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A pilot clinical study of D
9
-tetrahydrocannabinol in patients with
recurrent glioblastoma multiforme
M Guzma
´n*
,1
, MJ Duarte
2
, C Bla
´zquez
1
, J Ravina
2
, MC Rosa
2
, I Galve-Roperh
1
,CSa
´nchez
1
, G Velasco
1
and
L Gonza
´lez-Feria*
,2
1
Department of Biochemistry and Molecular Biology I, School of Biology, Complutense University, Madrid 28040, Spain;
2
Department of Neurosurgery,
Hospital Universitario de Canarias, La Laguna, Tenerife 38320, Spain
D
9
-Tetrahydrocannabinol (THC) and other cannabinoids inhibit tumour growth and angiogenesis in animal models, so their potential
application as antitumoral drugs has been suggested. However, the antitumoral effect of cannabinoids has never been tested in
humans. Here we report the first clinical study aimed at assessing cannabinoid antitumoral action, specifically a pilot phase I trial in
which nine patients with recurrent glioblastoma multiforme were administered THC intratumoraly. The patients had previously failed
standard therapy (surgery and radiotherapy) and had clear evidence of tumour progression. The primary end point of the study was
to determine the safety of intracranial THC administration. We also evaluated THC action on the length of survival and various
tumour-cell parameters. A dose escalation regimen for THC administration was assessed. Cannabinoid delivery was safe and could be
achieved without overt psychoactive effects. Median survival of the cohort from the beginning of cannabinoid administration was 24
weeks (95% confidence interval: 15–33). D
9
-Tetrahydrocannabinol inhibited tumour-cell proliferation in vitro and decreased tumour-
cell Ki67 immunostaining when administered to two patients. The fair safety profile of THC, together with its possible antiproliferative
action on tumour cells reported here and in other studies, may set the basis for future trials aimed at evaluating the potential
antitumoral activity of cannabinoids.
British Journal of Cancer (2006) 95, 197 – 203. doi:10.1038/sj.bjc.6603236 www.bjcancer.com
Published online 27 June 2006
&2006 Cancer Research UK
Keywords: cannabinoid; glioblastoma multiforme; pilot clinical study; antitumoral drug
The hemp plant Cannabis sativa L. produces approximately 60
unique compounds known as cannabinoids, of which D
9
-
tetrahydrocannabinol (THC) is the most important owing to its
high potency and abundance in cannabis (Gaoni and Mechoulam,
1964). D
9
-Tetrahydrocannabinol exerts a wide variety of biological
effects by mimicking endogenous substances – the so-called
endocannabinoids (Mechoulam and Hanus, 2000; Piomelli, 2003) –
that bind to and activate specific cell surface receptors. So far, two
cannabinoid-specific receptors have been cloned and characterised
from mammalian tissues (Howlett et al, 2002): CB
1
, particularly
abundant in the brain, and CB
2
, mainly expressed in the immune
system. One of the most active areas of current research in
the cannabinoid field is the study of the potential application
of cannabinoids as therapeutic agents. Among these possible
applications, cannabinoids have been known to exert palliative
effects in cancer patients since the early 1970s. The best established
of these effects is the inhibition of chemotherapy-induced nausea
and vomiting. Today, capsules of THC and its synthetic analogue
nabilone are approved in several countries for that purpose
(Guzma
´n, 2003; Hall et al, 2005). Other potential palliative effects
of cannabinoids in oncology – supported by phase III clinical trials
– include appetite stimulation and pain inhibition (Guzma
´n, 2003;
Hall et al, 2005). In addition, cannabinoids have been proposed as
potential antitumoral agents owing to their ability to inhibit the
growth and angiogenesis of various types of tumour xenografts
in animal models (Munson et al, 1975; Guzma
´n, 2003). However,
the antitumoral effect of cannabinoids has never been tested in
humans.
