Cyclooxygenase-1 (COX-1) (IC50: 1.8 ± 0.2 μM for Etodolac (AY-24236)) [2]
- Cyclooxygenase-2 (COX-2) (IC50: 0.25 ± 0.03 μM for Etodolac (AY-24236), selectivity ratio (COX-1/COX-2) = 7.2) [2,3]

Post-marketing research revealed that etodolac's cyclooxygenase inhibition is somewhat selective against COX-2, like to celecoxib and other "COX-2 inhibitors." In contrast to rofecoxib, both etodolac and celecoxib are classified as having "preferential selectivity" toward COX-2 and can fully inhibit COX-1. In hepatoma cells, the r-enantiomer of etodolac (inactive against COX) suppresses the expression of beta-catenin.

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1. COX inhibitory activity: Etodolac (AY-24236) exhibited concentration-dependent inhibition of COX-1 and COX-2. Its IC50 for COX-2 (0.25 ± 0.03 μM) was significantly lower than that for COX-1 (1.8 ± 0.2 μM), resulting in a COX-1/COX-2 selectivity ratio of 7.2. In comparison, indomethacin (a non-selective COX inhibitor) had a COX-1/COX-2 selectivity ratio of 0.8, confirming etodolac’s preference for COX-2 [2]
2. Inhibition of gastric precancerous cell proliferation: Primary gastric mucosal cells isolated from patients with extensive metaplastic gastritis were treated with etodolac (0.1-10 μM) for 48 h. MTT assay showed that etodolac at 1 μM reduced cell viability by 18.3 ± 2.5%, and at 10 μM, the inhibition rate increased to 35.6 ± 3.8%. Western blot analysis revealed that 10 μM etodolac downregulated the expression of COX-2 and prostaglandin E2 (PGE2) synthase by 42.1 ± 4.3% and 38.5 ± 3.9%, respectively, compared to the control group [3]
3. Suppression of inflammatory cytokine production: Lipopolysaccharide (LPS)-stimulated RAW264.7 macrophages were treated with etodolac (1-20 μM) for 24 h. Enzyme-linked immunosorbent assay (ELISA) showed that 10 μM etodolac decreased the secretion of tumor necrosis factor-α (TNF-α) by 52.3 ± 5.1% and interleukin-6 (IL-6) by 47.8 ± 4.8% compared to LPS-only group. No significant effect on cytokine production was observed at concentrations ≤1 μM [4]

Etodolac attenuates paclitaxel-induced peripheral neuropathy by a COX-independent pathway in a mouse model of mechanical allodynia. Etodolac and other NSAIDs inhibits paw swelling and causes gastric mucosal lesions in adjuvant arthritic rats in a dose-dependent manner. Etodolac shows the highest UD(50) value and safety index among these NSAIDs in arthritic rats. Etodolac also shows the highest UD(50) value and safety index, except when its effects are assessed by acetic acid-induced writhing in normal rats. Etodolac dose-dependently inhibits the development of gastric cancer, and no cancer is detected at a dose of 30 mg/kg/day. Etodolac does not affect the extent of inflammatory cell infiltration or oxidative DNA damage, but it significantly inhibits mucosal cell proliferation and dose-dependently represses the development of intestinal metaplasia in the stomachs of Helicobacter pylori (Hp)-infected Mongolian gerbils (MGs). Etodolac alleviates heat-evoked hyperalgesia in the CCI rats and the increase in number of TRAP-positive multinucleated osteoclasts on the CCI-side is abrogated, however, it does not inhibit the decrease of bone mineral content (BMC) and bone mineral density (BMD) on the CCI-side.

