EXTRACTION OF PHARMACEUTICALLY ACTIVE COMPONENTS FROM PLANT MATERIALS

Abstract

The invention relates to the extraction of pharmaceutically active components from plant materials, and more particularly to the preparation of a botanical drug substance (BDS) for incorporation in to a medicament. It also relates to a BDS of given purity, for use in pharmaceutical formulations. In particular it relates to BDS comprising cannabinoids obtained by extraction from cannabis.

Claims

1. (canceled)

2. A botanical drug substance obtained under subcritical conditions from botanical raw material from a high CBD containing cannabis plant, wherein said botanical drug substance is an extract derived from the high CBD cannabis plant by extracting with liquid CO2 and comprising at least 60% cannabinoid constituents together with non-cannabinoid constituents, wherein the cannabinoid constituents comprise at least 90% CBD and the non-cannabinoid constituents comprise terpenes, and reduced levels of hydrocarbon and triglyceride waxes and plant pigments, and wherein the extraction with liquid CO2 is conducted under subcritical conditions at a temperature in the range of from 5 to 15° C. and a pressure in the range of from 50 to 70 bar.

3. The botanical drug substance as claimed in claim 2 comprising no more than 4 ppb aflatoxin.

4. The botanical drug substance as claimed in claim 2 comprising no more than 20 ppm total heavy metals.

5. The botanical drug substance as claimed in claim 2 comprising no more than 15% w/w residual solvents.

6. The botanical drug substance as claimed in claim 5 wherein the residual solvent is ethanol.

7. The botanical drug substance as claimed in claim 2 comprising no more than 10.sup.5 cfu/g Total Viable Count (TVC), no more than 10.sup.4 cfu/g fungi, no more than 10.sup.3 cfu/g enterobacteria and other non gram negative organisms, and no detectable E. coli, Salmonella or S. aureus.

8. (canceled)

9. A botanical drug substance obtained from cannabis comprising at least 60% cannabinoids of which at least 85% is CBD, about 3% is THC and the remainder comprises other minor cannabinoids.

10. The botanical drug substance as claimed in claim 9, further mixed with a botanical drug substance obtained from cannabis comprising at least 60% cannabinoids of which at least 90% is THC, about 1.5% is CBD and the remainder comprises other minor cannabinoids.

11. A pharmaceutical formulation comprising the botanical drug substance of claim 2.

12. A pharmaceutical formulation comprising the botanical drug substance of claim 3.

13. A pharmaceutical formulation comprising the botanical drug substance of claim 4.

14. A pharmaceutical formulation comprising the botanical drug substance of claim 5.

15. A pharmaceutical formulation comprising the botanical drug substance of claim 6.

16. A pharmaceutical formulation comprising the botanical drug substance of claim 7.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0078] FIG. 1 illustrates loss of THC over time at 40° C. for standard THC botanical drug substance (BDS) and activated charcoal-treated THC BDS (purified BDS). Y-axis: amount of THC (expressed as percentage of t0 value), x-axis: time in months.

[0079] FIG. 2 illustrates loss of CBD over time at 40° C. for standard CBD botanical drug substance (BDS) and activated charcoal-treated CBD BDS (purified BDS). Y-axis: amount of CBD (expressed as percentage of t0 value), x-axis: time in months.

[0080] FIG. 3 illustrates formation of cannabinol (CBN) over time at 40° C. for standard THC botanical drug substance (BDS) and activated charcoal-treated THC BDS (purified BDS). Y-axis: amount of CBN (expressed as percentage of t0 value), x-axis: time in months.

EXAMPLES

Example 1—Development of a Process for Extraction of Cannabinoids from Cannabis Plants

Selection of Cannabis Chemovars

[0081] GW Pharma Ltd has developed distinct varieties of Cannabis plant hybrids to maximise the output of the specific chemical constituents, cannabinoids. Two types of plant are used; one chemovar produces primarily THC and a further chemovar produces predominately CBD. However alternative varieties can be obtained—see for example, Common cannabinoids phenotypes in 350 stocks of cannabis, Small and Beckstead, LLoydia vol 36b, 1973 p 144-156—and bred using techniques well known to the skilled man to maximise cannabinoid content.

[0082] Chemical and structural similarities exist between THC and CBD. Due to these similarities together with the botanic origin of the starting materials, each can be considered to be interchangeable with respect to the development of processes for extraction of cannabinoids.

[0083] Preferably, each Cannabis chemovar is processed and controlled separately to yield two distinct BDS's. However, it is possible to mix plant material from two or more chemovars or use a variety which will produce the desired ratio of given cannabinoids prior to extraction, and thus prepare a single BDS.

Production of Botanical Raw Material

[0084] BDS is prepared from extracts of Cannabis sativa L. (family Cannabidaceae). Cannabis sativa was described in the 1934 British Pharmacopoeia. Cannabis is grown under United Kingdom Home Office licence under the control of GW Pharma Ltd in the United Kingdom. Growing facilities are equipped with shades and full climatic control (temperature, humidity and high intensity lighting) so that several crops per year can be produced in almost identical growing conditions thus ensuring continuity of supply.

Cultivation:

[0085] Cannabis plants are propagated from cuttings taken from the mother plants, originating from a single seed source. Therefore a crop is produced through asexual propagation where the plants are all female. Propagation using cuttings controls genotype consistency.

[0086] The cuttings are rooted in compost supplied as pesticide free. The plants are watered and sustained release fertilizer is applied during the growing cycle. Through controlled growing conditions the plants take approximately 12 weeks to reach maturity.

[0087] The plants are irrigated throughout their growing cycle with potable quality water.

[0088] No synthetic herbicides or pesticides are used in the cultivation of Cannabis plants.

Compost:

[0089] Efficient cultivation of Cannabis necessitates the supply of a reliably uniform growing media.

