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Preparative Isolation of Cannabinoids from Cannabis sativa by Centrifugal Partition Chromatography

Authors:
  • Hazekamp Herbal Consulting

Abstract

A simple method is presented for the preparative isolation of seven major cannabinoids from Cannabis sativa plant material. Separation was performed by centrifugal partition chromatography (CPC), a technique that permits large‐scale preparative isolations. Using only two different solvent systems, it was possible to obtain pure samples of the cannabinoids; (−)‐Δ‐(trans)‐tetrahydrocannabinol (Δ‐THC), cannabidiol (CBD), cannabinol (CBN), cannabigerol (CBG), (−)‐Δ‐(trans)‐tetrahydrocannabinolic acid‐A (THCA), cannabigerolic acid (CBGA), and cannabidiolic acid (CBDA). A drug‐type and a fiber‐type cannabis cultivar were used for the isolation. All isolates were shown to be more than 90% pure by gas chromatography. This method makes acidic cannabinoids available on a large scale for biological testing. The method described in this report can also be used to isolate additional cannabinoids from cannabis plant material.
i
Cannabis; extracting the medicine
ii
Arno Hazekamp
Cannabis; extracting the medicine
Proefschrift Universiteit Leiden
ISBN 978-90-9021997-4
Printed by PrintPartners Ipskamp B.V., Amsterdam, The Netherlands
Paper cover: Cannabis Pur 100% (250 grams) from Grafisch Papier, The Nederlands.
Photo cover: Dutch medicinal cannabis, variety “Bedrocan”.
iii
Cannabis; extracting the medicine
Proefschrift
Ter verkrijging van
de graad van Doctor aan de Universiteit Leiden,
op gezag van de Rector Magnificus prof. mr. P. F. van der Heijden,
hoogleraar in de faculteit der Rechtsgeleerdheid,
volgens besluit van het College voor Promoties
te verdedigen op woensdag 5 september 2007
klokke 15.00 uur
door
Arno Hazekamp
Geboren op 15 maart 1976
te Bilthoven
iv
Promotiecommissie
Promotor Prof. dr. R. Verpoorte
Referent Dr. C. Giroud
(Institut Universitaire de Médecine Légale, Lausanne, Switzerland)
Overige leden Prof. dr. M. Danhof
Prof. dr. C. A. M. J. J. van den Hondel
Prof. dr. J. J. C. Scheffer
Dr. R. van der Heijden
The printing of this thesis was supported by grants of the following sponsors:
Storz & Bickel GmbH & Co. KG, Tuttlingen, Germany
Farmalyse BV, Zaandam, The Netherlands
Nationaal MS-fonds, Maassluis, The Netherlands
Multidisciplinary Association for Psychedelic Studies (MAPS), California, USA
Bedrocan BV, Veendam, The Netherlands
Mr. Michael Sautman, California, USA
v
Contents
Chapter 1 A general introduction to cannabis as medicine 1
Chapter 2 An evaluation of the quality of medicinal grade cannabis 25
in the Netherlands
Chapter 3 Preparative isolation of cannabinoids from Cannabis sativa 39
by centrifugal partition chromatography
Chapter 4 Quantitative analysis of cannabinoids from Cannabis sativa 53
using
1
H-NMR
Chapter 5 Synthesis and spectroscopic characterization of cannabinolic acid 63
Chapter 6 Chromatographic and spectroscopic data of cannabinoids from 71
Cannabis sativa L.
Chapter 7 Development and validation of a reversed-phase HPLC method for 91
the determination of major cannabinoids from medicinal grade
Cannabis sativa plant material
Chapter 8 Cannabis tea revisited: a systematic evaluation of the cannabinoid 107
composition of cannabis tea
Chapter 9 Structure elucidation of the tetrahydrocannabinol complex with 119
randomly methylated beta-cyclodextrin
Chapter 10 Evaluation of a vaporizing device (Volcano®) for the pulmonary 133
administration of tetrahydrocannabinol
Concluding remarks and perspectives 149
Summary 151
Samenvatting 157
References 165
Acknowledgements 177
Curriculum vitae 179
List of publications 181
1
CHAPTER 1
A general introduction to cannabis as medicine
• • •
Arno Hazekamp, Renee Ruhaak
• •
Leiden University, Department of Pharmacognosy, Gorlaeus Laboratories
Leiden, The Netherlands
Chapter 1
2
1.1 Cannabis as a medicine
It is hard to think of a medical topic that so strongly divides the research community as the
medicinal use of cannabis. It can probably be said that cannabis is the most controversial plant
in the history of mankind. But surely, if the plant Cannabis sativa would be discovered today,
growing in some remote spot of the world, it would be hailed as a wonder of nature; a new
miracle plant with the potential to treat anything ranging from headaches to neurological
disorders to cancer. It is therefore interesting to notice that, even after decades of research,
cannabis is probably most well known for causing anxiety, agitation and paranoia among
politicians, while its medicinal potential continues to be disputed.
Interestingly, delta-9-tetrahydrocannabinol (THC), the main component of the cannabis
plant, and one of the most renowned plant compounds of the world, is in fact already
acknowledged as a medicine. It has been available to patients since 1986 under the name
Marinol®, which is prescribed to treat nausea, pain and loss of appetite. So even if cannabis
was nothing more than an herbal receptacle of THC, it should at least be accepted as some
generic form of this registered medicine. However, on multiple levels (in vivo, in vitro, in
clinical trials) it is becoming increasingly clear that THC alone does not equal cannabis
[Williamson 2000; Russo 2003], pointing out that other components are necessary to explain
the claimed medicinal effects.
Cannabis has the potential to evolve into a useful and much needed medicine, but is seriously
obstructed by its classification as a dangerous narcotic. However, as shown in the case of the
opium plant (Papaver somniferum) and the opiates derived from it (e.g. morphine, codeine),
the distinction between a dangerous drug of abuse and a medicine can be made by proper,
unbiased and well conducted research. Hopefully this thesis can be a contribution to a more
rational approach to cannabis as a medicine.
1.2 The cannabis plant and its constituents
1.2.1 Forms of cannabis
Today, cannabis is the most commonly used psychoactive drug worldwide, together with
coffee and tobacco, and it is the single most popular illegal drug. Worldwide over 160 million
people are using cannabis regularly and these numbers are still rising [World Drug Report,
2006]. But what exactly is cannabis anyway? With such high popular demand, it is not
surprising that cannabis and its products are known under a large variety of names. Some of
the most widely used ones are defined here.
The commonly used term ‘marijuana’ or ‘marihuana’ traditionally describes the cannabis
plant when used as a recreational drug, and is frequently associated with the negative effects or
social impact of the drug (figure 1.1). ‘Weed’ is another name for cannabis when used as a
recreational drug. When the term ‘hemp’ is used, it usually refers to the use of cannabis as a
source of fiber, making the term ‘fiber-hemp’ therefore somewhat superfluous. Because of the
Introduction
3
inexact and unscientific nature of these
terms, they will not be used in this thesis.
