LTB4 receptor/BLT2 (IC50 = 100 nM)

The contractile response of lung parenchyma to LTB is competitively reduced by LY255283 (pA2=7.2)[2]. Significant inhibition of hyperinsulinemic 253 J-BV reversal of insulin resistance is shown with LY255283 (10 μM, 7 days) [4].

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Leukotriene B4 (LTB4) induces a number of functional changes in human neutrophils, including both superoxide release and CD11b/CD18 (Mo1)-mediated adherence to various substrates, such as keyhole limpet hemocyanin (KLH). These effects are both time- and concentration-dependent. Neutrophil adhesion was at least 10-fold more sensitive to the stimulatory action of LTB4 than superoxide production. Two LTB4 receptor antagonists, LY255283 (1-(5-ethyl-2-hydroxy-4-(6-methyl-6-(1H-tetrazol-5-yl)heptyloxy )- phenyl)ethanone) and the sodium salt of SC-41930 (7-[3-(4-acetyl-3-methoxy-2-propylphenoxy)-propoxy]-3,4-dihydro-8- propyl-2H- 1-benzopyran-2-carboxylic acid) were evaluated for effects on human neutrophil superoxide production and adhesion. Despite being more sensitive to LTB4-induced stimulation, neutrophil adhesion was at least 100-fold less sensitive to inhibition by LY255283 and SC-41930 than superoxide production. Both LTB4 receptor antagonists behaved similarly in these models. These compounds did not inhibit neutrophil responses induced by granulocyte/macrophage colony-stimulating factor (GM-CSF).[1]

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The actions of LY255283, a leukotriene (LT) B4 receptor antagonist, were examined on guinea pig lung. LTB4 and LY255283 displaced [3H]LTB4 from its binding site on lung membranes with pKi values of 9.9 and 7.0, respectively. In the functional correlate of the binding studies, LY255283 competitively reduced contractile responses of lung parenchyma to LTB4 (pA2 = 7.2). [2]

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In this study, immunohistochemical examination showed that the leukotriene B(4) receptor BLT2 is overexpressed in advanced malignant bladder cancers (human transitional cell carcinomas) in proportion to advancing stages, with high prognostic significance (p<0.001). Blockade of BLT2 with the specific antagonist LY255283 or siRNA knockdown significantly suppressed the invasiveness of highly aggressive 253J-BV bladder cancer cells [4].
In addition, the numbers of invasive 253 J-BV cells and the maximal invasion distances were higher than those of MCF-10A and SV-HUC-1 cells and were markedly reduced by treatment with LY255283, but not U75302 (Fig. 3A, right), suggesting that the loss of BLT2 signaling reduces the invasive potential of aggressive bladder cancer cells. Moreover, when we examined transmigration toward LTB4 and 12(S)-HETE, two BLT2 ligands known to be chemotactic factors [20], the chemotactic migration of 253 J-BV cells was significantly enhanced by LTB4 or 12(S)-HETE and blocked by treatment with LY255283 or siBLT2, but to a much lesser extent than with U75302 (Fig. 3B). Interestingly, the basal activity of 253 J-BV cells was significantly inhibited by treatment with LY255283 alone, i.e., without LTB4 or 12(S)-HETE; this is suggestive of autonomous basal activation of BLT2 signaling [4].

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NF-κB is a downstream target of the BLT2–ROS-linked cascade in 253 J-BV cells [4]
NF-κB and AP-1 are well-defined redox-regulated transcription factors that drive the transcription of a number of invasive genes during cancer progression. We used EMSA and immunofluorescence assays to investigate whether activation of NF-κB or AP-1 lies downstream of BLT2–Nox–ROS-linked signaling. LY255283 and DPI treatment of 253 J-BV cells inhibited NF-κB DNA binding activity (Fig. 5A). Also, we found that highly aggressive 253 J-BV cells showed intense nuclear fluorescence, reflecting the nuclear translocation of the NF-κB p65 subunit. Additionally, pretreatment with LY255283, but not U75302, markedly reduced nuclear levels of p65 NF-κB (Fig. 5B). In contrast, c-Jun was unaffected, indicating that BLT2 does not play a role in AP-1 signaling (data not shown). In addition, pretreatment with DPI (Fig. 5B) also significantly reduced NF-κB activation. Taken together, our results suggest that NF-κB lies downstream of the BLT2–Nox1/4–ROS cascade in highly aggressive 253 J-BV bladder cancer cells. Moreover, treatment with four different NF-κB inhibitors (SN-50, PDTC, Bay11-7082 or Bay11-7085) attenuated the invasiveness of 253 J-BV cells (Fig. 5C).

