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

Abstract

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.

Identification of an antigenic determinant of clavulanic acid responsible for IgE‐mediated reactions

“…To gain insight into the nature of the relevant determinants formed, we investigated the protein conjugation process using HSA as a model. We observed that covalent binding of CLV to HSA takes place in vitro and that this modification is concentration‐dependent, consistent with recent studies . The identification of the sites modified by CLV in HSA was carried out only for the conjugate formed at the lowest CLV concentration that allowed detection of protein modification, although still higher than that of the drug in vivo under therapeutic conditions .…”

“…Compared to penicillins, for which several antigenic determinants have been characterized, CLV reactivity is very complex and degradation pathways after its conjugation to proteins may lead to multiple possible determinants . We have hypothesized the formation of two main antigenic determinants (AD‐I and AD‐II) in conditions similar to those occurring in the clinical practice, based on preliminary reported data and the chemical reactivity of CLV , …”

“…As a consequence of increasing allergic reactions to CLV, there is much interest in elucidating the underlying mechanisms and structures involved in its immunological recognition . As other β‐lactams, CLV must bind covalently to carrier proteins to form conjugates with sufficient size to be recognized by the immune system and induce a response .…”

“…However, due to its high instability, the resulting structure rapidly degrades, leading to the formation of multiple fragments (Figure A): Acylprotein [1] undergoes the subsequent opening of the five‐member oxazolidine ring, forming the linear imine [2]. This intermediate [2] may follow different reactivity pathways: (a) Decarboxylation giving rise to derivative [3], which like molecule [2], can break down via the imine bond to yield the protein conjugated to the derived aldehyde, which we hypothesize as a possible antigenic determinant (hereinafter called AD‐I); (b) isomerization of both intermediates [2] and [3] to the imine [5], which would be hydrolyzed under biological conditions leading to the formation of the protein conjugated to the 3‐aminopropanamide, forming a second possible determinant that we have named AD‐II; and (c) isomerization to the corresponding enamine [4], which apart from decarboxylation, in principle, should not experience further fragmentation.…”

Amoxicillin inactivation by thiol-catalyzed cyclization reduces protein haptenation and antibacterial potency

“…In vitro, incubation of human serum albumin (HSA) with AX, results in the modification of several lysine residues of which, Lys190 and Lys195 appear to be the most reactive [8]. Interestingly, after oral administration of AX, only modified Lys190 has been found [11,12]. Apart from the chemical reactivity of the antibiotic and the protein residues, little is known about other factors influencing protein haptenation by AX in biological settings.Previous studies with the antibiotic sulfamethoxazole have shown that oxidative stress can potentiate the formation of protein adducts in cells [13].…”