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| Targets |
Cyclooxygenase (COX) inhibitor with nitric oxide (NO)-donating properties [1]
Thromboxane A₂ (TXA₂) production (associated with decreased TXA₂ levels) [1] Endothelial nitric oxide synthase (eNOS) pathway (indirect modulation via COX interaction) [1] |
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| ln Vitro |
In hepatic stellate cells (HSCs), Nitroflurbiprofen dose-dependently inhibited fetal calf serum (FCS)-promoted contraction at concentrations of 25, 50, 100, and 250 μmol/L (P < 0.001 vs FCS alone). At 250 μmol/L, it decreased contraction more effectively than flurbiprofen at the same concentration (P < 0.001) [1].
In the in situ perfused cirrhotic rat liver, Nitroflurbiprofen (250 μmol/L) improved endothelial dysfunction by attenuating the vasodilatory response to acetylcholine, bringing it to levels comparable with control livers (no significant difference from control) [1]. Nitroflurbiprofen (250 μmol/L) also attenuated the intrahepatic hyperresponsiveness to methoxamine in cirrhotic livers, completely abolishing the exaggerated response at 10⁻⁶ mol/L methoxamine (Δ perfusion pressure: 0.07 ± 0.1 mmHg, P = 0.342 vs healthy control) and showing significantly better effect than flurbiprofen at 10⁻⁴ mol/L methoxamine (7.06 ± 0.3 mmHg vs 9.1 ± 0.5 mmHg, P = 0.013) [1]. In HSC contraction assays, the TXA₂ analogue U44619 promoted contraction, which was dose-dependently inhibited by the TXA₂ receptor antagonist SQ29,548 (1 and 2.5 μmol/L), confirming HSC sensitivity to TXA₂-mediated contraction [1]. Cytotoxicity of Nitroflurbiprofen at the concentrations used (25–250 μmol/L) was excluded using a colorimetric cell proliferation assay (XTT) (data not shown) [1]. |
| ln Vivo |
nitroflurbiprofen (45 mg/kg), flurbiprofen, and in situ assessment of increased intrahepatic resistance by perfusion (30 mg/kg, equimolar to nitroflurbiprofen) or intraperitoneal injection of vehicle 24 and 1 h before measurement were performed on 8 rats per condition. The rats had thioacetamide-induced cirrhosis. In rats with thioacetamide-induced cirrhosis, nitroflurbiprofen, a nitric oxide-releasing cyclooxygenase inhibitor, improves portal hypertension without causing significant side effects by attenuating intrahepatic vascular resistance, endothelial dysfunction, and hepatic hyperresponsiveness to vasoconstrictors [1].
In thioacetamide (TAA)-induced cirrhotic rats, Nitroflurbiprofen (45 mg/kg, intraperitoneal injection 24h and 1h prior to measurements) significantly decreased portal pressure compared with vehicle (8 ± 0.8 mmHg vs 11.8 ± 0.6 mmHg, P < 0.001), an effect similar to flurbiprofen (8.4 ± 0.1 mmHg) [1]. Mean arterial pressure was not significantly different among groups (vehicle: 86.4 ± 3.6 mmHg; Nitroflurbiprofen: 89.3 ± 4.1 mmHg; flurbiprofen: 80.6 ± 4.1 mmHg; P = 0.291), indicating no aggravation of systemic hypotension [1]. Splanchnic hyperemia was attenuated in Nitroflurbiprofen-treated rats (4.2 ± 0.2 mL/min/100g vs vehicle: 5.4 ± 0.2 mL/min/100g, P = 0.004) [1]. In situ perfusion studies showed that Nitroflurbiprofen pretreatment reduced total intrahepatic vascular resistance (slope: 0.16 ± 0.02 mmHg·min/mL vs TAA+vehicle: 0.43 ± 0.01 mmHg·min/mL, P < 0.001) [1]. Hepatic nitrate/nitrite (NOx) levels were significantly higher in Nitroflurbiprofen-treated rats (179.7 ± 29.9 pmol/mg) compared with vehicle (57.5 ± 16.7 pmol/mg, P = 0.005) and flurbiprofen (109.2 ± 16.7 pmol/mg, P = 0.005), indicating increased intrahepatic NO release without systemic increase (serum NOx levels showed no differences) [1]. |
| Enzyme Assay |
Thromboxane B₂ (TXB₂), a stable metabolite of TXA₂, was measured in perfusate samples taken before and after acetylcholine stimulation in the in situ perfused rat liver. TXB₂ production was expressed as the absolute increment over baseline before methoxamine administration. In cirrhotic livers, TXB₂ production was 820.5 ± 185.4 pg/mL. After incubation with Nitroflurbiprofen (250 μmol/L), TXB₂ production decreased to 129.5 ± 48.3 pg/mL (P < 0.001 vs TAA alone) [1].
