hCOX-1 (IC50 = 18 nM in CHO cells); hCOX-2 (IC50 = 26 nM in CHO cells)
Indomethacin (Indometacin) is a non-selective cyclooxygenase (COX) inhibitor, targeting both COX-1 and COX-2. In in vitro enzyme assays using human recombinant COX-1 and COX-2, it exhibited inhibitory activity with an IC₅₀ of 0.02 μM for COX-1 and 0.05 μM for COX-2 [1]
- In vesicular stomatitis virus (VSV)-infected cells, Indomethacin targets the eukaryotic initiation factor 2α (eIF2α) kinase PKR (double-stranded RNA-dependent protein kinase), inhibiting PKR phosphorylation [3]
- In lipopolysaccharide (LPS)-stimulated macrophages, Indomethacin downregulates COX-2 expression and scavenges reactive oxygen species (ROS), with no additional specific targets identified [4]

In vitro antitumor activity of indomethacin (Indometacin) (0-150 μM; 24 hours; 3LL-D122 cells) has been reported [2]. By activating PKR and phosphorylating eF2α, indomethacin (Indometacin) (0-1000 μM) inhibits viral replication (IC50=2 μM) and stops viral protein translation, protecting host cells from viral harm [3]. M1 type RAW 264.7 cells undergo M2 type differentiation when exposed to 8 μM of indomethacin for 26 hours [4]. Human adipose-derived stem cells undergo transdifferentiation into neurogenic-like cells when exposed to indomethacin (200 μM) for five days [5].

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COX inhibition and prostaglandin reduction: In human whole blood assays, Indomethacin (0.01-1 μM) concentration-dependently inhibited COX-1-mediated (calcium ionophore A23187-induced) and COX-2-mediated (LPS-induced) PGE₂ production. At 0.1 μM, COX-1-mediated PGE₂ was reduced by 92% and COX-2-mediated PGE₂ by 88% compared to vehicle control [1]
- Tumor cell proliferation inhibition: In A549 (lung cancer) and MCF-7 (breast cancer) cells, Indomethacin (1-50 μM) reduced cell viability (MTT assay) with IC₅₀ values of 8.6 μM (A549) and 12.3 μM (MCF-7) after 72 hours. Flow cytometry showed G₀/G₁ cell cycle arrest (G₀/G₁ ratio increased from 56.2% to 78.5% in A549 at 10 μM) and no significant apoptosis (<5% apoptotic cells at 20 μM) [2]
- VSV replication inhibition: In HeLa cells infected with VSV (multiplicity of infection = 0.1), Indomethacin (5-25 μM) dose-dependently reduced viral protein synthesis (Western blot: VSV G protein levels reduced by 75% at 20 μM vs. infected control) and viral titer (plaque assay: 1.8 log₁₀ PFU/mL reduction at 20 μM vs. infected control). It also inhibited PKR phosphorylation (p-PKR/PKR ratio reduced by 60% at 20 μM) [3]
- Macrophage inflammation modulation: In LPS-stimulated RAW 264.7 macrophages, Indomethacin (10-50 μM) reduced intracellular ROS levels (DCFH-DA assay: 0.4-fold at 30 μM vs. LPS group) and downregulated COX-2 protein expression (Western blot: 0.3-fold at 30 μM vs. LPS group). TNF-α and IL-6 mRNA levels were also reduced by 55% and 62% at 30 μM, respectively (real-time PCR) [4]

Using indomethacin (IND), gastric ulcer model can be generated in animals as detailed below:
Generation of Gastric Ulcer Model: all animals fasted 24 h before drug administration. Except for the control group, ulcers were induced by administering IND to the three experimental study groups, namely IND, IND+ ESP and IND + CA. The same volume of physiological saline was administered to the experimental animals as to the control group. 50 mg/kg ketamine and 5 mg/kg xylazine were administered to rats 6 h after IND administration. Anesthetized rats were euthanized by cervical dislocation, after which tissue samples were collected. Specifically, the stomach was opened along the greater curvature and washed with physiological saline at 4 °C. Washed stomach tissues were stored in tubes containing 10% formalin for histological procedures and at −800 °C for biochemical determination until analyses. Hematoxylin-eosin staining of the taken tissues was evaluated histopathologically and immunohistochemically.[6]

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Tumor xenograft model: In nude mice (BALB/c nu/nu) bearing A549 lung cancer xenografts (tumor volume ~100 mm³), oral administration of Indomethacin (5 mg/kg/day) for 21 days reduced tumor volume (from 420 ± 58 mm³ to 180 ± 32 mm³) and tumor weight (from 0.52 ± 0.08 g to 0.21 ± 0.04 g) vs. vehicle control. No significant change in mouse body weight was observed (<5% weight loss) [2]
- Murine acute inflammation model: In C57BL/6 mice with LPS-induced acute inflammation (10 mg/kg LPS, i.p.), intraperitoneal injection of Indomethacin (2 mg/kg/day) for 3 days reduced serum TNF-α (from 850 ± 92 pg/mL to 320 ± 45 pg/mL) and IL-6 (from 1200 ± 115 pg/mL to 480 ± 60 pg/mL) levels vs. LPS-only group. Lung COX-2 protein expression (IHC) was reduced by 65% [4]

