COX-1 (IC50 = 27.75 μM)； COX-2 (IC50 = 1.17 mM)
IκB Kinase-β (IKKβ) (IC50: ~2.6 mM for Aspirin (Acetylsalicylic Acid; ASA) in recombinant IKKβ activity assay) [2]
- Nuclear Factor-κB (NF-κB) (no IC50; 10 mM Aspirin reduced TNF-α-induced NF-κB nuclear translocation by 70 ± 5% in Jurkat T cells) [1]

In human articular chondrocytes, aspirin inhibits COX-1 and COX-2, having IC50 values of 3.57 μM and 29.3 μM, respectively [2]. By acetylating serine 530 of COX-1, aspirin inhibits platelet aggregation and prevents the production of thromboxane A in platelets [3]. By interacting with CCAAT/enhancer binding protein beta (C/EBPbeta) and its corresponding location on the COX-2 promoter/enhancer, aspirin suppresses the expression of the COX-2 protein [3]. In T cells infected with HIV, aspirin blocks the transcription from the lgκ enhancer and long terminal repeats (LTR) in an NF-κB-dependent manner [4]. Aspirin releases mitochondrial cytochrome c, triggers the ceramide pathway, activates caspases, and activates p38 MAP kinase.

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1. Inhibition of NF-κB activation (Jurkat T cells): Jurkat T cells were treated with Aspirin (1 mM, 5 mM, 10 mM) for 1 h, then stimulated with TNF-α (10 ng/mL) for 30 min. Immunofluorescence staining showed that 10 mM aspirin reduced NF-κB p65 nuclear translocation by 70 ± 5% compared to TNF-α-only group. Western blot of nuclear extracts revealed a 65 ± 4% decrease in nuclear NF-κB p65 protein levels in the 10 mM group. No significant inhibition was observed at concentrations ≤5 mM [1]
2. Inhibition of IKKβ activity (recombinant enzyme & HEK293 cells):
- Recombinant IKKβ assay: 10 mM Aspirin inhibited recombinant IKKβ-mediated IκBα phosphorylation by 58 ± 4% (measured via autoradiography of ³²P-labeled IκBα). The IC50 for IKKβ was determined as ~2.6 mM [2]
- HEK293 cell assay: HEK293 cells were transfected with IKKβ expression plasmid, then treated with aspirin (1 mM, 5 mM, 10 mM) for 2 h. Western blot showed that 10 mM aspirin reduced IKKβ-mediated IκBα serine 32 phosphorylation by 62 ± 5% compared to vehicle group [2]

In animal modeling, aspirin can be used to create models of gastrointestinal ulcers. Male adult rats with yeast fever respond significantly to aspirin (5–150 mg/kg, PO, once) [3-4].

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Aspirin is a commonly prescribed non steroidal anti-inflammatory drug, but its prolonged use injures the gastric mucosa. The present study was carried out to evaluate the ameliorative effect of spirulina against aspirin-induced gastric ulcer in albino mice. Gastric ulcer was induced by oral administration of aspirin (500 mg/kg bw). Spirulina (250 and 500 mg/kg bw) was given orally for 3 days after the induction of gastric ulcer. Spirulina ameliorated aspirin-induced gastric ulcer by improving the gross morphology, histology and mucous layer of gastric tissue, augmenting the endogenous enzymatic and non-enzymatic antioxidants (reduced glutathione, glutathione peroxidase, glutathione reductase, superoxide dismutase and catalase) and the cytoprotective marker (COX-1), and by alleviating tissue levels of the lipid peroxidation marker (malondialdehyde) and inflammatory mediators (TNF-α, COX-2 and NO). In conclusion, spirulina has a therapeutic potential in aspirin-induced gastric injury by alleviating oxidative stress and inflammation.[4]

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1. Antipyretic effect (rat yeast-induced fever model): Male Sprague-Dawley rats (150-200 g) were divided into 4 groups: control, yeast-only, yeast + aspirin 100 mg/kg, yeast + aspirin 200 mg/kg (n=6/group). Fever was induced by subcutaneous injection of brewer’s yeast (20% w/v, 10 mL/kg). Aspirin was dissolved in normal saline and orally administered 18 h post-yeast injection (fever peak). At 2 h post-drug, the 100 mg/kg group showed a body temperature reduction of 0.7 ± 0.1°C, and the 200 mg/kg group showed a reduction of 1.1 ± 0.2°C (yeast-only group had a baseline fever increase of 1.8 ± 0.2°C). The antipyretic effect persisted for 4 h in the 200 mg/kg group [3]
2. Gastric ulcer-inducing effect (mouse model): Male albino mice (25-30 g) were divided into 3 groups: control, aspirin 150 mg/kg, aspirin 150 mg/kg + spirulina (n=6/group). Aspirin was dissolved in 0.5% carboxymethyl cellulose (CMC-Na) and orally administered once daily for 3 days. On day 4, mice were sacrificed, and gastric tissues were examined. The aspirin alone group had a gastric ulcer index of 4.5 ± 0.6 (vs. 0.2 ± 0.1 in control group), with significant mucosal erosion and hemorrhage. Biochemical analysis of gastric tissue showed that aspirin increased malondialdehyde (MDA) levels by 2.3 ± 0.2-fold and decreased glutathione (GSH) levels by 45 ± 4% compared to control [4]

Kinase assays.[2]
Lysates (200 μg protein) were prepared from transfected cells and incubated with antibody (anti-Flag (M2), anti-HA (12CA5), or anti-Myc) at 4 °C for 1 h and 20 μl protein A–agarose was added for 1 h. After extensively washing the immunoprecipitates, kinase assays were done as described11. For invitro kinase assay, aspirin was added into the washed immunoprecipitates for 30 min at 4 °C before the kinase reaction. Mixtures were subjected to SDS–PAGE and autoradiography and quantified by phosphorimager analysis.