Instead, the proper scientific name
“cannabis” will be consistently used to
describe the plant Cannabis sativa L. in all its
varieties.
When talking about cannabis for either
recreational or medicinal use, what is usually
referred to are the female flowers (‘flos’),
being the most potent part of the plant. The
dried resin obtained from these flowers is
generally known as ‘hash’, or ‘hashish’,
although a large variety of names exists. This
resin is the origin of the most important
bioactive components of the cannabis plant,
the ‘cannabinoids’, which will be the main
focus throughout this thesis.
Finally, ‘dronabinol’ is another name for the
naturally occurring (-)-trans-isomer of THC,
often used in a medical context in the
scientific and political literature, and adopted
by the World Health Organization.
1.2.2 The botany of cannabis
The basic material of all cannabis products is the plant Cannabis sativa L (figure 1.2). It is an
annual, usually dioecious, more reraly monoecious, wind-pollinated herb, with male and
female flowers developing on separate plants. It propagates from seed, grows vigorously in
open sunny environments with well drained soils, and has an abundant need for nutrients and
water. It can reach up to 5 meters (16 feet) in height in a 4 to 6 month growing season.
However, in modern breeding and cultivation of recreational cannabis, the preferred way to
propagate the plants is by cloning, using cuttings of a so-called ‘mother plant’. As this term
indicates, female plants are used for this purpose, as they produce significantly higher
amounts of psychoactive compounds than the male plants.
The sexes of Cannabis are anatomically indistinguishable before they start flowering, but after
that, the development of male and female plants varies greatly (figure 1.3). Shorter days (or
more accurately longer nights) induce the plant to start flowering [Clarke, 1981]. The female
plant then produces several crowded clusters of individual flowers (flowertops); a large one at
the top of the stem and several smaller ones on each branch, while the male flowers hang in
loose clusters along a relatively leafless upright branch. The male plants finish shedding pollen
Figure 1.1: Marihuana, the “assassin of youth”.
Assassin of Youth (1937) is a pre-WWII movie about
the negative effects of marijuana, reflecting the
hysterical anti-drug propaganda of its time.
Chapter 1
4
and die before the seeds in the female plants ripen four to eight weeks after being fertilized. A
large female can produce over one kilogram of seed. If the seed survives, it may germinate the
next spring.
Figure 1.2: Cannabis sativa L. Scientific drawing from Franz Eugen Köhler's Medizinal-Pflanzen. Published and
copyrighted by Gera-Untermhaus, FE Köhler in 1887 (1883–1914). The drawing is signed W. Müller.
Introduction
5
According to current botanical classification, Cannabis belongs with Humulus (hops) to the
family of Cannabinaceae (also Cannabaceae and Cannabidaceae [Frohne, 1973; Turner, 1980;
Schultes, 1980]. Despite this relationship, cannabinoids can only be found in Cannabis sativa.
In the genus Humulus and also in crafting experiments between Cannabis and Humulus no
cannabinoids have been found [Crombie, 1975; Fenselau, 1976]. The current systematic
classification of Cannabis is [Lehmann, 1995]:
Division Angiosperms
Class Dicotyledon
Subclass Archichlamydeae
Order Urticales
Family Cannabinaceae
Genus Cannabis
Species sativa L.
Because of centuries of breeding and selection, a large variation of cultivated varieties (or
cultivars) has been developed. Recently, more than 700 different cultivars were described
[Snoeijer, 2001] and many more are thought to exist. As a result, there has been extensive
discussion about further botanical and chemotaxonomic classification. So far, several
classifications of cannabis have been proposed: a classification into Cannabis sativa L., C.
indica LAM. and C. ruderalis JANISCH [Schultes, 1974; Anderson, 1974; Emboden, 1974] or
Cannabis sativa L. ssp. Sativa and ssp. Indica [Small, 1976a,b; Cronquist, 1981]. However, it is
becoming commonly accepted that Cannabis is monotypic and consists only of a single
species Cannabis sativa, as described by Leonard Fuchs in 16
th
century [Beutler, 1978; Lawi-
Berger, 1982a,b; Brenneisen, 1983].
To solve the controversy in a biochemical way, a first chemical classification was done by Grlic
[1968], who recognized different ripening stages. Fettermann [1971b] described different
phenotypes based on quantitative differences in the content of main cannabinoids and he was
the first to distinguish the drug- and fiber- type. Further extension and perfection of this
approach was subsequently done by Small and Beckstead [1973], Turner [1979] and
Brenneisen [1987]. It was found that a single plant could be classified into different
phenotypes, according to age. Although these chemotaxonomic classifications don’t strictly
define the contents of main cannabinoids for each chemotype, it does provide a practical tool
for classification. A final validation of Cannabis classification awaits further chemotaxonomic
and genetic research.
For forensic and legislative purposes, the most important classification of Cannabis types is
that into the fiber-type and the drug-type. The main difference between these two is found in
the content of the psychotropically active component 9-tetrahydrocannabinol (THC): a high
content of THC classifies as a drug-type cannabis, while a low THC content is found in fiber-
type cannabis. All cannabis varieties presently used for medicinal purposes belong to the drug-
type, because of their high content of the biologically active THC. But although fiber-type
Chapter 1
6
cannabis is commonly not used for medicinal or recreational purpose, it does contain
components that have been found to be biologically active, indicating that the distinction
between the two types has limited relevance for medicinal research into cannabis.
Figure 1.3: Photograph and drawing of male and female flowers of cannabis. Reprinted with permission of Ed
Rosenthal.
Introduction
7
1.2.3 History of cannabis as a useful plant
Cannabis most likely originates from Central Asia, as
archeological evidence indicates it was cultivated in China for
food and fiber already 10.000 years ago. Also in ancient Egyptian
mummies clues have been found for the use of cannabis as food
or medicine [Balabanova, 1992]. In fact, cannabis is one of the
oldest known medicinal plants and is described in almost every
ancient handbook on plant medicine, most comonly in the form
of a tincture or a tea [Zuardi, 2006; Grotenhermen, 2002]. Some
religions were closely related with the properties of the cannabis
plant. For example, in Hindu legend cannabis is believed to be
the favorite food of the god Shiva, because of its energizing
properties. As cannabis spread from Asia towards the West,
almost every culture came into contact with this miracle plant.
Nowadays, cannabis can be found in all temperate and tropical
zones, except in humid, tropical rainforests [Conert, 1992].
As a fiber plant cannabis produces some of the best and most durable fibers of natural origin.
For a long time in history these fibers were used to produce sails for sea-ships, paper,
banknotes and even the first Levi’s jeans. The oil of the hempseed has been suggested to be
well balanced in regards to the ratio of linoleic and linolenic acids for human nutrition.