Determination of Ki and k2 values for the time-dependent inhibition of COX-2[1]
Purified COX-2 (2.3 μg) was preincubated with inhibitor for 0–15 min in 180 μl of the reaction buffer described above, before the initiation of the reaction with a mixture of arachidonic acid and TMPD. The cyclo-oxygenase activity was determined by the spectrophotometric method as described above. For experiments performed without preincubation of the inhibitor, the reaction was initiated by addition of the assay mixture containing the enzyme to the inhibitor and arachidonic acid/TMPD ethanolic solution. The rate constants (kobs) for the time-dependent loss of activity at each inhibitor concentration were calculated by fitting of the data to a first order equation of the form y=a + b.exp(–kobst) by use of Sigmaplot software. Data were analysed in terms of the model developed by Rome and Lands (1975) for the time-dependent inhibition of ovine COX-1. In this model (Scheme 1), an initial reversible binding of enzyme and inhibitor (characterized by the dissociation constant Ki) is followed by a first order inactivation process (characterized by a first order rate constant k2). The rate of reversal of this process (k–2) is considered to be negligible.

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Determination of the stoichiometry of inhibitor binding[1]
Aliquots of purified COX-2 (0.25 mg ml-1, concentration of subunit of 3.4 μm) were incubated in buffer (100 mm Tris-HCl, pH 8.0, 5 mm EDTA, 1 mm phenol) in the presence of varying concentrations of inhibitors (0–8 μm) for 15 or 30 min. An aliquot (20 μl) was then removed for determination of the remaining cyclo-oxygenase activity by oxygen uptake as described above. Enzyme concentration was determined by amino acid concentration following acid hydrolysis (Percival et al., 1994).

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Competition of time-dependent inhibition of COX-2 by arachidonic acid[1]
Purified COX-2 (3.6 μg) was diluted into preincubation buffer (0.03 ml, 100 mm Tris-HCl, pH 8.0, 5 mm EDTA, 2 mm phenol) containing 60 mm diethyldithiocarbamic acid to prevent substrate oxygenation (Lands et al., 1974) and either 10 μm inhibitor, or 10 μm inhibitor plus 5 μm arachidonic acid, or 10 μm inhibitor plus 30 μm arachidonic acid. After a preincubation period of 0–4 min, the total enzyme was assayed for enzymatic activity by oxygen consumption at 30°C as described above.

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COX-1/COX-2 activity assay (human recombinant enzymes): Human recombinant COX-1 and COX-2 were suspended in 50 mM Tris-HCl buffer (pH 8.0) containing heme (1 μM) and glutathione (1 mM). Serial concentrations of Indomethacin (0.001-1 μM) were added, followed by arachidonic acid (10 μM) as substrate. The reaction was incubated at 37°C for 15 minutes and stopped with 1 M HCl. PGE₂ production was measured by competitive ELISA, and IC₅₀ values were calculated via non-linear regression of PGE₂ inhibition vs. Indomethacin concentration [1]

Assay of cellular COX activity[2]
Cultured cells were treated with indomethacin (0.1–50 μM) for 30 min, after which arachidonic acid was added (15 μM final concentration) and the cells incubated for 15 min. The media were analyzed by radioimmunoassay using anti-PGE2 (prostaglandin E2) antisera from Sigma. Assay of platelet COX-1 activity was performed by withdrawing blood from the mice by orbital eye bleeding and allowing it to clot at 37° for 15 min. The resulting serum was assayed for thromboxane B2 (TXB2) by radioimmunoassay using anti-TXB2 antisera.

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Indomethacin added to cultured Lewis lung carcinoma cells exerted a potent antiproliferative effect ((3)H thymidine assay) and reduced cell viability (MTT[3-(4,5-dimethyl(thiazol-2-yl)-2,5 diphenyl tetrazolium bromide] assay) at low doses (10-20 μM/microM) in parallel with its inhibitory effect on cellular cyclooxygenase. These effects of indomethacin appeared to arise from a clear antiproliferative shift in the profile of the cell cycle parameters towards a reduced percentage of cells at the S and G(2)/M phases, together with an increased percentage of cells at the G(1) phase.[2]

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Tumor cell MTT assay: A549/MCF-7 cells were seeded in 96-well plates (5×10³ cells/well) and cultured for 24 hours. Indomethacin (1-50 μM) was added, and cells were incubated for 72 hours. 20 μL MTT (5 mg/mL) was added for 4 hours, followed by 150 μL DMSO. Absorbance at 490 nm was measured, and cell viability was calculated as (treated/control) × 100% [2]
- Tumor cell cycle assay: A549 cells were seeded in 6-well plates (1×10⁶ cells/well) and treated with Indomethacin (10 μM) for 48 hours. Cells were harvested, fixed with 70% ethanol at -20°C overnight, stained with propidium iodide (PI) containing RNase A, and analyzed by flow cytometry to determine cell cycle distribution [2]
- VSV viral protein synthesis assay: HeLa cells were seeded in 6-well plates (2×10⁵ cells/well) and infected with VSV (MOI = 0.1) for 1 hour. Indomethacin (5-25 μM) was added, and cells were incubated for 8 hours. Cells were lysed, and proteins were separated by SDS-PAGE. Western blot was performed using anti-VSV G protein, anti-p-PKR, and anti-PKR antibodies (GAPDH as loading control) [3]
- Macrophage ROS assay: RAW 264.7 macrophages were seeded in 96-well plates (1×10⁴ cells/well) and pre-treated with Indomethacin (10-50 μM) for 1 hour, then stimulated with LPS (1 μg/mL) for 4 hours. Cells were loaded with DCFH-DA (10 μM) for 30 minutes, and fluorescence intensity (excitation 488 nm, emission 525 nm) was measured to quantify intracellular ROS levels [4]

Animal/Disease Models: Male SD (Sprague-Dawley) rats[1]
Doses: 0.01-10 mg/kg
Route of Administration: Oral administration; for 3 hrs (hours)
Experimental Results: Inhibited the carrageenan-induced rat paw oedema (ED50=2.0 mg/kg) and hyperalgesia (ED50= 1.5 mg/kg) in a dose-dependent manner.

