U.S. patent application number 15/279068 was filed with the patent office on 2017-03-23 for compositions comprising cannabinoids for treatment of nausea, vomiting, emesis, motion sickness or like conditions.
This patent application is currently assigned to GW Pharma Limited. The applicant listed for this patent is GW Pharma Limited. Invention is credited to Farideh Afshin Javid, Brian Anthony Whittle.
Application Number | 20170079933 15/279068 |
Document ID | / |
Family ID | 27665365 |
Filed Date | 2017-03-23 |
United States Patent
Application |
20170079933 |
Kind Code |
A1 |
Whittle; Brian Anthony ; et
al. |
March 23, 2017 |
COMPOSITIONS COMPRISING CANNABINOIDS FOR TREATMENT OF NAUSEA,
VOMITING, EMESIS, MOTION SICKNESS OR LIKE CONDITIONS
Abstract
Cannabinoids, in particular CBD and CBDA and their acid
derivatives are provided for use as an active pharmaceutical
substance in the treatment of nausea, vomiting, emesis, motion
sickness. In particular extracts of cannabis plants are presented
which are rich in these substances and suitable for pharmaceutical
use.
Inventors: |
Whittle; Brian Anthony;
(Hornsea, GB) ; Javid; Farideh Afshin;
(Huddersfield, GB) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
GW Pharma Limited |
Salisbury |
|
GB |
|
|
Assignee: |
GW Pharma Limited
Salisbury
GB
|
Family ID: |
27665365 |
Appl. No.: |
15/279068 |
Filed: |
September 28, 2016 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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13222114 |
Aug 31, 2011 |
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15279068 |
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10502822 |
Mar 2, 2005 |
8034843 |
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PCT/GB2003/000451 |
Feb 3, 2003 |
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13222114 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
A61K 31/192 20130101;
A61K 2236/10 20130101; A61K 36/185 20130101; A61P 1/08 20180101;
A61K 2236/37 20130101; A61K 31/05 20130101; A61K 2236/50 20130101;
A61K 36/185 20130101; A61K 2300/00 20130101 |
International
Class: |
A61K 31/05 20060101
A61K031/05; A61K 36/185 20060101 A61K036/185 |
Foreign Application Data
Date |
Code |
Application Number |
Feb 1, 2002 |
GB |
0202385.1 |
Mar 15, 2002 |
GB |
0206183.6 |
Claims
1.-30. (canceled)
31. A botanical drug substance (BDS) in the form of a subcritical
CO.sub.2 extract obtained from a high cannabidiol (CBD) containing
cannabis plant, wherein said BDS comprises cannabinoid constituents
and other chemical entities, wherein the cannabinoid constituents
comprise at least 90% CBD and the other chemical entities include
terpene or carotene rich volatile oils, said BDS having been
purified by winterisation in ethanol and cold filtration to remove
waxes and other non-specific lipid-soluble material, and any water
and ethanol have been removed by evaporation.
32. A crystalline cannabidiol (CBD) obtained by re-dissolving a BDS
as claimed in claim 31 in a suitable solvent, filtering to remove
insoluble material and then removing the solvent, such that the
crystalline CBD has a chromatographic purity of greater than 99% by
area normalization and contains less than 0.1% each of
tetrahydrocannabinol (THC) and cannabinol (CBN).
33. The crystalline cannabidiol (CBD) as claimed in claim 32,
wherein the suitable solvent is pentane.
34. A process of preparing a botanical drug substance (BDS) in the
form of a subcritical CO.sub.2 extract obtained from a high
cannabidiol (CBD) containing cannabis plant comprising the
following steps: i) Decarboxylate dry high CBD plant material by
heating for a sufficient time and temperature to decarboxylate CBDA
to CBD; ii) Extract with liquid carbon dioxide under subcritical
conditions of 50 to 70 bar and 5 to 15.degree. C. for up to 10
hours; iii) Removal of carbon dioxide by depressurization to
recover crude extract; iv) Winterization in ethanol and cold
filtration to remove waxes and other non-specific lipid-soluble
material; and v) Removal of ethanol and water by evaporation to
produce BDS.
35. The process as claimed in claim 34, wherein the dry high CBD
plant material is chopped to 2 to 3 mm prior to
decarboxylation.
36. The process as claimed in claim 34, wherein the plant material
is decarboxylated by heating at 100 to 150.degree. C.
37. The process as claimed in claim 34, wherein the winterization
step (iv) comprises chilling at -20.degree. C. .+-.5.degree. C. for
up to 52 hours.
38. The process as claimed in claim 34, wherein the ethanol and
water in step (v) is removed by evaporation under reduced
pressure.
39. A process of preparing crystalline cannabidiol (CBD) such that
the crystalline CBD has a chromatographic purity of greater than
99% by area normalization and contains less than 0.1% each of
tetrahydrocannabinol (THC) and cannabinol (CBN) from the BDS
obtained from claim 34, further comprising the additional steps of:
i) Re-dissolving the BDS obtained in a suitable solvent; ii)
Filtering to remove insoluble material; and iii) Removing the
solvent.
40. The process as claimed in claim 39, wherein the suitable
solvent is pentane.
Description
RELATED APPLICATIONS
[0001] This application is a continuation of U.S. patent
application Ser. No. 13/222,114, filed Aug. 31, 2011, which is a
continuation of U.S. patent application Ser. No. 10/502,822, filed
Mar. 2, 2005, which application is a national stage filing under 35
U.S.C. .sctn.371 of PCT International application
PCT/GB2003/000451, filed Feb. 3, 2003, which was published under
PCT Article 21(2) in English, the contents of each of which is
incorporated by reference herein in its entirety.
[0002] Functional vomiting is the forceful expulsion of gastric
contents produced by involuntary contraction of the abdominal
musculature. This occurs when the gastric fundus and lower
oesophageal sphincter are relaxed. Functional vomiting may be
accompanied by nausea (an unpleasant feeling that vomiting is about
to occur). Nausea is associated with altered physiological
activity, including gastric hypomotility, and increased
parasympathetic tone. Nausea may precede and accompany vomiting.
They represent the patient's awareness of afferent stimuli to the
medullary vomiting centre.
[0003] Physiological vomiting is a functional condition that occurs
in response to a number of factors affecting the vomiting centre.
It may also be triggered by peripheral factors such as ingestion of
toxins, disturbance of the vestibular system, peritoneal
inflammation and bowel obstruction. It may also occur in disorders
of delayed gastric emptying as, for example, in diabetes and
idiopathic gastroparesis.
[0004] Psychogenic vomiting may be self-induced or may occur
involuntarily in situations that are anxiety inducing, threatening
or in some way regarded as distasteful by the subject. It is also
possible that psychological factors leading to vomiting are
culturally determined (for example, eating exotic food may be
considered repulsive in the subject's own cultural group). Vomiting
may also express hostility as when children vomit during a temper
tantrum or in certain conversion disorders.
