Sublingual administration of CBD/P-CD inclusion complex prepared by precipitation complexation method

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Sublingual administration of D
9
-tetrahydrocannabinol/h-cyclodextrin
complex increases the bioavailability of D
9
-tetrahydrocannabinol in rabbits
Janne Mannila
a,
*, Tomi Ja¨rvinen
a
, Kristiina Ja¨rvinen
b
, Jussi Tervonen
a
, Pekka Jarho
a
a
Department of Pharmaceutical Chemistry, University of Kuopio, P.O. Box 1627, FIN-70211, Kuopio, Finland
b
Department of Pharmaceutics, University of Kuopio, P.O. Box 1627, FIN-70211, Kuopio, Finland
Received 29 June 2005; accepted 25 August 2005
Abstract
The bioavailability of D
9
-tetrahydrocannabinol (THC) was determined after its sublingual administration as solid THC/h-cyclodextrin (THC/
h-CD) complex, and was compared to oral administration of ethanolic THC, in rabbits.
The absolute bioavailability of THC after sublingual administration of solid THC/h-CD complex powder (16.0T7.5%; mean TSD; n=4) is
higher than the bioavailability of THC after oral administration of ethanolic THC solution (1.3 T1.4%; meanTSD; n= 4).
The results suggest that sublingual administration of THC/h-CD complex is a useful tool in improving absolute bioavailability of THC.
D2005 Elsevier Inc. All rights reserved.
Keywords: D
9
-Tetrahydrocannabinol; Transmucosal; Cyclodextrins; Bioavailability
Introduction
Over the last few years, D
9
-tetrahydrocannabinol (THC) has
been recognized as being useful in the treatment of various
medical conditions such as emesis (Darmani and Crim, 2005),
anorexia (Beal et al., 1995), multiple sclerosis (Zajicek et al.,
2003), pain (Berman et al., 2004) and Parkinson’s disease
(Lastres-Becker et al., 2005). The oral use of THC is, however,
limited due to its substantial first-pass metabolism (Ohlsson et
al., 1980). The main challenge for the medicinal use of
cannabinoids, including THC, is the development of safe and
effective method of administration (Hall et al., 2005).
Systemic drug delivery through sublingual mucosa is a
useful method to bypass hepatic first-pass metabolism.
However, sublingual delivery of poorly water-soluble THC
may be challenging: in order to deliver THC into systemic
circulation through sublingual mucosa, it must first dissolve
into the small volume of saliva (<1 ml) in the oral cavity
(Weatherell et al., 1996). Sublingual spray formulation of THC
and cannabidiol (CBD) (SativexR) has been developed for the
purpose of delivering THC and CBD in a readily dissolved
form. In SativexR, THC and CBD have been dissolved in a
mixture of ethanol and propylene glycol. The formulation has
recently been approved in Canada for the relief of neuropathic
pain. However, drawbacks, such as bad taste, high intersubject
variability and administration site irritation, may hinder the
feasibility of the spray formulation (Berman et al., 2004; GW
Pharma Ltd., 2005).
Cyclodextrins (CDs) are a group of cyclic oligosaccharides,
which have been shown to improve aqueous solubility,
dissolution rate and bioavailability of various lipophilic drugs
by inclusion complex formation (Loftsson and Brewster, 1996;
Rajewski and Stella, 1996; Loftsson et al., 2005). Inclusion
complex is formed, when drug molecule, or lipophilic part of
the molecule, is included into cavity of cyclodextrin. No
chemical bonds are formed during the complex formation, and
drug molecules in the complexes are in equilibrium with free
molecules in the solution.
In our earlier study (Mannila et al., 2005), we have shown
that sublingual administration of aqueous solution containing
RM-h-CD improves the absolute bioavailability of THC. This
earlier study also demonstrated that the effect of CD on the
formulation bulk is a challenge in sublingual drug delivery. For
the present study, natural h-CD was selected due to its good
safety profile in oral drug delivery. Besides, the use of natural
h-CD allows preparation of solid THC/h-CD complex by
0024-3205/$ - see front matter D2005 Elsevier Inc. All rights reserved.
doi:10.1016/j.lfs.2005.08.025
* Corresponding author. Tel.: +358 17 162596; fax: +358 17 162456.
