Evaluation of a Lymph Node Proliferation Assay for its Ability to Detect Pharmaceuticals with Potential to Cause Immune-Mediated Drug Reactions

Abstract

Hypersensitivity reactions to systemically administered drugs cannot be predicted using available preclinical models. This research is a collaborative project to evaluate the ability of the Lymph Node Proliferation Assay (LNPA) to predict systemic hypersensitivity caused by pharmaceuticals. The assay design is a modification of the Local Lymph Node Assay with the major modification being injection of the test substance subcutaneously to achieve a known systemic exposure to the drug. Fourteen compounds were evaluated in the LNPA. These were two clinically negative drugs (Metformin, phenobarbital), an assay positive control (streptozotocin), eight human hypersensitivity positive drugs (sulfamethoxazole, procainamide, clonidine, ofloxacin, nevirapine, abacavir, lamotrigine, zomepirac), and 3 investigational drugs (CM40874, CM40954 and CM40420), one of which caused hypersensitivity in primates. Hypersensitivity-positive drugs were classified as such based on at least two of three independent data sources: U.S. FDA postmarketing database, drug labeling information, and clinical trial data. All drugs were tested in multiple laboratories for a total of 2-12 evaluations per compound. The pure drug substance was used for testing if it could be obtained commercially, otherwise the marketed drug formulation was used. Neither of the negative control drugs showed a positive reaction in the test system. Four of the eight hypersensitivity positive drugs showed a mixed or positive reaction. Two of the three investigational compounds gave a positive response. A smaller number of LNPAs were run concurrently using footpad injection and evaluation of the popliteal lymph node and gave generally comparable results. Additional development may increase the reproducibility of the assay and facilitate detection of drugs that require metabolic activation to become allergenic, or drugs for which there is dose-limiting toxicity. The data suggest that this method might be useful as a first-line screen to identify candidate drugs that are more likely to cause a high prevalence of human drug hypersensitivity.

Development and partial validation of a mouse model for predicting drug hypersensitivity reactions

“…In 2005, Weaver et al (2005) published on the lymph node proliferation assay (LNPA), which is a combination of the PLNA and the local lymph node assay (LLNA). In this model, a compound is administered via subcutaneous injection in the nape of the neck (as opposed to the footpad), the draining lymph nodes are recovered and proliferation is measured using tritiated thymidine incorporation.…”

Route-dependent systemic and local immune effects following exposure to solutions prepared from titanium dioxide nanoparticles

“…The LNPA assay is a modification of the LLNA that is designed to test the ability of many substances, especially drugs, to induce immune-mediated systemic hypersensitivity (Weaver et al, 2005). In addition, the LNPA is recommended over the LLNA when evaluating the hypersensitivity potential of nanoparticles (Dobrovolskaia et al, 2009).…”

“…The assay was performed as described by Weaver et al (2005) with modification. Prior to exposure, mice were weighed, randomized, and tattooed for identification.…”

Characterization of the draining lymph node response in the mouse drug allergy model: A model for drug hypersensitivity reactions

“…A modification of the PLNA, i.e. the lymph node proliferation assay (LNPA), was tested by multiple labs to assess the potential immunogenic effects of systemically-administered drugs (Weaver et al, 2005). However, in that study, only a limited number of compounds were tested and the mechanisms of drug-induced immune responses were left unclear.…”

“…It has been shown that B-cell proliferation is responsible for the increased cellularity in the spleen of mice that have been infected by Babesia microti (Inchley, 1987). Measurement of cell proliferation is the endpoint most widely used for the LLNA and LNPA (Kimber et al, 2002;Weaver et al, 2005).…”