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Structure elucidation of the tetrahydrocannabinol complex with randomly methylated β-cyclodextrin


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The low aqueous solubility of the bioactive cannabinoid tetrahydrocannabinol (THC) is a serious obstacle for the development of more efficient administration forms. In this study the aqueous solubility of THC was tested in the presence of alpha-, beta- and gamma-CD, and randomly methylated beta-CD (RAMEB). It was found that only RAMEB was able to increase the aqueous solubility of THC to a significant level. A THC concentration of about 14 mg/ml was reached by using a 24% (187 mM) RAMEB solution, which means an increase in solubility of four orders of magnitude. The resulting THC/RAMEB complex was investigated through phase-solubility analysis, complemented by (1)H NMR, NOESY- and UV-studies in order to obtain details on the stoichiometry, geometry and thermodynamics of the complexation. The binding ratio of THC to CD was found to be 2:1, with the second THC molecule bound by non-inclusion interactions. Based on the obtained results a model for the complex structure is presented. Stability of the complex under laboratory room conditions was tested up to 8 weeks. Results show that complexation with RAMEB seems to be promising for the development of water-based THC formulations.
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Structure elucidation of the tetrahydrocannabinol complex
with randomly methylated -cyclodextrin
Arno Hazekamp, Rob Verpoorte
Department of Pharmacognosy, Leiden University, Institute of Biology, Leiden, The Netherlands
article info
Article history:
Received 20 January 2006
Received in revised form
28 June 2006
Accepted 2 July 2006
Published on line 6 July 2006
NMR spectroscopy
Physical characterization
The low aqueous solubility of the bioactive cannabinoid tetrahydrocannabinol (THC) is a
serious obstacle for the development of more efficient administration forms. In this study
the aqueous solubility of THC was tested in the presence of -, - and -CD, and randomly
methylated -CD (RAMEB). It was found that only RAMEB was able to increase the aqueous
solubility of THC to a significant level. A THC concentration of about 14mg/ml was reached
by using a 24% (187 mM) RAMEB solution, which means an increase in solubility of four orders
of magnitude. The resulting THC/RAMEB complex was investigated through phase-solubility
analysis, complemented by 1H NMR, NOESY- and UV-studies in order to obtain details on
the stoichiometry, geometry and thermodynamics of the complexation. The binding ratio
of THC to CD was found to be 2:1, with the second THC molecule bound by non-inclusion
interactions. Based on the obtained results a model for the complex structure is presented.
Stability of the complex under laboratory room conditions was tested up to 8 weeks. Results
show that complexation with RAMEB seems to be promising for the development of water-
based THC formulations.
© 2006 Elsevier B.V. All rights reserved.
1. Introduction
The Cannabis plant (Cannabis sativa L.) has a long history
of medicinal use and the main constituents, the cannabi-
noids, are under intensive study (Grotenhermen, 2002). At
present a number of medicines based on the biological activ-
ities of the cannabinoids are available, such as Marinol®
and Nabilone, and several more are expected to be intro-
duced in the near future. Among them are rimonabant, for
treatment of obesity (van Gaal et al., 2005), and the potent
analgesic ajulemic acid (Burstein et al., 2004). It seems clear
that the Cannabis plant still has highly relevant potential for
The main psychoactive cannabinoid 9-tetrahydrocann-
abinol (THC, Fig. 1a) has been shown to be clinically useful for
Corresponding author at: Leiden University, Department of Pharmacognosy, Gorlaeus Laboratories, Einsteinweg 55, 2333 CC Leiden, The
Netherlands. Tel.: +31 71 527 4784; fax: +31 71 527 4511.
E-mail address: (A. Hazekamp).
a large diversity of indications, including nausea and weight-
loss associated with chemotherapy and HIV/AIDS, spasms in
multiple sclerosis, chronic neuropathic pain and glaucoma
(Grotenhermen, 2002). However, the reduced bioavailability
of orally administered THC, due to low absorption and high
first-pass metabolism (Brenneisen et al., 1996), prompts the
development of more reliable administration forms, such as
aqueous THC solutions for inhalation, sublingual or injec-
tion purposes. However, the solubility of THC was reported
to be only 1–2 g/ml in a 0.9% NaCl solution (Garrett and
Hunt, 1974). Recently a water-based preparation of cannabis-
extract has been developed for sublingual use (Sativex®). How-
ever, it contains ethanol and propyleneglycol as solubilizing
agents, resulting in frequent irritation of the administration
site (Sativex product monograph, Bayer Healthcare, Canada).
0928-0987/$ – see front matter © 2006 Elsevier B.V. All rights reserved.
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european journal of pharmaceutical sciences 29 (2006) 340–347 341
Fig. 1 – (a) Structure of 9-tetrahydrocannabinol (THC). The
lettering of the rings is indicated. (b) General structure of
the cyclodextrins; -CD (n= 6), -CD (n= 7), -CD (n= 8). In
randomly methylated--CD a proportion of
hydroxyl-groups is substituted for methoxy-groups.
Clearly there is still a need for the development of a more opti-
mal preparation of aqueous THC.
Cyclodextrins (CDs) are natural cyclic oligosaccharides
constituted by six (-CD), seven (-CD) or eight (-CD) d-
glucose units (Fig. 1b). The three-dimensional structure of the
CD-ring is a truncated cone, with each of the -, -, and -
CDs having a different cavity volume. They can form inclusion
complexes with lipophilic guest molecules, thereby improving
their aqueous solubility, increasing stability and bioavailabil-
ity, and reducing side effects (Martin Del Valle, 2004). Various
modifications of the natural CDs have been developed, such
as the randomly methylated -CD (RAMEB) and hydroxypropyl
The use of cyclodextrins for the development of aque-
ous THC preparations seems to be promising. In a study
by Jarho et al. (1998), THC could be solubilized up to about
1 mg/ml, using a 40% HP--CD solution with addition of the
polymer hydroxypropylmethylcellulose. However, no further
details were reported on the chemical structure, stability or
kinetics of the complex. In another study complexation with
-CD has been shown to improve the chemical stability of
THC (Shoyama et al., 1983). Recently, Mannila et al. (2005)
demonstrated that complexation with RAMEB increases both
the aqueous solubility and dissolution rate of THC as well as
the related compound cannabidiol (CBD). These results also
showed that the sublingual administration of a THC/RAMEB
complex substantially increases the bioavailability of THC in
rabbits. Based on phase-solubility data a binding ratio of 1:2
(guest:CD) was suggested for the complex, but no further elu-
cidation of the structure was performed.