Pigs' lipopolysaccharide sensor ARDS is improved by LY255283 (3, 30 mg/kg), maybe as a result of PMN recruitment that ignites activators into the alveoli in a way similar to dancing anesthesia [3]. Results from LY255283 (2.5 mg/kg, ip) suggest that bladder cancer is largely caused by the BLT2-Nox-ROS-NF-κB cascade [4].

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LTB4 produced airway obstruction which was reduced by LY255283 administered i.v. (ED50 = 2.8 mg/kg) or orally (ED50 = 11.0 mg/kg). Contractile responses to histamine, LTD4 and the thromboxane mimetic, U46619, were not reduced by LY255283. The compound also did not inhibit cyclooxygenase or 5-lipoxygenase enzymes. We conclude that LY255283 selectively antagonized pharmacologic responses to LTB4 on lung tissue and appears to be a useful tool to investigate the role of LTB4 in pulmonary disease.[2]

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In control pigs, lipopolysaccharide induced hypoxemia, pulmonary hypertension, and neutrophil activation (increased CORE/MORE ratio). These changes were attenuated by LY255283, particularly when pigs were infused with the higher dose of the compound. The drug also blunted lipopolysaccharide-induced recruitment of PMNs in pulmonary air spaces, as assessed by bronchoalveolar lavage performed at 240 minutes, although the degree of pulmonary leukosequestration caused by lipopolysaccharide was not affected. Conclusions: In a dose-dependent fashion, LY255283 ameliorated lipopolysaccharide-induced ARDS in pigs, possibly by blocking the recruitment of activated PMNs into alveoli [3].

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BLT2 signaling is critical for metastatic colonization of highly aggressive bladder cancer cells [4]
We next used an assay called experimental metastasis to evaluate the in vivo effects of the depletion of BLT2 signaling on metastasis. We injected 1 × 106 untreated 253 J-BV cells or cells pretreated with LY255283 or U75302 into the lateral tail vein of 5-week-old athymic mice and then determined the number and size of metastatic nodules formed in the lung. Doses of 0.25 mg/kg U75302 or 2.5 mg/kg LY255283, which are similar to doses used previously, were administered intraperitoneally 3 and 5 days after injection of the 253 J-BV cells. By 12 weeks after injection, untreated tumor cells had formed 12–18 metastatic nodules per lung in all mice analyzed, and similar numbers of nodules were found in mice treated with U75302. In contrast, in mice treated with LY255283 only 0–3 nodules formed per lung (Fig. 6A, top), and histological analysis confirmed that the number of micrometastatic lesions was markedly reduced (Fig. 6A, bottom).

Cell viability assay[4]
Cell Types: 253 J-BV cells.
Tested Concentrations: 5 or 10 μM.
Incubation Duration: 7 days.
Experimental Results: Inhibiting BLT2 signaling attenuated the invasive migration of 253 J-BV cells.

Animal/Disease Models: Mice (injected with 253 J-BV cells) [4].
Doses: 2.5 mg/kg.
Route of Administration: IP injection 3 and 5 days after cell injection.
Experimental Results: Twelve weeks after injection, mice treated with LY255283 developed only 0-3 nodules per lung, and histological analysis confirmed a significant reduction in the number of micrometastatic lesions.

