Hepatic covalent adduct formation with zomepirac in the CD26-deficient mouse

BACKGROUND AND AIMS: Zomepirac (ZP), a non-steroidal anti-inflammatory drug (NSAID), has been reported to cause immune-mediated liver injury. In vivo, ZP is metabolized to a chemically reactive acyl glucuronide conjugate (ZAG) which can undergo covalent adduct formation with proteins. Such acyl glucuronide-derived drug-protein adducts may be important in the development of immune and toxic responses caused by NSAID. We have shown using immunoabsorptions that the 110 kDa CD26 (dipeptidyl peptidase IV) is one of the hepatic target proteins for covalent modification by ZAG. In the present study, a CD26-deficient mouse strain was used to examine protein targets for covalent modification by ZP/metabolites in the liver. METHODS AND RESULTS: The CD26-deficient phenotype was confirmed by immunohistochemistry, flow cytometry analysis, RT-PCR, enzyme assay and immunoblotting. Moreover, by using monoclonal antibody immunoblots, CD26 was not detected in the livers of ZP-treated CD26-deficient mice. Immunoblots using a polyclonal antiserum to ZP on liver from ZP-treated mice showed three major sizes of protein bands, in the 70, 110 and 140 kDa regions. Most, but not all, of the anti-ZP immunoreactivity in the 110 kDa region was absent from ZP-treated CD26-deficient mice. CONCLUSION: These data definitively showed that CD26 was a component of ZP-modified proteins in vivo. In addition, the data suggested that at least one other protein of approximately 110 kDa was modified by covalent adduct formation with ZAG.     

Anaphylactic reactions to zomepirac

Three cases of apparent anaphylactoid reactions to zomepirac sodium (Zomax) are reported. One patient initially appeared to have a dissecting abdominal aortic aneurysm with vascular collapse. The second patient experienced conjunctival pruritis after each of several doses of zomepirac before emergency department presentation with acute dyspnea and urticaria. The third patient had been admitted previously with a diagnosis of rule out myocardial infarction, which in retrospect was probably a zomepirac reaction. All three recovered uneventfully.     

Fast and efficient tritium labelling of the nonsteroidal anti‐inflammatory drugs naproxen,tolmetin, and zomepirac

Fast and efficient tritium labelling of the nonsteroidal anti‐inflammatory drugs naproxen, tolmetin and zomepirac is reported. Naproxen along with its (R)‐enantiomer were labelled by catalytic tritium–halogen exchange of the corresponding 5‐bromo derivatives providing [³H]naproxen with a specific activity of 25.4 Ci/mmol. Tolmetin and zomepirac were labelled by the hydrogen isotope exchange reaction using Crabtree's catalyst. This provided [³H]tolmetin and [³H]zomepirac with specific activities of 80.8 and 64.3 Ci/mmol, respectively. All compounds were obtained in high radiochemical purity (>98%). Copyright © 2005 John Wiley & Sons, Ltd.     

Reversible binding of tolmetin,zomepirac, and their glucuronide conjugates to human serum albumin and plasma

Acyl glucuronides of drugs and bilirubin have been shown in the past decade to be reactive metabolites undergoing acyl migration and irreversible binding. The latter reaction has been hypothesized to be facilitated by or to proceed through the formation of a reversible complex. Furthermore, it has been suggested that the decreased binding seen in patients with compromised excretory function may be due to competition by elevated plasma concentrations of the glucuronides. In these reversible binding studies, we characterized the extent and the “site” of binding of tolmetin, zomepirac, their glucuronides and isomeric conjugates. We also examined the displacement between the parent drugs and their glucuronide conjugates using a rapid ultrafiltration method. Tolmetin exhibited three classes of binding sites with a primary association constant of 1.7×10⁶ M⁻¹ (K_dl=0.60 μM). The primary association constant of zomepirac (1.16×10⁶ M⁻¹, K_dl=0.86 μM) is similar to that of tolmetin. The β 1 and α/β3 glucuronides of both compounds bind to a lesser extent than their parent aglycones. The isomeric glucuronide conjugates of both compounds showed much stronger binding than the β/1 conjugates. Of the four glucuronides investigated, tolmetin glucuronide-α/β3 isomer was bound by fatty acid free human serum albumin with the highest affinity (4.6×10⁵ M⁻¹, K_d=2.22 μM). Protein binding of the parent drugs and conjugates were decreased significantly at pH 5.0. In displacement studies, except for salicylate and acetylsalicylate, drugs known to bind to Sites I and II as well as the digitoxin and tamoxifen binding sites had little inhibitory effect on the binding of tolmetin, zomepirac, and their glucuronide conjugates. Supported in part by Grant GM 36633 from the National Institute of General Medical Sciences.     

