oxigenases/metabolismo , Ligação Proteica , Linfócitos T/metabolismo
12.
J Pharmacol Exp Ther ; 350(2): 387-402, 2014 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-24902585

RESUMO

Covalent protein modifications by electrophilic acyl glucuronide (AG) metabolites are hypothetical causes of hypersensitivity reactions associated with certain carboxylate drugs. The complex rearrangements and reactivities of drug AG have been defined in great detail, and protein adducts of carboxylate drugs, such as diclofenac, have been found in liver and plasma of experimental animals and humans. However, in the absence of definitive molecular characterization, and specifically, identification of signature glycation conjugates retaining the glucuronyl and carboxyl residues, it cannot be assumed any of these adducts is derived uniquely or even fractionally from AG metabolites. We have therefore undertaken targeted mass spectrometric analyses of human serum albumin (HSA) isolated from diclofenac patients to characterize drug-: derived structures and, thereby, for the first time, have deconstructed conclusively the pathways of adduct formation from a drug AG and its isomeric rearrangement products in vivo. These analyses were informed by a thorough understanding of the reactions of HSA with diclofenac AG in vitro. HSA from six patients without drug-: related hypersensitivities had either a single drug-: derived adduct or one of five combinations of 2-8 adducts from among seven diclofenac N-acylations and three AG glycations on seven of the protein's 59 lysines. Only acylations were found in every patient. We present evidence that HSA modifications by diclofenac in vivo are complicated and variable, that at least a fraction of these modifications are derived from the drug's AG metabolite, and that albumin adduction is not inevitably a causation of hypersensitivity to carboxylate drugs or a coincidental association.


Assuntos
Anti-Inflamatórios não Esteroides/metabolismo , Diclofenaco/metabolismo , Glucuronídeos/metabolismo , Espectrometria de Massas/métodos , Albumina Sérica/metabolismo , Adulto , Idoso , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Ligação Proteica
13.
Bioanalysis ; 6(7): 919-33, 2014 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-24806902

RESUMO

BACKGROUND: Mevalonic acid (MVA), as a product of 3-hydroxy-3-methylglutaryl coenzyme A reductase, represents a potential multipurpose biomarker in health and disease. A translational urinary MVA quantification method was developed, validated and used to demonstrate the diurnal variation of urinary MVA excretion in rats and healthy children. METHODS: Urinary MVA was converted to mevalonolactone at pH 2, extracted with ethyl acetate and quantified by reversed-phase liquid chromatography-tandem mass spectrometry. RESULTS: The assay had a dynamic range of 0.0156-10 µg/ml with precision <15% CV, accuracy 85-115% and was transferred between laboratories. Urinary MVA excretion in rats and healthy children displayed a diurnal variation consistent with the known diurnal variation of hepatic 3-hydroxy-3-methylglutaryl coenzyme A reductase activity. CONCLUSION: Urinary MVA can be quantified accurately over a wide dynamic range by a validated translational and transferable method with biomarker capability.


Assuntos
Biomarcadores/urina , Cromatografia de Fase Reversa/métodos , Hidroximetilglutaril-CoA Redutases/metabolismo , Ácido Mevalônico/urina , Espectrometria de Massas em Tandem/métodos , Calibragem , Humanos
14.
Chem Res Toxicol ; 27(4): 524-35, 2014 Apr 21.
Artigo em Inglês | MEDLINE | ID: mdl-24571427

