Specific probe substrate for cytochrome P450 3A4 enzyme and application of substrate
A technology of cytochrome and probe substrate, which is applied in the field of medical technology of the Ming Dynasty, can solve the problems of confusing the catalytic ability of cytochrome P4503A4 and 3A5, and the inability to clearly analyze the metabolic scavenging ability of cytochrome P4503A4, and achieve good ultraviolet absorption characteristics, detect sensitive effects
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Embodiment 1
[0032] Cinobufagin used to detect enzyme activity of human recombinant CYP3A4 and CYP3A4+cytochrome b5 system
[0033] Cinobufagin was used to detect the difference in the catalytic activity of two human recombinant single enzymes (the recombinant expression system contains cytochrome b5 and does not contain cytochrome b5), the specific steps are as follows:
[0034] (1) In 200 microliters of in vitro metabolic reaction system, 10 mM glucose-6-phosphate, 1 unit of glucose-6-phosphate dehydrogenase, and 4 mM MgCl 2 , the concentration of recombinant CYP3A4 containing cytochrome b5 was 0.05 mg / ml, the final concentration of cinobufagin was 30 μM, and pre-incubated at 38°C for 5 minutes;
[0035] (2) Add 20 μl NADP to the reaction system + (final concentration 1 mM) initial reaction;
[0036] (3) After 10 minutes, add 200 μl methanol and shake vigorously to terminate the reaction;
[0037] (4) Use a high-speed refrigerated centrifuge to centrifuge the above system for 10 min...
Embodiment 2
[0041] Determination of CYP3A4 enzyme activity in 12 individual cases of human liver microsomes with bufagenin
[0042] Twelve commercial samples of human liver microsomal samples from different individuals were purchased, and the enzyme activity of CYP3A4 in human liver samples was determined by using bufagenin. The specific operation procedure is as follows:
[0043] (1) In 200 microliters of in vitro metabolic reaction system, 10 mM glucose-6-phosphate, 1 unit of glucose-6-phosphate dehydrogenase, and 4 mM MgCl 2 , the concentration of human liver microsomes was 0.2 mg / ml, the final concentration of bufagenin was 30 μM, and pre-incubated at 37°C for 3 minutes;
[0044] (2) Add 20 μl NADP to the reaction system + (final concentration 1 mM) initial reaction;
[0045] (3) After 10 minutes, add 200 μl methanol and shake vigorously to terminate the reaction;
[0046] (4) Use a high-speed refrigerated centrifuge to centrifuge the above system for 10 minutes under the conditi...
Embodiment 3
[0049] Determination of CYP3A4 Enzyme Activity in Human Hepatocellular Carcinoma Cell BEL7402 by Bufalin
[0050] (1) Dilute BEL7402 cells with hepatocyte incubation solution, place in a 6-well culture plate, 4ml per well, put in a metal bath shaker, 80 r / min, 40 ℃ for 120 minutes with continuous shaking;
[0051] (2) Add bufalin to the culture plate, the final concentration is 50 μM;
[0052] (3) After 30 minutes, draw 200 μl of the incubation solution and place it in a -80°C ultra-low temperature refrigerator to stop the reaction;
[0053] (4) Add 200ul of methanol to the sample to precipitate protein, and use a high-speed refrigerated centrifuge to centrifuge the above system for 10 minutes under the condition of 20,000×g, then take the supernatant for UFLC-ESI-MS detection and analysis
[0054] UFLC-ESI-MS was used to detect bufalin and its metabolites, and the SIM mode was selected to detect the molecular ion peaks of bufalin and its metabolites [M+H] + ( m / z 387 ...
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