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

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