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The quality demands of the pharmaceutical industry require prescription medicines to be consistent in their active ingredient content. Achieving this, using raw cannabis as a feedstock, is especially challenging. The plant material is extremely inhomogeneous, and the ratios of active ingredients are affected by a range of factors. These include the genetics of the plant, the growing and storage conditions, the state of maturity at harvest, and the methods used to process and formulate the material. The reasons for this variability are described, with particular emphasis on the botanical considerations. To produce the complex botanical medicine Sativex®, which contains the cannabinoids Δ9–tetrahydrocannabinol (THC) and cannabidiol (CBD) and a range of other ingredients, GW Pharmaceuticals had to manage these variables. This medicine, for the treatment of spasticity due to multiple sclerosis, is the first cannabis-based medicine to be approved in the UK. The company's methodology for producing this and other chemotypes is described. Copyright © 2013 John Wiley & Sons, Ltd.
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The World Health Organisation has estimated that over 21 000 plant species are used for medicinal purposes. However, only about 100 of these are specifically grown for the pharmaceutical industry, and in each case this will be in an agricultural setting. Outdoor growing of pharmaceutical cannabis is fraught with difficulties. This highly regulated plant has a large retail cash value, and a unique cachet that makes it vulnerable to those with a curious, acquisitive or malicious intent. The genotypes used may have resulted from many years of plant breeding input. Intellectual property such as this deserves protection. To successfully overcome these security concerns, outdoor-grown pharmaceutical crops in the USA have necessitated tall steel perimeter fences, overlooked from security turrets. Even then, such outdoor crops would be vulnerable to the vagaries of the weather, and at the mercy of pests, disease, and contamination. Similar problems would face producers of illicit cannabis.
Much of the material consumed in past decades has been relatively low-potency outdoor-grown material, consisting of the upper leaves and seed-bearing female flowers. Alternatively it has been cannabis resin (hashish), collected from this material by a variety of cultural methods. Since the 1970s however, in the USA and in Canada especially, there has been a change to indoor growing to avoid the attention of the police and theft from rivals. A law-enforcement crackdown and large-scale eradication efforts may have inadvertently encouraged this. However, indoor growing has had other appeals. More recently, the Western recreational cannabis market has changed, with an increasing proportion of consumers preferring only unfertilized floral parts of the female cannabis plant. This more potent form is often called sinsemilla (from the Spanish sin semilla: without seeds) and most of it is grown indoors. In the more easily controlled indoor environment, the quality of this material is increasingly guaranteed. In the UK, organized crime groups have established so- called cannabis factories on a massive scale. Small producers of illicit cannabis for alleged medicinal use have also preferred indoor growing.
GW Pharmaceuticals has grown a limited number of outdoor cannabis crops in the UK, for research purposes. These have been in discreet Home Office licensed locations. Most crops have been CBD dominant, and only a few plants have been of a psychoactive THC chemotype. The company has also evaluated the growing of crops in a totally enclosed environment. However, for routine production, GW Pharmaceuticals has to date rejected both outdoor and factory-style growing. Since its inception in 1998 the company has overseen the propagation of approximately 1.5 million cannabis plants in a sophisticated glasshouse environment. This enables the provision of high-level security and allows year-round propagation in stable conditions. This uniformity facilitates the production of cannabis that meets the pharmaceutical industry's demand for quality, safety, and efficacy. Indeed, greater importance is placed on crop uniformity than on high yields. The lighting systems for indoor cannabis production have been calculated to consume one kilowatt hour of energy for each gram of dry cannabis inflorescence.[8, 9] Although the glasshouses are fitted with supplementary lighting, much less electrical energy is consumed than in a totally enclosed environment.
Even when grown in idealized conditions, cannabis is a highly variable material. To guarantee product uniformity the company has chosen to produce medicines from standardized extracts. This paper describes the propagation and processing of the company's cannabis. It also includes some description of cannabis anatomy and biochemistry, as this is necessary to explain the reasoning behind the growing methods.