RESUMO

Abacavir (ABC), a nucleoside-analogue reverse transcriptase inhibitor, is associated with severe hypersensitivity reactions that are thought to involve the activation of CD8+ T cells in a HLA-B*57:01-restricted manner. Recent studies have claimed that noncovalent interactions of ABC with HLA-B*57:01 are responsible for the immunological reactions associated with ABC. However, the formation of hemoglobin-ABC aldehyde (ABCA) adducts in patients exposed to ABC suggests that protein conjugation might represent a pathway for antigen formation. To further characterize protein conjugation reactions, we used mass spectrometric methods to define ABCA modifications in patients receiving ABC therapy. ABCA formed a novel intramolecular cross-linking adduct on human serum albumin (HSA) in patients and in vitro via Michael addition, followed by nucleophilic adduction of the aldehyde with a neighboring protein nucleophile. Adducts were detected on Lys159, Lys190, His146, and Cys34 residues in the subdomain IB of HSA. Only a cysteine adduct and a putative cross-linking adduct were detected on glutathione S-transferase Pi (GSTP). These findings reveal that ABC forms novel types of antigens in all patients taking the drug. It is therefore vital that the immunological consequences of such pathways of haptenation are explored in the in vitro models that have been used by various groups to define new mechanisms of drug hypersensitivity exemplified by ABC.


Assuntos
Proteínas Sanguíneas/metabolismo , Didesoxinucleosídeos/metabolismo , Infecções por HIV/tratamento farmacológico , Inibidores da Transcriptase Reversa/metabolismo , Sequência de Aminoácidos , Proteínas Sanguíneas/química , Didesoxinucleosídeos/uso terapêutico , Infecções por HIV/sangue , Humanos , Dados de Sequência Molecular , Inibidores da Transcriptase Reversa/uso terapêutico , Espectrometria de Massas em Tandem
15.
Org Biomol Chem ; 11(48): 8426-34, 2013 Dec 28.
Artigo em Inglês | MEDLINE | ID: mdl-24193242

RESUMO

The dibenz[b,f]azepine heterocyclic system and related molecules with a single 10,11-bond are important templates for well-prescribed drug molecules, notably carbamazepine (anticonvulsant), clomipramine and imipramine (antidepressants). We synthesised a range of halogenated carbamazepine analogues, in connection with metabolic and immunological studies, as probes for structure-metabolism and hypersensitive effects and have published on their metabolic behaviour. While a number of synthetic routes to such analogues are possible, we naturally sought short and efficient methods for our target compounds. In the following report we present an effective two-step synthesis of a range of dibenz[b,f]azepines from appropriate indoles via N-arylation, then acid-catalysed rearrangement, with a critical analysis of other approaches. We showed earlier that this route was effective for fluoro analogues and here present a broader review of its scope. The 5-(carboxamido) side chain of carbamazepine may be added in various ways, affording overall a convenient access to drug molecules.


Assuntos
Dibenzazepinas/síntese química , Indóis/química , Anticonvulsivantes/síntese química , Anticonvulsivantes/química , Carbamazepina/análogos & derivados , Carbamazepina/síntese química , Catálise , Dibenzazepinas/química , Halogenação
16.
Chem Res Toxicol ; 26(7): 1064-72, 2013 Jul 15.
Artigo em Inglês | MEDLINE | ID: mdl-23697900

RESUMO

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 in vitro 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 in vivo.


Assuntos
Células Apresentadoras de Antígenos/citologia , Células Apresentadoras de Antígenos/metabolismo , Didesoxinucleosídeos/metabolismo , Biotransformação , Linhagem Celular Transformada , Citosol/metabolismo , Didesoxinucleosídeos/química , Humanos , Cinética , Fígado/citologia , Espectrometria de Massas , Conformação Molecular , Oxirredução , Frações Subcelulares/química , Frações Subcelulares/metabolismo , Fatores de Tempo
18.
J Med Chem ; 55(22): 9773-84, 2012 Nov 26.
Artigo em Inglês | MEDLINE | ID: mdl-23088585

RESUMO

The anticonvulsant carbamazepine 1 is associated with adverse drug reactions (ADRs), including hepatotoxicity; oxidative metabolism of 1 has been implicated in the pathogenesis of the ADRs. We report the synthesis and evaluation of 2-monohalo and 2,8-dihalo analogues of 1 that were intended to minimize reactive metabolite formation via arene oxidation and 10,11-epoxidation. Halo analogues were obtained either by rearrangement of halogenated N-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 1. In human liver microsomes, difluoro analogue 5b underwent 10,11-epoxidation but gave no arene oxidation.


