Analysis of the Cannabinoid 1 Receptor Inverse Agonist, AM 251, on Bone
Density in a Murine Model of Bone Cancer
Eduardo Valenzuela, Alysia Lozano, Todd W. Vanderah, Ph.D
The University of Arizona Graduate College and Bio5 Institute, Department of
Pharmacology, Minority Health Disparities Summer Research Program Maria
Teresa Velez, Ph.D. Director
Introduction
Results
Bone cancer is a disease endured by many people all over the world. A decrease of cancer related pain is
a goal of bone cancer drug development. In the treatment of bone cancer, the inhibition of a group of
cells called osteoclasts that consume bone may play a central role. Under normal circumstances,
osteoclasts absorb bone at the same rate osteoblasts build bone. This results in constant bone
mass. Cancer cells present in bone gradually consume bone stimulating osteoclasts to reabsorp bone
further weakening bone strength and promoting pain. The bone is destroyed faster than it can be
replenished. In the treatment of bone cancer, the reduction of osteoclast numbers may lead to a decrease
in bone loss and the occurrence of fractures resulting in less pain.
The question that arises is, how can osteoclast numbers be reduced? Previous experimentation
has shown that the cannabinoid receptor CB1 plays a role in bone remodeling by regulating osteoclast
activity (Idris et al., 2005). The goal of this study was to observe if in fact the CB1 inverse agonist (a
ligand that causes a receptor to reverse its actions as compared to when it is activated) AM251, would
decrease osteoclast numbers resulting in decreased bone loss and a reduction of pain in mice suffering
from bone cancer.
Tactile Allodynia
0.3 mg/kg
0.3 mg/kg
Although AM251 at the dose tested here did not reduce bone
loss, it did reduce bone cancer related pain. The reduction in
pain behavior may be due to a reduction in tumor growth and
a trend towards a decrease in bone loss yet further doses
must be tested before such conclusions can be made.
Discussion
Methods
Model.- Previous studies have shown that the pain present in murine model of bone cancer is
very similar to the pain experienced in humans with the disease (Luger 2005). It is for this
reason that this model was used in this experiment.
Surgery.- 25,000 CCL-11 murine sarcoma cells in a 5 µl. of alpha minimal essential medium
consisting of 1% bovine serum albumin or 5 µl. of the medium alone (control) was injected
into drilled hole in the distal end of the right femur.
Injections.- Commencing on day 7 after surgery, mice were injected twice daily with a 0.3
mg/kg dosage of AM251 mixed with a vehicle solution that contained 10% Tween 80, 10%
dimethyl sulphoxide and 80% saline. Some mice were injected solely with the vehicle
(control).
Assessment of Pain
The animals underwent testing for tactile allodynia, spontaneous pain and movement evoked
pain before the surgery and 7, 10 and 14 days after surgery. All tests took place in a raised
plexiglass chamber with a wire gird floor.
-Tactile Allodynia.- Testing consisted of increasing logarithmically incremental thickness of
the filaments that were equivalent to the weights of 0.03 to 2.34 grams pressed for 2 to 3
seconds perpendicularly against the planar surface of the right rear paw. It was observed
whether or not the mouse withdrew the paw as a result. If the mouse responded, a filament of
less thickness was used until no response was recorded. After the initial response, the mouse
was tested with four more filaments.
-Spontaneous pain.- Guarding testing consisted of keeping track of the time the mouse
intentionally kept pressure off the right rear paw during a period of 2 minutes (except for
walking and other movement). The flinching testing consisted of counting the number of times
the mouse unintentionally removed his right rear paw off the wire bottom during a period of 2
minutes.
-Movement Evoked Pain.- The mice were observed walking and their right rear limb was
rated using the following scale.- 4 = normal use, 3 = limping, 2 = limping and guarding, 1 =
partial non use, 0 = complete lack of use.
Bone Destruction Determination.- A faxitron machine was used to take radiographs of
mice’s right femurs before surgery, 7 days, 10 days and 14 days after surgery. The amount of
bone loss was rated using the following scale.- 0 = normal, 1 = bone loss observed with no
fracture, 2 = full thickness unicortical bone loss indicating unicortical bone fracture,
3 = full thickness bicortical bone loss indicating bicortical bone fracture,
4 = billateral bone fracture.
Conclusion
Movement Evoked Pain
Rating.- 0 = no use of right hind limb at all, 1 = partial non use,
2=limp and guard, 3 = limp, 4 = normal use
Spontaneous Pain.- Flinching
0.3 mg/kg
Spontaneous Pain.- Guarding
0.3 mg/kg
The purpose of this experiment was to investigate possible
reduction of bone loss and reduction of bone cancer related pain
by the CB1 inverse agonist AM251. The fact that the data
shows that this drug did reduce behavioral signs of pain yet did
not significantly reduce bone loss may be due to the doses
needed to identify a significant change in bone loss. In order to
further test the hypothesis that a CB1 inverse agonist may
attenuate osteoclast activity higher doses must be tested. The
reduction in pain would suggest that AM251 may directly
inhibit pain fibers, reduce bone loss that was not detectable by
the faxitron or reduce the tumor burden. Further studies will
test these possibilities.
Future research should include investigating ways to
rebuild bone. One possible drug being explored is AM1241.
This drug is believed to increase the osteoblast numbers. The
combination of AM251 and AM1241 may result in a great
advance in significantly reducing bone loss and bone cancer
related pain in cancer patients.
References
Idris et al., Nat Med., 11(7): 774-779, 2005
Luger et. al., Journal of Pain and Symptom Management, 29 (5S):
32-46, 2005
Bone Destruction
0.3 mg/kg
Rating.- 0 = normal, 1 = bone loss observed less than half of 1/3 distal
end, 2= bone loss observed greater than half of 1/3 distal end,
3 = unicortical bone fracture, 4 = billateral fracture