However, there is growing evidence that the stoichiometry
of drug/cyclodextrin complexes cannot be derived exclusively
from simple phase-solubility studies, as it becomes increas-
ingly clear that they are highly oversimplified descriptions,
and ignore important aspects of the formation of cyclodextrin
complexes. Cyclodextrins are able to form both inclusion and
non-inclusion complexes. Self-association of surface-active
drugs, lipophilic drug molecules, and drug/cyclodextrin com-
plexes, as well as drug solubilization through non-inclusion
interactions with drug/cyclodextrin complex, will influence
both the shapes and mathematical interpretation of phase-
solubility diagrams (Loftsson et al., 2002, 2004). In severalcases
a different stoichiometry was obtained when using the phase-
solubility studies compared to the more reliable construc-
tion of a continuous variation (Job’s) plot using techniques
such as NMR, UV or potentiometry (reviewed by Loftsson
et al., 2004). Therefore, other techniques such as construc-
tion of Job’s plot, preferably in combination with theoretical
computer-simulated modelling are important complementary
data for determination of stoichiometry.
In this study the aqueous solubility of THC was tested fol-
lowing binding to -, - and -CD, and RAMEB and the most
efficient CD-type was selected for further study. The result-
ing complex of THC with RAMEB was investigated through
phase-solubility analysis, complemented by 1H NMR, NOESY
and UV studies in order to obtain details on the stoichiometry,
geometry and thermodynamics of the complexation. Based
on the obtained results a model for the complex structure
is presented. Stability of the complex under laboratory room
conditions was tested up to 8 weeks.
2. Materials and methods
2.1. Materials and chemicals
All solvents were analytical or HPLC-grade and were obtained
from Biosolve (Valkenswaard, The Netherlands). Deuteriated
solvents for NMR studies were from Eurisotop (Gif-sur-Yvette,
France). Cyclodextrins; -, -, - and randomly methylated
-CD (RAMEB) were purchased from Wacker Chemie GmbH
(Burghausen, Germany) and were used as received. RAMEB
was of pharmaceutical grade (Cavasol W7 M Pharma) and
had a degree of substitution of 1.7. The cannabinoids used in
this study were isolated and quantified according to a method
developed by our laboratory (Hazekamp et al., 2004a,b). Stock
solutions of cannabinoids and CDs were prepared in ethanol.
Water was of Millipore quality.
2.2. Assay of THC
THC concentrations were assayed by an HPLC-method. The
HPLC profiles were acquired on a Waters (Milford, MA, USA)
HPLC system consisting of a 626 pump, a 717 plus autosam-
pler and a 2996 diodearray detector (DAD), controlled by
Waters Millennium 3.2 software. Ten-microliter samples were
injected on a Vydac column (Hesperia, CA, USA) C18, type
218MS54 (4.6 mm ×250 mm, 5 m) fitted with a Waters Bon-
dapak C18 (2 mm ×20 mm, 50 m) guard column. The mobile
phase consisted of a mixture of methanol–water contain-
ing 25 mM of formic acid in gradient mode from 65 to 100%
methanol over 25min. Flow rate was adjusted to 1.5 ml/min.
All samples were analysed in duplicate or triplicate at
228 nm.
This method was successfully validated and showed good
linearity, reproducibility and accuracy between 10g/ml and
1 mg/ml. The method is stability indicating.
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342 european journal of pharmaceutical sciences 29 (2006) 340–347
2.3. General procedure for preparation of complexes
For preparation of complexes, ethanolic stock solutions of
CD and THC were mixed in appropriate ratios and samples
were evaporated to dryness under vacuum. Dried samples
were resuspended in unbuffered water, or methanol/water
(for some of the NMR studies) by ultrasonication (Lyng et al.,
2004), then left to equilibrate for 72 h in the dark at room tem-
perature under constant agitation. For the phase-solubility
study an excess amount of THC was added. After equilibra-
tion, undissolved THC was removed from the suspensions by
Intrinsic solubility (S0) of THC in pure water was deter-
mined by following the same protocol, but without addi-
tion of cyclodextrin. After equilibration, the water phase was
lyophilized and reconstituted in a small quantity of ethanol
for quantification of dissolved THC by HPLC.
2.4. Phase solubility study
Effects on the aqueous solubility of THC were studied using the
phase-solubility method (Higuchi and Conners, 1965). Excess
amounts of THC were mixed with ever increasing concen-
trations of CD. The tested CDs were -CD (4–50mM), -CD
(4–16 mM), -CD (4–40 mM), and RAMEB (8–187 mM). Com-
plex was prepared as described above and the solutions were
assayed for THC content by HPLC.
2.5. Job’s plots
The Job’s (continuous variation) plot of THC was determined
from 1H NMR and UV data, according to the continuous vari-
ation method (Job, 1928; Chankvetadze et al., 1998).
The NMR experiment was carried out as described below
with solutions of THC and RM--CD in unbuffered D2O/MeOD
(1:1, v/v). The total molar concentration of the two compo-
nents concentrations was kept constant at 6.36mM, but the
mole fraction of RAMEB {i.e., [RAMEB]/([RAMEB] + [THC])}var-
ied from 0.1 to 0.9. Chemical shift of proton signals was
observed for preparation of the plot.
Solutions of the same composition, but in unbuffered water
only, were used for UV-spectrophotometric determination of
the stoichiometry using the same method. The shift of max
around 275 nm of the UV-spectrum of THC was observed to
prepare the Job’s plot. Spectra were obtained with a Shimadzu
UV–vis 1240-mini spectrophotometer (0.1 nm resolution). Each
complex solution was measured in triplicate.
2.6. NMR-study of the THC–RAMEB interaction
The 1H NMR spectrum of pure THC in D2O could not be
determined due to its very low aqueous solubility. There-
fore, 1H NMR signal assignments for THC were performed
in D2O/MeOD (1:1). Also the Job’s plot was determined in
D2O/MeOD (1:1) in order to have enough signal strength at low
RAMEB concentration.
All spectra were recorded on a Bruker DPX-300 spectrom-
eter operating at 300 MHz for protons. Temperature was set
at 30 C. The peak of residual water (H2O) was used as inter-
nal reference at 4.80 ppm. For proton (1H) NMR, 128 scans were
recorded with the following parameters: 32K data points, pulse
width of 4.0 s and relaxation delay of 1s. FID’s were Fourier
transformed with LB of 0.5 Hz.
For two-dimensional (2D) nuclear Oberhauser effect spec-
troscopy (NOESY)-experiments measurements were per-
formed in D2O with 8 number of scans, 2K data points in
F2, relaxation delay 1s and mixing time 1s.In order to avoid
confusion in discussing the NMR results, protons of THC are
referred to in normal font type (H4), while protons of CD are
referred to in italic (H3).
