the therapeutic potential of agonists of the CB2 receptor has been most strongly demonstrated in animal models of inflammatory Fingolimod and neuropathic pain. Much of this data has been generated using the racemic mixture of the synthetic ligand AM1241. The in vitro selectivity of R,S AM1241 for CB2 vs CB1 receptors has been demonstrated to be approximately 80 collapse in binding studies, employing natively showing recombinant cell systems and cells. In pain efficacy reports, the action of R,S AM1241 at CB2 receptors has been shown often pharmacologically using CB2 selective antagonists, such as for instance AM630 or SR144528, or genetically, using animals lacking the CB2 receptor. Similarly, efficacy through CB1 receptor activation is eliminated through the use of both CB1 particular villain compounds or CB1 animals. In our report, we offer a comprehensive in vitro pharmacological characterization of R,S AM1241, measuring binding affinity and functional inhibition of forskolin stimulated cyclic adenosine ARN 509 monophosphate accumulation in CHO K1 cell lines overexpressing human, rat or mouse CB2. We show not just species specific ramifications of R,S AM1241, but in extending this research towards the separated enantiomers of R,S AM1241, we also show stereoisomer specific pharmacology for this artificial cannabinoid ligand both in vitro and in vivo. Techniques Cloning and cell culture CHO K1 Metastatic carcinoma cells expressing hCB1 and hCB2 receptors were cultured in Hams F12 medium containing penicillin /streptomycin, 10% foetal bovine serum and 400 mgml 1 G418. Mouse Carfilzomib and rat CB2 receptor open reading frame sequences were PCR amplified fromcommercially prepared spleen cDNA applying oligonucleotide primers spanning the start and stop sites developed from posted sequences and AF176350. Restriction websites were included in the Oprozomib Proteasome inhibitors collection of the PCR primers to facilitate cloning in to pcDNA3. 1. Transfection of CHO K1 cells was with Lipofectamine Plus based on the manufacturer s directions. Initial selection of transfectants was with 800 mgml 1 G418. Cell lines stably expressing mCB2 and rCB2 receptors were cultured in Dulbecco s altered Eagle s medium containing 10 percent FBS, penicillin /streptomycin, non essential proteins and 500 mgml 1 G418. All tissue tradition reagents were from Invitrogen. Chiral separation of R,S AM1241 Fingolimod The enantiomers of R,S AM1241 were divided by chiral HPLC on the 2 25cm Chiralcel OD column. S AM1241 eluted at 12. 2 min, and Dtc AM1241 eluted at 17. 26 min. Optical rotations were received with a Jasco G 1020 polarimeter with a cell. S AM1241: 25 N 461, Kiminas AM1241 25 N t401.