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 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 Introduction 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. 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. Tactile Allodynia 0.3 mg/kg Movement Evoked Pain 0.3 mg/kg 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 Results 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 Conclusion 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 Idris et al., Nat Med., 11(7): , 2005 Luger et. al., Journal of Pain and Symptom Management, 29 (5S): 32-46, 2005 Analysis of the Cannabinoid 1 Receptor Inverse Agonist, AM 251, on Bone Density in a Murine Model of Bone Cancer