2.7. Stability during storage
Solutions of the THC/RM--CD complex in unbuffered water
were stored at ambient temperature in tightly closed, clear
glass vials while exposed to natural light conditions in the
laboratory room. Initial THC concentration was 1mg/ml. After
1, 2, 4 and 8 weeks of storage, duplicate samples were taken
and analysed by HPLC for signs of decomposition.
3. Results
3.1. Complexation and phase solubility studies
It is most common to perform complexation studies such as
described here, in buffered aqueous solutions. However, it has
been shown that, in most cases, ionic strength has a negligible
effect on the binding of neutral molecules to CDs (Zia et al.,
2001). Furthermore, we found that pH changes in the range
of 5–9 had no effect on the solubilizing of THC by RAMEB. We
therefore concluded that it was possible to perform our com-
plexation studies in unbuffered pure water. Although treat-
ment of a THC/hydroxypropyl--CD complex with an ultra-
sonic bath was reported to result in some minor degradation
of THC (Jarho et al., 1998), such degradation was not observed
in our study after ultrasonication.
Testing of four different cyclodextrins showed that only the
use of RAMEB results in significant levels of solubilized THC.
At their highest tested concentrations, -CD (50 mM) and -
CD (16 mM) had a very slight solubilizing effect in the order
of 0.1 mM THC, but whether this was the result of inclusion
or some other mechanism was not further determined. Prac-
tically no THC was solubilized with the use of -CD (40 mM).
At the maximal RAMEB concentration tested (24%; 187 mM) a
THC concentration of 45 mM (14mg/ml) was reached, which
means an increase of aqueous solubility of THC of about four
orders of magnitude. The phase-solubility diagram is shown
in Fig. 2.
An Ap-type phase solubility diagram was obtained, which
suggests formation of a higher-order complex with respect to
cyclodextrin (i.e. 1:2 complex). Based on similar data, Mannila
et al. (2005) concluded earlier that THC forms a complex with
RAMEB in a 1:2 stoichiometric ratio. However, complemen-
tary data obtained in our study by preparing the Job’s plot of
THC/RAMEB showed the stoichiometry to be a 2:1 ratio of THC
The intrinsic solubility (S0) of THC in unbuffered water at
20 C was determined to be 2.3 M (0.7 g/ml). Subsequently,
the apparent stability constant was calculated from the
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european journal of pharmaceutical sciences 29 (2006) 340–347 343
Fig. 2 – Phase solubility diagram for THC in the presence of
RAMEB at 298 K. Datapoints are average values of duplicate
initial linear part of the phase-solubility diagram according
to Higuchi and Conners (1965):
K1:1 =slope/([S0](1slope)) M1
The value of K1:1 apparent was found to be 15,900 M1, which
is in accordance with the value (19,600 M1) reported earlier for
this complex based on phase-solubility study (Mannila et al.,
2005). The 2:1 binding constant obviously could not be deter-
mined from the diagram.
At higher concentrations of CD the diagram slightly curves
off. As pointed out by Higuchi and Conners (1965), negative
curvature diagrams reflect an alteration in the effective nature
of the solvent in the presence of high concentrations of the
host molecule (i.e. viscous and “non ideal” characteristic of
the solution), leading to a change in the complex formation
constant. Alternatively, it is possible that the formation of 2:1
complexes results in subsequent formation of micellar-like
structures. Such structures could precipitate from solution,
thereby lowering the THC concentration.
3.2. Determination of the stoichiometry
Two independent techniques were used for preparation of a
continuous variation plot in order to determine the stoichiom-
etry of the inclusion complex. The NMR results were obtained
for most of the THC peaks but for only some CD peaks (Me2,
Me6), mainly because of spectral overcrowding. Thus, the ratio
of CD and THC was varied while the sum of their concen-
trations was kept constant, and a continuous variation plot
was prepared. Using this method the value for ı reaches a
maximum at the stoichiometric point. The plot for the NMR-
peaks of THC undergoing the largest shifts is shown in Fig. 3a.
Results for the NMR-determination of CD are not shown, but
all results yielded 2:1 stoichiometry of THC to CD. In a single,
stable complex, the plot usually has a triangular form with a
maximum, while the formation of weak complexes results in
curved plots. The shape of the plot in Fig. 3a therefore suggests
that the studied complex is indeed not of the single (1:1 sto-
ichiometry) stable kind. For all ratios of THC:CD only a single
set of peaks was observed for THC, indicating a fast exchange
Fig. 3 – (a) Continuous variation plot for THC obtained from
the chemically induced shift displacement (CID) of selected
NMR proton signals of THC; H2 (), H4 (), H5(), H1(×).
(b) Continuous variation plot for THC obtained from UV
investigations. Datapoints are average values of triplicate
measurements. Error bars indicate standard error.
The very low solubility in water did not allow NMR studies
of the guest in pure water. Instead some studies had to be
carried out in a methanol/water mixture. Although it must
be noted that the addition of methanol possibly changes the
nature of the complex, the stoichiometry of 2:1 was confirmed
by the results of the UV determination, which was performed
in water only (Fig. 3b).
3.3. Chemically induced shift displacements (CID)
study of the complex
An updated assignment of signals for THC was recently pub-
lished by Choi et al. (2004). The signals in the obtained 1H
NMR spectrum of THC were well separated from the signals
of RAMEB, with the exception of the H10a signal. The signal of
H6was obscured by the signal of residual water in the deu-
teriated solvent.
NMR studies on RAMEB are difficult because it is not a
single pure compound, but rather a mixture of randomly
methylated molecules of -CD. As a result only some of
the NMR-signals for RAMEB could be unambiguously iden-
tified: Me2,Me6, and H1. Other signals were uncertain and
could not be used for interpretation. Therefore, it is hard
to make definitive conclusions about the orientation of
THC in the complex with CD. Peak assignment for RAMEB
was performed by using published data on RAMEB and
DM--CD (Ravichandran and Divakar, 1998; Correia et al.,
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344 european journal of pharmaceutical sciences 29 (2006) 340–347
Table 1 – 1H NMR chemical shift values for free and
complexed THC with RM--CD (equimolar ratio, total
concentration = 6.36 mM)
Proton signal Chemical shift (ppm) Shift in ppm
Free Complex
H2 6.14 6.00 0.14
H4 6.27 6.14 0.13
H6alpha 1.41 1.44 +0.03
H6beta 1.09 1.10 +0.01
H7 1.90 1.90 0
H8 2.16 2.16 0
H10 6.31 6.27 0.04
H11 1.68 1.67 0.01
H12.42 2.36 0.06
H21.55 a
H3/H41.29 a
H50.87 0.95 +0.08
aNot clear.