[0005] Nausea and vomiting may also be induced by cytotoxic
chemotherapy and radiotherapy. Post-operatively, patients may also
vomit and experience nausea, which may be attributable to the
anaesthetic and analgesic agents frequently administered
concurrently.
[0006] There are therefore peripheral and central mechanisms which
are involved in the expression of nausea and frank vomiting.
Existing therapies are available for the treatment of these
conditions but they have limitations, and there is a need for
alternative treatments, particularly where these can exert their
effect through a central nervous mechanism.
[0007] Investigation of a number of agents in a conscious animal
model in which motion sickness is induced has confirmed that
extracts of cannabis have an anti-emetic effect. Conventionally,
the anti-emetic effect of cannabis has been ascribed to
delta.sup.9-tetrahydrocannabinol (.DELTA..sup.9-THC). The use of a
whole animal, conscious model to explore this effect, and the
availability of cannabis extracts containing predominantly one
cannabinoid or another have allowed for more detailed analysis of
the contribution of specific cannabinoids.
[0008] Surprisingly, it has been found that the anti-emetic effect,
in a model of motion sickness in Suncus murinus (the Asian musk
(house) shrew), is greatest in high cannabidiol (CBD)(and/or its
acid form CBDA) containing extracts rather than in high
tetrahydrocannabinol (THC) (and/or its acid form THCA) containing
extracts (greater than 50% CBD more preferably greater than 80%
most preferably greater than 90% relative to other cannabinoids
present).
[0009] It is hypothesised that the results will extend to the
propyl variant of CBD, namely CBDV and its acid form CBDVA.
[0010] More particularly, the results were noted in extracts in
which the cannabinoids were predominantly in their acid form since
the extracts were prepared by a methanolic extraction and had not
been subjected to a decarboxylation step by, for example, heating.
It is possible therefore that the therapeutic effects noted are due
to the acid form of the cannabinoids. If it is the acid form of the
cannabinoid that is responsible for the observed therapeutic effect
this is particularly surprising since the acid forms of
cannabinoids have not hitherto been known to exhibit therapeutic
effects.
[0011] According to a first aspect of the present invention there
is provided a cannabis extract, rich in CBD and/or CBDA and/or the
propyl variants CBDV and/or CBDVA, for use in the manufacture of a
medicament for the treatment of nausea, vomiting, emesis, motion
sickness or like conditions.
[0012] By rich is meant greater than 2% w/w of CBD and/or CBDA
and/or the propyl variants CBDV and/or CBDVA, more particularly
greater than 5%, more preferably still greater than 7%.
[0013] According to another aspect of the present invention there
is provided the use of CBD and/or CBDV in the manufacture of a
medicament for the treatment of nausea, vomiting, emesis, motion
sickness or like conditions.
[0014] According to another aspect of the present invention there
is provided a cannabinoid acid for use as an active pharmaceutical
substance.
[0015] According to yet another aspect of the present invention
there is provided CBDA or CBDVA for use as an active pharmaceutical
substance.
[0016] Preferably the active pharmaceutical substance is present as
a medicament for the treatment of nausea, vomiting, emesis, motion
sickness or like conditions.
[0017] In one embodiment the CBD and/or CBDA and/or CBDV and/or
CBDVA are present with other cannabinoids as a mixture derived from
a plant extract (CMBE, cannabis based medicinal extract).
[0018] Plant extracts are preferred as, in addition to one or more
cannabinoids, they will contain other chemical entities that may
provide a beneficial effect either in their own right or in
combination with the one or more cannabinoids. Such other chemicals
include, for example, volatile oils e.g. terpene or carotene rich
volatiles. Known terpenes present in the CMBE include C.sub.10
terpenes, e.g. mycerene, and pinenes and C.sub.15 terpenes e.g.
caryophyllene.
[0019] Preferably the CBD or CBDV and/or the acids thereof are
present with THC or THCV and/or the acids thereof.
[0020] Alternatively the CBD or CBDV and/or the acids thereof are
substantially free (less than 10%, more prferably less than 5% and
most preferably less than 2% relative to other cannabinoids
present)from other cannabinoids.
[0021] In another embodiment the CBD or CBDV and/or the acids
thereof are synthetic.
[0022] The invention also extends to methods of treating nausea,
vomiting, emesis, motion sickness or like conditions with CBD or
CBDV and/or the acids thereof, either as the sole active ingredient
or in mixtures as plant extracts.
[0023] Whilst the observation has been made on an extract
administered intraperitoneally, the skilled man will appreciate
that a medicament can be prepared for administration by any
suitable means. These include, but are not limited to, solids, semi
solids, e.g. gels, liquids, sprays, aerosols, inhalers,
vapourisers, enemas, rectal suppositories and the like. The route
of administration need not be intraperitoneally but could be oral,
buccal, sublingual, or by any other suitable route e.g. the
respiratory tract, nasal tract and distal rectum.
[0024] A "plant extract" is an extract from a plant material as
defined in the Guidance for Industry Botanical Drug Products Draft
Guidance, August 2000, US Department of Health and Human Services,
Food and Drug Administration Centre for Drug Evaluation and
Research.
[0025] "Plant material" is defined as a plant or plant part (e.g.
bark, wood, leaves, stems, roots, flowers, fruits, seeds, berries
or parts thereof) as well as exudates.
[0026] The term "Cannabis plant(s)" encompasses wild type Cannabis
sativa and also variants thereof, including cannabis chemovars
which naturally contain different amounts of the individual
cannabinoids, Cannabis sativa subspecies indica including the
variants var. indica and var.kafiristanica, Cannabis indica and
also plants which are the result of genetic crosses, self-crosses
or hybrids thereof. The term "Cannabis plant material" is to be
interpreted accordingly as encompassing plant material derived from
one or more cannabis plants. For the avoidance of doubt it is
hereby stated that "cannabis plant material" includes dried
cannabis biomass.
[0027] In the context of this application the terms "cannabis
extract" or "extract from a cannabis plant", which are used
interchangeably encompass "Botanical Drug Substances (BDS)" derived
from cannabis plant material. A Botanical Drug Substance is defined
in the Guidance for Industry Botanical Drug Products Draft
Guidance, August 2000, US Department of Health and Human Services,
Food and Drug Administration Centre for Drug Evaluation and
Research as: "A drug substance derived from one or more plants,
algae, or macroscopic fungi. It is prepared from botanical raw
materials by one or more of the following processes: pulverisation,
decoction, expression, aqueous extraction, ethanolic extraction, or
other similar processes." A botanical drug substance does not
include a highly purified or chemically modified substance derived
from natural sources. Thus, in the case of cannabis, "botanical
drug substances" derived from cannabis plants do not include highly
purified, Pharmacopoeial grade cannabinoids.
