Diclofenac

Manufactured by Cayman Chemical

Sourced in United States

Diclofenac is a synthetic compound commonly used as a reference standard in pharmaceutical analysis. It is a non-steroidal anti-inflammatory drug (NSAID) with analgesic and antipyretic properties. Diclofenac is often utilized in research and quality control settings to assess the identity, purity, and potency of drug products containing this active ingredient.

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Lab products found in correlation

Indomethacin, by Merck Group (3 mentions) Sulindac sulfide, by Merck Group (2 mentions) Salubrinal, by Apexbio (2 mentions) Penicillin, by Thermo Fisher Scientific (2 mentions) Streptomycin, by Thermo Fisher Scientific (2 mentions) Celecoxib, by Merck Group (2 mentions) Acetaminophen, by Merck Group (2 mentions) Naproxen sodium, by Apexbio (2 mentions) Mccoy s 5a, by Thermo Fisher Scientific (2 mentions) M3 media, by INCELL (2 mentions) Sodium salicylate, by Merck Group (2 mentions) Hct116, by American Type Culture Collection (2 mentions) Metformin, by Cayman Chemical (2 mentions) Prostaglandin f2α, by Cayman Chemical (1 mentions) Xe991, by Cayman Chemical (1 mentions) Histamine, by Merck Group (1 mentions) Ml213, by Cayman Chemical (1 mentions) Nifedipine, by Merck Group (1 mentions) Adenosine, by Merck Group (1 mentions) Tamsulosin hydrochloride, by Merck Group (1 mentions)

12 protocols using diclofenac

Histamine and Pharmacological Compounds

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Histamine was purchased from Sigma-Aldrich (St. Louis, MO). ML213, UCL2077, diclofenac, Prostaglandin F_2α, bradykinin and XE991 were purchased from Cayman Chemical (Ann Arbor, MI).

Chen X., Li W., Hiett S.C, & Obukhov A.G. (2016). Novel Roles for Kv7 Channels in Shaping Histamine-Induced Contractions and Bradykinin-Dependent Relaxations in Pig Coronary Arteries. PLoS ONE, 11(2), e0148569.

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Preparation of Compound Stock Solutions

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100 mM stock solutions of all screened compounds were prepared in individual, sterile amber vials. All stock solutions were dissolved in sterile DMSO, except L-arginine, which was solubilized using dH₂O, due to solubility constraints. Sterile cell-culture grade DMSO, tamsulosin hydrochloride, nifedipine, isoproterenol, butoxamine, adenosine and L-arginine were obtained from Sigma Aldrich. PGE1, PGE2, rolipram, L-arginine, ondansetron, celecoxib, diclofenac, mirabegron, sildenafil, atropine, vardenafil and tiotropium were obtained from Cayman Chemical. Y-27632 dihydrochloride was obtained from R&D Systems. Stock solutions when unused were stored at −20 °C

Lee C.X., Cheah J.H., Soule C.K., Ding H., Whittaker C.A., Karhohs K., Burds A.A., Subramanyam K.S., Carpenter A.E., Eisner B.H, & Cima M.J. (2019). Identification and local delivery of vasodilators for the reduction of ureteral contractions. Nature biomedical engineering, 4(1), 28-39.

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Platelet Function Inhibition Assay

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The TP antagonist SQ 29,548 was purchased from Sigma (product no. NET936). Ramatroban and Diclofenac were purchased from Cayman Chemicals (Michigan, USA), L-670596 from Tocris Bioscience (Bristol, UK). The TP agonist U46619 was purchased from the Cayman Chemicals Company. Protease inhibitors and common chemicals were purchased from either Fisher or Sigma. The buffers and detergents were the same as those used previously [15] (link). Polyclonal antibody directed towards 1−15 amino acids of the N-terminal in TPα was purchased from Lifespan BioSciences (Washington, USA). PE-anti-CD41 antibody and FITC-anti-CD62P were purchased from Biolegend (California, USA). Nucleofection kit for Meg −01 cells was purchased from Lonza (Texas, USA).

Chakraborty R., Bhullar R.P., Dakshinamurti S., Hwa J, & Chelikani P. (2014). Inverse Agonism of SQ 29,548 and Ramatroban on Thromboxane A2 Receptor. PLoS ONE, 9(1), e85937.