One of the most devastating forms of cancer is glioblastoma
multiforme (grade IV astrocytoma), the most frequent class of
malignant primary brain tumours. Current standard therapeutic
strategies for the treatment of glioblastoma multiforme (surgical
resection and focal radiotherapy) are only palliative, and, as a
consequence, survival after diagnosis is normally 6 – 12 months
(Afra et al, 2002; Kleihues et al, 2002; Lonardi et al, 2005). A large
number of chemotherapeutic agents (e.g. alkylating agents such as
temozolomide and nitrosoureas such as carmustine) have also
been tested, but no remarkable improvement on patient survival
has been achieved as yet (Afra et al, 2002; Lonardi et al, 2005;
Reardon et al, 2006). Likewise, although dendritic cell- and
peptide-based immunotherapy strategies appear promising as
a safe approach to induce an antitumour immune response
(Yamanaka, 2006), no immunotherapy or gene therapy trial
performed to date has been significantly successful. It is therefore
essential to develop new therapeutic strategies for the management
of glioblastoma multiforme to obtain significant clinical results.
Revised 15 May 2006; accepted 5 June 2006; published online 27 June
2006
*Correspondence: Professor M Guzma
´n; E-mail: mgp@bbm1.ucm.es or
Professor L Gonza
´lez-Feria; E-mail: lgferia@yahoo.es
British Journal of Cancer (2006) 95, 197 – 203
&
2006 Cancer Research UK All rights reserved 0007 – 0920/06
$
30.00
www.bjcancer.com
Translational Therapeutics
We have previously shown that cannabinoids inhibit the growth
(Galve-Roperh et al, 2000; Sa
´nchez et al, 2001) and angiogenesis
(Bla
´zquez et al, 2003, 2004) of gliomas in animal models.
Remarkably, this antiproliferative effect seems to be selective for
brain-tumour cells as the survival of normal brain cells (astrocytes
(Go
´mez del Pulgar et al, 2002), oligodendrocytes (Molina-Holgado
et al, 2002) and neurons (Mechoulam et al, 2002)) is unaffected
or even favoured by cannabinoid challenge. On the basis of these
preclinical findings, we have conducted a pilot clinical study aimed
at assessing cannabinoid antitumoral action in patients with
recurrent glioblastoma multiforme.
PATIENTS AND METHODS
Patients
Nine patients with glioblastoma multiforme were enroled. All
patients had failed standard therapy, which included surgery and
external-beam radiotherapy (60 Gy), had clear evidence of tumour
progression on sequential magnetic resonance scanning, and had
a minimum Karnofsky performance score (KPS) of 60 (i.e. ability
to function independently). Two patients (Patients 2 and 5) had
received adjuvant temozolomide chemotherapy (two cycles of
150 –200 mg m
2
daily for 5 days; 28 days between the two
cycles), but temozolomide administration had finished 24 weeks
(Patient 2) or 8 weeks (Patient 5) before enrolment in the study.
Pregnant patients and patients with systemic diseases or active
infections were excluded. Additional requirements included
acceptable haematological and hepatic function (glutamate :
oxalacetate transaminase and glutamate : piruvate transaminase
o2.5 normal values, total bilirubin o1.5 normal values,
platelets 4100 000 mm
3
, neutrophils 41000 mm
3
, haemoglobin
410 g dl
1
) and no change in steroid administration protocol for
at least 2 weeks. All patients provided written informed consent
before entering the study. The protocol, progression and final
report of the study were approved by the Clinical Trials and Ethics
Committee of Hospital Universitario de Canarias and by the
Spanish Ministry of Health.
THC administration
THC was obtained from The Health Concept (Richelbach,
Germany) and was kindly provided by Mr Alfredo Dupetit.
Preparations contained 496.5% THC, o1.5% of its isomer
D
8
-THC, o0.5% butyl-THC and o0.5% propyl-THC.