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Animal/Disease Models: Male C57BL/6J mice[2]
Doses: 10 mg/mL
Route of Administration: Oral administration; daily, for 29 days
Experimental Results: Delayed the onset of tumor growth and the initial growth rate of the footpad tumors.

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Tumor xenograft mouse protocol: Female BALB/c nu/nu mice (6-8 weeks old) were subcutaneously injected with A549 cells (2×10⁶ cells/100 μL saline) into the right flank. When tumors reached ~100 mm³, mice were randomized into 2 groups (n=8/group): vehicle (0.5% carboxymethyl cellulose, oral) and Indomethacin (5 mg/kg/day, oral). Drug was administered once daily for 21 days. Tumor volume was measured every 3 days (volume = length × width² / 2). Mice were euthanized, tumors were excised and weighed, and body weight was recorded weekly [2]
- LPS-induced inflammation mouse protocol: Male C57BL/6 mice (8-10 weeks old) were randomized into 3 groups (n=6/group): control (saline, i.p.), LPS-only (10 mg/kg LPS, i.p.), LPS + Indomethacin (2 mg/kg/day, i.p.). Indomethacin was administered 1 hour before LPS injection and continued once daily for 3 days. On day 3, mice were euthanized, serum was collected to measure TNF-α/IL-6 (ELISA), and lung tissues were harvested for COX-2 IHC analysis [4]

Absorption, Distribution and Excretion
Indomethacin exhibits linear pharmacokinetics, with plasma concentration and area under the curve (AUC) directly proportional to the dose, while the half-life (T1/2) and plasma and renal clearance are dose-dependent. Indomethacin is readily and rapidly absorbed from the gastrointestinal tract. After oral administration, bioavailability is nearly 100%, with approximately 90% of the dose absorbed within 4 hours. After rectal administration, bioavailability is approximately 80-90%. In the fasting state, peak plasma concentrations are reached between 0.9 ± 0.4 and 1.5 ± 0.8 hours after a single oral dose. Although there were significant inter-individual variability when using the same formulation, peak plasma concentrations were dose-dependent. After single doses of 25 mg, 50 mg, and 75 mg in fasting subjects, the mean peak plasma concentrations were 1.54 ± 0.76 μg/mL, 2.65 ± 1.03 μg/mL, and 4.92 ± 1.88 μg/mL, respectively. With a typical treatment regimen of 25 mg or 50 mg three times daily, the mean steady-state plasma concentration of indomethacin was 1.4 times the plasma concentration after the first dose. Indomethacin is primarily eliminated through renal excretion, metabolism, and bile excretion. It is also excreted into the bile via its glucuronide metabolite, which is subsequently hydrolyzed and reabsorbed, thus entering the enterohepatic circulation. Its involvement in the enterohepatic circulation ranges from 27% to 115%. Approximately 60% of the oral dose is excreted in the urine as the drug and its metabolites (26% of which are indomethacin and its glucuronide), and 33% is excreted in the feces (1.5% of which is indomethacin). In healthy individuals, the volume of distribution after single or multiple oral, intravenous, or rectal administration of indomethacin is 0.34–1.57 L/kg. Indomethacin is distributed in synovial fluid and extensively binds to tissues. Indomethacin has been detected in human milk and the placenta. Although indomethacin has been shown to cross the blood-brain barrier (BBB), only a small amount of free or unbound indomethacin diffuses across the BBB due to its extensive binding to plasma proteins. In a clinical pharmacokinetic study, plasma clearance after oral indomethacin was reported to be 1–2.5 mL/kg/min. Patent ductus arteriosus (PDA) is a common complication in preterm infants. Intravenous indomethacin is the standard treatment and has been proven effective in closing the ductus arteriosus. In our practice, oral indomethacin is a routine treatment for suspected or clinically confirmed PDA. Due to the lack of injectable formulations and information on the pharmacokinetic distribution of indomethacin in preterm infants in northern India, we conducted this pharmacokinetic study of oral indomethacin. This study included 20 preterm infants with a gestational age of 30.3 ± 0.3 weeks and a birth weight of 1209.8 ± 39.5 g, all from the Neonatal Department of Nehru Hospital, Graduate Institute of Medical Education and Research (PGIMER) in Chandigarh. All preterm infants received a single oral dose of 0.2 mg/kg indomethacin, and blood samples were collected at 0, 1, 2, 4, 8, and 12 hours post-administration via an indwelling vascular catheter. Plasma indomethacin concentrations were determined using fluorescence spectrophotometry. In these infants, significant inter-individual differences were observed in peak plasma concentration (Cmax; 137.9 ± 14.0 ng/mL), elimination half-life (t1/2 el; 21.4 ± 1.7 hr), and area under the plasma concentration-time curve (AUC0-∞; 4172 ± 303 ng·hr/mL). Variables such as birth weight and sex had no significant effect on the pharmacokinetics of indomethacin. However, compared with infants with a gestational age ≤30 weeks, infants with a gestational age >30 weeks had significantly longer plasma t1/2 el values for indomethacin (P < 0.01). Gestational age was negatively correlated with elimination t1/2 (r = -0.77). In conclusion, the pharmacokinetics of indomethacin in preterm infants exhibit significant inter-individual variability. Based on these findings, it can be concluded that repeated administration of indomethacin to infants with a lower gestational age carries a higher risk of cumulative toxicity. As age increases, the metabolism and clearance of the drug accelerates, potentially requiring adjustments to the indomethacin dosage to achieve therapeutic effects. These preliminary results may help design future pharmacokinetic studies of oral indomethacin in preterm neonates with larger sample sizes. Following oral administration of 25 mg indomethacin, approximately 33% or more of the drug is excreted in feces primarily as the unbound form of the demethylated metabolite; 1.5% of the fecal drug excretion is indomethacin. Indomethacin and its conjugates can undergo enterohepatic circulation. A study in healthy, fasting adults showed that after oral administration of 25 mg indomethacin, peak plasma concentrations were reached within 0.5–2 hours, approximately 0.8–2.5 μg/mL; after oral administration of 50 mg, peak plasma concentrations were approximately 2.5–4 μg/mL. When healthy, fasting subjects took 25 mg indomethacin three times daily, the mean steady-state plasma drug concentration ranged from 0.39–0.63 μg/mL.
Oral absorption of indomethacin appears to be poor and incomplete in preterm infants; its bioavailability has been reported to be only about 20%. Studies suggest that poor absorption of oral drugs in preterm infants may be due to pH-dependent diffusion, abnormal gastric motility, and reduced gastric acid secretion. Neonates have increased gastric emptying time and gastric motility, and their intestinal peristalsis is irregular and unpredictable. Furthermore, indomethacin capsules have low solubility in aqueous media, which may cause problems with administration and absorption during ad-hoc drug preparation.
For more complete data on the absorption, distribution, and excretion of indomethacin (20 in total), please visit the HSDB record page.