[0028] "Botanical drug substances" derived from cannabis plants
include primary extracts prepared by such processes as, for
example, maceration, percolation, extraction with solvents such as
C1 to C5 alcohols (e.g. ethanol), Norflurane (HFA134a), HFA227 and
liquid carbon dioxide under pressure. The primary extract may be
further purified for example by supercritical or subcritical
extraction, vaporisation and chromatography. When solvents such as
those listed above are used, the resultant extract contains
non-specific lipid-soluble material. This can be removed by a
variety of processes including "winterisation", which involves
chilling to -20.degree. C. followed by filtration to remove waxy
ballast, extraction with liquid carbon dioxide and by
distillation.
[0029] Preferred "cannabis extracts" include those which are
obtainable by using any of the methods or processes specifically
disclosed herein for preparing extracts from cannabis plant
material. The extracts are preferably substantially free of waxes
and other non-specific lipid soluble material but preferably
contain substantially all of the cannabinoids naturally present in
the plant, most preferably in substantially the same ratios in
which they occur in the intact cannabis plant.
[0030] Botanical drug substances are formulated into "Botanical
Drug Products" which are defined in the Guidance for Industry
Botanical Drug Products Draft Guidance, August 2000, US
[0031] Department of Health and Human Services, Food and Drug
Administration Centre for Drug Evaluation and Research as: "A
botanical product that is intended for use as a drug; a drug
product that is prepared from a botanical drug substance."
[0032] "Cannabinoids" may be highly purified, Pharmacopoeial Grade
substances and may be obtained by purification from a natural
source or via synthetic means. The cannabinoids will include, but
are not limited to, tetrahydrocannabinoids, their precursors, alkyl
(particularly propyl) analogues, cannabidiols, their precursors,
alkyl (particularly propyl) analogues, and cannabinol.
[0033] In preferred embodiments of the invention the formulations
comprise extracts of one or more varieties of whole Cannabis
plants, particularly Cannabis sativa, Cannabis indica or plants
which are the result of genetic crosses, self-crosses or hybrids
thereof. The precise cannabinoid content of any particular cannabis
variety may be qualitatively and quantitatively determined using
methods well known to those skilled in the art, such as TLC or
HPLC. Thus, one may chose a Cannabis variety from which to prepare
an extract which will produce the desired ratio of CBD or CBDV to
THC or THCV. Alternatively, extracts from two of more different
varieties may be mixed or blended to produce a material with the
preferred cannabinoid ratio for formulating into a pharmaceutical
formulation.
[0034] The preparation of convenient ratios of CBD, CBDV, CBDA and
CBDVA containing medicines is made possible by the cultivation of
specific chemovars of cannabis. These chemovars (plants
distinguished by the cannabinoids produced, rather than the
morphological characteristics of the plant) can be bred by a
variety of plant breeding techniques which will be familiar to a
person skilled in the art. Suitable methods are given in Example 3.
Propagation of the plants by cuttings for production material
ensures that the genotype is fixed and that each crop of plants
contains the cannabinoids in substantially the same ratio.
[0035] Horticulturally, it is convenient to grow chemovars
producing e.g. CBD and CBDV as the predominant cannabinoid from
cuttings. This ensures that the genotype in each crop is identical
and the qualitative formulation (the proportion of each cannabinoid
in the biomass) is the same. From these chemovars, extracts can be
prepared by the similar method of extraction. Convenient methods of
preparing primary extracts include maceration, percolation,
extraction with solvents such as C1 to C5 alcohols (ethanol),
Norflurane (HFA134a), HFA227 and liquid carbon dioxide under
pressure. The primary extract may be further purified for example
by supercritical or subcritical extraction, vaporisation and
chromatography. When solvents such as those listed above are used,
the resultant extract contains non-specific lipid-soluble material.
This can be removed by a variety of processes including chilling to
-20.degree. C. followed by filtration to remove waxy ballast,
extraction with liquid carbon dioxide and by distillation.
Preferred plant cultivation and extract preparation methods are
shown in the Examples. The resulting extract is suitable for
incorporation into pharmaceutical preparations.
[0036] A detailed examination of the pharmacological differences
between CBD and THC has revealed significant differences in these
compounds and consequently the finding that CBD and/or its acid
CBDA appear to be responsible for the therapeutic effects noted was
surprising. THC is bound with high avidity to CB1 and CB2 receptors
in cerebral cortex and other sites; CBD is relatively inactive
against CB1 receptors and appears to have non-cannabinoid receptor
pharmacological actions in the central nervous system. Without
prejudice to the teaching of the invention, it is possible that the
anti-emetic effect of CBD and/or its acid CBDA is mediated via a
non-cannabinergic mechanism.
[0037] Table 1 below illustrates some of the differences between
these cannabinoids.
TABLE-US-00001 TABLE 1 Effect THC THCV CBD CBDV Reference CB.sub.1
(Brain receptors) ++ .+-. Pertwee et al, 1998 CB.sub.2 (Peripheral
+ - receptors) CNS Effects Anticonvulsant .dagger. -- ++ Carlini et
al, 1973 Antimetrazol - - GW Data Anti-electroshock - ++ GW data
Muscle Relaxant -- ++ Petro, 1980 Antinociceptive ++ + GW data
Catalepsy ++ ++ GW data Psychoactive ++ - GW data Antipsychotic -
++ Zuardi et al, 1991 Neuroprotective + ++ Hampson A J antioxidant
et al, 1998 activity* Antiemetic ++ - Sedation (reduced + + Zuardi
et al, spontaneous activity) 1991 Appetite stimulation ++ Appetite
suppression ++ Anxiolytic - ++ GW data Cardiovascular Effects
Bradycardia - + Smiley et al, 1976 Tachycardia + - Hypertension
.sctn. + - Hypotension .sctn. - + Adams et al, 1977
Anti-inflammatory .+-. .+-. Brown, 1998 Immunomodulatory/
anti-inflammatory activity Raw Paw Oedema - ++ GW data Test Cox 1
GW data Cox 2 GW data TNF.alpha. Antagonism + + ++ ++ Glaucoma ++ +
*Effect is CB1 receptor independent. .dagger. THC is pro convulsant
.sctn. THC has a biphasic effect on blood pressure; in naive
patients it may produce postural hypotension and it has also been
reported to produce hypertension on prolonged usage. GW Internal
Report No 002/000159.
[0038] Whilst it is known that THC can be used to control nausea
and vomiting pre-operatively the effect of other cannabinoids or
combinations or the effect of the acid forms present in, for
example, plant extracts was not hitherto known.