Patients underwent a surgical intervention aimed at resecting
and creating a cavity in the recurrent tumour. Biopsies were taken
and glioblastoma multiforme diagnosis was confirmed in all
cases. The tip (approx. 5 cm) of a silastic infusion catheter (9.6
French; 3.2 mm diameter) was placed into the resection cavity. The
infusion catheter was connected to a Nuport subclavicular
subcutaneous reservoir. Each day an aliquot of the THC solution
(100 mg ml
1
in ethanol) was dissolved in 30 ml of physiological
saline solution supplemented with 0.5% (w v
1
) human serum
albumin, and the resulting solution was filtered and transferred to
an opaque syringe. This process was performed at the Department
of Pharmacy of the Hospital Universitario de Canarias. Owing to
the high hydrophobicity of THC, we controlled by gas chromato-
graphy/mass spectrometry (see below) the actual concentration
of THC in the final solution. The THC solution was administered
to the patients for different times starting at days 3 – 6 after surgery
at a rate of 0.3 ml min
1
with a syringe pump connected to the
subcutaneous reservoir. In the case of Patients 1 and 2, who
received THC for 30 and 26 days, respectively, biopsies were also
taken after the THC-treatment period and various tumour-cell
parameters were evaluated (see below).
Patient monitoring
Patients underwent continuous physical, neurological, biochemical
and haematological examinations as well as frequent magnetic
resonance and computed tomography scans of the brain for the
detection of evidence of toxicity (haemorrhage, oedema, injury)
and monitoring of tumour progression. Magnetic resonance
imaging evaluations (T1-weighted gadolinium enhancement axial
images) measured enhanced tumour size, which is believed to
represent the viable portion of the tumour.
Determination of THC concentration
The plasma and urine concentration of THC was determined daily
in Patients 1 and 2 during the first 7 days of administration by
three different methods: a fluorescence polarisation immunoassay
kit (AxSYM Cannabinoid Assay, Abbott, Abbott Park, IL, USA;
detection limit, 50 ng ml
1
), a cloned enzyme donor immunoassay
kit (Microgenics CEDIA DAU Multi-Level THC, Microgenics,
Pleasanton, CA, USA; detection limit, 50 ng ml
1
) and gas
chromatography/mass spectrometry (determinations performed
at the Department of Toxicology, School of Medicine, Complutense
University, Madrid, Spain; detection limit, 10 ng ml
1
). These
procedures also detected the major THC metabolites 11-hydroxy-
THC and 11-nor-THC-9-carboxylic acid. In all the analyses
performed in both plasma and urine, the concentrations of THC
and its two metabolites were below the detection limits. Gas
chromatography/mass spectrometry (determinations performed at
the Institute of Toxicology, Ministry of Health, Tenerife) was also
used to determine the actual concentrations of THC in the final
solution inoculated to the patients.
Tumour cell cultures
Tumour biopsies were digested with collagenase (type Ia, Sigma, St
Louis, MO, USA) in Dulbecco’s modified Eagle’s medium (DMEM)
at 371C for 90 min and the supernatant was seeded in DMEM
containing 15% foetal calf serum and 1 mMglutamine. Cells were
kept in primary culture for about 2 weeks. Cells were subsequently
seeded for the experiments, and 24 h before cannabinoid addition
they were transferred to 0.5% serum DMEM. Cell viability was
determined by Trypan blue exclusion. Apoptosis was determined
by Hoechst 33258 staining and with a terminal deoxynucleotide
transferase-mediated deoxy uridine triphosphates-biotin nick-end
labelling (TUNEL) kit (Boehringer, Mannheim, Germany). THC as
well as SR141716 and SR144528 (kindly given by Sanofi-Aventis,
Montpellier, France) were prepared in dimethylsulphoxide (final
concentration: 0.1– 0.2% (v v
1
)). Control incubations had the
corresponding dimethylsulphoxide content. All determinations
were performed in triplicate.