Production of botanical medicines
In the vast majority of cases, plants grown for the pharmaceutical industry are intended for the extraction and purification of a single active pharmaceutical ingredient (API). Of GW Pharmaceuticals’ range of available cannabinoids, several show evidence of individual therapeutic potential, and these may eventually appear in medicines containing a single API. However, its medicine Sativex® is very different, this being a complex botanical drug, i.e. a well-characterized, multi-component standardized medicine extracted from plant sources.
When applying for permission to market this product in Europe, a Summary of Product Characteristics (SmPC) had to be submitted. For this medicine it stated that:
Each mL contains 38–44 mg and 35–42 mg of two extracts (as soft extracts) from Cannabis sativa L., folium cum flore (Cannabis leaf and flower) corresponding to 27 mg delta-9-tetrahydrocannabinol and 25 mg cannabidiol.
In addition to a number of cannabinoids, Sativex® also contains a range of other naturally occurring cannabis-derived terpenoids. Several of these are pharmacologically active. Many are considered to act synergistically, with the principal cannabinoids, in certain therapeutic applications.[12, 13] By growing the feedstock for this medicine in a glasshouse, in stable conditions, a consistent ratio of ingredients can be produced. Uniformity is especially important when synergists are involved, as relatively small variations in the ratios can have large effects on the overall activity.
The concentration and profile of pharmaceutically active ingredients derived from cannabis are affected by a range of factors, including its genetics, the plant part(s) used, the growth stage at harvest, the growing conditions, the manner of drying and storage, and the methods of processing (decarboxylation) and extraction. These are discussed in turn.
Genetics exert the greatest control of a plant's cannabinoid profile. This opens exciting possibilities for the plant breeder. Of the various cannabinoids in cannabis, Δ9–tetrahydrocannabinol (THC) and cannabidiol (CBD) are the most common. In fresh plant material these exist in the cannabinoid acid forms, Δ9–tetrahydrocannabidiolic acid (THCA) and cannabidiolic acid (CBDA). As the plant material ages, or is heated, the acid molecules lose a carboxyl moiety. Decarboxylation results in the conversion of the cannabinoid acids into their neutral forms (e.g. CBDA CBD). As is common, this paper hereafter refers to the cannabinoids in their neutral form only.
Compelling research performed by de Meijer et al. supports a hypothesis that the ability of a plant to efficiently produce THC and/or CBD is governed by the inheritance of either of two co-dominant genes, with the proposed names BT and BD. According to this hypothesis, a proportion of plants in a natural cannabis population will have inherited a BT gene from each parent, and these homozygous BTBT plants will produce an enzyme THC synthase. This enables them to biosynthesize THC in quantity, while producing CBD at near undetectable levels. Others will have only inherited the BD gene, and these homozygous BDBD genotypes will produce an enzyme CBD synthase, enabling them to efficiently produce CBD, and minimal THC. A third, heterozygous BTBD category will have inherited one of each gene, and will produce both enzymes, resulting in them biosynthesising a more even mixture of THC and CBD. However, in a natural setting, large variations of ratios between these two cannabinoids are found in heterozygous siblings of any one parental cross. Growing conditions can also introduce variations in THC:CBD ratio of heterozygous chemotypes. This is illustrated in Table 1, which compares the THC:CBD ratio in genetically identical batches of plants, grown in a glasshouse and outdoor UK environment. These were grown from cuttings, and originated from a single packet of seeds, retailed under the name Early Pearl. In this instance, glasshouse growing favoured THC biosynthesis.
Table 1. The relative proportions of THC and CBD synthesized in five heterozygous BTBD genotypes (n = 4 plants per genotype) derived from seed retailed as cv. Early Pearl. (*two-tailed t-test, p < 0.001)
| ||THC as % of THC + CBD|
|Field||33.1 ± 2.4||33.2 ± 0.2||48.9 ± 1.9||33.9 ± 0.0||33.3 ± 0.1||36.5|
|Glasshouse||38.8 ± 1.5||39.9 ± 0.6||57.4 ± 0.9||40.6 ± 0.6||38.6 ± 2.0||43.1*|
Both THC and CBD are synthesized from the precursor CBG (cannabigerol), which is a cannabinoid with its own pharmacology. Another cannabinoid called cannabichromene (CBC) is also produced from CBG in smaller quantities, and proportionally more of this is found in juvenile tissue.