[0090] The compost provides a soft texture, high air porosity, ready wetting, low conductivity and balanced nutrient supply.

The compost consists of peat and added natural minerals including lime (magnesium and calcium carbonates) to provide pH control of the compost during the growing cycle of the Cannabis plants.

[0091] The compost contains an adequate supply of essential minerals and a minimum of minerals with known adverse effects on the plants. Some minerals including manganese can be present in an insoluble form in compost and be released in a freely soluble form over time. Controlling compost pH and monitoring irrigation to avoid waterlogging will control soluble manganese levels. Compost pH is maintained above 5.5.

[0092] The compost is declared as pesticide free, as no pesticides or herbicides are added.

Fertiliser:

[0093] The compost contains fertiliser identifiable in two discrete forms, a base fertiliser and a slow release fertiliser. Additional slow release fertiliser is applied to the plants during growing.

Disease and Pest Control:

[0094] No artificial herbicides or pesticides are used during cultivation. Stringent hygiene conditions reduce ingress of pests and diseases.

[0095] By controlling the growing conditions, environmental stresses such as drought, insufficient light and unfavourable temperatures reduces the risk of disease.

[0096] Regular inspection of the plants during the growing cycle allows for the detection of any rogue plants and pests. Rogue male plants may arise, though weeds should be absent due to the controlled growing conditions and media. Frequent inspections and biological control methods are used to manage any pests and diseases that may occur.

Plant Collection:

[0097] Through strict control of growing conditions the Cannabis plants reach maturity in approximately 12 weeks. In the last weeks of growth, dense resinous flowers develop. By the end of approximately week 11 the cannabinoid biosynthesis has slowed markedly, and the plants are ready for harvest.

[0098] The entire plant is cut and dried in a temperature and humidity controlled environment.

[0099] Approximately 21° C.

[0100] Approximately 38-45% RH.

Dried plant is physically assessed for end-point.

[0101] THC and CBD are the principle bioactive constituents in the BDS. However, these constituents are present as biologically inactive carboxylic acids in the BRM.

[0102] THCA

[0103] CBDA

The acid forms slowly decarboxylate over time during drying. The leaves and flowers are stripped from the larger stems to provide the Botanical Raw Material (BRM).

Storage of BRM:

[0104] Under conditions of storage the loss on drying reaches equilibrium of approximately 10%. The storage conditions for the dried BRM will be dependent on the physical status of the BRM.

General storage conditions for BRM:

[0105] Protected from light.

[0106] Approximately 15-25° C. or −20° C.

[0107] Approximately 38-42% RH.

Summary-production of a BRM:
Typical BRM specification derived from a high CBD variety is illustrated in Table 2:

TABLE-US-00002 Test Method Specification Identification: A Visual Complies B TLC Corresponds to standard (for CBD & CBDA) C HPLC/UV Positive for CBDA Assay: In-house Not less than (NLT) 90% of CBDA + CBD (HPLC/UV) assayed cannabinoids by peak area Loss on Drying: Ph. Eur. Not more than (NMT) 15% Aflatoxin: UKAS* method NMT 4 ppb Microbial: Ph. Eur. TVC NMT 10.sup.7 cfu/g Fungi NMT 10.sup.5 cfu/g E. coli NMT 10.sup.2 cfu/g Foreign Matter: Ph. Eur. NMT 2% Residual Ph. Eur. Complies Herbicides and Pesticides: *United Kingdom Accreditation Service

Analytical Methods:

Identification by Visual:

[0108] Macroscopic characteristics allow the features of the Cannabis plant to be distinguished from potential adulterants and substitutes. It is a visual identification against a photographic standard.

Identification by TLC:

[0109] TLC uses both retention time and characteristic spot colour to effectively identify the variety of Cannabis. Laboratory samples are prepared for TLC analysis by extracting the dried herb. An aliquot is spotted onto a TLC plate, alongside reference samples for THC and CBD. Following exposure to Fast Blue B reagent, THC and THCA present as pink spots, while CBD and CBDA are orange in colour. Neutrals can be distinguished from the acids by comparison of the Rf value to that obtained for the standards. Identity is confirmed by comparison of Rf and colour of the sample spot, to that obtained for the appropriate standard.

Identification by HPLC:

[0110] HPLC uses retention time comparison of cannabinoids to effectively identify the variety of Cannabis. The reversed phase HPLC method is specific for CBD and CBDA, and therefore may be used as an identity test. Samples of biomass are extracted and centrifuged. Detection of all analytes is accomplished at 220 nm with additional confirmation of acidic analytes at 310 nm.

Assay (CBD+CBDA):

[0111] This assay is used to monitor the CBD and CBDA content in the plant. CBD and CBDA assay are determined using an HPLC method.

[0112] The efficiency of the decarboxylation process is determined by dividing the % content in terms of w/w of CBD by the total CBD+CBDA content.

Loss on Drying:

[0113] Loss on Drying is evaluated using Ph.Eur. test method.

Aflatoxin:

[0114] Aflatoxin is analysed using a United Kingdom Accreditation Service (UKAS) accredited method.

Microbial:

[0115] Microbiological quality is determined using Ph.Eur. methodology.

Foreign Matter:

[0116] Foreign Matter is evaluated using the Ph.Eur. test method. Flowers, leaves and side stems are spread out in a thin layer on a clean laboratory surface. Foreign Matter is separated by hand as completely as possible, and is weighed. Results are expressed as % w/w of Foreign Matter in the herbal biomass sample. Foreign Matter may comprise no more than 2% of the biomass.

Residual Herbicides and Pesticides:

[0117] The Cannabis plants are grown in a well controlled environment. No artificial herbicides or pesticides are used or needed during cultivation.