Furthermore, the oil because of this feature and the presence of gamma-linolenic acid, is ideal
as an ingredient for body oils and lipid-enriched creams [Oomah, 2002].
Despite the fact that cannabis was grown on a large scale in most countries, the abuse as a
narcotic remained uncommon in Europe or the United States untill relatively recently. People
were largely unaware of the psychoactive properties of cannabis and it is unlikely that early
cultivars, selected mainly for their fiber qualities, contained significant amounts of the
psychoactive compound THC. The medicinal use of cannabis was only introduced in Europe
around 1840, by a young Irish doctor, William O’Shaughnessy, who served for the East India
Trading Company in India, where the medicinal use of cannabis was widespread. Unlike the
European fiber cannabis, these Indian varieties did contain a reasonable amount of bioactive
compounds. In the following decades cannabis knew a
short period of popularity both in Europe and the United
States. At the top of its popularity, more than 28 different
medicinal preparations were available with cannabis as
active ingredient, which were recommended for
indications as various as menstrual cramps, asthma,
cough, insomnia, support of birth labor, migraine, throat
infection and withdrawal from opium use
[Grotenhermen, 2002].
However, difficulties with the supply from overseas and
Chapter 1
8
varying quality of the plant material made it difficult to prepare a reliable formulation of
cannabis. Because no tools existed for quality control it was impossible to prepare a
standardized medicine, so patients often received a dose that was either too low, having no
effect, or too high, resulting in serious side effects. Moreover, cannabis extract was not water-
soluble and could not be injected, while oral administration was found to be unreliable
because of its slow and erratic absorption. Because of such drawbacks the medicinal use of
cannabis increasingly disappeared in the beginning of the twentieth century. When finally a
high tax was imposed on all cannabis-based products (seeds and fibers excluded) and
increasingly restrictive legislation was introduced for cannabis abuse, the medicinal use of
cannabis gradually disappeared from all Western pharmacopoeias in the period from 1937
[Grotenhermen and Russo, 2002]. In contrast to the alkaloid drugs codeine and morphine,
which are derived from opium, isolation of the pure active
substances from cannabis was not achieved until the 1960s
[Gaoni, 1964a].
Only since the flower-power-time of the 1960s, the smoking of
cannabis as a recreational drug has become a widely known
phenomenon in the Western world. From then on, import of
stronger varieties from the tropics, combined with a growing
interest in breeding, initially most notably among American
Vietnam war veterans, led to a steady increase in psychoactive
potency. Contemporary recreational cannabis has increasingly
become a high-tech crop, grown indoors under completely
artificial conditions.
1.2.4 Cannabis constituents
With over 420 known constituents, Cannabis is one of the chemically best studied plants
[Turner, 1980; Ross, 1995]. Most interesting among these constituents are the secretions of
the head cells of glandular hairs (trichomes) distributed across the surface of the cannabis
plant (figure 1.4). Although trichomes can be found all over the male and female plants, they
are particularly concentrated at some parts of the female inflorescence. Solitary resin glands,
consisting of one or two dozen cells, most often form at the tips of slender trichome stalks
which form as extensions of the plant surface. These glands secrete an aromatic terpenoid-
containing resin with a very high content of cannabinoids, which collects under a thin waxy
membrane surrounding the secretory head cells. The secreted resin is largely segregated from
the secretory cells, which isolates the resin from the atmosphere as well as membrane bound
enzymes, protecting it from oxidative degradation and enzymatic change. A layer of abscission
cells at the base of each secretory head allows the gland to be easily removed [Kim, 2003].
The resin excreted by the trichomes contains a variety of constituents, any of which might play
a role in the biological activities of the cannabis plant. Among these are terpenoids, flavonoids
and cannabinoids. Because it would be too complex to study all these components in a single
Introduction
9
Figure 1.4: Microscope photograph and drawing of a cannabis resin gland, with secretory head cells visible
underneath the transparent cannabinoid- and terpenoid-rich resin.
Source: drawing from RC Clarke. Hashish! Los Angeles: Red Eye Press, 1998. Reprinted with permission.
PhD-project, this thesis is particularly focused on the cannabinoids. Hopefully the other
classes of compound will (again) receive their share of scientific attention in the near future.
The adaptational significance of the resin glands remains speculative. Although the resin gives
a certain defense against insect and fungal attack, cannabis crops are still vulnerable to attack
by a wide variety of pests, particularly under greenhouse conditions. Certainly, the
intoxicating effects of Cannabis resin have increased cannabis predation by humans, as well as
encouraged its domestication, thus dramatically widening its distribution. Recently, it has
been shown that the cannabinoids cannabigerolic acid (CBGA) and tetrahydrocannabinolic
acid (THCA) induce cell death via apoptosis in plant cells but also in insect cells. Furthermore,
formation of THCA is linked to hydrogen peroxide formation which may contribute to self-
defense of the Cannabis plant [Sirikantaramas, 2005]. These results strongly suggest that
cannabinoids act as plant defense compounds, like many other plant secondary metabolites.
An extensive review of cannabis constituents has been made [Turner, 1980; Ross, 1995].
Besides at least 66 cannabinoids, compounds that have been identified in cannabis products
are listed in table 1.1 [Grotenhermen, 2002].
Chapter 1
10
Table 1.1: An overview of compounds identified in cannabis.
120 terpenoids
50 hydrocarbons
34 sugars and related compounds
27 nitrogenous compounds
25 non-cannabinoid phenols
22 fatty acids
21 simple acids
21 flavonoids
18 amino acids
13 simple ketones
13 simple esters and lactones
12 simple aldehydes
11 proteins, glycoproteins and enzymes
11 steroids
9 elements
7 simple alcohols
2 pigments
1 vitamin
So far, more than 100 terpenoids have been found in cannabis, including 58 monoterpenoids,
38 sesquiterpenoids, one diterpenoid, two triterpenoids and four other terpenoids [Turner,
1980]. They can be studied after steam-distillation of cannabis material or by headspace-gas
chromatography, although large qualitative differences are seen between these two techniques
[Hood, 1973; Strömberg, 1974; Hendriks, 1978]. While cannabinoids are odorless, the volatile
mono- and sesquiterpenoids are the compounds that give cannabis its distinct smell. The
sesquiterpenoid β-caryophyllene-epoxide (figure 1.5), for example, is the main compound
that search-dogs are trained to recognize [Stahl, 1973]. Only one unusual terpenoid can be
found in cannabis: the monoterpenoid m-mentha-1,8(9)-dien-5-ol (figure 1.5). All others can
be found ubiquitously in nature. For this reason the terpenoids of cannabis did not receive
much scientific interest, until it was found that the terpenoid spectrum of cannabis products
can help in determining the origin of cannabis in custom seizures [Brenneisen, 1988].
