sPLA2/secretory phospholipase A2 (IC50 = 9 nM)

The RA-induced rise in MUC16 protein seen in cell lysates at both time intervals was entirely suppressed by Varespladib (LY-315920)(10 μM; 24 and 48 hours; HCjE cells) therapy [2]. Treatment with varaspladib (10 μM; 24 and 48 hours; HCjE cells) dramatically reduced the amount of MUC16 that was produced by RA by 100% at 24 hours and 99% at 48 hours [2].

---

Varespladib (LY-315920) is a potent, selective inhibitor of recombinant human, group IIA, nonpancreatic secretory PLA2 (sPLA2). In a chromogenic isolated enzyme assay, Varespladib (LY-315920) inhibited sPLA2 activity with an IC50 of 9 +/- 1 nM or 7.3 x 10(-6) mole fraction, which approached the stiochiometric limit of this assay. The true potency of LY315920 was defined using a deoxycholate/phosphatidylcholine assay with a mole fraction of 1.5 x 10(-6). LY315920 was 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 release of thromboxane A2 (TXA2) from isolated guinea pig lung bronchoalveolar lavage cells was inhibited by LY315920 with an IC50 of 0.79 microM. The release of TXA2 from these cells by N-formyl-methionyl-leucyl-phenylalanine or arachidonic acid was not inhibited. [1]

---

Having shown inhibition of RA-induced MUC16 upregulation using a broad spectrum PLA2 inhibitor, ArA, we sought to determine if a specific inhibitor of group IIA sPLA2, Varespladib (LY-315920), would affect the RA-induced MUC16 expression. We examined MUC16 mRNA expression levels by real time PCR in HCjE cultures treated for 24 and 48 hours with vehicle (DMSO), 100 nM RA, 100 nM RA plus 10 μm Varespladib (LY-315920) or 10 μm LY315920 alone. As shown in Figure 6, addition of 10 μm LY315920 significantly inhibited RA-induced MUC16 expression by 100% at 24 hours and 99% at 48 hours. As shown in Figure 7, the addition of the specific inhibitor for sPLA2-IIA, LY315920, results in complete inhibition of the RA-induced increase in MUC16 protein detected in cell lysates at both 24 (p<0.01) and 48 (p<0.0001) hours [2].

At an IC50 of 0.79 μM, Varespladib (LY-315920) therapy prevents human sPLA2-induced release of thromboxane A2 (TXA2) from isolated guinea pig lung bronchoalveolar lavage cells. 16.1 mg/kg is the ED50 for Varespladib (LY-315920)[1].
The i.v. administration of Varespladib (LY-315920), 5 min before harvesting the bronchoalveolar lavage cells, resulted in the inhibition of sPLA2-induced production of TXA2 with an ED50 of 16.1 mg/kg. Challenge of guinea pig lung pleural strips with sPLA2 produced contractile responses that were suppressed in a concentration-dependent manner by Varespladib (LY-315920) with an apparent KB of 83 +/- 14 nM. Contractile responses induced by arachidonic acid were not altered. Intravenous or oral administration of Varespladib (LY-315920) to transgenic mice expressing the human sPLA2 protein inhibited serum sPLA2 activity in a dose-related manner over a 4-h time course. Varespladib (LY-315920) is a potent and selective sPLA2 inhibitor and represents a new class of anti-inflammatory agent designated SPI. This agent is currently undergoing clinical evaluation and should help to define the role of sPLA2 in various inflammatory disease states [1].

---

ApoE−/− Standard Model [3]
The body weights of animals in control or A-002 (varespladib methyl/LY333013/S-3013-treated groups were similar at time 0 (vehicle: 27.2 ± 2.6, 30 mg/kg A-002: 26.7 ± 2.3, and 90 mg/kg A-002: 27.3 ± 2.5). Body weight increased over the 16 weeks on a Western diet by approximately 155%, 150%, and 141%, for the vehicle, 30 mg/kg A-002 (varespladib methyl/LY333013/S-3013, and 90 mg/kg A-002 groups, respectively (Fig. 2). There was no statistically significant difference in body weights between the groups using a repeated-measures 2-way ANOVA where time and treatment were the variables.