[0118] An equivalent BRM specification (compare table 2) is derived for a high THC variety and identical analytical methods followed, except that THC/THCA replaces CBD/CBDA.

Decarboxylation

[0119] THC and CBD are the principle bioactive constituents in Cannabis. However, these constituents are present as the biologically inactive carboxylic acids in Cannabis plants. In order to extract THC or CBD from cannabis plant material, it is necessary to convert the storage precursor compounds of THCA and CBDA into their more readily extractable and pharmacologically active forms. THC and CBD acids slowly decarboxylate naturally over time. The traditional way to increase rate of decarboxylation is by the application of heat. However, THCA is converted not only to THC, but also to another cannabinoid, cannabinol (CBN).

##STR00001##

[0120] The decarboxylation procedure is generally carried out within the preparation of the starting material or botanical raw material (BRM), prior to the initiation of the extraction process.

Laboratory Studies-Decarboxylation

[0121] Portions of milled dried plant material were subjected to heat (approximately 0.25 g with particle size 1-2 mm). A pilot scale experimental system was set up, with the objective of determining parameters for the optimal conversion of THCA or CBDA into THC and CBD respectively, with concomitant minimal loss of these ensuing compounds into their thermal degradation products, in the case of THC the formation of CBN.

Brief Description of Materials and Methods:

[0122] Portions (0.25 g) of milled (approximately 1-2 mm particle size) of both THCA and CBDA herbal materials were placed in 20-ml glass headspace vials and the vials sealed tightly with crimp capped Teflon-faced butyl rubber seals. Sealed vials were heated at one of three temperatures, for periods of up to 4 hrs as follows:

105° C., 120° C., 140° C. for 0.5, 1.0, 2.0 and 4.0 hours.

[0123] The heating was performed in an oven with forced air circulation. Oven conditions were shown to be accurate to within 0.5-1.0 degree at the three temperatures used.

[0124] After the heating process was complete representative samples of the decarboxylated herb were assayed using HPLC, GC and TLC techniques. Standards of THC, CBD and CBN were include in the HPLC and GC sequences.

Results and Discussions:

[0125] HPLC analysis of the solvent extracts was able to demonstrate the disappearance of either CBDA or THCA as a function of time at the two lower temperatures. At 140° C., the earliest time point samples at 0.5 hour contained only very modest levels of a peak eluting at the retention times of CBDA or THCA.

[0126] Tables 3 and 4 present HPLC data quantifying the conversion of CBDA or THCA into the free compounds; also presented is data showing the content of CBD or THC and the ratio of CBD/CBDA+CBD or THC/THCA+THC. The conversion of the carboxylic acid forms to the corresponding decarboxylated form can be monitored by comparing the decarboxylated/decarboxylated plus un-decarboxylated ratio with the absolute content of the decarboxylated compounds. Thus, when the ratio reaches a maximum value (>0.95), the earliest time/temperature point at which the content of THC or CBD is also maximal, should be optimal for the conversion process.

[0127] Thus, for CBD containing herb, 1 hour at 120° C. or 0.5 hour at 140° C., was appropriate.

[0128] This is confirmed by examination of the TLC chromatogram for the solvent extracts, CBDA is absent after 1 hour at 120° C. or at any time point at 140° C.

[0129] For THC there is a third criterion, formation of CBN, where it is desirable to minimise the formation of this compound during the thermal decarboxylation process. Table 5 provides Gas Chromatography (GC) data where a CBN/THC ratio can be derived. Taken into consideration, alongside the THC/THCA+THC ratio and the maximum THC content, minimal CBN formation occurs after 0.5 or 1.0 hour at 120° C. At 140° C., even 0.5 hour gives a higher content of CBN than either of the two lower time/temperature points.

[0130] Therefore laboratory studies demonstrate the optimum conditions for the decarboxylation of:

[0131] Chemovar producing primarily CBD is 1 hour at 120° C. or 0.5 hour at 140° C.

[0132] Chemovar producing primarily THC to minimise CBN formation, is 1 to 2 hours at 105° C. or 1 hour at 120° C.

Thin layer chromatography reveals that virtually all of the THCA has disappeared after 4 hours at 105° C. and after 1 hour at 120° C. No THCA is visible at any time point when the herb is heated at 140° C. A small amount of residual staining at this retention value on TLC and the presence at low levels of a peak coincident with THCA on HPLC analysis may indicate the presence of a minor cannabinoid rather than residual THCA.

TABLE-US-00003 TABLE 3 HPLC Data from Decarboxylation of CBDA Herbal Material CBD peak Time CBD/CBD + area/0.1 g Temperature (hours) CBDA of herb 105° C. Zero 0.15 4769 0.5 0.22 5262 1.0 0.86 5598 2.0 0.93 5251 4.0 0.98 5242 120° C. 0.5 0.91 5129 1.0 0.97 5217 2.0 0.99 5037 4.0 1.00 5200 140° 0.5 0.96 5440 1.0 1.00 5105 2.0 1.00 5157 4.0 1.00 5005

TABLE-US-00004 TABLE 4 HPLC Data from Decarboxylation of THCA Herbal Material THC peak Time THC/THC + area/0.1 g Temperature (hours) THCA of herb 105° C. Zero 0.17 992.9 0.5 0.87 5749 1.0 0.93 5273 2.0 0.98 7734 4.0 0.99 7068 120° C. 0.5 0.97 7189 1.0 0.99 6391 2.0 0.99 6500 4.0 1.00 5870 140° C. 0.5 1.00 6724 1.0 1.00 5981 2.0 1.00 5361 4.0 1.00 4787

TABLE-US-00005 TABLE 5 GC Data from Decarboxylation of THC Herbal Material Temperature Time (hours) CBN/THC (%) 105° C. Zero 2.4 0.5 3.5 1.0 4.2 2.0 3.7 4.0 5.6 120° 0.5 3.2 1.0 4.1 2.0 6.7 4.0 11.3 140° C. 0.5 5.7 1.0 13.0 2.0 17.5 4.0 23.8

[0133] The decarboxylation conditions for a batch scale of about 4 kg of botanical raw material (BRM) are as follows:

[0134] Approximately 4 kg of milled BRM (either THCA or CBDA) to be decarboxylated was initially heated to 105° C. and held at this temperature for about 15 minutes to evaporate off any retained water and to allow uniform heating of the BRM. The batch was then further heated to 145° C. and held at this temperature for 45 minutes to allow decarboxylation to be completed to greater than 95% efficiency.