Western blot
Tumour samples were homogenised and subjected to sodium
dodecyl sulphate–polyacrylamide gel electrophoresis, and proteins
were transferred from the gels onto polyvinylidene fluoride
membranes. The blots were incubated with antibodies raised
against residues 1–77 of the human CB
1
receptor (1 : 1000; kindly
given by Ken Mackie, University of Washington, Seattle, WA,
USA) or residues 1 – 99 of the human CB
2
receptor (1 : 1000;
Affinity Bioreagents, Golden, CO, USA). a-Tubulin (1 : 4000,
Sigma) was used as a loading control. Samples were finally
subjected to luminography with an enhanced chemiluminiscence
detection kit (Amersham Life Sciences, Arlington Heights, IL,
USA). Densitometric analysis of the blots was performed with
Multianalyst software (Bio-Rad Laboratories, Hercules, CA, USA).
Cannabinoid administration to cancer patients
M Guzma
´net al
198
British Journal of Cancer (2006) 95(2), 197 – 203 &2006 Cancer Research UK
Translational Therapeutics
Confocal microscopy
Sections of formalin-fixed, paraffin-embedded tumour samples
were stained with anti-CB
1
receptor (1 : 500; kindly given by Ken
Mackie), anti-CB
2
receptor (1 : 500; Affinity Bioreagents), anti-Ki67
(Lab Vision, Fremont, CA, USA) or anti-CD31 (1 : 400; Cymbus
Biotechnology, Hampshire, UK) antibodies as described (Bla
´zquez
et al, 2004). Slices were further incubated (1 h, room temperature,
darkness) with a secondary antibody– Alexa Fluor 594 (1 : 400;
Molecular Probes, Leiden, The Netherlands). Sections were
mounted with Mowiol mounting medium (Merck, Darmstadt,
Germany) containing YOYO-1 iodide (1 : 1000; Molecular Probes,
Leiden, The Netherlands) to stain cell nuclei. Ten to fifteen fields of
4–6 sections were analysed per tumour. Morphometric analysis
was performed with Metamorph-Offline software (Universal
Imaging, Downingtown, PA, USA).
Statistics
Survival was calculated as the median (95% confidence interval
(CI)) survival time of the cohort of patients from the surgical
operation of tumour relapse, and was represented as a Kaplan –
Meier curve (Figure 1B). Data on tumour-cell parameters (Figure 3)
are given as mean7s.d. and were analysed by analysis of variance
with a post hoc Student–Neuman–Keuls test.
0
0.5
Time after second surgery (weeks)
Surviving fraction
0 204060
1.0
B
GBM
diagnosis
First surgery Radiotherapy
Relapse
(all patients)
Pretreatment
tumour biopsy
THC treatment
(all patients)
Post-treatment
tumour biopsy
(Patients 1 and 2) Decease
days
±chemotherapy
Second surgery
(Patients 1 and 2)
A
15 days
Figure 1 Effect of THC administration on overall survival. (A) Schematic diagram of the clinical protocol. See text for further details. (B) Kaplan – Meier
survival curve of the cohort of patients from the surgical operation of tumour relapse. For comparison with survival upon administration of standard
chemotherapeutic drugs such as temozolomide and carmustine, see Dinnes et al (2002) and Brem et al (1995), respectively.
Table 1 Summary of patient characteristics, treatment and outcome
THC treatment
Patient
Age
(sex) KPS
Tumour
location
Recurrent-tumour
volume (cm
3
)
Total
days
Number
of cycles
Total dose
(mg)
Time between first and
second surgery (wk)
Time from second
surgery to death (wk)
1 47 (m) 90 L – O 120 30 2 1.46 70 19
2 58 (m) 80 R – T 69 26 4 1.29 63 18
3 35 (m) 80 L – T 40 64 6 3.29 9 53
4 67 (m) 70 R – T 76 11 1 0.81 23 24
5 51 (f) 70 R – P 41 15 2 1.13 28 17
6 64 (f) 90 R – T 58 10 1 0.80 8 9
7 69 (f) 90 L – T 43 21 3 1.68 112 29
8 51 (f) 90 R – F 52 10 1 1.60 24 49
9 55 (f) 70 L – T 76 11 1 1.28 38 24
Abbreviations: f ¼female; F ¼frontal; KPS ¼Karnofsky performance score; L¼left; m ¼male; O ¼occipital; P ¼parietal; R ¼right; T ¼temporal; wk ¼week.