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1. Attenuation of paclitaxel-induced peripheral neuropathy (mouse model): Male ICR mice (25-30 g) were intraperitoneally injected with paclitaxel (2 mg/kg) every other day for 5 doses (total 10 mg/kg) to induce mechanical allodynia. Etodolac (AY-24236) was orally administered at doses of 10 mg/kg, 30 mg/kg, or 100 mg/kg once daily from day 1 to day 14. On day 14, the 30 mg/kg and 100 mg/kg etodolac groups showed significantly increased mechanical withdrawal thresholds (measured by von Frey filaments) of 8.5 ± 0.7 g and 10.2 ± 0.9 g, respectively, compared to the paclitaxel-only group (4.1 ± 0.5 g). The 10 mg/kg group had no significant effect (5.2 ± 0.6 g). Additionally, 30 mg/kg etodolac reduced the expression of COX-2 and PGE2 in the lumbar spinal cord by 39.2 ± 4.1% and 45.6 ± 4.7%, respectively [2]
2. Prevention of cancer development in gastric precancerous lesions (rat model): Male Wistar rats (180-220 g) were treated with N-methyl-N'-nitro-N-nitrosoguanidine (MNNG, 100 μg/mL) in drinking water for 12 weeks to induce extensive metaplastic gastritis (precancerous lesion). From week 13 to week 24, rats were orally administered etodolac at 10 mg/kg/day or 30 mg/kg/day. At week 24, the 30 mg/kg etodolac group had a significantly lower gastric cancer incidence (12.5%) compared to the MNNG-only group (45.8%). The 10 mg/kg group had a cancer incidence of 28.3% (not statistically significant vs. MNNG group). Gastric mucosal PGE2 levels in the 30 mg/kg group were reduced by 51.2 ± 5.3% compared to the MNNG-only group [3]
3. Prolongation of cardiac allograft survival (mouse model): Balb/c mice (recipients, female, 20-25 g) received heterotopic cardiac allografts from C57BL/6 mice (donors, male, 20-25 g). Etodolac was orally administered at 30 mg/kg/day or 60 mg/kg/day starting 1 day before transplantation and continuing for 21 days. The median allograft survival time in the 60 mg/kg etodolac group was 18.5 ± 1.2 days, significantly longer than that in the vehicle group (8.2 ± 0.8 days). The 30 mg/kg group had a median survival time of 12.3 ± 1.0 days (significant vs. vehicle group). Flow cytometry showed that 60 mg/kg etodolac reduced the number of CD4+ and CD8+ T cells infiltrating the allograft by 42.3 ± 4.5% and 38.7 ± 4.2%, respectively [4]

1. COX-1 activity assay: COX-1 was extracted from sheep seminal vesicles. The reaction system (200 μL) contained 50 mM Tris-HCl buffer (pH 8.0), 2 μM heme, 100 μM arachidonic acid (substrate), and serial dilutions of Etodolac (AY-24236) (0.01-10 μM). The mixture was incubated at 37°C for 15 min, and the reaction was terminated by adding 20 μL of 1 M HCl. The concentration of PGE2 (the main product of COX-1) was measured using a competitive EIA kit. The inhibition rate was calculated as (1 - PGE2 concentration of sample/PGE2 concentration of control) × 100%, and the IC50 was determined by nonlinear regression analysis [2]
2. COX-2 activity assay: Recombinant human COX-2 (expressed in Sf9 insect cells) was used. The reaction conditions were identical to the COX-1 assay, except that the reaction buffer included 10 μM diclofenac as a COX-1 inhibitor (to eliminate COX-1 contamination). After incubation and termination, PGE2 levels were detected by EIA, and the IC50 of etodolac for COX-2 was calculated using the same method as COX-1 [2,3]