2002), in combination with the obtained results of 1H and
A definite increase of the water solubility was observed for
THC in the presence of RAMEB and addition of RAMEB to a
solution of THC (in D2O/MeOD) resulted in modification of the
1H NMR spectrum of THC. These changes of the NMR spectra of
THC can be understood in terms of the formation of inclusion
complexes, where a molecule of THC is positioned inside the
hydrophobic cavity. Examination of the observed chemically
induced shift displacements (CID, shown in Table 1) provided
information of the nature of guest–CD interaction because
protons that undergo the largest shift upon complexation are
considered to be most strongly involved in interactions lead-
ing to complexation.
The THC signals of H2, H4, H5and H1were most affected,
while that of H7, H8 and H11 underwent almost no displace-
ment. This indicates an inclusion of ring A and the alkyl side
chain of THC into the CD cavity, while ring C is not, or only
partially, included. It should be noted that H2, H4 and H1all
undergo an expected upfield shift upon inclusion, while the
H5signal showed a shift downfield. An explanation for this
could be that the alkyl side chain completely enters the CD
cavity and protrudes from the opposite opening, exposing H5
to the solvent. The moderate downfield shift that is observed
for H6could be explained by a change in the orientation of
the surrounding shell of water molecules upon inclusion, or
possibly by conformational changes in a non-included part of
the molecule. The relatively small ı values observed for all
signals indicate a relatively weak association.
Regarding the NMR spectrum of RAMEB, the presence of
THC is related to an upfield shift of Me2, which seems to sug-
gest its involvement in complexation. The associated small
upfield shift for H1, located on the outside of the CD-ring, is
possibly due to conformational changes in the CD-ring struc-
ture upon complexation. Data on Me6 was inconclusive. Shift
of any other signal could not be observed due to spectral over-
crowding in the NMR spectrum, so based on these data alone,
only limited conclusions can be made on the involvement
of CD-protons in complexation. More conclusive data could
be derived by studying THC complexation with DM--CD, but
such study was not performed as part of this work. Moreover,
there is the possibility that substituting RAMEB with DM--CD
might alter the nature of the complex.
3.4. NOESY-experiments
The NOESY spectrum of the complex dissolved in D2O(Fig. 4)
shows a variety of interactions between THC and CD protons.
These interactions confirmed the inclusion of at least one THC
molecule inside the cavity of RAMEB. Two signals of RAMEB
could be clearly identified (Me2 and Me6) and this proved to
provide enough information to elucidate the complex struc-
ture. The H1-signal (not shown) could be identified also, but
it shows no crosspeaks at all as this proton is present at the
outside of the CD-ring.
When it is assumed that a THC molecule is positioned
inside the cavity, two general orientations along the long axis
of THC are possible. A strong interaction between H3-, H4-
and H5-signals of the pentyl side chain of THC and Me6 of
CD indicate that the side chain protrudes through the pri-
mary opening. This orientation of THC brings H11 and H6
into proximity of Me2, which is confirmed by the presence of
the expected crosspeaks. A notable absence of crosspeaks is
observed for H7 and H8, while only very weak interactions are
Fig. 4 – Partial contour plot of a NOESY spectrum of the THC complex with RM--CD. Peaks of THC are identified on the top
of the figure, while peaks of CD are marked on the left. *Position of H2-signal.
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european journal of pharmaceutical sciences 29 (2006) 340–347 345
Fig. 5 – Proposed structure of the THC–RAMEB complex.
observed for H6a and H10a. This suggests that ring C remains
at least partially outside the CD cavity and this is in agreement
with the analysis of the observed chemical shift displace-
Based on the obtained NMR data a model for the inclusion
of THC into the RAMEB cavity can be suggested. The proposed
structure of the 1:1 complex can be understood from Fig. 5.
Although the THC side chain is included inside the complex,
there is a notable absence of crosspeaks betweenH1and H2of
THC with CD-protons. Likely, the presence of the more bulky
phenolic ring restricts the movement of the alkyl chain and
physically prevents the H1and H2protons to come into prox-
imity of the CD protons on the inside of the cavity. A similar
result was obtained for complexes of -CD with fusidate and
helvolate, which contain a side chain attached to a rigid (ring)
structure (Jover et al., 2003). The proposed structure also corre-
sponds with the suggestion, based on the study of chemically
induced shift displacements (CID), that H5is exposed to the
Because we propose a 2:1 binding of THC to CD, a second
THC molecule must be bound to the complex. This binding is
thought to be the result of non-inclusion interactions. It was
discussed above that an inclusion interaction exists between
H11 and H6, and Me6 of RAMEB. However, at the same time
H11 and H6show a clear interaction with Me2, which is
positioned at the other end of the CD cavity. This seemingly
incompatible data can be explained by the presence of the sec-
ond THC molecule at the primary opening of CD as shown in
Fig. 5. A non-inclusion interaction between protruding methyl
groups from both THC and RAMEB seems very plausible.
The proposed structure allows interaction between H5of
included THC with the free THC, possibly providing an alter-
native explanation for the observed positive CID value for H5.
However, no such crosspeaks were observed in the NOESY
spectrum, indicating this interaction, if present, must be very
3.5. Stability during storage
A solution of THC in ethanol will rapidly degrade under the
influence of light and air, resulting in formation of degrada-
tion products delta-8-THC and cannabinol (CBN) (Fairbairn
et al., 1976). However, storage of the complex dissolved in
unbuffered water under standard laboratory room conditions
(artificial light, temperature ±22 C) did not results in any sig-
nificant degradation of THC during the test period of 8 weeks.
Furthermore the THC concentration remained constant.
In general, stability studies are performed in buffered solu-
tions to get the most reliable results. However, in our case we
were interested in the behaviour of complex in unbuffered
water, as our research is focussed on the future preparation of
purely aqueous THC solutions with a minimum of additives.
For this reason water was not buffered in the stability test.
We believe this is possible because THC and CD have no effect
on pH upon dissolving in water, and we found that complex
formation was not influenced by pH in the range of pH 5–9.
4. Discussion
In this study it was found that out of four different
types of cyclodextrins tested, only randomly methylated -
cyclodextrin was able to increase the aqueous solubility of
THC to a significant level. A concentration of THC of about
14 mg/ml was reached by using a 24% (187mM) RAMEB solu-
tion. The binding ratio of THC to CD was found to be 2:1
by using both an NMR- and a spectrophotometric method.
However, such a complexation theoretically should result in
a linear phase-solubility diagram while in fact an Ap-type was
observed (this study; Mannila et al., 2005). The cavity of RAMEB
has a diameter that is somewhat smaller than that of natural
-CD (6 ˚
A) and this would allow inclusion of THC no further
than ring B. Based on spatial restrictions it seems unlikely
that RAMEB could accommodate two molecules of THC. This
seemingly incompatible data could be plausibly explained by
assuming the formation of a 1:1 inclusion complex with non-
inclusion interaction leading to a 2:1 complex. A similar struc-
ture was recently found for the complexation of ketoprofen
with -CD (Rozou et al., 2005).