Cannabinoid administration to cancer patients
M Guzma
´net al
199
British Journal of Cancer (2006) 95(2), 197 – 203&2006 Cancer Research UK
Translational Therapeutics
RESULTS
Patients
Nine patients (four men, five women) were enroled in the study
between March 2002 and November 2003 (Table 1). The cohort
had a mean age of 55 years and a moderately altered physical
performance (mean KPS: 81; most frequent symptoms on
enrolment: cephalalgia, alterations in higher cerebral functions,
long tract signs and epilepsy in six out of nine, four out of nine,
eight out of nine and three out of nine patients, respectively).
The recurrent tumours had medium – large size (mean estimated
volume of active tumour: 64 cm
3
) and had appeared after a period
expected for average glioblastoma multiforme progression (mean
interval between first and second surgery: 42 weeks). The cohort,
although small, was therefore considered representative of
recurrent glioblastoma multiforme routinely found in the clinical
practice, which would make the study unbiased towards patients
with better prognosis. The primary end point of the study was to
determine the safety of intracranial THC administration. We also
assessed THC action on the length of survival and various tumour-
cell parameters. The clinical protocol is summarised in Figure 1A.
Safety of the treatment
Patients were entered one by one in order to ascertain a dose
escalation regimen for THC administration based on the
appearance of psychoactive side effects. In Patient 1, the initial
daily dose of THC was 20 mg, which was progressively increased for
4 days until 100 mg, with which a very mild episode of euphoria
appeared. This effect was transient and difficult to interpret, as it
never repeated. The daily dose was subsequently set at 60 – 80 mg
and no further side effects appeared anymore during the first cycle
(19 days, 0.98 mg total THC) or the second cycle (11 days, 0.48 mg
total THC). A similar approach was used to define the adminis-
tration pattern of THC to other patients and no significant
psychoactive effects were evident, except for Patient 8, who had a
mild and transient episode of bulimia, hypothermia and euphoria.
Overall, the initial dose of THC administered to the patients was
20 –40 mg at day 1, increasing progressively for 2 – 5 days up to
80 –180 mg day
1
. The median duration of an administration cycle
was 10 days. Some patients received more than one THC cycle
(Table 1), and so the median duration of total THC administration
was 15 days (Figure 1A). Of interest, no significant alterations in
physical, neurological, biochemical and haematological parameters
could be ascribed to THC in any of the patients. All patients
experienced cerebral oedema during the study, as is typical for
postoperative craniotomy, and were treated with corticosteroids.
There was no apparent effect of THC on steroid requirement.
Progression and survival
Median survival from the surgical operation of tumour relapse was
24 weeks (95% CI: 15 – 33). Two of the patients (3 and 8) survived
for approximately 1 year (Table 1, Figure 1B).
Patient 3 (Table 1, Figure 2A) had an extremely aggressive
recurrent glioblastoma multiforme in the left temporal lobe that
was evident shortly after the extensive surgical resection of the
primary tumour. The recurrent tumour was marginally removed
and a total of six THC cycles was administered. During the first
three cycles, tumour growth was curbed for about 9 weeks. As the
patient showed a clear improvement of clinical symptoms (e.g.
dysphasia and cranial hypertension disappeared and haemiparesis
ameliorated), three more cannabinoid cycles were administered.
However, the KPS started to decline at week 21.
Patient 8 (Table 1, Figure 2B) had an actively growing recurrent
glioblastoma multiforme in the right frontal lobe that was partially
resected. One THC cycle was subsequently administered, although
in view of the high tolerance of the patient the cycle contained
more THC than those administered to other patients (Table 1).
