Assay of PAT-048 inhibition of ATX

Inhibition of plasma ATX activity by the selective ATX inhibitor PAT-048 (patent publication number WO 2012024620 A2; Amira Pharmaceuticals, San Diego, CA, USA) was evaluated by measuring the concentration of 20:4 LPA produced from the endogenous LPC in mouse plasma after incubating for 4 h at 37°C (concentrations of 18:1 and 18:0 LPA were also measured, with similar results). In brief, BALB/c blood was collected into heparinized Vacutainer tubes (BD Biosciences, San Diego, CA, USA) by cardiac puncture and centrifuged at 800 g for 10 min, to prepare the plasma. An aliquot of plasma was mixed with vehicle and precipitated immediately with ice-cold methanol to determine baseline concentrations of 20:4 LPA. For inhibition studies, 40 µl plasma was mixed with 1 µl PAT-048 or vehicle control (DMSO) and incubated at 37°C for 4 h before precipitating with 5 volumes of ice-cold methanol containing 17:0 LPA as the internal standard. The samples were incubated for 10 min on ice before centrifugation at 4000 g for 10 min at 4°C. The supernatant (150 µl) was mixed with 100 µl 90:10:0.1 water:acetonitrile:ammonium hydroxide then centrifuged at 4000 g for 10 min at 4°C. The concentration of 20:4 LPA was measured by LC-MS/MS by injecting 20 µl onto an Xbridge 2.1 × 50 mm, 5 μm, C8 column (Waters, Milford, MA, USA). Mobile phase A was 90:10:0.1 water:acetonitrile:ammonium hydroxide. Mobile phase B was 10:90:0.1 water:acetonitrile:ammonium hydroxide. Flow rate was 800 μl/min with initial conditions of 90% mobile phase A and 10% mobile phase B. Initial conditions were held for 0.5 min then mobile phase B concentration was increased linearly to 90% over 1 min to elute 20:4 LPA and its internal standard, 17:0 LPA. This condition (10% A, 90% B) was held for 0.5 min. Initial conditions were restored and held for 0.9 min to equilibrate the column. An API-4000 Q-trap mass spectrometer (AB Sciex) was operated in negative-ion multiple-reaction monitoring mode to detect 20:4 LPA and 17:0 LPA. The transitions monitored were 457.2 to 153.0 and 423.2 to 153.0 for 20:4 LPA and 17:0 LPA, respectively. Source conditions were optimized by infusing 20:4 LPA. Declustering potential was −60 and collision energy was −30.