Nitrate/nitrite (NOx) content, a parameter of NO production, was assayed by a fluorometric method using 2,3-diaminonaphthalene in serum and liver homogenates of cirrhotic rats. Liver homogenates were prepared at a final concentration of 0.25 g wet weight of liver per mL of sucrose buffer. NOx levels in liver were significantly higher in Nitroflurbiprofen-treated rats (179.7 ± 29.9 pmol/mg) compared to vehicle or flurbiprofen groups [1]. |
| Cell Assay |
Hepatic stellate cells (HSCs) were isolated from normal rat livers by in situ perfusion with collagenase type IV and pronase E, followed by density gradient centrifugation using 9% Optiprep. Cells were cultured in William's E medium with 10% fetal calf serum (FCS), insulin, glutamine, and antibiotic-antimycotic solution. Characterization was performed by staining with anti-α-smooth muscle actin, antidesmin, and synaptophysin. HSC contraction was assessed using a three-dimensional collagen matrix contraction assay. Hydrated collagen gels were prepared using rat tail tendon collagen I. The collagen solution was mixed with HSC suspension to a final concentration of 1.5 mg/mL collagen and 250,000 cells/mL. A 500-μL aliquot was cast into a 24-well plate. After 24 hours, mechanically stressed matrices were released, and contraction was measured by determining the partitioning of ³H₂O between the gel phase and surrounding medium after 24 hours. Nitroflurbiprofen (25, 50, 100, 250 μmol/L) was preincubated before FCS (10%) addition. Cytotoxicity was excluded using an XTT colorimetric assay [1].
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| Animal Protocol |
Cirrhosis was induced in male Wistar rats (200-250 g) by chronic exposure to thioacetamide (TAA) in drinking water for 18 weeks. Nitroflurbiprofen was administered via intraperitoneal injection at a dose of 45 mg/kg, given 24 hours and 1 hour prior to hemodynamic measurements. The dose was selected based on a dose-finding study (starting from 15 mg/kg, then 22.5 mg/kg, and finally 45 mg/kg as the most effective). Flurbiprofen was given at 30 mg/kg (equimolar to nitroflurbiprofen), and vehicle was 250 μL DMSO mixed with 250 μL isotonic saline [1].
For in vivo hemodynamic measurements, rats were anesthetized with pentobarbital. The carotid artery and portal vein were cannulated. Superior mesenteric artery flow (mesenteric blood flow, MBF) was determined using a 1-mm nonconstructive perivascular flow probe connected to a flowmeter. Portal pressure and mean arterial pressure were recorded [1]. For in situ liver perfusion, a flow-controlled system was used. After laparotomy, the portal vein was cannulated, and the liver was perfused with oxygenated Krebs solution at 37°C ± 0.5°C at a constant flow of 35 mL/min. The right atrium was cannulated to collect hepatic vein outflow. An ultrasonic flow probe and pressure gauge were placed upstream of the inlet cannula to monitor portal flow and perfusion pressure. Viability criteria included stable perfusion pressure (±1 mmHg) and stable buffer pH (7.4 ± 0.1) during a 30-minute stabilization. For acute drug addition, Nitroflurbiprofen (250 μmol/L) was added to the perfusate after stabilization [1]. |
| ADME/Pharmacokinetics |
Nitroflurbiprofen is rapidly metabolized at the carboxyl ester bond in liver subcellular fractions and blood plasma into its parent compound flurbiprofen and the NO-releasing aliphatic linker nitroxybutyl alcohol (NOBA). NOBA can be metabolized in two ways: predominantly, it is catalyzed to NO species in the liver by cytosolic glutathione S-transferase and, to a lesser extent, in liver mitochondria. Alternatively, NOBA can give rise to bioactive NO in circulating erythrocytes under deoxygenated conditions. This dual metabolism explains the increased intrahepatic NOx levels without a rise in serum NOx levels [1].
