Studies On Modulation Of Intestinal Peptide Transporter1Function By Pharmaceutical Excipients

Objectives:Oligopeptide transporter (PEPT1) is a low-affinity H+/peptide transporter that is located in the brush border membrane of the small intestine, driven by the presence of an inward H+gradient and a negative membrane potential. PEPT1transports various natural di/tri-peptides as well as a comprehensive peptide-mimetics. PEPT1is a high capacity, low affinity transporter, which generally lead to low oral bioavailability for most of its substrates. Function modulation of PEPT1with some non-toxicity and non-pharmaco-activity modulator agents is of great interest since the disposition of certain drugs may be potentially altered and such modulators have not been reported yet. From our previous researches, several excipients were found to enhance the absorption of certain PEPT1substrate. Therefore, pharmaceutical excipients were selected to investigate their enhancement effect on the intestinal PEPT1to search for some modulator agents. In this dissertation Glycylsarcosine (Gly-Sar), a typical PEPT1substrate, was used as a probe drug and Caco-2monolayer model, molecular biology techniques, and intestine absorption model in vivo were employed to study the effect of excipients on transmembrane transport and absorption of PEPT1substrates.Methods:Caco-2cell model was adopted to investigate the effects of pharmaceutical excipients on the cell activity with Triton X-100as a positive control. The concentration of excipients used in celluar uptake experiment was determined according to the MTT results. The uptake of Gly-Sar was measured by HPLC-MS/MS. Pharmaceutical excipients with strong enhancement effect on PEPT1were sieved by comparing the uptake ratio of Gly-Sar in Caco-2cell model. To investigate the mechanism of enhanced absorption of Gly-Sar, protein expression, mRNA expression, intracellular pH and membrane fluidity were determined after the administration of active excipients. Flow cytometry was used to investigate the effect of excipients (PEG400, Cremophor EL, Cremophor RH40, Tween20, Tween80) on membrane fluidity and intracellular pH with DPH or BCECF as a probe. To further verify the impact of active pharmaceutical excipients on the absorption of PEPT1substrates, the pharmacokinetic study was performed using fosinopril sodium as a probe. The concentration of fosinopril and its active metabolite fosinoprilat were measured simultaneously by HPLC-MS/MS. After the pharmacokinetic study, the expression of PEPT1in rat intestine was determined.Results:13kinds of excipients (Cremophor EL, Cremophor RH40, PEG400, PEG6000, Eudragit L100, Eudragit L100-55, Eudragit RL PO, HPMC E30, Tween20, Tween80, CMC-Na, CMS-Na and chitosan) can enhance the absorption of Gly-Sar, suggesting that these excipients have a negligible effect on the function of PEPT1. Remaining excipients (Poloxamer188, Poloxamer407, PEG800, PEG1500, PEG4000, PVP K30, Cyclodextrin, Transcutol P and Microcrystalline cellulose) showed no significant effect on the uptake of the probe. The Western blot results showed that the addition of PEG400, CMS-Na, Cremophor EL, Cremophor RH40, Tween20, Tween80, Eudragit L100and Eudragit L100-55increased the protein expression of PEPT1in Caco-2cell. The real-time PCR results showed that the active excipients did not significantly induce or inhibit gene expression of PEPT1. Therefore, the increased protein expression level is presumably due to the translocation of protein from a preformed cytoplasmic pool to the membrane. The intracellular pH was slightly lower than the control group, and the order of intracellular pH was Tween20group<Cremophor EL group<Tween80group <PEG400group≈Cremophor RH40group. Also, the membrane fluidity was all slightly increased by the above excipients. Therefore, the function of PEPT1was probably enhanced by these five excipients due to the increase of protein expression, membrane fluidity and H+gradient. The relative bioavailability of fosinopril with the addition of PEG400, Cremophor RH40, Cremophor EL, Tween20, Tween80was90.98%,725.27%,190.65%,240.00%and156.17%, respectively, while the relative bioavailability of fosinopril was171.25%,207.33%,253.33%,253.76%,200.05%, respectively.For fosinoprilat, the relative bioavailability were increased to more than1.7times by the above excipients. However, after administration of pharmaceutical excipients, protein expression of PEPT1in rat duodenum did not significantly changed, and the abundance of the jejunum and ileum PEPT1protein exhibited some downward expression which indicated that the intestinal absorption of PEPT1substrate was enhanced by the above excipients.Conclusions:In summary, fifteen kinds of excipients were selected for further study on the mechanism of enhancing absorption through PEPT1using Caco-2cell model, and then verified the PEPT1substrate absorption promoting effect of five kinds of activity excipients in vivo. The results showed that PEG400, Cremophor RH40, Cremophor EL, Tween20, Tween80have promoted PEPT1transporter function and further improved oral bioavailability of PEPT1substrates.The impact of pharmaceutical excipients on PEPT1function may be the translocation of PEPT1protein to cell membrane, changing of the hydrogen ion gradient and increasing membrane fluidity. These results indicate that active pharmaceutical excipients could enhance absorption of PEPT1substrates, which provided the theoretical and experimental basis of drug design and interaction between drug and excipients.