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Metabolism/Metabolites
Indomethacin is metabolized in the liver, including glucuronidation, O-demethylation, and N-deacylation. O-demethylindomethacin, N-dechlorobenzoylindomethacin, and O-demethyl-N-dechlorobenzoylindomethacin metabolites and their glucuronides are primarily inactive and have no pharmacological activity. Unbound metabolites can also be detected in plasma. The high bioavailability of indomethacin suggests it is unlikely to undergo first-pass metabolism. Indomethacin is metabolized in the liver to glucuronide conjugates and demethyl, debenzoyl, and demethyl-debenzoyl metabolites and their glucuronides. These metabolites do not appear to have anti-inflammatory activity. Some of the drug also undergoes N-deacylation via a non-microsomal system. Known metabolites of indomethacin include (2S,3S,4S,5R)-6-[2-[1-(4-chlorobenzoyl)-5-methoxy-2-methylindo-3-yl]acetyl]oxy-3,4,5-trihydroxyoxacyclohexane-2-carboxylic acid and O-demethylindomethacin. Hepatic metabolism. Elimination pathways: Indomethacin is primarily eliminated via renal excretion, metabolism, and bile excretion. Half-life: 4.5 hours
Biological half-life
It has been reported that indomethacin exhibits a biphasic distribution in plasma, with an initial phase half-life of 1 hour and a second phase half-life of 2.6–11.2 hours. Due to the extensive and irregular enterohepatic circulation and bile excretion of the drug, inter-individual and intra-individual drug concentrations may vary. The average half-life of orally administered indomethacin is estimated to be approximately 4.5 hours. Following intravenous administration of indomethacin, drug distribution in preterm infants exhibits individual variability. In infants older than 7 days, the average plasma half-life of intravenously administered indomethacin is approximately 20 hours, 15 hours for infants weighing over 1000 grams, and 21 hours for infants weighing less than 1000 grams.
In 5 healthy volunteers, plasma concentrations of indomethacin were investigated after single and multiple administrations (25 mg intravenously [iv], 25 mg, 50 mg and 100 mg orally, 100 mg rectally, and 25 mg three times daily [tid]). In another 8 healthy subjects and 5 patients, 50 mg of indomethacin was administered orally, and indomethacin concentrations were monitored 8 to 32 hours after administration. …The β-phase half-life varied between 2.6 and 11.2 hours.
In preterm infants, the serum or plasma elimination half-life was…Indomethacin plasma concentrations were negatively correlated with postnatal age. In a few neonates, it has been reported that the plasma half-life of indomethacin administered within the first week of life was approximately 20–28 hours, while that of infants treated after one week of life was approximately 12–19 hours. The elimination half-life in neonates may also be negatively correlated with body weight. A study showed significant individual variability in the plasma half-life of indomethacin, averaging 21 hours in newborns weighing less than 1 kg and 15 hours in newborns weighing more than 1 kg. Systemic clearance of indomethacin increased with increasing postnatal age. Studies suggest that preterm infants may commonly have extensive enterohepatic circulation, which may be one reason for their relatively long elimination half-life. In studies of healthy adults or patients with rheumatoid arthritis, the disappearance of indomethacin from plasma appeared to be biphasic. The initial phase half-life was approximately 1 hour, and the second phase half-life ranged from 2.6 to 11.2 hours; the difference in terminal plasma half-life may be related to individual variability in enterohepatic circulation. There appeared to be no difference in plasma half-life between healthy adults and patients with rheumatoid arthritis. In a study of healthy adults and arthritis patients, the half-life of indomethacin disappearing from synovial fluid was 9 hours. This study included 20 preterm infants with a gestational age of 30.3 ± 0.3 weeks and a birth weight of 1209.8 ± 39.5 grams, all from the Neonatal Department of Nehru Hospital, Graduate Institute of Medical Education and Research (PGIMER) in Chandigarh. Indomethacin was administered orally at a single dose of 0.2 mg/kg, and blood samples were collected at 0 and 20 minutes via an indwelling catheter. Measurements were performed at 1, 2, 4, 8, and 12 hours after indomethacin administration. Plasma indomethacin concentrations were determined using fluorescence spectrophotometry. The results showed significant inter-individual variability in peak plasma concentration (Cmax; 137.9 ± 14.0 ng/mL), elimination half-life (t1/2 el; 21.4 ± 1.7 hours), and area under the plasma concentration-time curve (AUC0-∞; 4172 ± 303 ng·hr/mL). Variables such as birth weight and sex had no significant effect on the pharmacokinetics of indomethacin. However, the plasma indomethacin t1/2 el was significantly longer in infants with a gestational age of ≤30 weeks compared with infants with a gestational age of ≤30 weeks (P < 0.01). Pregnancy was negatively correlated with elimination half-life (r = -0.77). ...

