Cannabidiol-dihydroartemisinin conjugates for ameliorating neuroinflammation with reduced cytotoxicity

Cannabidiol (CBD) and dihydroartemisinin (DHA) can alleviate neuroinflammatory responses. However, they show cytotoxicity, which severely limits their therapeutic windows. Therefore, there is a great need to develop neuroprotective agents with improved safety. Drug-drug conjugate is an emerging approach for enhancing therapeutic index. Herein, the development, synthesis, and the pharmacological characterization of CBD-DHA conjugates were performed. Meanwhile, the combination of CBD and DHA as separate entities was also quantitatively analyzed for direct comparison with CBD-DHA conjugates. In this study, BV-2 microglial cell line was used to mimic primary microglia and the effects of CBD, DHA, the combination of CBD and DHA, as well as CBD-DHA conjugates on LPS-activated signaling molecules and pro-inflammatory factors were assessed. The interaction of CBD and DHA in inhibiting LPS-induced nitric oxide (NO) production was found to be additive. In contrast, DHA was found to synergize with CBD in inhibiting BV-2 cellular viability which implies that the combination of CBD and DHA amplifies their cytotoxicity. CBD-DHA conjugate C3D eliminated the cytotoxicity associated with single CBD/DHA use without significantly compromising the anti-neuroinflammation activity. C3D was more potent than C2D and C4D in inhibiting LPS-induced NO and mRNAs of iNOS and IL-1β, which implies that the linker length is critical for CBD-DHA conjugates' anti-inflammatory activities. Further signaling characterizations showed that C3D inhibited LPS-induced NF-κB but not MAPKs activation in BV-2 cells, therefore blocking LPS-induced neuroinflammation. This work provides a good example that conjugated drug-drug approach may improve the therapeutic index by increasing the maximum tolerated concentration/dose compared to traditional combination strategy.