Category: Preclinical Development
Purpose: Metformin (MET) and berberine (BBR) have been proven to be effective as anti-diabetic treatments in clinical practice. In addition, both of them were reported to be able to individually alter the composition of human gut microbiota. Our previous studies indicated that co-administration of BBR with MET after oral route could lead to increased AUC0-∞ and decreased CL/F of MET. To further evaluate the potential mechanisms of the interactions between the two drugs in GI tract, role of rat intestinal content, human fecalase and selected bacteria in the GI metabolism of MET in presence and absence of BBR were investigated in this study.
Methods: In-vitro incubations of MET and/or BBR with rat intestinal contents (n=5 in each group), human fecalase (n=2 in each group) and bacteria (n=3 in each group) were conducted. To mimick the potential co-treatments of MET in absence and presence of BBR in our previous pharmacokinetics studies in rats, five treatments (G1R to G5R for rat intestinal content, G1H to G5H for human fecalase, G1B to G5B for bacteria) were included. Rat intestinal content was obtained by removing the whole gut content followed by homogenized with 0.9% cold saline under 4-degree Celsius. Human fecalase was prepared by suspending 12.5 g sample into 50 ml saline and 10% glycerol, followed by centrifuging to obtain aliquots. Bacteria of Bacteroides fragilis, Bifidobacterium pseudolongum, Clostridium perfringens, Staphylococcus aureus, Klebsiella pneumoniae, Escherichia coli, Enterococcus faecalis, Enterobacter cloacae, Lactobacillus fermentum were selected.
For incubation with the rat intestinal contents and human fecalase, G1R/H and G2R/H were incubated with MET and BBR alone at a final concentration of 10 μg/ml, respectively. In G3R/H, incubation was conducted with MET (10 μg/ml) followed by BBR (10 μg/ml) 2-h after, while incubation order was reversed in G4R/H. In G5R/H, samples were incubated with MET (10 μg/ml) and BBR (10 μg/ml) at the same time. For incubation with the selected bacteria (G1B to G5B), MET and/or BBR at the final concentration of 50 μM were incubated followed the same grouping as above. All above mixture were incubated at 37-degree Celsius, while the incubations with bacteria were conducted under anaerobic conditions. Samples were collected at various time points up to 24-h. To 50 µl of the collected incubation sample, 25 µl albuterol sulfate (internal standard for MET, 250 ng/ml), 50 µl caffeine (internal standard for BBR, 250 ng/ml) and 100 µl methanol were added for protein precipitation followed by 20 seconds vortex. After centrifugation of the mixture at 13,000 rpm for 15 min, the supernatants were collected for concentrations analyses of MET and BBR by our previously developed and validated LC/MS/MS methods.
Results: As demonstrated in Figure 1, incubation with rat intestinal content revealed that only 50% MET and 60% BBR remained after 24-h in G1R and G2R, respectively. Compared with G1R, 2-h post-dose (G3R) and pre-dose (G4R) treatment of BBR significantly inhibited the MET degradation in intestine with 62.84 ± 4.54% (G3R) and 63.06 ± 9.05% (G4R) of MET remained after 24-h incubation, respectively (p < 0.05). Percentage of MET remaining did not show significant difference between G1R and G5R. On the contrary to the changed content of MET, the amount of BBR remained after 24-h incubation in rat intestinal content was not affected by MET (G2R to G5R) (Figure 1). The corresponding incubations of MET and BBR with human fecalase (G1H-G5H) depicted similar trend in percentage remaining as that from rat intestinal content (G1R to G5R) (Figure 2). Moreover, significant impacts of the selected bacteria on MET and/or BBR metabolism were also found after 24-h in-vitro incubation (Figure 3). Compared with G1B, post-dose (G3B) and pre-dose (G4B) of BBR decreased the MET degradation significantly (1.26% - 25.67%) in most selected of bacteria (p < 0.05). Meanwhile, MET decreased the BBR degradation (1.32% - 25.36%) only in certain bacteria after 24-h incubation (p < 0.05).
Conclusion: Our study for the first time demonstrated that MET and BBR could be degraded in rat intestinal content, human fecalase and bacteria with similar trend. Two-hour pre-dose/post-dose with BBR could significantly decrease the degradation of MET, leading to its higher bioavailability in-vivo. Therefore, microbiota could play a significant role in the pharmacokinetics interactions between MET and BBR.
Yufeng Zhang– Student, Chinese University of Hong Kong, Hong Kong, Hong Kong
Mengbi Yang– Post-doc, School of Pharmacy, Faculty of Medicine, The Chinese University of Hong Kong, Hong Kong, Hong Kong
Lin Lin– Hong Kong, Hong Kong
Xiao Yang– Hong Kong, Hong Kong
Paul Kay Sheung Chan– Hong Kong, Hong Kong
Alice Pik Shan Kong– Division of Endocrinology, Department of Medicine and Therapeutics, The Chinese University of Hong Kong, Hong Kong, Hong Kong
Zhong Zuo– Director and Professor, School of Pharmacy, Faculty of Medicine, The Chinese University of Hong Kong, Hong Kong, Hong Kong