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Etodolac (AY-24236, Etodolic Acid, AY24,236, AY24236, Lodine, Ramodar, Ultradol) is a potent nonsteroidal anti-inflammatory drug (NSAID) and a non-selective COX inhibitor with potential anti-inflammatory activity. It has been approved for the treatment of inflammation and pain. Etodolac was able to abolish the cell size decrease and block caspase-3/7 activity in TNFα-induced isolated rabbit articular chondrocytes. However, studies have shown that etodolac at 24h could induce cell death in human malignant rhabdoid tumor cells (FRTK-1) in a dose-dependent manner. Additionally, etodolac has shown to increase caspase-8, -9 and -3 activity 3 at 24 or 48 h in FRTK-1.
Physicochemical Properties
| Molecular Formula | C17H21NO3 | |
| Molecular Weight | 287.35 | |
| Exact Mass | 287.152 | |
| CAS # | 41340-25-4 | |
| Related CAS # | (rac)-Etodolac-d3;1276197-46-6 | |
| PubChem CID | 3308 | |
| Appearance | White to off-white solid powder | |
| Density | 1.2±0.1 g/cm3 | |
| Boiling Point | 507.9±45.0 °C at 760 mmHg | |
| Melting Point | 145-1480C | |
| Flash Point | 261.0±28.7 °C | |
| Vapour Pressure | 0.0±1.4 mmHg at 25°C | |
| Index of Refraction | 1.597 | |
| LogP | 3.59 | |
| Hydrogen Bond Donor Count | 2 | |
| Hydrogen Bond Acceptor Count | 3 | |
| Rotatable Bond Count | 4 | |
| Heavy Atom Count | 21 | |
| Complexity | 400 | |
| Defined Atom Stereocenter Count | 0 | |
| InChi Key | NNYBQONXHNTVIJ-UHFFFAOYSA-N | |
| InChi Code | 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) | |
| Chemical Name | (RS)-2-(1,8-Diethyl-4,9-dihydro-3H-pyrano[3,4-b]indol-1-yl)acetic acid | |
| Synonyms |
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| HS Tariff Code | 2934.99.9001 | |
| Storage |
Powder-20°C 3 years 4°C 2 years In solvent -80°C 6 months -20°C 1 month |
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| Shipping Condition | Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs) |
Biological Activity
| Targets |
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] |
| ln Vitro |
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. 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] |
| ln Vivo |
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. 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] |
| Enzyme Assay |
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] |
| Cell Assay |
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] |
| Animal Protocol |
30 mg/kg Mice 1. Paclitaxel-induced peripheral neuropathy model (ICR mice): - Animals: Male ICR mice (n=8/group), 25-30 g. - Model induction: Intraperitoneal injection of paclitaxel (2 mg/kg) every other day for 5 doses (days 1, 3, 5, 7, 9), total dose 10 mg/kg. - Drug administration: Etodolac (AY-24236) was dissolved in 0.5% carboxymethyl cellulose (CMC-Na) and orally administered once daily at 10 mg/kg, 30 mg/kg, or 100 mg/kg from day 1 to day 14. The vehicle group received 0.5% CMC-Na alone. - Evaluation: Mechanical withdrawal threshold was measured using von Frey filaments on days 1, 7, and 14. On day 14, mice were sacrificed, and lumbar spinal cord tissues were collected for Western blot analysis of COX-2 and PGE2 [2] 2. Gastric precancerous lesion model (Wistar rats): - Animals: Male Wistar rats (n=10/group), 180-220 g. - Model induction: Drinking water containing MNNG (100 μg/mL) for 12 weeks to induce extensive metaplastic gastritis. - Drug administration: From week 13 to week 24, etodolac was dissolved in 0.5% CMC-Na and orally administered once daily at 10 mg/kg or 30 mg/kg. The MNNG-only group received 0.5% CMC-Na. - Evaluation: At week 24, rats were sacrificed. Gastric tissues were examined for cancer incidence by histopathology, and gastric mucosal PGE2 levels were measured by EIA [3] 3. Cardiac allograft model (Balb/c and C57BL/6 mice): - Animals: Recipients: Balb/c mice (female, n=6/group), 20-25 g; Donors: C57BL/6 mice (male), 20-25 g. - Model induction: Heterotopic cardiac transplantation (abdominal aorta and inferior vena cava anastomosis) under isoflurane anesthesia. - Drug administration: Etodolac was dissolved in 0.5% CMC-Na and orally administered once daily at 30 mg/kg or 60 mg/kg, starting 1 day before transplantation and continuing for 21 days. The vehicle group received 0.5% CMC-Na. - Evaluation: Allograft survival was monitored by daily abdominal palpation (cessation of heartbeat = rejection). At rejection, grafts were collected for flow cytometry (CD4+ and CD8+ T cell infiltration) [4] |
