Methazolamide

Manufactured by Merck Group

Sourced in United States

Methazolamide is a carbonic anhydrase inhibitor. It is used as a laboratory reagent for research purposes.

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Lab products found in correlation

6 protocols using methazolamide

Synthesis and Characterization of Methazolamide Conjugates

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Methazolamide, acivicin, isoniazid and methimazole were purchased from Sigma Chemicals (St. Louis, MO, USA). Dexamethasone, β-naphthoflavone, dimethylsulfoxide (DMSO), acetic acid and acetonitrile were obtained from Wako Pure Chemicals (Osaka, Japan). In quantitative studies, we employed purified Methazolamide. It was purified in our laboratory by HPLC as described in the next paragraph. Cysteine, cysteinylglycine, and glutathione conjugates of Methazolamide were prepared in our laboratory according to the method described in the preceding paper [27 (link)]. N-[3 (link)-Methyl-5-sulfo-1,3,4-thiadiazol-2(3H)-ylidene]acetamide (MSO), and N-(3-methyl-5-mercapto-Δ⁴-1,3,4-thiadiazol–2-yl)acetamide (MSH) were also synthesized in our laboratory [14 (link)]. Fig. (1) shows the chemical structure of these compounds with their code names as used in this text. Phosphate-buffered saline (PBS) and Dulbecco’s MEM were obtained from Nikken Bioscience (Kyoto, Japan). Fetal bovine serum (Cat #12-10378) was supplied by IRH Biosciences (Lenexa, KS, USA).

Sasabe T., Maeda S., Kishida K., Yamano M., Miwa Y, & Sugiyama T. (2016). The Metabolism of Methazolamide in Immortalized Human Keratinocytes, HaCaT Cells. Drug Metabolism Letters, 10(4), 295-305.

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Cell Viability Assay of Sulfonamides

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A cell viability assay was performed to identify the highest concentration of sulfonamides with no effect on cell viability. A number of 5 × 10⁴ REH cells or 5 × 10⁴ RAW 264.7 cells were seeded in 100 µL RPMI1460 + 10% h.i. + 1% P/S and cultured at 37 °C and 5% CO₂ overnight. The following day, cells were incubated with 10⁻³ M, 10⁻⁴ M, 10⁻⁵ M, 10⁻⁶ M, 10⁻⁷ M or 10⁻⁸ M methazolamide, furosemide and dorzolamide (all Sigma-Aldrich, St. Louis, MO, USA) for 24 h. Afterward, viability assays were carried out using CellTiter-Blue^® Cell Viability Assay (Promega, Madison, WI, USA), according to the manufacturer’s instructions.

Rump K., Koos B., Ziehe D., Thon P., Rahmel T., Palmowski L., Marko B., Wolf A., Witowski A., Bazzi Z., Bazzi M., Orlowski J., Adamzik M., Bergmann L, & Unterberg M. (2024). Methazolamide Reduces the AQP5 mRNA Expression and Immune Cell Migration—A New Potential Drug in Sepsis Therapy?. International Journal of Molecular Sciences, 25(1), 610.

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Purification and Characterization of MBP

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His-tagged maltose-binding protein (MBP) was expressed from a plasmid in E. coli and then purified through Ni-chelated Sepharose Fast Flow Resin (GE Healthcare) and HiLoad 16/60 Superdex 75 gel filtration column (GE Healthcare). The protein was exchanged into assay buffer (120 mM NaCl, 20 mM NaH₂PO₄/Na₂HPO₄, pH 7.4) by dialysis. Both carbonic anhydrases were obtained from a commercial vendor (h-CA I (Sigma C4396) and b-CA II (Sigma C2522)). All protein concentrations were determined with Quick Start^TM Bradford Protein Assay Kit (Bio-Rad, catalog no. 5000201).
Ligands were all obtained from commercial vendors, as follows: maltose (EMD Millipore 105910), acetazolamide (Sigma 97582), methazolamide (Sigma SML0720), sulfanilamide (Sigma 46874), trifluoromethanesulfonamide (Sigma 638455), and 4-nitrophenyl acetate (Sigma N8130).

Bai N., Roder H., Dickson A, & Karanicolas J. (2019). Isothermal Analysis of ThermoFluor Data can readily provide Quantitative Binding Affinities. Scientific Reports, 9, 2650.

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Neuroprotective Chemical Compounds Evaluation

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Methazolamide (MTZ, N-[5-(aminosulfonyl)-3-methyl-1,3,4-thiadiazol-2(3H)-ylidene]-acetamide), melatonin (MEL, N-acetyl-5-methoxy tryptamine), Trolox (6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid), and sulforaphane (SFN, 1-isothiocyanato-4-(methylsulfinyl)-butane) were procured from Sigma. The PI3K inhibitors Wortmannin and LY294002 were from Millipore-Sigma (Burlington, MA) and Cell Signaling (Danvers, MA), respectively. The GSK-3 inhibitor SB216763 was purchased from Cell Signaling.

Sotolongo K., Ghiso J, & Rostagno A. (2020). Nrf2 activation through the PI3K/GSK-3 axis protects neuronal cells from Aβ-mediated oxidative and metabolic damage. Alzheimer's Research & Therapy, 12, 13.

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Methazolamide Treatment for Subarachnoid Hemorrhage

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Methazolamide was purchased from Sigma-Aldrich, St. Louis, MO. The SAH + Methazolamide group received 20 mg/kg of Methazolamide intraperitoneally 30 min after SAH induction, administered once every 12 h for no more than 7 days. The drug was dissolved in phosphate-buffered saline (PBS 0.1 mol/L, pH 7.4) that containing 3% DMSO. The SAH + vehicle group received the same volume of PBS intraperitoneally 30 min after SAH induction.

Li M., Wang W., Mai H., Zhang X., Wang J., Gao Y., Wang Y., Deng G., Gao L., Zhou S., Chen Q, & Wang X. (2016). Methazolamide improves neurological behavior by inhibition of neuron apoptosis in subarachnoid hemorrhage mice. Scientific Reports, 6, 35055.

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N-in-one Permeability Assay Protocol

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We chose 32 clinically used drugs for the N-in-one platform. Stock solutions of drugs (1 or 10 mg/mL) were prepared in either phosphate-buffered saline (PBS) or dimethyl sulfoxide (DMSO). The compounds were acetazolamide (Sigma, St. Louis, MO) in PBS, Because the compounds exhibited varying quantitation limits, they were prepared at 2 different stock concentrations: 1 and 10 mg/mL. Most (n ¼ 22) of the compounds belonged to the 1 mg/mL group, while 10 mg/mL was used for the following 10 compounds: aztreonam, bromfenac, dexamethasone, diclofenac, indomethacin, levocabastine, methazolamide, prednisolone, quinidine, and tizanidine. The cassette mix was prepared by diluting the stock solutions 1:100 with balanced salt solution (BSS Plus, Alcon Laboratories, Ft. Worth, TX). Another 1:10 dilution with BSS Plus was performed in the apical side of the diffusion cell to reach final drug concentrations in the permeability assays (1 or 10 mg/mL). The final DMSO concentration was 1.6% in the apical side.

Ramsay E., Ruponen M., Picardat T., Tengvall U., Tuomainen M., Auriola S., Toropainen E., Urtti A, & Del Amo E.M. (2017). Impact of Chemical Structure on Conjunctival Drug Permeability: Adopting Porcine Conjunctiva and Cassette Dosing for Construction of In Silico Model. Journal of pharmaceutical sciences, 106(9).

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