Effects Of C-18 Epimers Of Glycyrrhizic Acid And Their Hydrolysis Products On P-glycoprotein

Glycyrrhizic acid (GL) is the main active component of licorice (Radix Glycyrrhiza), one molecule of GL can be transformed to two molecule of glucuronic acid and one molecule of glycyrrhetinic acid (GA) after hydrolyzation. Base on the defferent configuration of C18g-H bond of triterpene saponin mother nucleus, GL has two different epimers:α-GL andβ-GL and they can be hydrolyzed to correspondingα-GA andβ-GA. They were not distinguished at most previous researches. But the difference of C-18 epimers of glycyrrhizic acid has attracted more and more attention after magnesium isoglycyrrhetate was marketed in 2006. The effects of C-18 epimers of glycyrrhizic acid and their hydrolysis products on P-glycoprotein (P-gp) were introduced in this paper.OBJECTIVEStudy the effects of C-18 epimers of glycyrrhizic acid and their hydrolysis products on the function and expression of P-gp and investigate that whether there are stereoselectivity on them. Confirm the experimental result of function and expression by studing the changes of transmembrane transport of P-gp substrates on Caco-2 cell monolayers. Study the effects of glycyrrhizin preparations on the pharmacokinetics of talinolol, initially investigate possible drug interaction between glycyrrhizin preparations and P-gp substrates.METHODS1. Separation and determination of 18a-GL and 18β-GL in glycyrrhizin preparations by RP-HPLCThe chromatographic analysis was carrid out on a Phenyl-Hexyl Column (4.6 mm×250 mm,5μm) with the mobile phase consisting of 10 mmol·L-1 ammonium acetate (pH 7.75)-acetonitrile (83:17 v/v) at a flow-tate of 1.0 mL·min-1. The detection wavelength was 250 nm. 2. Cell culture and establishment of Caco-2 cell monolayer modelCaco-2 cell and ECV304 cell were cultured with MEM (Modified Eagle's Medium) and were applied to study the effects of GL and GA epimers on the function and expression of P-gp. MTT (Methyl thiazolyl tetrazolium) assay was applied to determine the maximum non-cytotoxic dose of each test drugs to ensure the activity of cells during the test and to consult the maximum test concentration of each drugs.3. Rho-123 uptake experiments assay the the function of P-gpECV304 cell was used as negative cell control. Verapamil was used as positive control of the inhibitory effect on the function of P-glycoprotein. Flowcytometry were used to study the effects of C-18 epimers of GL and GA on the uptake of rhodamine-123 which is a substrate of P-gp in Caco-2 cell.4. Real-time PCR assay MDR1 mRNACyclosporin A was used as positive control of up regulation effect on the expression in mRNA level of MDR1 gene. Caco-2 cell without drug dealing was used as negative control. Real-time PCR was used to measure the expression of MDR1 mRNA in Caco-2 cell and to study the effects of C-18 epimers of GL and GA products on the expression of MDR1 mRNA.5. Flow cytometry and Western blot assay P-gp proteinCyclosporin A was used as positive control of up regulation effect on the expression of P-gp protein. Caco-2 cell without drug dealing was used as negative control. Flow cytometry (FCM) and Western blot were applied to study the effects of C-18 epimers of GL and GA on the expression of P-gp protein, respectively.6. P-gp transport experiments on Caco-2 monolayersAppropriate Caco-2 monolayers were established in TranswellTM plates. ELISA Reader was applied to detect the concentration of Rho-123 in transfer fluids. The bi-directional transports of Rho-123 after treated by instantaneous action and incubation for 72 h ofα-GA andβ-GA were studied. HPLC was applied to detect the concentration of talinolol in transfer fluids. The efflux of talinolol in Caco-2 monolayers after treated for 72 h byα-GA andβ-GA was also investigated.7. Effects of glycyrrhizin preparation on the pharmacokinetics of talinololA two-cycle double crossover design was used.14 subjects were randomized into 2 groups. One group administrated the glycyrrhizin preparation for 6 day (3 times one day,3 tablets once) and then administrated talinolol one tablet on the 7th day (Test Grop). Another group administrated the placebo for 6 day (3 times one day,3 tablets