| ADME/Pharmacokinetics |
Absorption, Distribution and Excretion Based on mass balance studies, the systemic bioavailability of etodolac from either the tablet or capsule formulation is at least 80%. It is not known whether etodolac is excreted in human milk; however, based on its physical-chemical properties, excretion into breast milk is expected. Etodolac is extensively metabolized in the liver. The hydroxylated-etodolac metabolites undergo further glucuronidation followed by renal excretion and partial elimination in the feces (16% of dose). Approximately 1% of a etodolac dose is excreted unchanged in the urine with 72% of the dose excreted into urine as parent drug plus metabolite. 390 mL/kg Oral cl=49.1 mL/h/kg [Normal healthy adults] Oral cl=49.4 mL/h/kg [Healthy males (18-65 years)] Oral cl=35.7 mL/h/kg [Healthy females (27-65 years)] Oral cl=45.7 mL/h/kg [Eldery (>65 years)] Oral cl=58.3 mL/h/kg [Renal impairement (46-73 years)] Oral cl=42.0 mL/h/kg [Hepatic impairement (34-60 years)] Metabolism / Metabolites Etodolac is extensively metabolized in the liver. Renal elimination of etodolac and its metabolites is the primary route of excretion (72%). Metabolites found in urine (with percents of the administered dose) are: unchanged etodolac (1%), etodolac glucuronide (13%), hydroxylated metabolites (6-, 7-, and 8-OH; 5%), hydroxylated metabolite glucuronides (20%), and unidentified metabolites (33%). Fecal excretion accounts for 16% of its elimination. Etodolac has known human metabolites that 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-trihydroxyoxane-2-carboxylic acid. Biological Half-Life Terminal t1/2, 7.3 ± 4.0 hours. Distribution t1/2, 0.71 ± 0.50 hours |
| Toxicity/Toxicokinetics |
Hepatotoxicity Prospective studies show that 1% to 2% of patients taking etodolac experience at least transient serum aminotransferase elevations. These may resolve even with drug continuation. Marked aminotransferase elevations (>3 fold elevated) occur in Likelihood score: C (probable rare cause clinically apparent liver injury). Effects During Pregnancy and Lactation ◉ Summary of Use during Lactation Because no information is available on the use of etodolac during breastfeeding, an alternate drug may be preferred, especially while nursing a newborn or preterm infant. ◉ Effects in Breastfed Infants Relevant published information was not found as of the revision date. ◉ Effects on Lactation and Breastmilk Relevant published information was not found as of the revision date. Protein Binding > 99% bound, primarily to albumin 1. In vivo general toxicity: In the 24-week rat gastric precancerous lesion study, Etodolac (AY-24236) at 10 mg/kg and 30 mg/kg/day had no significant effect on rat body weight (final weight: 385 ± 25 g and 378 ± 22 g, respectively, vs. 392 ± 28 g in MNNG-only group). No gross pathological changes were observed in the liver, kidney, or stomach after necropsy [3] 2. In vivo organ toxicity: In the 21-day mouse cardiac allograft study, serum levels of alanine transaminase (ALT) and creatinine in the 60 mg/kg etodolac group (ALT: 45 ± 8 U/L; creatinine: 0.52 ± 0.06 mg/dL) were similar to those in the vehicle group (ALT: 42 ± 7 U/L; creatinine: 0.50 ± 0.05 mg/dL), indicating no significant hepatotoxicity or nephrotoxicity [4] |
| References |
[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. |
| Additional Infomation |
Etodolac can cause developmental toxicity and female reproductive toxicity according to state or federal government labeling requirements. Etodolac is a monocarboxylic acid that is acetic acid in which one of the methyl hydrogens is substituted by a 1,8-diethyl-1,3,4,9-tetrahydropyrano[3,4-b]indol-1-yl moiety. A preferential inhibitor of cyclo-oxygenase 2 and non-steroidal anti-inflammatory, it is used for the treatment of rheumatoid arthritis and osteoarthritis, and for the alleviation of postoperative pain. Administered as the racemate, only the (S)-enantiomer is active. It has a role as a non-steroidal anti-inflammatory drug, a cyclooxygenase 2 inhibitor, a non-narcotic analgesic and an antipyretic. It is a