Studies On The Inhibitory Effect Of Pharmaceutical Excipients On P-glycoprotein Drug Pump

P-glycoprotein (P-gp), a 170 kDa ATP dependent membrane transporter, acts as a drug efflux pump. P-gp can exports structurally diverse hydrophobic compounds from serosa to mucosa and thus reduce the absorption and bioavailability of a wide range of drugs which are substrates for this membrane transporter. Inhibiting the drug efflux pump can increase the absorption of some orally drugs and decrease the renal clearance and hepatic-clearance. Many early P-gp modulator agents, such as verapamil and propafenone, often result in unacceptable toxicity that limits their clinical use. Therefore, to develop some P-gp modulators with less toxicity and non-pharmaco-activity is very important and pharmaceutical scientists all over the world have focus on this research field. In this dissertation pharmaceutical excipients were selcted to investigate their inhibitory effect on P-glycoprotein to search for some P-gp modulator agents with non-toxicity and non-pharmaco-activity. In this study celiprolol was used as the P-gp probe drug and Caco-2 monolayer model, everted gut sacs experiment in vitro, intestinal perfusion in situ and intestine absorption model in vivo were employed to study the effect of P-gp efflux pump on transmembrane transport and absorption of celiprolol. Pharmaceutical excipients with strong inhibitory effect on P-gp were sieved by comparing the inhibitory tensity of pharmaceutical adjuvant sorts and concentration.Caco-2 cell monolayer model has gained enormous popularity as a convenient and high-effective in vitro model for absorption mechanism study of oral drugs and high throughput screening method for drug candidates. The integrity of Caco-2 cell monolayer was dectected by testing the TEER value by Millicell-ERS equipment and phenol permeation experiment. Caco-2 cell monolayer model was then established to perform the bi-directional transport experient of celiprolol with verapamil as positive control. The concentration of celiprolol was determined by HPLC and apparent permeability coefficient(Papp) and efflux ratio (ER) value were calculated according to drug transmembrane transport amount. Then the inhibitory effect of pharmaceutical excipients on P-gp was evaluated with the parameters of Papp and ER. The results showed that Papp of celiprolol transport from apical to basolateral was far lower than that of basolateral to apical direction. With P-gp inhibitor verapamil, Papp of celiprolol transport from apical to basolateral increased by 30% and that from basolateral to apical decreased by 50%, which indicates that efflux protein P-gp plays a vital role in the transepithelial transport of celiprolol. Pharmaceutial adjuvants F-68, Cremophor EL, Cremophor RH40, T-80, PEG200, PEG400, PEG4000,β-CD, HP-β-CD, PVP K30, TritonX 100 and S-40 can also enhance Papp of apical to basolateral direction and reduce Papp of basolateral to apical direction. With the concentration of T-20, T-60, PEG1000, PEG2000, PEG6000 and PVP K10 increased, they would exhibit similar effect. But Lecithin almost has no influence on Papp of both direction transport, while SDS showed great cytotoxicity which result in abnormal alteration in this parameter. Furthermore, ER value was analyzed with SPSS software to elucidate the inhibitory effect strength of pharmaceutical adjuvants on P-gp. The result showed that F-68, Cremophor EL, Cremophor RH40, T-20, T-60, T-80, PEG200, PEG400,β-CD, HP-β-CD and S-40 could significantly inhibit excretion cross Caco-2 monolayers and enhance absorption. In conclusion, these pharmaceutical excipients had an ability to inhibit the P-gp efflux pump obviously in Caco-2 cell monolayer model.Everted gut sacs in vitro was used for the study of the transport function of P-gp with probe drug celiprolol. Jejunal segment and colon segment of rat intestine were studied in the absorption model and efflux model in vitro, respectively. With verapamil set as positive control, the transport characteristic of celiprolol under the influence of low, medium and high concentration of pharmaceutical adjuvants F-68, Cremophor EL, Cremophor RH40, T-60, T-80, PEG200, PEG400, PEG1000, HP-β-CD and S-40 was investigated to assess the contribution of P-gp on celiprolol absorptive promotion and efflux suppression. The transport samples from mucosal to serosal side and serosal to mucosal side in the present of various concentrations of pharmacrutical excipients were dectected by HPLC. The results of everted gut sac absorption model processed by SPSS show that F-68, Cremophor RH40, PEG400 and T-80 could significantly promote intestinal absorption while only at the higher concentration Cremophor EL, T-60, PEG200, PEG1000 and S-40 could increase absorptive transport across intestinal mucous membrane. However, there is no significant influence of HP-β-CD on M→S absorption. Furthermore, the results of excretive model in vitro showed that F-68, Cremophor EL, Cremophor RH40, T-60, T-80, PEG200, PEG400 and S-40 could inhibit the efflux of Celiprolol at the low, middle and high concentration while PEG1000 and HP-β-CD inhibit the P-gp efflux pump at much higher concentration.Rat intestinal perfusion model in situ was widespreadly used not only for oral application and absorption of drugs and