Figure 1.5: Two special constituents of the cannabis plant
β-caryophyllene-epoxide m-mentha-1,8(9)-dien-5-ol
O
HH
HO
Introduction
11
1.3 Cannabinoids
1.3.1 Cannabinoids defined
Cannabinoids are considered to be the main biologically active constituents of the cannabis
plant. In spite of the fact that THC is often erroneously referred to as the ‘active ingredient’ of
cannabis preparations, currently at least 66 different cannabinoids have been described. The
most important ones are shown in figure 1.6. Mechoulam and Gaoni [1967] defined
cannabinoids as: the group of C
21
compounds typical of and present in Cannabis sativa,
including their carboxylic acids, analogs, and transformation products. But from this rather
restricted pharmacognostic definition, considerable expansion is now required. A modern
definition will put more emphasis on synthetic chemistry and on pharmacology, and would
also include related structures or any other compound that affects cannabinoid receptors.
This, however, creates several chemical subcategories of cannabinoids. In this thesis, the focus
will be exclusively on the (phyto)cannabinoids, occurring naturally in the cannabis plant.
Chemically, the (phyto)cannabinoids belong to the terpenophenols, which are very common
in nature. Cannabinoids are accumulated in the glandular hairs described above, where they
typically make up more than 80% of the subcuticular secretion. In general all plant parts can
contain cannabinoids, except for the seeds. The traces of cannabinoids found in seeds are most
likely a result of contamination with cannabis resin from the flowers [Lawi-Berger, 1982; Ross,
2000]. Essentially there are no qualitative differences in cannabinoid spectrum between plant
parts, only quantitative differences [Fetterman, 1971b; Field, 1980]. The highest cannabinoid
concentrations (in % of dry weight plant material) can be found in the bracts of the flowers
and fruits. In the foliage leaves the content is lower, and in the stems and, even more so, the
roots the content is very low [Hemphill, 1980]. Cannabis grown outdoors generally has lower
levels of cannabinoids when compared to indoor grown plants. When grown under artificial,
high yielding conditions, cannabis flowering parts can be obtained with a resin content of up
to 25-30%, mainly consisting of THC (in the form of its acidic precursor THCA, see below).
This high abundance of a single type of secondary metabolite is virtually unparalleled in the
plant kingdom.
Interestingly, THC, the psychotropically active principle of cannabis, contains no nitrogen
atom and therefore is no alkaloid. This is rare amongst the psychotropically active
compounds.
Chapter 1
12
Tetrahydrocannabinolic acid Tetrahydrocannabinol Delta-8-tetrahydrocannabinol
(THCA) (THC) (delta-8-THC)
Cannabidiolic acid Cannabidiol Tetrahydrocannabivarin
(CBDA) (CBD) (THV)
Cannabigerolic acid Cannabigerol
(CBGA) (CBG)
Cannabinolic acid Cannabinol
(CBNA) (CBN)
Cannabichromenic acid Cannabichromene
(CBCA) (CBC)
Cannabicyclolic acid Cannabicyclol
(CBLA) (CBL)
O
OH
COOH
HO
OH
COOH
OH
HO
COOH
O
OH
COOH
OH
O
COOH
OH
O
COOH
O
OH
O
OH
HO
OH
OH
HO
O
OH
OH
O
OH
O
O
OH
Figure 1.6: Structures of the cannabinoids most commonly found in cannabis plant materials
Introduction
13
1.3.2 Biosynthesis
For the chemical numbering of cannabinoids 5 different nomenclature systems have been
used so far [Eddy, 1965], but the most commonly used system nowadays is the dibenzopyran
numbering, which is also adopted by Chemical Abstracts. In Europe the monoterpenoid
system based on p-cymene has also been widely used. As a result, the main psychoactive
cannabinoid delta-9-THC is sometimes described as delta-1-THC in older manuscripts. In
this thesis, the dibenzopyran numbering is consistently used, therefore THC is fully described
as (-)-trans-
9
-tetrahydrocannabinol (figure 1.7).
Figure 1.7: Two most commonly used numbering systems for the cannabinoids. The dibenzopyran system is
used in this thesis.
In all biosynthetic pathways for cannabinoids that were postulated until 1964 ,CBD or CBDA
was regarded as key intermediate, which was built from a monoterpene, and olivetol or
olivetolic acid, respectively. Other cannabinoids were then derived from this common
precursor. However, Gaoni and Mechoulam [1964b] showed that CBG is the precursor of
CBD, which was biosynthesized through the condensation of geranylpyrophosphate (GPP),
and olivetol or olivetolic acid. Subsequently, they concluded that CBD, THC and CBN all
derive from CBG and differ mainly in the way this precursor is cyclized [Mechoulam, 1965;
1967; 1970; 1973]. Shoyama [1970; 1975] further concluded that neither the free phenolic
forms of the cannabinoids nor CBNA were produced by the living plant. Instead, he
postulated a biosynthetic pathway based on geraniol and a polyketoacid. The same conclusion
was reached by Turner and Hadley [1973] after study of African cannabis types. This
biosynthetic pathway could explain the different contents of cannabinoids in cannabis
products of different origins and the occurrence of homologues and derivatives.
Currently, the hypothesis that the C
10
-terpenoid moiety is biosynthesized via the
deoxyxylulose phosphate pathway, and the phenolic moiety is generated by a polyketide-type
reaction sequence is widely accepted. More specifically, incorporation studies with
13
C-labeled
2'
3'
5'
2
4'
9
8
4
35
6
1
10
1'
6'
7
Dibenzopyran-numbering Monoterpene-numbering based on p-cymene
O
1
2
4
10
3
5
6
6a
10a
7
8
9
11
12
13
A
B
C
Chapter 1
14
glucose have shown that geranyl diphosphate (GPP) and the polyketide olivetolic acid are
specific intermediates in the biosynthesis of cannabinoids, leading to the formation of CBGA
(figure 1.8) [Fellermeier, 1998; Fellermeier, 2001]. Further biosynthetic pathways of
cannabinoid production have finally become clear by identification and subsequent cloning of
the responsible genes [Taura, 1995b; Taura, 1996; Morimoto, 1998]. A major structural
variation for the cannabinoids is found in the alkyl sidechain of the olivetolic acid moiety:
although the pentyl (C5)-sidechain is usually present, also shorter sidechains can be found,
ranging from C4 to C1. It is interesting to note that free olivetolic acid has never been detected
in cannabis plant material.