Plasma cholesterol levels at the beginning of the study were not significantly different between groups. However, after 1 month of twice a day treatment with A-002 (varespladib methyl/LY333013/S-3013, either 30 or 90 mg/kg doses, total cholesterol was significantly decreased (Fig. 3) compared with the control group. This effect remained consistent throughout the 4 months of treatment. Plasma cholesterol changed after 4 months of diet per treatment by +15%, −10%, and −12% for vehicle, 30 mg/kg A-002, and 90 mg/kg A-002 groups, respectively (Fig. 3). There was no apparent dose-response effect on plasma cholesterol concentrations.

Treatment with A-002 (varespladib methyl/LY333013/S-3013 had a significant effect on plaque content expressed as percent occupancy of the aortic luminal surface by atherosclerotic plaques. Vehicle-treated mice had approximately 12.6% ± 0.7% plaque coverage, whereas mice treated with 30 mg/kg A-002 twice a day had 6.3% ± 0.6% plaque coverage and mice treated with 90 mg/kg A-002 twice a day had 6.7% ± 0.8% plaque coverage. These represent significant decreases in plaque content in each of the A-002 treatment groups compared with the treatment group receiving only the formulation vehicle (P < 0.05) (Fig. 4).

---

Angiotensin II ApoE−/− Model [3]
Angiotensin II formulated in water twice a day or in 5% acacia twice a day resulted in similar aortic plaque coverage (18% ± 3.3% and 14.4% ± 4.8%, respectively, Fig. 5). A-002 (varespladib methyl/LY333013/S-3013 (30 mg/kg) significantly reduced the plaque coverage of the aorta (8% ± 3% vs. 18% ± 3.3% observed with angiotensin II infusion without drug treatment, P < 0.025). The background amount of atherosclerosis in the absence of angiotensin II (3.8% ± 0.6%, subcutaneous saline pump and water twice a day) was significantly lower than that with angiotensin II infusion (18% ± 3.3%, P < 0.025). Representative in face images are shown in figure 6.

Aortic aneurysm rate was assessed in each group of mice. In the absence of angiotensin II infusion, no aneurysms were observed. Infusion of angiotensin II formulated in water resulted in a 25% incidence of aneurysm and infusion of angiotensin II formulated in the acacia vehicle caused a 22.2% incidence of aneurysm. A-002 (varespladib methyl/LY333013/S-3013 treatment (30 mg/kg twice a day) in the mice infused with angiotensin II formulated in acacia prevented aneurysm formation completely (Prob>ChiSq = 0.0096) (Table 1).

sPLA2 Inhibition [3]
The intrinsic activity of Varespladib (LY-315920)/A-001 on the inhibition of sPLA2 group V and X enzymes was measured according to a chromogenic method described elsewhere.

---

Phospholipase A2 Inhibitor Treatment [2]
To investigate whether RA regulation of MUC16 is associated with sPLA2, the effect of the broad spectrum PLA2 inhibitor, aristolochic acid (ArA), on MUC16 mRNA levels was determined in HCjE cells cultured as above with 100 nM RA plus 100 μM of ArA, the inhibitor or vehicle (DMSO) alone for 24 and 48 hours. These experiments were followed up by testing the effect of an inhibitor specific for group IIA secretory phospholipase A2, Varespladib (LY-315920). HCjE cells were treated with 100 nM RA, 100 nM RA plus 10 μm Varespladib (LY-315920), the inhibitor or vehicle (DMSO) alone for 24 and 48 hours. MUC16 mRNA and protein levels were determined by real-time PCR and Western blot analysis, respectively. The experiments were performed twice for both inhibitors, each experiment being done in duplicate.

Western Blot Analysis[2]
Cell Types: HCjE cells
Tested Concentrations: 10 μM
Incubation Duration: 24 hrs (hours) and 48 hrs (hours)
Experimental Results: Dramatically inhibited the RA-induced MUC16 protein expression at both time points.

---

RT-PCR[2]
Cell Types: HCjE cells
Tested Concentrations: 10 μM
Incubation Duration: 24 hrs (hours) and 48 hrs (hours)
Experimental Results: Dramatically inhibited RA-induced MUC16 expression by 100% at 24 hrs (hours) and 99% at 48 hrs (hours).