It has been suggested that 1:1 drug/cyclodextrin inclu-
sion complexes form water-soluble non-inclusion complexes
with additional drug molecules to give rise to Ap-type phase-
solubility diagrams (Loftsson et al., 2002). This has been shown
with acridine/dimethyl--CD (Correia et al., 2002), where it
was concluded that a real 1:1 inclusion complex was formed,
while a second molecule of acridine probably interacts with
the DM--CD, but it remains outside the cavity. We speculate
that this is also the case for the THC/RAMEB complex.
If the studied complex is indeed of a 2:1 ratio, then what
would be the structure of such a complex? The NMR data
seems well capable of suggesting a 1:1 complex structure as
the chemical shift displacement values in NMR indicate the
strength of host–guest interactions which are responsible for
the equilibrium constant. This usually is an indication for the
stability of the complex. In the case of THC, the chemical shift
displacement was found to be relatively low, leading to the
conclusion that the complex of THC with RAMEB is a weak
one. This was also suggested by the curved shape of the NMR
Job’s plot.
From the obtained NMR data it was concluded that THC
forms a complex through inclusion of rings A and B, with the
pentyl side chain partly protruding from the primary opening
of RAMEB. Ring C seems to be only partially included due to
steric hindrance presented by the methyl groups in positions
6and 11. In order to even better allow the proposed inclusion
of THC inside the CD cavity, the side chain can adopt a folded
conformation inside the -CD cavity. A similar folded configu-
Author's personal copy
346 european journal of pharmaceutical sciences 29 (2006) 340–347
ration was found for the flexible side chain of bile salts (Ramos
Cabrer et al., 1999, 2003). In several studies it was shown that
alkyl side chains, because of their lipophilic character, are the
preferred substituent of the guest molecule for inclusion into
the cavity, provided they are accessible for interaction with the
CD molecule (Ravichandran and Divakar, 1998; Ramos Cabrer
et al., 1999, 2003; Zhang et al., 2002).
The formation of a 2:1 complex by binding of a second THC
molecule to the 1:1 complex through non-inclusion interac-
tions was supported by NMR data. A weak binding between
THC and RAMEB was suggested by the obtained data (CID
values, NMR Job’s plot). However, the apparent 1:1 stability
constant was relatively high. This supports the idea of a sec-
ond THC molecule, strengthening or stabilizing binding of the
included molecule. Unfortunately, binding constant of the 2:1
complex could not be calculated from the obtained data.
Although the use of RAMEB highly increased aqueous sol-
ubility of THC, only a very weak solubilization was observed
when THC was mixed with unsubstituted -CD. Apparently
the presence of methyl groups is needed for inclusion of THC
in the cavity, which is a further indication that complexa-
tion leading to formation of the 2:1 complex is mostly due to
hydrophobic interactions between THC and these non-polar
methyl groups.
The water concentration of THC that can be achieved by
the use of CDs is in a suitable range for possible clinical
or analytical applications. In a preliminary study we found
that several other major cannabinoids could be solubilized
as well in the presence of RAMEB. Studied cannabinoids
included 9-tetrahydrocannabinolic acid (THCA), cannabinol
(CBN), cannabidiol (CBD) and cannabigerol (CBG). Without CDs
present, all of these compounds were practically insoluble
in pure water. However, real inclusion could not be proven
by these experiments and complementary studies have to be
performed. Clearly the CD complexation of THC and possibly
other cannabinoids are a promising way for producing water-
based solutions of cannabinoids without the need for addition
of other solubilizers or organic solvents. Hopefully the results
obtained in this study will be a contribution to the further
development of cyclodextrin studies with the cannabinoids.
The authors are grateful to Farmalyse BV, The Netherlands
for providing the high quality THC and other cannabinoids
that were needed for our study. The van Leersum fund, The
Netherlands, is acknowledged for providing us with the funds
for obtaining the spectrophotometer.
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... Cyclodextrins possess a similar structure to alkyl-derived oligosaccharide released from the plant cell wall during a fungal infection; thus they act as elicitors for the production of secondary metabolites. More investigations are needed on the effect of cyclodextrins on the synthesis of non-polar cannabinoids in cannabis suspension cultures [65,70]. ...
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Phytocannabinoids are a structurally diverse class of bioactive naturally occurring compounds found in angiosperms, fungi, and liverworts and produced in several plant organs such as the flower and glandular trichrome of Cannabis sativa, the scales in Rhododendron, and oil bodies of liverworts such as Radula species; they show a diverse role in humans and plants. Moreover, phytocannabinoids are prenylated polyketides, i.e., terpenophenolics, which are derived from isoprenoid and fatty acid precursors. Additionally, targeted productions of active phytocannabinoids have beneficial properties via the genes involved and their expression in a heterologous host. Bioactive compounds show a remarkable non-hallucinogenic biological property that is determined by the variable nature of the side chain and prenyl group defined by the enzymes involved in their biosynthesis. Phytocannabinoids possess therapeutic, antibacterial, and antimicrobial properties; thus, they are used in treating several human diseases. This review gives the latest knowledge on their role in the amelioration of abiotic (heat, cold, and radiation) stress in plants. It also aims to provide synthetic and biotechnological approaches based on combinatorial biochemical and protein engineering to synthesize phytocannabinoids with enhanced properties.
... In most cases the stoichiometry of the complexes was derived from Job ś plots, while the orientation and stacking of both solutes within the CD cavity was derived from molecular modeling [46][47][48] . There are also reports of 2:1 complexes, in which one solute molecule was included in the CD cavity, while the second one was coordinated externally [50,51] . Neither these studies nor the abovementioned publications on 2:1 aggregates provided evidence for the presence of two complexes with different stoichiometries in solution. ...