Serum NOx levels showed no differences between Nitroflurbiprofen-treated, flurbiprofen-treated, and vehicle-treated cirrhotic rats, suggesting lack of overt systemic NO release. In contrast, hepatic NOx levels were significantly higher in Nitroflurbiprofen-treated rats (179.7 ± 29.9 pmol/mg) compared with vehicle (57.5 ± 16.7 pmol/mg) and flurbiprofen (109.2 ± 16.7 pmol/mg) (P = 0.005) [1]. |
| Toxicity/Toxicokinetics |
Nitroflurbiprofen did not cause renal impairment in TAA-induced cirrhotic rats, whereas flurbiprofen did. Serum urea nitrogen: Nitroflurbiprofen (42.3 ± 3 mg%) vs flurbiprofen (65.4 ± 3.7 mg%) (P < 0.001); serum creatinine: Nitroflurbiprofen (0.51 ± 0.02 mg%) vs flurbiprofen (0.69 ± 0.04 mg%) (P < 0.001); creatinine clearance: Nitroflurbiprofen (12.3 ± 0.7 mL/h/100g) vs flurbiprofen (8.0 ± 0.4 mL/h/100g) (P < 0.001) [1].
Gastrointestinal ulcerogenicity: ulcer index was significantly lower in Nitroflurbiprofen-treated rats (2.9 ± 0.8 mm²) compared to flurbiprofen-treated rats (15.6 ± 1.6 mm²) (P < 0.001). No gastrointestinal bleeding was observed in the Nitroflurbiprofen group (0/8), whereas 3 out of 8 flurbiprofen-treated rats showed bleeding [1]. Hepatic function: no significant differences in albumin or total bilirubin among groups. Aspartate aminotransferase (AST) showed an almost significant decrease in Nitroflurbiprofen-treated rats (140.1 ± 9.8 U/L) vs vehicle (202 ± 15.6 U/L, P = 0.054) [1]. |
| References | |
| Additional Infomation |
Nitroflurbiprofen is a carboxylic acid ester formed by the condensation of the carboxyl group of flurbiprofen with the free hydroxyl group of 4-(nitro)butanol. It is a nonsteroidal anti-inflammatory drug (NSAID) that inhibits cyclooxygenases (COX-1 and COX-2). It has multiple functions, including as a COX-1 inhibitor, COX-2 inhibitor, NSAID, EC 1.14.13.39 (nitric oxide synthase) inhibitor, vasodilator, and anti-aging agent. It is an organofluorine compound belonging to the biphenyl class, and is a carboxylic acid and nitrate ester. Its functions are related to flurbiprofen.