Animal/Disease Models: Male Hartley guinea pigs (300-500 g)[1]
Doses: 3 mg/kg, 10 mg/kg, and 30 mg/kg
Route of Administration: intravenous (iv) injection (pharmacokinetic/PK study)
Experimental Results: Consistent inhibition of sPLA2 activity in BAL fluid was observed. decreased the human sPLA2-induced generation of TXA2 on BAL cells from guinea pigs.

Plasma Levels and Dose Selection [3]
Figure 1 shows plasma levels of Varespladib (LY-315920)/A-001 in serum after a single oral dose of A-002 (varespladib methyl/LY333013/S-3013. Levels of Varespladib (LY-315920)/A-001 were detectable in all samples from dosed mice at 10, 30, and 90 mg/kg. The 2 highest doses, 30 and 90 mg/kg A-002 (varespladib methyl/LY333013/S-3013, reached concentrations of Varespladib (LY-315920)/A-001 in plasma that were greater than the IC50 values for sPLA2 groups V and X, respectively, throughout the dosing period.

---

Plasma Levels [3]
Plasma levels of A-001/Varespladib (LY-315920) (the active moiety of A-002 (varespladib methyl/LY333013/S-3013) were measured in a single-dose experiment in ApoE−/− mice. Animals were dosed by oral gavage as explained above with 10, 30, or 90 mg/kg ofA-002 (varespladib methyl/LY333013/S-3013. Blood was sampled at different time points using the tail clip method. Plasma was collected and stored at −70°C for analysis of the presence of A-001/Varespladib (LY-315920) by high-performance liquid chromatography-mass spectrometry. Plasma from dosed animals (25 μL) was combined with 5 μL of 20 μM LY329722 (internal standard), 25 μL of isopropanol, and 50 μL of acetonitrile in a polypropylene microfuge tube. The mixture was vortexed, incubated for 10 minutes at 37°C, and centrifuged at 12,000g at ambient temperature. Supernatant (65 μL) was decanted into 430 μL of water and 5 μL of formic acid in an 800-μL glass autosampler vial. The vial was capped, vortexed, and placed in a Surveyor autosampler. Injections (25 μL) were made onto a 2.1 × 100 mm, 5 μm, C8 Discovery reversed-phase column equipped with a 2.1 × 4 mm Phenomenex C8 guard column. Initial chromatography conditions were set to 95% mobile phase A (0.1% formic acid in 5% isopropanol) and 5% mobile phase B (0.1% formic acid in acetonitrile containing 5% isopropanol). At 1-minute postinjection, a gradient elution from 5% B to 60% B over 6 minutes gave retention times of 6.07 and 6.47 minutes for A-002 (varespladib methyl/LY333013/S-3013 and internal standard, respectively. Analytes were detected by electrospray, atmospheric pressure ionization, ion trap mass spectrometry. Capillary voltage was 4.5 kV and capillary temperature was 310°C. Nitrogen sheath flow was set at 75 arbitrary units. Collision-induced dissociation was optimized at 35% normalized collision energy. Transitions selective for the tandem mass spectrometric detection of A-001/Varespladib (LY-315920) and internal standard resulted from the loss of ammonia and carbon monoxide radical and were 381→336 m/z and 395→350 m/z, respectively. Peak areas for A-001 and internal standard were used to compute response factors and unknowns were interpolated against a linear calibration curve of A-001 spiked into pooled mouse plasma obtained from nondosed animals.

[1]. 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 Mar;288(3):1117-24.

[2]. Effect of retinoic acid on gene expression in human conjunctival epithelium: secretory phospholipase A2 mediates retinoic acid induction of MUC16. Invest Ophthalmol Vis Sci. 2005 Nov;46(11):4050-61.

[3]. Varespladib (A-002), a Secretory Phospholipase A2 Inhibitor, Reduces Atherosclerosis and Aneurysm Formation in ApoE−/− Mice J Cardiovasc Pharmacol. 2009 Jan;53(1):60-5.