[0039] The applicants studied the effect of other cannabinoids as
cannabis extracts, and particularly extracts containing
predominantly CBD or its acid form CBDA in Suncus murinus and in
which an emetic response can be induced by a motion stimulus.
Compounds which are effective in this test have therapeutic benefit
in the treatment of motion-induced nausea and vomiting, and also
these conditions when induced by other pathways in the peripheral
and central nervous systems.
[0040] The applicant has determined that, for example, extracts in
which the content of CBD and/or CBDA is 2-20% w/w, and the content
of THC and or THCA is 0.1-2% w/w are particularly beneficial.
[0041] The invention is further illustrated with reference to the
accompanying figures and examples:
[0042] FIG. 1 is a TLC from the methanolic extract of the high CBD
chemovar G5 (M16). It shows significant CBD and CBDA peaks at
around 6 and 7 minutes and lesser amounts of THC and THCA at around
10 and 18 minutes.
[0043] FIG. 2 is the TLC the methanolic extract of the high THC
chemovar G2 (M6). It shows significant THC and THCA peaks at around
10 and 18 minutes along with lesser amounts of CBD and CBDA at
around 6 and 7 minutes.
[0044] The figures shown in FIGS. 1 and 2 are quantitative.
[0045] FIG. 3 is a thin layer chromatography plate showing the
methanolic extracts, and by way of comparison BDS (decarboxylated
and extracted by sub critical liquid CO.sub.2). The results confirm
that the methanolic extracts comprise a high proportion of the
respective cannabinoids THC and CBD in what was later shown to be
their acid forms.
[0046] FIG. 4 illustrates the results obtained for G5, the upper
trace being for the BDS (decarboxylated and extracted by sub
critical liquid CO.sub.2) and the lower trace for the methanolic
extract. The CBD peak at 35 minutes and the THC peak at 38 minutes
are marked.
[0047] FIG. 5 illustrates the results obtained for G2, the upper
trace being for the BDS (decarboxylated and extracted by sub
critical liquid CO.sub.2) and the lower trace for the methanolic
extract. The CBD peak at 35 minutes and the THC peak at 37 minutes
are marked.
EXAMPLE 1
[0048] Extracts of cannabis can be prepared by a number of solvent
extraction techniques, including the use of organic solvents alone
or in admixture with water and under sub critical or supercritical
conditions. Cannabinoids may be present in cannabis biomass as free
cannabinoids and as the corresponding acidic precursors.
Conventional methods of preparation involve the total extraction of
free cannabinoids and precursors with solvents such as lower alkyl
alcohols, particularly methanol. In this example, total extracts of
a high THC, and a high CBD-containing chemovar were made using
methanol.
[0049] Biomass from each chemovar was separately extracted in a
column with methanol at room temperature, and the pooled percolate
was collected. Solvent was removed by evaporation in a rotary
evaporator at a temperature not exceeding 43.degree. C. The
resulting high THC and high CBD extracts were dispersed in 5%
Polysorbate 80/normal saline and a Polysorbate/saline vehicle was
used as control. The high THC extract (M6) contained more than 10%
of THC and/or THCA and less than 1% of CBD and/or CBDA. The high
CBD extract (M16) contained more than 7.3% of CBD and/or CBDA and
less than 2% of THC and/or THCA.
[0050] A preliminary HPLC analysis of the methanolic extracts (and
comparative data for a botanical drug substance (BDS), when
prepared by sub critical CO.sub.2 extraction after decarboxylation)
is given in the table 2 below:
TABLE-US-00002 TABLE 2 Analyte Comparative Comparative Methanolic
Decarboxylated Decarboxylated extract from a CO.sub.2 extract from
CO.sub.2 extract CBD rich THC rich from CBD rich chemovar chemovar
chemovar Extract % w/w % w/w % w/w THC N.D. 64.2% 2.9% THCA 1.4%
N.D. N.D. CBD 6.1% 1.1% 70.2% CBDA 49.9% N.D. N.D CBN N.D. 1.0%
N.D.
[0051] As would be expected, the acid forms of the cannabinoid
predominate in the methanolic extract of the non-decarboxylated
herb.
[0052] For the methanolic extracts the % w/w of the principal
cannabinoid (sum of acid+neutral forms) is lower than found in the
equivalent BDS. Again, this is to be expected due to the lower
selectivity of the methanol, as compared to liquid CO.sub.2. Thus,
the methanolic extraction gives rise to the extraction of more
non-target material and a diluting of the active content of the
extract.
[0053] The content of the methanolic extract was confirmed by TLC
analysis and chromatogram traces for the high THC and high CBD
extracts are shown in FIGS. 1 and 2.
[0054] The traces in FIGS. 1 and 2 indicate the acid form of the
cannabinoid to be the principal component observed, with smaller
amounts of the corresponding neutral cannabinoid also being
detected.
[0055] Samples of the methanolic extracts were run on a TLC plate
along with some cannabinoid markers, and by way of comparison
botanic drug substance (BDS) (decarboxylated and extracted by sub
critical liquid CO.sub.2). The results are illustrated in FIG.
3.
[0056] Further analysis of the methanolic extracts, and by way of
comparison the BDS (decarboxylated and extracted by sub critical
liquid CO.sub.2), using gas chromatography indicates the presence
of terpenes at around the 14 to 18 minute mark. It should be noted
that because G.C. is operated at around 250.degree. C. any acid
form of the CBD and THC present in the extracts will be
decarboxylated and appear on the trace as CBD or THC. The results
are illustrated in FIGS. 4 and 5.
[0057] The most significant difference between the lower traces of
FIGS. 4 and 5 is the difference between the THC and CBD peaks.
EXAMPLE 2
[0058] Adult Suncus murinus (30-89 g bodyweight) of either sex were
injected with either G2 (M6) or G5 (M16) at a range of doses from
1.0, 2.0 or 4.0 mg/kg (or vehicle), intraperitoneally, 30 minutes
prior to a motion stimulus (1 Hz 40 mm amplitude of shaking for 10
minutes). The animals were observed for any overt behavioural
change. A number of emetic episodes and the latency of onset were
recorded. Data were expressed as the mean.+-.s.e.m., group size n=6
and statistically analysed using ANOVA, followed by Bonferronni
Dunnett's T test. The results are summarised in table 3 below.
TABLE-US-00003 TABLE 3 Effect of Cannabis Extracts given
Intraperitoneally on Frequency of Latency to First Emesis (means
.+-. s.e.m.) Dose Emetic Episodes, mean .+-. s.e.m. Treatment mg/kg
Latency (secs) P value Number P Value Vehicle 103.0 + 25.3 12.4 +
2.4 control M16 1.0 206.8 .+-. 33.9 <0.05 6.3 .+-. 1.4 <0.05
High CBD 2.0 250.5 .+-. 84.5 <0.05 6.6 .+-. 2.4 <0.05
chemovar 4.0 Increase N5 Increase N5 M6 1.0 .dagger. NDDC NDDC High
THC 2.0 NDDC NDDC chemovar 4.0* <100 >18 <0.05 .dagger.
