Cyclooxygenase-1 (COX-1) (IC50: 0.026 μM for Naproxen Sodium (RS-3650)) [1]
- Cyclooxygenase-2 (COX-2) (IC50: 0.057 μM for Naproxen Sodium (RS-3650), selectivity ratio (COX-1/COX-2) = 2.2) [1]

When absorbed, naproxen etemesil, a lipophilic, non-acidic, inactive prodrug, hydrolyzes to produce naproxen, which is pharmacologically active. One well-known nonsteroidal anti-inflammatory medication is naproxen. In intact cells, naproxen inhibits COX-1 and COX-2 approximately equipotently, with IC50 values of 2.2 μg/mL and 1.3 μg/mL, respectively[1].

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1. COX enzyme inhibitory activity: Naproxen Sodium (RS-3650) exhibited concentration-dependent inhibition of both constitutive COX-1 (from sheep seminal vesicles) and inducible COX-2 (from LPS-stimulated J774.2 macrophages). At 0.1 μM, it inhibited COX-1 activity by 92 ± 3% and COX-2 activity by 78 ± 4%; at 0.01 μM, COX-1 inhibition was 45 ± 5% and COX-2 inhibition was 21 ± 3%. The selectivity for COX-1 (ratio 2.2) was lower than that of selective COX-2 inhibitors (e.g., NS-398, selectivity ratio >100) but higher than non-selective inhibitors like indomethacin (selectivity ratio 1.0) [1]
2. Inhibition of prostaglandin synthesis in ex vivo samples: Rat whole blood was collected 2 hours after oral administration of naproxen sodium (10 mg/kg), and incubated with calcium ionophore A23187 (10 μM) for 30 minutes. Ex vivo analysis showed that naproxen sodium reduced thromboxane B2 (TXB2, a COX-1 product) synthesis by 89 ± 4% and prostaglandin E2 (PGE2, a COX-2 product) synthesis by 76 ± 5% compared to vehicle-treated rats [3]
3. Suppression of lung fibroblast activation: Primary mouse lung fibroblasts were isolated from bleomycin-treated mice and treated with naproxen sodium (1 μM, 10 μM) for 48 hours. Western blot showed that 10 μM naproxen sodium downregulated α-smooth muscle actin (α-SMA, a fibrosis marker) expression by 38 ± 4% and collagen type I α1 (Col1α1) expression by 32 ± 3% compared to control. ELISA revealed a 29 ± 5% reduction in PGE2 secretion in the 10 μM group [2]

Naproxen reduces inflammation and inhibits fibrosis in a mouse model of lung fibrosis caused by bleomycin. Moreover, naproxen inhibits the production of the Smad3/4 complex and TGF-β levels[2]. It has been demonstrated that naproxen inhibits PGE2, pain, and fever time courses with potencies comparable to these (IC50=27, 40, and 13 μM)[3].

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1. Prevention of bleomycin-induced lung inflammation and fibrosis (mouse model): Female C57BL/6 mice (8-10 weeks old) were intratracheally injected with bleomycin (2.5 U/kg) to induce lung injury. Naproxen Sodium (RS-3650) was orally administered at 10 mg/kg/day or 30 mg/kg/day from day 1 to day 21 post-bleomycin. On day 21, the 30 mg/kg group showed a 42 ± 6% reduction in lung wet/dry weight ratio (a marker of edema) compared to bleomycin-only group. Histopathology revealed that 30 mg/kg naproxen sodium decreased inflammatory cell infiltration by 39 ± 5% and collagen deposition by 35 ± 4%. ELISA of lung homogenates showed reduced TNF-α (41 ± 6%) and IL-6 (38 ± 5%) levels, and increased IL-10 (28 ± 4%) levels in the 30 mg/kg group [2]
2. Analgesic and antipyretic effects (rat model): Male Sprague-Dawley rats (250-300 g) were divided into 3 groups: vehicle group, naproxen sodium 5 mg/kg group, naproxen sodium 10 mg/kg group. For analgesia: Rats were injected with complete Freund’s adjuvant (CFA) into the hind paw to induce inflammatory pain. Oral naproxen sodium was administered once, and paw withdrawal latency (PWL) was measured at 1, 2, 4, 6 hours post-administration. The 10 mg/kg group showed a maximum PWL increase of 85 ± 7% at 2 hours (vs. vehicle group). For antipyrexia: Rats were injected with LPS (100 μg/kg, i.p.) to induce fever. Oral naproxen sodium (10 mg/kg) reduced body temperature by 1.2 ± 0.2°C at 4 hours post-administration (vs. vehicle group) [3]
3. Inhibition of in vivo prostaglandin synthesis (rat model): After oral administration of naproxen sodium (10 mg/kg), rat serum TXB2 levels were measured at 1, 2, 4, 6 hours. The maximum inhibition of TXB2 (91 ± 3%) occurred at 2 hours, and the inhibition rate remained above 70% for 6 hours. Serum PGE2 levels were maximally inhibited by 82 ± 4% at 2 hours, with inhibition lasting for 4 hours [3]

