Delta9technologies

How are the cannabinoids made in the cannabis plant?

Figure 1. Metaboic Pathway for the production THCA and CBDA as well as the degradation to THC, CBD, and CBN.

The most unique aspect of the cannabis plant is its ability to produce medicinal cannabinoid molecules such as THC and CBD. In addition to the well known cannabinoids there are many other cannabinoids such as CBC, THCV, and CBG. Cannabigerol, or CBG, is the first cannabinoid-like molecule derived in the plant and serves as a building block for THC, CBD, CBC, and THCV. The quantity of each cannabinoid produced in the plant is directly related to the expression cannabinoid synthase proteins, and this is determined by the genetics of the plant. As shown in Figure 1 above, CBDA synthase catalyzes the reaction from CBGA to CBDA. Likewise, THCA, THCVA, and CBCA are produced from CBGA and the corresponding cannabinoid synthase. The acid-containing cannabinoids slowly degrade into THC and CBD upon standing under normal conditions. This process is rapidly accellerated upon heating and can cause THC to degrade into an inactive form, CBN, if excessive heat is applied. Below, there is an entire section outlining the medicinal properties of each cannabinoid wth links to peer-reviewed medical journals.

What are they looking for in urine samples for drug tests?

Figure 2. Metaboic pathway for the degradation of THC in humans. 11-nor-9-carboxy-THC is the metabolite of interest in drug tests

If you are like me, then you have probably wondered what scientists are looking for in urine samples to determine whether or not someone has consumed cannabis. Ironically, it is not THC, but rather an inactive more water soluble metabolite known as 11-nor-9-carboxy-THC (Figure 2). However, the first degradation step involves the conversion of THC to 11-hydroxy-THC as shown above. Interestingly, the intermediate 11-hydroxy-THC is 5-fold more potent than the parent cannabinoid, THC. This oxidative-mediated pathway is likely due to cytochrome P-450 monooxegenases, which can be up- or down-regulated depending on each individuals genetics. The varying effect cannabis-infused edibles can have on people can be attributed to the accumulation of the more highly potent metabolite, 11-hydroxy-THC. Additionally, the ratio of 11-hydroxy to 11-nor-9-carboxy-THC can be used to indicate the amount of time that has past since subject was intoxicated.

Therapeutic value of the cannabinoids (adapted from Mechoulam2009)

THCA-A: The two acidic forms of delta-9-THC, Delta-9-THCA-A and Delta-9-THCA-B, are the most abundant cannabinoids generally found in dried flowers of cannabis. Delta-9-THCA-A, first extracted by Korte and coworkers (1965), was isolated as a pure compound in 1967 by Nishioka’s group. In 1969, Mechoulam and coworkers reported the isolation of Delta-9-THCA-B. Delta-9 THCA is a potent TRPA1 agonist and TRPM8 antagonist [1] and has been shown to exert anti-proliferative [2] and anti-spasmodic [3] actions. As mentioned previously, THCA is not psychoactive and must be converted to THC by heating the sample.

THC: Isolated in 1964 by Gaoni and Mechoulam at the Weizmann Institute in Rehovot, delta-9-THC is the primary psychotropic ingredient of Cannabis. It is a partial agonist at CB1 and CB2 receptors (Ki approx. 20–40 nM). Delta-9-THC also activates PPAR-g (at nanomolar concentrations) and TRPA1 (at micromolar concentrations) [4]. It is therapeutically used as an antiemetic and to boost appetite in AIDS patients. A Cannabis based-extract with approx 1:1 ratio of delta-9-THC and CBD (Sativex) is marketed in Canada for the symptomatic relief of neuropathic pain in adults with multiple sclerosis and as an adjunctive analgesic treatment for adult patients with advanced cancer [5].

CBDA: The acidic cannabinoids originating from the plant decarboxylate over time to yield the better known neutral cannabinoids. None of the cannabinoid acids possess psychotropic activity [6]. The first cannabinoid acid to be discovered, cannabidiolic acid (CBDA), was isolated in 1955 by Krejci and Santavy. Together with CBD, CBDA is the main component of glandular hairs (up to 15%). In fresh plant material, 95% of CBD exists as its acid. It is a selective COX-2 inhibitor [7], TRPA1 and TRPV1 agonist and TRPM8 antagonist in the low micromolar range [1, 2]. It exerts anti-proliferative actions [2].

