Cyclooxygenase-2 (COX-2). [2]
Cyclooxygenase-1 (COX-1). [2]
For murine COX-2, IC₅₀ = 0.05 μM (50 nM); for murine COX-1, IC₅₀ > 10 μM. [2]

High selectivity for inducible COX-2 (IC50=1 μM) over COX-1 (IC50>100 μM) is exhibited by SC-58125 (0.001-100 μM) [1]. Time-dependent, SC-58125 (10 μM; 20-140 seconds) reaches half-maximal inhibition at 20 seconds and completes inhibition in 1 minute [1]. In vitro, SC-58125 (25-100 μM; 3 d) suppresses the development of LLC and HCA-7 cells [3]. In LLC cells, SC-58125 (100 µM; 12 hours) causes G2 arrest [3]. In HCA-7 cells, SC-58125 (25-100 μM; 3 d) lowers p34cdc2 levels [3]. In HCA-7 and LLC cells, SC-58125 (100 µM; 24 or 72 hours) does not cause apoptosis [3].

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SC-58125 potently and selectively inhibited murine recombinant COX-2 with an IC₅₀ of 0.05 μM, while showing no inhibition of murine recombinant COX-1 at concentrations up to 10 μM (IC₅₀ > 10 μM). A similar profile of potency and selectivity was observed for the human recombinant enzymes. [2]

Established colorectal cancer xenografts in mice are inhibited in growth by SC-58125 (10 mg/kg; intraperitoneally every 48 hours) [3]. In mice, tumor PGE2 levels can be decreased by SC-58125 (10 mg/kg; single intraperitoneal injection) [3]. Mice's tumor levels of COX-1 and COX-2 proteins are unaffected by SC-58125 (10 mg/kg; single intraperitoneal injection) [3].

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In the carrageenan-induced rat paw edema model, SC-58125 (administered orally) suppressed the edematous response observed 3 hours after carrageenan injection with an ED₅₀ of 8.0 mg/kg. [2]
SC-58125 demonstrated analgesic activity in the inflammatory hyperalgesia model, blocking the hyperalgesic response with an ED₅₀ of 8.0 mg/kg. [2]
In the rat air pouch model, SC-58125 completely suppressed carrageenan-induced pouch exudate prostaglandin synthesis with an ED₅₀ of 0.1 mg/kg, while having no effect on stomach prostaglandin production at doses up to 10 mg/kg (ED₅₀ > 10 mg/kg). [2]

The coding regions of mouse COX-1 and COX-2 were subcloned into the baculovirus expression vector pVL1393. Recombinant baculoviruses were isolated by transfecting plasmid DNA with linearized baculovirus DNA into SF9 insect cells. Cells expressing COX-1 or COX-2 were homogenized. SC-58125 (0.001 – 10 μM) was preincubated with homogenates (2–10 μg protein) for 10 minutes prior to the addition of arachidonic acid (10 μM). PGE₂ formed during a 10-minute incubation was detected by ELISA. [2]

Cell proliferation assay[3]
Cell Types: HCA-7 and LLC Cell
Tested Concentrations: 0, 25, 50, 100 μM
Incubation Duration: 3 days
Experimental Results: Cell number and MTT activity were diminished in a dose-dependent manner in both cell lines .

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Cell cycle analysis[3]
Cell Types: LLC Cell
Tested Concentrations: 100 μM
Incubation Duration: 12 hrs (hours)
Experimental Results: The number of cells containing 4n DNA content increased in a dose- and time-dependent manner. Mitotic numbers are diminished.

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Western Blot Analysis[3]
Cell Types: HCA-7 Cell
Tested Concentrations: 0, 25, 50, 100 μM
Incubation Duration: 3 days
Experimental Results: Even at the lowest concentration, a dose-dependent decrease in p34cdc2 activity was induced with a strong inhibitory effect.

