Studies On Biological Metabolic Reaction Of Berberine And Bioactivities Of Its Metabolites

Berberine (BBR), an isoquinoline natural product isolated from herbs such as Coptis chinensis, has been used in China for several decades as an anti-diarrhea drug. Our previous studies showed that BBR was a novel LDLR-up-regulator and cholesterol-lowering agent with a mechanism on the post-transcriptional level, which is distinct from that of statins. In addition, we also found that InsR was another key target for its hypoglycemic effect. These results indicate that BBR was a potential drug for the treatment of metabolic syndrome (MS). However, the metabolic enzymes of BBR and bioactivities of BBR metabolites are still unknown. To address this issue, the present study was performed to investigate the active metabolites of BBR and to identify the CYP450 (cytochrome P450) isoenzymes that are responsible for the production of BBR metabolites. Next, we take the active metabolite as a parent compound for the pro-drug study, in order to obtain effective candidate compounds for MS, with independent intellectual property and better bioavailability.We have designed a series of in vitro and in vivo experiments to study the I-phase metabolites. In the animal experiment, we used 200 mg/kg of BBR to treat the Wistar rats orally. We identified 4 metabolites, M1 (berberrubine), M2 (thalifendine), M3 (demethyleneberberine) and M4 (jatrorrhizine). For the in vitro studies, we investigated respectively in the rat liver homogenate, S9, liver microsomes, human liver microsomes, insect microsomes expressing human P450, primary hepatocyte, and Bel-7402 and HepG2 cell line. We found metbolites M2 and M3 of BBR in all the in vitro systems.In order to further identify P450 isozymes responsible for BBR's metabolism, we firstly used computer assistant docking software, by which we found that CYP2D6, CYP1A2 and CYP3A4 were the top three. Subsequently, two biological experiments, recombinant P450 incubation and inhibitory assay were developed to verify the screening P450 isozymes. Next, we ranked these P450 isozymes with the method of total normalized rate (TNR). For BBR to M2, CYP1A2, CYP2D6 and CYP3A4 are responsible for 78.38%, 18.97%and 2.65%of total transformation, respectively. And for M3 formation, CYP3A4 for 38.43%, CYP1A2 for 31.18%and CYP2D6 for 30.39%, respectively.To deeply understand the mechanisms and the chemical entities of BBR's multiple effects, we studied the activities of LDLR mRNA and InsR mRNA up-regulating, AMPK acivating in BBR and its metabolites M1-M4. M1-M4 remained bioactivities, and BBR in its original form showed the highest activity among these chemical entities. We speculated that BBR combined its major metabolites M1-M4 in modulating cellular energy metabolism in vivo. Besides, among the metabolites, Ml showed the best activity, very close to BBR. And the exposed 9-OH in Ml was better convenient for synthesizing pro-drugs, which could improve the bioavailability of BBR.Therefore, we semi-synthesized a series of M1 pro-drugs by 9-OH modification. By screening the plasma hydrolysis behavior and animal anti-hyperlipidemic experiment, we found 5g (Ml palmitate) for the first time which transformed BBR to a novel MS drug with better bioavailability and biactivity.In summary, evidences from subcellular, cellular level to animal; from biological study, computer technology, to chemical design all prove that BBR was bio-metabolized in vivo. It was metabolized at least through the action of human hepatocyte CYP2D6, CYP1A2 and CYP3A4. The phase I metabolites of BBR remained to be active in on the targets with significantly reduced potency. Designing pro-drugs of BBR's active metabolite M1 was one of the most effective approaches to improve BBR's bioavailability and discover novel MS drugs which are effective and low toxic independently.