Cannabis Background
Well, um, let’s see, where do we begin? Cannabinoids are a biosynthetically unique group of compounds found in the cannabis plant. Cannabis plants can make a wide variety of cannabinoids, among them are cannabigerol (CBG) tetrahydrocannabinol (THC) and cannabidiol (CBD) as well as their acidic forms of cannabigerolic acid (CBGA), tetrahydrocannabinolic acid (THCA) and cannabidiolic acid (CBDA). Cannabis has a long history of being used for a variety of purposes going back to at least 4000 BC. Different strains of cannabis have been used for textiles as well as the therapeutic benefits associated with cannabis. As a textile cannabis has been used for fiber, clothing, and building material. Therapeutic uses for cannabis have been recorded in Sanskrit and Hindi literature going back to 1400-2000 BC.
They weren’t the only societies using cannabis however, Greek physicians were also known to have prescribed cannabis therapeutically in the first centuries AD. Later, Dr. O’Shaughnessy was one of the first physicians to advocate for the therapeutic use of cannabis in the Western world in 1839, recommending its use as an analgesic and sleep aid. Throughout the late 1800’s and early 1900’s the therapeutic use of cannabis became more prevalent until the Marihuana Tax Act of 1937 started heavily taxing those that used cannabis therapies. In 1970 Cannabis became listed as a Schedule 1 substance.
CBD Vs. THC
While many different types of cannabinoids exist, the most studied and understood of these are THC and CBD as well as their acidic forms THCA and CBDA. THC is the compound in some strains of cannabis that is responsible for the euphoric “high” feeling that is associated with consuming these varieties of cannabis. CBD is commonly referred to as the therapeutic or “good” cannabinoid found in some strains of cannabis, however this is a common mistake as both THC and CBD as well as the other cannabinoids have shown to have a variety of therapeutic uses. CBD and THC both have very similar structures, however, one of the key differences between them is the way in which they interact with the endocannabinoid receptors.
Indica Vs. Sativa
The laws surrounding cannabis have changed greatly in recent years with there being a large difference between how cannabis is regulated from state to state as well as how the regulation of “hemp” vs. “marijuana” is handled. C. sativa is the nomenclature introduced by Linnaeus in 1753 to describe non-psychoactive cannabis used to make textiles. C. indica is the nomenclature introduced by Lamarck in 1785 to describe the psychoactive varieties of cannabis found throughout Afghanistan and India. Following the legalization of cannabis in some states, these terms became so frequently used incorrectly that they now have no consistent and relevant meaning in determining the difference between cannabis varieties. It is now accepted that cannabis strains with less that 0.3% THC are considered hemp while cannabis strains containing larger amounts of THC are considered marijuana. By this generalized breakdown, CBD varieties of cannabis are hemp and the THC varieties are marijuana.
Endocannabinoid System
Both CBD and THC interact with the receptors in the endocannabinoid system. The endocannabinoid system is a system of endogenous cannabinoids and the receptors that they work upon. There are two types of endocannabinoid receptors known as CB1 and CB2 that are widely dispersed throughout the body with CB1 receptors being expressed mostly in the central nervous system and CB2 receptors being expressed in the peripheral nervous system. Both the CB1 and CB2 receptors are G-coupled protein receptors, a common type of receptor that is embedded in the membrane of the cell. The endocannabinoid system plays a role in a number of regulatory processes including metabolism, nociception/feeling regulation, as well as the regulation of emotions including stress. THC and CBD differ greatly in how they affect the endocannabinoid system. THC is an agonist of both CB1 and CB2 receptors while CBD does not bind to either of the receptors as strongly. CBD does however have a low affinity at both the CB1 and CB2 receptors and this is likely the main mechanism through which CBD interacts with the endocannabinoid system.
Natural Endocannabinoids
The endogenous endocannabinoid ligands are N-arachidonoylethanolamine (anandamide) and 2-arachidonoylglycerol. Endocannabinoids are responsible for binding to the CB1 and CB2 receptors which results in potassium channels opening and the closing of calcium channels. When the receptors are activated there is an inhibition in the release of certain neurotransmitters including: GABA, acetylcholine, dopamine, serotonin, glutamate, noradrenaline and others. While CBD only binds very weakly to the CB1 or CB2 receptors, it has been reported to act either as an agonist, an inverse agonist or as an antagonist at both receptors depending on the cell type. Furthermore CBD has the ability to act as an allosteric modulator as well, a feature of cannabinoids often referred to as the entourage effect. CBD has also shown action on the endocannabinoid system by inhibiting the degrading enzyme FAAH known to break down anandamide. In this way CBD helps increase the body’s amount of this natural endocannabinoid signaling molecule.
So Where Do Cannabinoids Come From?
The biosynthesis of cannabinoids involves both the acetate and the methylerythritol pathways (MEP). The product of the MEP that gets used is geranyl pyrophosphate which is formed from a condensation reaction of two isoprene units. The product of the acetate pathway that gets used is olivetolic acid which comes from the hexanoyl-CoA and three molecules of malonyl-CoA. The addition product of olivetolic acid with geranyl pyrophosphate is cannabigerolic acid (CBGA).
The formation of either CBDA or THCA from cannabigerolic acid is done via a cyclization reaction in which the isoprene tail of CBGA undergoes variable cyclization patterns. In addition to the schemes shown that produce CBDA and THCA the chain can cyclize to other conformations generating other cannabinoids including cannabichromene (CBC). The cyclization is FAD dependent and is driven by the formation of a carbocation. It is the final cation quenching step that determines which cannabinoid will be produced.