Tumour volume did not stabilise and followed a continuous
increase, but the patient’s clinical symptoms largely improved (e.g.
cephalalgia and hallucinations disappeared and motor deficit
attenuated). However, the KPS started to decline at week 20.
Patient 5 (Table 1, Figure 2C) was one of the patients who
seemed not to respond to THC, at least regarding expected length
of survival. The right parietal-lobe recurrent tumour was slightly
removed, and after the first THC cycle, tumour volume kept
constant for 5 weeks. During that period, haemiparesis improved
and the KPS did not decrease, but tumour progression and clinical
symptoms rapidly worsened thereafter despite the administration
of a second THC cycle.
THC action on tumour cells
To gain further insight into how THC may affect tumour growth,
we determined various cellular parameters in the tumours. The
expression of cannabinoid receptors in tumour biopsies was
examined by Western blot (Figure 3A) and immunofluorescence
(Figure 3B and C). The tumours from the nine patients expressed
different amounts of CB
1
and CB
2
receptors, but no correlation was
found between receptor-type expression and survival (data not
shown). Because cannabinoid receptors are known to desensitise
upon prolonged occupancy (Howlett et al, 2002), it is conceivable
that this may hamper the efficacy of long-term treatments. We
therefore determined CB receptor expression after THC adminis-
tration to two patients. Data from Patients 1 and 2 showed a slight
decrease in CB
1
receptor expression and no change in CB
2
receptor
expression (Figure 3A), which might reflect a predominant binding
of THC to the former protein or its higher susceptibility to
desensitisation.
We next tested the functionality of cannabinoid receptors in the
inhibition of tumour cell growth. For this purpose, we isolated
tumour cells from glioblastoma biopsies, and observed that THC
decreased the number of viable cells in the cultures. This effect
relied on CB receptor activation as the CB
1
antagonist SR141716
together with the CB
2
antagonist SR144528 prevented cannabinoid
action (Figure 3D). THC growth-inhibiting action was due at least
in part to apoptosis, as determined by Hoechst 33258 and TUNEL
staining (Figure 3D). Likewise, in Patients 1 and 2, THC treatment
in vivo was associated with reduced tumour-cell proliferation
(Ki67 immunostaining) (Figure 3E). D
9
-Tetrahydrocannabinol
administration tended to decrease tumour vascularisation (CD31
immunostaining) in those two patients, but the effect was not
statistically significant (Figure 3F).
DISCUSSION
Here we report the first clinical study aimed at evaluating
cannabinoid antitumoral action. Owing to obvious ethical and
legal reasons, this pilot study was conducted in a cohort of
terminal patients harbouring actively growing recurrent tumours.
Although the use of cannabinoids in medicine may be limited by
their well-known psychotropic effects, it is generally believed that
cannabinoids display a fair drug safety profile and that their
potential adverse effects are within the range of those accepted for
other medications, especially in cancer treatment (Guzma
´n, 2003;
Hall et al, 2005; Iversen, 2005). In line with this idea, THC delivery
in our study was safe and could be achieved without overt
psychoactive effects. As the possible antitumoral action of nabilone
has never been evaluated in preclinical trials, THC was the unique
cannabinoid receptor agonist available for the present human
study. Nonetheless, most likely THC is not the most appropriate
cannabinoid agonist for future antitumoral strategies owing to its
high hydrophobicity, relatively weak agonistic potency and ability
Cannabinoid administration to cancer patients
M Guzma
´net al
200
British Journal of Cancer (2006) 95(2), 197 – 203 &2006 Cancer Research UK
Translational Therapeutics
to elicit CB
1
-mediated psychoactivity. Unfortunately, the current
synthetic cannabinoid agonists that have been reported to exert
antitumoral actions in animal models and that could theoretically
circumvent – at least in part – the pharmacokinetic and
pharmacodynamic limitations of THC (e.g. WIN-55,212-2, a more
potent and less hydrophobic CB
1
/CB
2
-mixed agonist (Galve-
Roperh et al, 2000), and JWH-133, a more potent CB
2
-selective
agonist (Sa
´nchez et al, 2001)) are still very far from the clinical
application owing to the lack of thorough preclinical toxicology
studies.
