The Effects Of Efflux Transporters And Metabolic Enzymes On Bioavailability Barrier Of Diterpene

Background and Objection:During the disposal process of drug absorption and metabolism, there is a bioavailability barrier composed of efflux transporters and metabolic enzymes. Physiologically, this barrier can protect the body from limiting the invasion of xenobiotics, especially toxicants.Diterpene compounds, such as toxic Aconitum alkaloids and effective andrographolide, have been used widely in clinic.The Aconitum species are widely used because of their excellent effects against rheumatism, rheumatoid arthritis, and other forms of inflammation Aconitum alkaloids are also highly toxic, with a relatively narrow margin of safety; there have been frequent poisonings. This herb must be properly processed to decrease its toxicity by hydrolyzing AC, MA, HA, respectively to benzyolaconine (BAC), aconine, benzoylmesaconine (BMA), mesaconine, benzoylhypaconine (BHA).Andrographolide has a wide range of pharmacological effects, such as lowering blood pressure, inhibiting platelet aggregation, antithrobotic, anti-ischemic cell and anti cancer.Therefore, this study investigates the bioavailability barrier characteristics and their molecular mechanism of Aconitum alkaloids and andrographolide based on the efflux transporters and metabolic enzymes, thereby to understand their relationship scientifically, to minimize drug toxicity and increase the efficacy of drugs.Methods and materials:Pharmacokinetics was evaluated in rats. Intestinal disposition was determined using a single-pass rat intestinal perfusion model and the cultured Caco-2cells and MDR1-MDCKII cells.Results:1. Pharmacokinetics study of Aconitum alkaloidsWe found that after intravenous administration of a mixture of AC, MA and HA, all eight alkaloids were found in rat blood. The results indicated that AC, MA and HA were metabolized (mostly hydrolyzed). For AC, MA and HA, the times for concentration to diminish by one-half (T1/2β) were90.77±22.71,155.25±66.43,185.29±46.2min, respectively; For their metabolites (BAC, BMA, BHA, aconine and mesaconine), the T1/2β were525.71±323.76,407.02±179.91,552.43±141.88,685.48±494.28, and399.11±150.84min, respectively; These results suggested that the parent compound (which is more toxic than its corresponding secondary metabolite) would be eliminated more rapidly.2. Transport of AC, MA, HA, BAC, BMA, BHA, aconine and mesaconine in cultured Caco-2cells and MDR1-MDCKII cellsThe transport of1μM Aconitum alkaloids across a Caco-2cell monolayer from A to B or from B to A side was investigated. The Papp values of AC, MA, and HA from A to B were (7.63±1.16×10-7) cm/s,(8.24±0.45×10-7) cm/s, and (21.5±0.72×10-/) cm/s, respectively. The Papp values of AC, MA, and HA from B to A were significantly (P<0.05) higher than those from A to B; the Efflux ratio values of AC, MA, and HA were34.6±4.2,29.7±2.1and15.6±2.1, respectively. The Papp values of BAC, BMA, and BHA from B to A were approximately4fold higher than those from A to B [(5.02±.28×10-7) cm/s,(5.84±2.37×10-7) cm/s, and (9.17±1.49×10-/) cm/s, respectively]. Their efflux ratio values were5.2±1.4,4.5±2.4and4.4±0.9respectivelyTheir The Papp values of aconine and mesaconine from B to A were not significantly different from those from A to B [(12.1±3.01×10-7) cm/s and (10.4±2.27×10-/) cm/s, respectively] and the Er values were1.1±0.4and1.1±0.5.Transport studies were performed in the presence of P-gp inhibitors (cyclosporine A or verapamil) to determine the effect of P-gp on the transport of Aconitum alkaloids. Under10μM cyclosporine A or50μM verapamil, efflux phenomena were (P<0.05) almost completely inhibited and Er decreased to1. The Er values of BAC, BMA, and BHA decreased more in the presence of cyclosporine A than in verapamil. No differences were observed between aconine and mesaconine. Consistent with the permeability results, intracellular accumulations of AC, MA, and HA also significantly (P<0.05) increased