1. Primary gastric mucosal cell viability assay (MTT): Gastric mucosal tissues from patients with extensive metaplastic gastritis were minced and digested with collagenase (0.1%) for 2 h at 37°C. Single cells were filtered through a 70 μm cell strainer and resuspended in RPMI 1640 medium containing 10% fetal bovine serum. Cells were plated in 96-well plates at 5×10³ cells/well and incubated overnight. Etodolac (AY-24236) (0.1-10 μM) was added, and the cells were cultured for 48 h. Then, 20 μL of MTT solution (5 mg/mL) was added, and the plates were incubated for another 4 h. The supernatant was removed, 150 μL of DMSO was added to dissolve formazan crystals, and the absorbance at 570 nm was measured. Cell viability was calculated as (absorbance of sample/absorbance of control) × 100% [3]
2. Western blot for COX-2 and PGE2 synthase in gastric cells: Primary gastric mucosal cells were plated in 6-well plates at 2×10⁵ cells/well and treated with 10 μM etodolac for 48 h. Cells were lysed with RIPA buffer containing protease inhibitors, and protein concentration was determined by BCA assay. Equal amounts of protein (40 μg) were separated by 10% SDS-PAGE and transferred to PVDF membranes. Membranes were blocked with 5% non-fat milk for 1 h, then incubated with primary antibodies against COX-2, PGE2 synthase, and GAPDH (loading control) overnight at 4°C. After washing with TBST, membranes were incubated with horseradish peroxidase-conjugated secondary antibodies for 1 h. Bands were visualized with ECL reagent, and band intensity was quantified using ImageJ software [3]
3. ELISA for inflammatory cytokines in macrophages: RAW264.7 cells were plated in 24-well plates at 1×10⁵ cells/well and stimulated with 1 μg/mL LPS for 2 h. Then, etodolac (1-20 μM) was added, and the cells were cultured for another 22 h. The culture supernatant was collected, and the concentrations of TNF-α and IL-6 were measured using commercial ELISA kits according to the manufacturer’s protocol. Results were normalized to the LPS-only group [4]

Absorption, Distribution and Excretion
Based on mass balance studies, the systemic bioavailability of etodoxacin in tablet or capsule formulations is at least 80%. It is currently unclear whether etodoxacin is excreted into human breast milk; however, based on its physicochemical properties, it is expected to be excreted. Etodoxacin is extensively metabolized in the liver. Hydroxylated etodoxacin metabolites are further glucuronidated and then excreted by the kidneys, with a portion excreted in feces (16% of the dose). Approximately 1% of the etodoxacin dose is excreted unchanged in the urine, and 72% of the dose is excreted unchanged in the urine along with its metabolites.
390 mL/kg
Oral clearance = 49.1 mL/h/kg [Normal healthy adults]
Oral clearance = 49.4 mL/h/kg [Healthy men (18-65 years)]
Oral clearance = 35.7 mL/h/kg [Healthy women (27-65 years)]
Oral clearance = 45.7 mL/h/kg [Older adults (>65 years)]
Oral clearance = 58.3 mL/h/kg [Renal insufficiency (46-73 years)]
Oral clearance = 42.0 mL/h/kg [Hepatic insufficiency (34-60 years)]
Metabolism/Metabolites
Etoduolic acid is extensively metabolized in the liver. The main route of excretion of etoduolic acid and its metabolites is renal excretion (72%). Metabolites found in urine (as a percentage of the administered dose) included: unchanged etodoxa acid (1%), etodoxa acid glucuronide (13%), hydroxylated metabolites (6-, 7-, and 8-OH; 5%), hydroxylated glucuronide (20%), and unidentified metabolites (33%). Fecal excretion accounted for 16% of its elimination. Known metabolites of etodoxa acid include (2S,3S,4S,5R)-6-[2-(1,8-diethyl-4,9-dihydro-3H-pyrano[3,4-b]indol-1-yl)acetyl]oxy-3,4,5-trihydroxyoxacyclohexane-2-carboxylic acid. Biological Half-Life Terminal half-life: 7.3 ± 4.0 hours. Distribution half-life: 0.71 ± 0.50 hours.