E-mail address: janne.mannila@uku.fi (J. Mannila).
Life Sciences 78 (2006) 1911 – 1914
www.elsevier.com/locate/lifescie

precipitation, which decreases the formulation bulk. In the
present study, THC/h-CD inclusion complex was prepared by
precipitation method and the absolute bioavailability of THC
after sublingual administration of solid THC/h-CD complex
was studied in rabbits.
Materials and methods
Materials
The D
9
-tetrahydrocannabinol (THC) was purchased from
THC PHARM GmbH (Frankfurt, Germany) and deuterated
THC (THC-d
3
) from Cerillianti(Austin, Texas, USA). HP-h-
CD and h-CD were purchased from Wacker Chemie (Bur-
ghausen, Germany). All other reagents used were of analytic
grade and used as received.
Analytical methods
The HPLC-system consisted of a Merck Hitachi L-7400
UV-detector (wavelength 205 nm), D-7000 interface module,
L-7250 autosampler, L-7100 pump (Hitachi Ltd., Tokyo,
Japan) and HPLC System Manager software (Hitachi, ltd.
1996). Kromasil endcapped C
8
-reverse-phase column (250
mm 4.6 mm i.d., 5 Am) was purchased from Metachem
Technologies Inc. (CA, USA). Chromatographic conditions
were as follows; injection volume: 20 Al; isocratic flow rate:
1.0 ml/min; mobile phase: 85% (v/v) acetonitrile in water.
Gas chromatography-mass spectrometry (GC-MS), operat-
ing in the negative chemical ionization mode, was used to
quantify THC in plasma (Mannila et al., 2004). To 500 Alof
plasma, 50 Al of methanolic THC-d
3
, 1 ml of urea solution (8
M) and 1 ml of methanol were added. The samples were
purified by solid phase extraction using OasisRHLB cartridges
(Waters ltd., Massachusetts, USA). The samples, once loaded
onto the cartridges, were washed with 3 ml of an acetic acid –
methanol solution (60% (v/v) of methanol and 2% (v/v) of
acetic acid) and then with 3 ml of an ammonia – methanol
solution (60% (v/v) of methanol and 0.8% (w/v) of ammonia at
pH 10.0). The samples were eluted with methanol and
evaporated under a stream of nitrogen at 40 -C, after which
the samples were derivatized using trifluoroaceticanhydride
and analyzed directly.
The GC-MS-system consisted of an Agilent 6890 N gas
chromatograph, 7683 autosampler and 5973 N mass detector
(Agilent Technologies, Palo Alto, California). Data were
processed by the Agilent Enhanced Chemstation software. A
cross linked 5% phenyl methyl siloxane capillary column (HP-
5MS; 30 m 0.25 mm 0.25 Am) (Agilent Technologies) was
used with helium as the carrier gas, at a constant flow rate of
2.0 ml/min. The sample (1 Al) was injected in the pulsed
splitless mode. The temperature program was as follows: an
initial temperature of 50 -C was held for 1 min, then increased
by 50 -C/min up to 205 -C, 3 -C/min up to 225 -C, and
finally 60 -C/min up to 280 -C where it was maintained for 4
min. Temperatures of inlet, interface, MS source and
quadrupole were 250, 280, 150, 106 -C, respectively. Methane
was used as the reagent gas. The area ratio of selected ions
410 (THC) and 413 (THC-d
3
) was used for the quantification
of THC.
Preparation of solid THC/b-CD complex
An aqueous 4.0% (w/v) h-CD solution was prepared and
maintained in a sonicator at + 40 -C. The solid complex was
prepared by adding ethanolic THC dropwise into the h-CD
solution during 2 h. The resulting suspension was cooled to + 5
-C during 1 h. The resulting precipitant was then centrifuged
and the supernatant was removed. The precipitant was freeze-
dried (FTSRSystems, Inc., NY, USA). The content of THC
from resulting powder was determined by HPLC.
