生物活性

U-73122 is an inhibitor of phospholipase C, phospholipase A2, and 5-LO (5-lipoxygenase). Inhibits agonist-induced platelet aggregation with IC50 values of 1-5 μM. This compound affects the phospholipases by inhibiting the hydrolysis of PPI (phosphatidylinositol) to IP3 (inositol triphosphate), which in turn leads to a drop in cytosolic Ca2+. In SK-N-SH neuroblastoma cells, U-73122 inhibits agonist-induced down-regulation of muscarinic receptors. Potently inhibits human polymorphonuclear neutrophil adhesion on biological surfaces (IC50 < 50 nM) and exhibits antinociceptive activity in vivo. Activates TRPM4 and inhibits TRPM3 channels. In addition, it is a useful tool to investigate receptor-mediated PI turnover in signal transduction. U-73122 is a potent inhibitor of human neutrophil adhesion to biological surfaces (IC50 = 50 nM) as well as adhesion-dependent granule exocytosis and oxidative burst. U-73343 is useful as a negative control for investigations of U-73122 phospholipase C antagonism and its cellular consequences.

体外研究

体内研究

激酶实验

Mixed Micellar Phospholipase C (PLC) Assay[1]

The activity of hPLCs in cell-free systemswas evaluated. Briefly, for Dodecyl maltoside (DDM) mixed micellar assay ³H-labeled(~30,000 dpm) and unlabeled PIP₂ (50 μM)were reconstituted in a 1mM DDM solution (final assay concentration 0.5mM) andmixed with assay buffer containing 40 mM HEPES (pH 7.4), 480 mM KCl, 40 mMNaCl, 8 mM EGTA, 4 mM MgCl₂, and 7.6 mM CaCl₂ in a finalvolume of 100 μl. Compounds at desired concentrations, and purified hPLC3 in 1% fattyacid-free BSA and 10 mM HEPES (pH 7.0), were subsequently added to the mixture.To initiate assays, samples were moved to a 37 °C water bath and incubated for2–10 min such that less than 15% of total substrate was hydrolyzed. Atdesignated times, reactions were stopped by addition of 750 μlof CHCl₃/MeOH/HCl (40:80:1), followed by the addition of 100μlof water, 250 μl of CHCl3, and 250 μl of 0.1 M HCl. Samples were vortexed and centrifuged at 3000 rpmfor 10 min at 4 °C. The amount of [³H] inositol phosphates formedwas measured by liquid scintillation counting of the upper phase (500 μl)in a Packard Tri Carb 4000 Series spectrophotometer.

For experiments in a cholate-mixed micellarassay, purified PLC isozymes were incubated with 10μM U73122 inthe presence of 3 μM cholate for 10 min at 32 °C. Subsequently, this mixture was addedto [³H] PIP₂ (30,000 dpm) and unlabeled PIP₂ (50μM) that were dried and resuspended in 0.5% cholate for a finalvolume of 60μl. After incubation at 32°C at time intervals between 0 and 10 min,reactions were stopped by the addition of 200μl of 10%(v/v) trichloroacetic acid and 100μl of10 mg/ml BSA to precipitateuncleaved lipids and protein. After centrifugation of the reaction mixture,soluble [³H] inositol phosphates in the supernatant were quantifiedusing liquid scintillation counting.

细胞实验

Cell cultures[2]

Human promonocytic cell line U937 cells werecultured in RPMI 1640 medium containing 10% FBS. The cells were maintained at37 °C in a 5% CO₂ humidified incubator. U937 cells were in vitrodifferentiated into macrophages dU937 by induction withphorbol-12-myristate-13-acetate (PMA) at a concentration of 100 nM for 48 h.

Peritoneal macrophages from ICR mice platedat ~3 × 10⁶ cells/well in 6-well plates were cultured overnight at37 °C and subsequently washed three times with PBS to remove nonadherent cells.The attached cells were used as peritoneal macrophages.

Adhesionof U937 cells induced by PMA

Monocytes U937 seeded in 24-well plates wereinduced into macrophages using PMA along with the treatment of DMSO solvent orPLC inhibitor U73122 at indicated concentrations for 48 h. After extensive washingwith PBS, the pictures of adherent cells were taken by light microscopy. Theuntreated U937 cells were collected by spin down and suspended with 1640 medium,and used for the control of undifferentiated cells. Adherent cells were furtherqualitatively analyzed with MTT assay. U937 cells seeded in 96-well plates weretreated with PMA in the presence of DMSO solvent or PLC inhibitor U73122 atindicated concentrations for 48 h. After three washings with PBS the adherentcells were qualitatively evaluated with MTT assay. The mean optical density ofthe PMA treatment wells was assigned as a value of 100%.