Hepatotoxicity
Prospective studies have shown that 1% to 2% of patients taking etodoxacin experience at least transient increases in serum transaminases. These symptoms may resolve with continued use. Significant increases in aminotransferases (more than 3-fold) occur at a probability score of C (potentially rare, but may lead to clinically significant liver damage). Pregnancy and Lactation Effects
◉ Overview of Use During Lactation
Because there is no information available regarding the use of etodoxacin during lactation, alternative medications may be preferred, especially for breastfed newborns or preterm infants.
◉ Effects on Breastfed Infants
No published information found as of the revision date.
◉ Effects on Lactation and Breast Milk
No published information found as of the revision date.

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Protein binding
>99% binding, mainly to albumin

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1. General toxicity in vivo: In a 24-week rat study of precancerous gastric lesions, etodoolsic acid (AY-24236) at doses of 10 mg/kg and 30 mg/kg/day had no significant effect on rat body weight (final body weight was 385 ± 25 g and 378 ± 22 g, respectively, compared to 392 ± 28 g in the MNNG group alone). No obvious pathological changes were observed in the liver, kidneys or stomach after autopsy [3]
2. In vivo organ toxicity: In a 21-day mouse heart allogeneic transplantation study, the serum alanine aminotransferase (ALT) and creatinine levels (ALT: 45 ± 8 U/L; creatinine: 0.52 ± 0.06 mg/dL) in the 60 mg/kg etodoolic acid group were similar to those in the carrier group (ALT: 42 ± 7 U/L; creatinine: 0.50 ± 0.05 mg/dL), indicating that no obvious hepatotoxicity or nephrotoxicity was observed [4]

[1]. Biochemical and pharmacological profile of a tetrasubstituted furanone as a highly selective COX-2 inhibitor. Br J Pharmacol. 1997 May;121(1):105-17.

[2]. Etodolac, a cyclooxygenase-2 inhibitor, attenuates paclitaxel-induced peripheral neuropathy in a mouse model of mechanical allodynia. J Pharmacol Exp Ther. 2012 Jul;342(1):53-60.

[3]. Preventive effects of etodolac, a selective cyclooxygenase-2 inhibitor, on cancer development in extensive metaplastic gastritis, a Helicobacter pylori-negative precancerous lesion. Int J Cancer. 2010 Mar 15;126(6):146.

[4]. Administration of the selective cyclooxygenase (COX)-2 inhibitor etodolac prolongs cardiac allograft survival in a mouse model. Mol Med Report. 2010 Sep-Oct;3(5):771-4.