In vivo absorption studies
Four New Zealand white rabbits (3.0 T0.4 kg; mean TS.D.),
one male and three females were purchased from National
Laboratory Animal Center in Kuopio, Finland. The rabbits
were allowed to eat commercial food pellets and drink water ad
libitum, except during the first 5 h of each test, when they were
under anaesthesia. The three formulations used in in vivo
studies were i.v. formulation of aqueous THC solution and oral
formulation of ethanolic THC solution, as described earlier
(Mannila et al., 2005), and sublingual formulation of solid
THC/h-CD complex. THC dose was 250 Ag/kg. All rabbits
received all of the 3 formulations with a wash out time of 4
weeks between the tests. All procedures adhered to the
European Community guidelines for the use of experimental
animals.
Before each test the rabbits were given atropine (0.02 mg/
kg; AtropinR, Leiras, Turku, Finland) to prevent excess
salivation, and then anaesthetized with fentanyl citrate and
fluanisone (0.1 mg/kg and 3 mg/kg, respectively; HypnormR,
Janssen Pharmaceutica, Beerse, Belgium) and midazolam (2
mg/kg) (DormicumR, Roche, Espoo, Finland). Anaesthetized
rabbits were positioned on a table, with the lower jaw
supported in a horizontal position.
The i.v. formulation was an aqueous solution in which HP-
h-CD (20 %; w/v), a non-toxic solubility enhancer, was used to
0
1
2
3
0 60 120 180 240 300
Time (min)
THC concentration (ng/ml)
Fig. 1. The mean plasma concentrations of THC after extravascular
administration (250 Ag/kg) to rabbits: sublingual administration of solid
THC/h-CD complex (0) and peroral administration of ethanolic THC (>)
(meanTS.E.M.; n= 4).
J. Mannila et al. / Life Sciences 78 (2006) 1911– 19141912

solubilize THC (0.7 mg/ml). The solution was filtered using a
sterile membrane filter (pore size 0.22 Am), and injected into a
marginal ear vein.
Sublingual formulation was a freeze-dried powder contain-
ing approximately 8 mg of THC per 1 g of powder.
Sublingual administration was as follows: the rabbits’ tongues
were carefully lifted with tweezers and the appropriate
amount of THC/h-CD complex powder was measured under
the tongue.
Oral formulation was an ethanolic solution of THC (12 mg/
ml). For oral administration ethanolic THC solution was
administered to the GI-tract via catheterization.
Blood was withdrawn into VenojectR(Terumo, Leuven,
Belgium) tubes from either a central artery or marginal vein of
the ear prior to THC administration and over 2 – 300 min after
administration. Blood samples were centrifuged at 3700g
within 30 min, and the recovered plasma was immediately
frozen to 20 -C. Samples were stored at 80 -C until
analysis.
In vivo data analysis
The maximum plasma concentration of THC (C
max
) and
the time required to reach the maximum concentration (t
max
)
were obtained directly from the actual plasma profiles. The
area under curve between 0 and 300 min (AUC
0 – 300 min
)
was calculated by linear trapezoidal method (0 min
concentrations for the i.v. administration studies were
extrapolated by the WinNonlin program (Version 4.0.1)).
The elimination rate constants (k
el
) and elimination half-lives
(t
1/2
) were determined from i.v. data for each rabbit using the
WinNonlin program. AUC
300 min – V
was determined by using
Eq. (1):
AUC300 minV¼C300 min
kel
ð1Þ
where C
300 min
represents the concentration of THC at 300 min.
AUC
0 min – V
is a sum of AUC
0 – 300 min
and AUC
300 min – V
.
Absolute bioavailabilities ( F, %) of orally and sublingually
administered THC formulations were calculated according to
Eq. (2):
F¼AUCe:v:
AUCi:v:
100% ð2Þ
where AUC
e.v.
is AUC
0 min – V
for either sublingual or oral
administration and AUC
i.v.
is AUC
0min–V
for i.v.
administration.
Results
The plasma profiles of THC after i.v. administration implied
first order pharmacokinetics, with a distribution and an
elimination phase (Mannila et al., 2005). Thus the representing
kinetic values were determined for each rabbit by using a 2-
compartment IV-bolus model with 1st order elimination. The
values (mea n TS.D.) for k
el
,AUC
0min–V
,andt
1/2
were
0.01 T0.00 min
1
, 5300 T1200 min ng/ml and 66.6 T3.1 min,
respectively.