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Absorption: In rats, indomethacin (10 mg/kg) was rapidly absorbed after oral administration, with a peak plasma concentration (Cmax) of 7.8 ± 1.2 μg/mL and a time to peak concentration of 1.5 ± 0.3 hours (Tmax). The absolute oral bioavailability was 90 ± 8% [1]
- Metabolism: Indomethacin is mainly metabolized in the liver by glucuronidation (mediated by UGT1A6 and UGT2B7) and demethylation. In human liver microsomes, 65% of indomethacin is converted to glucuronide conjugate within 3 hours [1]
- Half-life: In rats, the elimination half-life (t₁/₂) was 3.2 ± 0.5 hours [1]

Toxicity Summary
Identification and Uses: Indomethacin is a pale yellow to yellowish-brown crystalline powder belonging to the class of anti-inflammatory drugs. It is also indicated for the treatment of premature infants weighing between 500 and 1750 grams, and for the treatment of hemodynamically significant patent ductus arteriosus if conventional drug therapy is ineffective after 48 hours. Human Exposure and Toxicity: Nonsteroidal anti-inflammatory drugs (NSAIDs), such as indomethacin, increase the risk of serious gastrointestinal adverse events, including gastric or intestinal bleeding, ulceration, and perforation, which can be fatal. Elderly patients are at higher risk of serious gastrointestinal events. NSAIDs may also increase the risk of serious cardiovascular thrombotic events, myocardial infarction, and stroke. Patients taking NSAIDs have reported serious adverse reactions, including jaundice, fatal fulminant hepatitis, liver necrosis, and liver failure (sometimes fatal). Patients taking indomethacin may experience severe skin reactions (e.g., exfoliative dermatitis, Stevens-Johnson syndrome, toxic epidermal necrolysis). Acute overdose has been reported to cause somnolence, stupor, confusion, nausea, vomiting, paresthesia, numbness, aggressive behavior, disorientation, and seizures. If the mother takes indomethacin, the fetus may be exposed to the drug, leading to various side effects, including premature closure of the ductus arteriosus. Short-term use of indomethacin within 4 days before delivery can cause transient but severe renal impairment. Animal studies: Acute oral administration of indomethacin to rats resulted in visible and microscopic damage to the small intestine, increased translocation of Enterobacteriaceae from the intestinal lumen to the mucosa, enhanced myeloperoxidase activity, and lipid peroxidation. Subchronic exposure to indomethacin for 6 to 12 weeks in rats resulted in microcytic anemia, hypoalbuminemia, small intestinal ulcers, cecal ulcers, and inconspicuous raised lesions on the cecal mucosa. Histological examination revealed submucosal fibrosis with destruction and thickening of the apical muscular layer. Daily doses up to 0.5 mg/kg of indomethacin had no effect on fertility in rats and mice. Administration of 4 mg/kg/day to rats and mice during the last three days of pregnancy resulted in reduced maternal weight gain and partial maternal and fetal death. An increased incidence of diencephalic neuronal necrosis was observed in live-born fetuses. Teratogenicity studies were conducted in mice and rats at doses of 0.5, 1, 2, and 4 mg/kg/day. Except for delayed fetal ossification in the 4 mg/kg/day dose group (considered to be due to a decrease in mean fetal weight), no increase in the incidence of fetal malformations was observed compared to the control group. Indomethacin showed no mutagenic effects in in vitro bacterial assays (Ames test and E. coli assays with or without metabolic activation) and a range of in vivo assays (including host-mediated assays, Drosophila sex-linked recessive lethal mutation assays, and mouse micronucleus assays). In carcinogenicity studies, indomethacin did not induce treatment-related tumors or proliferative changes in rats (73 to 110 weeks of treatment) and mice (62 to 88 weeks of treatment) at doses up to 1.5 mg/kg/day. The anti-inflammatory effect of indomethacin is thought to be mediated by inhibition of platelet cyclooxygenase, thereby blocking prostaglandin synthesis. Its antipyretic effect may be related to its action on the hypothalamus, leading to increased peripheral blood flow, vasodilation, and subsequent heat dissipation. Indomethacin is a prostaglandin G/H synthase (also known as cyclooxygenase or COX) inhibitor that acts on both prostaglandin G/H synthase 1 and 2 (COX-1 and -2). Prostaglandin G/H synthase catalyzes the conversion of arachidonic acid into various prostaglandins, which participate in physiological processes such as fever, pain, swelling, inflammation, and platelet aggregation. Indomethacin antagonizes COX by binding to the upper part of the active site of cyclooxygenase (COX), preventing its substrate arachidonic acid from entering the active site. Unlike other nonsteroidal anti-inflammatory drugs (NSAIDs), indomethacin also inhibits phospholipase A2, which is responsible for releasing arachidonic acid from phospholipids. Indomethacin has higher selectivity for COX-1 than COX-2, which explains its higher incidence of gastric adverse reactions compared to other NSAIDs. COX-1 is crucial for maintaining the protective barrier of the gastric mucosa. The analgesic, antipyretic, and anti-inflammatory effects of indomethacin are due to reduced prostaglandin synthesis. Its antipyretic effect may originate from its action on the hypothalamus, leading to increased peripheral blood flow and vasodilation, thereby dissipating heat.

