Efficient synthesis of complex cannabinoids is possible while avoiding marijuana cultivation.
Cannabinoids have great potential to treat a wide variety of diseases, leading to tremendous interest among pharmaceutical companies in accessing pharmaceutically pure cannabinoids for the development of novel therapies. Several companies are developing cannabinoid-based treatments, including Vitality Biopharma, GW Pharmaceuticals, Zynerba Pharmaceuticals, INSYS Therapeutics, Teewinot Life Sciences Corporation, and Nemus Bioscience.
Some cannabinoids bind to the same receptors that are bound by endocannabinoids, which are naturally produced in human cells. The human endocannabinoid system (ECS) is a group of endogenous cannabinoid receptors located in the mammalian brain and throughout the central and peripheral nervous systems that are controlled by neuromodulatory lipids, according to Richard Peet, executive vice-president and research director at Teewinot Life Sciences Corporation. “The ECS is involved in a variety of physiological processes, including pain sensation, appetite, memory, and mood. It is for this reason that cannabinoids frequently influence various physiological functions when administered to humans,” he notes.
Cannabinoids such as delta-9-tetrahydrocannabinol (THC), cannabidiol (CBD), cannabichromene (CBC), tetrahydrocannabivarin (THCV), and cannabidivarin (CBDV) have many potential uses, such as the treatment of nausea associated with chemotherapy, epilepsy, appetite control, diabetes, and inflammation, among many others.
Why biocatalysis for cannabinoid synthesis?
Cannabinoids are synthesized in the Cannabis plant via biosynthetic pathways consisting of several enzyme-catalyzed steps. The Cannabis plant produces only small quantities of many of these cannabinoids including the varin series of cannabinoids (THCV, CBDV, and cannabichromevarin [CBCV]), and cost-effective chemical synthesis of these molecules is not available, according to Peet.
“Traditional chemical manufacture of these complex, chiral molecules typically requires several steps and elaborate purification from impurities,” observes James Lalonde, senior vice-president of research and development for Codexis. Chemical synthesis of cannabinoids such as THC also generates copious amounts of organic waste, takes many weeks to complete at the kilogram scale, and is very expensive, adds Peet. “Practical methods for chemical synthesis for many of the 111 known cannabinoids have not been developed,” he states. Enzymes that have been evolved in nature, in contrast, can synthesize these molecules cleanly with the exclusion of impurity-generating side reactions, according to Lalonde.
Purification of cannabinoids from the Cannabis plant is also not practical for production of large quantities of pharmaceutically pure cannabinoids. Extraction of targeted cannabinoids from the more than 100 related compounds in the plant is laborious and time consuming, according to Lalonde. “The Cannabis plant must be grown for three to four months and requires large quantities of water and energy inputs. Then obtaining pharmaceutically pure cannabinoids from plant tissue is very expensive, because many cannabinoids are produced in very small quantities in the Cannabis plant,” notes Peet.
The use of enzymes ex vivo allows the synthesis of targeted single distinct compounds, rather than a complex mixture. “Production of these compounds can be controlled for purity and regulatory controls in the same manner as any active pharmaceutical. In addition, the biocatalytic process is independent from the plant growth cycle and the constraints of marijuana cultivation. Scale up of the synthesis of rarer, but desirable compounds would also be made possible in an enzymatic, ex vivo process in which enzymes that catalyze formation only of the desired compounds could be cloned and purified in high yield,” Lalonde explains.
New practical biosynthetic processes
Teewinot has developed biosynthetic processes for the production of large quantities of cannabinoids including the varin series of cannabinoids. “Compared to chemical syntheses, these processes are less expensive, more efficient, and produce minimal amounts of chemical waste. As a result, biosynthetic processes make possible the production of pharmaceutically pure cannabinoids in much larger quantities than is practical through plant cultivation and cannabinoid extraction,” Peet asserts.
The company uses bioinformatics and metabolomics to facilitate the development of synthetic biology and biocatalytic routes to cannabinoids. Genes in the Cannabis plant, as well as chemical compounds in plant tissues, are identified, characterized, and quantified if relevant. “By cloning specific gene sequences from the plant and incorporating them into microorganisms, we are able to develop new expression systems and processes for the production of naturally-occurring and specific cannabinoids,” Peet explains. Specifically, Teewinot has cloned genes from the Cannabis plant that code for cannabinoid biosynthetic enzymes and incorporated them into yeast and other microorganisms.
