The great divide: Separation between in vitro and in vivo effects of PSNCBAM-based CB₁ receptor allosteric modulators

While allosteric modulators of the cannabinoid type-1 receptor (CB₁) continue to be developed and characterized, the gap between the in vitro and in vivo data is widening, raising questions regarding translatability of their effects and biological relevance. Among the CB₁ allosteric modulators, PSNCBAM-1 has received little attention regarding its effects in vivo. Recently, pregnenolone was reported to act as an allosteric modulator of CB₁, blocking THC's effects in vitro and in vivo, highlighting the potential of CB₁ allosteric modulators for treatment of cannabis intoxication. We investigated the pharmacological effects of PSNCBAM-1 and two structural analogs, RTICBM-15 and -28, as well as pregnenolone, in both signaling and behavioral assays including [³⁵S]GTPγS binding, the cannabinoid tetrad and drug discrimination. While the CB₁ allosteric modulator PSNCBAM-1 attenuated THC-induced anti-nociception and its structural analog RTICBM-28 reduced THC's potency in drug discrimination, most cannabinoid effects in mice were unaffected. In contrast to the mouse studies, PSNCBAM-1 and analogs insurmountably antagonized CP55,940- and THC-stimulated [³⁵S]GTPγS binding and exhibited negative binding cooperativity with [³H]SR141716 with similar apparent affinities. Notably, RTICBM-28, which contains a cyano substitution at the 4-chlorophenyl position of PSNCBAM-1, exhibited enhanced binding cooperativity with CP55,940. In contrast to previous findings, pregnenolone did not block THC's effects in drug discrimination or [³⁵S]GTPγS. These data further highlight the difficulty in translating pharmacological effects of CB₁ allosteric modulators in vivo but confirm the established pharmacology of PSNCBAM-1 and analogs in molecular assays of CB₁ receptor function.