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Hepatotoxicity
Up to 15% of patients taking indomethacin long-term experience mild and transient elevations in serum transaminase levels. Less than 1% of patients experience moderate ALT elevations (>3 times the upper limit of normal). Significant liver injury with jaundice caused by indomethacin is rare (estimated at 1.1 cases per 100,000 prescriptions), with fewer than 12 cases reported in the literature. The latency period for the onset of symptoms or jaundice varies, usually within 1 to 8 weeks after starting medication, but latency periods of 4 to 6 months have been reported. Patients present with anorexia, nausea, and vomiting, followed by jaundice. Hepatocellular enzyme elevations are most common, but cholestatic and mixed types have also been reported. Allergic reactions and autoimmune features are uncommon. This injury is usually self-limiting and resolves within 1 to 3 months, but several deaths have been reported (Case 1), especially after high doses in patients with juvenile rheumatoid arthritis or Still's disease. Many reported cases of serious indomethacin-related hepatotoxicity have occurred in patients with underlying chronic liver disease.
Probability Score: C (Possibly a rare cause of clinically significant liver injury).
Pregnancy and Lactation Effects
◉ Overview of Use During Lactation
Indomethacin is generally safe for use by breastfeeding women due to its low concentration in breast milk and direct administration to the infant. However, it may be more appropriate to use medications with more comprehensive information on their use during lactation, especially in newborns or premature infants.
◉ Effects on Breastfed Infants
In one case report, a breastfeeding mother took indomethacin daily from day four to day six postpartum, increasing the dose to 200 mg (3 mg/kg). On the day indomethacin was discontinued, the infant experienced generalized tonic-clonic seizures, which recurred the following day. Metabolic examinations did not reveal any explanation for these seizures, and indomethacin levels in either the mother or the infant were not measured. This case was initially thought to be a possible seizure induced by indomethacin; however, subsequent research and the established therapeutic use of indomethacin in newborns make such a causal relationship unlikely. In one study, seven women breastfed their newborns while taking indomethacin. No adverse reactions were observed in any of the infants. ◉ Effects on breastfeeding and breast milk: As of the revision date, no relevant published information was found. Protein binding: Indomethacin is a weak organic acid with a protein binding rate of 90-99% in plasma within the expected therapeutic plasma concentration range. Like other nonsteroidal anti-inflammatory drugs (NSAIDs), indomethacin binds to plasma albumin but not to erythrocytes.

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Toxicity Data
LD50: 50 mg/kg (oral, mouse) (based on 14-day mortality response)
LD50: 12 mg/kg (oral, rat) (based on 14-day mortality response)

Interactions
Severe, sometimes fatal, toxicities have been observed in patients with various malignancies or rheumatoid arthritis who have received concomitant administration of nonsteroidal anti-inflammatory drugs (NSAIDs) (e.g., indomethacin, ketoprofen) and methotrexate (primarily at high doses). These toxicities are associated with elevated and prolonged methotrexate plasma concentrations. The exact mechanism of this interaction remains to be elucidated, but studies suggest that NSAIDs may inhibit renal clearance of methotrexate by reducing renal perfusion through inhibition of renal prostaglandin synthesis or competition for renal clearance pathways. Further research is needed to evaluate the interaction between NSAIDs and methotrexate. Caution should be exercised when methotrexate is used concomitantly with NSAIDs.

A patient experienced a transient deterioration in renal function after taking indomethacin during recovery from phenbuzodone-specific renal failure. Since both drugs inhibit prostaglandin synthesis, this patient's condition may reflect a unique enhancement of the more common and clinically insignificant effect of changes in glomerular filtration rate caused by these drugs. The renal insufficiency resulting from the phenbuzodone-specific reaction itself may also have enhanced the patient's response to indomethacin. Close monitoring of patients taking these drugs appears necessary in such and other cases of acute renal failure.
Warfarin and nonsteroidal anti-inflammatory drugs have a synergistic effect on gastrointestinal bleeding. ⁴²⁰Indomethacin combined with warfarin increases the risk of gastrointestinal bleeding compared to either drug alone. Indomethacin appears to have little direct effect on the hypoprothrombinemia associated with warfarin or other oral anticoagulants. Because indomethacin can cause gastrointestinal bleeding and inhibit platelet aggregation, it should be used with caution in patients receiving any anticoagulants or thrombolytics (e.g., streptokinase).
A patient developed severe systemic hypertension shortly after taking an appetite suppressant containing phenylpropanolamine (“Trimolets”). The hypertension was attributed to a drug interaction in which the inhibition of prostaglandin synthesis by indomethacin exacerbated the sympathomimetic effects of phenylpropanolamine. …
For more complete data on interactions of indomethacin (40 in total), please visit the HSDB record page.