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Experimental and spontaneous metastasis assays and morphological and histological analyses [4]
Male nude mice were inoculated between 5 and 8 weeks of age for experimental or spontaneous metastasis assays. Cultured 253 J-BV cells (1 × 106 cells) were pretreated with BLT antagonists for 24 h to ensure the inhibition of BLT signaling and then briefly treated with 0.025% trypsin and 0.1% EDTA in Hanks’ balanced salt solution (HBSS). The cells were then resuspended in HBSS and, within 1 h, were injected in a 0.1-ml volume into the lateral tail vein using a 30-gauge needle. For inhibitor experiments, dimethyl sulfoxide (DMSO), 0.25 mg/kg U75302, or 2.5 mg/kg LY255283 was intraperitoneally injected 3 and 5 days after injection of cells. The mice were maintained under aseptic barrier conditions until sacrifice 12 weeks after cell injection (n = 3 in each group). To identify experimental pulmonary metastases, the number of lung surface metastasis nodules larger than 0.2 mm in diameter was counted after euthanasia.
To assay spontaneous metastasis, mice were anesthetized with ketamine and xylazine, after which a lower midline incision was made, and viable tumor cells (2 × 106 cells in 0.05 ml) in HBSS were implanted in the bladder wall. The formation of a bulla indicated a satisfactory injection. The bladder was then returned to the abdominal cavity, and the abdominal wall was closed with a single layer of metal clips. For inhibitor experiments, 14 days after the surgery DMSO or the aforementioned dose of LY255283 or U75302 (n = 4 for each group) was injected intraperitoneally three times with intervals of 5 days between injections. The mice were killed and necropsied 9 weeks after tumor cell implantation. The primary tumors were removed and weighed, and the presence of metastases (liver) was determined grossly and microscopically. The livers and bladders were dissected and fixed in 4% formalin, processed, and embedded in paraffin.

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Eighteen hours before being studied, pigs were injected with lipopolysaccharide (20 micrograms/kg). From 0 to 60 minutes, pigs received either Ringer's lactate solution (n = 5) or lipopolysaccharide (250 micrograms/kg). Among the pigs that were infused with lipopolysaccharide, nine received no other treatment, six received a low dose of LY255283 (30 mg/kg loading dose; 3 mg/kg-hr infusion), and six received a high dose of LY255283 (30 mg/kg loading dose; 30 mg/kg-hr). In vivo PMN activation was assessed with an automated chemiluminescence assay wherein results are expressed as CORE/MORE (i.e., the ratio of complement-opsonized zymosan receptor expression on circulating cells [CORE] divided by the maximal complement-opsonized zymosan receptor expression induced by incubating the cells in vitro with LTB4 or platelet-activating factor [MORE]). [3]

[1]. Effects of two leukotriene B4 (LTB4) receptor antagonists (LY255283 and SC-41930) on LTB4-induced human neutrophil adhesion and superoxide production. Prostaglandins Leukot Essent Fatty Acids. 1991 Aug;43(4):267-71.

[2]. Pulmonary actions of LY255283, a leukotriene B4 receptor antagonist. Eur J Pharmacol. 1992 Nov 13;223(1):57-64.

[3]. LY255283, a novel leukotriene B4 receptor antagonist, limits activation of neutrophils and prevents acute lung injury induced by endotoxin in pigs. Surgery. 1993 Aug;114(2):191-8.

[4]. BLT2 promotes the invasion and metastasis of aggressive bladder cancer cells through a reactive oxygen species-linked pathway. Free Radic Biol Med. 2010 Sep 15;49(6):1072-81.

1-[5-ethyl-2-hydroxy-4-[6-methyl-6-(2H-tetrazol-5-yl)heptoxy]phenyl]ethanone is an aromatic ketone.

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Background: Polymorphonuclear neutrophils (PMNs) have been implicated in the pathogenesis of the adult respiratory distress syndrome (ARDS). Because leukotriene B4 (LTB4) is a potent activator of PMNs, we sought to determine whether LY255283, an LTB4 receptor antagonist, could block PMN activation and lung injury in a porcine model of lipopolysaccharide-induced ARDS. [3]

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Aggressive bladder cancer is a major cause of morbidity and mortality. Despite the fact that metastatic disease results in death in the majority of bladder cancer cases, the molecular events regulating the invasive phenotype of aggressive bladder cancer are not well understood. In this study, immunohistochemical examination showed that the leukotriene B(4) receptor BLT2 is overexpressed in advanced malignant bladder cancers (human transitional cell carcinomas) in proportion to advancing stages, with high prognostic significance (p<0.001). Blockade of BLT2 with the specific antagonist LY255283 or siRNA knockdown significantly suppressed the invasiveness of highly aggressive 253J-BV bladder cancer cells. Moreover, our results demonstrated that BLT2 mediates invasiveness through a signaling pathway dependent on NAD(P)H oxidase (Nox) 1- and Nox4-induced generation of reactive oxygen species (ROS) and subsequent NF-kappaB stimulation. Metastasis of 253J-BV cells in mice was also dramatically suppressed by inhibition of BLT2 or its signaling. These findings suggest that a BLT2-Nox-ROS-NF-kappaB cascade plays a critical role in bladder cancer invasion and metastasis. [4]