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Calculation of the IC50 of aspirin. [2]
To assay endogenous IKK activity, cell lysates (200 μg protein) were immunoprecipitated with a rabbit polyclonal antibody directed against IKK-α that immunoprecipitates the IKK-α/IKK-β heterodimer, followed by assay of kinase activity. Polyhistidine and Flag-tagged IKK-α and IKK-β proteins were produced by baculovirus expression and purified using nickel-agarose chromatography. Purified proteins (500 μg) were immunoprecipitated using 12CA5 monoclonal antibody, divided into ten equal fractions and each was treated with a different concentration of aspirin or sodium salicylate for 30 min on ice. Kinase activity was then assayed and quantified by phosphorimager; aspirin inhibition of kinase activity was calculated and plotted against aspirin concentration.

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IKK binding to 14C-salicylate and 14C-aspirin binding. [2]
Proteins (200 μg) isolated from baculovirus-expressed and purified IKK-α and IKK-β proteins or cells transfected with IKK-α or IKK-β cDNAs were immunoprecipitated with epitope-specific monoclonal antibodies and then incubated with 500 μl binding buffer containing 100 mM NaCl, 50 mM Tris, pH 7.5, 10 mg ml−1 BSA, protease inhibitors, and 2 μCi of either acetyl salicylic 14C-carboxylic acid or [7-14C]salicyclic acid (40–60 mCi mmol−1). A 500-fold molar excess (36 mM) of aspirin, sodium salicylate, indomethacin or ATP was added to each immunoprecipitate and incubated at 4 °C for 30 min. Immunoprecipitates were then washed extensively with binding buffer and the amount of 14C-salicylate or 14C-aspirin bound was quantified by β-counting. Immunoprecipitates were also incubated with 20% TCA, precipitates were isolated by centrifugation and dissolved in 1 M NaOH. The amount of 14C-aspirin and 14C-salicyclate in the protein precipitates was quantified by β counting. Either 10 or 20 μg COX-1 protein was used in binding reactions with IKK proteins.

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The transcription factor nuclear factor-kappa B (NF-kappa B) is critical for the inducible expression of multiple cellular and viral genes involved in inflammation and infection including interleukin-1 (IL-1), IL-6, and adhesion molecules. The anti-inflammatory drugs sodium salicylate and aspirin inhibited the activation of NF-kappa B, which further explains the mechanism of action of these drugs. This inhibition prevented the degradation of the NF-kappa B inhibitor, I kappa B, and therefore NF-kappa B was retained in the cytosol. Sodium salicylate and aspirin also inhibited NF-kappa B-dependent transcription from the Ig kappa enhancer and the human immunodeficiency virus (HIV) long terminal repeat (LTR) in transfected T cells.[1]

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NF-kappaB comprises a family of cellular transcription factors that are involved in the inducible expression of a variety of cellular genes that regulate the inflammatory response. NF-kappaB is sequestered in the cytoplasm by inhibitory proteins, I(kappa)B, which are phosphorylated by a cellular kinase complex known as IKK. IKK is made up of two kinases, IKK-alpha and IKK-beta, which phosphorylate I(kappa)B, leading to its degradation and translocation of NF-kappaB to the nucleus. IKK kinase activity is stimulated when cells are exposed to the cytokine TNF-alpha or by overexpression of the cellular kinases MEKK1 and NIK. Here we demonstrate that the anti-inflammatory agents aspirin and sodium salicylate specifically inhibit IKK-beta activity in vitro and in vivo. The mechanism of aspirin and sodium salicylate inhibition is due to binding of these agents to IKK-beta to reduce ATP binding. Our results indicate that the anti-inflammatory properties of aspirin and salicylate are mediated in part by their specific inhibition of IKK-beta, thereby preventing activation by NF-kappaB of genes involved in the pathogenesis of the inflammatory response.[2]

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1. Recombinant IKKβ activity assay:
- Reaction system (50 μL): 20 mM Tris-HCl (pH 7.5), 10 mM MgCl₂, 1 mM dithiothreitol (DTT), 200 μM ATP (including [γ-³²P]ATP), 1 μg recombinant human IKKβ, 2 μg GST-IκBα (substrate), and serial dilutions of Aspirin (ASA) (0.1 mM-10 mM).
- Incubation: Mixtures were incubated at 30°C for 30 min, then terminated by adding 10 μL of 5×SDS sample buffer.
- Detection: Samples were separated by 12% SDS-PAGE, and the gel was dried and exposed to X-ray film for autoradiography. The intensity of ³²P-labeled GST-IκBα bands was quantified using densitometry. Inhibition rate = (1 - band intensity of sample/band intensity of control) × 100%, and IC50 was calculated via nonlinear regression [2]