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Analytical Standards for Environmental Analysis

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LC-MS grade methanol and water were obtained from VWR, Darmstadt, Germany. Quercetin dehydrate (≥95%, high performance liquid chromatography (HPLC)) was purchased from Enzo Life Sciences GmbH, Lörrach, Germany. Diclofenac (>99%) was obtained from Cayman Chemical Company, Ann Arbor, Michigan, MI, USA. Glyphosate (100 quality level, HPLC), gabapentin (200 quality level, HPLC), monuron (100 quality level, HPLC), chloridazon (100 quality level, HPLC), carbetamide (100 quality level, HPLC), metobromuron (100 quality level, HPLC), sotalol (≥98%), quinoxyfen (100 quality level, HPLC), metconazol (100 quality level, HPLC) and fenofibrate (≥99%) were obtained from Sigma, Darmstadt, Germany. Metformin (300 quality level, HPLC) was obtained from Fluka, Buchs, Switzerland. Furthermore, chlorbromuron (99.24%) and diazinon (99.53%) were obtained from Dr. Ehrenstorfer, Augsburg, Germany. Carbamazepine, 2,3-dihydro-2,3-dihydroxycarbamazepine, 10,11-dihydro-10,11-dihydroxy-carbamazepine, 10,11-dihydro-10-hydroxy-carbamazepine, 9-acridine carboxaldehyde and carbamazepine-10,11-epoxide were kindly provided by the German Research Center for Environmental Health, Comparative Microbiome Analysis (COMI), Helmholtz Centrum of Munich, Munich, Germany.

Wahman R., Sauvêtre A., Schröder P., Moser S, & Letzel T. (2020). Untargeted Metabolomics Studies on Drug-Incubated Phragmites australis Profiles. Metabolites, 11(1), 2.

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Diclofenac Blocks PGE2 in dTHESCs

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A nonselective COX inhibitor, diclofenac (Cayman Chemical), was used to block PGE₂ production in dTHESCs in vitro to generate CM supernatants for later THP-1 experimentation. Briefly, a 6-well dish containing 1 × 10⁶ dTHESCs in 1 mL was treated for 1 h with 3 μg/mL diclofenac or solvent control (0.1% ethanol) to prime the stromal cells before DMEM/F12^+/+ was changed and diclofenac was added again for 48 h with fresh media. CM supernatants were harvested, frozen, and assayed for the presence of PGE₂ by ELISA. Following dTHESC CM supernatant generation, THP-1 cells were PMA treated and plated at 1 × 10⁵ per well in a 96-well plate (200 μL) and rested for 1 h prior to pretreatment with diclofenac or solvent control-containing CM supernatant. After 1 h with CM supernatants, THP-1 cells were treated or not with 1 μg/mL LPS for 24 h. Supernatants were collected and stored at −20°C for ELISA analysis.

Rogers L.M., Anders A.P., Doster R.S., Gill E.A., Gnecco J.S., Holley J.M., Randis T.M., Ratner A.J., Gaddy J.A., Osteen K, & Aronoff D.M. (2018). Decidual stromal cell-derived PGE2 regulates macrophage responses to microbial threat. American journal of reproductive immunology (New York, N.Y. : 1989), 80(4), e13032.

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Comprehensive in vitro Metabolism Assay

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ADB-FUBINACA and diclofenac standards were purchased from Cayman Chemical (Ann Arbor, MI, USA) and Toronto Research Chemicals (Toronto, Canada) respectively. LC-MS grade water, methanol, and formic acid (Optima™ LC/MS) were acquired from Fisher Scientific (Fair Lawn, NJ, USA), and trypan blue and LC-MS grade acetonitrile from Sigma-Aldrich^® (St. Louis, MT, USA). Distilled water was produced by an ELGA PURELAB^® Ultra Analytic purifier (Siemens Water Technologies, Lowell, MA, USA). Fifty-donor pooled HLM, ten-donor-pooled cryopreserved human hepatocytes, InVitroGRO™ hepatocyte thawing (HT) medium, and InVitroGRO™ Krebs-Henseleit buffer (KHB) were obtained from BioreclamationIVT (Westbury, NY, USA). Solutions A (NADPH regeneration system) and B (glucose-6-phosphate dehydrogenase) were purchased from BD Biosciences (San Jose, CA, USA).

Carlier J., Diao X., Wohlfarth A., Scheidweiler K, & Huestis M.A. (2017). In Vitro Metabolite Profiling of ADB-FUBINACA, A New Synthetic Cannabinoid. Current Neuropharmacology, 15(5), 682-691.

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Ras-MAPK Signaling Pathway Modulation

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Docosahexaenoic acid, and diclofenac were obtained from Cayman Chemical (Ann Arbor, MI, USA). Indomethacin, naproxen, piroxicam, resazurin, horseradish peroxidase-labeled mouse anti-β-actin, anti-K-Ras, anti-N-Ras, and anti-H-Ras antibodies were purchased from Sigma-Aldrich Chemical Co. (St. Louis, MO, USA). Antibodies specific for Bax, Bim, Mcl-1, Bcl-xL, PARP, cleaved PARP, pro-caspase 3, pro-caspase 7, cleaved-caspase 3, phospho-MEK1/2-Ser217/221, phospho-p44/42 MAPK (Erk1/2)-Thr202/Tyr204, phospho-p90RSK-Ser380, phospho-Akt (Ser473), Akt (pan), horseradish peroxidase-labeled anti-mouse, and anti-rabbit immunoglobulins were obtained from Cell Signaling Technology (Beverly, MA, USA). The Ras Activation Assay Kit was purchased from Cytoskeleton Inc. (Denver, CO, USA).