Only rarely do natural sources of cannabis contain molecules other than THC and CBD as the dominant cannabinoid. An example was the discovery of a hemp cannabis plant having CBG as the main cannabinoid. Large variations in terpene content within cannabis populations have also been shown to be genetically linked.[19-22] Chemotypes have been bred that are almost totally devoid of cannabinoids, but still retain varying arrays of other terpenes.
Naming of parts and terpenoid biosynthesis
Cannabis is generally an annual dioecious species. Male plants are relatively short lived, their final few weeks being devoted to maximum pollen production. In a natural setting the females last longer, supporting the development of abundant fertile seeds. The cannabinoids and other terpenes are found in varying ratios in all aerial parts of the species, but female flowers are the main source of most cannabinoids. These flowers are clustered together in large numbers in indeterminate inflorescences. These, by definition, are flower clusters within which the tips never produce a true terminal flower. If grown in the absence of pollen, the flowering period of the resultant seed-free female cannabis (sinsemilla) is extended. New flowers continue to develop and unnaturally large inflorescences are formed (Figure 1). Within these unpollinated inflorescences cannabinoid biosynthesis is advantageously prolonged. Modern plant breeding techniques have made it possible to breed new parental crosses where both parents are female. Male cannabis plants now play little or no part at GW Pharmaceuticals in cannabinoid production or breeding.
Before giving an account of the ways of optimising cannabis growing systems for pharmaceutical applications, it is perhaps helpful to report when, where and possibly why the cannabinoids are synthesized.
The cannabinoids are examples of secondary metabolites, which are defined as organic compounds not directly involved in the normal structural growth, development, or reproduction of an organism. It is widely accepted that these cannabinoids are predominantly, if not entirely, synthesized and sequestered in microscopic structures called capitate or glandular trichomes. Most of the essential oils (monoterpenes and sesquiterpenes) found in cannabis are also located in these structures. Trichomes appear to play a defensive role, protecting the plant against herbivores and environmental stresses. The cannabinoids and accompanying essential oils play a vital part in this. Plants typically defend the parts of the structure of greatest importance in species survival, and in cannabis, like many species, these are the reproductive tissues. Hence, this is where the majority of glandular trichomes are found.
Plants throughout the Plant Kingdom have been said to face the dilemma, whether to grow or defend. Those plants that invest heavily in biosynthesis of defensive secondary metabolites predictably have less remaining energy to synthesize the primary metabolites required for vigorous vegetative growth, which can be vital in the competitive struggle for light, water and mineral nutrients. Within a natural population of siblings, plants will vary widely in the balance of energy allocated to primary or secondary metabolite synthesis. This variation is itself a useful defence strategy, as the optimum ratio would vary from place to place and season to season according to competitive, predatory and environmental pressures. In the hands of the plant breeder, the degree of variation may be reduced but some will remain.
GW Pharmaceuticals has routinely sought genotypes with the maximum yield or purity of a secondary metabolite. Of these, genotypes with unfavourable traits (e.g. disease susceptibility, excessive height, hermaphrodite tendencies) have been rejected. From the most appropriate parental crosses, the highest performers have been identified, and cuttings taken from them. The propagation of cannabis from cuttings guarantees the genetic uniformity of all plants produced from the same source. As expected, when seedlings from a single parent cross were compared to those derived from cuttings of a single genotype, the latter has shown significantly improved uniformity. Propagation from cuttings remains the favoured option.