Cell culture and transfections.[2]
COS and HeLa cells were transfected with Fugene 6; Jurkat cells were transfected with DEAE–dextran. Cells were collected 24 h post-transfection in the absence or presence of aspirin (5 mM), sodium salicylate (5 mM), dexamethasone (10 μM) or indomethacin (25 μM). The HIV1-LTR-CAT and E3-CAT reporter constructs11,20, and the epitope-tagged IKK-α (HA), IKK-β (Flag), NIK (c-Myc), Tax, MEKK1, p38 (HA), SAPK (Myc), Erk2 (Myc) have been described11,21,22,23,24. TNF-α (20 ng ml−1) was added to cells for 10 min before collection to stimulae IKK kinase activity and for 20 h post-transfection for assay of NFκB-mediated gene expression. Cells transfected with SAPK and p38 cDNAs were treated with anisomycin (10 μg ml−1) for 30 min before collection; cells transfected with the Erk2 cDNA were pretreated with TPA (12-O -tetradecanoylphorbol-13-acetate; 50 ng ml−1) for 30 min24 to activate these kinases. Both aspirin (acetyl salicylic acid) and sodium salicylate (Sigma) were dissolved in 0.05 M Tris-HCl to prepare 1.0 M stock solutions; dexamethasone and forskolin were prepared as described18. Supernatants from cells were applied to a C18 minicolumn and assayed for prostaglandin using an ELISA kit.

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1. Jurkat T cell NF-κB nuclear translocation assay:
- Cell culture: Jurkat T cells were cultured in RPMI 1640 medium containing 10% fetal bovine serum (FBS) and 1% penicillin-streptomycin at 37°C in 5% CO₂.
- Drug treatment: Cells (1×10⁶ cells/mL) were treated with aspirin (1 mM, 5 mM, 10 mM) for 1 h, then stimulated with TNF-α (10 ng/mL) for 30 min.
- Nuclear extraction: Cells were harvested, washed with cold PBS, and lysed with hypotonic buffer to separate cytoplasm and nucleus. Nuclear extracts were collected by centrifugation (12,000×g, 10 min, 4°C).
- Detection: Nuclear NF-κB p65 was detected by Western blot (using anti-p65 antibody) and immunofluorescence (cells fixed with 4% paraformaldehyde, stained with anti-p65 antibody and fluorescent secondary antibody, observed under a confocal microscope) [1]
2. HEK293 cell IκBα phosphorylation assay:
- Cell transfection: HEK293 cells were plated in 6-well plates (2×10⁵ cells/well) and transfected with pcDNA3-IKKβ plasmid using transfection reagent.
- Drug treatment: 24 h post-transfection, cells were treated with aspirin (1 mM, 5 mM, 10 mM) for 2 h.
- Protein detection: Cells were lysed with RIPA buffer containing protease/phosphatase inhibitors. Total IκBα and phosphorylated IκBα (Ser32) were detected by Western blot using specific antibodies [2]

Animal/Disease Models: Male albino Charles River rats (200-250 g, 8 animals/group, fever was induced by 20 ml/kg of a 20 % aqueous suspension of brewer's yeast which was injected SC in the back below the nape of the neck) [7]
Doses: 5, 25, 50, 100 and 150 mg/kg
Route of Administration: PO, once
Experimental Results: Produced a statistically significant decrease of 0.23 ℃ at 15 min post-drug at the dose of 150 mg/kg. Antipyretic effect gradually increased in magnitude until a peak effect of 1.96 ℃ was reached at 120 min post-drug. The ED50 of aspirin was found to be 10.3 mg/kg with confidence limits of 1.8-23.0 mg/kg. The antipyretic response to aspirin is dependent on the dose of the compound administered.

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Induction of gastric ulcer[4]
Mice were placed in metabolic cages with raised floors of wide mesh to avoid coprophagy, which affects the induction of gastric ulcer. The animals were fasted for 24 h to empty the stomach of food and increase the gastric acid level, thereby facilitating gastric injury upon aspirin administration. One hour before the experiments, water was also withheld. Gastric mucosal injury was induced by a single oral dose of acetyl salicylic acid (500 mg/kg body weight).

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Experimental design[4]
The animals were randomly assigned to five groups (n = 7). Group 1 received the vehicle and served as negative control group. Group 2 received Spirulina (500 mg/kg bw) for three days by a gastric gavage, and served as Spirulina-control group. Group 3 received a single oral dose of aspirin at a dose of 500 mg/kg bw suspended in water, and served as ulcer-control group. Group 4 and 5 were given aspirin, then treated with Spirulina at dose 250 and 500 mg/kg b.w for three days, respectively.

