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european journal of pharmaceutical sciences 29 (2006) 340–347
available at www.sciencedirect.com
journal homepage: www.elsevier.com/locate/ejps
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
Keywords:
Cyclodextrins
Cannabis
Tetrahydrocannabinol
Complexation
Solubility
NMR spectroscopy
Physical characterization
abstract
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
medicine.
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: ahazekamp@rocketmail.com (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.
doi:10.1016/j.ejps.2006.07.001
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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
(HP)--CD.
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
centrifugation.
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
to RAMEB.
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
measurements.
initial linear part of the phase-solubility diagram according
to Higuchi and Conners (1965):
K1:1 =slope/([S0](1−slope)) M−1
The value of K1:1 apparent was found to be 15,900 M−1, which
is in accordance with the value (19,600 M−1) 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
regime.
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
H6␣was 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
NOESY NMR.
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 H6␣could 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-
ments.
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
solvent.
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
weak.
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.
Acknowledgements
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.
references
Brenneisen, R., Egli, A., ElSohly, M.A., Henn, V., Spiess, Y., 1996.
The effect of orally and rectally administered delta
9-tetrahydrocannabinol on spasticity: a pilot study with 2
patients. Int. J. Clin. Pharm. Ther. 34, 446–452.
Burstein, S.H., Karst, M., Schneider, U., Zurier, R.B., 2004. Ajulemic
acid: a novel cannabinoid produces analgesia without a
“high”. Life Sci. 75 (12), 1513–1522.
Chankvetadze, B., Pintore, G., Burjanadze, N., Bergenthal, D.,
Strickmann, D., Cerri, R., Blanschke, G., 1998. Capillary
electrophoresis, nuclear magnetic resonance and mass
spectrometry studies of opposite chiral recognition of
chlorpheniramine enantiomers with various cyclodextrins.
Electrophoresis 19, 2101–2108.
Choi, Y.H., Hazekamp, A., Peltenburg-Looman, A.M.G., Fr´
ed´
erich,
M., Erkelens, C., Lefeber, A.W.M., Verpoorte, R., 2004. NMR
assignments of the major cannabinoids and
cannabiflavonoids isolated from flowers of Cannabis sativa.
Phytochem. Anal. 15 (6), 345–354.
Correia, I., Bezzenine, N., Ronzani, N., Platzer, N., Beloeil, J.C.,
Doan, B.T., 2002. Study of inclusion complexes of acridine
with beta- and (2,6-di-O-methyl)-beta-cyclodextrin by use of
solubility diagrams and NMR spectroscopy. J. Phys. Org. Chem.
15, 647–659.
Fairbairn, J.W., Liebmann, J.A., Rowan, M.G., 1976. The stability of
cannabis and its preparations on storage. J. Pharm.
Pharmacol. 28, 1–7.
van Gaal, L.F., Rissanen, A.M., Scheen, A.J., Ziegler, O., Rossner, S.,
2005. Effects of the cannabinoid-1 receptor blocker
rimonabant on weight reduction and cardiovascular risk
factors in overweight patients: 1-year experience from the
RIO-Europe study. Lancet 365, 1389–1397.
Garrett, E.R., Hunt, C.A., 1974. Physiochemical properties,
solubility, and protein binding of delta
9-tetrahydrocannabinol. J. Pharm. Sci. 63,
1056–1064.
Grotenhermen, F., 2002. Effects of cannabis and cannabinoids. In:
Grotenhermen, F., Russo, E. (Eds.), Cannabis and
Cannabinoids. Haworth Press, New York, pp. 55–67.
Hazekamp, A., Simons, R., Peltenburg-Looman, A., Sengers, M.,
van Zweden, R., Verpoorte, R., 2004a. Preparative isolation of
cannabinoids from Cannabis sativa by centrifugal partition
chromatography. J. Liq. Chromatogr. 27, 2421–2439.
Hazekamp, A., Choi, Y.H., Verpoorte, R., 2004b. Quantitative
analysis of cannabinoids from Cannabis sativa using 1HNMR.
Chem. Pharm. Bull. 52 (6), 718–721.
