In vitro activity: Ampiroxicam (<150 μM) dose-dependently decreases the proliferation of Panc-1 cells. Ampiroxicam (50 μM) results in decreased expression of Sp1, Sp3, Sp4, and VEGFR1 proteins in Panc-1 cells and L3.6pl cells as determined by Western blot analysis. Ampiroxicam (50 μM) results in increased phosphorylation of MAPK1/2 in Panc-1 cells and L3.6pl cells.
Kinase Assay: Panc-1 cells are plated in DME/F12 medium with 5% fetal bovine serum and treated on the next day with vehicle (0.1% DMSO) or various concentrations of Ampiroxicam. Cells are counted at the indicated times with a Coulter Z1 cell counter. Each experiment is done in triplicate, and results are expressed as means, with error bars representing 95% confidence intervals (CIs).

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Ampiroxicam (CP 65703) at 50 μM for 48 hours did not decrease Sp1, Sp3, or Sp4 protein levels in Panc-1 cells as determined by immunoblot analysis [1].
Ampiroxicam (CP 65703) at 50 μM for 48 hours also had no effect on Sp1, Sp3, or Sp4 expression in L3.6pl pancreatic cancer cells [1].
Treatment with Ampiroxicam (CP 65703) at 50 μM for 48 hours did not reduce VEGF protein expression in Panc-1 cells compared to DMSO control [1].
Ampiroxicam (CP 65703) at 50 μM for 12 hours did not decrease VEGF mRNA levels in Panc-1 cells as measured by semiquantitative RT-PCR [1].
In Panc-1 cells transfected with pVEGF1 or pVEGF2 (VEGF promoter constructs), Ampiroxicam (CP 65703) at 50 μM did not significantly decrease luciferase activity, indicating no inhibition of VEGF promoter transactivation [1].
Similar lack of effect on VEGF promoter transactivation was observed in L3.6pl cells treated with Ampiroxicam (CP 65703) at 50 μM [1].
Ampiroxicam (CP 65703) at concentrations 20-80 μM did not decrease luciferase activity in Panc-1 cells transfected with pVEGF2 [1].
In electrophoretic mobility shift assays, nuclear extracts from Panc-1 or L3.6pl cells treated with Ampiroxicam (CP 65703) at 50 μM for 48 hours showed no reduction in Sp protein binding to a GC-rich VEGF promoter oligonucleotide [1].
Immunocytochemistry showed that Ampiroxicam (CP 65703) at 50 μM for 48 hours did not block secreted VEGF protein staining in Panc-1 cells [1].
Ampiroxicam (CP 65703) at 150 μM for 48 hours did not induce degradation of Sp1, Sp3, or Sp4 proteins nor cause PARP cleavage (apoptosis marker) in Panc-1 cells [1].
Ampiroxicam (CP 65703) inhibited Panc-1 cell proliferation at higher concentrations (e.g., 150 μM) but with lower potency compared to tolfenamic acid; at 50 μM, growth inhibition was minimal [1].
In L3.6pl cells, Ampiroxicam (CP 65703) at 25, 50, and 100 μM inhibited cell proliferation but to a lesser extent than tolfenamic acid at the same concentrations [1].
In Panc-28 cells, Ampiroxicam (CP 65703) at 25, 50, and 100 μM also inhibited cell proliferation less effectively than tolfenamic acid [1].

Ampiroxicam inhibits the stretching response in mice induced by phenylbenzoquinone (PBQ) with maximum protective effect (MPE) of 2 mg/kg. Ampiroxicam inhibits swelling in a dose-responsive manner in the rat foot edema (RFE) assay with ED50 of 28 mg/kg at single oral dose and 7.8 mg/kg at 5 daily oral dose. Ampiroxicam blocks primary and secondary lesion development in rat adjuvant arthritis with ED50 of 2.2 mg/kg and 0.5 mg/kg, respectively. Ampiroxicam (3.2 mg/kg) leads to a plasma concentration of 12 μg/mL at a Tmax of 2 hours for piroxicam derived from ampiroxicam in rats. Ultraviolet-A (UVA)-irradiated 1% Ampiroxicam sensitized in guinea pigs shows positive reaction in the patch testing to UVA-irradiated 1% Ampiroxicam and 1% thiosalicylate (TOS). Concentration of Ampiroxicam is easily reduced by the increase in UVA irradiation doses, as compared with that of piroxicam.