NDDC no detectable difference from control *Increase in vomiting,
reduction in latency
[0059] The results for the high CBD producing chemovar extract
(M16) shown in Table 3 are not unlike U-shaped dose response curves
noted for some other pharmacological actions of cannabinoids.
[0060] The lack of effect of the high THC producing chemovar
extract in this test system is surprising. Indeed, at high doses
there appears to be an increase in vomiting and reduction in
latency. It was confirmed that neither M6 nor M16 had emetic
activity in their own right. These data further emphasise the
differences noted in the pharmacological effects of THC and CBD,
which have been investigated by the applicant.
EXAMPLE 3
Growing of Medicinal Cannabis
[0061] Plants are grown as clones from germinated seeds, under
glass at a temperature of 25.degree. C. .+-.1.5.degree. C. for 3
weeks in 24 hour daylight; this keeps the plants in a vegetative
state. Flowering is induced by exposure to 12 hour day length for
8-9 weeks.
[0062] No artificial pesticides, herbicides, insecticides or
fumigants are used. Plants are grown organically, with biological
control of insect pests.
[0063] The essential steps in production from seed accession to
dried Medicinal Cannabis are summarised as follows:
##STR00001##
EXAMPLE 4
Determination of Cannabinoid Content in Plants and Extracts
[0064] Identity by TLC
[0065] a) Materials and methods [0066] Equipment Application device
capable of delivering an accurately controlled volume of solution
i.e. 1 .mu.l capillary pipette or micro litre syringe. [0067] TLC
development tank with lid [0068] Hot air blower [0069] Silica gel G
TLC plates (SIL N-HR/UV254), 200 .mu.m layer with fluorescent
indicator on polyester support. [0070] Dipping tank for
visualisation reagent.
TABLE-US-00004 [0070] Mobile phase 80% petroleum ether 60:80/20%
Diethyl ether. Visualisation reagent 0.1% w/v aqueous Fast Blue B
(100 mg in 100 ml de-ionised water). An optional method is to scan
at UV 254 and 365 nm.
[0071] b) Sample preparation [0072] i) Herbal raw material
[0073] Approximately 200 mg of finely ground, dried cannabis is
weighed into a 10 ml volumetric flask. Make up to volume using
methanol:chloroform (9:1) extraction solvent. [0074] Extract by
ultrasound for 15 minutes. Decant supernatant and use directly for
chromatography. [0075] ii) Herbal drug Extract
[0076] Approximately 50 mg of extract is weighed into a 25 ml
volumetric flask. Make up to volume using methanol solvent. Shake
vigorously to dissolve and then use directly for
chromatography.
[0077] c) Standards
[0078] 0.1 mg/ml delta-9-THC in methanol.
[0079] 0.1 mg/ml CBD in methanol.
[0080] The standard solutions are stored frozen at -20.degree. C.
between uses and are used for up to 12 months after initial
preparation.
[0081] d) Test solutions and method [0082] Apply to points
separated by a minimum of 10 mm. [0083] i) either 5 .mu.l of herb
extract or 1 .mu.l of herbal extract solution as appropriate,
[0084] ii) 10 .mu.l of 0.1 mg/ml delta-9-THC in methanol standard
solution, [0085] iii) 10 .mu.l of 0.1 mg/ml CBD in methanol
standard solution. [0086] Elute the TLC plate through a distance of
8 cm, then remove the plate. Allow solvent to evaporate from the
plate and then repeat the elution for a second time (double
development). [0087] The plate is briefly immersed in the Fast Blue
B reagent until the characteristic red/orange colour of
cannabinoids begins to develop. The plate is removed and allowed to
dry under ambient conditions in the dark. [0088] A permanent record
of the result is made either by reproduction of the image by
digital scanner(preferred option) or by noting spot positions and
colours on a tracing paper.
[0089] Assay THC, THCA, CBD, CBDA and CBN by HPLC [0090] a)
Materials and methods [0091] Equipment: HP 1100 HPLC with diode
array detector and autosampler. The equipment is set up and
operated in accordance with in-house standard operating procedures
(SOP1ab037)
TABLE-US-00005 [0091] HPLC column Discovery C8 5 .mu.m, 15 .times.
0.46 cm plus Kingsorb ODS2 precolumn 5 .mu.m 3 .times. 0.46 cm.
Mobile Phase Acetonotrile:methanol:0.25% aqueous acetic acid
(16:7:6 by volume) Column Operating 25.degree. C. Temperature Flow
Rate 1.0 ml/min Injection Volume 10 .mu.l Run time 25 mins
Detection Neutral and acid cannabinoids 220 nm (band width 16 nm)
Reference wavelength 400 nm/bandwidth 16 nm Slit 4 nm Acid
cannabinoids are routinely monitored at 310 nm (band width 16 nm)
for qualitative confirmatory and identification purposes only. Data
capture HP Chemistation with Version A7.01 software
[0092] b) Sample preparation [0093] Approximately 40 mg of Cannabis
Based Medicinal Extract is dissolved in 25 ml methanol and this
solution is diluted to 1 to 10 in methanol. This dilution is used
for chromatography. [0094] 0.5 ml of the fill solution, contained
within the Pump Action Sublingual Spray unit, is sampled by glass
pipette. The solution is diluted into a 25 ml flask and made to the
mark with methanol. 200 .mu.l of this solution is diluted with 800
.mu.l of methanol. [0095] Herb or resin samples are prepared by
taking a 100 mg sample and treating this with 5 or 10 ml of
Methanol/Chloroform (9/1 w/v). The dispersion is sonicated in a
sealed tube for 10 minutes, allowed to cool and an aliquot is
centrifuged and suitably diluted with methanol prior to
chromatography.
[0096] c) Standards
[0097] External standardisation is used for this method. Dilution
of stock standards of THC, CBD and CBN in methanol or ethanol are
made to give final working standards of approximately accurately
0.1 mg/ml. The working standards are stored at -20.degree. C. and
are used for up to 12 months after initial preparation.
[0098] Injection of each standard is made in triplicate prior to
the injection of any test solution. At suitable intervals during
the processing of test solutions, repeat injections of standards
are made. In the absence of reliable CBDA and THCA standards, these
compounds are analysed using respectively the CBD and THC standard
response factors.
[0099] The elution order has been determined as CBD, CBDA, CBN, THC
and THCA. Other cannabinoids are detected using this method and may
be identified and determined as necessary.