Poku R.A., Jones K.J., Van Baren M., Alan J.K, & Amissah F. (2020). Diclofenac Enhances Docosahexaenoic Acid-Induced Apoptosis in Vitro in Lung Cancer Cells. Cancers, 12(9), 2683.

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In Vitro Anti-Inflammatory Assays

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The chemicals λ-carrageenan, Diclofenac sodium and carboxy methyl cellulose (CMC) were purchased from Sigma-Aldrich Chemical Co. in Mumbai, India. Cyclooxygenase (COX) Inhibitory Screening Assay Kit, Lipoxygenase (5-LOX) Inhibitor Screening Assay Kit, Secretory Phospholipase A2 (sPLA2) (Type V) Inhibitor Screening Assay Kit, Diclofenac, Nordihydroguaiaretic acid (NGDA) and Arachidonic Thioester Phosphatidylcholine (TEPC) were procured from Cayman Chemical Company (Tallinn, Estonia).

George A., Chinnappan S., Chintamaneni M., Kotak C V., Choudhary Y., Kueper T, & Radhakrishnan A.K. (2014). Anti-inflammatory effects of Polygonum minus (Huds) extract (Lineminus™) in in-vitro enzyme assays and carrageenan induced paw edema. BMC Complementary and Alternative Medicine, 14, 355.

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Spinal Cord Injury Treatment Study

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Male C57BL/6 mice (8 weeks old; 25-30 g) were obtained from National Laboratory Animal Center (Taipei, Taiwan). The animals were acclimatized to the new environment one week and then randomly assigned into the following groups (6 mice/ group): 1. control group (no injury, PBS-treated, oral gavage), 2. injury group (with drop-mass injury, PBS-treated, oral gavage), 3. injury+ Diclofenac group (with drop-mass injury, Diclofenac-treated, 10 mg/kg BW, oral gavage), and 4. injury+ betulin group (with drop-mass injury, betulin-treated, 10 mg/kg BW, oral gavage). Diclofenac and betulin powder were purchased from the Cayman Chemical (Ann Arbor, Michigan, USA). All animals were received humane care following the guidelines of the Institutional Animal Care and Use Committee of Changhua Christian Hospital (CCH-AE-110-012). The “resource equation” approach was used to calculate the number of animals 16 (link), 17 . Based on this approach, the acceptable range of the error degrees of freedom (DF) in an analysis of variance (ANOVA) is between 10 to 20. DF = Total number of animals - Total number of groups. In this study, DF is 20 that located in the acceptable range.

Lu Y.H., Huang Y.F., Hsieh C.P., Chen J.K., Chen H.Y, & Chuang S.M. (2024). Betulin Accelerated the Functional Recovery of Injured Muscle in a Mouse Model of Muscle Contusion. International Journal of Medical Sciences, 21(1), 37-44.

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Culturing and Treating CRC Cell Lines

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Human CRC cell lines, including HCT116, RKO and HT29 (purchased from American Type Culture Collection) and derivatives, were cultured in McCoy’s 5A (Gibco; ThermoFisher) modified media supplemented with 10% defined FBS (Hyclone), 100 U/mL penicillin, and 100 μg/mL streptomycin (Invitrogen). NCM356 non-transformed colonic epithelial cells (INCELL) and derivatives were cultured in M3 Media (INCELL). DR5 KO HCT116 and RKO cells were described previously [49 (link)].
Cell lines were authenticated by genotyping and analysis of protein expression by Western blotting, and routinely checked for Mycoplasma contamination by PCR. All cell lines were maintained at 37°C and an atmosphere of 5% CO₂.
For drug treatment, cells were plated in 12-well plates at 20-30% density 24 hr prior to treatment. Chemical agents, including sulindac sulfide (Merck), indomethacin (Sigma), celecoxib (Sigma), acetaminophen (Sigma), metformin (Cayman Chemical), diclofenac (sodium salt; Cayman Chemical), sodium salicylate (Sigma), and salubrinal (Apex Bio), were prepared as DMSO stock solutions, except for Naproxen sodium (Apex Bio), which was dissolved in dH₂O. Stock solution of each agent was diluted to appropriate concentrations in cell culture medium, and then added to cells.

Fletcher R., Tong J., Risnik D., Leibowitz B., Wang Y.J., Concha-Benavente F., DeLiberty J.M., Stolz D.B., Pai R.K., Ferris R.L., Schoen R.E., Yu J, & Zhang L. (2021). Non-steroidal anti-inflammatory drugs induce immunogenic cell death in suppressing colorectal tumorigenesis. Oncogene, 40(11), 2035-2050.

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