d that AM1241 exhibited inconsistent functional efficacies. In ERK activation assays, AM1241 exhibited Gi/o dependent partial agonist activity at the CB2 receptor, stimulating ERK activation at a level lower than that of CP 55,940. In c-Met inhibitor drug contrast, AM1241 was an apparent c-Met inhibitor drug antagonist in FLIPR assays, blocking the CP 55,940 evoked calcium influx through the CB2 receptor, similar to the effect observed with SR144528. In cyclase assays, AM1241 produced inconsistent efficacies that were dependent upon the assay conditions used. When a higher forskolin concentration was used to stimulate the adenylyl cyclase, AM1241 failed to produce a change in efficacy.
However, AM1241 reversed the effects of agonist CP 55,940 and inverse agonist SR144528 in a concentration dependent manner, demonstrating that AM1241 behaved as a neutral antagonist.
When assays were performed using lower forskolin concentrations, AM1241 consistently exhibited agonist efficacy, reducing the cAMP level, as did CP 55,940. The divergence c-Met pathway inhibitor among functional properties of AM1241 c-Met pathway inhibitor in various in vitro assays and the lack of robust CB2 agonist efficacies may suggest that AM1241 is a protean agonist at the CB2 receptor. In contrast, the agonist CP 55,940 and inverse agonist SR144528 exhibited consistent functional efficacies across different assay systems. In order to provide a direct comparison to preclinical animal studies, a racemic mixture of AM1241 has been used in the current studies.
In addition, it has been shown by Uveges et al.
that the individual enantiomers exhibit similar potencies and efficacies at the human CB2 receptor in cyclase assays compared with those of the racemic mixture, indicating that neither enantiomer is likely to produce confounding functional properties. CP 55,940 failed to show agonist activity in cyclase assays in native cell lines such as HuT 78 that expresses the human CB2 receptor gene. In contrast, the recombinant HEK cell line used in the current studies expresses the human CB2 receptor at a high level, allowing readily detection of both agonists and antagonists.
According to the current receptor activation theory, the increased receptor availability in recombinant systems increases the absolute number of receptors activated by agonist ligands, leading to significant augmentation of the signalling pathway as well as the detection capability of the assay system, resulting in significant amplification in maximal agonist efficacies.
Concomitantly, the higher level of receptor expression in recombinant systems increases the total number of spontaneously activated receptors regardless of the presence of agonist ligands, and thus the assay system displays higher levels of constitutive activity. The level of spontaneous activation of receptors is dependent upon multiple factors the thermodynamic nature of receptor conformational changes from quiescent state to active state, the cellular background, such as repertoires of G proteins, GPCR binding and effector proteins, as well as the conditions under which the cells were grown and the assays performed. In contrast to native systems where the receptors are quiescent in general and do not display significant constitutive activity, recombinant systems often show variable levels of constitutive activity. Such constitut