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MTT assay: Cells were seeded at 2 × 10⁴ cells/well in 96-well plates and grown for 24 hours. SC-58125 or DMSO in 1% FBS-containing OptiMEM media was added. At indicated times, 10 μL MTT (5 mg/mL) was added and incubated for 4 hours. Formazan crystals were solubilized with 10% SDS in 0.01 N HCl overnight at 37°C, and optical density was measured at 570 nm. [3]
Apoptosis assays: TUNEL assay was performed using the in situ cell death detection kit (POD) for in situ evaluation and the APO-Direct kit for quantitative flow cytometric evaluation. For flow cytometry, cells were harvested, fixed in 1% paraformaldehyde, permeabilized with 70% ethanol, and fragmented DNA was end-labeled with FITC-conjugated dUTP. DNA laddering was assessed by electrophoresis on 1.5% TPE agarose gels. Annexin V staining was also performed. [3]
Flow cytometric cell cycle analysis: Cells were trypsinized, fixed in ice-cold ethanol, stained with propidium iodide (PI) staining solution (0.5 mg/mL RNase A, 20 μg/mL PI in PBS), and analyzed on a FACScan flow cytometer with ModFit software. [3]
BrdU labeling: Cells were pulsed with 10 μM BrdU for 45 minutes, washed, and then treated with SC-58125. Cells were harvested and processed for dual-parameter BrdU/PI flow cytometry using the In Situ Cell Proliferation Kit (FLOUS). [3]
p34ᶜᵈᶜ² mRNA analysis: Total RNA was isolated using Tri-Reagent. cDNA microarrays (GENEFILTERS) were hybridized with [³³P]-labeled cDNA probes. Differential expression was confirmed by Northern blotting using a [α-³²P]-labeled p34ᶜᵈᶜ² cDNA probe. [3]
p34ᶜᵈᶜ² protein analysis: Western blot analysis was performed using p34ᶜᵈᶜ²-specific antisera. [3]
p34ᶜᵈᶜ² kinase assay: Histone H1 kinase activity was measured using immunoprecipitated p34ᶜᵈᶜ². [3]

Animal/Disease Models: Athymic SD (SD (Sprague-Dawley)) mice injected with HCA-7 cells [3]
Doses: 10 mg/kg
Route of Administration: intraperitoneal (ip) injection every 48 hrs (hrs (hours)); at the time of tumor implantation or after 2 and 4 weeks.
Experimental Results: significant Dramatically reduce tumor growth rate.

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Carrageenan-induced paw edema and hyperalgesia: Male Sprague-Dawley rats were used. SC-58125 was suspended in 0.5% methylcellulose in water and administered orally by gavage 1 hour prior to carrageenan injection (0.1 ml of 1% carrageenan in saline into the hindpaw). Paw volume was measured at intervals to assess edema. Hyperalgesia was measured as the withdrawal latency to a radiant heat source applied to the hindpaw. Measurements were taken 3 hours after carrageenan injection. [2]
Rat air pouch model: An air cavity was produced by subcutaneous injection of sterile air into the back of male rats. Carrageenan (1% solution) was injected into the pouch. Animals were fasted for 17 hours and then pretreated with SC-58125 (0.001 – 10 mg/kg, orally) 1 hour prior to carrageenan administration. After 6 hours, pouch exudates were collected for prostaglandin determination. Stomachs were excised for visual examination of gastric glandular damage and processed for tissue prostaglandin production. [2]
Gastric toxicity study: Rats were fasted for 16 hours prior to administration of SC-58125 (0.03 – 30 mg/kg) by gavage. Five hours after administration, stomachs were removed and inspected for gastric glandular mucosal damage with a stereomicroscope. No visible gastric lesions were observed at doses up to 400 mg/kg (60 times the ED₅₀ for edema). [2]
Intestinal toxicity study: Fed rats were dosed once with a suspension of SC-58125 (400 or 600 mg/kg) intragastrically. After 72 hours, animals were euthanized, and the abdominal cavity was opened to assess the presence of adhesions. No intestinal lesions were observed at doses up to 200 mg/kg. [2]