Growth stage, trichome distribution, and changing cannabinoid profile
Three forms of so-called glandular trichome are found on female cannabis; viz. capitate sessile (Figure 2), capitate stalked (Figure 3), and bulbous (Figure 4). This last type is thought to have a minimal role in secondary metabolite production. The capitate sessile and capitate stalked trichomes both develop a glandular head (or resin head) incorporating a disc of secretory cells at the base. Above the secretory cells, and below the trichome's outer membrane, is a chamber within which the secretory cells sequester a resinous mixture that includes cannabinoids and essential oils. Capitate sessile trichomes are found on all aerial parts of the plant, and due to their relatively small size and number, they only make a modest contribution to the overall cannabinoid content of the plant. Capitate stalked trichomes are much larger, and are found in dense populations on the floral tissue and immediately surrounding bracts, as shown in Figure 5. (Bracts are specialized leaves, only found within the inflorescence.)
Capitate sessile and stalked trichomes differ significantly in their cannabinoid profile and yield. Their monoterpene and sesquiterpene profiles also differ greatly. As a result of the differing populations of sessile and stalked trichomes, floral tissue and foliage vary greatly in secondary metabolite content. Perhaps the most important feature is the much higher cannabinoid content associated with the innermost inflorescence material.
The cannabinoid profile undergoes rapid changes in the early phases of plant growth, (as reviewed by de Meijer et al.). The enzymes synthesising THC or CBD have a very similar turn-over rate (kcat), and affinity (Km) for their substrate. However, the properties described for CBC synthase were quite different, this enzyme showing a lower Km (23 μM instead of 134 and 137 μM of the other two synthases) and a lower turnover number (kcat = 0.04 s-1 against 0.19 s-1 and 0.20 s-1 of CBD and THC synthase). In the early growth stages, when CBG (the precursor of THC, CBD and CBC synthesis) is in short supply, CBC synthesis is most efficient. However, as CBG concentration increases, so does the efficiency of THC and CBD biosynthesis, soon exceeding that of CBC. In the later stages of plant growth, CBG synthesis slows and its relative proportion within the cannabinoid profile decreases. The ratio of THC to CBD in heterozygous BTBD plants can prove unstable, being affected by plant age as well as growing conditions. Variation in THC and CBD ratio in Sativex® is avoided by including only the homozygous chemotypes in the formulation. These are routinely harvested after a fixed period of growth.
Provision of stable bright lighting conditions is vitally important to the production of uniform, cannabinoid-rich cannabis. Supplementary lighting within the GW Pharmaceuticals’ glasshouses raises irradiance levels whenever natural lighting conditions are below a tolerable minimum. During winter, the lamps operate permanently during the day. Over the course of one year, approximately half the light energy within the glasshouse is from electric lamps.
Few herbaceous plant species produce higher concentrations of secondary metabolites than cannabis. Terpenoid molecules, like those in cannabis, are more expensive per gram to biosynthesize than most other carbon-based secondary metabolites, requiring up to three times more energy per unit weight than glucose. The combination of high secondary metabolite concentration, and high biosynthetic energy demand, means that cannabis must have access to abundant light energy, to achieve sufficient levels of photosynthesis. This has been achieved by the installation of a supplementary lighting system that delivers 55 W m-2 photosynthetically active radiation. This is well above the maximum levels typically found in UK glasshouses producing food or ornamental crops.
Photosynthesis and resultant growth is markedly affected by temperature. Cannabis plants originating from different agro-climatic zones worldwide exhibit varying optimum temperatures for photosynthesis, this ranging between 25°C and 35°C. Insect pests also have an optimum. These factors, as well as staff welfare, dictate the glasshouse growing conditions.
All plants at GW Pharmaceuticals are grown in pots and hand-watered until roots are established. Thereafter watering is automated. Hydroponic growing systems were initially considered. These by definition grow plants without the use of soil, peat or a similar fertile substrate. Instead, roots are supported in an inert medium (e.g. gravel, glass fibre or expanded clay pebbles), or suspended in a liquid, with nutrients steadily added. (Ancient Greek derivation of hydroponic; hydro = water, Ponos = god of hard labour and toil). Hydroponic growing systems were rejected as overly complicated and hard work. Such systems do not appear to increase cannabis productivity or potency.