Assuntos
Anticonvulsivantes/farmacologia , Carbamazepina/análogos & derivados , Halogênios/química , Hepatócitos/efeitos dos fármacos , Microssomos Hepáticos/efeitos dos fármacos , Animais , Anticonvulsivantes/síntese química , Carbamazepina/farmacologia , Células Cultivadas , Cromatografia Líquida de Alta Pressão , Cromatografia Líquida , Glutationa/metabolismo , Hepatócitos/metabolismo , Humanos , Masculino , Microssomos Hepáticos/metabolismo , Estrutura Molecular , Oxirredução , Ratos , Ratos Wistar , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz , Relação Estrutura-Atividade
19.
J Med Chem ; 54(19): 6443-55, 2011 Oct 13.
Artigo em Inglês | MEDLINE | ID: mdl-21888440

RESUMO

Dispiro-1,2,4,5-tetraoxanes and 1,2,4-trioxolanes represent attractive classes of synthetic antimalarial peroxides due to their structural simplicity, good stability, and impressive antimalarial activity. We investigated the reactivity of a series of potent amide functionalized tetraoxanes with Fe(II)gluconate, FeSO(4), FeSO(4)/TEMPO, FeSO(4)/phosphatidylcholine, and heme to gain knowledge of their potential mechanism of bioactivation and to compare the results with the corresponding 1,2,4-trioxolanes. Spin-trapping experiments demonstrate that Fe(II)-mediated peroxide activation of tetraoxanes produces primary and secondary C-radical intermediates. Reaction of tetraoxanes and trioxolanes with phosphatidylcholine, a predominant unsaturated lipid present in the parasite digestive vacuole membrane, under Fenton reaction conditions showed that both endoperoxides share a common reactivity in terms of phospholipid oxidation that differs with that of artemisinin. Significantly, when tetraoxanes undergo bioactivation in the presence of heme, only the secondary C-centered radical is observed, which smoothly produces regioisomeric drug derived-heme adducts. The ability of these tetraoxanes to alkylate the porphyrin ring was also confirmed with Fe(II)TPP and Mn(II)TPP, and docking studies were performed to rationalize the regioselectivity observed in the alkylation process. The efficient process of heme alkylation and extensive lipid peroxidation observed here may play a role in the mechanism of action of these two important classes of synthetic endoperoxide antimalarial.


Assuntos
Antimaláricos/síntese química , Compostos Ferrosos/química , Heme/química , Peróxidos/síntese química , Fosfatidilcolinas/química , Compostos de Espiro/síntese química , Alquilação , Antimaláricos/química , Antimaláricos/farmacologia , Modelos Moleculares , Testes de Sensibilidade Parasitária , Peróxidos/química , Peróxidos/farmacologia , Plasmodium berghei/efeitos dos fármacos , Plasmodium falciparum/efeitos dos fármacos , Compostos de Espiro/química , Compostos de Espiro/farmacologia , Estereoisomerismo , Relação Estrutura-Atividade , Tetraoxanos/síntese química , Tetraoxanos/química , Tetraoxanos/farmacologia
20.
Org Lett ; 13(20): 5592-5, 2011 Oct 21.
Artigo em Inglês | MEDLINE | ID: mdl-21939196

RESUMO

Efficient procedures for the synthesis of benzo-fluorinated dibenz[b,f]azepines (iminostilbenes) from fluorinated isatins or indoles using a number of ring-expansion reactions are described. A range of mono- and difluorinated analogues is accessible, and the syntheses can deliver gram quantities of the final products, which are precursors of fluoro analogues of the important anticonvulsant carbamazepine.


Assuntos
Acridinas/química , Azepinas/síntese química , Hidrocarbonetos Fluorados/química , Hidrocarbonetos Fluorados/síntese química , Indóis/química , Isatina/análogos & derivados , Isatina/química , Anticonvulsivantes/síntese química , Anticonvulsivantes/química , Azepinas/química , Carbamazepina/análogos & derivados , Carbamazepina/síntese química , Carbamazepina/química , Catálise , Estrutura Molecular
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