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Panc-1, L3.6pl, and Panc-28 cells were cultured in DME/F12 medium with 5% fetal bovine serum. For immunoblot analysis, cells were treated with 0.1% DMSO (vehicle control) or Ampiroxicam (CP 65703) at indicated concentrations (e.g., 50 μM or 150 μM) for 24 or 48 hours. Whole-cell lysates were prepared, and equal amounts of protein (60 μg per lane for Sp proteins, 100 μg for VEGF) were subjected to SDS-PAGE on 10%-12.5% gels, transferred to membranes, and probed with primary antibodies against Sp1, Sp3, Sp4, VEGF, PARP, or β-tubulin, followed by HRP-conjugated secondary antibody. Band intensities were quantified by laser scanning densitometry [1].
For reverse transcription-polymerase chain reaction (RT-PCR), Panc-1 cells were treated with 0.1% DMSO or Ampiroxicam (CP 65703) at 50 μM for 12 hours. Total RNA was isolated, reverse transcribed with oligo(dT) primers, and amplified using VEGF-specific primers and GAPDH as a control. PCR products were resolved on 2% agarose gels containing ethidium bromide and visualized by UV transillumination [1].
For transfection and luciferase reporter assays, Panc-1 or L3.6pl cells were cultured in six-well plates until 50%-60% confluent, then transfected with pVEGF1 or pVEGF2 (VEGF promoter-luciferase constructs) using Lipofectamine. After transfection, cells were treated with 0.1% DMSO, Ampiroxicam (CP 65703) (e.g., 50 μM or 20-80 μM), or other NSAIDs for 24 or 48 hours. Luciferase activity was measured in whole-cell lysates and normalized to β-galactosidase activity [1].
For electrophoretic mobility shift assay (EMSA), nuclear extracts from Panc-1 or L3.6pl cells treated with 0.1% DMSO or Ampiroxicam (CP 65703) at 50 μM for 48 hours were isolated. A 32P-labeled double-stranded oligonucleotide containing the GC-rich region (-66 to -47) of the VEGF promoter was incubated with nuclear extracts (3 μg protein) and poly(dI-dC). After incubation, protein-DNA complexes were resolved by native polyacrylamide gel electrophoresis and detected by autoradiography. For supershift assays, antibodies against Sp1, Sp3, or Sp4 were added [1].
For cell proliferation assays, Panc-1, L3.6pl, or Panc-28 cells were plated in DME/F12 medium with 5% fetal bovine serum, then treated the next day with 0.1% DMSO or various concentrations of Ampiroxicam (CP 65703) (e.g., 25, 50, 100, or 150 μM). Cells were counted at days 2, 4, and 6 using a Coulter cell counter. Each experiment was done in triplicate [1].
For immunocytochemistry, Panc-1 cells were cultured on chamber slides, treated with 0.1% DMSO or Ampiroxicam (CP 65703) at 50 μM for 48 hours. Cells were fixed in methanol, blocked with goat serum, and incubated with rabbit anti-VEGF polyclonal antibody (1:200 dilution), followed by FITC-conjugated goat anti-rabbit antibody (1:1000 dilution). Nuclear counterstaining was performed with DAPI-containing mounting medium. Fluorescence images were captured [1].

Dissolved in 0.1% methylcellulose; 3.2 mg/kg; p.o. Rats with adjuvant arthritis

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J Natl Cancer Inst.2006 Jun 21;98(12):855-68;Agents Actions.1993 Jul;39(3-4):157-65.

Amipiroxacin is a benzothiazide drug, a 1-[(ethoxycarbonyl)oxy]ethyl ether derivative of piroxicam. As a prodrug of piroxicam, it is used to relieve pain and inflammation caused by musculoskeletal diseases such as rheumatoid arthritis and osteoarthritis. Amipiroxacin possesses various pharmacological activities, including as a prodrug, analgesic, nonsteroidal anti-inflammatory drug (NSAID), antirheumatic drug, and prostaglandin intraperoxidase (EC 1.14.99.1) inhibitor. It is a benzothiazide, sulfonamide, aminopyridine, monocarboxylic acid amide, etopolate, and acetal compound. Its structure is closely related to that of piroxicam.

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Ampiroxicam (CP 65703) is an oxicam class NSAID that was used as a negative control in this study because it did not induce degradation of Sp1, Sp3, or Sp4 proteins in pancreatic cancer cells, in contrast to tolfenamic acid. The lack of activity of Ampiroxicam (CP 65703) in all assays (Sp protein expression, VEGF expression, promoter transactivation, DNA binding) supports the conclusion that Sp protein degradation is a specific mechanism for the antipancreatic cancer activity of tolfenamic acid and not a general property of all NSAIDs [1].

C20H21N3O7S

447.46

447.11

99464-64-9

2176

White to off-white solid powder

1.4±0.1 g/cm3

159-161?C

1.630

3.12

1

9

8

31

808

0

LSNWBKACGXCGAJ-UHFFFAOYSA-N

InChI=1S/C20H21N3O7S/c1-4-28-20(25)30-13(2)29-18-14-9-5-6-10-15(14)31(26,27)23(3)17(18)19(24)22-16-11-7-8-12-21-16/h5-13H,4H2,1-3H3,(H,21,22,24)

Carbonic acid, ethyl 1-((2-methyl-3-((2-pyridinylamino)carbonyl)-2H-1,2-benzothiazin-4-yl)oxy)ethyl ester, S,S-dioxide

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)

Solubility in Formulation 1: ≥ 2.5 mg/mL (5.59 mM) (saturation unknown) in 10% DMSO + 40% PEG300 + 5% Tween80 + 45% 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 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 (5.59 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.)