[0135] The heating time for CBDA BRM was extended to 55 minutes at 145° C. as it became apparent from results that CBDA was slightly more resistant to decarboxylation than THCA. This difference between CBD and THC would be even more pronounced at commercial scale batches. The THC BRM heating time was retained at 145° C. for 45 minutes.

[0136] The conditions used in pilot scale closely reflect those conditions determined as optimal from the laboratory studies. The differences can be explained by slower and less efficient heat transfer via the containers and through the BRM at the increased batch size for the pilot scale.

[0137] Tables 6 and 7 provide data to demonstrate the efficiency of decarboxylation measured in terms of content of the biologically active cannabinoid, THC or CBD.

TABLE-US-00006 TABLE 6 Decarboxylation Efficiency for CBD BRM Batch % Efficiency of Number Decarboxylation CBD Specification >95% A 98.8 B 99.5 C 98.3 D 100.0 E 100.0 F 100 G 96.9 H 100.0

[0138] Increase in batch size of CBD BRM from approximately 4 kg to 6 kg resulted in a need to increase decarboxylation time. The decarboxylation time at 145° C. was increased from 55 minutes to 90 minutes.

TABLE-US-00007 TABLE 7 Batch % Efficiency of Number Decarboxylation THC Specification >95% I 99.4 J 97.3 K 98.5 L 100.0 M 97.8 N 99.9 O 100.0

Overview of Extraction Process:

[0139] The BDS is extracted from decarboxylated BRM using liquid carbon dioxide methodology. This involves continuously passing liquefied carbon dioxide through the chopped biomass, which is contained in a high-pressure vessel. The crude extract is dissolved in ethanol, cooled to a low temperature then filtered to remove precipitated constituents such as waxes. Removing ethanol and water in vacuo produces BDS containing either high concentrations of CBD or THC, depending on the biomass used.

Flow diagram of typical extraction process:

Extraction No. 1

[0140] The first stage in the manufacturing process is Extraction using liquid CO.sub.2 under sub-critical conditions.

[0141] Experiments indicated that both THC and CBD could be extracted from Cannabis plant material in high efficiency using sub-critical CO.sub.2 at low temperature, of approximately 10° C.±5° C. using a pressure of approximately 60 bar±10 bar.

The Table 8 below shows comparative data generated for a BDS rich in THC

TABLE-US-00008 Temp % w/w wax % thc w/w post Charge No Pressure bar ° C. removed winterisation Ac1202 400 60 8.2 67.2 Ac1205 400 60 6.1 67.0 Ac1206 400 60 6.1 68.0 Three runs 60 10 2.2-4.8 59.9-73.7 Ave about 3 Ave 65%

[0142] From the results it can be seen that there is loss of selectivity, as indicated by the high wax burden under super critical conditions. Whilst winterisation can remove larger amounts of wax, processing is difficult as, for example, filters block.

Similar results were obtained with CBD.

[0143] Preferred conditions for liquid CO.sub.2 extraction are as follows:

Decarboxylated botanical raw material is packed into a single column and exposed to liquid CO.sub.2 under pressure.

[0144] Batch size: Approximately 60 kg

[0145] Pressure: 60 bar±10 bar

[0146] Temperature: 10° C.±5° C.

[0147] Time: Approximately 8 hours

[0148] CO.sub.2 mass flow 1250 kg/hr±20%.

[0149] Preferred process parameters for production of BDS are: extraction time >10 hours, CO.sub.2 pressure 50-70 bar, extraction temp 5-15° C., CO.sub.2 mass 167 kg/kg BRM.

[0150] Following depressurisation and venting off of the CO.sub.2 the crude BDS extract is collected into sealed vessels. The original BRM reduces to approximately 10% w/w of crude BDS extract. The crude BDS extract is held at −20° C.±5° C.

[0151] The crude BDS extract contains waxes and long chain molecules. Removal is by “winterisation” procedure (extraction 2), whereby the crude BDS extract is warmed to e.g. 40° C.±4° C. to liquefy the material. Ethanol is added in the ratio of 2:1 ethanol volume to weight of crude BDS extract. The ethanolic solution is then cooled to −20° C.±5° C. and held at this temperature for approximately 48 hours.

[0152] On completion of the winterisation the precipitate is removed by cold filtration through a 20 μm filter.

Extraction No. 2

[0153] The second stage in the manufacturing process is Extraction No. 2, referred to as “winterisation” using ethanol.

Crude BDS extract is produced from Extraction No. 1 that contains constituents, such as waxes. Ethanol effectively extracts long chained molecules from the crude extract.

Studies:

[0154] It was found by warming the crude BDS extract to approximately 40° C. the mixing ability of the crude extract with solvent was improved.

[0155] It was preferred to chill the “winterisation” solution to −20° C. for about 48 hours.

[0156] Preferred process parameters for production of BDS are: extraction temp 36-44° C., ratio ethanol:product approx. 2:1, freezer temp −25° C. to −15° C., time 48-54 hours.

Filtration

[0157] The ethanolic solution produced in the second extraction stage requires filtration to remove the resulting precipitation.