1. COX-1 activity assay: COX-1 was extracted from sheep seminal vesicle microsomes. The reaction system (1 mL) contained 50 mM Tris-HCl buffer (pH 8.0), 2 μM heme, 100 μM arachidonic acid (substrate), and serial dilutions of Naproxen Sodium (RS-3650) (0.001-1 μM). The mixture was incubated at 37°C for 10 minutes, then the reaction was terminated by adding 100 μL of 1 M HCl. The concentration of PGE2 (COX-1 product) was measured using a radioimmunoassay (RIA) kit. The inhibition rate was calculated as (1 - PGE2 concentration of sample/PGE2 concentration of control) × 100%, and IC50 was determined by nonlinear regression [1]
2. COX-2 activity assay: COX-2 was prepared from LPS-stimulated J774.2 macrophages (incubated with LPS 1 μg/mL for 16 hours). The reaction system was identical to COX-1 assay, except that the buffer included 10 μM indomethacin (a COX-1 inhibitor) to exclude COX-1 activity. After incubation and termination, PGE2 was detected by RIA, and IC50 of naproxen sodium for COX-2 was calculated using the same method as COX-1 [1]

1. Primary mouse lung fibroblast culture and activation assay: Lung tissues from bleomycin-treated mice (day 7 post-bleomycin) were minced and digested with collagenase (0.2%) and hyaluronidase (0.1%) at 37°C for 2 hours. The cell suspension was filtered through a 70 μm strainer, and fibroblasts were purified by differential adhesion (plated for 1 hour, non-adherent cells removed). Fibroblasts were cultured in DMEM + 10% fetal bovine serum (FBS) and plated in 6-well plates at 1×10⁵ cells/well. Cells were treated with naproxen sodium (1 μM, 10 μM) for 48 hours. Cells were lysed for Western blot (detection of α-SMA, Col1α1) or culture supernatant was collected for ELISA (PGE2 detection) [2]
2. Ex vivo whole blood prostaglandin synthesis assay: Rat whole blood (0.5 mL) was collected in heparinized tubes 2 hours after oral naproxen sodium administration. Blood samples were incubated with calcium ionophore A23187 (10 μM) at 37°C for 30 minutes to stimulate prostaglandin synthesis. The reaction was terminated by centrifugation (3000×g, 10 minutes), and plasma was collected. Plasma TXB2 and PGE2 concentrations were measured by RIA [3]

Dissolved in 0.9% NaCl; 2.5, 10 or 25 mg/kg; i.v. or i.p. injection
Male Sprague-Dawley rats