Teewinot refers to its cannabinoid biosynthetic processes as CannSynthesis. Using its synthetic biology and biocatalytic processes, the company can now biosynthetically produce 18 different cannabinoids that have the identical chemical structures to those produced in the plant, including tetrahydrocannabinolic acid (THCA), THC, tetrahydrocannabivarinic acid (THCVA), THCV, cannabidiolic acid (CBDA), CBD, cannabidivarinic acid (CBDVA), CBDV, cannabichromenic acid (CBCA), CBC, cannabichromevarinic acid (CBCVA), cannabichromevarin (CBCV), cannabigerolic acid (CBGA), cannabigerol (CBG), cannabigerovarinic acid (CBGVA), cannabigerovarin (CBGV), cannabinol (CBN), and cannabicyclol (CBL), according to Peet.
For example, Teewinot produces the Cannabis enzymes THCA synthase and CBDA synthase in a microorganism. THCA synthase converts the substrate CBGA into THCA and/or CBCA, while CBDA synthase converts the substrate CBGA into CBDA and/or CBCA. During biocatalysis, both reactions proceed in a pH-dependent manner in a bioreactor. The CBGA used in this biocatalytic process can be produced in any number of ways, including synthetic biology or chemical synthesis. Alternatively, the entire process can be done in a microorganism by means of synthetic biology.
Teewinot can also produce next-generation, proprietary cannabinoid pro-drugs and cannabinoid analogs, many of which are novel and have never been produced in large quantities in pharmaceutically pure form. “Some of these compounds have improved efficacy and bioavailability and other beneficial characteristics that improve therapeutic outcomes,” Peet notes. He adds that it is now possible to test the efficacy of these compounds in the treatment of a wide variety of diseases, including inflammatory bowel disease, Crohn’s, epilepsy, cancer, migraine, fibromyalgia, and many others, according to Peet. For instance, in 2016, Teewinot licensed its patent-protected biosynthetic processes to Nemus Biosciences for use in the manufacture of its THC pro-drug for treatment of glaucoma.
The company expects to undertake GMP production of cannabinoids using biocatalysis within the next three to six months, and is in conversations with a large number of pharmaceutical companies seeking to purchase cannabinoids produced by its biosynthetic processes for use in pre-clinical and clinical trials.
In March 2017, Teewinot signed a letter of intent with Noramco, a producer of controlled-substance APIs, to commercially produce cannabinoids using its CannSynthesis technology. The agreement provides Noramco access to Teewinot’s patent-protected processes for the production of pure cannabinoids using biocatalysis and the company’s assistance with rapid implementation of the technology.
Noramco will create 10-15 cannabinoid reference standards and evaluate the feasibility of using the technology to produce Dronabinol on a commercial scale. Dronabinol is known for its effectiveness in pain management, as an antiemetic, in the control of vomiting, as an appetite stimulant in the treatment of AIDS and for reducing the side effects of chemotherapy.
Education is important
Although biosynthetic processes like those developed by Teewinot are new and improved methods of production that reduce cost and production time, increases purity, and allow for the synthesis of a much greater diversity of molecules that can be incorporated into drug formulations, the approach is different from traditional extraction or chemical synthesis. As a result, Teewinot has found that bringing this new technology to market has created the need to educate researchers and others in the pharmaceutical cannabinoid field. “Educating researchers includes describing the broad array of cannabinoids that can now reliably be manufactured, tested, and put into clinical trials for development of potential new therapies,” Peet comments.
He does note, however, that because synthetic biology and biocatalysis are common pharmaceutical manufacturing processes, pharmaceutical companies and regulators are generally familiar with the technical aspects of these methods.
This article was originally published by Pharmaceutical Technology, Vol. 41, No. 6, June 2017, Pages: 20–22. When referring to this article, please cite it as C. Challener, “Accessing Cannabinoids Using Biocatalysis," Pharmaceutical Technology 41 (6) 2017.