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].

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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]

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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].

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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).

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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.

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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.

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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 Concentration and Dosage Selection [3] Figure 1 shows the plasma concentrations of Varespladib (LY-315920)/A-001 after a single oral administration of A-002 (varespladib methyl/LY333013/S-3013). Varespladib (LY-315920)/A-001 was detectable in samples from all treated mice at doses of 10, 30, and 90 mg/kg. At the two highest doses, 30 and 90 mg/kg A-002 (varespladib methyl/LY333013/S-3013), the plasma concentrations of Varespladib (LY-315920)/A-001 were higher than the IC50 value. Throughout the administration period, the sPLA2 V and X groups were tested separately.

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Plasma levels[3]
In a single-dose experiment in ApoE−/− mice, plasma levels of A-001/Varespladib (LY-315920) (A-002 (varespladib methyl/LY333013/S-3013), the active ingredient), were determined. As described above, animals were administered 10, 30, or 90 mg/kg of A-002 (varespladib methyl/LY333013/S-3013) by gavage. Blood samples were collected at different time points using the tail-cutting method. Plasma was collected and stored at −70°C for analysis of the presence of A-001/Varespladib. LY-315920 was analyzed by high-performance liquid chromatography-mass spectrometry (HPLC-MS). Plasma (25 μL) from the treated animals was mixed with 5 μL of 20 μM LY329722 (internal standard), 25 μL of isopropanol, and 50 μL of acetonitrile in a polypropylene microcentrifuge tube. After vortexing, the mixture was incubated at 37°C for 10 min, followed by centrifugation at 12,000 g at room temperature. The supernatant (65 μL) was transferred to an 800 μL glass autosampler vial containing 430 μL of water and 5 μL of formic acid. The vial was capped, vortexed, and placed in a Surveyor autosampler. The sample (25 μL) was injected into a 2.1 × 100 mm, 5 μm, C8 Discovery reversed-phase column equipped with a 2.1 × 4 mm Phenomenex C8 guard column. The initial chromatographic conditions were set as follows: mobile phase A was 95% (0.1% formic acid in 5% isopropanol solution), and mobile phase B was 5% (0.1% formic acid in acetonitrile solution containing 5% isopropanol). After injection, a 6-minute gradient elution was performed, increasing the proportion of mobile phase B from 5% to 60%, resulting in retention times of 6.07 min for A-002 (varespladib methyl/LY333013/S-3013) and 6.47 min for the internal standard. The analytes were detected using electrospray ionization mass spectrometry (ESI). The capillary voltage was 4.5 kV, and the capillary temperature was 310°C. The nitrogen sheath flow rate was set to 75 arbitrary units. The optimized value for collision-induced dissociation (CID) was 35% (normalized). Collision energy was also specified. The selective transitions for A-001/Varespladib (LY-315920) and the internal standard used in tandem mass spectrometry detection originated from the loss of ammonia and carbon monoxide radicals, respectively, with m/z values of 381→336 and 395→350. The peak areas of A-001 and the internal standard were used to calculate the response factor. The response factor for unknown samples was calculated by interpolation using a linear calibration curve of A-001 added to mixed plasma from untreated mice.

[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 belongs to the indole class of compounds with the structure 1H-indole, substituted at positions 1, 2, 3, and 4 with benzyl, ethyl, oxaloyloxy, and carboxymethoxy groups, respectively. It is an orally secreted phospholipase A2 inhibitor with anti-inflammatory activity. It can be used as an EC 3.1.1.4 (phospholipase A2) inhibitor, anti-inflammatory drug, and antidote. It belongs to the indole, benzene, aromatic ether, dicarboxylic acid monoamide, monocarboxylic acid, and primary carboxamide classes. It is the conjugate acid of varespladib (1-). Varespladib has been investigated for the treatment and prevention of sickle cell disease, vascular occlusive crisis, and acute coronary syndrome. LY315920 is a potent, selective recombinant human group IIA non-pancreatic secretory PLA2 (sPLA2) inhibitor. In the chromogenic separase assay, the IC50 of LY315920 inhibiting sPLA2 activity was 9 ± 1 nM or 7.3 × 10⁻⁶ molar fraction, close to the stoichiometric limit of this assay. The true potency of LY315920 was determined by a deoxycholic acid/phosphatidylcholine assay with a molar fraction of 1.5 × 10⁻⁶. LY315920 reduced the activity of pancreatic sPLA2 in human IB group by 40-fold and had no activity against cytoplasmic PLA2 or constitutive and inducible cyclooxygenases. LY315920 inhibited the release of thromboxane A2 (TXA2) from guinea pig bronchoalveolar lavage cells induced by human sPLA2, with an IC50 of 0.79 μM. The release of TXA2 from these cells induced by N-formylmethionylleucylphenylalanine or arachidonic acid was not inhibited. Intravenous injection of LY315920 5 minutes before bronchoalveolar lavage cell collection inhibited sPLA2-induced TXA2 production, with an ED50 of 16.1 mg/kg. Stimulation of pleural strips in guinea pig lungs with sPLA2 elicited a contractile response, which was inhibited by LY315920 in a concentration-dependent manner, with an apparent KB value of 83 ± 14 nM. Arachidonic acid-induced contractile responses were unaffected. Intravenous or oral administration of LY315920 to transgenic mice expressing human sPLA2 protein inhibited serum sPLA2 activity in a dose-dependent manner over 4 hours. LY315920 is a potent and selective sPLA2 inhibitor, representing a new class of anti-inflammatory drugs named SPI. This drug is currently undergoing clinical evaluation and is expected to help elucidate the role of sPLA2 in various inflammatory diseases. [1]

