P-gp-mediated efflux

W., Hilgendorf, C., Spahn-Langguth, H., Wunderli-Allenspach, H., Merkle, H. P., Langguth, P., P-glycoprotein (P-gp) mediated efflux in Caco-2 cell monolayers the influence of culturing conditions and drug exposure on P-gp expression levels, J. Pharm. Sci. 1998, 87, 757-762. 

A more recent example of this technique has been the study on human absorption characteristics of fexofenadine [109], Fexofenadine has been shown to be a substrate for P-gp in the in vitro cell lines its disposition is altered in knockout mice lacking the gene for MDRla, and co-administration of P-gp inhibitors (e.g. ketoconazole and verapamil) was shown to increase the oral bioavailability of fexofenadine [110-113], Hence, it is suggested that the pharmacokinetics of fexofenadine appears to be determined by P-gp activity. In the human model, the intestinal permeability estimated on the basis of disappearance kinetics from the jejunal segment is low, and the fraction absorbed is estimated to be 2% [114], Co-administration of verapamil/ketoconazole did not affect the intestinal permeability estimates however, an increased extent of absorption (determined by de-convolution) was demonstrated. The increased absorption of fexofenadine was not directly related to inhibition of P-gp-mediated efflux at the apical membrane of intestinal cells as intestinal Peff was unchanged. Furthermore, the effect cannot be explained by inhibition of intestinal based metabolism, as fexofenadine is not metabolised to any major extent. It was suggested that this may reflect modulation of efflux transporters in hepatocyte cells, thereby reducing hepatobiliary extraction of fexofenadine. 

Deferme S, Mols R, Van Driessche W, Augustijns P (2002b) Apricot extract inhibits the P-gp mediated efflux of talinolol. J Pharm Sci 91 2539-2548. 

Combination of several descriptors believed to be important for oral absorption have been used in various multivariate analysis studies [26]. The general trend is that a combination of size/shape and a hydrogen bond descriptor, sometimes in combination with log D, has good predictive value. At present such models do not account for the biological function of the membrane, such as P-gp-mediated efflux. 

Other mechanisms of interaction have also been reported, such as altered activity of other enzymes within the CYP450 family (14—17). Moreover, GFJ may also inhibit the intestinal P-glycoprotein (P-gp)-mediated efflux transport of drugs such as cyclosporine to increase its oral bioavailability (18-21). GFJ and other fruit juices have recently been shown to be potent in vitro inhibitors of a number of organic anion-transporting polypeptides (OATPs) (22,23). 

Quercetin has been found to inhibit P-gp-mediated efflux of ritonavir in Caco-2 cells (47), to reduce the oxidation of acetaminophen in rat liver microsomes and HepG2 cells (48), and to inhibit the metabolism of midazolam and quinidine in human liver microsomes (49). It did not have an effect on CYP3A4-mediated metabolism and P-gp-mediated transport of saquinavir (41). Rutin was demonstrated to moderately increase the uptake of idar-ubicin in an isolated perfused rat lung model, and also the outflow recovery of the major metabolite idarubicinol, possibly by affecting P-gp (45). Nobe-litin and tangeretin were shown to inhibit OATP-B-mediated uptake of estrone-3-sulfate into human embryonic kidney cells (23). 

The permeability of two peptides, P-gp substrate and non P-gp substrate, across caco-2 cells in the presence or absence of polysorbate 80 and cremophor EL, commonly used surfactants in pharmaceutical formulations, was investigated. The permeability of the P-gp substrate peptide across caco-2 cells was enhanced in the presence of polysorbate 80 and cremophor EL, whereas the non-P-gp substrate peptide was not affected by these surfactants [94]. Another commonly used lipidic excipient that has been shown to inhibit P-gp mediated efflux is D-a-tocopheryl polyethylene glycol 1000 succinate (TPGS) [95]. The insertion of a known CYP3A4 and P-gp inhibitor to the formulation is another approach to elevate bioavailability. 

Substrates bind to P-gp while they are associated with the plasma membrane this process is possibly the most important aspect of P-gp-mediated efflux activity to appreciate. By using fluorescent dye esters, it was shown that P-gp interacts with its substrates within the plasma membrane. As these dye esters cross the membranes, esterases quickly hydrolyze the esters to their free acid form in the cytoplasm. Cells expressing P-gp showed no accumulation of the free acid dye in the cytoplasm clearly illustrating that P-gp can efflux substrates directly from the membrane (129). Additionally, P-gp can bind to substrates at the inner leaflet—cytosolic interface as demonstrated in studies with the P-gp substrate rhodamine 123 (133). It was shown that P-gp does not influence drug concentration in the exofacial leaflet (134), thus implying that P-gp only binds compounds from either within the inner leaflet or at the inner leaflet—cytosolic interface. These findings clearly show that the behavior of the substrate/inhibitor within the lipid barrier is likely to be a primary determinant of P-gp-mediated efflux activity. This separates P-gp from traditional transporters in which binding of the substrate to the active site in an enzyme-like fashion is the primary determinant of transport activity. 

The Role of the Plasma Membrane in P-gp-Mediated Efflux Activity... 

Unlike most transporters, the composition and physical state of the plasma membrane and the interaction of the substrate with the plasma membrane are important determinants of P-gp-mediated efflux activity. A discussion of these phenomena is helpful to aid in further understanding of the nature of P-gp efflux activity. 

Figure 2 Apically directed P-gp-mediated efflux of drugs across intestinal epithelium and synergistic interactions of P-gp with CYP3A4 in attenuating the absorptive transport. Heavy arrows versus light arrows indicate relative magnitudes of the flux. This exemplifies an elimination mechanism that a dual substrate of P-gp and CYP3A4 may encounter in the enterocyte. Conceivably, the metabolite may or may not be a substrate for P-gp (as drawn, it is a substrate). Abbreviation P-gp, P-glycoprotein.

The effect of P-gp-mediated efflux activity on excretion has been clearly shown through experiments with vinblastine and paclitaxel in mdrla(—/—) mice. The results of these experiments have shown how P-gp-mediated efflux activity accelerates tissue clearances and also systemic clearances of its substrates. Additionally, these studies have highlighted the role of the intestine in elimination. While the role of intestinally expressed P-gp in limiting absorption is recognized, these experiments have helped elucidate its role in making the intestine a significant route of elimination. 

CGP41251 is the A-benzyl derivative of staurosporine and appears to have some affinity for protein kinase C along with an ability to inhibit P-gp-mediated efflux (299). There have been few clinical studies performed with this agent to date. 

---