once) and then administrated talinolol one tablet on the 7th day (Control Grop). Before the administration of the preparation and at 0.25,0.5,0.75,1,1.5, 2,2.5,3,4,6,8,12,16 and 24 h after the administration of the preparation,5 mL of the venous blood was taken respectively. The blood samples were centrifuged in a centrifugal tube with heparin to separate the plasma, which was stored at-70℃until testing. HPLC was used to measure the concentration of talinolol in human plasma. DAS (Drug and Statistics 2.1.1) and SPSS 13.0 were used in the statistical analysis.RESULTS AND CONCLUSION1. The composition ratios ofα-GL andβ-GL in different glycyrrhizin preparations were different. Quality specifications about the composition ratios of epimers of GL should be established.2. The effects ofα-GL andβ-GL on P-gp show an opposite trend. At middle and high concentrations (10μmol·L-1,60μmol·L-1), a-GL inhibited the function of P-gp and with on dose dependent while P-GL induced the function of P-gp at three test concentrations with no dose dependent too; At middle and high concentrations (10μmol·L-1,60μmol·L-1),α-GL down-regulated the expression of MDR1 mRNA. At high concentrations (60μmol·L-1),β-GL up-regulated the expression of MDR1 mRNA; At high concentrations (60μmol·L-1),β-GL induced the expression of P-gp protein whileα-GL has no effect on the expression of P-gp protein at three test concentrations. The effects ofα-GL andβ-GL on the expression of MDR1 mRNA and CYP3A mRNA showed the same trend. The character that epimers of GL act on CYP3A and P-gp show similar stereoselectivity whether relate to PXR need further study.3. The actions on P-gp betweenα-GA andβ-GA may with different degree. The effects ofα-GA andβ-GA on P-gp were different from their corresponding parent bodies (GL). At middle and high concentrations (1μmol·L-1,10μmol·L-1),α-GA induced the function of P-gp and with dose dependent whileβ-GA also induced the function of P-gp at high concentration (10μmol·L-1); At high concentrations (10μmol·L-1), a-GA up-regulated the expression of MDR1 mRNA while P-GA has no effect on the expression of MDR1 mRNA at three test concentrations;α-GA up-regulated the expression of P-gp protein at high concentrations (10μmol·L-1) whileβ-GA has no effect on the expression of P-gp protein at three test concentrations. The difference between the effects of glycyrrhizin and glycyrrhetinic acid need further study.4. In the transport experiments of P-gp substrates on Caco-2 monolayers, high concentration (10μmol·L-1)α-GA induced the efflux of Rho-123 both in the instantaneous action test and the incubation for 72 h test. High concentration (10μmol·L-1)β-GA induced the efflux of Rho-123 only in the incubation for 72 h test. The transports from AP to BL were not affected by any test drugs. The efflux test of talinolol from BL to AP got the similar results with those of Rho-123. The inducibility ofα-GA was stronger than that ofβ-GA. Glycyrrhizin can up-regulate MDR1 mRNA, induce the excretion of xenobiotics, it may be one of the detoxification mechanism of licorice.5. The pharmacokinetics experiments of talinolol suggested that no adverse reaction was observed and glycyrrhizin did not affect the pharmacokinetics of talinolol on the whole. But maybe the small number of samples or individual difference leaded to the results with no significance. The administration time of glycyrrhizin maybe too short or the dose of glycyrrhizin maybe too small, then P-gp in intestinal tract was not induced. Although talinolol was used as a probe drugs in many researches, the accuracy of the assay need further confirmation. In the light of result of this research,α-GA may be stronger thanβ-GA to induce P-gp. Effects of α-GL preparations on pharmacokinetics of P-gp substrates need further study. Because the clinical application of a-GL preparations are not long enough, when a-GL preparations used in conjunction with other durgs, drug interaction should be observed.According to the results of our research and other references, effects of epimers on P-gp may be different even though only configurations are different between the two compounds. This information deserves attention in drug research and development.