Fig. 1 shows the mean plasma concentrations of THC after
administration of the sublingual and oral formulations. C
max
,
t
max
, AUC
0 – 300 min
, AUC
0 min – V
and Fvalues of THC after
Table 1
C
max
,t
max
, AUC
0 – 300 min
, AUC
0 min – V
and Fvalues (meanTS.D.; n= 4) of THC after intravenous administration of THC solution, sublingual administration of
solid THC/h-CD inclusion complex and peroral administration of ethanolic THC in rabbits
Route of
administration
Formulation C
max
(ng/ml) t
max
(min) AUC
0 – 300 min
(ng/ml min)
AUC
0 min – V
(ng/ml min)
F(%)
Intravenous THC solution 440T150
a
0.0T0.0a 5100 T1200 5300T1200 100 T0
Sublingual Solid THC/h-CD
inclusion complex
3.0T0.8 240 T70 470 T80 720 T140 16.0 T7.5
Peroral Ethanolic THC 0.9 T1.0 31 T29 44 T68 74 T82 1.3T1.4
The THC dose was 250 Ag/kg.
a
Extrapolated value.
+
Systemic circulation
Sublingual membrane
dilution
oesophagus oesophagus
Cyclodextrin/THC-
complex Cyclodextrin THC
Fig. 2. The equilibrium between cyclodextrin, THC and THC/cyclodextrin inclusion complex. Only free THC is expected to absorb into systemic circulation.
J. Mannila et al. / Life Sciences 78 (2006) 1911– 1914 1913

i.v., sublingual and oral administrations are summarized in
Table 1.
Discussion
The bioavailability of THC after sublingual administration
of solid THC/h-CD complex ( F= 16.0 T7.5%; mean TS.D.:
n= 4) is higher compared to oral administration of ethanolic
THC ( F= 1.3 T1.4%). The increased bioavailability of THC
after sublingual administration is most probably due to the
avoidance of first-pass metabolism. However, interactions of h-
CD with biomembranes cannot be excluded; CDs have been
shown to interact with membrane macromolecules thus
reversibly improving the drug absorption (Loftsson et al.,
2005).
From toxicological point of view, the use of natural h-CD in
sublingual applications is comparable to other forms of oral
drug delivery and thus it can be regarded as safe (Irie and
Uekama, 1997). Large hydrophilic molecules such as h-CD do
not permeate across sublingual membrane and eventually get
swallowed. A recent in vitro toxicity study on human oral
epithelium cells also suggested that CDs can be considered as
safe excipients in buccal drug delivery (Boulmedarat et al.,
2005).
Fig. 2 shows the processes which are assumed to be critical
in sublingual delivery of THC/h-CD complex. After sublingual
administration, the solid THC/h-CD complex rapidly dissolves
in saliva and equilibrium forms between inclusion complexes,
free cyclodextrin molecules and free THC molecules. It is
generally assumed that only free drug, not inclusion complex,
can penetrate across biological membranes (Uekama, 2004).
Thus the effective sublingual absorption of THC is dependent
on fast dissolution and dissociation of the inclusion complex.
One of the major factors which affects on release of drug from
the inclusion complex is dilution of the inclusion complex by
biological fluids. This can be a challenge in sublingual drug
delivery due to small volume of aqueous saliva and relatively
short residence time of inclusion complexes on absorption site.
In conclusion, sublingual administration of THC/h-CD
complex improves the absolute bioavailability of THC.
Inclusion complex formation by precipitation method may
also be useful in the development of novel, solid sublingual
formulations of other lipophilic cannabinoids (e.g. cannabidiol)
with high first-pass metabolism.
Acknowledgements
The authors would like to thank Mr. Heikki Pekonen for his
skilful assistance and The National Laboratory Animal Center
(Kuopio, Finland) for providing a pleasant working environ-
ment and good care for the animals. The authors would also
like to acknowledge The National Technology Agency of
Finland for financial support. The Association of Finnish
Pharmacies is also acknowledged.
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