5-LO

5-Lipoxygenase (5-LO) is a crucial enzyme in the synthesis of the bioactive leukotrienes (LTs) from arachidonic acid (AA), and inhibitors of 5-LO are thought to prevent the untowarded pathophysiological effects of LTs. In this study, we present the molecular pharmacological profile of the novel nonredox-type 5-LO inhibitor CJ-13,610 that was evaluated in various in vitro assays. In intact human polymorphonuclear leukocytes (PMNL), challenged with the Ca(2+)-ionophore A23187, CJ-13,610 potently suppressed 5-LO product formation with an IC(50)=0.07 microm. Supplementation of exogenous AA impaired the efficacy of CJ-13,610, implying a competitive mode of action. In analogy to ZM230487 and L-739.010, two closely related nonredox-type 5-LO inhibitors, CJ-13,610 up to 30 microm failed to inhibit 5-LO in cell-free assay systems under nonreducing conditions, but inclusion of peroxidase activity restored the efficacy of CJ-13,610 (IC(50)=0.3 microm). In contrast to ZM230487 and L-739.010, the potency of CJ-13,610 does not depend on the cell stimulus or the activation pathway of 5-LO. Thus, 5-LO product formation in PMNL induced by phosphorylation events was equally suppressed by CJ-13,610 as compared to Ca(2+)-mediated 5-LO activation. In transfected HeLa cells, CJ-13,610 only slightly discriminated between phosphorylatable wild-type 5-LO and a 5-LO mutant that lacks phosphorylation sites. In summary, CJ-13,610 may possess considerable potential as a potent orally active nonredox-type 5-LO inhibitor that lacks certain disadvantages of former representatives of this class of 5-LO inhibitors. [1]

Determination of 5-LO product formation in cell-free systems [1]
For the determination of 5-LO activity in cell homogenates, 7.5 × 106 freshly isolated PMNL were resuspended in PBS containing 1 mM EDTA, sonicated (3 × 10 s) at 4°C, and 1 mM ATP was added. For determination of the activity of recombinant isolated 5-LO, partially purified 5-LO (0.5 μg in 5 μl) was added to 1 ml of a 5-LO reaction mix (PBS, pH 7.4, 1 mM EDTA, 25 μg ml−1 phosphatidylcholine, 1 mM ATP, and 20 μg ml−1 γ-globulin). Samples of either cell homogenates or partially purified 5-LO were supplemented with DTT (1 mM), GSH (1 mM), GPx-1 (30 mU), and CJ-13,610 as indicated. After 5–10 min at 4°C, samples were prewarmed for 30 s at 37°C and 2 mM CaCl2 and AA at the indicated concentrations were added to start 5-LO product formation. The reaction was stopped after 10 min at 37°C by the addition of 1 ml ice-cold methanol and the formed metabolites were analyzed by HPLC as described for intact cells.

Determination of 5-LO product formation in intact cells [1]
For assays of intact cells, 7.5 × 106. freshly isolated PMNL or 2 × 106 HeLa cells were finally resuspended in 1 ml PGC buffer. After preincubation with the indicated compounds at 37°C, 5-LO product formation was started by the addition of the indicated stimuli plus exogenous AA as indicated. After 10 min at 37°C, the reaction was stopped with 1 ml of methanol and 30 μl of 1 N HCl, 200 ng prostaglandin B1, and 500 μl of PBS were added. Formed 5-LO metabolites were extracted and analyzed by HPLC as described (Werz & Steinhilber, 1996). 5-LO product formation is expressed as ng of 5-LO products per 106 cells that includes LTB4 and its all-trans isomers, 5(S),12(S)-di-hydroxy-6,10-trans-8,14-cis-eicosatetraenoic acid (5(S),12(S)-DiHETE), and 5(S)-hydro(pero)xy-6-trans-8,11,14-cis-eicosatetraenoic acid (5-H(p)ETE). Cysteinyl LTs (LTC4, D4, and E4) were not detected and oxidation products of LTB4 were not determined.