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1. Bleomycin-induced lung injury mouse model:
- Animals: Female C57BL/6 mice (8-10 weeks old, 20-22 g), n=24, randomly divided into 4 groups: control group, bleomycin group, bleomycin + naproxen sodium 10 mg/kg group, bleomycin + naproxen sodium 30 mg/kg group (n=6/group).
- Model induction: Mice were anesthetized with isoflurane, and bleomycin (2.5 U/kg, dissolved in 50 μL normal saline) was intratracheally injected; control group received 50 μL normal saline.
- Drug administration: Naproxen Sodium (RS-3650) was dissolved in 0.5% carboxymethyl cellulose (CMC-Na). From day 1 to day 21 post-bleomycin, drug groups received daily oral gavage (volume: 10 μL/g body weight); control and bleomycin groups received 0.5% CMC-Na.
- Sample collection: On day 21, mice were sacrificed, lungs were harvested to measure wet/dry weight ratio, and part of lung tissue was fixed in 4% paraformaldehyde for histopathology, while the rest was homogenized for ELISA [2]
2. Inflammatory pain and fever rat model:
- Animals: Male Sprague-Dawley rats (250-300 g), n=18, randomly divided into 3 groups: vehicle group, naproxen sodium 5 mg/kg group, naproxen sodium 10 mg/kg group (n=6/group).
- Model induction: For pain: 100 μL complete Freund’s adjuvant (CFA) was injected into the right hind paw; for fever: 100 μg/kg LPS (dissolved in normal saline) was intraperitoneally injected.
- Drug administration: Naproxen sodium was dissolved in 0.5% CMC-Na. For pain: Single oral gavage 24 hours after CFA injection; for fever: Single oral gavage 1 hour after LPS injection. Vehicle group received 0.5% CMC-Na.
- Sample collection: For pain: PWL was measured at 1, 2, 4, 6 hours post-drug; for fever: Body temperature was measured every hour for 6 hours. At 2 hours post-drug, blood was collected to measure TXB2 and PGE2 levels [3]

1. Pharmacokinetic parameters in rats: Sprague-Dawley rats were orally administered naproxen sodium (RS-3650) (10 mg/kg), and the following parameters were determined by high performance liquid chromatography (HPLC): - Maximum plasma concentration (Cmax): 42.3 ± 3.5 μg/mL - Time to peak concentration (Tmax): 1.2 ± 0.2 h - Plasma half-life (t1/2): 2.8 ± 0.3 h - Area under the plasma concentration-time curve (AUC0-∞): 185.6 ± 12.4 μg·h/mL - Oral bioavailability: 91.2 ± 4.6% (compared to intravenous naproxen sodium 5 mg/kg) [3] 2. Tissue distribution: After oral administration (10 mg/kg), the highest detectable concentration of naproxen in the liver was 68.5 ± 3.5 μg/mL at Tmax (1.2 h). The highest concentration was 5.2 μg/g in the kidney (45.3 ± 3.8 μg/g) and the highest in the lung (39.2 ± 4.1 μg/g). The peak plasma concentration (Tmax) was 42.3 ± 3.5 μg/mL, indicating moderate tissue permeability [3].

1. General toxicity in mice: In a 21-day lung injury study, naproxen sodium (RS-3650) at 10 mg/kg and 30 mg/kg/day had no significant effect on mouse body weight (final body weight was 22.5 ± 1.2 g and 21.8 ± 1.1 g, respectively, compared to 22.1 ± 1.3 g in the bleomycin group). Serum alanine aminotransferase (ALT) and creatinine levels in the 30 mg/kg group (ALT: 48 ± 5 U/L; creatinine: 0.51 ± 0.04 mg/dL) were similar to those in the control group (ALT: 45 ± 4 U/L; creatinine: 0.49 ± 0.03 mg/dL), indicating no hepatotoxicity or nephrotoxicity [2]. 2. Toxicity in rats: No abnormal behaviors (e.g., somnolence, diarrhea) were observed after a single oral administration of naproxen sodium (10 mg/kg). Serum AST (52 ± 6 U/L) and urea nitrogen (15.3 ± 1.2 mg/dL) levels were within the normal range and showed no significant difference from the control group [3]. 3. Plasma protein binding rate: In rat plasma, naproxen sodium had a high protein binding rate (99.2 ± 0.3%), which was determined by ultrafiltration [3].

[1]. Selectivity of nonsteroidal antiinflammatory drugs as inhibitors of constitutive and inducible cyclooxygenase. Proc Natl Acad Sci U S A. 1993 Dec 15;90(24):11693-7.

[2]. Prevention of bleomycin-induced lung inflammation and fibrosis in mice by naproxen and JNJ7777120 treatment. J Pharmacol Exp Ther. 2014 Nov;351(2):308-16.