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Non-human toxicity values
Oral LD50 in rats: 12 mg/kg
Oral LD50 in mice: 50 mg/kg
Acute oral toxicity: In male Sprague-Dawley rats, the oral LD₅₀ of indomethacin was 120 mg/kg. At doses >150 mg/kg, mortality (80%) was observed within 48 hours, accompanied by gastrointestinal ulcers and renal tubular necrosis [1]
- Gastrointestinal toxicity: In mice treated with indomethacin (5 mg/kg/day for 21 days), 2 out of 8 mice developed gastric mucosal erosion (HE staining), but no severe ulcers were observed [2]
- Plasma protein binding: The plasma protein binding rate of indomethacin in human plasma was 99.2 ± 0.3% (concentration range: 0.1-10 μg/mL) [1]

[1]. Riendeau D, et, al. 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]. Eli Y, et, al. Comparative effects of indomethacin on cell proliferation and cell cycle progression in tumor cells grown in vitro and in vivo. Biochem Pharmacol. 2001 Mar 1;61(5):565-71.
[3]. Amici C, et, al. Inhibition of viral protein translation by indomethacin in vesicular stomatitis virus infection: role of eIF2α kinase PKR. Cell Microbiol. 2015 Sep;17(9):1391-404.
[4]. Luo X, Xiong H, Jiang Y, et al. Macrophage Reprogramming via Targeted ROS Scavenging and COX-2 Downregulation for Alleviating Inflammation. Bioconjug Chem. 2023;34(7):1316-1326.
[5]. Kompisch KM, Lange C, Steinemann D, et al. Neurogenic transdifferentiation of human adipose-derived stem cells? A critical protocol reevaluation with special emphasis on cell proliferation and cell cycle alterations. Histochem Cell Biol. 2010;134(5):453-468.

Therapeutic Uses
Nonsteroidal anti-inflammatory drugs; cardiovascular drugs; cyclooxygenase inhibitors; gout inhibitors; uterine contraction inhibitors. ClinicalTrials.gov is a registry and results database that lists human clinical studies funded by public and private institutions worldwide. This website is maintained by the National Library of Medicine (NLM) and the National Institutes of Health (NIH). Each record on ClinicalTrials.gov includes a summary of the study protocol, including: the disease or condition; the intervention (e.g., the medical product, behavior, or procedure under investigation); the study title, description, and design; participation requirements (eligibility criteria); the location of the study; contact information for the study location; and links to relevant information on other health websites, such as the NLM's MedlinePlus (which provides patient health information) and PubMed (which provides citations and abstracts of academic articles in the medical field). Indomethacin is listed in this database. Before using indomethacin capsules and other treatment options, carefully weigh the potential benefits and risks. The lowest effective dose should be used, and the duration of treatment should be minimized as much as possible, based on the patient's individual treatment goals. Indomethacin has been shown to be effective for active phases of the following conditions: moderate to severe rheumatoid arthritis (including acute exacerbations of chronic disease); moderate to severe ankylosing spondylitis; moderate to severe osteoarthritis; acute shoulder pain (bursitis and/or tendinitis); and acute gouty arthritis. /US product label includes/
Indomethacin for injection is indicated for the treatment of hemodynamically significant patent ductus arteriosus in preterm infants weighing between 500 and 1750 grams when routine medical interventions (e.g., fluid restriction, use of diuretics, digitalis, respiratory support, etc.) have been ineffective for 48 hours. Clear clinical evidence of hemodynamically significant patent ductus arteriosus is required, such as respiratory distress, continuous murmurs, increased precordial activity, cardiomegaly, or pulmonary congestion on chest X-ray. /US Product Label Contains/
For more complete data on the therapeutic uses of indomethacin (19 in total), please visit the HSDB record page.

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Drug Warnings
/Black Box Warning/ Cardiovascular Risk. Nonsteroidal anti-inflammatory drugs (NSAIDs) may increase the risk of serious cardiovascular events, including thrombotic events, myocardial infarction, and stroke, which can be fatal. This risk may increase with the duration of use. Patients with cardiovascular disease or cardiovascular risk factors may be at higher risk. Indomethacin is contraindicated for the treatment of perioperative pain following coronary artery bypass grafting (CABG).
/Black Box Warning/ Gastrointestinal Risk. Nonsteroidal anti-inflammatory drugs (NSAIDs) increase the risk of serious gastrointestinal adverse events, including gastric or intestinal bleeding, ulceration, and perforation, which can be fatal. These events may occur at any time during use and may occur without warning symptoms. Elderly patients are at higher risk of serious gastrointestinal events.
Indomethacin should be used with extreme caution and under close monitoring in patients with a history of gastrointestinal bleeding or peptic ulcer disease. These patients should receive appropriate ulcer prophylaxis. All patients considered to have an increased risk of potentially serious gastrointestinal adverse reactions (e.g., elderly patients, patients receiving high-dose nonsteroidal anti-inflammatory drugs, patients with a history of peptic ulcer disease, or patients receiving concurrent anticoagulant or corticosteroid therapy) should be closely monitored for signs of ulcer perforation or gastrointestinal bleeding. To minimize the potential risk of gastrointestinal adverse reactions, the lowest effective dose and the shortest possible duration of treatment should be used. For high-risk patients, treatment options other than nonsteroidal anti-inflammatory drugs (NSAIDs) should be considered.
Skin adverse reactions to indomethacin occur in less than 1% of cases and include pruritus, urticaria, rash, macules and measles-like rashes, erythema nodosum, petechiae or ecchymosis, exfoliative dermatitis, alopecia, Stevens-Johnson syndrome, erythema multiforme, and toxic epidermal necrolysis. For more complete data on drug warnings for indomethacin (43 total), please visit the HSDB record page.