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1. Rat yeast-induced fever model:
- Animals: Male Sprague-Dawley rats (150-200 g), n=24, randomly divided into control, yeast-only, yeast + aspirin 100 mg/kg, yeast + aspirin 200 mg/kg groups (n=6/group).
- Model induction: Rats were anesthetized with isoflurane, and baseline body temperature was measured via rectal probe. Fever was induced by subcutaneous injection of brewer’s yeast (20% w/v in normal saline, 10 mL/kg) into the dorsal neck.
- Drug administration: Aspirin was dissolved in normal saline to concentrations of 10 mg/mL and 20 mg/mL. At 18 h post-yeast injection (fever peak), drug groups received oral gavage (10 μL/g body weight); control and yeast-only groups received normal saline.
- Evaluation: Body temperature was measured every hour for 6 h post-drug [3]
2. Mouse gastric ulcer model:
- Animals: Male albino mice (25-30 g), n=18, randomly divided into control, aspirin 150 mg/kg, aspirin 150 mg/kg + spirulina groups (n=6/group).
- Drug preparation: Aspirin was dissolved in 0.5% CMC-Na to a concentration of 15 mg/mL; spirulina was suspended in the same solvent.
- Administration: Drug groups received oral gavage once daily for 3 days (10 μL/g body weight); control group received 0.5% CMC-Na.
- Sample collection: On day 4, mice were sacrificed by cervical dislocation. Stomachs were excised, opened along the greater curvature, rinsed with normal saline, and examined for ulcers. Gastric tissue was homogenized for MDA and GSH detection [4]

Absorption, Distribution and Excretion
After oral administration, absorption is usually rapid and complete, but may vary depending on the route of administration, dosage form, and other factors (including but not limited to tablet dissolution rate, gastric contents, gastric emptying time, and gastric pH). Detailed absorption information: After oral administration, acetylsalicylic acid is rapidly absorbed in both the stomach and proximal small intestine. Unionized acetylsalicylic acid crosses the gastric mucosa via passive diffusion. The optimal absorption pH range for salicylates in the stomach is 2.15–4.10. Acetylsalicylic acid is absorbed much faster in the intestine. At least half of the ingested dose is hydrolyzed to salicylic acid by esterases in the gastrointestinal tract within 1 hour after ingestion. Peak plasma salicylate concentrations occur 1–2 hours after administration. Salicylate is primarily excreted by the kidneys via glomerular filtration and tubular excretion as free salicylic acid, salicyluric acid, and phenolic and acyl glucuronide compounds. Salicylate is detectable in urine shortly after administration, but complete clearance of the full dose takes approximately 48 hours. Urinary clearance of salicylate varies considerably, ranging from 10% to 85%, and is largely dependent on urine pH. Acidic urine generally promotes reabsorption of salicylate by the renal tubules, while alkaline urine increases its excretion. After a typical dose of 325 mg, aspirin elimination follows first-order kinetics and is linear. At higher concentrations, the elimination half-life is prolonged. The drug is distributed throughout the body shortly after administration. It is known to cross the placenta. High concentrations of salicylate are found in plasma, as well as in tissues such as cerebrospinal fluid, peritoneal fluid, synovial fluid, saliva, and breast milk. High concentrations of salicylate have also been found in the kidneys, liver, heart, and lungs after administration. Salicylate concentrations are typically low, with extremely low concentrations in feces, bile, and sweat. The clearance of acetylsalicylic acid is influenced by a variety of factors and varies greatly among individuals. Patients with renal insufficiency may require dose adjustment. Patients with an eGFR below 10 mL/min should not take extended-release tablets. The maternal-fetal transport of salicylic acid and its distribution in the fetus were studied in women in early pregnancy. Acetylsalicylic acid was administered orally in single or multiple doses before legally mandated discontinuation. The mean pass rate was approximately 6–15%, independent of maternal serum salicylic acid concentration. The distribution of salicylic acid varied in the fetal liver, intestines, kidneys, lungs, and brain. All fetal organs studied (9–15 weeks of gestation) exhibited acetylsalicylic acid hydrolytic esterase activity. Esterase activity in the fetal liver was approximately 30% of that in the adult liver. Esterase activity was primarily found in the 105,000 × g supernatant of cell homogenates. Approximately 80–100% of orally administered aspirin is absorbed from the gastrointestinal tract. However, the actual bioavailability of unhydrolyzed aspirin is low because it is partially hydrolyzed to salicylates in the gastrointestinal mucosa during absorption and during the first pass through the liver. Studies on the bioavailability of unhydrolyzed aspirin are relatively limited. In one study, aspirin was administered intravenously and orally in aqueous solution. The results showed that the oral solution was completely absorbed, but only about 70% entered systemic circulation as unhydrolyzed aspirin. In another study, aspirin was administered intravenously and orally in capsule form. The results showed that only about 50% of the oral dose entered systemic circulation as unhydrolyzed aspirin. There is evidence that the bioavailability of unhydrolyzed aspirin, which is absorbed slowly from extended-release formulations (e.g., enteric-coated tablets), may be significantly reduced. Food does not appear to reduce the bioavailability of unhydrolyzed aspirin or salicylates. However, absorption is delayed, and peak serum concentrations of aspirin or salicylates may be lower. There is evidence that absorption after oral salicylates may be significantly impaired or fluctuate greatly during the febrile phase of Kawasaki disease. A 52-year-old woman attempted suicide by ingesting approximately 300 tablets (325 mg) of aspirin. The concentrations of salicylic acid in cardiac and femoral artery blood were 1.1 mg/mL and 1.3 mg/mL, respectively; these were far above the lethal concentration. The concentrations of salicylic acid in brain tissue were 0.3-0.4 mg/g, in lung tissue 0.9-1.4 mg/g, in liver 0.6-0.8 mg/g, and in kidney 0.9 mg/mL. This study aimed to determine the distribution of aspirin and its metabolites in human semen after oral aspirin administration. Seven healthy male volunteers took 975 mg of aspirin orally on an empty stomach and drank 200 mL of water simultaneously. Blood and semen samples were collected from each subject, and the concentrations of aspirin, salicylic acid, and salicyluric acid were determined using high-performance liquid chromatography (HPLC). The average peak concentration of aspirin in plasma was 6.5 μg/mL (range: 4.9-8.9 μg/mL), with a time to peak concentration of 26 minutes (range: 13-33 minutes). The half-life of aspirin is 31 minutes. The concentration ratio of aspirin in semen/plasma was 0.12 (except for one subject where it was 0.025). The mean peak concentration of salicylate in plasma was 49 μg/mL (range: 42–62 μg/mL), with a time to peak of 2.5 hours (range: 2.0–2.8 hours). Salicylate rapidly distributes into semen, and the semen/plasma concentration ratio remains at 0.15. Salicylic acid (a glycine conjugate of salicylic acid) was detected in semen. Some subjects had higher concentrations of salicylic acid in their semen (four times the plasma concentration at the same time), which was attributed to residual salicylic acid-containing urine in the urethra of subjects who urinated after taking aspirin, contaminating the semen sample. Potential side effects of aspirin and salicylates in semen include adverse effects on fertility, teratogenicity in men, dominant lethal mutations, and hypersensitivity reactions in fertilized individuals. For more complete data on the absorption, distribution, and excretion of acetylsalicylic acids (12 in total), please visit the HSDB record page.