preparations, but also for transport mechanism across biomembrane. This model was established and performed using celiprolol Krebs solution containing phenol to indicate the water ingredient alteration. To study the effect of 10 pharmaceutical excipients on the efflux function of P-gp the concentration of celiprolol in the effluent solution was detected and parameters absorptive rate constant (ka) and effective permeability coefficients (Peff) were calculated. The results processed by SPSS indicated that F-68, Cremophor EL, Cremophor RH40, T-60, T-80, PEG200, PEG400 and S-40 could increase ka and Peff which showed that these pharmaceutical excipients facilitate absorption process via inhibiting P-gp, but these parameters were not affected by PEG1000 and HP-β-CD.Intestinal absorption experiment in vivo was set up to detect the concentration of celiprolol by collecting blood plasma samples. This in vivo model was used for detecting whether 10 pharmaceutical excipients such as F-68, Cremophor EL and T-80 had the ability of inhibiting P-gp efflux pump at the low, middle, high concentration. The plasma drug concentration-time data was processed by pharmacokinetic software DAS. The inhibitory effect on P-gp in vivo was investigated by the pharmacokinetics parameters of celiprolol after the administration of different doses of pharmaceutical excipients. From the concentration-time curve, it could be obtained that the drug concentration in plasma raised to a distinct extent with coadministration of F-68, Cremophor EL, Cremophor RH40, T-80, PEG200, PEG400 and S-40 while that in plasma maintain at the same level of negative control with coadministration of T-60, PEG1000 and HP-β-CD. From the pharmacokinetics parameters analyzed by DAS, the half life time (t1/2) of celiprolol prolonged and clearance rate (CL) decreased after giving pharmaceutical excipients. Statistical analysis results of the area under the curve (AUC) showed that high concentration group of F-68, Cremophor RH40, PEG200, PEG400 and S-40 and medium, high concentration group of Cremophor EL and T-80 could increase the absorption of P-gp substrate celiprolol. But these three pharmaceutical adjuvants T-60, PEG1000 and HP-β-CD do not affect the pharmacokinetic process in vivo.Based on the screening resuluts of different models, four pharmaceutical excipients F-68, Cremophor EL, T-80 and PEG400 were selected to investigate the influence of intestinal P-gp expression. The relationship between P-gp inhibitor and protein expression in intestine was studied by BCA protein assay kit, SDS-PAGE with gel image analysis technique and western blot analysis. In contrast with control group, experimental groups coadministration with F-68, Cremophor EL, T-80 or PEG400 increased the expression of P-gp dramatically. Thus efflux protein P-gp could be induced by its substrate and inhibitors.To investigate the effect of four pharmaceutic excipients on the absorption of ganciclovir, Caco-2 cell model and intestinal absorption experiment in vivo were exploited to value the extent of absorption promotion. The results in cell model showed that Papp in AP→BL direction increased and that in BL→AP direction decreased, which resulted in that ER reduced significantly. From the statistical analysis results, there exist significant difference between negative control group and verapamil group or medium and high concentration group of pharmaceutical excipients inhibitor F-68, Cremophor EL, T-80 and PEG400 while other groups showed no significant difference. Moreover, intestinal absorption experiment in vivo discovered that only low and medium concentration group of Cremophor EL, low concentration group of T-80 and PEG400 put significant effect on AUC of ganciclovir while other groups showed their enhancement effectiveness with no significant difference.In conclusion, 20 pharmaceutical excipients were choosed to investigate the inhibitory effect on P-gp in Caco-2 model and then 10 adjuvants were selected for animal research. Furthermore, research on intestinal P-gp expression and ganciclovir transport feature were studied with the presence and absence of F-68, Cremophor EL, T-80 and PEG400. The results indicated that several pharmaceutical exicipients such as nonionic surfactant can reverse the efflux function of P-gp and improve the absorption and bioavailability of P-gp substrate celiprolol and ganciclovir, but these inhibitors also enhance the expression level of P-gp. Thus, excipient inhibitors could ameliorate absorprion and pharmacokinetic characters in vivo of some certain drugs via reversing the intestinal efflux function of P-gp not by inhibiting P-gp expression but probably by inhibiting ATP enzyme activity, competitive binding function site and P-gp conformational change.Pharmaceutical excipients were used as a vehicle of water-soluble, liposoluble and even indissolvable drugs with low toxicity and satisfactory biocompatibility. Furthermore, it provides direction and method to decrease efflux of cytotoxic drugs from the cancer cells, promote the effect of clinical therapeutic efficacy, recover the sensitivity of the cancer cells to the cytotoxic drugs, and improve the characteristics of pharmacokinetics of some drugs such as increasing the absorption of some orally drugs, decreasing the renal clearance and hepat-clearance.