T‐036 was synthesized at Neuroscience Drug Discovery Unit in Takeda Pharmaceutical Company Limited. For in vivo experiment, T‐036 was reconstituted with a 0.5% (w/v) methylcellulose solution (Fujifilm Wako) and was used at the indicated doses. The drug was administered to mice at 10 ml/kg body weight.
Methylcellulose solution
Manufactured by Fujifilm
Sourced in Japan
Methylcellulose solution is a viscous liquid composed of methylcellulose, a water-soluble, nonionic cellulose ether. It is used as a thickening, suspending, and binding agent in various laboratory and industrial applications.
Automatically generated - may contain errors
Lab products found in correlation
High phosphorus diet, by CLEA Japan (4 mentions)
Calcium assay kit, by Fujifilm (4 mentions)
Retsch mm300 tissuelyser, by Qiagen (4 mentions)
Dba 2 mouse, by CLEA Japan (2 mentions)
Risperidone, by LGC (2 mentions)
Polyethylene glycol 400, by Fujifilm (2 mentions)
N n dimethylacetamide, by Fujifilm (2 mentions)
Minocycline, by Merck Group (1 mentions)
Tween 80, by Merck Group (1 mentions)
0.5 ml insulin syringe, by Terumo (1 mentions)
Dasatinib, by Fujifilm (1 mentions)
Dasatinib, by LC Laboratories (1 mentions)
Ala11d leu15 orexin b, by Bio-Techne (1 mentions)
Angiotensin 2, by Peptide Institute (1 mentions)
γ oryzanol, by Fujifilm (1 mentions)
Tadalafil, by Cayman Chemical (1 mentions)
Mk 801 5r 10s 5 methyl 10 11 dihydro 5h dibenzo a d cyclohepten 5 10 imine hydrogen maleate, by Merck Group (1 mentions)
Corn starch, by Oriental Yeast (1 mentions)
Calcium carbonate, by Fujifilm (1 mentions)
Sodium dihydrogen phosphate dihydrate, by Fujifilm (1 mentions)
19 protocols using methylcellulose solution
1
Synthesis and In Vivo Administration of T-036
2
Anti-Calcification Compound Evaluation
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Example 5
A 5/6 nephrectomized DBA/2 mouse (male, 8 weeks old) is purchased from CLEA Japan, Inc. This mouse is loaded with 1.2% high-phosphorus diet (Oriental Yeast Co., Ltd.). Each test compound suspended in a 0.5% methylcellulose solution (powder purchased from Wako Pure Chemical Industries, Ltd. is adjusted to 0.5% with Otsuka distilled water) is administered orally twice daily for three months. After three months, the animal is sacrificed, and the kidney is sampled. The tissue sample is freeze-dried (FREEZE DRYER, FRD-50M, Iwaki Asahi Techno Glass Corp.). Then, 10% formic acid (undiluted solution purchased from Kishida Chemical Co., Ltd. is adjusted to 10% with Milli-Q water) is added to the tissue sample, which is then homogenized using QIAGEN Retsch MM300 TissueLyser (Qiagen N. V.). The homogenate is centrifuged, and the supernatant is used as a sample. The calcium concentration in the sample is measured as absorbance (ABS 612 nm, Microplate reader, model plus 384, Molecular Devices, LLC) using Calcium assay kit (Wako Pure Chemical Industries, Ltd.) to calculate the amount of calcium in the tissue.
The compound of the present invention exhibits an excellent anti-calcification effect and is useful as a pharmaceutical drug for the treatment or prophylaxis of ectopic calcification.
