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 vespladib sodium (10 μM; 24 and 48 hours; HCjE cells) therapy [2]. With an inhibition rate of 100% at 24 hours and 99% at 48 hours, vespladib sodium (10 μM) administration can strongly suppress RA-induced MUC16 expression in HCjE cells [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].

An IC50 of 0.79 μM is observed for the inhibition of human sPLA2-induced thromboxane A2 (TXA2) release from guinea pig lung bronchial lavage cells upon varespladibodium administration. Varespladib sodium has an ED50 of 16.1 mg/kg[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].

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 Cell
Tested Concentrations: 10 μM
Incubation Duration: 24 hrs (hours), 48 hrs (hours)
Experimental Results: RA-induced MUC16 protein expression was Dramatically inhibited at both time points.

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

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

[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. 1.

[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.

Varespladib sodium is an intravenously administered secretory phospholipase A2 (sPLA2) inhibitor with orphan drug designation. Varespladib sodium is currently undergoing clinical trials for the prevention of acute chest pain syndrome in the treatment of sickle cell disease. Varespladib sodium belongs to the indole class of compounds with the structure 1H-indole, substituted at positions 1, 2, 3, and 4 by benzyl, ethyl, oxalamido, and carboxymethoxy groups, respectively. It is an orally administered secretory phospholipase A2 inhibitor with anti-inflammatory effects. 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 and selective inhibitor of recombinant human group IIA non-pancreatic secretory PLA2 (sPLA2). In a chromogenic dissociation enzyme assay, LY315920 showed an IC50 of 9 ± 1 nM or 7.3 × 10⁻⁶ molar fraction for inhibiting sPLA2 activity, 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 group IB by 40-fold and showed no activity against cytoplasmic PLA2, constitutive, or inducible cyclooxygenases. LY315920 inhibited the release of thromboxane A2 (TXA2) from bronchoalveolar lavage cells induced by human sPLA2, with an IC50 of 0.79 μM. N-formylmethionylleucylphenylalanine or arachidonic acid-induced TXA2 release from these cells 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. The contractile response induced by arachidonic acid was 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 genes in ocular surface epithelial cells that respond to vitamin A using gene chip analysis. The study subjects were human conjunctival epithelial (HCjE) cell lines cultured with all-trans retinoic acid (RA). The results showed that secretory phospholipase A2 group IIA (sPLA2-IIA) was the most significantly upregulated gene by RA, followed by membrane-associated mucin MUC16, but the upregulation time was later. Since arachidonic acid products produced by the PLA2 family—arachidic acid—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 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 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 the ArA or sPLA(2)-IIA specific inhibitor LY315920, RA-induced MUC16 mRNA 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 promotes the maintenance of the ocular surface moist phenotype. [2]

C21H19N2NAO5

402.375736474991

402.119

C, 62.68; H, 4.76; N, 6.96; Na, 5.71; O, 19.88

172733-42-5

Varespladib;172732-68-2

23674730

Off-white to light yellow solid powder

1.749

1

5

8

29

596

0

CCC1=C(C2=C(N1CC3=CC=CC=C3)C=CC=C2OCC(=O)[O-])C(=O)C(=O)N.[Na+]

XZZUHXILQXLTGV-UHFFFAOYSA-M

InChI=1S/C21H20N2O5.Na/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);/q;+1/p-1

sodium;2-(1-benzyl-2-ethyl-3-oxamoylindol-4-yl)oxyacetate

Varespladib sodium; 172733-42-5; UNII-F6M52CDT0W; LY315920-Na; Varespladib Sodium [USAN]; LY315920-Sodium salt; F6M52CDT0W; Varespladib (sodium);

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Note: Please store this product in a sealed and protected environment, avoid exposure to moisture.

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

May dissolve in DMSO (in most cases), if not, try other solvents such as H2O, Ethanol, or DMF with a minute amount of products to avoid loss of samples

Note: Listed below are some common formulations that may be used to formulate products with low water solubility (e.g. < 1 mg/mL), you may test these formulations using a minute amount of products to avoid loss of samples.

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Injection Formulation 1: DMSO : Tween 80： Saline = 10 : 5 : 85 (i.e. 100 μL DMSO stock solution → 50 μL Tween 80 → 850 μL Saline)
*Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH ₂ O to obtain a clear solution.
Injection Formulation 2: DMSO : PEG300 ：Tween 80 : Saline = 10 : 40 : 5 : 45 (i.e. 100 μL DMSO → 400 μLPEG300 → 50 μL Tween 80 → 450 μL Saline)
Injection Formulation 3: DMSO : Corn oil = 10 : 90 (i.e. 100 μL DMSO → 900 μL Corn oil)
Example: Take the Injection Formulation 3 (DMSO : Corn oil = 10 : 90) as an example, if 1 mL of 2.5 mg/mL working solution is to be prepared, you can take 100 μL 25 mg/mL DMSO stock solution and add to 900 μL corn oil, mix well to obtain a clear or suspension solution (2.5 mg/mL, ready for use in animals).

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Injection Formulation 4: DMSO : 20% SBE-β-CD in saline = 10 : 90 [i.e. 100 μL DMSO → 900 μL (20% SBE-β-CD in saline)]
*Preparation of 20% SBE-β-CD in Saline (4°C，1 week): Dissolve 2 g SBE-β-CD in 10 mL saline to obtain a clear solution.
Injection Formulation 5: 2-Hydroxypropyl-β-cyclodextrin : Saline = 50 : 50 (i.e. 500 μL 2-Hydroxypropyl-β-cyclodextrin → 500 μL Saline)
Injection Formulation 6: DMSO : PEG300 : castor oil : Saline = 5 : 10 : 20 : 65 (i.e. 50 μL DMSO → 100 μLPEG300 → 200 μL castor oil → 650 μL Saline)
Injection Formulation 7: Ethanol : Cremophor : Saline = 10： 10 : 80 (i.e. 100 μL Ethanol → 100 μL Cremophor → 800 μL Saline)
Injection Formulation 8: Dissolve in Cremophor/Ethanol (50 : 50), then diluted by Saline
Injection Formulation 9: EtOH : Corn oil = 10 : 90 (i.e. 100 μL EtOH → 900 μL Corn oil)
Injection Formulation 10: EtOH : PEG300：Tween 80 : Saline = 10 : 40 : 5 : 45 (i.e. 100 μL EtOH → 400 μLPEG300 → 50 μL Tween 80 → 450 μL Saline)

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Oral Formulation 1: Suspend in 0.5% CMC Na (carboxymethylcellulose sodium)
Oral Formulation 2: Suspend in 0.5% Carboxymethyl cellulose
Example: Take the Oral Formulation 1 (Suspend in 0.5% CMC Na) as an example, if 100 mL of 2.5 mg/mL working solution is to be prepared, you can first prepare 0.5% CMC Na solution by measuring 0.5 g CMC Na and dissolve it in 100 mL ddH2O to obtain a clear solution; then add 250 mg of the product to 100 mL 0.5% CMC Na solution, to make the suspension solution (2.5 mg/mL, ready for use in animals).

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Oral Formulation 3: Dissolved in PEG400
Oral Formulation 4: Suspend in 0.2% Carboxymethyl cellulose
Oral Formulation 5: Dissolve in 0.25% Tween 80 and 0.5% Carboxymethyl cellulose
Oral Formulation 6: Mixing with food powders

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Note: Please be aware that the above formulations are for reference only. InvivoChem strongly recommends customers to read literature methods/protocols carefully before determining which formulation you should use for in vivo studies, as different compounds have different solubility properties and have to be formulated differently.

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