CBD: CBD a major non-psycotropic cannabinoid, was first isolated in 1940 by Adams and coworkers, but its structure and stereochemistry were determined in 1963 by Mechoulam and Shvo. CBD exerts a plethora of pharmacological effects, mediated by multiple mechanisms. It has been clinically evaluated in anxiety, psychosis, and movement disorders, and to relieve neuropathic pain in patients with multiple sclerosis (in combination with delta-9-THC as a 1:1 mixture, i.e. Sativex) [8].

CBN: Isolated in 1896 by Wood and colleagues in Cambridge, CBN represents the first natural cannabinoid to be obtained in pure form. Its correct structure was later determined by Adams and colleagues in 1940. It was initially—and incorrectly—assumed to be the active psychotropic ingredient of Cannabis. It is a relatively minor constituent in fresh Cannabis because it is a product of delta-9-THC oxidation. CBN content increases as delta-9-THC degrades in storage. It is a weak CB1 and CB2 partial agonist, with approximately 10% of the activity of delta-9-THC. It has potential therapeutic application in diseases in which cannabinoid receptors are up-regulated [4].

Delta-8-THC: In general, delta-8-THC is regarded as an artifact because it results from the isomerization of delta-9-THC. Delta-8-THC concentration in cannabis is usually minuscule, and it does not contribute significantly to the activity of the plant extract. Delta-8-THC is considered less expensive to prepare and more stable than delta-9-THC. The pharmacology of delta-8-THC is similar to that of delta-9-THC, although it may be less active [3]. It is as active as delta-9-THC in antiemetic studies, although it is not marketed (apparently for purely commercial reasons).

Delta-9-THCV: Delta-9-THCV was detected in 1970 by Edward Gil and colleagues from a tincture of Cannabis BPC (then a licensed medicine in the UK). It is particularly abundant in Pakistani hashish. Delta-9-THCV antagonises the effects of delta-9-THC effects at low doses (<3 mg/kg), but it acts as a CB1 agonist at higher doses (10 mg/kg) in mice [4, 9]. Delta-9-THCV shares the ability of synthetic CB1 antagonists to reduce food intake in mice [10]. It has also shown promise in the treatment of diabetes and Parkinsons disease.

CBC: The discovery of CBC, a non-psychotropic cannabinoid, was independently reported by Claussen and coworkers, and Gaoni and Mechoulam in 1966. CBC, together with delta-9-THC, is the major cannabinoid in freshly harvested dry-type material. CBC is nearly 2.5-times more toxic than delta-9-THC [3]. CBC exerts anti-inflammatory, antimicrobial and modest analgesic activity [3, 12, 13]. It is a potent TRPA1 agonist and weak anandamide reuptake inhibitor [2, 11].

CBG: Non-psychotropic cannabinoid obtained in 1964 by Gaoni and Mechoulam when they separated a hexane extract of hashish on Florisil. It exerts anti-proliferative and antibacterial activity. It is a potent TRPM8 antagonist [1], a TRPV1, TRPA1 and cannabinoid agonist, and an anandamide reuptake inhibitor in the low micromolar range [1,2].

Cannabinoid Nomenclature

Scientific nomenclature for chemical structures can be confusing, but is necessary to appropriately describe complex molecules such as the cannabinoids. Delta-9-tetrahydrocannabinol (THC) contains 21 carbons arranged in a tricyclic phenol-containing system as shown in the Figure. Notice that the double bond moiety in delta-9-tetrahydrocannabinol contains a double bond listed numerically at position 9. Its name is derived from cannabinol (CBN), but differs with regard to four hydrogens (tetrahydro) attached to the ring. Delta 9 indicates the position of the double bond. THC, Delta-9 Tetrahydrocannabinol.

There is also a positional isomer known as delta-8-tetrahydrocannabinol. This isomer contains the double bond in the 8-position, and is generally detected in samples that have been heated or stored for extended periods of time. In contrast to THC, cannabidiol (CBD) uses an entirely different numbering scheme. For example, position-9 of the THC molecule is labeled position 1 in CBD.

Figure 3. Numbering scheme used to describe the cannabinoids.

The Future Outlook of the Medicinal Cannabis Industry

What will the future landscape of the marijuana industry look like? In my humble view, it might not resemble the wild west grass roots industry that it currently is. Despite the allure of this romanticized image, the greatest advances in recent years have been the identification of CBDA and THCA synthase [14, 15]. These enzymes are responsible for converting an endogenous convergent intermediate (cannbigerolic acid, CBGA) to the well known cannabinoids CBDA and THCA. These compounds are recognized as precursors to the compounds that account for the plants medicinal value. As of 2005, Shoyama crystallized THCA synthase making it possible to obtain X-ray crystallographic information about the structure and function of the enzyme. Not long after this scientific breakthrough, CBDA synthase was also purified and characterized.