3
Nephrectomized Mouse Model for Ectopic Calcification
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Example 5
A 5/6 nephrectomized DBA/2 mouse (male, 8 weeks old) was purchased from CLEA Japan, Inc. This mouse was loaded with 1.2% high-phosphorus diet (Oriental Yeast Co., Ltd.). Each test compound suspended in a 0.5% methylcellulose solution (powder purchased from Wako Pure Chemical Industries, Ltd. was adjusted to 0.5% with Otsuka distilled water) was administered orally twice daily for three months. After three months, the animal was sacrificed, and the kidney was sampled. The tissue sample was freeze-dried (FREEZE DRYER, FRD-50M, Iwaki Asahi Techno Glass Corp.). Then, 10% formic acid (undiluted solution purchased from Kishida Chemical Co., Ltd. was adjusted to 10% with Milli-Q water) was added to the tissue sample, which was then homogenized using QIAGEN Retsch MM300 TissueLyser (Qiagen N.V.). The homogenate was centrifuged, and the supernatant was used as a sample. The calcium concentration in the sample was measured as absorbance (ABS 612 nm, Microplate reader, model plus 384, Molecular Devices, LLC) using Calcium assay kit (Wako Pure Chemical Industries, Ltd.) to calculate the amount of calcium in the tissue.
The compound of the present invention exhibits an excellent anti-calcification effect and is useful as a pharmaceutical drug for the treatment or prophylaxis of ectopic calcification.
4
Pharmacological Formulation for Preclinical Studies
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Pregabalin was dissolved in 5% Tween 80 (Sigma Aldrich) in saline formulated for intraperitoneal (i.p.) delivery of 3, 10, and 30 mg/kg in a volume of 3 mL/kg. EMA401 and minocycline (Sigma Aldrich) were suspended in 0.5% methyl cellulose solution (Wako) for oral administration (p.o.) of 10 mg/kg and 80 mg/kg respectively, in a volume of 5 mL/kg.
5
Anti-Calcification Compound Evaluation
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Example 5
A 5/6 nephrectomized DBA/2 mouse (male, 8 weeks old) is purchased from CLEA Japan, Inc. This mouse is loaded with 1.2% high-phosphorus diet (Oriental Yeast Co., Ltd.). Each test compound suspended in a 0.5% methylcellulose solution (powder purchased from Wako Pure Chemical Industries, Ltd. is adjusted to 0.5% with Otsuka distilled water) is administered orally twice daily for three months. After three months, the animal is sacrificed, and the kidney is sampled. The tissue sample is freeze-dried (FREEZE DRYER, FRD-50M, Iwaki Asahi Techno Glass Corp.). Then, 10% formic acid (undiluted solution purchased from Kishida Chemical Co., Ltd. is adjusted to 10% with Milli-Q water) is added to the tissue sample, which is then homogenized using QIAGEN Retsch MM300 TissueLyser (Qiagen N. V.). The homogenate is centrifuged, and the supernatant is used as a sample. The calcium concentration in the sample is measured as absorbance (ABS 612 nm, Microplate reader, model plus 384, Molecular Devices, LLC) using Calcium assay kit (Wako Pure Chemical Industries, Ltd.) to calculate the amount of calcium in the tissue.
The compound of the present invention exhibits an excellent anti-calcification effect and is useful as a pharmaceutical drug for the treatment or prophylaxis of ectopic calcification.
6
Anti-Calcification Effect of Compound
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Example 5
A ⅚ nephrectomized DBA/2 mouse (male, 8 weeks old) was purchased from CLEA Japan, Inc. This mouse was loaded with 1.2% high-phosphorus diet (Oriental Yeast Co., Ltd.). Each test compound suspended in a 0.5% methylcellulose solution (powder purchased from Wako Pure Chemical Industries, Ltd. was adjusted to 0.5% with Otsuka distilled water) was administered orally twice daily for three months. After three months, the animal was sacrificed, and the kidney was sampled. The tissue sample was freeze-dried (FREEZE DRYER, FRD-50M, Iwaki Asahi Techno Glass Corp.). Then, 10% formic acid (undiluted solution purchased from Kishida Chemical Co., Ltd. was adjusted to 10% with Milli-Q water) was added to the tissue sample, which was then homogenized using QIAGEN Retsch MM300 TissueLyser (Qiagen N.V.). The homogenate was centrifuged, and the supernatant was used as a sample. The calcium concentration in the sample was measured as absorbance (ABS 612 nm, Microplate reader, model plus 384, Molecular Devices, LLC) using Calcium assay kit (Wako Pure Chemical Industries, Ltd.) to calculate the amount of calcium in the tissue.
The compound of the present invention exhibits an excellent anti-calcification effect and is useful as a pharmaceutical drug for the treatment or prophylaxis of ectopic calcification.