Figure 1.8: Biosynthetic pathway for the production of the cannabinoids
OH
HO
COOH
HO
OH
COOH
OH
O
COOH
O
OH
COOH
OH
HO
COOH
OPP
Geranyl diphosphate
(GPP)
Olivetolic acid
Cannabigerolic acid
(CBGA)
Tetrahydrocannabinolic acid
(THCA)
Cannabichromenic acid
(CBCA)
Cannabidiolic acid
(CBDA)
OH
HO
COOH
HO
OH
COOH
OH
O
COOH
O
OH
COOH
OH
HO
COOH
OPP
Geranyl diphosphate
(GPP)
Olivetolic acid
Cannabigerolic acid
(CBGA)
Tetrahydrocannabinolic acid
(THCA)
Cannabichromenic acid
(CBCA)
Cannabidiolic acid
(CBDA)
Introduction
15
The main biosynthetic steps are shown in figure 1.8. Based on this pathway, cannabinoids are
produced by the cannabis plant as carboxylic acids, where the substituent at position 2 is a
carboxyl moiety (–COOH). Consequently, in fresh plant material almost no neutral
cannabinoids can be found, but theoretically all cannabinoids are present in this acidic form.
However, the carboxyl group is not very stable and is easily lost as CO
2
under influence of heat
or light, resulting in the corresponding neutral cannabinoid. In this way the acidic precursor
THCA can be converted into the psychoactive THC, which is the reason why all forms of
(recreational) cannabis consumption include some form of heating of the material (i.e.
smoking, vaporizing, making tea or baked products).
1.3.3 Classifications of cannabinoids
Although more than 60 cannabinoids are known, it should not be concluded that all
cannabinoids are detectable in all cannabis products. They were identified over several decades
of cannabis research, studying many different cannabis products and different and sometimes
rare types of cannabis plants from a variety of origins and qualities.
The main cannabinoid types that are usually detected in each breeding strain or cultivar of
cannabis are THC, CBD, CBN, CBG and CBC. However, there can be an enormous variation
in their quantitative ratios. The different chemical types of cannabinoids have been well
described [Turner, 1980, ElSohly 1983] and will therefore not be extensively discussed here.
However, understanding how the cannabinoids are (chemically) related to each other is
important when studying cannabis samples, as degradation and changes in the cannabinoid
profile might occur as a result of storage or breeding conditions, variations in preparation of
medicines, mixing with other components (e.g. tobacco when smoking), heating etc. For the
phytochemical work in this thesis, the cannabinoids can most conveniently be divided in three
groups (see also figure 1.9):
1) cannabinoids produced by metabolism of the plant (acidic cannabinoids);
2) cannabinoids present in the plant resulting from decarboxylation (neutral
cannabinoids);
3) cannabinoids occurring as artefacts by degradation (e.g.: oxidation, isomerization,
UV-light).
The group of cannabinoids that occur as a result of degradative conditions deserve some
special attention, because their presence is largely the result of variable and unpredictable
conditions during all stages of growing, harvest, processing, storage and use. As a result, a
well-defined cannabis preparation may change rapidly into a product with significantly
different biological effects. Particularly in samples that have been stored for an extended
period, CBN can be found in relatively large amounts. Cannabinoids of the CBN type are not
formed by biosynthesis, but rather by oxidative degradation of THC- and CBD types. Also the
types
8
-THC and CBL are not naturally occurring, but artifacts. The isomerization of
9
-
Chapter 1
16
THC to
8
-THC is well documented [Mechoulam, 1970; Mechoulam, 1973; Razdan, 1973].
Since
8
-THC is more thermostable than
9
-THC, it will accumulate during heating of
9
-
THC. The cannabinoid CBL arises by exposure of CBC to UV-radiation, leading to
crosslinking of two double bonds in the molecule [Crombie, 1968].
Figure 1.9: Relationships between the major cannabinoids found in cannabis plant materials. Three different
groups are distinguished: cannabinoids produced by biosynthesis of the plant; cannabinoids resulting from natural
decarboxylation of acidic cannabinoids; degradation products resulting from various influences, such as UV-light,
oxydation or isomerization. Arrows indicate the routes of conversion.
1.3.4 Studying cannabinoids
Medicines based on natural products are usually hard to study. Plant materials may contain
many (structurally) closely related compounds, and often it is unclear what the active
ingredient is, if indeed there is only one. Sometimes the biologically active components of the
plant have only been partially characterized (e.g. Ginkgo biloba, St. John’s Wort, Hypericum
perforatum, Echinacea purpurea). Because of this complexity of medicinal plants, some
important conditions for reliable study of natural products are: the availability of analytical
methods that can study the components without sample degradation; reference standards of
the compounds of interest; and a clear overview of physicochemical, spectroscopic and
chromatographic properties of the sample components.
For the study of cannabinoids, the analytical methods that are available have recently been
extensively reviewed by Raharjo [2004]. By far the most commonly used chromatographic
methods have been high performance liquid chromatography (HPLC) and gas
chromatography (GC). The use of GC, commonly coupled to flame ionization detection
(FID) or mass (MS)-detection, permits the analysis of a large variety of cannabinoids with
very high resolution. However, a major disadvantage of GC is in the fact that the acidic
cannabinoids can not be analyzed without prior derivatization to protect the labile carboxyl
function. Because it is hard to perform a quantitative derivatization for all components in a
complex mixture, GC analysis has only limited value when studying the authentic
composition of cannabis products. When analyzing cannabinoids in their authentic form,
HPLC is the preferred method. Making use of a UV- or photodiode-array detector (PDA),
cannabinoids can be efficiently analyzed without causing degradation of sample components.
Biosynthesis
THC
A
CBD
A
CBG
A
CBC
A
Decarboxylation THC CBD CBG CBC
Degradation
CBNA CBN Delta-8-THC CBL CBLA
Introduction
17
However, it is difficult to separate all major cannabinoids in a single run. To overcome this
problem, the use of mass-detection (LC-MS) to distinguish between overlapping
chromatographic peaks is becoming increasingly important [Stolker, 2004; Hazekamp, 2005].
Independent of the method used for cannabinoid analysis, reliable standards are needed for
the compounds to be studied, in order to allow high quality, quantitative research on the
pharmacological and medicinal aspects of cannabis. However, at the time the work for this
thesis was started, only a few of the major cannabinoids were commercially available (THC,
CBD, CBN and
8
-THC). Even the cannabinoid present in the highest concentration in any
drug-type cannabis plant, THCA, had not been made commercially available yet. Without a
doubt, this lack of reference standards is a great obstacle for a detailed study and
understanding of cannabis.
Although spectroscopic and chromatographic data have been published for most known
cannabinoids during isolation and identification experiments (see Turner et al. [1980] for an
overview), they are scattered over a huge amount of scientific papers. Moreover, standardized
data obtained under identical analytical conditions have not been reported yet. This is
regrettable, because when studying a complex phytomedicine like cannabis, it is important to
communicate about the subject in a standardized way. After all, differences in analytical
methods, or in the interpretation of results make it hard to discuss the science behind
cannabis. Such differences can be prevented by the development of validated methods, which
are agreed upon by all scientists involved. For other important drugs (such as cocaine, opioids,
LSD) such standardized methods have been developed and cross-validated between
laboratories, commonly resulting in official Pharmacopoeia texts. For cannabis, such a text
has not been available since several decades.
