FABP1 controls hepatic transport and biotransformation of Δ9-THC

Abstract

The increasing use of medical marijuana highlights the importance of developing a better understanding of cannabinoid metabolism. Phytocannabinoids, including ∆⁹-tetrahydrocannabinol (THC), are metabolized and inactivated by cytochrome P450 enzymes primarily within the liver. The lipophilic nature of cannabinoids necessitates mechanism(s) to facilitate their intracellular transport to metabolic enzymes. Here, we test the central hypothesis that liver-type fatty acid binding protein (FABP1) mediates phytocannabinoid transport and subsequent inactivation. Using X-ray crystallography, molecular modeling, and in vitro binding approaches we demonstrate that FABP1 accommodates one molecule of THC within its ligand binding pocket. Consistent with its role as a THC carrier, biotransformation of THC was reduced in primary hepatocytes obtained from FABP1-knockout (FABP1-KO) mice. Compared to their wild-type littermates, administration of THC to male and female FABP1-KO mice potentiated the physiological and behavioral effects of THC. The stark pharmacodynamic differences were confirmed upon pharmacokinetic analyses which revealed that FABP1-KO mice exhibit reduced rates of THC biotransformation. Collectively, these data position FABP1 as a hepatic THC transport protein and a critical mediator of cannabinoid inactivation. Since commonly used medications bind to FABP1 with comparable affinities to THC, our results further suggest that FABP1 could serve a previously unrecognized site of drug-drug interactions.

Figure 1

Overview of the major THC metabolic pathway. (A) Chemical structures of relevant cannabinoids and CYP2C-generated THC metabolites. (B) Schematic of FABP1-mediated transport of THC for subsequent metabolism by intracellular CYP450 enzymes.

Figure 2

Binding of cannabinoids to FABP1. DAUDA displacement curves and Ki values for (A) THC, (B) 11-OH-THC, (C) THC-COOH, (D) AJA, and (E) CBD are shown.

Figure 3

Structure of THC bound to FABP1. (A) Structure of FABP1 containing a single molecule of THC within the binding cavity (PDB: 6MP4). (B) Structure of the THC binding site showing side chains for residues positioned within 4.5 Å. Density surrounding THC is shown from a simulated annealing 2mF_o − DF_c OMIT map contoured at 0.8 σ. (C) Structural overlay of THC and palmitic acid (PDB: 3STM) bound to FABP1 showing the distinct binding sites of the molecules with altered positions of Met74 and Phe50.

Figure 4

Computational modeling. (A) Overlay of docked poses for THC (green), 11-OH-THC (pink), THC-COOH (light green), AJA (blue), and CBD (tan). Green molecular surface for THC. Protein residues hidden for clarity. (B) Molecular surface of FABP1 zoomed in on the metabolite THC-COOH highlighting solvent exposure of the carboxylic acid moiety.

Figure 5

THC biotransformation by primary mouse hepatocytes. (A) Time-course of ex vivo THC biotransformation to 11-OH-THC in hepatocytes derived from WT and FABP1-KO mice. (B) 5–60 minute AUC analysis of 11-OH-THC formation by cultured primary hepatocytes. (C) In vitro conversion of the luciferin-H substrate by crude hepatic microsomes or BSA control in the presence or absence of the CYP2C9 inhibitor sulphaphenazole (30 µM). (D) In vitro THC biotransformation by hepatic microsomes or by BSA control. *p < 0.05 by t-test.

Figure 6

Time-course of THC-induced hypothermia. (A) Male and (B) female wild-type or FABP1-KO mice were treated with vehicle or 10 mg/kg THC. Bar indicates range where statistical significance was reached comparing the THC-treated groups. *p < 0.05 by two-way ANOVA with Bonferroni’s post-hoc test. n = 5–9 animals per group.

Figure 7

Home cage locomotor activity. Spontaneous motility in males (A,B) and females (C,D) over a 12 hour period following administration of vehicle (A,C) or 10 mg/kg THC (B,D). AUC analysis of the resulting motility curves in males (E) and females (F). *p < 0.05; **p < 0.01 by t-test. n = 9–12 animals per group.