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This perspective was adapted from a Career Achievement Award talk given at the International Cannabinoid Research Society Symposium in Bukovina, Poland on June 27, 2016. As a biochemist working in the neurosciences, I was always fascinated with neurotransmitter inactivation. In 1993 we identified an enzyme activity that breaks down anandamide. We called the enzyme anandamide amidase, now called FAAH. We and other laboratories developed FAAH inhibitors that were useful reagents that also proved to have beneficial physiological effects and until recently, new generations of inhibitors were in clinical trials. Nearly all neurotransmitters are water soluble and as such, require a transmembrane protein transporter to pass through the lipid membrane for inactivation inside the cell. However, using model systems, we and others have shown that this is unnecessary for anandamide, an uncharged hydrophobic molecule that readily diffuses across the cellular membrane. Interestingly, its uptake is driven by the concentration gradient resulting from its breakdown mainly by FAAH localized in the endoplasmic reticulum. We identified the FABPs as intracellular carriers that “solubilize” anandamide, transporting anandamide to FAAH. Compounds that bind to FABPs block AEA breakdown, raising its level. The cannabinoids (THC and CBD) also were discovered to bind FABPs and this may be one of the mechanisms by which CBD works in childhood epilepsy, raising anandamide levels. Targeting FABPs may be advantageous since they have some tissue specificity and do not require reactive serine hydrolase inhibitors, as does FAAH, with potential for off-target reactions. At the International Cannabis Research Society Symposium in 1992, Raphe Mechoulam revealed that his laboratory isolated an endogenous lipid molecule that binds to the CB1 receptor (cannabinoid receptor type 1) and this became the milestone paper published in December of that year describing anandamide (AEA, Devane et al., 1992). As to be expected, this discovery raised the issues of AEA's synthesis and breakdown.

This perspective was adapted from a Career Achievement Award talk given at the International Cannabinoid Research Society Symposium in Bukovina, Poland on June 27, 2016. As a biochemist working in the neurosciences, I was always fascinated with neurotransmitter inactivation. In 1993 we identified an enzyme activity that breaks down anandamide. We called the enzyme anandamide amidase, now called FAAH. We and other laboratories developed FAAH inhibitors that were useful reagents that also proved to have beneficial physiological effects and, until recently, new generations of inhibitors were in clinical trials. Nearly all neurotransmitters are water soluble and, as such, require a transmembrane protein transporter to pass through the lipid membrane for inactivation inside the cell. However, using model systems, we and others have shown that this is unnecessary for anandamide, an uncharged hydrophobic molecule that readily diffuses across the cellular membrane. Interestingly, its uptake is driven by the concentration gradient resulting from its breakdown mainly by FAAH localized in the endoplasmic reticulum. We identified the FABPs as intracellular carriers that solubilize anandamide, transporting anandamide to FAAH. Compounds that bind to FABPs block AEA breakdown, raising its level. The cannabinoids (THC and CBD) also were discovered to bind FABPs and this may be one of the mechanisms by which CBD works in childhood epilepsy, raising anandamide levels. Targeting FABPs may be advantageous since they have some tissue specificity and do not require reactive serine hydrolase inhibitors, as does FAAH, with potential for off-target reactions.