Filter size is preferably 20 μm.
Preferred process parameters for production of BDS are: total filtration time >6 hours.

Evaporation

[0158] The final stage of the manufacturing process is the removal of ethanol and any water that may be present.

Preferably this is carried out by heating at 60° C.±2° C. to give a vapour temperature of 40° C.±2° C. under a vacuum of 172 mbar±4 mbar. The distillation under these conditions continues until there is little or no visible condensate. Reducing the vacuum further, in stages, down to approximately 50 mbar, completes water removal. On completion the BDS is transferred into sealed stainless steel containers and stored in a freezer at −20° C.±5° C.
Preferred process parameters for production of BDS are: evaporation vapour temperature 38-42° C., vacuum pressure removal of ethanol 167-177 mbar, vacuum pressure removal of water 70-75 mbar 62-58 mbar 52-48 mbar, time <8 hours.

Characterisation of BDS

[0159] The THC BDS is a brown, viscous, semi-solid extract consisting of at least 60% cannabinoids constituents. The cannabinoid constituents include at least 90% THC, about 1.5% CBD with the remainder being made up of other minor cannabinoids.

[0160] The chemical composition of Cannabis has been thoroughly studied with over 400 compounds identified (Hendricks et al., 1975; Turner et al., 1980). More than 60 cannabinoids have been identified, with CBDA and THCA (the CBD and THC pre-cursors) being the most abundant. Generally, the non-cannabinoid constituents comprise up to 50% of extracts, depending on the extraction process. Chemical classes identified include alkanes (25-30 carbon chain), nitrogenous compounds, amino acids, sugars, aldehydes, alcohols and ketones, flavanoids, glycosides, vitamins, pigments and terpenes. About 95 mono- and sesqui-terpenes have been identified in Cannabis and are responsible for the characteristic odour.

[0161] Considerable work has been carried out to completely elucidate the structure of both CBD and THC (summarised in the above papers) and both have been prepared synthetically. Pure THC has been successfully isolated in sufficient quantity from the BDS to be used as reference material for identification and quantification.

Impurities:

[0162] The BDS substance is a selective extract from dried decarboxylated leaves and flowering heads of specific chemovars of Cannabis sativa. A range of over 400 compounds, including over 60 cannabinoids, have been found in Cannabis plants (Turner 1980). As these are naturally occurring it is not considered necessary to deem any of these components as impurities. The major impurities therefore occur in four areas, pesticides introduced during the growing process, aflatoxins, any new products formed by decarboxylation and the materials other than the cannabinoids, which make up the BDS.

[0163] The growing process is closely controlled using GAP guidelines and takes place in a climate controlled indoor growing environment. No pesticides are applied to the crops during growth, all pest control being managed by biological means. No pesticides are incorporated in the growing medium.

To ensure that no pesticide residues are introduced into the product the growing medium is periodically tested for pesticides known to be used by the growing medium supplier.

[0164] Once the plant material has been harvested and dried further samples are periodically tested using a general pesticide screen to ensure no contamination of the crop has occurred Potential impurities are adequately controlled at the BRM stage.

[0165] Although the growing conditions are carefully controlled to prevent this, the raw material has the potential for microbiological contamination resulting in aflatoxins in the product. The BRM and the BDS are therefore tested periodically for aflatoxins content.

[0166] The naturally occurring form of THC in the freshly grown plant is the acid THCA, although small quantities of the neutral THC do occur. Before extraction the THCA is decarboxylated by heating to yield the neutral THC. The process is efficient but a small amount of THCA remains and this is monitored during the final testing of the BDS. Thermal degradation of the THCA and THC during the decarboxylation process is possible to yield CBNA and CBN. These are monitored in the BDS.

[0167] The non-cannabinoid components that make up the ballast portion of the BDS include hydrocarbon and triglyceride waxes, plant pigments and terpenes. These are common components of many other extracts of medicinal plants and are considered to be of little toxicological and pharmacological significance. The range of other components present is wide but they are generally present in only small quantities

[0168] The quantity of ballast is reduced by the winterisation process which precipitates the waxes. The ballast materials are considered to be a diluent of the active constituents and are not assayed or controlled.

TABLE-US-00009 TABLE 9 Specification for the control of BDS high in CBD: Test Test Method Limits Appearance In-House Brown viscous semi-solid Identification: A TLC Spots have characteristic R.sub.f and colours, compared with CBD standard B HPLC/UV Positive for CBD CBD content In-house NLT 55% w/w of extract (HPLC-UV) Related In-house cannabinoids: (HPLC/UV) THC content NMT 7.5% of the CBD content Others (total) NMT 5% of the CBD content Aflatoxin: TEA NMT 4 ppb Total Heavy Metals: Ph. Eur. NMT 20 ppm Residual solvents: In-house Ethanol NMT 5% w/w Microbial: Ph. Eur. TVC NMT 10.sup.5 cfu/g Fungi NMT 10.sup.4 cfu/g Other NMT 10.sup.3 cfu/g enterobacteria & certain other gram negative organisms E. coli Absent in 1 g Salmonella Absent in 10 g S. aureus Absent in 1 g

Analytical Procedures

Identification, Assay and Related Cannabinoids:

[0169] The content of THC, CBD and Cannabinol (CBN) in the BRM and BDS, are quantitatively determined by extraction with methanol or methanol/chloroform (9:1). Reverse-phase High Performance Liquid Chromatography (HPLC) with UV detection at 220 nm is the method of quantification. All analysis must be performed under amber light because the compounds of interest are known to be light sensitive.

