4 research outputs found
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
Abacavir Forms Novel Cross-Linking Abacavir Protein Adducts in Patients
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 <i>in vitro</i> 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 <i>S</i>-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 <i>in vitro</i> models that have been used by various groups to define new mechanisms
of drug hypersensitivity exemplified by ABC
β‑Lactam Antibiotics Form Distinct Haptenic Structures on Albumin and Activate Drug-Specific T‑Lymphocyte Responses in Multiallergic Patients with Cystic Fibrosis
β-Lactam
antibiotics provide the cornerstone of treatment
for respiratory exacerbations in patients with cystic fibrosis. Unfortunately,
approximately 20% of patients develop multiple nonimmediate allergic
reactions that restrict therapeutic options. The purpose of this study
was to explore the chemical and immunological basis of multiple β-lactam
allergy through the analysis of human serum albumin (HSA) covalent
binding profiles and T-cell responses against 3 commonly prescribed
drugs; piperacillin, meropenem, and aztreonam. The chemical structures
of the drug haptens were defined by mass spectrometry. Peripheral
blood mononuclear cells (PBMC) were isolated from 4 patients with
multiple allergic reactions and cultured with piperacillin, meropenem,
and aztreonam. PBMC responses were characterized using the lymphocyte
transformation test and IFN-γ /IL-13 ELIspot. T-cell clones
were generated from drug-stimulated T-cell lines and characterized
in terms of phenotype, function, and cross-reactivity. Piperacillin,
meropenem, and aztreonam formed complex and structurally distinct
haptenic structures with lysine residues on HSA. Each drug modified
Lys190 and at least 6 additional lysine residues in a time- and concentration-dependent
manner. PBMC proliferative responses and cytokine release were detected
with cells from the allergic patients, but not tolerant controls,
following exposure to the drugs. 122 CD4+, CD8+, or CD4+CD8+ T-cell
clones isolated from the allergic patients were found to proliferate
and release cytokines following stimulation with piperacillin, meropenem,
or aztreonam. Cross-reactivity with the different drugs was not observed.
In conclusion, our data show that piperacillin-, meropenem-, and aztreonam-specific
T-cell responses are readily detectable in allergic patients with
cystic fibrosis, which indicates that multiple β-lactam allergies
are instigated through priming of naïve T-cells against the
different drug antigens. Characterization of complex haptenic structures
on distinct HSA lysine residues provides a chemical basis for the
drug-specific T-cell response