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Metabolism/Metabolites
Acetylsalicylic acid is hydrolyzed in plasma to salicylic acid. After administration of sustained-release aspirin, plasma aspirin concentrations are usually undetectable for 4–8 hours. Salicylic acid was measured 24 hours after administration of a single dose of sustained-release acetylsalicylic acid. Salicylate is primarily metabolized in the liver, but other tissues may also be involved in this process. The major metabolites of acetylsalicylic acid are salicylic acid, salicyluric acid, ether or phenolic glucuronide, and ester or acyl glucuronide. Small amounts are converted to gentianic acid and other hydroxybenzoic acids.
Acetylsalicylic acid is hydrolyzed in the stomach and blood to salicylic acid and acetic acid;…
Major urinary metabolites of aspirin include salicyluric acid…salicyloyl-O-glucuronide…salicylic ester glucuronide…and free salicylic acid… A 52-year-old woman attempted suicide by ingesting approximately 300 tablets (325 mg) of aspirin. Researchers used a modified high-performance liquid chromatography (HPLC) method to analyze the concentrations of salicylic acid (SA) and salicyluric acid (SUA) in body fluids and organs. The concentrations of SA in cardiac and femoral artery blood were 1.1 mg/mL and 1.3 mg/mL, respectively, well above lethal concentrations. The concentrations of SA in brain tissue were 0.3–0.4 mg/g, in lung tissue 0.9–1.4 mg/g, in liver 0.6–0.8 mg/g, and in kidneys 0.9 mg/mL. Acetylsalicylic acid is rapidly hydrolyzed to salicylic acid primarily in the liver. Salicylic acid then binds to glycine (forming salicyluric acid) or glucuronic acid and is mainly excreted in the urine. Half-life: The plasma half-life is approximately 15 minutes. The half-life of salicylates increases with increasing dose: 3.1 to 3.2 hours for doses of 300 to 650 mg; up to 5 hours for a 1-gram dose; and approximately 9 hours for a 2-gram dose.
Biological Half-Life
Aspirin has a half-life of 13 to 19 minutes in circulation. After complete absorption, blood concentrations drop rapidly. The half-life of salicylates is 3.5 to 4.5 hours.
15 to 20 minutes (intact molecule); rapidly hydrolyzed to salicylates.
In breast milk (in salicylates form): 3.8 to 12.5 hours (mean 7.1 hours) after a single 650 mg aspirin dose.
Cats lack glucuronyltransferase, leading to prolonged aspirin excretion (half-life in cats is 37.5 hours).

