Qi Chen
(Key Laboratory of Drug-Targeting and Drug Delivery System of the Education Ministry and Sichuan Province, Sichuan Engineering Laboratory for Plant-Sourced Drug and Sichuan Research Center for Drug Precision Industrial Technology, West China School of Pharmacy, Sichuan University, Chengdu 610041, China.)
Background: CYP3A4 and 3A7 specifically catalyze the tertiary oxidation of deoxychoate (DCA) and glycodeoxycholate (GDCA). The oxidation rates fit well with Hill kinetic model at low substrate concentrations (DCA 1-400 μM, GDCA 30-250 μM), but gradually decrease as substrate levels increase, eventually leading to complete enzyme deactivation above the critical micelle concentration (CMC). We hypothesized that this abnormal kinetics is associated with microsomal vesicle disruption induced by their submicellar assembly.
Methods: We performed DCA/GDCA oxidation assays using human liver microsomes (HLM) and recombinant CYP3A4/3A7. Bilirubin was added as a guest in molecular assembly to assess its effect on oxidation rates of DCA/GDCA. Transmission electron microscopy (TEM) examined microsomal vesicle changes with varying DCA concentrations. UV, NMR, and UV/fluorescence (bilirubin as a probe) analyzed submicellar assembly of DCA/GDCA. Phenacetin/dextromethorphan oxidation and DCA/GDCA sulfation assays explored membrane dependency of abnormal kinetics.
Results: Spectroscopic analysis determined the CMCs of both DCA and GDCA at about 1000 μM in 0.1M PBS, with evidence suggesting small aggregate formation below the CMC. TEM revealed that DCA did not damage microsomal vesicles at submicellar levels but disrupted membranes when concentrations reached 1000 μM. Submicellar DCA/GDCA did inhibit their own CYP3A-catalyzed oxidation, while their assembly with bilirubin (50 μM) did not affect oxidation rates. Finally, submicellar DCA/GDCA inhibited vesicle-bound CYP1A2 and CYP2D6, but not cytosolic sulfurtransferase, confirming that abnormal kinetics is dependent on vesicle membrane.
Conclusions: Submicellar DCA and GDCA inhibit their own CYP3A-catalyzed oxidation via vesicle membrane-dependent mechanisms, with their self-assembly potentially linked to the abnormal kinetics.
Cuitong Wang
(Key Laboratory of Drug-Targeting and Drug Delivery System of the Education Ministry and Sichuan Province, Sichuan Engineering Laboratory for Plant-Sourced Drug and Sichuan Research Center for Drug Precision Industrial Technology, West China School of Pharmacy, Sichuan University, Chengdu 610041, China.)
Bin Cheng
(Key Laboratory of Drug-Targeting and Drug Delivery System of the Education Ministry and Sichuan Province, Sichuan Engineering Laboratory for Plant-Sourced Drug and Sichuan Research Center for Drug Precision Industrial Technology, West China School of Pharmacy, Sichuan University, Chengdu 610041, China.)
Qi Chen
(Key Laboratory of Drug-Targeting and Drug Delivery System of the Education Ministry and Sichuan Province, Sichuan Engineering Laboratory for Plant-Sourced Drug and Sichuan Research Center for Drug Precision Industrial Technology, West China School of Pharmacy, Sichuan University, Chengdu 610041, China.)
Wei Wei
(Key Laboratory of Drug-Targeting and Drug Delivery System of the Education Ministry and Sichuan Province, Sichuan Engineering Laboratory for Plant-Sourced Drug and Sichuan Research Center for Drug Precision Industrial Technology, West China School of Pharmacy, Sichuan University, Chengdu 610041, China.)
Wushuang Zeng
(1 Key Laboratory of Drug-Targeting and Drug Delivery System of the Education Ministry and Sichuan Province, Sichuan Engineering Laboratory for Plant-Sourced Drug and Sichuan Research Center for Drug Precision Industrial Technology, West China School of Pharmacy, Sichuan University, Chengdu 610041, China. 2 Chengdu Cynogen Bio-pharmaceutical Tech. Co., Ltd., Chengdu, China.)
Lanlan Gui
(1 Key Laboratory of Drug-Targeting and Drug Delivery System of the Education Ministry and Sichuan Province, Sichuan Engineering Laboratory for Plant-Sourced Drug and Sichuan Research Center for Drug Precision Industrial Technology, West China School of Pharmacy, Sichuan University, Chengdu 610041, China. 2 Chengdu Cynogen Bio-pharmaceutical Tech. Co., Ltd., Chengdu, China.)
Yixuan Wang
(Key Laboratory of Drug-Targeting and Drug Delivery System of the Education Ministry and Sichuan Province, Sichuan Engineering Laboratory for Plant-Sourced Drug and Sichuan Research Center for Drug Precision Industrial Technology, West China School of Pharmacy, Sichuan University, Chengdu 610041, China.)
Yutong Wang
(Key Laboratory of Drug-Targeting and Drug Delivery System of the Education Ministry and Sichuan Province, Sichuan Engineering Laboratory for Plant-Sourced Drug and Sichuan Research Center for Drug Precision Industrial Technology, West China School of Pharmacy, Sichuan University, Chengdu 610041, China.)
Ke Lan
(1 Key Laboratory of Drug-Targeting and Drug Delivery System of the Education Ministry and Sichuan Province, Sichuan Engineering Laboratory for Plant-Sourced Drug and Sichuan Research Center for Drug Precision Industrial Technology, West China School of Pharmacy, Sichuan University, Chengdu 610041, China. 2 Chengdu Cynogen Bio-pharmaceutical Tech. Co., Ltd., Chengdu, China.)
