Definition of the Nature and Hapten Threshold of the ß-Lactam Antigen Required for T Cell Activation In Vitro and in Patients.

Covalent modification of protein by drugs may disrupt self-tolerance, leading to lymphocyte activation. Until now, determination of the threshold required for this process has not been possible. Therefore, we performed quantitative mass spectrometric analyses to define the epitopes formed in tolerant and hypersensitive patients taking the ß-lactam antibiotic piperacillin and the threshold required for T cell activation. A hydrolyzed piperacillin hapten was detected on four lysine residues of human serum albumin (HSA) isolated from tolerant patients. The level of modified Lys541 ranged from 2.6 to 4.8%. Analysis of plasma from hypersensitive patients revealed the same pattern and levels of modification 1-10 d after the commencement of therapy. Piperacillin-responsive skin-homing CD4+ clones expressing an array of Vß receptors were activated in a dose-, time-, and processing-dependent manner; analysis of incubation medium revealed that 2.6% of Lys541 in HSA was modified when T cells were activated. Piperacillin-HSA conjugates that had levels and epitopes identical to those detected in patients were shown to selectively stimulate additional CD4+ clones, which expressed a more restricted Vß repertoire. To conclude, the levels of piperacillin-HSA modification that activated T cells are equivalent to the ones formed in hypersensitive and tolerant patients, which indicates that threshold levels of drug Ag are formed in all patients. Thus, the propensity to develop hypersensitivity is dependent on other factors, such as the presence of T cells within an individual's repertoire that can be activated with the ß-lactam hapten and/or an imbalance in immune regulation.

Amoxicillin and Clavulanate Form Chemically and Immunologically Distinct Multiple Haptenic Structures in Patients.

Amoxicillin-clavulanate (AC) is one of the most common causes of drug induced liver injury (DILI). The association between AC-DILI and HLA alleles and the detection of drug-specific T cells in patients with AC-DILI indicate that the adaptive immune system is involved in the disease pathogenesis. In this study, mass spectrometric methods were employed to characterize the antigen formed by AC in exposed patients and the antigenic determinants that stimulate T cells. Amoxicillin formed penicilloyl adducts with lysine residues on human serum albumin (HSA) in vitro, with K190 and K199 being the most reactive sites. Amoxicillin-modified K190 and K199 have also been detected in all patients, and more extensive modification was observed in patients exposed to higher doses of amoxicillin. In contrast, the binding of clavulanic acid to HSA was more complicated. Multiple adducts were identified at high concentrations in vitro, including those formed by direct binding of clavulanic acid to lysine residues, novel pyrazine adducts derived from binding to the degradation products of clavulanic acid, and a cross-linking adduct. Stable adducts derived from formylacetic acid were detected in all patients exposed to the drug. Importantly, analysis of hapten-protein adducts formed in the cell culture medium revealed that the highly drug-specific T-cell responses were likely driven by the markedly different haptenic structures formed by these two drugs. In this study, the unique haptenic structures on albumin in patients formed by amoxicillin and clavulanic acid have been characterized and shown to function as chemically distinct antigens which can stimulate separate, specific T-cell clones.

Observational infant exploratory [(14)C]-paracetamol pharmacokinetic microdose/therapeutic dose study with accelerator mass spectrometry bioanalysis.