in two directions in the presence of P-gp inhibitors. Under cyclosporine A or verapamil alone, intracellular amounts of AC transported from A to B increased from2pmol to58pmol or60pmol, those of MA increased from1pmol to44pmol or45pmol, and those of HA increased from3pmol to90pmol or44pmol. Intracellular amounts of BAC, BMA, and BHA also increased after treatment with P-gp inhibitors. In contrast, no changes in the intracellular amounts of aconine and mesaconine were observed.Consistent with the results of the Caco-2monolayer studies,1μM AC, MA, or HA in MDRl-MDCKII cells showed significantly (P<0.05) greater permeabilities in the B to A direction compared with those in the A to B direction. The Er values of AC, MA, and HA in MDRl-MDCKII cells were much higher than those in MDCKII cells (22.2±2.6versus1.8±0.5,27.9±5.3versus1.5±0.2, and10±1.3versus1.4±0.3, respectively). The Er values of BAC, BMA, BHA, aconine and mesaconine in MDRl-MDCKII cells were equal to those MDCKII cell lines. Intracellular accumulations of the Aconitum alkaloids in the MDR1-MDCKII and MDCKII cell lines were also measured. Intracellular amounts of AC, MA, and HA in the MDR1-MDCKII line were significantly (P<0.05) less in both directions compared with those in MDCKII cells. No significant differences were observed in the intracellular amounts of BAC, BMA, BHA, aconine, and mesaconine in the two cell lines. Taken together, P-gp was demonstrated to mediate the transport of AC, MA and HA.Transport studies were carried out in the presence of a Bcrp inhibitor (Ko143) to determine the role of Bcrp in the transport of Aconifum alkaloids. Under5μM Ko143, the Er values of AC, MA, HA and BAC greatly decreased to close to2(P<0.05). Under Ko143, intracellular amounts of AC transported from A to B increased from2pmol to71pmol, those of MA increased from1pmol to47pmol, and those of HA increased from3pmol to112pmol. Taken together. Bcrp may mediate the transport of AC, MA and HA.Transport studies were performed in the presence of an Mrp2inhibitor (MK571) to determine the effect of Mrp2on the transport of Aconitum alkaloids. Under20μM MK571, the Er values of AC, MA, HA, BAC, BMA, and BHA greatly decreased (P<0.05); In the presence of MK571, intracellular accumulations of AC, MA, and HA from A to B increased from2pmol to81pmol, from1pmol to48pmol, and from3pmol to101pmol. respectively. Intracellular amounts of BAC, BMA, and BHA also significantly increased from A to B and from B to A. No significant increase was detected in aconine and mesaconine. Taken together, Mrp2may mediate the transport of AC, MA, HA, BAC, BMA and BHA.3. The CYP metabolism of Aconitum alkaloidsMA could be transformed into at least nine metabolites in human liver mi crosomes. The metabolic pathways were demethylation, dehydrogenation, hydro xylation, and demethylation-dehydrogenation. Results showed that the inhibitor of CYP3A had a strong inhibitory effect; the inhibitors of CYP2C8,2C9,2C19, and2D6had modest inhibitory effects; whereas inhibitors of CYP1A2a nd2E1had no obvious inhibitory effects on MA metabolism. Recombinant hu man cytochrome P450isoforms CYP3A4and3A5contributed greatly to the f ormation of MA metabolites, and CYP2C8,2C9, and2D6played a minor rol e in the formation of MA metabolites.HA was transformed into11metabolites in vitro in human liver microso mes. The main metabolic pathways of HA were demethylation, demethylation-d ehydrogenation, hydroxylation, and didemethylation. The primary contributing C YP isoforms were CYP3A4and3A5. CYP2C19,2D6and2E1are secondary contributors, while CYP1A2and CYP2C8contributions were rarely observed.4. The absorption and metabolism of andrographolide (AP)After the oral administration of AP (120mg/kg), the Cmax was0.23±0.05μg/ml, which was observed at29.75±0.5min; thereafter, it declined with a T1/2β of142.30±34.48min, and the AUC0-∞were29.45±3.73μg·min/ml. After i.v. injection of AP at24mg/kg, the T1/2β, clearance, and