Varespladib is a member of the class of indoles that is 1H-indole substituted by benzyl, ethyl, oxamoyl, and carboxymethoxy groups at positions 1, 2, 3, and 4, respectively. It is an oral secretory phospholipase A2 inhibitor and exhibits anti-inflammatory effects. It has a role as an EC 3.1.1.4 (phospholipase A2) inhibitor, an anti-inflammatory drug and an antidote. It is a member of indoles, a member of benzenes, an aromatic ether, a dicarboxylic acid monoamide, a monocarboxylic acid and a primary carboxamide. It is a conjugate acid of a varespladib(1-).
Varespladib has been investigated for the treatment and prevention of Sickle Cell Disease, Vaso-occlusive Crisis, and Acute Coronary Syndrome.

---

LY315920 is a potent, selective inhibitor of recombinant human, group IIA, nonpancreatic secretory PLA2 (sPLA2). In a chromogenic isolated enzyme assay, LY315920 inhibited sPLA2 activity with an IC50 of 9 +/- 1 nM or 7.3 x 10(-6) mole fraction, which approached the stiochiometric limit of this assay. The true potency of LY315920 was defined using a deoxycholate/phosphatidylcholine assay with a mole fraction of 1.5 x 10(-6). LY315920 was 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 release of thromboxane A2 (TXA2) from isolated guinea pig lung bronchoalveolar lavage cells was inhibited by LY315920 with an IC50 of 0.79 microM. The release of TXA2 from these cells by N-formyl-methionyl-leucyl-phenylalanine or arachidonic acid was not inhibited. The i.v. administration of LY315920, 5 min before harvesting the bronchoalveolar lavage cells, resulted in the inhibition of sPLA2-induced production of TXA2 with an ED50 of 16.1 mg/kg. Challenge of guinea pig lung pleural strips with sPLA2 produced contractile responses that were suppressed in a concentration-dependent manner by LY315920 with an apparent KB of 83 +/- 14 nM. Contractile responses induced by arachidonic acid were not altered. Intravenous or oral administration of LY315920 to transgenic mice expressing the human sPLA2 protein inhibited serum sPLA2 activity in a dose-related manner over a 4-h time course. LY315920 is a potent and selective sPLA2 inhibitor and represents a new class of anti-inflammatory agent designated SPI. This agent is currently undergoing clinical evaluation and should help to define the role of sPLA2 in various inflammatory disease states. [1]

---

Purpose: How vitamin A contributes to the maintenance of the wet-surfaced phenotype at the ocular surface is not well understood. This study sought to identify vitamin A-responsive genes in ocular surface epithelia using gene microarray analysis of cultures of a human conjunctival epithelial (HCjE) cell line grown with all-trans-retinoic acid (RA). The analysis showed that secretory phospholipase A(2) group IIA (sPLA(2)-IIA) was the gene most upregulated by RA, followed by the membrane-associated mucin MUC16 at a later time point. Since eicosanoids, the product of arachidonic acid generated by the PLA(2) family, have been shown to increase mucin production, this study sought to determine whether sPLA(2) mediates the RA induction of MUC16.

Methods: HCjE cells were cultured with or without RA for 3, 6, 24, and 48 hours. Complementary RNA prepared from RNA of the HCjE cells was hybridized to human gene chips and analyzed using commercial software. Microarray data on mucin expression were validated by real-time PCR. To investigate whether sPLA(2) is associated with RA-induced MUC16 upregulation, HCjE cells were incubated with RA and the broad-spectrum PLA(2) inhibitor aristolochic acid (ArA) or the specific sPLA(2)-IIA inhibitor LY315920, followed by analysis of MUC16 mRNA and protein by real-time PCR and Western blot analysis.

Results: After RA addition, 28 transcripts were upregulated and 6 downregulated by more than twofold (P < 0.01) at both 3 and 6 hours (early phase). Eighty gene transcripts were upregulated and 45 downregulated at both 24 and 48 hours (late phase). Group IIA sPLA(2), significantly upregulated by 24 hours, and MUC16 were the most upregulated RNAs by RA at 48 hours. sPLA(2) upregulation by RA was confirmed by Western blot analysis. When HCjE cells were incubated with RA plus ArA or specific inhibitor of sPLA(2)-IIA, LY315920, the RA-induced MUC16 mRNA was significantly reduced (P < 0.01).