During a screening of cyclodextrins (CDs) as chiral selectors for the separation of daclatasvir (DCV) and its enantiomer by capillary electrophoresis (CE), an unusual phenomenon for CDs was observed, that is two peaks with a plateau in between using γ-CD as chiral selector. The same result was encountered when enantiopure DCV was injected or when analyzing a sample containing enantiopure DCV and γ-CD in a CD-free background electrolyte. Peak coalescence was observed at 45°C and at a pH above 3.5. Two peaks with a plateau were also observed for DCV stereoisomers as well as a structural analog. However, only a single peak was detected if one or both amino acid moieties of DCV were lacking. Nuclear magnetic resonance (NMR) experiments including Nuclear Overhauser effect-based methods showed that in solution DCV adopted a folded conformation in which the isopropyl side chain of the valine residues pointed toward the aromatic rings of DCV. Moreover, NMR unequivocally demonstrated the simultaneous formation of DCV-γ-CD inclusion complexes with 1:1 and 2:1 stoichiometry, which was corroborated by mass spectrometry. In both complexes, DCV also adopted a folded structure. The RSSR-diastereomer of DCV as well as an analog lacking one of the amino acid moieties also formed 1:1 and 2:1 complexes with γ-CD although a plateau was only observed in the case of the RSSR-diastereomer. As shown by CE-MS, both DCV–γ-CD complexes surprisingly comigrated as the first peak, while the second migrating peak represents non-complexed DCV.
... Such a solubilization behaviour is also observed for other cannabinoids [63]. Another example is the solubility profile of dexamethasone where the γ-CD inclusion complex has only a limited solubility and therefore, this complexation does not lead to an increase of the concentration of the drug. ...
Cyclodextrins are widely used in pharmacy, chemistry and other scientific disciplines, due to their unique properties which are consequences of the special geometries of these compounds. The cyclic arrangements of glucopyranose rings form structures where small and medium-sized molecules can be included. This inclusion reaction is of high interest, because it may change the physico-chemical properties of the guest molecules and allows the application of the involved compounds like drugs for a better delivery. Another important feature is the fact, that a large number of cyclodextrin derivatives is existing, with different affinities to the guest molecules, different thermodynamic properties and consequently a broad variety of applications. In the present review a short overview will be given about the various structures, the applications, in particular as drug carriers. © 2020 Science Society of Thailand under Royal Patronage. All rights reserved.
... Among the main reasons, the low temperatures used in the heating process originated a conversion into decarboxylated analogs unpredictable and incomplete, with the presence of THCA-A and CBDA in decoction preparations [16,24]. Moreover, the conversion of THCA-A into THC is limited in boiling water, also due to the result of saturation of the water phase with THC, whilst THCA-A is more hydrophilic and soluble [25]. Concerns remain whether stable and therapeutic cannabinoid levels are achievable in real-life situations, when the preparation of the decoction is performed by the patient at home, in non-standard conditions. ...
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Purpose: The recent release of a medical cannabis strain has given a new impulse for the study of cannabis in Italy. The National Health Service advises to consume medical cannabis by vaporizing, in decoction or oil form. This is the first study that explores the pharmacokinetics and tolerability of a single oral dose of cannabis as decoction (200 ml) or in olive oil (1 ml), as a first step to improve the prescriptive recommendations. Methods: This is a single-center, open-label, two-period crossover study designed to assess the pharmacokinetics and tolerability of oral cannabis administered to 13 patients with medication overuse headache (MOH). A liquid chromatography tandem-mass spectrometry (LC-MS/MS) method was conducted for the quantification of THC, CBD, 11-OH-THC, THC-COOH, THC-COOH-glucuronide, THCA-A, and CBDA. Blood pressure, heart rate, and a short list of symptoms by numerical rating scale (NRS) were assessed. Results: Decoctions of cannabis showed high variability in cannabinoids content, compared to cannabis oil. For both preparations, THCA-A and CBDA were the most widely absorbed cannabinoids, while THC and CBD were less absorbed. The most important differences concern the bioavailability of THC, higher in oil (AUC0-24 7.44, 95% CI 5.19, 9.68) than in decoction (AUC0-24 3.34, 95% CI 2.07, 4.60), and the bioavailability of CBDA. No serious adverse events were reported. Conclusions: Cannabis decoction and cannabis oil showed different pharmacokinetic properties, as well as distinct consequences on patients. This study was performed in a limited number of patients; future studies should be performed to investigate the clinical efficacy in larger populations.
Conference Paper
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Cannabis sativa L. is an important herbaceous species originated in Central and East Asia. It also has a wide distribution range from the Caspian and Himalayas to China and Siberia. Cannabis is an annual and dicotyledonous flowering plant, commonly known as hemp. It is usually dioecious, but can also be monoecious. Throughout history, it has been counted among five major grains and used in the field of textiles, rope, paper and energy. Since the ancient times, some parts of cannabis plant have been utilized in folk medicine as well as an important source of textile fiber. This study presents the analysis of the micromorphology and anatomy, seed size variation and morphological features of the seeds of both drug-and fiber-type Cannabis according to the published literatures. Based on the size of seeds, these two types of Cannabis were easily distinguishable, and comparatively bigger sized seeds were observed in the fiber type. On the other hand, Cannabis sativa L. is the only valid name of the cannabis plant that is given to different taxa in different studies.
Cannabis sativa L, commonly known as Hemp, shows numerous health and economic benefits with commercial applications such as in seed production, fiber, oil, and pharmaceutical uses. Hemp oil has been widely acknowledged to have a panoply of health benefits, such as cholesterol lowering properties and decreasing high blood pressure, owing to the presence of two essential fatty acids (linoleic acid and α-linolenic). Given the economic and industrial importance of hemp, the present study was designed in an endeavor to select and compare the best cultivar and region to increase both quantity and quality of hemp oil from Iran. Seeds of two native populations from Iran (Fars and Yazd provinces) and one foreign variety from France (Fedora17, as an industrial hemp cultivar) with its progenies (Fedora17-2) were cultivated in research fields of three locations (Gilan, Golestan, and Alborz province) in Iran. The mature seeds of cultivated plants were collected and their oils were extracted by Soxhlet apparatus and analyzed by Gas Chromatograph. The seed yield was 288.70 to 3182.60 kg ha⁻¹ and oil yield were 0.08 to 0.95 t ha⁻¹ in the studied sites. Also, the range of 1000- seed weight was 5.20 to 15.28 g. The oil content of different varieties varied from 15.46 to 36.63%. Based on the results, the highest seed yield, 1000-seed weight, and oil yield belonged to native populations. It was also determined that Alborz was significantly higher in terms of oil production, 1000-seed weight, and seed and oil yield per hectare compared with the other two regions. The most abundant fatty acid in the samples was linoleic acid, (56.80 to 63.98%), and almost identical in all cultivars and areas studied. The range of linoleic acid and α-linolenic in three regions were 57.55 to 63.98% and 7.57 to 22.91%, respectively. In addition, the palmitic acid content of foreign varieties was higher than that of the native populations in all three locations. Since the interfaces in the production and synthesis of fatty acids in plants are influenced by variation in temperature, light and moisture amount and farming conditions so the percentage and kind of fatty acids in hemp oil can be varied in different regions.
