Fluoroketone inhibition of Ca(2+)-independent phospholipase A2 through binding pocket association defined by hydrogen/deuterium exchange and molecular dynamics.

The mechanism of inhibition of group VIA Ca(2+)-independent phospholipase A(2) (iPLA(2)) by fluoroketone (FK) ligands is examined by a combination of deuterium exchange mass spectrometry (DXMS) and molecular dynamics (MD). Models for iPLA(2) were built by homology with the known structure of patatin and equilibrated by extensive MD simulations. Empty pockets were identified during the simulations and studied for their ability to accommodate FK inhibitors. Ligand docking techniques showed that the potent inhibitor 1,1,1,3-tetrafluoro-7-phenylheptan-2-one (PHFK) forms favorable interactions inside an active-site pocket, where it blocks the entrance of phospholipid substrates. The polar fluoroketone headgroup is stabilized by hydrogen bonds with residues Gly486, Gly487, and Ser519. The nonpolar aliphatic chain and aromatic group are stabilized by hydrophobic contacts with Met544, Val548, Phe549, Leu560, and Ala640. The binding mode is supported by DXMS experiments showing an important decrease of deuteration in the contact regions in the presence of the inhibitor. The discovery of the precise binding mode of FK ligands to the iPLA(2) should greatly improve our ability to design new inhibitors with higher potency and selectivity

Intrathecal Delivery of Ketorolac Loaded In Situ Gels for Prolonged Analgesic and Anti-Inflammatory Activity in Vertebral Fracture

Purpose: To develop biodegradable, polymeric in situ gels based on sodium alginate and hydroxypropyl methylcellulose for intrathecal delivery of ketorolac tromethamine (KT) for effective management of pain and inflammation in vertebral fracture.Method: Ion activated in situ gels were used as implants and were prepared from sodium alginate and hydroxypropyl methylcellulose. The fabricated gels were evaluated for visual appearance, clarity, pH, gelling capacity, drug content, viscosity (using Brookfield viscometer), in vitro drug release (using a fabricated KC cell) and in vivo analgesic and anti-inflammatory activity (by intrathecal administration of in situ gel near the fractured vertebra in a rat model).Results: The physicochemical properties (visual appearance, clarity, pH, gelling capacity, drug content and viscosity) of in situ gels were acceptable for therapeutic use. KT-loaded gels demonstrated high drug encapsulation efficiency (98.3 - 103.3 %). Further, KT-loaded gels exhibited viscosity in the range of 1.11 to 6 cps at 50 rpm and shear thinning property (rheology testing). Additionally, the gels demonstrated 84.43 to 96.98 % drug release at the end of 12 h. In particular, in situ gels prepared from 1.2 % alginate/0.4 % HPMC (G7) exhibited excellent analgesic (54.28 %) and anti-inflammatory activity (51.6 % inhibition of rat paw edema) in the animal model of vertebral fracture.Conclusion: The formulated in situ gels can potentially be used as implants for the treatment of patients with vertebral fracture.Keywords: Ketorolac, Orthopaedic implant, Extended release, Analgesic, Anti inflammation, Vertebral fractur