Impact of excipients on the absorption of P-glycoprotein substrates in vitro and in vivo
Introduction
P-glycoprotein (P-gp) is a membrane transporter that actively “pumps” xenobiotics out of cells. It is expressed in the apical membranes of the epithelial cells of the intestine and, due to its action, drugs that have been absorbed by these cells can be pumped back into the lumen of the intestine. Given the very broad specificity of P-gp, then its activity in the intestine may reduce the oral bioavailability of a wide range of drugs. However, there is not agreement on the significance of P-gp in modulating the absorption of orally administered drugs. For example, based largely on human pharmacokinetic data, Chiou and colleagues claim that for a number of common drugs that are known P-gp substrates, the role of P-gp is not important (Chiou et al., 2001). It is clear that there can be drug interactions centered on intestinal P-gp. Thus, in human volunteers, the oral bioavailability of digoxin was significantly increased when talinolol was co-administered orally, but not when it was administered intravenously, suggesting an interaction at the intestinal level (Westphal et al., 2000). Many NCEs are poorly absorbed and are substrates of P-gp, and there is considerable interest in trying to increase the bioavailability of oral drugs that are P-gp substrates particularly anticancer and anti-HIV drugs. The oral administration of competing low molecular weight compounds, such as verapamil or cyclosporine, can enhance the oral bioavailability of P-gp substrate drugs, but such compounds have themselves pharmacological activity. Thus, they may also interact with P-gp everywhere that it is present, as is also the case with the new generation of low molecular weight P-gp inhibitors currently appearing and in development. So the challenge for oral drug administration is to find inhibitors of P-gp that do not have such drawbacks. A number of excipients, which are commonly added to pharmaceutical formulations, may disrupt the function of intestinal P-gp and thus enhance the intestinal permeability of a substrate drug. Vitamin E-TPGS (d-α-tocopheryl polyethylene glycol 1000 succinate) is a good example, as it increases the absorption flux of amprenavir (Yu et al., 1999), and has been characterized as an inhibitor of P-gp-mediated drug transport in the human intestinal Caco-2 cell monolayers and other cell lines (Dintaman and Silverman, 1999, Bogman et al., 2003). It has been shown to enhance the bioavailability of cyclosporine in human volunteers (Chang et al., 1996) and of colchicine in rats when the drugs were administered orally (Bittner et al., 2002). Many of the in vitro studies on the intestinal absorption of P-gp substrates have been carried out using the Caco-2 cell monolayer, but these cells tend to show variations in P-gp expression depending upon the culture conditions (Anderle et al., 1998) and it is suggested that they overexpress P-gp (Collett et al., 1999). In our study, we have used the improved rat everted gut sac for the in vitro studies. This model uses tissue culture medium (TC 199) as the incubating medium, ensuring high viability and maximal functionality of the tissue as previously demonstrated by various criteria (Barthe et al., 1998a). There is a large surface available for absorption, and good mixing of substrates and excipients and the technique has already proved to be a very useful in vitro tool to evaluate the role of P-gp in drug absorption (Barthe et al., 1998b, Carreno-Gomez and Duncan, 2000). Initial studies in our laboratory using this technique have shown that the excipients/surfactants Cremophor EL and Polysorbate 80 (Tween 80) could enhance net digoxin absorption (Cornaire et al., 2000). In the current study, we have greatly extended the range of surfactants/excipients tested as well as using a second P-gp substrate drug (celiprolol) and carrying out studies in vivo in rats. Excipients were selected because they are currently used in pharmaceutical formulations and/or had been demonstrated to modulate P-gp activity in other systems. Some were selected because they were amphiphilic or lipophilic but soluble or dispersible in water. We first used digoxin as a model drug because it has clearly been shown to be a P-gp substrate and there is also a considerable amount of data available on its pharmacokinetics. However, digoxin is metabolized by the enzyme CYP3A from rat liver (Salphati and Benet, 1999), and as this enzyme is present in the intestinal epithelium, there is the possibility of first-pass metabolism during absorption by the GI tract. Hence, we decided to use celiprolol as a second model drug because it is a P-gp substrate that is not metabolized in the rat intestine, and P-gp has been implicated in its absorption in man (Karlsson et al., 1993, Engman et al., 2001). The rationale of this work was to screen the excipients in vitro for absorption enhancement with the model drugs digoxin and celiprolol. The most promising excipients were subsequently taken forward for in vivo studies.
Section snippets
Materials
[]Digoxin (250 μCi/mmol) and Ready Safe™ scintillation fluid were obtained from NEN-Life Sciences (Le Blanc-Mesnil, France) and from Beckman Instruments (Gagny, France), respectively. d-[1-]Mannitol (55 mCi/mmol) was from American Radiolabelled Chemicals (St Louis, MO, USA). The excipients, purchased by the Galenic Research and Preformulation Department of UPSA Laboratories (Rueil-Malmaison, France), were: Cremophor EL, Solutol HS 15 (BASF), Softigen 767, Imwitor 742, Imwitor 370 (Hüls);
Effect of excipients on digoxin transport and LDH release by everted gut sacs
Among the selected excipients, propylene glycol, PEG 400, Plurol oleic, Labrafil M1944CS, Gelucire 44/14, Imwitor 370, Sucroester 7, and Sucroester 15 had no significant effect on digoxin uptake into the sac contents at the three tested concentrations of excipient (data not shown).
On the other hand, as shown in Table 1, Cremophor EL, TPGS, Solutol HS 15, Sucrose monolaurate, Acconon E, Polysorbate 20, Polysorbate 80, Labrasol, and Softigen 767 all showed enhancement of the digoxin uptake into
Discussion
Within recent years a number of studies have suggested that several common pharmaceutical surfactants/excipients can modulate the activity of the efflux transporter P-gp, and possibly other transporters. Thus, the concept of all excipients being “inactive” has been challenged, and the idea that they were largely inert molecules used to control the stability and solubility of drugs has had to be reassessed (Wandel et al., 2003). In trying to evaluate the potential of such excipients to enhance
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