Introduction: The concept of a cannabis ‘entourage effect’ was first coined as a hypothetical afterthought in 1998. Since then, multiple scientific reviews, lay articles, and marketing campaigns have promoted the effect as a wholly beneficial manifestation of polypharmacy expected to modulate the therapeutic effects of cannabis and its derivatives. There is reason to wonder at the authenticity of such claims. Areas covered: A broad definition of the entourage effect is presented, followed by brief summaries of the nature of cannabis polypharmacy and the commonly cited contributing phytochemicals, with special attention to their attendant adverse effects. A critical analysis is then offered of the primary literature that is often portrayed as suggestive of the effect in existing reviews, with further studies being drawn from PubMed and Google Scholar searches. A final discussion questions the therapeutic value of the entourage effect and offers alternate perspectives on how it might be better interpreted. Expert opinion: Claims of a cannabis entourage effect invoke ill-defined and unsubstantiated pharmacological activities which are commonly leveraged toward the popularization and sale of ostensible therapeutic products. Overestimation of such claims in the scientific and lay literature has fostered their misrepresentation and abuse by a poorly regulated industry.
Rosmarinic acid (RA) is a phenolic compound with remarkable antioxidant activity; however it presents low bioavailability. Cyclodextrin (CD) derivatives, such as hydroxypropyl-β-CD (HPβCD) and methyl-β-CD (MβCD), have been used to solve the low bioavailability of drugs, increasing their solubility or permeability. The aim of the present study was to investigate the complexation of RA with two derivative CDs, HPβCD and MβCD, and the resulting antioxidant activity. A phase-solubility study was performed to determine the stoichiometric ratio and apparent stability constant, and electrospray ionization coupled to mass spectroscopy (ESI–MS) was used to confirm the stoichiometric ratio. The freeze-dried (FD) complexes were characterized by liquid chromatography (LC), differential scanning calorimetry (DSC), Fourier-transform infrared spectroscopy (FTIR), scanning electron microscopy (SEM), and nuclear magnetic resonance (NMR). The antioxidant activity of RA and the FD complexes was evaluated by the DPPH method. A 2:1 (RA:CD) stoichiometric ratio and strong apparent constant stability were found for both complexes using a phase-solubility study, and ESI-MS spectra confirmed a signal corresponding to the 2:1 complex. The FD complexes showed high yield and RA content. The results of DSC, FTIR and SEM demonstrate changes in the physical-chemical characteristics of RA, suggesting its interaction with CDs. These interactions were confirmed by NMR, which revealed the formation of inclusion and non-inclusion complexes. The antioxidant activity of the RA:HPβCD and RA:MβCD FD complexes were higher than that of RA. The results suggest that FD complexes can be a technological approach to the development of formulations containing high content of RA with a view to increasing antioxidant potential.
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Book chapter published in the famous Handbook of Cannabis (2016) by Roger Pertwee. This was chapter 18 and it focused on self-medication of patients with cannabis: their motivations, obstacles, preferences and what we can learn from that.
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In recent years, the Cannabis plant (Cannabis sativa L.) has been rediscovered as a source of new medicines around the world. Despite the fact that a number of registered medicines have been developed on the basis of purified cannabis components, there is a rapid increasing acceptance and use of cannabis in its herbal form. Licensed producers of high quality cannabis plants now operate in various countries including The Netherlands, Canada, Israel, and Australia, and in many US states. The legal availability of cannabis flowers allows to prescribe and prepare different cannabis galenic preparations by pharmacists. It is believed that synergy between cannabis components, known as “entourage effect”, may be responsible for the superior effects of using herbal cannabis versus isolated compounds. So far, only a few cannabis components have been properly characterized for their therapeutic potential, making it unclear which of the isolated compounds should be further developed into registered medicines. Until such products become available, simple and accessible galenic preparations from the cannabis plant could play an important role. In cannabis, phytochemical and pharmacological attention has been attributed mainly to four major cannabinoids (Δ9- tetrahydrocannabinol, cannabidiol, cannabigerol and cannabichromene) and to terpene components. This means a basic knowledge of these compounds and their bioavailability in different administration forms is useful for producers as well as prescribers of galenic preparations. This work will outline the most important aspects of cannabinoids and terpenes, and their behaviors during preparation and use of various administration forms including vaporizing, cannabis oils and extracts, tea, and skin creams.
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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.
Investigation is made of the preparation of the inclusion complex of Δ9-tetrahydrocannabinol (1) with β-cyclodextrin (β-CD) in solid form by the precipitation method. In order to estimate the inclusion complex formation, various spectral data (uv, cd, and emission spectra) have been studied. It has been estimated that the inclusion complex is highly stable under various conditions [light of 10,000 lux, oxygen existence, and high temperature (120°)] compared to 1 alone. The extensive application for a drug delivery system is also discussed.
The complexation behavior of two bile saltssodium cholate (NaC) and sodium deoxycholate (NaDC)with β-cyclodextrin (β-CD), 6-deoxy-6-amino-β-cyclodextrin (β-CDNH2), and dimer I (N,N‘-bis(6-deoxy-β-cyclodextrin)pyromellic acid diamide) was studied by NMR techniques. Complexes formed between β-CD and β-CDNH2 with NaC and NaDC have 1:1 and 2:1 (host:guest) stoichiometries, respectively. Complexes with β-CDNH2 show higher equilibrium constants than those with β-CD because of the electrostatic effect of the protonated amine group. Dimer I showed 1:2 and n:n stoichiometries with NaC and NaDC, respectively. ROESY spectra stated that bile salts enter first with their 5-C ring forward the inner cavity by the side of the secondary hydroxyl groups of cyclodextrins. In the complexes formed with β-CDNH2, the steroid body of the bile salt enters deeper in the cavity, while the carboxylated side chain is extended toward the protonated amine group at C-6, allowing an electrostatic interaction between both groups. In the case of the 2:1 stoichiometry, the second cyclodextrin complexes ring A of the steroid body.
