Inhibitiory Effect Of Quinone Derivatives On Carboxylesterase 2

ObjectiveThe purpose of the experiment is to investigate the effects of Quinone derivatives on the hydrolytic activity of Carboxylesterase 2（hCE2）in vitro and to estimate the Drug-Drug interaction（DDI）risk of hypericin and shikonin in vivo.Further research on hypericin and shikonin with strong inhibition has reached the goal of reducing toxicity and increasing efficiency during clinical use.Therefore,it provides a theoretical basis for the clinical development of a new combination drug strategy to ensure the safety and effectiveness of drug use.MethodsEnzymatic reaction was carried out in vitro using a Phase I metabolic buffer containing human liver microsomes（HLM）（pH 7.4）,inhibitors and fluorescent substrates at 37°C to simulate the condition of human physiological environment.The dimethyl sulfoxide（DMSO）was used instead of the inhibitor as a negative control.The residual activity（%）of the enzyme was calculated by measuring the fluorescence intensity of the product using a synergistic H1multifunctional microplate reader and high performance liquid chromatography.A broad spectrum of strong inhibitor bis（p-nitrophenyl）phosphate was used as a positive control to verify the reliability of the experimental results.The inhibition type,half-inhibitory concentration（IC50）and inhibition constant（Ki）of different Quinone derivatives for hCE2 were determined by further inhibition kinetic experiments.The intensity of inhibition of shikonin and hypericin in living cells was verified by cell fluorescence imaging experiments.The in vivo drug use risk was predicted by in vivo-in vitro extrapolation（IV-IVE）.ResultsThe initial screening of FD as a substrate revealed that ubidecarenone,chrysophanic acid,emodin,rhein,aloe emodin,emodin-3-methyl ether,1,3-dihydroxy-2-methylanthraquinone had no significant inhibitory effect on hCE2.The alkaloids of hypericin,1,2-dihydroxy-9,10-anthracenedione,purpurin,obtusifolin,aloin and the naphthoquinones of5-hydroxy-1,4-naphthalenedione,vitamin K1 and shikonin exhibited different degrees of inhibitory activity against hCE2.The IC₅₀ of the hydrolysis activity of hypericin,1,2-dihydroxy-9,10-anthracenedione,purpurin,obtusifolin,aloin on hCE2 were:0.89±0.142μM、80.40±0.213μM、26.40±0.318μM、86.89±0.245μM、23.10±0.318μM.Among the naphthoquinones,the IC₅₀ of the5-hydroxy-1,4-naphthalenedione,vitamin K1 and shikonin were 41.93±0.323μM、42.66±0.421μM、7.210±0.596μM,respectively.The inhibitory effects of hypericin and shikonin on hCE2 activity were further determined using another two hCE2 substrates.The IC₅₀ of the fluorescent substrate NCEN was26.6±0.262μM and 43.3±0.742μM,respectively.The IC₅₀ values measured with the clinical drug irinotecan（CPT-11）as the substrate were 108.7±0.253μM and 220±0.163μM,respectively.Combined with the determination results of three substrates,the Ki values of Hypericin for hCE2 hydrolysis were:1.57μM,21.9μM,55.6μM;the K_i values of shikonin for hCE2 hydrolysis activity were:9.43μM,42.9μM,250μM.When FD and NCEN were used as substrates,hypericin showed non-competitive inhibition,while with CPT-11 as a substrate,it was competitive inhibition.However,the inhibition type of shikonin measured by the three substrates was non-competitive inhibition.In addition,cell experiments demonstrated that hypericin and shikonin can significantly inhibit hCE2 activity in living cells.In vivo predictions show that both hypericin and shikonin can increase the area under the curve（AUC）of the three substrates in different degrees.ConclusionsCompared with other Quinone derivatives,hypericin and shikonin have stronger inhibitory effects on hCE2.When the half-inhibitory concentration(IC₅₀)and the inhibition constant（Ki）of the hydrolysis activity of hypericin and shikonin on hCE2 were determined by combining the three substrates,it was found that hypericin inhibited hCE2 more than shikonin.Moreover,the inhibitory effects of both are still present in living cells and have a high risk of DDI in vivo.Combined with the maximum concentration of hypericin and shikonin in the human intestinal tract,it is concluded that both as potent inhibitors of hCE2 can effectively inhibit the hydrolysis of hCE2 in the intestine to reduce the use of CPT-11.Delayed diarrhea caused by a large accumulation of the active product SN-38 hydrolyzed by hCE2 in the intestinal tract.It not only achieves synergistic anti-tumor effects,but also alleviates the symptoms of delayed diarrhea after taking CPT-11.At the same time,the risk of drug use in the body is predicted,and the safe dose of the drug is accurately judged.