Chromogenic assay

Varespladib was evaluated in the assay containing 0.96 mM racemic 1,2-bis(thioheptanoyl)-1,2-dideoxyphosphatidylcholine, 0.27 mM Triton X-100, and 0.12 mM 5,5’-dithiobis[2-nitrobenzoic acid] (DTNB). Concentration-response curves were generated with 16 nM recombinant human sPLA2 for 30 mins at 40 ℃ in a microtiter plate format. IC50 values were determined as well as mole fraction for 50% inhibition (Xi50). Mole fraction represented a dimensionless number derived by dividing the IC50 value by the concentration of lipid in the assay mixture. IC50 values were determined in triplicate.

Cell lines

BAL cells

Preparation method

The solubility of this compound in DMSO is >8.175 mg/mL. General tips for obtaining a higher concentration: Please warm the tube at 37 ℃ for 10 minutes and/or shake it in the ultrasonic bath for a while. Stock solution can be stored below - 20 ℃ for several months.

Reacting condition

0.1 ~ 3 μM

Applications

In BAL cells stimulated with human sPLA2, Varespladib (0.1 ~ 3 μM) inhibited the formation of thromboxane in a concentration-dependent manner, with an IC50 value of approximately 8 × 10-7 M. In control experiments, Varespladib (3 μM) showed no effect on formyl-methionyl-leucyl-phenylalanine (a chemotactic peptide)-induced thromboxane release. Similarly, arachidonic acid-induced generation of thromboxane A2 was not inhibited by prior exposure to Varespladib.

Animal models

Transgenic mice expressing human sPLA2 protein

Dosage form

0.3 ~ 3 mg/kg; i.v. or p.o.

Applications

In transgenic mice expressing human sPLA2 protein, Varespladib (0.3 ~ 3 mg/kg, i.v.) inhibited serum sPLA2 activity. However, the inhibition effect of Varespladib gradually decreased over the 4-hr observation period. Oral administration of Varespladib to transgenic mice leaded to similar dose-dependent inhibition on serum sPLA2 activity.

Other notes

Please test the solubility of all compounds indoor, and the actual solubility may slightly differ with the theoretical value. This is caused by an experimental system error and it is normal.

IC50: 9 ± 1 nM or 7.3 x 10-6 mole fraction for sPLA2 activity

Phospholipases (PLAs) produce rate-limiting precursors in the various types of biologically active lipid biosynthesis. Increased levels of human nonpancreatic secretory phospholipase A2 (hnps-PLA2) have been detected in several pathological conditions. LY315920 was selected for evaluation clinically as an hnps-PLA2 inhibitor.

In vitro: The true potency of LY315920 was defined to be a mole fraction of 1.5 x 10-6 using a deoxycholate/phosphatidylcholine assay. LY315920 was found to be 40-fold less active against human, group IB, pancreatic sPLA2 and was inactive against cytosolic PLA2 and the constitutive and inducible forms of cyclooxygenase. Human sPLA2-induced thromboxane A2 release from isolated guinea pig lung bronchoalveolar lavage cells was inhibited by LY315920 with the IC50 of 0.79 μM [1].

In vivo: The i.v. administration of LY315920, 5 min before the bronchoalveolar lavage cells harvest, led to the inhibition of sPLA2-induced production of TXA2 with an ED50 of 16.1 mg/kg. [2].

Clinical trials: Varespladib methyl, an oral prodrug of LY-315920, effectively reduced LDL-C and inflammatory biomarkers in ACS patients treated with conventional atorvastatin therapy. There were no treatment differences in clinical cardiovascular events [2].

References:
[1] Snyder DW, Bach NJ, Dillard RD, Draheim SE, Carlson DG, Fox N, Roehm NW, Armstrong CT, Chang CH, Hartley LW, Johnson LM, Roman CR, Smith AC, Song M, Fleisch JH.  Pharmacology of LY315920/S-5920, [[3-(aminooxoacetyl)-2-ethyl-1- (phenylmethyl)-1H-indol-4-yl]oxy] acetate, a potent and selective secretory phospholipase A2 inhibitor: A new class of anti-inflammatory drugs, SPI. J Pharmacol Exp Ther. 1999;288(3):1117-24.
[2] Rosenson RS, Hislop C, Elliott M, Stasiv Y, Goulder M, Waters D.   Effects of varespladib methyl on biomarkers and major cardiovascular events in acute coronary syndrome patients. J Am Coll Cardiol. 2010;56(14):1079-88.