Calcium assay kit

Example 5

[Figure (not displayed)]

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.

Example 5

[Figure (not displayed)]

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.

Example 5

[Figure (not displayed)]

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.