This perspective was adapted from a Career Achievement Award talk given at the International Cannabinoid Research Society Symposium in Bukovina, Poland on June 27, 2016. As a biochemist working in the neurosciences, I was always fascinated with neurotransmitter inactivation. In 1993 we identified an enzyme activity that breaks down anandamide. We called the enzyme anandamide amidase, now called FAAH. We and other laboratories developed FAAH inhibitors that were useful reagents that also proved to have beneficial physiological effects and until recently, new generations of inhibitors were in clinical trials. Nearly all neurotransmitters are water soluble and as such, require a transmembrane protein transporter to pass through the lipid membrane for inactivation inside the cell. However, using model systems, we and others have shown that this is unnecessary for anandamide, an uncharged hydrophobic molecule that readily diffuses across the cellular membrane. Interestingly, its uptake is driven by the concentration gradient resulting from its breakdown mainly by FAAH localized in the endoplasmic reticulum. We identified the FABPs as intracellular carriers that “solubilize” anandamide, transporting anandamide to FAAH. Compounds that bind to FABPs block AEA breakdown, raising its level. The cannabinoids (THC and CBD) also were discovered to bind FABPs and this may be one of the mechanisms by which CBD works in childhood epilepsy, raising anandamide levels. Targeting FABPs may be advantageous since they have some tissue specificity and do not require reactive serine hydrolase inhibitors, as does FAAH, with potential for off-target reactions. At the International Cannabis Research Society Symposium in 1992, Raphe Mechoulam revealed that his laboratory isolated an endogenous lipid molecule that binds to the CB1 receptor (cannabinoid receptor type 1) and this became the milestone paper published in December of that year describing anandamide (AEA, Devane et al., 1992). As to be expected, this discovery raised the issues of AEA's synthesis and breakdown.

Knowledge of the endogenous cannabinoid system has expanded greatly during the past years. After the discovery of the cannabinoid receptors, of their endogenous agonists and of the proteins for their synthesis and inactivation, significant progress has been made towards the understanding of the role of the endocannabinoid system in vital functions. Subsequently, an increasing number of papers has been published on the biochemistry and pharmacology of endocannabinoids. This article overviews the endocannabinoid signalling system with focus on its biochemical aspects. In particular we review the mechanisms for the biosynthesis and inactivation of the endocannabinoids, as well as the various molecular targets for some of the endocannabinoids described so far.

1. (-)-Cannabidiol (CBD) is a non-psychotropic component of Cannabis with possible therapeutic use as an anti-inflammatory drug. Little is known on the possible molecular targets of this compound. We investigated whether CBD and some of its derivatives interact with vanilloid receptor type 1 (VR1), the receptor for capsaicin, or with proteins that inactivate the endogenous cannabinoid, anandamide (AEA). 2. CBD and its enantiomer, (+)-CBD, together with seven analogues, obtained by exchanging the C-7 methyl group of CBD with a hydroxy-methyl or a carboxyl function and/or the C-5' pentyl group with a di-methyl-heptyl (DMH) group, were tested on: (a) VR1-mediated increase in cytosolic Ca(2+) concentrations in cells over-expressing human VR1; (b) [(14)C]-AEA uptake by RBL-2H3 cells, which is facilitated by a selective membrane transporter; and (c) [(14)C]-AEA hydrolysis by rat brain membranes, which is catalysed by the fatty acid amide hydrolase. 3. Both CBD and (+)-CBD, but not the other analogues, stimulated VR1 with EC(50)=3.2 - 3.5 microM, and with a maximal effect similar in efficacy to that of capsaicin, i.e. 67 - 70% of the effect obtained with ionomycin (4 microM). CBD (10 microM) desensitized VR1 to the action of capsaicin. The effects of maximal doses of the two compounds were not additive. 4. (+)-5'-DMH-CBD and (+)-7-hydroxy-5'-DMH-CBD inhibited [(14)C]-AEA uptake (IC(50)=10.0 and 7.0 microM); the (-)-enantiomers were slightly less active (IC(50)=14.0 and 12.5 microM). 5. CBD and (+)-CBD were also active (IC(50)=22.0 and 17.0 microM). CBD (IC(50)=27.5 microM), (+)-CBD (IC(50)=63.5 microM) and (-)-7-hydroxy-CBD (IC(50)=34 microM), but not the other analogues (IC(50)100 microM), weakly inhibited [(14)C]-AEA hydrolysis. 6. Only the (+)-isomers exhibited high affinity for CB(1) and/or CB(2) cannabinoid receptors. 7. These findings suggest that VR1 receptors, or increased levels of endogenous AEA, might mediate some of the pharmacological effects of CBD and its analogues. In view of the facile high yield synthesis, and the weak affinity for CB(1) and CB(2) receptors, (-)-5'-DMH-CBD represents a valuable candidate for further investigation as inhibitor of AEA uptake and a possible new therapeutic agent.