In conclusion, a lot of data on cannabis and the cannabinoids have been published, but their
value is only limited. There is a clear need to put all the pieces of the cannabis puzzle together
and come up with reliable, validated results.
1.4 Cannabinoids as active compounds
1.4.1 Mechanisms of cannabinoid action
Until the discovery of specific cannabis receptors, the biochemical mode of action of
cannabinoids was much disputed. Because of their lipophilic character, cannabinoids can
penetrate cellular membranes by diffusion. Initially, possible explanations for cannabinoid
activity included unspecific membrane binding resulting in fluidity- and permeability changes
of neural membranes, the inhibition of acetylcholine-synthesis, an increase in the synthesis of
catecholamines, and an interaction with the synaptosomal uptake of serotonin [Dewey, 1986;
Pertwee, 1988]. However, it was established in the mid 1980s that cannabinoid activity is
highly stereoselective [Mechoulam, 1992], indicating the existence of a receptor mediated
mechanism.
Chapter 1
18
The first reliable indications that cannabinoids act through receptors came when it was shown
that cannabinoids can act as inhibitors of the adenylate cyclase second messenger pathway in
brain tissue and neuroblastoma cell lines. This activity was dose-dependent, stereospecific, and
could be modulated by pertussistoxin [Howlett, 1985, 1986, 1987; Devane, 1988; Bidaut-
Russell, 1990]. Finally, a stereospecific G-protein-coupled cannabinoid receptor (CB-1) was
found and cloned [Matsuda, 1990].
The CB-1 receptor is most clearly present in the central nervous system, but it is also found in
certain peripheral organs and tissues. Amongst others, it inhibits adenylate cyclase activity and
the opening of N-type calcium channels [Mackie, 1992]. Shortly after that, a second,
periferous cannabinoid receptor (CB-2) was found with a possible role in immunological
processes [Munro, 1993]. It is primarily expressed by immune tissues like leukocytes, spleen
and tonsils, and it shows a different selectivity than centrally acting CB-1. So far, the
physiological roles of CB-2 receptors are proving difficult to establish, but at least one of these
seems the modulation of cytokine release (Molina-Holgado, 2003). Surprisingly, there is only
a mere 45% homology between the CB-1 and CB-2 receptors.
Based on the observation that all natural cannabinoids are highly lipid soluble, an attempt was
made to isolate endogenous ligands for the cannabinoid receptors from fatty tissues of
animals. Finally, a single compound could be isolated from porcine brain tissue, with a high
affinity for the CB1 receptor, named anandamide (arachidonic acid ethanolamine) [Devane,
1992]. Later, a related compound was isolated from canine gut with an affinity for
cannabinoid receptors; 2-arachidonyl glycerol (2-AG, see figure 1.10)) [Mechoulam, 1995]. In
recent years, a large variety of compounds with endocannabinoid activity have been isolated
or synthesized [Mechoulam, 1998; Pertwee, 2006b], interestingly all having an eicosanoid
structure. Cannabinoid receptors and their endogenous ligands together constitute what is
referred to as the endogenous cannabinoid (endocannabinoid) system.
Figure 1.10: Structures of the two major endocannabinoids
Not all of the effects of cannabinoids can be explained by receptor-mediated effects, and it is
believed that at least some effects are non-specific and caused through membrane turbation
[Makriyannis, 1995], or by binding to yet unknown targets in the cell. It has been found in
isolated blood vessel preparations that some endocannabinoids can activate vanilloid
receptors on sensory neurons [Zygmunt, 1999], which raises the possibility that
endocannabinoids are endogenous agonists for vanilloid receptors [Pertwee, 2005]. These
receptors might therefore be putatively regarded as CB-3 receptors. The cannabinoid signaling
N
O
OH
O
O
OH
OH
Anandamide 2-arachidonylglycerol
Introduction
19
system is teleologically millions of years old, as it has been found in mammals, fishes, and
invertebrates down to very primitive organisms, such as the hydra [De Petrocellis, 1999].
Indeed, there are indications that CB receptors are evolutionary related to the vanilloid
receptors [McPartland, 2002].
1.4.2 Therapeutic potential
Cannabis preparations have been employed in the treatment of numerous diseases, with
marked differences in the available supporting data. Clinical studies with single cannabinoids
(natural or synthetic) or whole plant preparations (e.g. smoked cannabis, encapsulated
extract) have often been inspired by positive anecdotal experiences of patients using crude
cannabis products for self-treatment. The antiemetic [Dansak, 1997], appetite enhancing
[Plasse, 1991], analgesic [Noye, 1974] and muscle relaxant effects [Clifford, 1983], and the
therapeutic use in Tourette’s syndrome [Muller-Vahl, 1999] were all discovered or
rediscovered in this manner. Incidental observations have also revealed therapeutically useful
effects. The discovery of decreased intraocular pressure with THC administration, potentially
useful in the treatment of glaucoma, was made serendipitously during a systematic
investigation of healthy cannabis users [Hepler, 1971]. However, anecdotes as to the efficacy
of Cannabis or THC in indications that have not been confirmed in controlled studies have to
be judged with caution.
Although most known cannabinoids have been tested to describe their relative potency in
comparison to THC (in receptor binding assays or in THC specific assays), up to very recently
virtually nothing was known about their own biological activities. However, testing non-THC
cannabinoids as serious candidates for new leads, can sometimes lead to completely counter-
intuitive results, as shown in the case of THV. Its potency is about ¾ of that of THC in
classical in vitro assays, [Turner, 1980; Hollister, 1974], while only very recently in vivo testing
showed THV to be rather an antagonist of THC activity [Thomas, 2005]. And although CBN
was initially considered an inactive degradation product of THC, it was later found to have
some interesting activities of its own [Herring, 2001; Jan, 2002]. And even while, in potent
plant material, THCA can be present at levels of more than 20% of dry weight, its activities
remained unstudied for decades. The therapeutic value of the acidic cannabinoid THCA as an
immuno-modulating agent has only been discovered very recently [Verhoeckx, 2006], and its
effect has been patented. Examples like these show that the study of medicinal cannabis should
include the whole array of cannabinoids present, as far as possible [McPartland, 2001].
The therapeutic potential of cannabinoids can be further clarified by pointing out the central
physiological importance of the endocannabinoid system, and its homology to, and
interaction with the endorphin system. In addition to the role as modulator of food intake, the
cannabinoid system is involved in several physiological functions and might be related to a
general stress-recovery system. This variety of effects was concisely summarized by Di Marzo
et al.
[1998], who stated that cannabinoids help you 'feel less pain, control your movement,
relax, eat, forget (posttraumatic), sleep, and protect your neurons'. The activation of the
Chapter 1
20
endogenous cannabinoid system could represent a crucial and important component for each
of these functions. One yet unproven but intriguing idea is that endocannabinoids may set the
“analgesic tone” of the body, with the level of their production acting as a kind of pain
thermostat. It is likely that such a system relies on the combined activities of a range of
compounds. Strategies to modulate endocannabinoid activity include inhibition of re-uptake
into cells and inhibition of their degradation to increase concentration and duration of action.