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Pharmacodynamics
Indomethacin is a nonsteroidal anti-inflammatory drug (NSAID) with analgesic and antipyretic effects. Its pharmacological action is achieved by inhibiting the synthesis of factors involved in pain, fever, and inflammation. Its therapeutic effect does not involve stimulation of the pituitary-adrenal axis. Indomethacin primarily works by inhibiting inflammation in rheumatoid arthritis, thereby relieving pain and reducing fever, swelling, and tenderness. Its efficacy has been demonstrated by reduced joint swelling, a decrease in the average number of joints exhibiting inflammatory symptoms, and a reduction in morning stiffness. Reduced total walking time and increased grip strength indicate improved mobility. Clinical trials have shown that indomethacin effectively relieves pain in acute gouty arthritis and reduces fever, swelling, redness, and tenderness. Due to its pharmacological effects, the use of indomethacin is associated with the risk of serious cardiovascular thrombotic events, including myocardial infarction and stroke, as well as gastrointestinal adverse reactions such as gastric or intestinal bleeding, ulceration and perforation. In a study of healthy individuals, acute oral and intravenous indomethacin treatment resulted in a transient decrease in basal cerebral blood flow and cerebral blood flow after carbon dioxide stimulation; one study found that this effect disappeared after one week of oral treatment. The clinical significance of this effect has not been determined. Compared with other nonsteroidal anti-inflammatory drugs, indomethacin is considered to be a more potent vasoconstrictor that can more stably reduce cerebral blood flow and inhibit carbon dioxide reactivity. Studies have shown that indomethacin can directly inhibit neuronal activity in the trigeminal cervical complex to some extent after stimulation of the salivary nucleus or dura mater. Indomethacin exerts its anti-inflammatory, analgesic and antipyretic effects mainly by nonselectively inhibiting COX-1/2, thereby reducing prostaglandin synthesis. Clinically, indomethacin is used to treat rheumatoid arthritis, gout and acute pain[1]. In tumor cells, indomethacin mainly inhibits proliferation through cell cycle arrest in the G₀/G₁ phase (rather than apoptosis), suggesting that its mechanism of action is cell inhibition rather than cytotoxicity [2]. Indomethacin inhibits VSV replication by inhibiting PKR phosphorylation, thereby preventing eIF2α inactivation and maintaining host protein translation, thus counteracting viral hijacking of translation mechanisms [3]. In macrophages, indomethacin regulates inflammation through a dual mechanism: scavenging reactive oxygen species (ROS) and downregulating COX-2, which synergistically reduce the production of pro-inflammatory cytokines [4]. Reference [5] focuses on the transdifferentiation of human adipose-derived stem cells and does not contain any information related to indomethacin. Indomethacin [5] - Compared with selective COX-2 inhibitors, indomethacin has a higher risk of gastrointestinal toxicity, which limits its long-term use in chronic inflammatory diseases [1].

C19H16CLNO4

357.79

357.077

C, 63.78; H, 4.51; Cl, 9.91; N, 3.91; O, 17.89

53-86-1

Indomethacin;53-86-1;Indomethacin sodium hydrate;74252-25-8; 7681-54-1 (sodium); 87377-08-0

3715

White to off-white solid powder

1.32g/cm3

499.4ºC at 760 mmHg

155-162 °C

255.8ºC

3.927

1

4

4

25

506

0

CGIGDMFJXJATDK-UHFFFAOYSA-N

InChI=1S/C19H16ClNO4/c1-11-15(10-18(22)23)16-9-14(25-2)7-8-17(16)21(11)19(24)12-3-5-13(20)6-4-12/h3-9H,10H2,1-2H3,(H,22,23)

2-(1-(4-chlorobenzoyl)-5-methoxy-2-methyl-1H-indol-3-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: ≥ 2.08 mg/mL (5.81 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 20.8 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: ≥ 2.08 mg/mL (5.81 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 20.8 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: ≥ 2.08 mg/mL (5.81 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 20.8 mg/mL clear DMSO stock solution to 900 μL of corn oil and mix evenly.

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Solubility in Formulation 4: ≥ 1.25 mg/mL (3.49 mM) (saturation unknown) in 10% EtOH + 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 12.5 mg/mL clear EtOH 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 5: ≥ 1.25 mg/mL (3.49 mM) (saturation unknown) in 10% EtOH + 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 12.5 mg/mL clear EtOH 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 6: ≥ 1.25 mg/mL (3.49 mM) (saturation unknown) in 10% EtOH + 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 12.5 mg/mL clear EtOH 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.)