Compounds and Methods for Treatment of Visceral Pain
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example 1
on of Compounds
[0581]The preparation of compounds of Formulas Ia-Id, including Compound A, is described in International Patent Application No. PCT / US2010 / 002360, published as International Publication No. WO2011 / 025541, the entire contents of which are incorporated herein by reference in their entirety.
[0582]The preparation of crystal forms of Compound A, including the anhydrous, non-solvated crystalline form, is described in International Patent Application No. PCT / US2012 / 026506, published as International Publication No. WO2012 / 116276, the entire contents of which are incorporated herein by reference in their entirety.
example 2
us Time-Resolved Fluorescence (HTRF®) Assay For Direct cAMP Measurement
[0583]A: CB2 Assay
[0584]Compounds were screened for agonists and inverse agonists of CB2 receptor (e.g., human CB2 receptor) using the HTRF® assay for direct cAMP measurement (Gabriel et al., ASSAY and Drug Development Technologies, 1:291-303, 2003) in recombinant CHO-K1 cells stably transfected with the CB2 receptor. CHO-K1 cells were obtained from ATCC® (Manassas, Va.; Catalog # CCL-61). An agonist of the CB2 receptor was detected in the HTRF® assay for direct cAMP measurement as a compound which decreased cAMP concentration. An inverse agonist of the CB2 receptor was detected in the HTRF® assay for direct cAMP measurement as a compound which increased cAMP concentration. The HTRF® assay also was used to determine EC50 values for CB2 receptor agonists and inverse agonists.
[0585]B: CB1 Assay
[0586]Compounds were also screened for agonists and inverse agonists of the CB1 receptor (e.g., human CB1 receptor) using H...
example 3
r β-Arrestin Assay
[0598]A: CB2 Assay
[0599]Compounds were screened for agonists of the human CB2 receptor using the DiscoveRx PathHunter β-arrestin assay which measures the β-arrestin binding to the CB2 receptor upon its activation. CB2 was cloned into the pCMV-PK vector (DiscoveRx, Fremont, Calif.; catalog #93-0167) and transfected into the CHO-K1 EA-Arrestin parental cell line (DiscoveRx, Fremont, Calif.; catalog #93-0164). CHO-K1 positive clones stably expressing the CB2—ProLink fusion protein were identified by their responses to the CB2 agonist CP55,940. Clone #61 was chosen for its big agonist window and homogenous expression as detected by anti-HA flow cytometry.
[0600]Principle of the Assay:
[0601]The PathHunter-arrestin assay measures the interaction of β-arrestin with activated GPCRs using Enzyme Fragment Complementation (Yan et al., J. Biomol. Screen. 7: 451-459, 2002). A small, 42 amino acid β-galactosidase fragment, Prolink, is fused to the c-terminus of a GPCR, and β-arre...
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