CBD concentrations have unknowingly been bred out of California marijuana and have led to a world-wide initiative to identify CBD-rich strains of cannabis. The CBD project is rapidly growing in popularity and has already identified numerous CBD rich srains to aid cultivators to the appropriate genetics to express this phenotype. The scientific community now knows that the expression of CBDA synthase is upregulated in CBD-rich strains. Adcvances in molecular cloning techniques have successfully transformed the genes for these enzymes into yeast and other plant strains. For example, transgenic tobacco hairy roots were shown to produce THCA when fed the starting material CBGA. While we have all envisioned fruits and vegetables that produce THC, the time may well be upon us. Who knows, soon you may be able to eat produce that can bake you. Despite the many potential applications, perhaps the most applicable use of this technology will be to produce pure THCA and CBDA that can be recombined in precise quantities. The true value of the cannabinoids will be realized when a complete picture of the synergistic relationship shared between them has been fully characterized.

References:

0. Mechoulam, R. et al. (2009) Non-psychotropic plant cannabinoids: new therapeutic opportunities from an ancient herb. Trends Pharmacol. Sci. 30, 515-527.

1. De Petrocellis, L. et al. (2008) Plant-derived cannabinoids modulate the activity of transient receptor potential channels of ankyrin type-1 and melastatin type-8. J. Pharmacol. Exp. Ther. 325, 1007–1015.

2. Ligresti, A. et al. (2006) Antitumor activity of plant cannabinoids with emphasis on the effect of cannabidiol on human breast carcinoma. J. Pharmacol. Exp. Ther. 318, 1375–1387.

3.Turner, C.E. (1980) Constituents of Cannabis sativa L. XVII. A review of the natural constituents. J. Nat. Prod. 43, 169–234.

4. Pertwee, R.G. (2008) The diverse CB1 and CB2 receptor pharmacology of three plant cannabinoids: delta9-tetrahydrocannabinol, cannabidiol and delta9-tetrahydrocannabivarin. Br. J. Pharmacol. 153, 199–215.

5. Russo, E.B. et al. (2007) Cannabis, pain, and sleep: lessons from therapeutic clinical trials of Sativex, a cannabis-based medicine. Chem. Biodivers. 4, 1729–1743.

6. Mechoulam, R. and Hanus, L. (2000) A historical overview of chemical research on cannabinoids. Chem. Phys. Lipids. 108, 1–13.

7. Takeda, S. et al. (2009) Cannabidiol-20,60-Dimethyl Ether, a Cannabidiol Derivative, is a highly potent and selective 15- lipoxygenase inhibitor. Drug. Metab. Dispos. 37, 1733–1737.

8. Russo, E.B. et al. (2007) Cannabis, pain, and sleep: lessons from therapeutic clinical trials of Sativex, a cannabis-based medicine. Chem. Biodivers. 4, 1729–1743.

9. Pertwee, R.G. et al. (2007) The psychoactive plant cannabinoid, delta9- tetrahydrocannabinol, is antagonized by delta-8-and delta-9-tetrahydrocannabivarin in mice in vivo. Br. J. Pharmacol. 150, 586–594.

10. Riedel, G. et al. (2009) Synthetic and plant-derived cannabinoid receptor antagonists show hypophagic properties in fasted and nonfasted mice. Br. J. Pharmacol. 156, 1154–1166.

11. Davis, W.M. and Hatoum, N.S. (1983) Neurobehavioral actions of cannabichromene and interactions with delta 9-tetrahydrocannabinol. Gen. Pharmacol. 14, 247–252.11.

12. Turner, C.E. and Elsohly, M.A. (1981) Biological activity of cannabichromene, its homologs and isomers. J. Clin. Pharmacol. 21, 283S–291S.

13. Appendino, G. et al. (2008) Antibacterial cannabinoids from Cannabis sativa: a structure-activity study. J. Nat. Prod. 71, 1427–1430.

14. Supaart, S.; Futoshi, T.; Satoshi, M.; Yukihiro, S. Recent Advances in Cannabis sativa Research: Biosynthetic Studies and Its Potential in Biotechnology. Current Pharmaceutical Biotechnology 2007, 8, 237-243.

15. Taura, F.; Sirikantaramas, S.; Shoyama, Y.; Shoyama, Y.; Morimoto, S. Phytocannabinoids in Cannabis sativa: Recent Studies on Biosynthetic Enzymes. Chemistry & Biodiversity 2007, 4, 1649-1663.