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Subcellular localization of 5-LO [1]
Subcellular localization of 5-LO was investigated as described previously (Werz et al., 2002a). In brief, freshly isolated PMNL (3 × 107) in 1 ml PGC buffer were incubated at 37°C for 10 min with the indicated stimuli and chilled on ice. Nuclear and non-nuclear fractions were obtained after cell lysis by 0.1% NP-40. Aliquots of these fractions were analyzed for 5-LO protein by SDS–PAGE and immunoblotting using anti-5-LO antiserum (AK7, 1551; affinity purified on a 5-LO column). Proteins were visualized by alkaline phosphatase-conjugated IgGs using nitroblue tetrazolium and 5-bromo-4-chloro-3-indolylphosphate as substrates.

[1]. Molecular pharmacological profile of the nonredox-type 5-lipoxygenase inhibitor CJ-13,610. Br J Pharmacol. 2004;142(5):861-868.

Intracellular 5-lipoxygenase (5-LO) is activated in response to external stimuli, a process involving the translocation of 5-LO from its soluble region to the nuclear membrane, where it co-localizes with FLAP (Peters-Golden & Brock, 2001). This process is mediated by increased intracellular Ca2+ levels and/or phosphorylation of 5-LO by mitogen-activated protein kinase-activated protein kinase (MAPKAP) at Ser-271 and by ERK1/2 at Ser-663 (Werz et al., 2000; 2002a, 2002b). Studies have shown that in certain cell types, 5-LO activated by stimuli inducing phosphorylation is Ca2+-independent (Werz et al., 2002a; Burkert et al., 2003). The potency of the non-redox 5-lipoxygenase (5-LO) inhibitors ZM230487 and L-739.010 depends on the stimulatory and activation pathways that induce 5-LO product synthesis (Fischer et al., 2003). ZM230487 and L-739.010 effectively inhibit Ca2+-mediated 5-LO activation in polymorphonuclear leukocytes (PMNLs), but inhibition of phosphorylation-induced 5-LO product synthesis requires 10 to 100-fold higher inhibitor concentrations (Fischer et al., 2003). Conversely, in intact PMNLs, the potency of CJ-13,610 is not diminished when 5-LO is activated by phosphorylation. Furthermore, CJ-13,610 showed no significant difference in potency against phosphorylated wild-type or non-phosphorylated mutant S217A/S663A-5-LO in HeLa cells. It is noteworthy that some diseases associated with elevated 5-LO product levels, such as inflammatory responses, allergic asthma, various cancers, and atherosclerosis, are associated with elevated cellular phosphorylation status (Hajjar & Pomerantz, 1992; Johnson & Lapadat, 2002), and phosphorylation status determines the sensitivity of 5-LO to non-redox inhibitors. Since the efficacy of CJ-13,610 does not depend on the phosphorylation status of 5-LO, targeting 5-LO with this drug may indeed be beneficial for the treatment of these diseases. Although the therapeutic potential of CJ-13,610 may be somewhat limited by elevated peroxide levels, thus its efficacy is somewhat limited, this compound is clearly superior to previous representatives of such 5-LO inhibitors, which have failed to obtain routine approval due to poor clinical efficacy. [1]

C22H23N3O2S

393.51

393.151

C, 67.15; H, 5.89; N, 10.68; O, 8.13; S, 8.15

179420-17-8

179420-27-0

9821945

White to off-white solid powder

198 - 200 °C

5.015

1

4

5

28

532

0

VPTONMHDLLMOOV-UHFFFAOYSA-N

InChI=1S/C22H23N3O2S/c1-16-24-11-12-25(16)18-5-7-19(8-6-18)28-20-4-2-3-17(15-20)22(21(23)26)9-13-27-14-10-22/h2-8,11-12,15H,9-10,13-14H2,1H3,(H2,23,26)

4-[3-[4-(2-methylimidazol-1-yl)phenyl]sulfanylphenyl]oxane-4-carboxamide

179420-17-8; CJ-13610; CJ-13,610; CJ 13610; 4-(3-((4-(2-Methyl-1H-imidazol-1-yl)phenyl)sulfanyl)phenyl)tetrahydro-2H-pyran-4-carboxamide; CHEMBL195309; 2H-Pyran-4-carboxamide, tetrahydro-4-[3-[[4-(2-methyl-1H-imidazol-1-yl)phenyl]thio]phenyl]-; 5275PJ1C59;

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)

DMSO : ~10 mg/mL (~25.41 mM)

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.)