7
Passive Cutaneous Anaphylaxis Model in Mice
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ICR mice (5- to 6-week-old) were sensitized with 20 μL of anti-DNP-IgE/PBS (25 μg/mL) by intradermal injection in the ears with a 0.5-mL insulin syringe (Terumo, Tokyo, Japan). After 24 h, passive cutaneous anaphylaxis (PCA) reactions were induced by intravenous injection of 200 μL of DNP-BSA/PBS (0.5 mg/mL) containing 1% Evans blue. Samples or vehicle (water) were orally administered by feeding needle 1 h before antigen challenge. Ume extract was dissolved in water and TL was suspended in 0.5% methyl cellulose solution (Wako, Osaka, Japan). Mice were sacrificed under anaesthesia 30 min after antigen challenge, and their ears were removed.
For measurement of Evans blue extravasation, mouse ears were punched by an 8-mm biopsy punch (Kai Medical, Gifu, Japan) and punched ears containing extravasated dye were dissolved in 800 μL of 1 N KOH at 37 °C for 24 h. An equal amount of phosphoric acid/acetone (5:13, v/v) was mixed with ears dissolved in KOH and filtered by a 0.45-μm filter. The absorbance of the extract was measured at 620 nm. For histological observation, remaining ears were fixed in 10% formalin and stained with toluidine blue (paraffin-embedded tissue sections, 4-μm thick).
For measurement of Evans blue extravasation, mouse ears were punched by an 8-mm biopsy punch (Kai Medical, Gifu, Japan) and punched ears containing extravasated dye were dissolved in 800 μL of 1 N KOH at 37 °C for 24 h. An equal amount of phosphoric acid/acetone (5:13, v/v) was mixed with ears dissolved in KOH and filtered by a 0.45-μm filter. The absorbance of the extract was measured at 620 nm. For histological observation, remaining ears were fixed in 10% formalin and stained with toluidine blue (paraffin-embedded tissue sections, 4-μm thick).
8
Synthesis and Characterization of Fatty Acids and Pharmaceuticals
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T-3601386, AM-1638 [14 (link), 15 (link)] and fasiglifam were synthesized at Takeda Pharmaceutical Company, Limited. (Kanagawa, Japan). Alpha-linolenic, linoleic, palmitic and γ-linolenic acid were purchased from FUJIFILM Wako (Osaka, Japan), Cayman Chemical (Ann Arbor, MI) or Sigma (St Louis, MO). Sibutramine, liraglutide, and CCK-8 were purchased from Alexis Biochemicals (San Diego, CA), Novo Nordisk pharma (Tokyo, Japan), and Peptide Institute, Inc. (Osaka, Japan), respectively. Compounds were dissolved in dimethyl sulfoxide for the in vitro study and were suspended or dissolved in 0.5% methylcellulose solution (FUJIFILM Wako) for oral dosing. Liraglutide and CCK-8 were dissolved in saline.
9
Dasatinib Treatment for Leukemic Engraftment
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After substantial engraftment of leukemic cells was confirmed by BM aspiration, mice (n = 2 per group) were treated with dasatinib (LC laboratories, Woburn, MA) or vehicle. dasatinib was formulated in 0.5% (w/v) methylcellulose solution (Wako, Japan) and delivered orally at 35 mg/kg for 5 days (Monday to Friday) on/2 days off. Leukemic cell chimerism was monitored by flow cytometry analysis of BM aspirates, as described above. Once leukemic cell chimerism in BM reached <5%, the dasatinib dose was reduced to 20 mg/kg. After chimerism had recovered to >90%, mice were sacrificed by cervical dislocation, the femur, tibia, ilium, and brain were removed and mechanically dispersed, and leukemic cells were then isolated by Ficoll–Hypaque density gradient centrifugation and used for FISH analysis.
10
Synthesis and Oral Administration of MR1916
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MR1916 was synthesized in house and used as the free base in all experiments. MR1916 and risperidone (Toronto Research Chemicals Inc.) were suspended in 0.5% methylcellulose solution (Wako Pure Chemical Industries, Ltd.) and orally administered at a volume of 5 mL/kg.
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