Chromatography Equipment and Conditions:

[0170] Equipment Agilent (HP)1100 HPLC system with variable wavelength UV detector or diode array detector. [0171] HPLC Column Discovery C8 5 μm 15 cm×0.46 cm [0172] Pre-Column Kingsorb C18 5 μm 3 cm×0.46 cm [0173] Mobile Phase Acetonitrile:Methanol:0.25% w/v acetic acid (16:7:6 by volume) [0174] Column Temp 25° C. [0175] Flow Rate 1.0 ml min.sup.−1 [0176] Detection 220 nm 600 mA f.s.d. Second wavelength 310 nm [0177] Injection Volume 100 μl [0178] Run Time 20-25 minutes (may be extended for samples containing small amount of late-eluting peaks) [0179] Elution Order CBD, CBDA, Δ.sup.9 THCV, CBN, Δ.sup.9 THC, CBC, Δ.sup.9 THCA

Standard Preparation:

[0180] Stock standard solutions of CBD, CBN and Δ.sup.9 THC in methanol at approximately 1 mg ml.sup.−1 are stored at −20° C.

[0181] Diluted working standards (0.1 mg/ml for Δ.sup.9 THC and CBD and 0.01 mg/ml for CBN) are prepared in methanol from the stock standards and stored at −20° C. (maximum period of twelve months after initial preparation). After preparation, standard solutions must be aliquoted into vials to reduce the amount of standard exposed to room temperature. Prior to use in an HPLC sample assay, the required number of standard vials are removed and allowed to equilibrate to room temperature.

Sample Preparation:

[0182] In all preparations, alternative weights and volumes may be used to give the same final dilutions.

Botanical Raw Material

[0183] Accurately weigh approximately 100 mg of chopped dried homogeneous material into a 10 ml volumetric flask.

[0184] Disperse material in methanol:chloroform (9:1 v/v) and make to volume in the same solvent.

[0185] Extract sample in an ultrasonic bath for 15 minutes.

[0186] Centrifuge an aliquot at 3000 rpm for about 2 minutes.

[0187] Dilute 1000 of the supernatant to 1 ml with methanol in a suitable HPLC sample vial. (Further dilution may be required if the principal cannabinoid concentration is outside the linear working range).

Decarboxylated Botanical Raw Material:

As for Botanical Raw Material.

Botanical Drug Substance:

[0188] Accurately weigh approximately 80 mg of BDS into a 50 ml volumetric flask. [0189] Dissolve BDS and make up to volume with methanol. [0190] Dilute 100 μl of the prepared supernatant to lml with methanol in a suitable HPLC auto sampler vial.

Chromatography Procedure:

[0191] Samples are placed in the autosampler rack in the order entered into the sequence list on the Agilent chemstation.

Standard solutions are used to provide quantitative and retention time data. These may be typically injected in duplicate or triplicate prior to the injection of any sample solutions and then singularly at suitable intervals during the run, with a maximum of 10 test samples in between standards.

Chromatography Acceptance Criteria:

[0192]

TABLE-US-00010 TABLE 10 Retention time windows and Relative Retention Time (RRT) to Δ.sup.9THC for each analyte: Retention Time Cannabinoid (Minutes) RRT(THC) CBD 5.1-5.8 0.58 CBN 7.4-8.3 0.83 Δ.sup.9 THC  9.0-10.0 1.00 CBDA 5.5-6.2 0.615 Δ.sup.9 THCV 5.9-6.6 0.645 CBC 11.6-12.8 1.30 Δ.sup.9 THCA 14.6-16.0 1.605

TABLE-US-00011 TABLE 11 Peak Shape (Symmetry Factor according to British Pharmacopoeia method): Cannabinoid Symmetry Factor CBD <1.30 CBN <1.25 Δ.sup.9 THC <1.35

Calculation:

Botanical Raw Material:

[0193] The following equation is used to obtain a result for the purity of the principal cannabinoid as a % of the currently assayable cannabinoids (CBD, CBDA, CBN, Δ.sup.9 THC & Δ.sup.9 THCA) in the batch:

For high Δ.sup.9 THC material:

[00001]$\%.Math.\mathrm{THC}=\frac{\mathrm{peak}.Math..Math.\mathrm{area}.Math..Math.\mathrm{sum}.Math..Math.\mathrm{of}.Math..Math.\mathrm{THC}\&.Math..Math.\mathrm{THCA}}{\mathrm{peak}.Math..Math.\mathrm{area}.Math..Math.\mathrm{sum}.Math..Math.\mathrm{of}.Math..Math.\mathrm{assayable}.Math..Math.\mathrm{cannabinoids}}\times 100$

For high CBD material, CBD & CBDA replace THC & THCA in the top line of the equation.

Decarboxylated Botanical Raw Material:

[0194] The following equation is used to calculate the efficiency of the decarboxylation process:

For high Δ.sup.9 THC material:

[00002]$\%.Math..Math.\mathrm{decarboxylation}.Math..Math.\mathrm{efficiency}=\frac{\mathrm{Peak}.Math..Math.\mathrm{area}.Math..Math.\mathrm{of}.Math..Math.\mathrm{THC}}{\mathrm{Peak}.Math..Math.\mathrm{area}.Math..Math.\mathrm{sum}.Math..Math.\mathrm{of}.Math..Math.\mathrm{THC}\&.Math..Math.\mathrm{THCA}}\times 100$

For high CBD material, CBD & CBDA replace THC & THCA in the equation.

Botanical Drug Substance:

[0195] The following equations are used to calculate the concentration of drug substance sample, the individual sample cannabinoid concentration, the % content of the assayable cannabinoids in the drug substance, the quantity of principal cannabinoid as a % of currently assayable cannabinoids and the amount of principal cannabinoid in the whole weight of extracted drug substance.