Before
second surgery second surgery
After
Before
second surgery second surgery
After
Before
second surgery second surgery
After
Week 4 Week 18
Week 3 Week 32
Week 3 Week 15
Week 29
0
25
50
75
Time after second surgery (weeks)
Tumour volume (cm3)
0102030
Time after second surgery (weeks)
0102030
Time after second surgery (weeks)
0 5 10 15
100
Patient 3
Patient 8
Patient 5
A
0
20
40
60
Tumour volume (cm3)
80
B
0
20
40
60
Tumour volume (cm3)
80
C
Figure 2 Effect of THC administration on tumour growth. Tumour growth plots and gadolinium-enhanced T1-weighted magnetic resonance scans after
the second surgery in three patients. Arrows indicate the THC administration cycles. (A) Patient 3, scans before and after surgery of tumour relapse as well
as after the second, fourth and sixth THC cycle (weeks 4, 18 and 29, respectively). (B) Patient 8, scans before and after surgery of tumour relapse as well as
after the THC cycle (week 3) and at week 32. (C) Patient 5, scans before and after surgery of tumour relapse as well as after the fist THC cycle (week 3) and
at week 15.
Cannabinoid administration to cancer patients
M Guzma
´net al
201
British Journal of Cancer (2006) 95(2), 197 – 203&2006 Cancer Research UK
Translational Therapeutics
This is not only the first clinical study to assess cannabinoid
antitumoral action but also the first human study in which a
cannabinoid is administered intracranially. This route of admin-
istration was used to mimic our preclinical studies in rodents
(Galve-Roperh et al, 2000) and has been previously used for
the delivery of other cytotoxic drugs such as carmustine to
patients with malignant brain tumours (Brem et al, 1995).
Nonetheless, we note that a non-invasive, less traumatic (e.g. oral)
route would be more desirable in the clinical practice. Although
intratumoral delivery may allow a high local concentration of the
drug in situ, in the case of large tumours such as those treated
in the present study, the local perfusion through a catheter placed
at one point of the tumour constitutes an obvious limitation of
the technique. Further studies should assess the distribution
pattern of the THC solution within the tumour as well as within
the whole brain.
A
Pre-THC
100 82± 5
(P=0.07)
CB1
CB2
CB1
CB2
CB1
CB2
-tubulin
Zone 1
Zone 2
Zone 3
100 103±2
Post-THC
Vehicle
THC
Hoechst TUNEL
SR1+SR2 1.0 M
D
Vehicle
THC 0.5
M
THC 1.0
M
THC 2.5
M
THC 2.5 M
Cell viability (%)
0
20
40
60
80
100
Pat 1
Pat 2
Pat 3
Mean
P=0.16*
P=0.18*
P=0.02*
P=0.01 P=0.36
P=0.01#
CD31
F
Pre-THC
Post-THC
Blood vessel area
(% of total area)
0
5
10
Ki67
E
Pre-THC
Post-THC
Pre-THC
Post-THC
Pre-THC
Post-THC
Ki67+ cells (%)
0
5
10
15
BPhase
contrast
Phase
contrast
Phase
contrast
Immuno
fluorescence
C
Figure 3 Effect of THC administration on tumour cells. (A) Western blot analysis of CB
1
and CB
2
receptor expression in three different tumour zones of
Patient 1 (left panel) and in tumour biopsies of Patient 1 before and after THC treatment (right panel). Optical density values relative to those of loading
controls (a-tubulin) are given for Patients 1 and 2 in arbitrary units. (B) Immunostaining of CB
1
and CB
2
receptors (red) in a tumour biopsy of Patient 1.