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Carrageenan-induced paw edema and hyperalgesia: Male Sprague-Dawley rats were used. SC-58125 was suspended in 0.5% methylcellulose in water and administered orally by gavage 1 hour prior to carrageenan injection (0.1 ml of 1% carrageenan in saline into the hindpaw). Paw volume was measured at intervals to assess edema. Hyperalgesia was measured as the withdrawal latency to a radiant heat source applied to the hindpaw. Measurements were taken 3 hours after carrageenan injection. [2]
Rat air pouch model: An air cavity was produced by subcutaneous injection of sterile air into the back of male rats. Carrageenan (1% solution) was injected into the pouch. Animals were fasted for 17 hours and then pretreated with SC-58125 (0.001 – 10 mg/kg, orally) 1 hour prior to carrageenan administration. After 6 hours, pouch exudates were collected for prostaglandin determination. Stomachs were excised for visual examination of gastric glandular damage and processed for tissue prostaglandin production. [2]
Gastric toxicity study: Rats were fasted for 16 hours prior to administration of SC-58125 (0.03 – 30 mg/kg) by gavage. Five hours after administration, stomachs were removed and inspected for gastric glandular mucosal damage with a stereomicroscope. No visible gastric lesions were observed at doses up to 400 mg/kg (60 times the ED₅₀ for edema). [2]
Intestinal toxicity study: Fed rats were dosed once with a suspension of SC-58125 (400 or 600 mg/kg) intragastrically. After 72 hours, animals were euthanized, and the abdominal cavity was opened to assess the presence of adhesions. No intestinal lesions were observed at doses up to 200 mg/kg. [2]

Following intraperitoneal administration of 10 mg/kg SC-58125 in tumor-bearing mice, tumor PGE₂ levels were significantly reduced within 2 hours, with maximal inhibition at 12 hours. PGE₂ levels remained low for 24 hours and then began to increase, reaching 50% of initial levels within 36 hours. This indicates that the drug is well distributed to the tumor site and effectively inhibits COX-2 activity. [3]

SC-58125 did not cause gastric glandular mucosal lesions at oral doses up to 400 mg/kg, which is 60 times the ED₅₀ for edema. In the gastric toxicity study, no lesions were observed at doses up to 30 mg/kg. [2]
SC-58125 did not cause intestinal lesions at doses up to 200 mg/kg. [2]
SC-58125 had no effect on stomach prostaglandin production at doses up to 10 mg/kg (ED₅₀ > 10 mg/kg), in contrast to indomethacin which inhibited stomach PGs with an ED₅₀ of 0.4 mg/kg. [2]

[1]. A single amino acid difference between cyclooxygenase-1 (COX-1) and -2 (COX-2) reverses the selectivity of COX-2 specific inhibitors. J Biol Chem. 1996 Jun 28; 271(26): 15810-4.

[2]. Pharmacological and biochemical demonstration of the role of cyclooxygenase 2 in inflammation and pain. Proc Natl Acad Sci U S A. 1994 Dec 6; 91(25): 12013-7.

[3]. A cyclooxygenase-2 inhibitor (SC-58125) blocks growth of established human colon cancer xenografts. Neoplasia. Sep-Oct 2001; 3(5): 428-36.

SC-58125 is a pyrazole compound with the structure 1H-pyrazole, substituted at the 5-position with a 4-fluorophenyl group, at the 1-position with a 4-(methanesulfonyl)phenyl group, and at the 3-position with a trifluoromethyl group. It is a selective cyclooxygenase-2 inhibitor with anticancer activity. It can be used as both a cyclooxygenase-2 inhibitor and an antitumor drug. SC-58125 belongs to the pyrazole class, organofluorine compounds, and sulfone class of compounds.

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SC-58125 (1-[(4-methylsulfonyl)phenyl]-3-trifluoromethyl-5-(4-fluorophenyl)pyrazole) is a selective cyclooxygenase-2 (COX-2) inhibitor. Its discovery provided evidence that selective inhibition of COX-2 could produce anti-inflammatory and analgesic effects without the gastric toxicity associated with nonselective NSAIDs. The study demonstrated that COX-2 is induced at the site of inflammation, while COX-1 is constitutively expressed in tissues such as stomach and kidney. [2]

C17H12N2O2F4S

384.34798

384.055

162054-19-5

115239

Off-white to pink solid powder

1.4±0.1 g/cm3

512.6±50.0 °C at 760 mmHg

263.8±30.1 °C

0.0±1.3 mmHg at 25°C

1.573

3.86

0

7

3

26

577

0

CS(=O)(=O)C1=CC=C(C=C1)N2C(=CC(=N2)C(F)(F)F)C3=CC=C(C=C3)F

JHBIMJKLBUMNAU-UHFFFAOYSA-N

InChI=1S/C17H12F4N2O2S/c1-26(24,25)14-8-6-13(7-9-14)23-15(10-16(22-23)17(19,20)21)11-2-4-12(18)5-3-11/h2-10H,1H3

5-(4-fluorophenyl)-1-(4-methylsulfonylphenyl)-3-(trifluoromethyl)pyrazole

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 : ~250 mg/mL (~650.45 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.)