For high Δ.sup.9 THC material:

[00003]$\mathrm{Concentration}.Math..Math.\mathrm{of}.Math..Math.\mathrm{drug}.Math..Math.\mathrm{substance}.Math..Math.\mathrm{sample}=\frac{\mathrm{Weight}.Math..Math.\mathrm{of}.Math..Math.\mathrm{sample}}{\mathrm{Dilution}.Math..Math.\mathrm{factor}}$

Where dilution factor=50×10=500

[00004]$\mathrm{Sample}.Math..Math.\mathrm{THC}.Math..Math.\mathrm{concentration}=\frac{\mathrm{THC}.Math..Math.\mathrm{standard}.Math..Math.\mathrm{conc}\times \mathrm{mean}.Math..Math.\mathrm{THC}.Math..Math.\mathrm{sample}.Math..Math.\mathrm{area}}{\mathrm{mean}.Math..Math.\mathrm{THC}.Math..Math.\mathrm{standard}.Math..Math.\mathrm{area}}$$\%.Math..Math.w.Math.\text{/}.Math.w.Math..Math.\mathrm{THC}.Math..Math.\mathrm{content}.Math..Math.\mathrm{of}.Math..Math.\mathrm{drug}.Math..Math.\mathrm{substance}=\frac{\mathrm{THC}.Math..Math.\mathrm{sample}.Math..Math.\mathrm{concentration}}{\mathrm{drug}.Math..Math.\mathrm{substance}.Math..Math.\mathrm{sample}.Math..Math.\mathrm{concentration}}\times 100$

[0196] CBD and CBN can be substituted into all of these equations instead of Δ.sup.9 THC to obtain quantitative results for both. Δ.sup.9 THCA and CBDA are also calculated using the standard concentrations for Δ.sup.9 THC or CBD in the absence of specific reference standards of their own.

[0197] Related Substances are defined as the sum of the mean % w/w values for CBN, Δ.sup.9 THCA and CBDA.

[00005]$\mathrm{THC}.Math..Math.\mathrm{as}.Math..Math.\%.Math..Math.\mathrm{of}.Math..Math.\mathrm{total}.Math..Math.\mathrm{assayable}.Math..Math.\mathrm{cannabinoids}=\frac{\%.Math..Math.w.Math.\text{/}.Math.w.Math..Math.\mathrm{THC}.Math..Math.\mathrm{contect}}{\mathrm{sum}.Math..Math.\mathrm{of}.Math..Math.\%.Math..Math.w.Math.\text{/}.Math.w.Math..Math.\mathrm{of}.Math..Math.\mathrm{all}.Math..Math.\mathrm{assayable}.Math..Math.\mathrm{cannabinoids}}\times 100$

[0198] The total amount of Δ.sup.9 THC present in the whole drug substance extract is obtained.

Example 2—Investigation of the Stabilisation of Botanical Drug Substance (BDS) by Partial Purification Using Activated Charcoal

[0199] Results from stability studies on THC formulations indicate that THC in the form of BDS is unstable even at storage temperatures as low as 5° C. This contrasts with the behaviour of the purified THC (Dronabinol USP) in Marinol soft gel capsules, for which a shelf life of 2 years at cool ambient temperature is accepted. It should also be noted that the shelf life of THC standard solutions in methanol supplied by Sigma-Aldrich is claimed to be 4 years when stored refrigerated and protected from light.

[0200] This apparent discrepancy between the stability of BDS (THC) and purified THC prompted speculation that some component of BDS was destabilising the principal cannabinoid.

[0201] A solution to this problem would be to purify the BDS (THC) to yield high purity, preferably crystalline cannabinoid. However, the additional processing costs incurred on transforming BDS to pure cannabinoid would substantially increase the cost of finished pharmaceutical products incorporating the cannabinoid.

[0202] Hence, the applicant sought to develop a simple purification step which would produce BDS with enhanced stability but which did not increase processing costs to a prohibitive extent.

[0203] The applicant has determined that a charcoal clean-up step may be conveniently carried out in close conjunction with the “winterisation” process by passing the ethanolic winterisation solution through a filter bed to remove precipitated waxes and then directly through a charcoal column in a single step and that the use of activated charcoal significantly improves shelf life.

Experimental Detail

[0204] Solutions of either BDS (THC) or BDS (CBD) at a concentration of 100 mg/ml in absolute ethanol BP were passed through a column packed with activated charcoal and the eluate collected. These were then diluted with further absolute ethanol to achieve a concentration of ca. 25 mg/ml cannabinoid. The solution was then transferred into a 10 ml type AX1 (i.e. amber glass) vial and crimp sealed. These samples were designated charcoal purified BDS.

[0205] Samples of the BDS (THC) and BDS (CBD) solutions which had not been passed through the charcoal column were similarly diluted to give a cannabinoid concentration of 25 mg/ml and were then sealed in an amber glass vial of the same type. These samples were designated “standard BDS” and served as a control for the stability study.

[0206] The vials containing standard BDS (“std” BDS) and charcoal purified BDS of each type were stored in a stability incubator at 40° C. and samples then periodically withdrawn over the period

1-12 months for HPLC analysis of cannabinoid content and TLC profiling.
Normal phase TLC analysis employed the following conditions:

Stationary Phase: Silica Gel G

[0207] Mobile Phase: 80:20 hexane/acetone
Development: 2×8 cm i.e. double development

Visualisation: Dip in 0.1% w/v Fast Blue B (aq)

[0208] Reverse phase TLC analysis employed the following conditions:
Stationary Phase: C18 coated Silica Gel
Mobile Phase: 6:7:16 0.25% v/v acetic acid (aq)/methanol/acetonitrile
Development: 2×8 cm i.e. double development

Visualisation: Dip in 0.1% w/v Fast Blue B (aq)

[0209] For each sample a volume of solution containing approximately 5 μg total cannabinoid was applied to the TLC plate.