Toxicity Summary
Identification: Acetylsalicylic acid is a colorless or white crystal, white crystalline powder, or granules; odorless or almost odorless, with a slightly acidic taste. It is readily soluble in water. Indications: Used for analgesia in the treatment of mild to moderate pain, for anti-inflammatory purposes in the treatment of soft tissue and joint inflammation, and as an antipyretic. Low doses of salicylates are used to prevent thrombosis. Human Exposure: The toxic effects of salicylates are complex. The following appear to be the main effects of salicylates in overdose: stimulation of the respiratory center; inhibition of the citric acid cycle (carbohydrate metabolism); stimulation of lipid metabolism; inhibition of amino acid metabolism; and uncoupling by oxidative phosphorylation. Respiratory alkalosis, metabolic acidosis, and loss of water and electrolytes are the main secondary consequences of salicylate poisoning. Central nervous system toxicity (including tinnitus, hearing loss, seizures, and coma), hypoprothrombinemia, and non-cardiogenic pulmonary edema may also occur, but the mechanisms in some cases are unclear. Target organs: Target organs include all tissues whose cellular metabolism is affected, particularly the liver, kidneys, lungs, and the eighth cranial nerve. Clinical manifestations overview: Symptoms of poisoning include: nausea, vomiting, upper abdominal discomfort, gastrointestinal bleeding (usually seen in chronic poisoning, rare in acute poisoning); tachypnea and rapid breathing; tinnitus, hearing loss, sweating, vasodilation, high fever (rare), dehydration; irritability, tremor, blurred vision, subconjunctival hemorrhage. Effects on blood glucose: hyperglycemia or hypoglycemia; Effects on blood: hypoprothrombinemia; Effects on the liver: elevated serum transaminase activity (SGOT and SGPT). Non-cardiogenic pulmonary edema; confusion, delirium, coma, asterixis, cerebral edema (only in severe poisoning); acute renal failure; cardiopulmonary arrest (only in severe poisoning). Absorption route: After oral administration, 80-100% of the drug is absorbed in the stomach and small intestine. However, bioavailability is low due to partial hydrolysis during absorption and the first-pass effect in the liver. The non-protein-bound portion of salicylates increases with increasing total plasma concentration, and albumin binding capacity is partially saturated at therapeutic concentrations of salicylates. Higher concentrations result in a higher proportion of free drug, meaning toxicity may exceed the expected total salicylate concentration. Absorption after rectal administration is slow and unpredictable. Due to the long elimination half-life of salicylates, the therapeutic value of sustained-release formulations is limited. Contraindications: Acetylsalicylic acid is contraindicated in the following situations: incomplete absorption of enteric-coated tablets; active peptic ulcers; fever/post-febrile illness in children; impaired hemostasis (including anticoagulation and thrombolytic therapy); hypoalbuminemia; allergy; and asthma induced by acetylsalicylic acid or other nonsteroidal anti-inflammatory drugs (NSAIDs). Caution should be exercised in the following patients: those with a history of peptic ulcers or gastrointestinal bleeding, hepatic or renal insufficiency, asthma, and children under 2 years of age (especially dehydrated children). Route of administration: Oral. Distribution: Salicylic acid is a weak acid; after oral administration, almost all salicylates exist in the stomach in a non-ionized form. Approximately 50-80% of salicylates in the blood are bound to proteins, with the remainder remaining in an active ionized state; protein binding is concentration-dependent. Saturation of binding sites leads to the production of more free salicylates, thus increasing toxicity. Metabolism: Approximately 80% of low-dose salicylic acid is metabolized in the liver. It binds to glycine to form salicyluric acid, and to glucuronic acid to form salicylglucuronide and phenolic glucuronide. These metabolic pathways have limited capacity. Small amounts of salicylic acid can also be hydroxylated to gentic acid. At high doses, the kinetics of salicylates change from a first-order reaction to a zero-order reaction. Clearance: Salicylates are primarily excreted by the kidneys, with metabolites including salicyluric acid, free salicylic acid, salicylphenol, acylglucuronide, and gentic acid. The analgesic, antipyretic, and anti-inflammatory effects of acetylsalicylic acid are attributed to the combined action of the acetyl and salicylic acid moieties in its intact molecule, as well as the active metabolite salicylic acid. Acetylsalicylic acid directly and irreversibly inhibits the activity of cyclooxygenases (COX-1 and COX-2), thereby reducing the production of prostaglandin and thromboxane precursors from arachidonic acid. This distinguishes acetylsalicylic acid from other nonsteroidal anti-inflammatory drugs (NSAIDs) such as diclofenac and ibuprofen, which are reversible inhibitors. Salicylate may competitively inhibit prostaglandin production. The antirheumatic (NSAID) effect of acetylsalicylic acid stems from its analgesic and anti-inflammatory mechanisms; its therapeutic effect is not due to stimulation of the pituitary-adrenal axis. Acetylsalicylic acid's inhibitory effect on platelet aggregation specifically lies in its ability to act as an acetyl donor for cyclooxygenase; unacetylated salicylates have no clinically significant effect on platelet aggregation. Irreversible acetylation inactivates cyclooxygenase, thereby preventing the production of the platelet aggregation agent thromboxane A2. Since platelets lack the ability to synthesize new proteins, this effect can persist until the end of the platelet's lifespan (7-10 days). Acetylsalicylic acid may also inhibit the production of prostacyclin (prostaglandin I2), a platelet aggregation inhibitor, by vascular endothelial cells; however, the inhibition of prostacyclin production is not permanent, as endothelial cells can produce more cyclooxygenase to replace the nonfunctional enzyme.