AIMS: The aims of the study were to compare [(14)C]-paracetamol ([(14)C]-PARA) paediatric pharmacokinetics (PK) after administration mixed in a therapeutic dose or an isolated microdose and to develop further and validate accelerator mass spectrometry (AMS) bioanalysis in the 0-2 year old age group. METHODS: [(14)C]-PARA concentrations in 10-15 µl plasma samples were measured after enteral or i.v. administration of a single [(14)C]-PARA microdose or mixed in with therapeutic dose in infants receiving PARA as part of their therapeutic regimen. RESULTS: Thirty-four infants were included in the PARA PK analysis for this study: oral microdose (n = 4), i.v. microdose (n = 6), oral therapeutic (n = 6) and i.v. therapeutic (n = 18). The respective mean clearance (CL) values (SDs in parentheses) for these dosed groups were 1.46 (1.00) l h(-1), 1.76 (1.07) l h(-1), 2.93 (2.08) l h(-1) and 2.72 (3.10) l h(-1), t(1/2) values 2.65 h, 2.55 h, 8.36 h and 7.16 h and dose normalized AUC(0-t) (mg l(-1) h) values were 0.90 (0.43), 0.84 (0.57), 0.7 (0.79) and 0.54 (0.26). CONCLUSIONS: All necessary ethical, scientific, clinical and regulatory procedures were put in place to conduct PK studies using enteral and systemic microdosing in two European centres. The pharmacokinetics of a therapeutic dose (mg kg(-1)) and a microdose (ng kg(-1)) in babies between 35 to 127 weeks post-menstrual age. [(14)C]-PARA pharmacokinetic parameters were within a two-fold range after a therapeutic dose or a microdose. Exploratory studies using doses significantly less than therapeutic doses may offer ethical and safety advantages with increased bionalytical sensitivity in selected exploratory paediatric pharmacokinetic studies.

Mass spectrometric characterization of circulating covalent protein adducts derived from a drug acyl glucuronide metabolite: multiple albumin adductions in diclofenac patients.

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.

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

UNLABELLED: 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 ß-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.

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 S-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.

Mass spectrometric characterization of circulating and functional antigens derived from piperacillin in patients with cystic fibrosis.

A mechanistic understanding of the relationship between the chemistry of drug Ag formation and immune function is lacking. Thus, mass spectrometric methods were employed to detect and fully characterize circulating Ags derived from piperacillin in patients undergoing therapy and the nature of the drug-derived epitopes on protein that can function as an Ag to stimulate T cells. Albumin modification with piperacillin in vitro resulted in the formation of two distinct haptens, one formed directly from piperacillin and a second in which the dioxopiperazine ring had undergone hydrolysis. Modification was time and concentration dependent, with selective modification of Lys(541) observed at low concentrations, whereas at higher concentrations, up to 13 out of 59 lysine residues were modified, four of which (Lys(190), Lys(195), Lys(432), and Lys(541)) were detected in patients' plasma. Piperacillin-specific T lymphocyte responses (proliferation, cytokines, and granzyme B release) were detected ex vivo with cells from hypersensitive patients, and analysis of incubation medium showed that modification of the same lysine residues in albumin occurred in situ. The antigenicity of piperacillin-modified albumin was confirmed by stimulation of T cells with characterized synthetic conjugates. Analysis of minimally modified T cell-stimulatory albumin conjugates revealed peptide sequences incorporating Lys(190), Lys(432), and Lys(541) as principal functional epitopes for T cells. This study has characterized the multiple haptenic structures on albumin in patients and showed that they constitute functional antigenic determinants for T cells.

Direct analysis of pharmaceutical tablet formulations using Matrix-Assisted Laser Desorption/Ionisation Mass Spectrometry Imaging.

Matrix-Assisted Laser Desorption/Ionisation Mass Spectrometry Imaging (MALDI MSI) has been used to directly analyse a range of tablets in order to assess the homogeneity of the active drug compound throughout the excipients contained within the tablets studied. The information gained from the imaging experiments can be used to improve and gain a greater understanding of the manufacturing process; such knowledge will enable improvements in finished product quality to make safer and more efficacious tablet formulations. Commercially available and prescription tablet formulations have been analysed, including aspirin, paracetamol, sildenafil citrate (Viagra(R)) and a batch of tablets in development (tablet X: placebo; 1 mg; 3 mg and 6 mg). MALDI MSI provides semi-quantitative information that is related to ion abundance, therefore Principal Component Analysis (PCA), a multivariate analysis technique, has been used to differentiate between tablets containing different amounts of active drug ingredient. Aspects of sample preparation have also been investigated with regard to tablet shape and texture. The results obtained indicate that MALDI MSI can be used effectively to analyse the spatial distribution of the active pharmaceutical component (API) in pharmaceutical tablet formulations.