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We further confirmed the effect of BLT2 inhibition on the metastatic phenotype of aggressive bladder cancer cells by carrying out orthotopic metastasis assays. For these assays, we initially injected 253 J-BV cells into the bladder wall, as described under Materials and methods. Beginning 14 days later, three intraperitoneal injections of DMSO, LY255283, or U75302 (n = 4 per group) were administered with 5-day intervals between injections. We then analyzed tumor growth and the metastatic phenotype. As expected, all four mice treated with LY255283 showed a great reduction in metastasis (Fig. 6B). This is in contrast to findings in the untreated and U75302-treated mice, which developed large tumors in their bladders within 9 weeks as well as small metastatic nodules (< 0.2 mm diameter) in their livers (Fig. 6B). Taken together, these in vivo findings further confirm that BLT2 signaling plays a critical role in the metastasis of 253 J-BV bladder cancer cells.
TCC of the urinary bladder is responsive to conventional chemotherapeutic agents; however, the response is often short-lived, as chemoresistance can develop rapidly. Despite an initial chemotherapeutic response, most patients with advanced or metastatic TCC of the bladder die from progression of their disease (median survival, < 2 years). Thus, the development of new therapeutic agents that improve the outcome for patients with advanced bladder cancer is urgently needed. Here we show that a BLT2-linked cascade is critical for invasion and for the metastatic phenotype of aggressive bladder cancer. Notably, expression of BLT2, but not BLT1, was elevated in proportion to the tumor stage in bladder cancer specimens and metastatic bladder cancer cells. In addition, the level of BLT2 expression had high prognostic significance (p < 0.001). The fact that inhibition of BLT2 signaling by LY255283 or siBLT2 suppressed the invasive and metastatic potential in highly metastatic bladder cancer 253 J-BV cells (Fig. 2, Fig. 3, Fig. 6) suggests that the BLT2-linked cascade may be specifically required for invasion and metastasis in advanced bladder cancers in vivo [3].

C19H28N4O3

360.450624465942

360.216

C, 63.31; H, 7.83; N, 15.54; O, 13.32

117690-79-6

122023

Off-white to light yellow solid powder

1.2±0.1 g/cm3

573.4±60.0 °C at 760 mmHg

160-162 °C

300.6±32.9 °C

0.0±1.6 mmHg at 25°C

1.553

4.04

2

6

10

26

447

0

O(C1C=C(C(C(C)=O)=CC=1CC)O)CCCCCC(C1N=NNN=1)(C)C

WCGXJPFHTHQNJL-UHFFFAOYSA-N

InChI=1S/C19H28N4O3/c1-5-14-11-15(13(2)24)16(25)12-17(14)26-10-8-6-7-9-19(3,4)18-20-22-23-21-18/h11-12,25H,5-10H2,1-4H3,(H,20,21,22,23)

1-[5-ethyl-2-hydroxy-4-[6-methyl-6-(2H-tetrazol-5-yl)heptoxy]phenyl]ethanone

LY-255283; LY255283; LY255,283; LY-255,283; LY 255,283; UNII-H037W1I5AL; (1-(5-Ethyl-2-hydroxy-4-(6-methyl-6-(1H-tetrazol-5-yl)heptyloxy)phenyl)ethanone); DTXSID30151872; CGS 23356; ...; 117690-79-6; LY 255283

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 (e.g. under nitrogen), 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)

DMSO : ~100 mg/mL (~277.43 mM)

Solubility in Formulation 1: 2.5 mg/mL (6.94 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: ≥ 2.5 mg/mL (6.94 mM) (saturation unknown) in 10% DMSO + 90% (20% SBE-β-CD in Saline) (add these co-solvents sequentially from left to right, and one by one), clear solution.
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 900 μL of 20% SBE-β-CD physiological saline solution and mix evenly.
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.

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Solubility in Formulation 3: ≥ 2.5 mg/mL (6.94 mM) (saturation unknown) in 10% DMSO + 90% Corn Oil (add these co-solvents sequentially from left to right, and one by one), clear solution.
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 900 μL of corn oil and mix evenly.

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