Abstracts

Rationale: (–)-Cannabidiol is the primary ingredient in marihuana that has been show to prevent or suppress seizures.  Unlike delta-9-tetrahydrocannabinol (delta-9-THC), cannabidiol does not bind to the cannabinoid receptor-1 in the brain and does not cause euphoria.  Cannabidiol is rapidly metabolized by several cytochrome P450 enzymes to eight different mono-hydroxylated metabolites (1).  The goal of this study was to determine the in vitro clearance of cannabidiol in incubations with pooled human and pooled dog liver microsomes as there is limited information known about interspecies differences in cannabidiol metabolism. Methods: (–)Cannabidiol (500 ng/mL) was incubated with pooled human (n=50 donors; HLM) and pooled beagle dog (n=5 donors; CLM) liver microsomes (HLM and CLM, 0. 5 mg protein/ml, Xenotech) with 1 mM NADPH and 5 mM MgCl2 in a 50 mM K phosphate buffer, pH 7.4 for 5, 15, 30, 45, 60, and 90 min. Incubations were stopped with 500 µL of acetonitrile, and centrifuged at 13,000Xg to remove precipitate protein. The disappearance of cannabidiol was determined by HPLC-MS/MS on a Shimadzu Prominence 8050 tandem mass spectrometer. Chromatographic separation of cannabidiol and metabolites was done under gradient conditions with 0.2% NH4OH (A) and 100% methanol (B)with a Phenomenex Kinetex EVO C18 100 x 2.1mm, 2.6 µ column at a flow rate of 0.4 mL/min at 40°C.  A 4 min gradient from 65% B to 95% B was run with a 1 min hold at 95% MeOH followed by re-equilibration. Several hydroxylated metabolites were observed at (M+16–H]–, but baseline separation of the metabolites was not possible Results: The rate of disappearance was 2.26 ng/min/mg protein in HLMs and 0.58 ng/min/mg protein in CLMs.  The estimated half-life was 24 min in HLM vs. 198 min in CLMs indicating significantly slower clearance in dog liver. Conclusions: The rate of oxidative metabolism of (-)-cannabidiol was significantly slower in dog liver microsomes compared to human liver microsomes. Samara et al. identified 36 metabolites by GC-MS in dog urine and observed that metabolites oxidized at the 6alpha and 6beta-positions were most abundant, whereas 7-hydroxylation is a dominant pathway for delta-9-THC and cannabidiol metabolism in humans, suggesting that dogs have lower 7-hydroxylase activity (2) and a reduced metabolic rate compared to humans. Jiang R, Yamaori S, Takeda S, Yamamoto I, Watanabe K. Life Sciences, 2011; 89:165-171Samara E, Bialer M, and Harvey DH, Drug Metab. Disp., 1990; 18:571-579 Funding: Funded by the Epilepsy Foundation and the Patricia L Nangle Fund and MacMillan Innovative Epilepsy Research and Education Fund.