Conclusions: The RA-associated upregulation of membrane-associated mucin MUC16 at late phase appears to be through sPLA(2)-IIA. Upregulation of this hydrophilic membrane-associated mucin may be one of the important mechanisms by which vitamin A facilitates maintenance of the wet-surfaced phenotype on the ocular surface. [2]

---

The family of secretory phospholipase A2 (sPLA2) enzymes has been associated with inflammatory diseases and tissue injury including atherosclerosis. A-001 is a novel inhibitor of sPLA2 enzymes discovered by structure-based drug design, and A-002 (varespladib methyl/LY333013/S-3013 is the orally bioavailable prodrug currently in clinical development. A-001 inhibited human and mouse sPLA2 group IIA, V, and X enzymes with IC50 values in the low nM range. A-002 (1 mg/kg) led to high serum levels of A-001 and inhibited PLA2 activity in transgenic mice overexpressing human sPLA2 group IIA in C57BL/6J background. In addition, the effects of A-002 on atherosclerosis in 2 ApoE−/− mouse models were evaluated using en face analysis. (1) In a high-fat diet model, A-002 (30 and 90 mg/kg twice a day for 16 weeks) reduced aortic atherosclerosis by 50% (P < 0.05). Plasma total cholesterol was decreased (P < 0.05) by 1 month and remained lowered throughout the study. (2) In an accelerated atherosclerosis model, with angiotensin II-induced aortic lesions and aneurysms, A-002 (30 mg/kg twice a day) reduced aortic atherosclerosis by approximately 40% (P < 0.05) and attenuated aneurysm formation (P = 0.0096). Thus, A-002 was effective at significantly decreasing total cholesterol, atherogenesis, and aneurysm formation in these 2 ApoE−/− mouse models.[3]

C21H20N2O5

380.39

380.137

C, 66.31; H, 5.30; N, 7.36; O, 21.03

172732-68-2

Varespladib sodium;172733-42-5;Varespladib methyl;172733-08-3

155815

White to yellow solid powder

1.3±0.1 g/cm3

667.9±65.0 °C at 760 mmHg

357.7±34.3 °C

0.0±2.1 mmHg at 25°C

1.630

2.45

2

5

8

28

589

0

CCC1=C(C2=C(N1CC3=CC=CC=C3)C=CC=C2OCC(=O)O)C(=O)C(=O)N

BHLXTPHDSZUFHR-UHFFFAOYSA-N

InChI=1S/C21H20N2O5/c1-2-14-19(20(26)21(22)27)18-15(9-6-10-16(18)28-12-17(24)25)23(14)11-13-7-4-3-5-8-13/h3-10H,2,11-12H2,1H3,(H2,22,27)(H,24,25)

2-((3-(2-amino-2-oxoacetyl)-1-benzyl-2-ethyl-1H-indol-4-yl)oxy)acetic acid

A002; A 002; LY315920; Varespladib; 172732-68-2; LY315920; Varespladib (LY315,920); LY-315,920; S-5920; VAREPLADIB; 2Q3P98DATH; LY-315920; LY 315920; A-002;

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)

Solubility in Formulation 1: 2.5 mg/mL (6.57 mM) in 10% DMSO + 40% PEG300 + 5% Tween80 + 45% Saline (add these co-solvents sequentially from left to right, and one by one), suspension solution; with sonication.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 25.0 mg/mL clear DMSO stock solution to 400 μL PEG300 and mix evenly; then add 50 μL Tween-80 to the above solution and mix evenly; then add 450 μL normal saline to adjust the volume to 1 mL.
Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH₂ O to obtain a clear solution.

---

Solubility in Formulation 2: 30% PEG400+0.5% Tween80+5% propylene glycol:30 mg/mL

---

View More

Solubility in Formulation 3: 1.5 mg/mL (3.94 mM) in 17% Polyethylene glycol 12-hydroxystearate in Saline (add these co-solvents sequentially from left to right, and one by one), suspension solution; with ultrasonication.
Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH₂ O to obtain a clear solution.

---

 (Please use freshly prepared in vivo formulations for optimal results.)