| The oral administrated drugs have ubiquitous gastrointestinal first pass effect, and intestinal metabolism in the gastrointestinal tract occurred before it is absorbed into the blood circulation, which then results in the decreased bioavailability. For certain drugs, the first pass effects in the gastrointestinal tract exceeded in the liver. P-glycoprotein transporter protein (P-gp), phase I metabolic enzyme cytochrome P450 enzymes (CYP450) and phase Ⅱ metabolic enzymes in urine glycoside phosphotransferase glucose (UGT) are the most important proteins related to gastrointestinal first pass effect. In this study, P-gp, CYP450 and UGT were regulated by herbal extracts or pharmaceutical excipients to inhibit the intestinal metabolism and enhance the oral bioavailability of drug which was the substrate of these proteins. We aimed to modulate the gastrointestinal first pass effect of orally administrated drugs so as to avoid the fast metabolism by intestinal enzymes and investigate the inhibitory mechanism to related metabolic enzymes by herbal extracts or pharmaceutical excipients.Firstly, inhibitory effects of pharmaceutical excipients on P-gp, CYP450 and UGT were screened in vitro. The results showed that most of the non-ionic surfactants and polymer-based excipients exhibited greatest inhibitory activity for the three types of proteins. Meanwhile, some pharmaceutical excipients without inhibitory activity were also screened in order to develop a negative control formulation.We chose the Semen Ziziphi Spinosae with the major pharmacologically active ingredients of flavonoid to modulate the P-gp activity because many pharmaceutical excipients had been reported to affect the activity of P-gp. Our results showed that extracts of Semen Ziziphi Spinosae (STS) extacted by n-butanol had inhibitory activity against P-gp compared with the positive control of verapamil in vitro. The STS (50μg/mL) can significantly decrease the efflux rate (ER) of rhodamine 123 from 4.99 ± 0.53 to 2.22 ± 0.23, which was comparable with verapamil (1.34 ±0.15) in Caco-2 cell monolayers transport model. The pharmacokinetic results of rhodamine 123 in vivo showed that the combination of STS can significantly increase the maximum concentration (Cmax) and the area under the curve of concentration (AUC0-t) by 177% and 223%, respectively. Further study showed that it was the total flavonoids but not the total saponins contained in the STS extracts which played the role of inhibition activity for the P-gp and the possible mechanism was by inhibiting the activity of P-gp ATPase. Our results indicated that the Semen Ziziphi Spinosae could modulate the P-gp activity in gastrointestinal tract, which suggested that potential risks might exist when combination of Semen Ziziphi Spinosae and the P-gp substrate drugs.For CYP450 metabolic modulation, pharmaceutical excipients with or without inhibitory activity against CYP450 were used to prepare two kinds of self-microemulsion drug delivery system (SMEDDS) with purified active fraction of Ginseng, DOS-1227, (with the main components of PPT and PPD, which can be metabolized by CYP450 in the gastrointestinal tract). There were no significant differences between the two SMEDDS formulations in terms of physicochemical properties, release properties in vitro, cellular uptake and intestinal absorption of the lymphatic system, but the pharmacokinetics results showed that the SMEDDS contained inhibitory excipients can increased the AUC0-∞ of PPT and PPD by 8.85-fold and 4.22-fold compared to free drug, and 3.95-fold and 2.21-fold compared to the formulation without inhibitory excipients. Our results showed that the inhibitory pharmaceutical excipients containing SMEDDS could improve the bioavailability of PPT and PPD by in inhibiting the CYP450 mediated metabolism in the intestine.For the UGT regulation, as the quality of submicroemulsion was easily controlled and can be simply prepared, so we choose resveratrol as a model drug to prepared submicroemulsion of F68-N (F68 used as emulsifier which have no inhibitory activity) and Lab-N (labrasol used as emulsifier which have inhibitory activity). These two kinds of resveratrol-loaded submicroemulsion had similar physicochemical properties, cell cytotoxicity, cellular uptake, phagocytic pathways, lymphatic system and the intestinal absorption, but the Lab-N could significantly inhibit the metabolism of resveratrol glucuronide compared with the F68-N in everted sacs of rat gut study. Pharmacokinetics results showed that Lab-N could improve the bioavailability of resveratrol by 6.12-fold compared to free drug, 5.60-fold compared to F68-N, and it could inhibit the resveratrol metabolites by 46.5%. The blank Lab-N physically mixed with resveratrol can not inhibit the intestinal glucuronidation of resveratrol, indicating that resveratrol if not be encapsulated into submicroemulsion could not regulate the activity of UGT, and the reason may be that pharmaceutical excipients and resveratrol were not absorbed synchronously. Our research showed that it is feasible to modulate the UGT activity by UGT inhibitory excipients containing formulations and the oral bioavailability of resveratrol could be been improved by UGT metabolism inhibition.There was only a single pharmaceutical excipient having UGT inhibiting activity in Lab-N, so we evaluated whether there were the synergic effects when various pharmaceutical excipients were applied together in vitro. Our results showed that three excipients (labrasol, labrafil and RH40) had greater inhibitory effects than used alone (P<0.05). And the effects of pharmaceutical excipients on UGT enzyme kinetics metabolism displayed that labrasol, labrafil and RH40 appeared to be competitive inhibitors for UGT1A1 and mixed-competitive inhibitors for UGT1A9.Finally, in order to verify the synergic effects among pharmaceutical excipients in vivo and to prepare a more suitable formulation for oral administration with a stronger inhibitory activity to UGT, we prepared two kinds of resveratrol-loaded SMEDDS which contained all of the pharmaceutical excipients with or without UGT inhibitory activity. And then we investigated the metabolic properties of these two kinds SMEDDS in vitro and in vivo. Results showed that there were no significant difference physicochemical properties, cell cytotoxicity, cellular uptake, phagocytic pathways, lymphatic system and the intestinal absorption between these two kinds of RES loaded SMEDDS. However, the results of everted sacs rat gut showed that SMEDDS containing inhibitory excipients could significantly inhibit glucuronidation resveratrol metabolism, and pharmacokinetics studies in vivo showed that SMEDDS with inhibitory excipients could improve the bioavailability of resveratrol by 12.72-fold compared to free drug, and by 6.51-fold compared to SMEDDS without inhibitory excipients. Our study showed that pharmaceutical excipients contained in SMEDDS are capable of increasing the oral bioavailability of resveratrol by modulating the activity of UGT, and the combination of different inhibitory excipients may exert synergistic effects in vivo.In summary, in this paper Semen Ziziphi Spinosae was applied to modulate the P-gp activity, increasing the oral bioavailability of its substrates rhodamine 123. At the same time, the inhibitory pharmaceutical excipients against CYP450 and UGT were used to prepare with submicroemulsion and/or SMEDDS. In vivo studies showed that submicroemulsion and SMEDDS containing inhibitory excipients could improve oral bioavailability and inhibit the intestinal metabolism. These results provided a new theoretical basis and a technical reference in the development field of oral administrated drugs.
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