[0100] d) Test solutions
[0101] Diluted test solutions are made up in methanol and should
contain analytes in the linear working range of 0.02-0.2 mg/ml.
[0102] e) Chromatography Acceptance Criteria:
[0103] The following acceptance criteria are applied to the results
of each sequence as they have been found to result in adequate
resolution of all analytes (including the two most closely eluting
analytes CBD and CBDA) [0104] i) Retention time windows for each
analyte: [0105] CBD 5.4-5.9 minutes [0106] CBN 7.9-8.7 minutes
[0107] THC 9.6-10.6 minutes [0108] ii) Peak shape (symmetry factor
according to BP method) [0109] CBD<1.30 [0110] CBN<1.25
[0111] THC<1.35 [0112] iii) A number of modifications to the
standard method have been developed to deal with those samples
which contain late eluting impurity peaks e.g method CBD2A extends
the run time to 50 minutes. All solutions should be clarified by
centrifugation before being transferred into autosampler vials
sealed with teflon faced septum seal and cap. [0113] iv) The
precolumn is critical to the quality of the chromatography and
should be changed when the back pressure rises above 71 bar and/or
acceptance criteria regarding retention time and resolution, fall
outside their specified limits.
[0114] f) Data Processing
[0115] Cannabinoids can be subdivided into neutral and acidic-the
qualitative identification can be performed using the DAD dual
wavelength mode. Acidic cannabinoids absorb strongly in the region
of 220 nm-310 nm. Neutral cannabinoids only absorb strongly in the
region of 220 nm.
[0116] Routinely, only the data recorded at 220 nm is used for
quantitative analysis.
[0117] The DAD can also be set up to take UV spectral scans of each
peak, which can then be stored in a spectral library and used for
identification purposes.
[0118] Data processing for quantitation utilises batch processing
software on the Hewlett Packard Chemstation.
[0119] a) Sample Chromatograms
[0120] HPLC sample chromatograms for THC and CBD Herbal Drug
extracts are provided in the accompanying Figures.
EXAMPLE 5
Preparation of the Herbal Drug Extract
[0121] A flow chart showing the process of manufacture of extract
from the High-THC and High-CBD (non acid and acid forms) chemovars
is given below:
EXAMPLE 6
[0122] High CBD cannabis was grown under glass at a mean
temperature of 21+2.degree. C., RH 50-60%. Herb was harvested and
dried at ambient room temperature at a RH of 40-45% in the dark.
When dry, the leaf and flower head were stripped from stem and this
dried biomass is referred to as "medicinal cannabis".
[0123] Medicinal cannabis was reduced to a coarse powder (particles
passing through a 3 mm mesh) and packed into the chamber of a
Supercritical Fluid Extractor. Packing density was 0.3 and liquid
carbon dioxide at a pressure of 600 bar was passed through the mass
at a temperature of 35.degree. C. Supercritical extraction is
carried out for 4 hours and the extract was recovered by stepwise
decompression into a collection vessel. The resulting green-brown
oily resinous extract is further purified. When dissolved in
ethanol BP (2 parts) and subjected to a temperature of -20.degree.
C. for 24 hours a deposit (consisting of fat-soluble, waxy
material) was thrown out of solution and was removed by filtration.
Solvent was removed at low pressure in a rotary evaporator. The
resulting extract is a soft extract which contains approximately
60% CBD with up to 4% tetrahydrocannabinol, within a total of other
cannabinoids of 6%. Extracts were made using THCV and CBDV
chemovars using the general method described above.
[0124] A high THC chemovar was similarly treated and yielded an
extract containing approximately 60% THC and approximately 6% of
other cannabinoids of which 1-2% is cannabidiol and the remainder
is minor cannabinoids including cannabinol. Quantitative yield was
9% w/w based on weight of dry medicinal cannabis.
[0125] A person skilled in the art will appreciate that other
combinations of temperature and pressure (in the range +10.degree.
C. to 35 .degree. C. and 60-600 bar) can be used to prepare
extracts under supercritical and subcritical conditions.
EXAMPLE 7
Preparation of CBDA
[0126] Summary of process:
[0127] Yield:
[0128] 100 g of G5 chemovar yields approx 5 g of purified CBDA.
[0129] Characteristics:
[0130] Pale yellow crystalline solid
[0131] Melting Point =45-48.degree. C.
[0132] Chromatographic purity=94% CBDA by area normalisation
[0133] *CBD 3%.
[0134] THCA non detected i.e. <0.1%
[0135] THC non detected i.e. <0.1%
[0136] Material slowly decarboxylates in solution
[0137] CBDA.fwdarw.CBD+CO.sub.2 * As CBDA does not co-elute with
CBD during processing of the extract in the low pressure column
chromatography method employed, the detected CBD is likely to be
formed from the breakdown of the CBDA during processing and
analysis. This undesirable decarboxylation of the purified material
might be minimised by manipulation of CBDA at sub-ambient
temperatures.
EXAMPLE 8
Purification of CBD
Overview of Process
[0138] Starting from freshly harvested plant material the process
comprises drying and decarboxylation of the plant material,
optional treatment (e.g. milling) of the dried plant material to
reduce the particle size (preferably to less than 2000 .mu.m),
extraction with liquid carbon dioxide, ethanolic precipitation to
reduce the amount of non-target material, clean-up of the crude
ethanolic extract by passage through activated charcoal, removal of
solvent (ethanol) to produce a CBD-enriched fraction, and
re-crystallisation of CBD from pentane.
Plant Material
[0139] GW Pharma Ltd has developed distinct varieties of Cannabis
plant hybrids to maximise the output of the specific chemical
constituents, cannabinoids. A "high CBD" chemovar designated G5
produces >90% total cannabinoid content as CBD (naturally
occurring in the plant in the form of CBDA). Alternative "high CBD"
varieties can be obtained--see for example, Common cannabinoids
phenotypes in 350 stocks of cannabis, Small and Beckstead, Lloydia
vol 36b , 1973, p144-156 -and bred using techniques well known to
the skilled man to maximise cannabinoid content.
Solvents
[0140] All solvents used in the isolation and analysis of CBD (e.g.
n-pentane) were, unless otherwise stated, of chromatographic or
A.R. grade.
Standards
[0141] Reference materials from Sigma were used as standards in the
analysis of extracts, intermediates and finished products, these
were: .DELTA..sup.9 THC in methanol BN 10601/B (ca. 1 mg/ml) and
CBD in methanol BN 10601/C (ca. 1 mg/ml).
Preparation of a Cannabidiol-Containing Extract
[0142] A cannabidiol-containing extract is prepared from a "high
CBD" cannabis chemovar according to the following process:
[0143] Prepare ethanolic solution of botanical drug substance as
follows:
##STR00002##
[0144] Extraction using liquid CO.sub.2 is carried out under
sub-critical conditions at a temperature of approximately
10.degree. C. .+-.5.degree. C. using a pressure of approximately 60
bar .+-.10 bar. Decarboxylated plant material is packed into a
single column and exposed to liquid CO.sub.2 under pressure for
approximately 8 hours, CO.sub.2 mass flow 1250 kg/hr .+-.20%.