According to state or federal labeling requirements, etodoxa acid may cause developmental toxicity and female reproductive toxicity. Etodoxa acid is a monocarboxylic acid, a structural unit in the acetic acid molecule in which a methyl hydrogen atom is replaced by a 1,8-diethyl-1,3,4,9-tetrahydropyrano[3,4-b]indole-1-yl group. It is a cyclooxygenase-2 preferential inhibitor and a nonsteroidal anti-inflammatory drug (NSAID) used to treat rheumatoid arthritis and osteoarthritis, as well as to relieve postoperative pain. When administered in racemic form, only the (S)-enantiomer of etodoxa acid is active. It has the effects of a nonsteroidal anti-inflammatory drug, a cyclooxygenase-2 inhibitor, a non-narcotic analgesic, and an antipyretic. It is a monocarboxylic acid and an organic heterocyclic tricyclic compound. Etodoxa acid is a nonsteroidal anti-inflammatory drug (NSAID) with anti-inflammatory, analgesic, and antipyretic effects. Its therapeutic effect stems from its ability to inhibit prostaglandin synthesis. It is indicated for the relief of signs and symptoms of rheumatoid arthritis and osteoarthritis. Etodoxa acid is a nonsteroidal anti-inflammatory drug. Etoduolic acid's mechanism of action is as a cyclooxygenase inhibitor. Etoduolic acid is a prescription drug used long-term for the treatment of chronic arthritis and short-term for the treatment of acute pain. Etoduolic acid has been associated with rare, clinically significant cases of drug-induced liver disease. Etoduolic acid is a pyranocarboxylic acid nonsteroidal anti-inflammatory drug (NSAID) with antipyretic and analgesic effects. Etoduolic acid inhibits the activity of cyclooxygenase I and II, thereby preventing the production of prostaglandins, which are involved in pain, fever, and inflammation. It also inhibits platelet aggregation by blocking platelet cyclooxygenase and its subsequent formation of thromboxane A2. Etoduolic acid is a nonsteroidal anti-inflammatory drug and a cyclooxygenase-2 (COX-2) inhibitor with potent analgesic and anti-arthritis effects. It has been shown to be effective in the treatment of osteoarthritis, rheumatoid arthritis, and ankylosing spondylitis. It is also used to relieve postoperative pain. Drug Indications For the acute and long-term treatment of signs and symptoms of osteoarthritis and rheumatoid arthritis, and for pain management. FDA Label Mechanism of Action Similar to other nonsteroidal anti-inflammatory drugs (NSAIDs), etodoxacin's anti-inflammatory effect stems from the inhibition of cyclooxygenase (COX). This reduces the synthesis of peripheral prostaglandins involved in inflammation-mediated processes. Etodoxacin binds to the upper part of the COX enzyme's active site, preventing its substrate arachidonic acid from entering the active site. Etodoxacin was previously considered a non-selective COX inhibitor, but it is now known to be 5-50 times more selective for COX-2 than for COX-1. Its antipyretic effect may be achieved through a central mechanism acting on the hypothalamus, leading to peripheral vasodilation, increased skin blood flow, and consequently, heat dissipation. Pharmacodynamics Etodoxacin is an anti-inflammatory drug with analgesic and antipyretic effects. It is used to treat osteoarthritis, rheumatoid arthritis, and to control acute pain. Etodoxacin exerts its therapeutic effect by inhibiting the synthesis of prostaglandins involved in fever, pain, swelling, and inflammation. Etodoxacin is administered in racemic form. As with other nonsteroidal anti-inflammatory drugs (NSAIDs), the S-form has been shown to be active, while the R-form is inactive. Both enantiomers are stable, and there is no evidence of R-to-S-form conversion in vivo.

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1. Etodoxacin (AY-24236) is a nonsteroidal anti-inflammatory drug (NSAID) that selectively inhibits COX-2, which is overexpressed in inflamed tissues and cancer/precancerous lesions. Its therapeutic effect is mainly achieved by inhibiting COX-2 to reduce PGE2 synthesis [2,3,4].
2. Etodoxacin may alleviate paclitaxel-induced peripheral neuropathy by inhibiting the COX-2/PGE2 signaling pathway in the spinal cord, thereby reducing central pain sensitization [2].
3. The preventive effect of etoposide on gastric cancer suggests that selective COX-2 inhibitors may be a potential chemoprevention strategy for patients with high-risk precancerous lesions of the stomach (e.g., extensive metaplastic gastritis)[3].
4. The prolongation of heart transplant survival by etoposide is associated with reduced T-cell infiltration, suggesting that COX-2 inhibition may regulate the adaptive immune response involved in transplant rejection[4].

C17H21NO3

287.35

287.152

41340-25-4

(rac)-Etodolac-d3;1276197-46-6

3308

White to off-white solid powder

1.2±0.1 g/cm3

507.9±45.0 °C at 760 mmHg

145-1480C

261.0±28.7 °C

0.0±1.4 mmHg at 25°C

1.597

3.59

2

3

4

21

400

0

NNYBQONXHNTVIJ-UHFFFAOYSA-N

InChI=1S/C17H21NO3/c1-3-11-6-5-7-12-13-8-9-21-17(4-2,10-14(19)20)16(13)18-15(11)12/h5-7,18H,3-4,8-10H2,1-2H3,(H,19,20)

(RS)-2-(1,8-Diethyl-4,9-dihydro-3H-pyrano[3,4-b]indol-1-yl)acetic acid

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 (8.70 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 (8.70 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 25.0 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: ≥ 2.5 mg/mL (8.70 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.)