Making the Pieces: Geranyl Pyrophosphate
Geranyl Pyrophosphate is a product of the methylerythritol pathway. One molecule of dimethylallyl pyrophosphate (DMAPP) combines with one molecule of isopentenyl pyrophosphate (IPP) to form geranyl pyrophosphate (GPP). The reaction proceeds when the pyrophosphate acts as a leaving group creating the carbocation on DMAPP this gets attacked by the electrons in the double bond of IPP. The result of the addition product has a carbocation on the tertiary carbon which gets satisfied by the loss of a proton on the adjacent carbon forming the double bond. The GPP formed in the MEP pathway will be used in the formation of CBDA and THCA following the addition of GPP to olivetolic acid, a product in the acetate pathway. GPP is also used to make a number of the terpenes commonly found in cannabis as well such as limonene, myrcene, linalool and more. C13 labeling studies have shown that the GPP used in the formation of cannabinoids is derived from the MEP rather than the mevalonate pathway.
Making the Pieces: Olivetolic Acid
Olivetolic Acid is a product of the acetate pathway. It involves the only known polyketide synthase cyclase in plants. The synthesis begins with hexanoyl-CoA as the starter unit with three malonyl-CoA extender units being added. These reactions are carried out via the polyketide synthase where each addition of malonyl-CoA is driven by the decarboxylation that occurs on malonyl-CoA forming the enol. The enol then attacks the carbonyl carbon of the polyketide chain bound to the enzyme. The cyclization is carried out via Olivetolic Acid Cyclase (OAC).
The Only Cyclase of Its Type You Say? Take A Look!
The mechanism begins with a tyrosine amino acid side chain deprotonating the histidine amino acid side chain. The histidine amino acid side chain then removes the hydrogen on the alpha carbon between the two carbonyl carbons resulting in the formation of an enolate. The enolate then attacks the carbonyl carbon positioned such that the result is the formation of a six membered ring. Following the formation of the six membered ring there is a spontaneous aromatization that leads to the aromatic ring. Hydrolysis from the SCoA results in the formation of Olivetolic Acid.
Active Site of Olivetolic Acid Cyclase
The active site of olivetolic acid creates a pocket for the pentyl chain to rest in. There is also hydrogen bonding from histidine and tyrosine side chain residues holding the substrate in place. In the mechanism, the histidine and tyrosine side chains are involved in proton transfers that facilitate the cyclization reaction.
Formation of Cannabigerolic Acid
The formation of Cannabigerolic Acid “the mother cannabinoid” involves the product of the MEP pathway (GPP) and the product of the acetate pathway (olivetolic acid) in an addition reaction. The enzyme responsible for the addition of GPP onto olivetolic acid is olivtolate geranyltransferase. The addition is driven by the pyrophosphate acting as a leaving group resulting in the carbocation. The carbocation gets attacked by the electrons from the aromatic ring attaching the alkyl chain and forming CBGA. CBGA is commonly referred to as the “mother cannabinoid” as it leads to a variety of cannabinoids including THCA, CBDA and CBCA which along with CBGA are the most prevalent cannabinoids in most varieties of cannabis.
Cyclization of CBGA forming CBDA or THCA
The cyclization of CBGA can lead to the formation of a variety of cannabinoids including CBDA and THCA. The reaction begins with FAD accepting a hydride and generating a carbocation. The carbocation isomerizes such that the positive charge is spread out via resonance. The electrons from the double bond attack the positive charge forming a six membered ring with the carbocation now on the tertiary carbon. From here the positive charge can be satisfied by deprotonation of the adjacent carbon resulting in CBDA or by attack from the oxygen resulting in THCA.
Cannabis Inflammation Responses
CBD is involved in a number of pathways that affect natural inflammatory responses. CBD has shown to increase Ca+ levels in mast cells which play a role in chronic airway inflammation which in turn degranualizes the mast cells reducing inflammation. In animal models CBD has been shown to have a non cannabinoid receptor mediated effect on inflammation by enhancing adenosine signaling thus acting as an A2A receptor antagonist. Arachidonic acid release is increased with both THC and CBD which has displayed downstream effects such as increasing lipoxin A4 and 15d-PGJ2 which both display anti inflammatory properties. A key pathway by which CBD initiates a response to inflammation is through the inhibition of COX2.
CBDA Selective Inhibition of COX2
Both CBDA and THCA are COX2 inhibitors with CBDA being nearly 50 times as effective at COX2 inhibition than THCA. Both CBDA and THCA contain salicylic acid moieties which have been shown to be nonselective inhibitors of both COX1 and COX2. CBDA has a unique structure that allows it to act more selectively, having a 9 times greater effect on COX2 than on COX1. The selective inhibition of COX2 is significant in that while COX1 and COX2 both play a role in inflammation, COX1 is also responsible for a number of other important cellular functions. Traditional Non-Steroidal Anti-Inflammatories (NSAIDS) are nonselective COX inhibitors and it is the inhibition of COX1 which is widely linked to the negative side effects associated with NSAIDS.
Summary of Cannabis
Cannabinoids are unique to the cannabis plant with over 100 different cannabinoids having been characterized. The biosynthesis of cannabinoids includes the only known polyketide cyclase synthase in plants. While the classification of cannabis as a controlled substance has limited the availability of cannabis to researchers, cannabinoids have displayed therapeutic properties for a variety of conditions. Various cannabinoids have exhibited therapeutic potential on their own but it is commonly believed that the therapeutic profile of cannabinoids becomes increased when used in combination, a phenomenon referred to as the entourage effect.