[0145] Following depressurisation and venting off of the CO.sub.2
the crude BDS extract is collected into sealed vessels. The crude
BDS extract is held at -20.degree. C. .+-.5.degree. C.
[0146] The crude BDS extract contains waxes and long chain
molecules. Removal is by "winterisation", whereby the crude BDS
extract is warmed to e.g. 40.degree. C. .+-.4.degree. 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.degree. C. .+-.5.degree. C. and held at this
temperature for approximately 48 hours.
[0147] On completion of the winterisation the precipitate is
removed by cold filtration through a 20 .mu.m filter, to give an
ethanolic solution of the BDS.
[0148] Preliminary charcoal clean-up may be carried out by passing
the ethanolic BDS solution (400-500 mg/ml) through a disposable
plastic column (130 mm.times.27 mm i.d.) packed with activated
charcoal (decolourcarb DCL GDC grade, from Sutcliffe Speakman
Carbons, 15.4 g per unit). Absolute ethanol B.P. (Hayman) is used
as the solvent.
[0149] Ethanol and any water that may be present are removed by
rotary evaporation or thin film evaporation under reduced
pressure(60.degree. C. .+-.2.degree. C., with vapour at 40.degree.
C. .+-.2.degree. C./172 mbar and 72 mbar .+-.4 mbar) to produce a
CBD-rich extract.
Solvent Re-Crystallisation
[0150] The CBD-rich extract is re-dissolving in a suitable solvent
(e.g. n-pentane) and filtered to remove insoluble material. Solvent
is then removed, e.g. by rotary evaporation, to produce crystalline
CBD. All steps are carried out according to standard laboratory
procedures, such as would be known to those skilled in the art.
[0151] Product Characteristics
[0152] Yield:
[0153] 3 g of CBD BDS yields approx 1 g of purified CBD.
[0154] Characteristics:
[0155] White crystalline solid.
[0156] Chromatographic purity >99% CBD by area
normalization.
[0157] Chromatographic purity superior to commercially available
CBD Sigma standard (refer to FIGS. 1 and 3).
[0158] THC non detected i.e. <0.1%
[0159] CBN non detected i.e. <0.1%
[0160] Identity confirmed by HPLC, GC and TLC retention behaviour
compared to CBD Sigma standard.
[0161] Assay vs both Sigma CBD std in range 98.0-102.0% Melting
Point=64-66 .degree. C. (literature value=66-67.degree. C.)
[0162] HPLC Analysis
[0163] The composition of the isolated products may be determined
by HPLC analysis.
[0164] A typical HPLC assay for .DELTA..sup.9 THC, .DELTA..sup.9
THCA, CBD, CBDA and CBN may be carried out as follows:
[0165] a) Materials and methods
[0166] Chromatography Equipment and conditions:
TABLE-US-00006 Equipment Agilent (HP)1100 HPLC system with variable
wavelength UV detector or diode array detector. HPLC Column
Discovery C8 5 .mu.m 15 cm .times. 0.46 cm Pre-Column Kingsorb C18
5 .mu.m 3 cm .times. 0.46 cm Mobile Phase
Acetonitrile:Methanol:0.25% w/v acetic acid (16:7:6 by volume)
Column Temp 25.degree. C. Flow Rate 1.0 ml min-1 Detection 220 nm
600 mA f.s.d. Second wavelength 310 nm Injection 10 .mu.l Volume
Run Time 20-25 minutes (may be extended for samples containing
small amount of late-eluting peaks) Elution Order CBD, CBDA,
.DELTA..sup.9 THCV, CBN, .DELTA..sup.9 THC, CBC, .DELTA..sup.9
THCA
[0167] b) Sample preparation
[0168] Samples of "pure" cannabidiol are diluted in methanol prior
to HPLC analysis. Optimal dilutions may be determined
empirically.
[0169] Herbal cannabis samples are prepared by taking a 100 mg
sample and treating this with 5 or 10 ml of Methanol/Chloroform
(9/1 w/v). The dispersion is sonicated in a sealed tube for 10
minutes, allowed to cool and an aliquot is centrifuged and suitably
diluted with methanol prior to chromatography.
[0170] c) Standards
[0171] Stock standard solutions of CBD, CBN and .DELTA..sup.9 THC
in methanol at approximately 1 mg ml.sup.-1 are stored at
-20.degree. C. Diluted working standards (0.1 mg/ml for
.DELTA..sup.9 THC and CBD and 0.01 mg/ml for CBN) are prepared in
methanol from the stock standards and stored at -20.degree. 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.
[0172] Injection of each standard is made in triplicate prior to
the injection of any test solution. At suitable intervals during
the processing of test solutions, repeat injections of standards
are made. In the absence of reliable CBDA and .DELTA..sup.9 THCA
standards, these compounds are analysed using respectively the CBD
and .DELTA..sup.9 THC standard response factors.
[0173] d) Test solutions
[0174] Diluted test solutions are made up in methanol and should
contain analytes in the linear working range of 0.02-0.2 mg/ml.
[0175] e) Chromatography Acceptance Criteria:
[0176] The following acceptance criteria are applied to the results
of each sequence as they have been found to result in adequate
resolution of all analytes (including the two most closely eluting
analytes CBD and CBDA)
TABLE-US-00007 TABLE 4 Retention time windows and Relative
Retention Time (RRT) to .DELTA..sup.9 THC for each analyte
Retention time Cannabinoid (minutes) RRT (THC) CBD 5.1-5.8 0.58 CBN
7.4-8.3 0.83 .DELTA..sup.9 THC 9.0-10.0 1.00 CBDA 5.5-6.2 0.615
.DELTA..sup.9 THCV 5.9-6.2 0.645 CBC 11.6-12.8 1.30 .DELTA..sup.9
THCA 14.6-16.0 1.605
TABLE-US-00008 TABLE 5 Peak Shape (Symmetry Factor according to
British Pharmacopoeia method) Cannabinoid Symmetry factor CBD
<1.30 CBN <1.25 .DELTA..sup.9 THC <1.35
[0177] f) Data Processing
[0178] Cannabinoids can be subdivided into neutral and acidic-the
qualitative identification can be performed using the DAD dual
wavelength mode. Acidic cannabinoids absorb strongly in the region
of 220 nm-310 nm. Neutral cannabinoids only absorb strongly in the
region of 220 nm.
[0179] Routinely, only the data recorded at 220 nm is used for
quantitative analysis.