AUC0-∞were157.60±74.09min,109.24±8.99ml/min/kg, and220.83±18.26μg·min/ml, respectively. Thus, the absolute Foral of AP was2.67%.To determine the reasons for AP’s poor oral bioavailability, vectorial transport experiments were used to estimate the absorption characteristics of AP. Transport of AP at10μM from apical side (A) to basolateral side (B) or from basolateral side (B) to apical side (A) were investigated in Caco-2cell model at pH7.4. The transport across Caco-2monolayers from B to A was significantly higher than that from A to B (P<0.05). The Papp value of AP from B to A (4.94×10-5cm/s) was approximately4-fold higher than that from A to B (1.14×10-5cm/s). However, absorptive permeability of1.14×10-5cm/s suggested that this compound has high permeability and that poor absorption should not be the main reason for its poor bioavailability.To further investigate the transport mechanism of AP, permeability studies were carried out in MDR1-MDCKII cells, which stably and functionally overexpress human MDR1(PgP), and their wild-type parental cell line MDCKII, which expresses constitutive canine PgP, but at a level much lower than that in the recombinant MDR1-MDCKII cells. The transport data across these two MDCKII cell lines are shown in Fig.4. As expected, the efflux ratio of AP was significantly higher in MDR1-MDCKII cells as compared to parental MDCKII cells at10μM (4vs1).AP was very rapidly degraded in the blank duodenum (about70%in an hour) and jejunum blank perfusates, and was moderately degraded (10%in an hour) in the blank ileum and colon perfusates. Significant differences (P<0.05) were seen in the amounts of AP absorbed from different regions of the intestine when40μM AP was perfused. The apparent dimensionless effective permeability (P*eff) of AP was higher in the upper small intestine (i.e., duodenum and jejunum) than in the terminal ileum and colon. Owing to the presence of significant metabolism, permeability decreased somewhat when amounts metabolized were corrected for in the calculation of permeability. However, the previously observed trend in region-dependent permeability did not change. To understand the reasons for the observed regional difference in permeability, absorption of AP was determined in the presence of the P-gp efflux transporter inhibitor verapamil (100μM) or/and breast cancer resistance protein (Bcrp) inhibitor dipyridamole (50μM). The P*ef value of AP was significantly increased (P<0.05) in the ileum and colon, but not in duodenum and jejunum by verapamil. Dipyridamole did not significant affect the P*eff value of AP in any of the four regions of the intestine. However, verapamil together with dipyridamole significantly increased the P*eff value and amount absorbed of AP in the ileum and colon (P<0.01), but again not in the duodenum and jejunum. In the presence of these two inhibitors, the regional dependent absorption of AP disappeared.The amounts of AP measured in bile samples were affected by inhibitors. Biliary excretion of AP in the presence of these inhibitors was significantly higher than those when AP was perfused alone after60min.One major metabolite of AP (M) was found in duodenum and jejunum perfusates, with a retention time of1.191min. The perfusate was loaded onto an ODS column (2×10cm,10um) and washed using a solvent of HaO/MeOH. The M (about2mg) was eluted using a solvent of HbO/MeOH (9:1). The high resolution mass spectrum showed the molecular weight of M was413.1657and its molecular formula was calculated as C20H29O7S. It displayed very similar signals with14-Deoxy-12-sulfo-andrographolide. The amount of M formed in the duodenum was two times higher than in jejunum (P<0.05).Conclusions:1.Metabolic enzymes (such as CYP) and efflux transporters ((P-gp, Bcrp, Mrp2) constituted the bioavailability barrier of Aconitum alkaloids, and Protection of this barrier can prevent the occurrence of toxicity.2.P-gp and SULT enzyme constituted the bioavailability barrier of AP, and suppression the barrier effect, can improve its bioavailability.