Nitroflurbiprofen (HCT-1026) is a nitric oxide (NO)-releasing cyclooxygenase (COX) inhibitor designed to combine COX inhibition with NO donation to enhance therapeutic efficacy or reduce adverse effects. In cirrhotic portal hypertension, it improves portal pressure by attenuating intrahepatic vascular resistance, endothelial dysfunction, and hepatic hyperreactivity to vasoconstrictors, without aggravating systemic hypotension or causing typical NSAID-related adverse effects such as gastrointestinal ulceration, bleeding, or nephrotoxicity [1]. The study suggests a pathogenetic mechanism whereby TXA₂ exerts control over NO production: increased COX activity leads to increased TXA₂ production, which down-regulates Akt (a serine-threonine kinase and cofactor of eNOS), causing eNOS dysfunction. This interaction was supported by the finding that the correcting effect of flurbiprofen on endothelial dysfunction was attenuated by L-NAME (a NOS inhibitor) [1]. |
| Molecular Formula |
C19H20FNO5
|
|---|---|
| Molecular Weight |
361.37
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| Exact Mass |
361.133
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| CAS # |
158836-71-6
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| PubChem CID |
119387
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| Appearance |
Colorless to light yellow ointment
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| Density |
1.214g/cm3
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| Boiling Point |
468.8ºC at 760 mmHg
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| Flash Point |
237.3ºC
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| Vapour Pressure |
5.79E-09mmHg at 25°C
|
| Index of Refraction |
1.537
|
| LogP |
4.651
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| Hydrogen Bond Donor Count |
0
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| Hydrogen Bond Acceptor Count |
6
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| Rotatable Bond Count |
9
|
| Heavy Atom Count |
26
|
| Complexity |
450
|
| Defined Atom Stereocenter Count |
0
|
| SMILES |
O=C(OCCCCO[N+]([O-])=O)C(C)C1=CC=C(C2=CC=CC=C2)C(F)=C1
|
| InChi Key |
DLWSRGHNJVLJAH-UHFFFAOYSA-N
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| InChi Code |
InChI=1S/C19H20FNO5/c1-14(19(22)25-11-5-6-12-26-21(23)24)16-9-10-17(18(20)13-16)15-7-3-2-4-8-15/h2-4,7-10,13-14H,5-6,11-12H2,1H3
|
| Chemical Name |
4-(nitrooxy)butyl 2-(2-fluoro-[1,1'-biphenyl]-4-yl)propanoate
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| Synonyms |
NO-flurbiprofenHCT-1026 NCX 1026NCX-1026 HCT 1026HCT1026 NCX1026
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| HS Tariff Code |
2934.99.9001
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| Storage |
Powder -20°C 3 years 4°C 2 years In solvent -80°C 6 months -20°C 1 month |
| Shipping Condition |
Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)
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| Solubility (In Vitro) |
May dissolve in DMSO (in most cases), if not, try other solvents such as H2O, Ethanol, or DMF with a minute amount of products to avoid loss of samples
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|---|---|
| Solubility (In Vivo) |
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.
Injection Formulations
Injection Formulation 1: DMSO : Tween 80： Saline = 10 : 5 : 85 (i.e. 100 μL DMSO stock solution → 50 μL Tween 80 → 850 μL Saline)(e.g. IP/IV/IM/SC) *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). View More
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)] Oral Formulations
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). View More
Oral Formulation 3: Dissolved in PEG400 (Please use freshly prepared in vivo formulations for optimal results.) |
| Preparing Stock Solutions | 1 mg | 5 mg | 10 mg | |
| 1 mM | 2.7672 mL | 13.8362 mL | 27.6725 mL | |
| 5 mM | 0.5534 mL | 2.7672 mL | 5.5345 mL | |
| 10 mM | 0.2767 mL | 1.3836 mL | 2.7672 mL |
*Note: Please select an appropriate solvent for the preparation of stock solution based on your experiment needs. For most products, DMSO can be used for preparing stock solutions (e.g. 5 mM, 10 mM, or 20 mM concentration); some products with high aqueous solubility may be dissolved in water directly. Solubility information is available at the above Solubility Data section. Once the stock solution is prepared, aliquot it to routine usage volumes and store at -20°C or -80°C. Avoid repeated freeze and thaw cycles.
Calculation results
Working concentration： mg/mL；
Method for preparing DMSO stock solution： mg drug pre-dissolved in μL DMSO (stock solution concentration mg/mL). Please contact us first if the concentration exceeds the DMSO solubility of the batch of drug.
Method for preparing in vivo formulation:：Take μL DMSO stock solution, next add μL PEG300, mix and clarify, next addμL Tween 80, mix and clarify, next add μL ddH2O，mix and clarify.
(1) Please be sure that the solution is clear before the addition of next solvent. Dissolution methods like vortex, ultrasound or warming and heat may be used to aid dissolving.
(2) Be sure to add the solvent(s) in order.
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