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Objective: How vitamin A maintains the moist phenotype of the ocular surface is unclear. This study aimed to identify vitamin A-responsive genes in ocular surface epithelial cells using gene chip analysis. The study used human conjunctival epithelial (HCjE) cell lines cultured with all-trans retinoic acid (RA). Analysis showed that secretory phospholipase A2 group IIA (sPLA2-IIA) was the most significantly upregulated gene by RA, followed by membrane-associated mucin MUC16, but with a later upregulation. Since arachidonic acid products—arachidic acid—produced by the PLA2 family have been shown to increase mucin production, this study aimed to investigate whether sPLA2 mediates RA-induced MUC16 expression. Methods: HCjE cells were cultured in RA-containing or RA-free media for 3, 6, 24, and 48 hours. Complementary RNA prepared from HCjE cell RNA was hybridized to a human gene chip 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 upregulation of MUC16, HCjE cells were incubated with RA and either the broad-spectrum PLA(2) inhibitor aristolochic acid (ArA) or the specific sPLA(2)-IIA inhibitor LY315920. MUC16 mRNA and protein expression were then analyzed by real-time PCR and Western blot. Results: After RA treatment, 28 transcripts were upregulated at 3 and 6 hours (early stage), and 6 transcripts were downregulated by more than 2-fold (P < 0.01). At 24 and 48 hours (late stage), 80 gene transcripts were upregulated, and 45 gene transcripts were downregulated. sPLA(2) was significantly upregulated at 24 hours in the IIA group, while MUC16 was the most significantly upregulated RNA after 48 hours of RA treatment. Western blot analysis confirmed the upregulatory effect of RA on sPLA(2). When HCjE cells were incubated with RA in combination with ArA or sPLA(2)-IIA specific inhibitor LY315920, RA-induced MUC16 mRNA expression was significantly reduced (P < 0.01). Conclusion: The upregulation of membrane-associated mucin MUC16 induced by RA in the later stage appears to be mediated by sPLA(2)-IIA. This upregulation of hydrophilic membrane-associated mucin may be one of the important mechanisms by which vitamin A maintains the moist phenotype of the ocular surface. [2] Secretory phospholipase A2 (sPLA2) enzyme family is associated with inflammatory diseases and tissue damage, including atherosclerosis. A-001 is a novel sPLA2 enzyme inhibitor discovered through structure-based drug design, while A-002 (varespladib methyl/LY333013/S-3013) is a highly bioavailable oral prodrug currently under clinical development. A-001 inhibits the sPLA2 IIA, V, and X histoenzymes in humans and mice with IC50 values in the low nanomolar range. In transgenic mice overexpressing human sPLA2 IIA in a C57BL/6J background, A-002 (1 mg/kg) increased serum A-001 concentration and inhibited PLA2 activity. In addition, the effect of A-002 on atherosclerosis in two ApoE−/− mouse models was assessed using surface analysis. (1) In a high-fat diet model, A-002 (30 and 90 mg/kg, twice daily for 16 weeks) reduced the degree of aortic atherosclerosis. The degree of atherosclerosis was reduced by 50% (P < 0.05). Plasma total cholesterol was significantly reduced within 1 month (P < 0.05) and remained at a low level throughout the study period. (2) In the accelerated atherosclerosis model induced by angiotensin II in aortic lesions and aneurysms, A-002 (30 mg/kg, twice daily) reduced the degree of aortic atherosclerosis by about 40% (P < 0.05) and alleviated aneurysm formation (P = 0.0096). Therefore, A-002 can effectively and significantly reduce total cholesterol, atherosclerosis and aneurysm formation in these two 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.

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

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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.

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