Caroline Earnshaw

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

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 β‐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. (HEPATOLOGY 2013;)

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 Lys541 observed at low concentrations, whereas at higher concentrations, up to 13 out of 59 lysine residues were modified, four of which (Lys190, Lys195, Lys432, and Lys541) 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 Lys190, Lys432, and Lys541 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.

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 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.

Observational infant exploratory [14C]‐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.

T Cell Responses to Drugs and Drug Metabolites

Understanding the chemical mechanisms by which drugs and drug metabolites interact with cells of the immune system is pivotal to our knowledge of drug hypersensitivity as a whole.In this chapter, we will discuss the currently accepted mechanisms where there is scientific and clinical evidence to support the ways in which drugs and their metabolites interact with T cells. We will also discuss bioanalytical platforms, such as mass spectrometry, and in vitro test assays such as the lymphocyte transformation test that can be used to study drug hypersensitivity; the combination of such techniques can be used to relate the chemistry of drug antigen formation to immune function. Ab initio T cell priming assays are also discussed with respect to predicting the potential of a drug to cause hypersensitivity reactions in humans in relation to the chemistry of the drug and its ability to form haptens, antigens and immunogens in patients.

Antigen exposure required for T cell activation

Background A high frequency of hypersensitivity reactions to -lactam antibiotics are observed in patients with cystic fibrosis. -lactam antibiotics form protein conjugates in vitro and in vivo, and a core group of lysine residues of human serum albumin have been shown to be penicilloylated by mass spectrometric methods. Furthermore, protein/ peptide conjugates have been shown to stimulate T cells isolated from patients with -lactam hypersensitivity. However, the threshold level of protein conjugation required to trigger immune responses has not been studied. Method Thus, we focused on piperacillin, a commonly used drug in patients with cystic fibrosis, to (a) quantify the piperacillin antigens formed in patients using mass spectrometry and a synthetic piperacillin-modified albumin peptide (539ATK(Pip)EQLK545) as a standard and (b) determine the quantity of the piperacillin protein adducts formed at the time of T cell activation. Plasma was collected prior to commencing treatment and after a 14 day treatment course from 10 patients and the level of piperacillin-modified albumin in patient plasma was measured. Piperacillin-specific CD4+ T-cell clones were generated from hypersensitive patients by serial dilution and cultured with soluble drug or antigen presenting cells pulsed with piperacillin for 1-48h for the analysis of drug-specific proliferative responses. At each time-point piperacillin albumin binding was quantified. Results Piperacillin-modified lysine was detected in human serum albumin in all 10 patients; the level of piperacillinmodified lysine 541 was found to range from 2.6 to 6.5%. Antigen presenting cells pulsed with piperacillin for 1 and 4h did not stimulate a strong T-cell proliferative response and this coincided with low levels of albumin modification. In contrast, antigen presenting cells pulsed with piperacillin for 24 or 48h activated all of the clones. Quantitative analysis of incubation medium revealed that approximately 3% of lysine 541 was modified after 24h. Conclusion In conclusion, these data quantify for the first time, the level of piperacillin albumin binding in drug exposed patients and in vitro at drug antigen concentrations that activate piperacillin-specific T-cells.