The effect of plant cannabinoids interacting with such an endocannabinoid system could be
on multiple levels, other than receptor binding alone. Some of such interactions have already
been described [Watts, 2004].
The endocannabinoid system that is responsible for our physiological response to cannabis
use is in many respects analogous to the endorphin system. It is widely known that opioids
and cannabinoids share several pharmacological effects, including antinociception,
hypothermia, inhibition of locomotor activity, hypotension, and sedation [Cichewicz, 2004].
Furthermore, crosstalk between the two systems has been shown [Corchero, 2004].
Cannabinoids and opioids both produce analgesia through a G-protein-coupled mechanism,
and the analgesic effect of THC is, at least in part, mediated through opioid receptors,
indicating an intimate connection between cannabinoid and opioid signaling pathways in the
modulation of pain perception [Cichewicz, 2004]. Although both cannabinoids and opioids
are accompanied by undesirable side effects at high doses, it was found that THC can enhance
the potency of opioids such as morphine, thereby dramatically reducing the dose needed for
pain control [Williams, 2006].
In the past, opium abuse led to the study of the physiological effects of opium constituents,
which in turn prompted the discovery of opioid receptors. The result was one of our most
significant medicines in use today: morphine. The story of cannabis has been exactly
analogous to the opium story, up to the point of discovery of the endocannabinoid system.
However, there seems to be a reluctance to make the final step and turn cannabinoids into real
medicine. A review by the US Institute of Medicine has commented on how little we know
about cannabinoids in comparison with opiates [Joy, 1999]. However, the brain has more
CB1- than opioid-receptors. The analogy between the history of research into the two groups
suggests good reason for optimism about the future of cannabinoid drug development
[Vigano, 2005; Pertwee, 2006].
1.4.3 Cannabis medicines
A major obstacle in the development of cannabinoid-based drugs has been the low water
solubility of the cannabinoids [Garrett, 1974], which makes it difficult to develop effective
formulations for human use [Hazekamp, 2006]. Nevertheless, an increasing number of
pharmaceutical companies start to pick up the idea of cannabinoids or their antagonists as
therapeutic drugs. At present a number of medicines based on the biological activities of the
cannabinoids are available, such as Marinol, Nabilone, and Sativex. Marinol (dronabinol,
synthetic
9
-THC) and Cesamet (nabilone,
a THC-derivative) are registered for the indication
Introduction
21
of nausea and vomiting associated with cancer chemotherapy. Marinol is also approved for
anorexia and cachexia in HIV/AIDS. Although there are some clear indications that some
effects may vary according to the fact if a cannabinoid is taken alone, or in combination with
other cannabinoids, virtually no work has been done on the activities of combined
cannabinoids. One important exception is the clinical testing of combinations of THC and
CBD in the medicinal product Sativex [Russo, 2006], which is currently registered only in
Canada.
Several new cannabinoid-based products are expected to be introduced in the near future.
Among them are Rimonabant (Acomplia, by Sanofi-Aventis) [van Gaal, 2005], and the potent
analgesic ajulemic acid [Burstein, 2004]. Rimonabant was developed based on the observation
that cannabis consumption commonly leads to an insatiable feeling of hunger, also known as
‘the munchies’. Rimonabant is an antagonist of the CB1 receptor, and causes the opposite to
occur. To be launched in the near future, it is expected to become a major drug in the fight
against obesity. Ajulemic acid (AJA) is a synthetic analog of the human THC metabolite,
THC-11-oic acid. Although the mechanism of AJA action remains largely unknown, it has
potent analgesic and anti-inflammatory activity, without the psychotropic action of THC.
Unlike the nonsteroidal anti-inflammatory drugs, AJA is not ulcerogenic at therapeutic doses,
making it a promising anti-inflammatory drug.
Although it seems clear that the Cannabis plant still has a highly relevant potential for
medicine, it is also clear that the medicinal use of cannabis is not a panacea. Cannabis, as any
other medicine, can have its side effects, especially when consumed in high amounts. But a
widely expressed opinion on the unwanted actions of cannabis and THC has been formulated
in a 1999 report of the US Institute of Medicine on the medical use of cannabis: ”Marijuana is
not a completely benign substance. It is a powerful drug with a variety of effects. However,
except for the harms associated with smoking, the adverse effects of marijuana use are within
the range of effects tolerated for other medication” [Joy, 1999]. The toxic properties of
cannabis are mostly dependent on the content of cannabinoids. The toxicity of cannabis drugs
and cannabinoids is considered to be generally low, and comparable to socially accepted
psychoactive products like coffee, alcohol and tobacco [Hollister, 1986]. So even though the
role of cannabinoids in modern therapeutics remains uncertain, there are enough clues to
realize it would be irrational not to explore it further.
In general, there are 5 major concerns about cannabis use: 1) the unabated increase in use, 2)
the constant decrease of the age of first use, 3) the increased risk of psychosis in vulnerable
people, 4) the constant increase of cannabis heavy users searching help for quitting cannabis
use, and 5) the increased risk of driving accidents. However, these worries should not prevent
any scientific research on cannabis use in medicine. Instead, a clear distinction must be made
between therapeutic and recreational use.
Chapter 1
22
1.5 Cannabis and the law
1.5.1 Political cannabis
Starting from 1954, the World Health
Organization (WHO) has claimed
that cannabis and its preparations no
longer serve any useful medical
purpose and are therefore essentially
obsolete. Up to that moment,
cannabis legislation had been based
on a large number of conventions,
causing considerable confusion in the
execution of treaties. Under pressure
of increasing reports that cannabis was
a drug dangerous to society, it was proposed to combine all in single convention, the draft of
which was finally accepted by the United Nations in 1961. In following years several
complementary treaties were made to strengthen it. Under the “Single Convention on
Narcotic Drugs” cannabis and its products were defined as dangerous narcotics with a high
potential for abuse and no accepted medicinal value. It reflected the belief that cannabis was a
dangerous narcotic with a threat that was equal to the most dangerous opiates, as it was
strongly believed that cannabis use could serve as stepping stone to the use of such drugs.
Since the Single Convention, the potential danger of cannabis abuse by recreational users has
been much higher on the political agenda then any of its benefits as a source for fiber, food or
medicines (figure 1.11). Nowadays it may be hard to believe, but according to the American
president Nixon, cannabis was a secret weapon of the communists, being spread by the Jews to
destabilize the Western world. This sense of cannabis-related fear has been the base for the
legislation that is currently seriously obstructing the rediscovery of cannabis as a medicine.