[3]. Pharmacokinetic-pharmacodynamic modeling of the inhibitory effects of naproxen on the time-courses of inflammatory pain, fever, and the ex vivo synthesis of TXB2 and PGE2 in rats.

Naproxen sodium is an organic sodium salt composed of equimolar amounts of naproxen (1-) anion and sodium anion. It is a non-narcotic analgesic, cyclooxygenase 2 inhibitor, cyclooxygenase 1 inhibitor, antipyretic, and nonsteroidal anti-inflammatory drug (NSAID). It contains the naproxen (1-) molecule. Naproxen sodium is the sodium salt form of naproxen, which belongs to the arylaceous acid class of NSAIDs and has anti-inflammatory, analgesic, and antipyretic effects. Naproxen sodium reversibly and competitively inhibits cyclooxygenase (COX), thereby blocking the conversion of arachidonic acid into pro-inflammatory prostaglandins. This inhibits the production of prostaglandins involved in pain, inflammation, and fever. It is an anti-inflammatory drug with analgesic and antipyretic effects. Naproxenic acid and its sodium salt are used to treat rheumatoid arthritis and other rheumatic or musculoskeletal diseases, dysmenorrhea, and acute gout. See also: naproxen (containing active ingredient); diphenhydramine hydrochloride; naproxen sodium (ingredient); naproxen sodium; pseudoephedrine hydrochloride (ingredient)... See more... 1. Naproxen sodium (RS-3650) is a nonsteroidal anti-inflammatory drug (NSAID) that preferentially inhibits COX-1. Its anti-inflammatory, analgesic, and antipyretic effects are achieved by inhibiting cyclooxygenase (COX) to reduce prostaglandin synthesis [1,3]. 2. The preventive effect of naproxen sodium against bleomycin-induced pulmonary fibrosis is related to its dual action: inhibiting COX-mediated PGE2 synthesis to reduce inflammation, and inhibiting pulmonary fibroblast activation (downregulating α-SMA and Col1α1) to reduce collagen deposition [2]. 3. Naproxen sodium has a high oral bioavailability (91.2%) and a moderate half-life (2.8 hours) in rats, supporting its clinical application as an oral formulation for the treatment of acute and chronic inflammatory diseases. Its high plasma protein binding rate (99.2%) may help prolong its in vivo efficacy, but attention should be paid to potential drug interactions with other drugs with high protein binding rates [3].

C14H13NAO3

252.24

252.076

26159-34-2

(±)-Naproxen;23981-80-8;Naproxen;22204-53-1;Naproxen etemesil;385800-16-8

23681059

White to off-white solid powder

403.9ºC at 760 mmHg

250-251ºC

154.5ºC

3.01E-07mmHg at 25°C

1.701

0

3

3

18

282

1

C[C@@H](C1=CC2=C(C=C1)C=C(C=C2)OC)C(=O)[O-].[Na+]

CDBRNDSHEYLDJV-FVGYRXGTSA-M

InChI=1S/C14H14O3.Na/c1-9(14(15)16)10-3-4-12-8-13(17-2)6-5-11(12)7-10;/h3-9H,1-2H3,(H,15,16);/q;+1/p-1/t9-;/m0./s1

2-Naphthaleneacetic acid, 6-methoxy-alpha-methyl-, sodium salt, (S)-

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Note: Please store this product in a sealed and protected environment, avoid exposure to moisture.

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: ≥ 0.5 mg/mL (1.98 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 5.0 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: ≥ 0.5 mg/mL (1.98 mM) (saturation unknown) in 10% DMSO + 90% (20% SBE-β-CD in 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 5.0 mg/mL clear DMSO stock solution to 900 μL of 20% SBE-β-CD physiological saline solution and mix evenly.
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.

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Solubility in Formulation 3: ≥ 0.5 mg/mL (1.98 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 5.0 mg/mL clear DMSO stock solution to 900 μL of corn oil and mix evenly.

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Solubility in Formulation 4: 120 mg/mL (475.74 mM) in PBS (add these co-solvents sequentially from left to right, and one by one), clear solution; with ultrasonication.

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 (Please use freshly prepared in vivo formulations for optimal results.)