A cell-based test system for the assessment of pharmacokinetics of NOD1 and NOD2 receptor agonists

Agonists of nucleotide oligomerization domain (NOD) 1 and NOD2 receptors represent a promising class of immunostimulants and immunological adjuvants. Here, we describe a cell-based test system to assess their pharmacokinetics. In this system, NOD1 and NOD2 agonist concentrations in sera are determined using a reporter cell line, 293Luc, which contains an NF-κB-inducible luciferase reporter construct and naturally expresses NOD1 and NOD2. The 293Luc cells dose-dependently respond to different NOD1 and NOD2 agonists in the nanomolar to low-micromolar concentration range. To verify that the NF-κB-inducing activity of serum samples is due to the administered agonist and not to secondarily induced endogenous molecules, a 293Luc-derived NOD1/NOD2 double-knockout clone is used. Within-run and between-run precisions of the system are <15% and <20%, respectively. Applicability of the novel assay is illustrated by studying pharmacokinetics of two specific NOD2 agonists (N‑acetyl‑d‑glucosaminyl‑N‑acetyl‑d‑muramyl‑l‑alanyl‑d‑isoglutamine and N‑glycolyl‑d‑muramyl‑l‑alanyl‑d‑isoglutamine) and a specific NOD1 agonist (N‑acetyl‑d‑glucosaminyl‑N‑acetyl‑d‑sorbitolamine‑d‑lactoyl‑l‑alanyl‑d‑isoglutamyl‑meso‑diaminopimelic acid). In summary, the test system described here can potentially be used to assess pharmacokinetics of NOD1 and NOD2 agonists in different animal species.