Nuclei are stained in green. (C) Immunostaining of CB
1
and CB
2
receptors (green) in tumour cells obtained from Patient 1. (D) THC-induced apoptotic
death of tumour cells obtained from Patients 1 – 3. Cells were incubated for 48 h with THC and/or 1.0 mMSR141716 (SR1) plus 1.0 mMSR144528 (SR2).
Statistical comparison vs vehicle (*) or vs 2.5 mMTHC alone (#) is given. Arrows point to Hoechst-stained fragmented nuclei or to TUNEL-positive nuclei in
cells from Patient 1 treated with 2.5 mMTHC. (E,F) Tumour cell proliferation (Ki67 immunostaining, panel E) and tumour vascularisation (CD31
immunostaining, panel F) as determined by confocal microscopy in Patient 1 (J) and Patient 2 (K) before and after THC treatment. Insets in panel E show
higher-magnification micrographs. Cell nuclei are stained in green. Representative micrographs of Patient 1 biopsies are shown.
Cannabinoid administration to cancer patients
M Guzma
´net al
202
British Journal of Cancer (2006) 95(2), 197 – 203 &2006 Cancer Research UK
Translational Therapeutics
Owing to the characteristics of this study the effect of THC on
patient survival was unclear, and an evaluation of survival would
require a larger trial with a different design. In this context, pilot
placebo-controlled trials for recurrent glioblastoma multiforme
with temozolomide, the current benchmark for the management
of malignant gliomas, showed a slight impact on overall length of
survival (median survival ¼24 weeks; 6-month survival ¼46 –
60%) (Dinnes et al, 2002; Nagasubramanian and Dolan, 2003).
Likewise, the efficacy of a biodegradable polymer impregnated
with carmustine was evaluated in patients with recurrent high-
grade gliomas requiring re-operation (Brem et al, 1995). The
median survival was 31 weeks, but it should be noted that in that
study one-third of the treated patients had tumours with better
prognosis than glioblastoma multiforme, for example, oligoden-
drogliomas and anaplastic astrocytomas. Recurrent glioblastoma
multiforme is an extremely rapid and lethal disease, and trials in
newly diagnosed tumours have allowed a clear improvement in the
therapeutic efficacy of temozolomide and carmustine through the
development of various administration regimes (Lonardi et al,
2005; Stupp et al, 2005; Reardon et al, 2006). It is therefore
conceivable that better outcomes could also be obtained with
cannabinoid-based therapies in newly diagnosed gliomas.
Most of the experiments performed so far in animal models of
cancer have evidenced a tumour growth-inhibiting action of
cannabinoids (Guzma
´n, 2003). However, a few studies have shown
that THC may induce proliferation of tumour cells in vitro (Hart
et al, 2004) and in vivo (Zhu et al, 2000; McKallip et al, 2005). The
latter was attributed to a cannabinoid-induced inhibition of
host antitumour immunity and was evident in models in which
xenografted tumour cells did not express significant levels of
cannabinoid receptor, therefore disabling cannabinoid receptor-
mediated tumour-cell killing. The present study clearly supports
that THC does not facilitate tumour growth nor decreases patient
survival, at least in our cohort of brain tumour patients expressing
cannabinoid receptors.
In view of the fair safety profile of THC, together with its
possible antiproliferative action on tumour cells reported here
and in other studies (Guzma
´n, 2003), it would be desirable that
additional trials – on this and other types of tumours – were run
to determine whether cannabinoids – as single drugs or in
combination with established antitumoral drugs – could be used,
other than for their palliative effects, to inhibit tumour growth. In
particular, our next goal is to evaluate the efficacy of THC in
patients with newly diagnosed gliobastoma multiforme.
ACKNOWLEDGEMENTS
We are indebted to all our hospital and university colleagues as
well as to Mr Alfredo Dupetit for providing support and assistance
to this work. This work was supported by grants from Fundacio
´n
Cientı
´fica de la Asociacio
´n Espan
˜ola Contra el Ca
´ncer, Spanish
Ministry of Education and Science (SAF2003/00745) and Funda-
cio
´n Ramo
´n Areces.
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