Higuchi, T., Conners, K.A., 1965. Phase solubility techniques. Adv.
Anal. Chem. Instrum. 4, 117–212.
Jarho, P., Pate, D.W., Brenneisen, R., J¨
arvinen, T., 1998.
Hydroxypropyl--cyclodextrin and its combination with
hydroxypropyl-methylcellulose increases aqueous solubility
of 9-tetrahydrocannabinol. Life Sci. 63 (26), 381–384.
Job, P., 1928. Formation and stability of inorganic complexes in
solution. Ann. Chim. (Paris) 9, 113–203.
Jover, A., Budal, R.M., Al-Soufi, W., Meijide, F., V´
azquez Tato, J.,
Yunes, R.A., 2003. Spectra and structure of complexes formed
by sodium fusidate and potassium helvolate with - and
␥-cyclodextrin. Steroids 68, 55–64.
Loftsson, T., Magn ´
usd ´
ottir, A., M´
asson, M., Sigurj ´
onsd ´
ottir, J.F.,
2002. Self-association and cyclodextrin solubilization of
drugs. J. Pharm. Sci. 91, 2307–2316.
Loftsson, T., M´
asson, M., Brewster, M.E., 2004. Mini review;
self-association of cyclodextrins and cyclodextrin complexes.
J. Pharm. Sci. 93, 1091–1099.
Lyng, S.M.O., Passos, M., Fontana, J.D., 2004. Bixin and
␣-cyclodextrin inclusion complex and stability tests. Process
Biochem. 39, 100–113.
Mannila, J., Jarvinen, T., Jarvinen, K., Tarvainen, M., Jarho, P., 2005.
Effects of RM-beta-CD on sublingual bioavailability of
delta(9)-tetrahydrocannabinol in rabbits. Eur. J. Pharm. Sci. 26
(1), 71–77.
Martin Del Valle, E.M., 2004. Cyclodextrins and their uses: a
review. Process Biochem. 39, 1033–1046.
Ramos Cabrer, P., Alvarez-Parrilla, E., Mejide, F., Seijas, J.A.,
Rodr´
ıguez N ´
u˜
nez, E., V´
azquez Tato, J., 1999. Complexation of
sodium cholate and sodium deoxycholate by -cyclodextrin
and derivatives. Langmuir 15, 5489–5495.
Author's personal copy
european journal of pharmaceutical sciences 29 (2006) 340–347 347
Ramos Cabrer, P., Alvarez-Parrilla, E., Al-Soufi, W., Mejide, F.,
Rodr´
ıguez N ´
u˜
nez, E., V´
azquez Tato, J., 2003. Complexation of
bile salts by natural cyclodextrins. Supramol. Chem. 15 (1),
33–43.
Ravichandran, R., Divakar, S., 1998. Inclusion of ring A of
cholesterol inside the -cyclodextrin cavity: evidence from
oxydation reactions and structural studies. J Inclusion
Phenom. Mol. Recogn. Chem. 30, 253–270.
Rozou, S., Michalaes, S., Antoniadou-Vyza, E., 2005. Study of
structural features and thermodynamic parameters,
determining the chromatographic behaviour of
drug–cyclodextrin complexes. J. Chromatogr. A 1087 (1–2),
86–94.
Shoyama, Y., Morimoto, S., Nishioka, I., 1983. Cannabis. Part XV.
Preparation and stability of
delta-9-tetrahydrocannabinol-beta-cyclodextrin inclusion
complex. J. Nat. Prod. 46, 633–637.
Zhang, X., Gramlich, G., Wang, X., Nau, W.M., 2002. A joint
structural, kinetic, and thermodynamic investigation of
substituent effects on host–guest complexation of bicyclic
azoalkanes by -cyclodextrin. J. Am. Chem. Soc. 124 (2),
254–263.
Zia, V., Rajewski, R.A., Stella, V.J., 2001. Effect of cyclodextrin
charge on complexation of neutral and charged substrates:
comparison of (SBE)7M-beta-CD to HP-beta-CD. Pharm. Res.
18, 667–673.