Flowcytometry assay

U937 cells seeded in 6-well plates weretreated with PMA in the presence of DMSO solvent or PLC inhibitor U73122 atindicated concentrations for 48 h. Adherent cells were detached with 2mM EDTA.Cells of both suspended and adherent were stained with antibody CD163-FITC andsubjected to flow cytometry analysis on FACS Calibur. The data was analyzedwith software WinMDI version 2.9.

Westernblot analysis

U937 cells in 60-mmdishes were inducedintomacrophages by PMA (100 nM) in the absence or presence of U73122 at variousconcentrations of 0.1, 1 and 5 μM. At 48 h post-stimulation, both attached andunattached cells were lysed using lysis buffer. The cell lysates were collectedand boiled for 10 min and stored at −80 °C for further analysis. Cell lysateswere separated on 10% SDS-polyacrylamide gels and transferred to apolyvinylidene difluoride (PVDF) membrane. After blocking with 5% nonfat milkin Tris-buffered saline (TBS) buffer containing 0.05% Tween 20, the membrane wasincubated with respective primary antibodies, followed by HRP-conjugatedsecondary antibodies in the blocking reagent. After extensive washing withTBST, immune reactive bands were analyzed by film exposure after enhancedchemiluminescence (ECL) reaction. Densitometry analysis was performed with ImageJ 1.45 s ,with results expressed as relative fold change vs. negative controlband.

动物实验

Animals[3]

The animals were maintained on a 12-hlight/dark cycle, the room temperature was set at 64°-84°F, and the humidityset at 30 to 70%. They were fed food and water ad libitum.

Carrageenan-Induced Paw Edema in Rats

Male Sprague- Dawley rats (Ace Animals),weighing ca. 200 g, were fasted overnight. Then, 100μl of a 0.5%carrageenan solution was injected into the subplantar tissue of one hind paw 1h after drug or vehicle pretreatment. Paw volume displacement was measuredusing a mercury plethysmograph at 1, 3, and 5 h after induction ofinflammation, and the edema was expressed as an increase in paw volume due tocarrageenan injection. Indomethacin (10 mg/kg p.o.), a cyclooxygenase (COX)inhibitor, was used as a control.

LPS-Induced Peritonitis and MacrophageAccumulation in Mice

Swiss-Webster mice (ACE Animals) were dosedintraperitoneally with test compound in sterile saline. Thirty minutes later, theywere injected intraperitoneally with 1 ml of LPS (5 μg/ml) to induceperitonitis. The mice were dosed with U73122 30 mg/kg i.p. on day 0, and oncedaily for a total of nine doses. The animals were then sacrificed in a CO2chamber on day 9 and the peritoneal cavities lavaged with 3 ml of Dulbecco’sPBS to collect cells. Lavage samples were analyzed by differential cell countsusing the same procedure as described in the canine subcutaneous chamber model.Percentage of inhibition was evaluated by comparison with the increase inmacrophage count between the baseline control group and the positive controlgroup.

TPA-Induced Ear Edema in Mice

TPA-induced ear edema was performed. TPA(1.0 μg) dissolved in 20 μl of acetone was applied to the dorsal surface of the right ear ofmice. U73122 was administered intravenously before the TPA administration. Sixhours later, the animals were sacrificed and 7-mm biopsy punches were takenfrom each ear. The punches were weighed and the difference between treated and untreatedears determined. The percentage of inhibition was calculated by comparing thedifference in ear weight of vehicle-treated with compound-treated mice.

不同实验动物依据体表面积的等效剂量转换表（数据来源于FDA指南）

例如，已知某工具药用于小鼠的剂量为88 mg/kg , 则用于大鼠的剂量换算方法：将88 mg/kg 乘以小鼠的Km系数（3），再除以大鼠的Km系数（6），得到该药物用于大鼠的等效剂量44 mg/kg。

参考文献

[1] Klein RR, Bourdon DM, Costales CL, et al. Direct activation of human phospholipase C by its well known inhibitor u73122. J Biol Chem. 2011;286(14):12407-12416.
[2] Zhu L, Yuan C, Ma Y, Ding X, Zhu G, Zhu Q. Anti-inflammatory activities of phospholipase C inhibitor U73122: Inhibition of monocyte-to-macrophage transformation and LPS-induced pro-inflammatory cytokine expression. Int Immunopharmacol. 2015;29(2):622-627.
[3] Hou C, Kirchner T, Singer M, Matheis M, Argentieri D, Cavender D. In vivo activity of a phospholipase C inhibitor, 1-(6-((17beta-3-methoxyestra-1,3,5(10)-trien-17-yl)amino)hexyl)-1H-pyrrole-2,5-di one (U73122), in acute and chronic inflammatory reactions. J Pharmacol Exp Ther. 2004;309(2):697-704.

体内溶解度
4 mg/mL

Clinical Trial Information ( data from http://clinicaltrials.gov )

注：以上所有数据均来自公开文献，并不保证对所有实验均有效，数据仅供参考。

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