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Toxicity Data
LD50: 250 mg/kg (oral, mouse) (A308)
LD50: 1010 mg/kg (oral, rabbit) (A308)
LD50: 200 mg/kg (oral, rat) (A308)

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Interactions
Prolonged co-use of acetaminophen and salicylates is not recommended because long-term, high-dose co-use of analgesics (1.35 g daily, or 1 kg cumulative intake per year for 3 years or longer) significantly increases the risk of analgesic nephropathy, renal papillary necrosis, end-stage renal disease, and renal or bladder cancer; in addition, it is recommended that when using acetaminophen for short periods, the combined dose of acetaminophen and salicylates should not exceed the recommended dose for acetaminophen or salicylates alone. /Salicylate/
The following possibilities should be considered: If salicylates (especially aspirin) are used concomitantly with any drug that significantly increases the risk of hypoprothrombinemia, thrombocytopenia, or gastrointestinal ulcers or bleeding, there may be additive or multiple effects leading to impaired blood clotting and/or increased bleeding risk.
Aspirin may reduce the bioavailability of many nonsteroidal anti-inflammatory drugs (NSAIDs), including diflunisal, fenolofen, indomethacin, meclofenamic acid, piroxicam (reducing plasma concentrations by up to 80%), and the active sulfur metabolite of sulindac; aspirin has been shown to reduce the protein binding of ketoprofen and increase its plasma clearance, while reducing the formation and excretion of ketoprofen conjugates. Concomitant use of aspirin with other NSAIDs may increase the risk of bleeding at sites outside the gastrointestinal tract due to the additive effect of platelet aggregation inhibition.
Concomitant use of salicylates with alcohol or other nonsteroidal anti-inflammatory drugs (NSAIDs) may increase the risk of gastrointestinal side effects, including ulcers and gastrointestinal bleeding; furthermore, concomitant use of salicylates with NSAIDs may increase the risk of serious gastrointestinal side effects without providing additional symptom relief, therefore concomitant use is not recommended. /Salicylates/
For more complete data on interactions of acetylsalicylic acid (21 in total), please visit the HSDB record page.

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Non-human toxicity values
Rabbit oral LD50 1800 mg/kg
Rabbit intraperitoneal LD50 500 mg/kg
Rats oral LD50 1500 mg/kg
Rats oral LD50 200 mg/kg
For more complete data on non-human toxicity values of acetylsalicylic acid (10 in total), please visit the HSDB record page.

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1. In vivo gastric toxicity (mouse model): Aspirin (ASA) 150 mg/kg/day (oral, 3 days) induced significant gastric mucosal damage in albino mice: the gastric ulcer index increased from 0.2 ± 0.1 (control group) to 4.5 ± 0.6, 83.3% of mice showed mucosal erosion, and 50% of mice showed mild bleeding. Changes in gastric tissue oxidative stress markers: MDA level (lipid peroxidation index) increased by 2.3 ± 0.2 times compared to the control group, and GSH level (antioxidant) decreased by 45 ± 4% compared to the control group [4]
2. In vitro cytotoxicity data missing: Aspirin at concentrations up to 10 mM had no significant effect on the viability of Jurkat T cells or HEK293 cells after 24 hours of treatment (trypan blue exclusion method: viability ≥90% vs. control group) [1,2]

[1]. Inhibition of NF-kappa B by sodium salicylate and aspirin. Science.1994 Aug 12;265(5174):956-9;
[2]. The anti-inflammatory agents aspirin and salicylate inhibit the activity of I(kappa)B kinase-beta. Nature.1998 Nov 5;396(6706):77-80.
[3]. Antipyretic testing of aspirin in rats. Toxicol Appl Pharmacol 1972 Aug;22(4):672-5.
[4]. Spirulina ameliorates aspirin-induced gastric ulcer in albino mice by alleviating oxidative stress and inflammation. Biomed Pharmacother. 2019 Jan:109:314-321.

Therapeutic Uses

Nonsteroidal anti-inflammatory drugs; cyclooxygenase inhibitors; fibrinolytic agents; platelet aggregation inhibitors.
Salicylate salts are indicated for the relief of myalgia, musculoskeletal pain, and other non-rheumatic inflammatory symptoms, such as sports injuries, bursitis, joint bursitis, tendinitis, and nonspecific acute tenosynovitis. /Included on US product label/
Salicylate salts are indicated for the relief of symptoms of acute and chronic rheumatoid arthritis, juvenile idiopathic arthritis, osteoarthritis, and related rheumatic diseases. Aspirin is usually the first-line treatment and may be the first-line treatment for patients who can tolerate long-term high-dose therapy. These medications do not affect the progression of rheumatoid arthritis. Depending on the disease being treated and the patient's response, concomitant use of corticosteroids or disease-modifying antirheumatic drugs may be necessary. /Included on US product label/
Salicylate salts are also used to reduce arthritis complications associated with systemic lupus erythematosus. /Salicylate salts; Not included on US product label/
For more complete data on the therapeutic uses of acetylsalicylic acid (12 in total), please visit the HSDB record page.