Induction and maintenance of flowering
Cannabis is generally described as a short-day plant. Such species naturally commence flower formation in autumn, when specialized photoreceptor proteins called phytochromes induce a response to the seasonal increase in night length. Indoor cannabis growers manipulate this response and generally commence plant growth in a long day length of between 18 and 24 h per day. It is an almost ubiquitous and well-documented practise to then induce and maintain flowering by switching the day length to a regime (photoperiod) of 12-h days and 12-h nights. This artificial autumn equinox is achieved in summer by closing the glasshouse blinds to exclude ingress of natural lighting for more than 12 h/day. Conversely, in winter, lamps extend the naturally dark days.
About a week after the reduction in day length, the first flowers are visible. Stem and foliar development slows and has all but stopped after three weeks in short days. However floral development continues apace, only showing signs of slowing after about eight weeks. A survey of 200 cannabis varieties showed that 88% had a recommended growth period in short day length of seven to nine weeks and most GW Pharmaceuticals’ genotypes, including those for Sativex® production, fall within this range. The glasshouse bench is typically restocked with the next crop within 24 h of harvest. Benches in the flowering zone can thus host over six crops a year.
The development and changing floral THC content of Sativex® THC plants during the flowering process is shown in Figures 6 and 7. These report the yields of experimental plants harvested at weekly intervals, after the switch to a 12-h regime. Most commercial cannabis varieties tested have shown a similar pattern. Once sufficiently developed (after three weeks in short days), foliar and floral tissues were separated and assessed for THC content (the foliar fraction included outer non-resinous bract material). The figures show that the potency (THC concentration) of the floral tissue was fairly stable through the flowering process, but yield increased steadily. THC chemotype plants for Sativex® production are normally harvested after eight weeks in short day length when, like those in Figure 6, they typically produce an average yield of 400 g m-2 of floral material combined with 200 g m-2 of foliage. This is a little lower than the 500 g m-2 of floral typically achieved in an indoor growth room setting where electrical consumption is higher. However, as stated earlier, yield is less important than crop uniformity and quality. Those plants in Figure 6 contained approximately 14% and 2% THC, respectively. The foliage therefore contained only 7% of the total cannabinoid content. Before being supplied to licensed dispensaries in the USA or the Netherlands, cannabis foliage would normally be removed and discarded. For Sativex® it is retained and the active ingredients extracted.
Figure 6. The mean weight of foliar and floral material (+/− sd, n = 4) in batches of plants (Sativex® THC chemotype) harvested between 0 and 9 weeks after placement in 12-h day length.
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Figure 7. The THC concentration (+/− sd, n = 4) of manicured inflorescence material from plants (Sativex® THC chemotype) sampled at weekly intervals, four to nine weeks after placement in short days.
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Pest and disease control
The Good Agricultural Practise Guidelines, which dictate quality standards in medicinal crop growing and processing, permit limited pesticide use. GW Pharmaceuticals has avoided this so far by a dual approach of prevention and cure. Rigorous cleaning regimes are routine and growing conditions that favour infections and infestations are avoided. Those pests that do arrive are dealt with by beneficial insects and mites that eat or parasitize the invader. Plants not meeting required quality standards are destroyed.
Drying and storage
To minimize bacterial and fungal spoilage, crops are cut at the base and promptly dried in warm, dehumidified and ventilated conditions. When grown for illicit use, consumers increasingly demand material only containing unpollinated female floral material. As stated earlier, although the secondary metabolite content of the foliage contributes relatively little, GW Pharmaceuticals reaps both the floral and foliar tissue to make Sativex®. This flower and leaf mixture is referred to as the botanical raw material (BRM). The discarded stems are shredded and composted. This BRM is milled and frozen prior to processing.
The conversion of the cannabinoid acids (THCA, CBDA, etc.) into neutral cannabinoids requires the removal of the carboxyl group. This is achieved by uniformly heating the dried material. Controlled conditions and timings ensure that this is adequately achieved.
Feedstock quality control
Sativex® has to exceed minimum quality standards, which apply to all phases of drug production, commencing with the growing process. A computerized glasshouse management system aims to provide fixed irradiance levels and temperatures. It also monitors and records the conditions. Rejection of the BRM may result from conditions falling outside set limits. In order to achieve a fully standardized formulation, GW Pharmaceuticals employs a range of analytical technologies to demonstrate batch-to-batch uniformity. As a result of these technologies, GW is able to standardize the formulation across the extracts as a whole, not simply by reference to their key active components.