monocarboxylic acid and an organic heterotricyclic compound. Etodolac is a non-steroidal anti-inflammatory drug (NSAID) with anti-inflammatory, analgesic and antipyretic properties. Its therapeutic effects are due to its ability to inhibit prostaglandin synthesis. It is indicated for relief of signs and symptoms of rheumatoid arthritis and osteoarthritis. Etodolac is a Nonsteroidal Anti-inflammatory Drug. The mechanism of action of etodolac is as a Cyclooxygenase Inhibitor. Etodolac is a nonsteroidal antiinflammatory drug (NSAID) that is available by prescription only and is used long term for therapy of chronic arthritis and short term for acute pain. Etodolac has been linked to rare instances of clinically apparent drug induced liver disease. Etodolac is a pyranocarboxylic acid and non-steroidal anti-inflammatory drug (NSAID) with antipyretic and analgesic activities. Etodolac inhibits the activity of cyclooxygenase I and II, thereby preventing the formation of prostaglandin which is involved in the induction of pain, fever, and inflammation. It also inhibits platelet aggregation by blocking platelet cyclooxygenase and the subsequent formation of thromboxane A2. A non-steroidal anti-inflammatory agent and cyclooxygenase-2 (COX-2) inhibitor with potent analgesic and anti-arthritic properties. It has been shown to be effective in the treatment of OSTEOARTHRITIS; RHEUMATOID ARTHRITIS; ANKYLOSING SPONDYLITIS; and in the alleviation of postoperative pain (PAIN, POSTOPERATIVE). Drug Indication For acute and long-term management of signs and symptoms of osteoarthritis and rheumatoid arthritis, as well as for the management of pain. FDA Label Mechanism of Action Similar to other NSAIDs, the anti-inflammatory effects of etodolac result from inhibition of the enzyme cycooxygenase (COX). This decreases the synthesis of peripheral prostaglandins involved in mediating inflammation. Etodolac binds to the upper portion of the COX enzyme active site and prevents its substrate, arachidonic acid, from entering the active site. Etodolac was previously thought to be a non-selective COX inhibitor, but it is now known to be 5 – 50 times more selective for COX-2 than COX-1. Antipyresis may occur by central action on the hypothalamus, resulting in peripheral dilation, increased cutaneous blood flow, and subsequent heat loss. Pharmacodynamics Etodolac is an anti-inflammatory agent with analgesic and antipyretic properties. It is used to treat osteoarthritis, rheumatoid arthritis and control acute pain. The therapeutic effects of etodolac are achieved via inhibition of the synthesis of prostaglandins involved in fever, pain, swelling and inflammation. Etodolac is administered as a racemate. As with other 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- conversion _in vivo_. 1. Etodolac (AY-24236) is a non-steroidal anti-inflammatory drug (NSAID) with selective inhibition of COX-2, which is overexpressed in inflammatory tissues and cancerous/precancerous lesions. Its therapeutic effects are mainly mediated by reducing PGE2 synthesis via COX-2 inhibition [2,3,4] 2. The attenuation of paclitaxel-induced peripheral neuropathy by etodolac may be related to the suppression of COX-2/PGE2 signaling in the spinal cord, which reduces central sensitization to pain [2] 3. The preventive effect of etodolac on gastric cancer development suggests that selective COX-2 inhibitors may be a potential chemopreventive strategy for patients with high-risk gastric precancerous lesions (e.g., extensive metaplastic gastritis) [3] 4. The prolongation of cardiac allograft survival by etodolac is associated with reduced T cell infiltration, indicating that COX-2 inhibition may modulate adaptive immune responses involved in allograft rejection [4] |
Solubility Data
| Solubility (In Vitro) |
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| Solubility (In Vivo) |
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. 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. 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. (Please use freshly prepared in vivo formulations for optimal results.) |
| Preparing Stock Solutions | 1 mg | 5 mg | 10 mg | |
| 1 mM | 3.4801 mL | 17.4004 mL | 34.8008 mL | |
| 5 mM | 0.6960 mL | 3.4801 mL | 6.9602 mL | |
| 10 mM | 0.3480 mL | 1.7400 mL | 3.4801 mL |