Do assay parameters affect the inter-assay comparability of cannabinoid CB₁ receptor affinity and activation...

Reported cannabinoid receptor type 1 (CB₁) affinities and activities vary substantially across studies, complicating cross-study comparisons. The present study aims to systematically and statistically evaluate whether assay-related parameters influence reported CB₁ binding affinity (K_i) and potency (EC₅₀). Our investigation was based on curated CB₁ affinity (n = 271) and potency (n = 169) datasets which contained repeated measurements of ten compounds, respectively. Analysis of these two datasets were conducted using linear mixed-effects models with multiple imputation to quantify the effects of experimental conditions while accounting for repeated measurements per compound. For affinity (pK_i), variability was primarily driven by compound-specific effects, with only minor contributions from assay parameters. Only small but significant effects were observed for CHO cells compared to HEK cells. Opposed to this, potency (pEC₅₀) variability was influenced by assay parameters such as assay readout, assay type and cell membrane type. Incubation time demonstrated a small but inconsistent association with potency values but not affinity values, while incubation temperature showed no robust effect within the observed range. Our findings indicate that CB₁ affinity measurements are largely comparable across studies under standardized assay conditions, whereas potency estimates are more context-dependent and should be interpreted in light of the assay used, particularly when derived from assays targeting distinct signaling pathways.