title: Investigating the TNP-OVA and direct popliteal lymph node assays for the detection of immunostimulation by drugs associated with anaphylaxis in humans.
abstract: 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.
authors.affiliation: Drug Safety Evaluation, Pfizer Global Research and Development, Groton Laboratories, Groton, CT 06340, USA.
authors.forename: Bradford W
authors.initials: BW
authors.lastname: Gutting
authors.forename: Lawrence W
authors.initials: LW
authors.lastname: Updyke
authors.forename: David E
authors.initials: DE
authors.lastname: Amacher
mesh_terms: D000276:Adjuvants, Immunologic / Q000633:toxicity*; D000707:Anaphylaxis / Q000139:chemically induced*; D000818:Animals; D000894:Anti-Inflammatory Agents, Non-Steroidal / Q000276:immunology / Q000633:toxicity*; D004008:Diclofenac / Q000276:immunology / Q000633:toxicity; D004306:Dose-Response Relationship, Immunologic; D005260:Female; D005897:Glafenine / Q000276:immunology / Q000633:toxicity; D006241:Haptens; D006801:Humans; D007073:Immunoglobulin E / Q000096:biosynthesis; D023441:Local Lymph Node Assay; D008198:Lymph Nodes / Q000187:drug effects* / Q000276:immunology / Q000473:pathology; D051379:Mice; D008807:Mice, Inbred BALB C; D009929:Organ Size / Q000187:drug effects; D010047:Ovalbumin / Q000276:immunology / Q000633:toxicity*; D013047:Specific Pathogen-Free Organisms; D014046:Tolmetin / Q000031:analogs & derivatives* / Q000276:immunology / Q000633:toxicity; D018675:Toxicity Tests
chemical_list: D000276:Adjuvants, Immunologic; D000894:Anti-Inflammatory Agents, Non-Steroidal; D006241:Haptens; C038600:trinitrophenyl-ovalbumin; D004008:Diclofenac; D007073:Immunoglobulin E; D005897:Glafenine; C020549:zomepirac; D010047:Ovalbumin; D014046:Tolmetin