Accurate analysis of cannabis plants for cannabinoid content is exceedingly difficult. As found in forensic studies, cannabis is an extremely inhomogeneous material. Even within one plant, the potency of the floral material will vary widely. Because of this, when herbal cannabis samples supplied to Dutch dispensaries have been analyzed, THC levels in any one product (based upon a single variety) have been found to vary by several per cent. This variability is recognized by the Office for Medicinal Cannabis (OMC), the Netherlands government office responsible for the production of cannabis for medical and scientific purposes. The herbal cannabis material Bedrocan, which is supplied to Dutch dispensaries, is described by the OMC as containing 18% THC (specifications: 15.5–21.0%). Another herbal material, Bediol, is described as containing approximately 6% THC and approximately 7.5% CBD. The plant material grown for Sativex® manufacture predictably exhibits variability in content. A limited range of analytical tests are performed on the plant material. However, when the feedstock is converted into an extract, the resultant BDS is a much more uniform product. This undergoes a detailed analysis, quantifying a large range of ingredients.[35, 36] Further tests are performed on the final formulated botanical drug product (BDP) using reverse phase high performance liquid chromatography (HPLC). This incorporates a C18 analytical column with and ultra-violet detection at 220 nm. The method can simultaneously quantify both acid and neutral cannabinoids.
Production of the botanical drug substance and botanical drug product (BDP)
To produce a cannabis extract, batches of dried plant material are immersed in liquid carbon dioxide at extremely high pressure. The ingredients dissolving in this solvent are then separated and purified.
Sativex® is formulated by incorporating BDSs containing THC and CBD in an accurately measured ratio. The only incipients are ethanol, propylene glycol and peppermint oil, the latter being added to improve palatability.
By blending two BDSs, a uniform THC:CBD ratio is assured. As reported by Robson, in this copy of the journal, one reason why the cannabinoid-based medicines first introduced to the west by William O'Shaughnessey met decline, was variability in potency of the product. O'Shaughnessy's medicines used hashish (cannabis resin) as a starter material. This is a highly variable product, the THC:CBD ratio being affected by many factors, but most importantly the genetics of the plants used to make it. THC is much less stable than CBD, and so with time the THC:CBD ratio within the material decreases and conversely increasing quantities of the THC degradant cannabinol (CBN) are formed. CBN is a cannabinoid with its own pharmacology. Whilst the cannabinoid ratio within the original hashish-derived products can only be guessed, the likely variability can be appreciated by looking at the ratio of THC, CBD, and CBN in modern resin. Figure 8 shows the ratios in samples of resin seized by police in England in 2004/2005.[16, 38] This highly variable product is what many patients in the UK have relied upon, in the absence of a prescribed cannabinoid-based medicine. (In this study, the resin samples were analyzed by gas chromatography, as described by de Meijer et al. This technique decarboxylates cannabinoid acids, so all cannabinoids are shown in their neutral forms only.
Production of single API medicines
A complex medicine like Sativex® is markedly different to another cannabinoid-based medicine – Marinol. This contains just one active ingredient – synthetic THC, and this is formulated as a capsule in sesame seed oil for oral administration. Medicines containing a single cannabinoid remain a distinct future possibility for GW Pharmaceuticals. For these, uniform growing conditions will be less of a necessity.
Specific chemotypes (chemical genotypes) have been specifically bred to supply GW Pharmaceuticals with high yields of the rarer cannabinoids, as well as THC and CBD. These include chemotypes where the dominant cannabinoid is CBG or CBC. The production of a chemotype dominant in tetrahydrocannabivarin (THCV) was achieved using plants that produced cannabigerivarin (CBGV) rather than CBG as the main intermediate cannabinoid. Chemotypes dominant in CBGV, cannabidivarin and cannabichromivarin are under development at GW Pharmaceuticals. A range of diseases are seen to respond favourably to these cannabinoids and an era of promising drug development has arrived.