Results and Discussion

[0210] The ethanolic solutions of std BDS (THC) and std BDS (CBD) are a fairly intense yellow. Passage of the BDS solutions through the activated charcoal effectively decolourised the solutions, presumably by the adsorption of plant pigments co-extracted with the cannabinoids during the preparation of BDS from cannabis herb by liquid CO.sub.2 extraction.

[0211] The HPLC analysis results for the different BDS solutions are tabulated below as Table 12 and are also presented in graphical form (FIGS. 1-3). All data is reported as % of the t0 assay. CBN values are included for the BDS (THC) solutions as this compound has been identified as a marker of thermal degradation of THC in previous stability studies.

TABLE-US-00012 TABLE 12 Cannabinoid Assay Values for Std and Purified BDS Solutions over the Period 1-12 Months at 40° C. Months Solution Cannabinoid 1 4 6 12 Std BDS THC  97.3%  92.4% 85.3% 74.0% (THC) CBN .sup. 104% .sup. 119%  133%  154% Purified THC 102.9% 107.4% 96.0% 88.6% BDS (THC) CBN   94% .sup. 111%  111%  120% Std BDS CBD 100.3% 103.6% 93.3% 91.0% (CBD) Purified CBD 101.0% 100.7% 97.2% 96.9% BDS (CBD)

[0212] From the above data it is quite clear that for both BDS (THC) and BDS (CBD) there is some component of the ballast, which can be removed by charcoal, which is destabilising the cannabinoids.

[0213] Comparison of the levels of degradation reached after 12 months at 40° C. for the std BDS and the corresponding charcoal purified BDS indicate that for both the THC and the CBD extracts the charcoal purification increases the resistance to thermal degradation by over 50%.

[0214] For BDS (THC), the level of CBN is seen to increase as a function of the principal cannabinoid lost (FIG. 3). As observed for other formulations containing THC, the level of CBN is again confirmed to be a marker of thermal degradation.

[0215] Comparison between cannabinoid regions of HPLC chromatograms of standard BDS (CBD) and purified BDS (CBD) samples after 12 months at 40° C. (data not shown) revealed no significant information. However, similar comparison of HPLC chromatograms of the standard and purified BDS (THC) after degradation was informative.

[0216] The CBN was at a higher level in the more highly degraded unpurified standard BDS, but a second significant degradation product was also observed, which is again present in both samples but which is more abundant in the more degraded sample. The spectrum of this degradation product was again essentially identical to that of CBN and on the basis of this and the retention time appeared to be one of the CBN analogues.

Conclusion

[0217] Significant improvement in resistance to thermal degradation is achieved by a simple charcoal treatment.

Example 3—Effect of Addition of Organic Modifier on CO.SUB.2 .Extraction of Cannabis Plant Material

[0218] The following example describes an investigation into the effect of the addition of a polar co-solvent on the characteristics of an extract produced from cannabis plant material (G5 chemovar) using liquid CO.sub.2 extraction, and illustrates the difference in selectivity obtained using sub-critical vs super-critical CO.sub.2 extraction.

Experimental Detail

[0219] Extraction experiments were carried out using a 1 litre capacity CO.sub.2 extraction apparatus. Food grade CO.sub.2 and BP grade absolute ethanol were employed as solvents.

[0220] A batch of G5 cannabis (a high CBD chemovar) was used. The CBD content was 7.3% w/w after decarboxylation. Analysis of the cannabinoid content of the extracts was carried out by HPLC.

Results and Discussion.

[0221] The data relating to the composition of the final extract obtained after a 4 hour extraction time under the specified conditions is presented below in Table 13:

TABLE-US-00013 TABLE 13 Composition and Yield Data for Extracts Produced under Different Extraction Conditions. Extraction % w/w % CBD % Recovery Sample Conditions Extract (w/w) of CBD AC470 10° C./60 BAR  8.4% 63.6% 72.9% AC471 40° C./100 BAR 10.7% 54.4% 79.5% AC472 40° C./100 BAR + 10.3% 64.6% 91.0% 2% ETHANOL

[0222] The recovery efficiency is based on the CBD available in decarboxylated plant material charged to the vessel for each extraction.

[0223] The results illustrate that changing the extraction conditions from sub-critical to super-critical increases the solvating power of the CO.sub.2 and results in a higher recovery of the available CBD. However, the supercritical CO.sub.2 can now solubilise a wider range of compounds and the extraction of these additional compound has the effect of diluting the concentration of CBD in the extract to such an extent that it is now lower than that obtained for the sub-critical extraction. Consequently, the marginal additional recovery of available CBD from the raw material would not outweigh this disadvantage and demonstrates the use of supercritical conditions is not desirable.

[0224] The addition of 2% w/w absolute ethanol to supercritical CO.sub.2 as a modifier increases the recovery of the available CBD to >90%. Presumably the relatively polar cannabinoid is more soluble in the extract of increased polarity.

[0225] Interestingly, the concentration of CBD in the extract is increased slightly by the addition of polar modifier. This would seem to indicate that the co-extractable non-cannabinoid material present in the plant material is less polar than the target cannabinoid and hence the extraction of this material (the “ballast”) is deselected when polarity is increased.

Thus, extraction of cannabis plant material with supercritical CO.sub.2+2% w/w ethanol provides an increase in recovery of the target active with no attendant penalty of loss of selectivity.
In summary:
1. A switch from sub-critical to super-critical conditions produces little advantage in terms of overall recovery of cannabinoid from the raw material but does result in the disadvantage of reducing the active content of the extract.
2. The addition of 2% absolute ethanol modifier to supercritical CO.sub.2 results in a significant improvement in the recovery of cannabinoid from the raw material with no penalty of dilution of active content by co-extracted material.

[0226] Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the invention described herein. Such equivalents are intended to be encompassed by the following claims.

[0227] All references disclosed herein are incorporated by reference in their entirety.