Anaphylactoid reaction to zomepirac

Circulatory collapse and respiratory arrest occurred in a young woman shortly after taking 200 mg of zomepirac for relief of headache. She had no history of drug hypersensitivity and had tolerated zomepirac without adverse effects 18 months prior to this reaction. The patient was successfully treated and suffered no sequelae.     

Reaction of Acyl Glucuronides with Insulin In Vitro: Identification of an Imine Mechanism by Electrospray lonization Mass Spectrometry

Pharmaceutical Research -     

Investigating the TNP-OVA and direct popliteal lymph node assays for the detection of immunostimulation by drugs associated with anaphylaxis in humans

Using current animal models, it is not possible to identify low-molecular-weight compounds (LMWCs) that are likely to be associated with anaphylaxis. It is generally accepted that the ultimate effector mechanism involves drug-induced IgE antibody. The objective of the present study was to determine if diclofenac, zomepirac and glafenine, which are associated with anaphylaxis in humans, have immunostimulating potential in the murine TNP-OVA (trinitrophenyl-ovalbumin) popliteal lymph node assay (PLNA), and more specifically to determine if the immunostimulation caused by these LMWCs results in IgE antibody production. These LMWCs were chosen because both zomepirac and glafenine were removed from the market due to high association with anaphylaxis, and diclofenac, which remains on the market, is frequently associated with anaphylaxis. In addition to conducting a TNP-OVA PLNA, the immunostimulating potential of these compounds was examined in the direct PLNA. When co-administered with TNP-OVA, all three LMWCs caused dose-dependent (0.25, 0.50, 1.00 and 1.25 mg) increases in popliteal lymph node (PLN) weight and cellularity that were observed beginning with the 0.25-mg dose. In addition, beginning with the 0.25-mg dose, all three compounds caused dose-dependent increases in TNP-OVA specific IgM and IgG(1) antibody-forming cells (AFCs). Diclofenac induced an isotype switch and caused a dose-dependent increase in the number of IgE AFCs with no detectable IgG(2a) AFCs and minimal high-dose-only IgG(2b) AFCs. Zomepirac induced IgE, IgG(2a) and IgG(2b) AFCs following the injection of 0.50 mg only, and glafenine induced IgE, IgG(2a) and IgG(2b) AFCs following the injection of 0.50-1.00 mg. In the direct PLNA, diclofenac caused dose-dependent increases in PLN weight and cellularity that were observed beginning with dose of 0.50 mg, whereas zomepirac failed to increase any PLN parameter and glafenine only increased the PLN weight. These results suggest that diclofenac, zomepirac and glafenine are immunostimulating LMWCs in the TNP-OVA PLNA with the potential to induce IgE antibody against a co-administered hapten-conjugate. Furthermore, these results suggest that the TNP-OVA PLNA offered significant advantages over the direct PLNA. Although it is not realistic to suggest that a single assay, based on a low number of test compounds, can identify all LMWCs with the potential to cause anaphylaxis in humans, these observations do demonstrate the potential utility of the PLNAs in examining LMWC-induced immunomodulation and support further development and investigation of the assays.