Even today, under US law, possession of only several grams of cannabis can lead to
imprisonment for life. The distinction between medicinal and recreational use is thereby made
only in a handful of US States.
It can be observed that new scientific insights on cannabis are only slowly and reluctantly
incorporated into new legislation. However, in the coming years, a large variety of scientific
and clinical data is expected to become available, further showing the physiological effects of
cannabinoids and the endocannabinoid system. And in several Western countries important
obstacles for a real acceptance of medicinal cannabis have already been addressed, as serious
steps are taken towards decriminalization of cannabis use or even providing medicinal
cannabis products to patients [GW pharmaceuticals, 2003; Duran, 2005; Sibald, 2005; Irvine,
2006]. These shifts constitute the first steps away from the dominant drug policy paradigm
advocated by the United States, which is punishment-based prohibition, and it signals that the
Single Convention may start to reach its expiry date. The legislation that follows it will depend
Figure 1.11: Medicinal cannabis: requested by a large
group of patients, but feared by the authorities.
Introduction
23
for a large part on the quality of the research available. However, good arguments will finally
not be enough; what is most needed is a change in mentality [Reinarman, 2004]; in politics,
but also in the way research is conducted.
1.5.2 The Dutch situation
The Netherlands have known a liberal drug policy already for several decades, so it is not
surprising that the Dutch have been among the first to approach the discussion on medicinal
cannabis in a practical way. In the 1990s, it was increasingly acknowledged that a considerable
group of people was using cannabis for medicinal purposes, obtained through the illicit
market. Simultaneously, a growing number of Dutch health officials judged that, although
scientific proof on the effectiveness of cannabis might still be insufficient, the perceived
dangers of cannabis use no longer outweighed its potential beneficial effects to certain groups
of chronically ill patients. However, its unofficial status made it impossible to make any
guarantees on the quality, consistency, or origin of the cannabis found in the illicit market.
Therefore, in order to supply these patients with a safe and reliable source of high quality
cannabis, the Office of Medicinal Cannabis (OMC) was established in March 2000. It started
acting as a national agency on 1 January 2001. The OMC is the organization of the Dutch
Government which is responsible for the production of cannabis for medical and scientific
purposes, and is in full agreement with international law. After an initial preparation period,
medical grade cannabis (in the form of dried female flowertops) finally became available in
Dutch pharmacies in September 2003, on prescription only. Based on the availability and
quality of clinical data and scientific literature, a selection of indications was made by the
OMC for treatment with its medicinal grade cannabis [OMC, 2006].
Right from the start, a reliable source of high quality cannabis materials was considered crucial
for the success of the Dutch medicinal cannabis program. Therefore, skilled breeders were
contracted for the cultivation of plants under highly standardized conditions, resulting in a
product with a very consistent composition. The whole process of growing, processing and
packaging of the plant material are performed according to pharmaceutical standards, and
supervised by the OMC. The quality is guaranteed through regular testing by certified
laboratories. Besides supplying high quality cannabis to medicinal users, the OMC also
provides the same material for research and development of medicinal preparations based on
cannabis constituents.
The availability of reliable cannabis of consistent quality has proven to be crucial to perform
good research, as it opened up the way for long term quantitative studies on cannabis and its
constituents on a national level. Currently, a variety of laboratories and research groups
cooperate for quality control, fundamental research and clinical development. Cannabis
research in The Netherlands is blooming, with a clear focus on scientific outcome, rather than
on repression of cannabis use. It is exactly these conditions that have made the work for this
thesis possible.
Chapter 1
24
1.6 Outline of this thesis
This thesis is written from an analytical, phytochemical point of view, and deals primarily with
biochemical aspects of medicinal cannabis. Because, after all, the cannabinoids are widely
considered to be the most important (but not the only!) active components of the cannabis
plant, the work has been focused on them. And since of all the cannabinoids, THC is the best
studied, this cannabinoid became the focus of several chapters in this thesis. However, the
main purpose of this thesis is to bring cannabis, as a whole, back into focus.
The work for this thesis was performed in The Netherlands, which has a well known tradition
of accepting cannabis as a recreational drug. Although this makes studying the medicinal
aspects of cannabis much easier, it is also confusing because the distinction between the two
can not always be clearly made. In chapter 2 it is shown how to make a difference between
medicinal and recreational cannabis, and why a regulated source of high grade cannabis is
needed for any pharmaceutical research to succeed.
Once the necessity of medicinal cannabis is established, quantitative research can begin. In
chapter 3 a method is developed for purification of the major cannabinoids from plant
material, which is the starting point for the production of standards. In chapter 4 a method is
then described to prepare solutions of cannabinoids reference standards. Unfortunately, one
potentially important cannabinoid, CBNA, could not be isolated, so a separate method was
developed to produce it by partial chemical synthesis. The procedure is described in chapter 5.
All cannabinoid standards were then characterized by their chromatographic and
spectroscopic properties. Consequently, chapter 6 provides cannabis researchers with a
synoptic overview of the analytical characteristics of the main cannabinoids. But it is clear that
even good quality cannabinoid standards can not be used if no method is available for their
reliable analysis. For this purpose, an HPLC-DAD method was developed and validated
according to the most recent pharmaceutical requirements, as described in chapter 7.
Cannabis as a medicine is consumed in a variety of forms and by different routes. A large
proportion of medicinal cannabis users prefers to consume it as a tea, but almost nothing has
been published on the characteristics of such tea. Therefore the parameters involved in tea-
making were systematically studied in chapter 8. Although generally, the easiest way of
administering a medicine is orally, the low water solubility of the cannabinoids makes this
route of administration rather unconvenient. In chapter 9, we studied the use of cyclodextrins
for improving the aqueous solubility as well as the stability of THC and other cannabinoids.
The most efficient administration route of cannabis is inhalation (smoking). To decrease the
exposure to toxic compounds of cannabis smoke, we evaluated the use of a vaporizer device,
that can evaporate the active components of the cannabis plant for inhalation, in chapter 10.
As a result of these studies, we now have a much better understanding of the cannabis plant,
its main active components the cannabinoids, and its galenic formulations and routes of
administration.
25
CHAPTER 2
An evaluation of the quality of medicinal grade cannabis
in the Netherlands
• • •
Arno Hazekamp, Pieter Sijrier, Rob Verpoorte
• •
Leiden University, Department of Pharmacognosy, Gorlaeus Laboratories
Leiden, The Netherlands
Published in Cannabinoids 2006, 1(1): 1-9
Abstract
Since 2003, medicinal grade cannabis is provided in the Netherlands on prescription through
pharmacies. Growing, processing and packaging of the plant material are performed according
to pharmaceutical standards and are supervised by the official Office of Medicinal Cannabis
(OMC). The quality is guaranteed through regular testing by certified laboratories. However,
in the Netherlands a tolerated illicit cannabis market exists in the form of so-called
‘coffeeshops’, which offers a wide varie