The rate and extent of glass binding of Δ9 -tetrahydrocannabinol in aqueous solution depend on the surface area and pretreatment of glass and the concentration of the drug. A total of 20 and 40% at 0.1 and 0.05 μg/ml, respectively, was bound to 50-ml volumetric flasks but could be minimized by silyl pretreatment of the glass. The drug rapidly diffused into plastics, and 70–97% was taken up by the rubber closures used for plasma vials. These bindings precluded classical methods of solubility determination, so spectral and particle-size counting determinations, which observed those concentrations at which true solution was terminated, were used. The aqueous solubility was a linear function of both the ethanol concentration (increasing) at constant ionic strength and the square root of the ionic strength (decreasing) at constant ethanol concentration. The salting-out coefficient was of high magnitude and typical solubilities were 2.8 mg/liter in water and 0.77 mg/liter in 0.15 M NaCl at 23°. The bindings also precluded the use of the classical methods of equilibrium dialysis and ultrafiltration to determine the protein binding of tetrahydrocannabinol. A method of variable plasma concentrations was devised, so protein binding was determined from the pseudoplasma concentrations of the drug after the separation of the pseudoplasma from the red blood cells added to form pseudoblood with known concentrations of Δ9 -tetrahydrocannabinol. This use of the competition between the high partitioning of drug between red blood cells with plasma water (D = 12.5) and the binding to plasma protein permitted an estimate of 97% binding which was not drug concentration dependent. The spectrophotometric pKa' of Δ9 -tetrahydrocannabinol was 10.6. Δ9 -Tetrahydrocannabinol degraded readily in acid solutions. Subsequent to a rapid loss, the kinetics appeared to be first-order and specific hydrogen-ion catalyzed. Concomitantly, small amounts of Δ8 -tetrahydrocannabinol were produced, as were two GLC observable products, P2 and P3, and the rate of their appearance appeared to parallel the rate of Δ9 -tetrahydrocannabinol degradation. A peak, P1, also appeared almost instantaneously but did not parallel drug degradation.
Previous molecular modeling studies, in our laboratory, have shown that some esters of type RCOO(CH2)nC5H5N+Cl− are potentially active against Alzheimer's disease. We have also demonstrated that acridine, which has strong anticholinesterase activity appears to be a suitable R substituent. The main obstacle to the possible pharmaceutical application of these compounds is their limited solubility in water, which is due to the poor aqueous solubility of acridine itself (0.26 mM). Inclusion complexation with cyclodextrins may overcome this problem. Solubility diagrams and NMR spectroscopy were used to study the inclusion of acridine (Acr) within β-cyclodextrin (βCD) and heptakis(2,6-di-O-methyl)cyclomaltoheptose (DMβCD). A 1:1 complex was formed for the Acr–βCD system and both 1:1 and 2:1 complexes for the Acr–DMβCD system (apparent Ka 215 ± 20 and 1150 ± 100 M−1, respectively). Data from 1H NMR studies corrected the assignment of the acridine H1, H8 and H4, H5 protons in D2O, which have been erroneously assigned in previous publications. 1H spin–lattice relaxation times T1 measured with selective, non-selective and null inversion characterize the dynamics of the inclusion complexes. Geometric features of the host–guest inclusion complexes were inferred from intermolecular dipolar interactions obtained by 2D adiabatic off-resonance ROESY experiments. A more detailed picture of the structure of the inclusion complexes was obtained by combining NMR structural data and molecular modeling (docking, dynamics under NMR constraints and energy calculations). Copyright © 2002 John Wiley & Sons, Ltd.
Phase-solubility diagrams are frequently used to calculate stoichiometry of drug/cyclodextrin complexes. Linear diagrams (AL-type systems) are thought to indicate that the complexes are first order with respect to cyclodextrin and first or higher order with respect to the drug. Positive deviation from linearity (AP-type systems) are thought to indicate formation of complexes that are first order with respect to the drug but second or higher order with respect to cyclodextrin. The phase solubility of several different compounds, i.e., cholesterol, ibuprofen, diflunisal, alprazolam, 17β-estradiol and diethylstilbestrol, and various charged and uncharged cyclodextrins was investigated. Phase-solubility diagrams of cholesterol in aqueous cyclodextrin solutions were all of AP type. However, the phase-solubility diagrams obtained with charged cyclodextrins could not be fitted to complexes of second or higher order with respect to cyclodextrin. The phase-solubility diagrams of ibuprofen and diflunisal were of AL type with slope greater than unity indicating formation of 2:1 drug/cyclodextrin complexes. However, Job's plots and space filling docking studies indicated that 1:1 complexes were formed. These and other observations show that stoichiometry of drug/cyclodextrin complexes cannot be derived from simple phase-solubility studies. Furthermore, the results indicate that drug/cyclodextrin complexes can self-associate to form water-soluble aggregates, which then can further solubilize the drug through non-inclusion complexation. © 2002 Wiley-Liss, Inc. and the American Pharmaceutical Association J Pharm Sci 91:2307–2316, 2002
The disposition of cholesterol inside the -cyclodextrin cavity(-CD) was deduced from oxidation of cholesterol secondary alcoholgroups by Ca(OCl)2 and H2O2 in thepyridine–acetic acid system. The amount of cholest-4-ene-3-one formedwas found to be proportional to the concentration of -cyclodextrin,resulting in 56.1% of ketone. The oxidation rate was enhanced by-cyclodextrin and its methyl, polymer and 1 : 1copper(II)–-cyclodextrin derivatives. Detailed investigationsinvolving UV-visible, 13C- and 1H-NMR(T1, 1D NOE and ROESY) spectroscopic studies were carried out.A binding constant value of 15,385 1500 M-2 wasobtained for the 2 : 1heptakis-2,6-di-O-methyl--cyclodextrin(DM-CD) : cholesterolcomplex in chloroform from UV studies. Proton and solid state13C-CP MAS spectra of the -CD–cholesterol mixtureshowed large magnitude shifts for the protons from the wider end of the-CD cavity as well as those of ring A and ring B of cholesterol. Both1D NOE and ROESY measurements indicated the proximity between ring A andring B protons of cholesterol and the wider end protons of -CD andDM-CD. Besides, analysis of c,i and tau;m from T1measurements showed not only a lowering of rotational motions but a value of 0.016–0.048 for some of the cholesterol protons, typical of aweak complex. Based on these studies, a probable structure for the 2 : 1complex involving two molecules of -CD/DM-CD was proposed withportions of ring A and ring B being present inside the wider end of the-CD/DM-CD cavity and ring D and the side chain attached atposition 17, projecting into the wider end of the secondCD/DM-CD molecule.
Bixin or 6,6′-diapo-ϕ,ϕ′-carotenodioic acid (mono)-methyl ester, isolated and purified as the major and water insoluble carotenoid from “urucum” (Bixa orellana, L.) seeds, was submitted to complexation with a natural cyclodextrin model (α-CD) using both column percolation and sonication. This water-soluble product was analyzed by spectrophotometry and 1H NMR to confirm complex formation as well as protection for the carotenoid from the effects of light and air or the combination of both. Also evaluated was the capability for free or complexed bixin as quencher/scavenger of free radicals such as α-α-diphenyl-β-picrylhydrazide (DPPH) and its degradation time course when challenged with ozone generated directly in the pigment solution or indirectly in the surrounding environment. The results showed that the complexed form of bixin is more resistant than free bixin to the damage caused by light and air or their combination besides and shows improved water solubility as required for novel formulations of medical or pharmaceutical interest.