The peripheral cannabinoid receptor 1 antagonist VD60 efficiently inhibits carbon tetrachloride-intoxicated hepatic fibrosis progression

Abstract

This study investigated a peripheral selective CB₁ antagonist 3,4,22-3-demethoxycarbonyl-3-hydroxylmethyl-4-deacetyl-vindoline 3,4-thionocarbonate (VD60) that efficiently inhibited hepatic fibrosis with lower psychological side effects. A competitive radiolabeled ligand binding experiment and 3′-5′-cyclic adenosine monophosphate (cAMP) response element-driven luciferase analysis were performed to evaluate the antagonistic activity of VD60. Cell viability and collagen production were examined in the human hepatic stellate cell (HSC) line LX-2 and primary cultured rat HSCs. The antifibrotic effects of VD60 were investigated in a CCl₄-induced liver fibrosis mouse model. The concentration of VD60 in the blood and the brain was determined by high-performance liquid chromatography–mass spectrum analysis. Furthermore, the potential underlying mechanisms of VD60 were investigated by Western blot. VD60 selectively competed with the radiolabeled CB₁ agonist to bind to CB₁. VD60 antagonized CB₁ agonist-induced Akt phosphorylation and increased the accumulation of intracellular cAMP. VD60 strongly reduced the expression of α₂(I) pro-collagen mRNA and exerted potent antiproliferative effects on primary HSCs and LX-2 cells. The inhibition of reactive oxygen species production and phosphorylation of Akt, extracellular-signal-regulated kinase (ERK), and Smad3 may explain the underlying mechanisms behind the antiproliferative effect of VD60. Moreover, the in vivo antifibrotic activity of VD60 was confirmed in a CCl₄-induced liver fibrosis mouse model. Most importantly, the concentration of VD60 in the peripheral blood was much higher than in the brain, suggesting that VD60 could act as a novel peripheral CB₁ antagonist to efficiently inhibit hepatic fibrosis and could be used as a lead compound with low brain side effects in peripheral antifibrotic agents.