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Drug Warning
Taking aspirin may cause Reye's syndrome in children and adolescents with acute febrile illnesses, especially influenza and chickenpox. It is recommended that salicylates not be started in febrile children or adolescents until such illnesses have been ruled out. Furthermore, it is recommended that long-term salicylate treatment be discontinued if these patients develop fever, and treatment should not be resumed until an underlying condition that could cause Reye's syndrome has been identified and resolved. Other forms of salicylate poisoning may also be more common in children, especially those with fever or dehydration. Close monitoring of serum salicylate levels is recommended for children with Kawasaki disease. During the early febrile phase of the illness, aspirin absorption is impaired; it may be extremely difficult to reach the anti-inflammatory plasma salicylate concentrations required for treatment. Furthermore, absorption improves as the febrile phase passes; if the dose is not adjusted, salicylate poisoning may occur. Patients receiving high-dose salicylates may require increased vitamin K intake. Salicylate
If salicylate poisoning leads to impaired kidney function, potassium lost from cells can accumulate in the extracellular fluid, potentially causing potassium poisoning.
For more complete data on drug warnings for acetylsalicylic acid (21 in total), please visit the HSDB record page.

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Pharmacodynamics
Effects on Pain and Fever: Acetylsalicylic acid interferes with the production of prostaglandins throughout the body by targeting cyclooxygenase-1 (COX-1) and cyclooxygenase-2 (COX-2). Prostaglandins are potent irritants that have been shown to cause headaches and pain when injected into the body. Prostaglandins increase the sensitivity of pain receptors as well as substances such as histamine and bradykinin. By interfering with the production and release of prostaglandins during inflammation, this drug blocks the action of prostaglandins on pain receptors, thereby preventing pain symptoms. Acetylsalicylic acid is considered an antipyretic because it interferes with the production of prostaglandin E1 in the brain. Prostaglandin E1 is a known potent pyrogenic factor. Effects on Platelet Aggregation: The mechanism by which acetylsalicylic acid inhibits platelet aggregation is by interfering with the production of thromboxane A2 in platelets through the inhibition of COX-1. Thromboxane A2 is an important lipid responsible for platelet aggregation, which can lead to thrombosis and increase the risk of future heart disease or stroke. Regarding cancer prevention: In recent years, the role of acetylsalicylic acid in preventing various malignant tumors has been studied. Typically, acetylsalicylic acid participates in interfering with various cancer signaling pathways and can sometimes induce or upregulate tumor suppressor genes. Multiple studies have shown that long-term use of aspirin (ASA) has benefits in preventing various cancers, including stomach cancer, colorectal cancer, pancreatic cancer, and liver cancer. Related research is ongoing.

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1. Aspirin (acetylsalicylic acid; ASA) is a classic nonsteroidal anti-inflammatory drug (NSAID) with anti-inflammatory, antipyretic, and antiplatelet effects. Its anti-inflammatory mechanism involves a dual pathway: inhibition of IKKβ to block NF-κB activation (reducing the production of pro-inflammatory cytokines), and acetylation of cyclooxygenase (COX) to reduce prostaglandin synthesis (not found in the relevant literature) [1,2]
2. In terms of antipyretics, aspirin lowers body temperature by inhibiting the synthesis of central prostaglandins (its effect of reducing yeast-induced fever in rats at oral doses of 100-200 mg/kg has been confirmed), but its clinical use in children is limited due to the risk of Reye's syndrome (not mentioned in the relevant literature, therefore not considered) [3].
3. Gastric toxicity is the main adverse reaction of aspirin: it induces gastric mucosal erosion and ulceration by increasing oxidative stress (increasing MDA levels and decreasing GSH levels) and inhibiting the synthesis of gastric mucosal prostaglandins (as demonstrated by gastric lesions in mouse models), which limits its long-term use [4].

C9H8O4

180.16

180.042

C, 60.00; H, 4.48; O, 35.52

50-78-2

Aspirin;50-78-2; 50-78-2; 69-46-5 (calcium); 62952-06-1 (lysine); 23413-80-1 (Aspirin Aluminum); 552-98-7 (lithium); Deuterated Aspirin 921943-73-9; 97781-16-3

2244

White to off-white solid powder

1.3±0.1 g/cm3

321.4±25.0 °C at 760 mmHg

134-136 °C(lit.)

131.2±16.7 °C

0.0±0.7 mmHg at 25°C

1.551

1.19

1

4

3

13

212

0

BSYNRYMUTXBXSQ-UHFFFAOYSA-N

InChI=1S/C9H8O4/c1-6(10)13-8-5-3-2-4-7(8)9(11)12/h2-5H,1H3,(H,11,12)

2-acetyloxybenzoic 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: ≥ 10 mg/mL (55.51 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 100.0 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: ≥ 10 mg/mL (55.51 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 100.0 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: ≥ 10 mg/mL (55.51 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 100.0 mg/mL clear DMSO stock solution to 900 μL of corn oil and mix evenly.

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Solubility in Formulation 4: 4% DMSO +PBS: 10mg/mL

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