[0180] The DAD can also be set up to take UV spectral scans of each
peak, which can then be stored in a spectral library and used for
identification purposes.
[0181] Data processing for quantitation utilises batch processing
software on the Hewlett Packard Chemstation.
[0182] g) calculation:
[0183] Chromatographic purity of cannabinoid samples is calculated
as a % of total cannabinoid content by area normalization.
[0184] Capillary Gas Chromatography (GC) Analysis
[0185] a) Chromatography equipment and conditions
TABLE-US-00009 Equipment Agilent (HP) 5890 or 6890 GLC system with
HP7673 Autosampler and FID detector GLC column SE54(EC5) 30 m
.times. 0.32 mm i.d. (Alltech) phase thickness 0.25 .mu.m Flow rate
Constant pressure (10.3 psi). Normal initial flow rate 34 cm
sec.sup.-1 (2.0 ml min.sup.-1) Column oven 70.degree. C. initially
then ramp 5.degree. C. min.sup.-1 to 250.degree. C. Hold at
250.degree. C. for 15 minutes. Injector temp 250.degree. C.
Detector temp 325.degree. C. Injection Vol 1 .mu.l, split ratio
2.5:1 Run time 45 minutes Fuel gases Hydrogen 40 ml min.sup.-1 Air
450 ml min.sup.-1 Helium 45 ml min.sup.-1
[0186] b) Standard preparation
[0187] Stock standard solutions of CBD, CBN and .DELTA..sup.9 THC
in methanol at approximately 1 mg ml.sup.-1 are stored at
-20.degree. C. Diluted working standards (0.1 mg/ml for
.DELTA..sup.9 THC and CBD and 0.01 mg/ml for CBN) are prepared in
methanol from the stock standards and stored at -20.degree. C.
(maximum period of twelve months after initial preparation). Allow
an aliquot pipetted into an autosampler vial to equilibriate to
room temperature prior to use in a GC assay.
[0188] c) Sample preparation
[0189] Samples of final products, i.e. "pure" cannabidiol, are
diluted in methanol prior to HPLC analysis. Optimal dilutions may
be determined empirically.
[0190] Cannabis plant material samples are prepared by taking 100
mg chopped dried material and treating this with 5 or 10 ml of
Methanol/Chloroform (9:1 v/v). Extract the sample in an ultrasonic
bath for 15 minutes and allow to stand in the dark for 18
hours.
[0191] d) Chromatography procedure
[0192] Standard solutions are used to provide quantitative and
retention time data. These can be typically injected in 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.
TABLE-US-00010 TABLE 6 Retention times THCV 33.7-34.5 minutes CBD
35.6-36.3 minutes .DELTA..sup.9 THC 37.2-38.1 minutes CBN 38.5-39.1
minutes
[0193] TLC analysis
[0194] The qualitative composition of final products and starting
materials may also be monitored by TLC.
[0195] TLC uses both retention time and characteristic spot colour
to effectively identify the cannabinoid/cannabinoid acid components
in a complex mixture. Methanolic solutions of the final products
and starting material, plus standards, are prepared for TLC. An
aliquot is spotted onto a TLC plate, alongside suitable reference
samples (e.g. for at least .DELTA..sup.9 THC and CBD). Following
exposure to Fast Blue B reagent, THC and THCA present as pink
spots, while CBD and CBDA are orange
[0196] 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.
[0197] A typical TLC protocol is as follows:
[0198] a) Materials and methods
[0199] Equipment:
[0200] Application device capable of delivering an accurately
controlled volume of solution i.e 1 .mu.l capillary pipette or
micro litre syringe.
[0201] TLC development tank with lid
[0202] Hot air blower
[0203] Silica gel G TLC plates (SIL N-HR/UV254), 200 .mu.m layer
with fluorescent indicator on polyester support.
[0204] Dipping tank for visualisation reagent.
TABLE-US-00011 Mobile phase 80% petroleum ether 60:80/20% Diethyl
ether. Visualisation reagent 0.1% w/v aqueous Fast Blue B salt BN
(Sigma Corp) (100 mg in 100 ml de-ionised water). An optional
method is to scan at UV 254 and 365 nm.
[0205] b) Sample preparation
[0206] i) Herbal raw material
[0207] Approximately 200 mg of finely ground, dried cannabis is
weighed into a 10 ml volumetric flask. Make up to volume using
methanol:chloroform (9:1) extraction solvent.
[0208] Extract by ultrasound for 15 minutes. Decant supernatant and
use directly for chromatography.
[0209] ii) Final products
[0210] The final products (crystalline CBD) are dissolved in
methanol to a suitable concentration (which may be determined
empirically) then used directly for chromatography. All sample
preparations should produce a final concentration of about 0.5
mg/ml.
[0211] iii) Botanical drug substance
[0212] Accurately weigh approximately 50 mg of botanical drug
substance into a 25 ml volumetric flask. Dissolve to make volume
with HPLC grade methanol.
[0213] c) Standards
[0214] 0.1 mg/ml .DELTA..sup.9-THC in methanol (Sigma).
[0215] 0.1 mg/ml CBD in methanol (Sigma).
[0216] The standard solutions are stored frozen at -20.degree. C.
between uses and are used for up to 12 months after initial
preparation.
[0217] d) Test solutions and method
[0218] Apply to points separated by a minimum of 10 mm.
[0219] i) either 5 .mu.l of herb extract or 1 .mu.l of pure
cannabinoid/enriched extract solution or 1 .mu.l of diluted column
eluate as appropriate,
[0220] ii) 5 .mu.l of 0.1 mg/ml .DELTA..sup.9-THC in methanol
standard solution,
[0221] iii) 5 .mu.l of 0.1 mg/ml CBD in methanol standard
solution.
[0222] Dry the prepared plate with a hot air blower.
[0223] Place the base of the TLC plate in a development tank
containing the mobile phase and saturated with vapour.
[0224] Elute the TLC plate through a distance of 8 cm, then remove
the plate. Allow solvent to evaporate from the plate and then
repeat the elution for a second time (double development). Remove
plate and allow it to dry in air.
[0225] The entire plate is briefly immersed in the Fast Blue B
reagent until the characteristic red/orange colour of cannabinoids
begins to develop. The plate is removed and allowed to dry under
ambient conditions in the dark.
[0226] Cannabinoids will give an orange-purple colour:
TABLE-US-00012 Cannabidiol CBD orange (fastest running)
.DELTA..sup.9 Tetrahydrocannabinol THC pink Cannabinol CBN purple
Cannabichromene CBC pink purple Cannabigerol CBG orange
.DELTA..sup.9 tetrahydrocannabivarin THCV purple
[0227] The corresponding acids form streaks of the same colour as
the neutral component spots. The acids run at lower R.sub.f.
* * * * *