Rhodamine transport

Both functional assays [vincristine transport (381) and rhodamine 123 transport (383)] and biochemical assays involving immunohistochemical analysis (381,384) have confirmed the expression of P-gp in the luminal membrane of BMECs cultured on polycarbonate membranes. Additionally, immunohistochemical methods showed the expression of P-gp in BMEC to be constant and at a high level in five- to seven-day-old old primary cultures (384). Like many other barrier-forming cells, BMECs appear to express other efflux proteins, for example, RT-PCR and immunoblot analysis have shown the presence of MRP1 in rat BMECs (385,386). Functional evidence has also been presented to confirm the expression of MRP1 in BMECs (387). 

Although the competition of two substrates for the same P-gp normally results in an inhibitory effect on the P-gp-mediated transport of the substrates, stimulation of P-gp-mediated efflux transport has been reported in some cases. The P-gp-mediated doxombicin efflux out of multidrug-resistant HCT-15 colon cells was significantly increased by some flavonoids (13). Similarly, rhodamine 123 and Hoechst 33342 stimulated the rate of P-gp-mediated transport of each other in P-gp-enriched plasma membrane vesicles isolated from Chinese hamster ovary CHRB30 cells (14). Interestingly, Hoechst 33342 transport was increased by daunorubicin and doxombicin, while rhodamine 123 transport was inhibited by daunombicin and doxombicin (14). These results strongly suggest that molecular mechanisms of P-gp interaction are quite complex and cannot be predicted readily. 

Ernst, R., Kueppers, P., Klein, C.M., Schwarzmueller, T., Kuchler, K., and Schmitt, L. (2008) A mutation of the El-loop selectively affects rhodamine transport by the yeast multidrug ABC transporter PdrS. Proceedings ofthe National Academy of Sciences ofthe United States of America, 105, 5069-5074. 

Guminski Y, Grousseaud M, Cugnasse S et al (2009) Synthesis of conjugated spermine derivatives with 7-nitrobenzoxadiazole (NBD), rhodamine and bodipy as new fluorescent probes for the polyamine transport system. Bioorg Med Chem Lett 19 2474—2477... 

Rege and colleagues have studied the effect of nonionic surfactants (e.g., Cremophor EL, Tween 80, and TPGS) on membrane transporters [116], Similar results for vitamin E TPGS using talinolol and rhodamine 123 as P-gp substrates have been reported by Bogman et al. ([32] and Collnot and coworkers [40], respectively. 

We then used this Caco-2 cell assay to categorize representative fluoroquinolone drug substance permeability [50], The drugs demonstrated some concentration-dependent permeability indicative of active drug transport. Based upon comparison to labetalol, ciprofloxacin was classified as a LP drug, whereas levofloxacin, lomefloxacin, and ofloxacin were classified as HP drugs, which matched their human in vivo bioavailabilities. All four fluoroquinolone drugs were subject to efflux transport (ciprofloxacin > lomefloxacin > rhodamine 123 > levofloxacin > ofloxacin). 

Figure 18-15 Tracks of two molecules of 20 pM rhodamine 6G in silica gel observed by fluorescence integrated over 0.20-s periods at 0.78-s intervals. Some points are not connected, because the molecule disappeared above or below the focal plane in the 0.45-ixnrvthick film and was not observed in a particular observation interval. In the nine periods when molecule A was in one location, it might have been adsorbed to a particle of silica. An individual molecule emits thousands of photons in 0.2 s as the molecule cycles between ground and excited states. Only a fraction of these photons reaches the detector, which generates a burst of —10-50 electrons. [From K. s. McCain, D. C. Hanley, andJ. M. Harris. "Single-Molecule Fluorescence Trajectories tor Investigating Molecular Transport in Thin Silica Sol-Gel Films," Anal.

P 67] Simulations were made following experiments made previously [156], Therein 0.11 mM Rhodamine B solutions in 20 mM carbonate buffer were mixed with the same carbonate buffer. For the buffer solution, the physical properties of water were approximated. For Rhodamine B, a diffusion coefficient of 2.8 10-6 cm2 s-1 was taken. Electroosmotic flow was applied for liquid transport. For all of the walls in the domain the electroosmotic (EO) mobility was set to 3.4 10-4 cm2 V-1 s 1, which corresponds to a zeta potential (Q of-44.1 mV. The electric field in the outlet channel was 1160 V cm-1. The Reynolds number was 0.22. The electric field strength was set low in order to decrease diffusive (pre-)mixing prior to the groove structure. 

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. 

BMECs have been used to study various aspects of the P-gp-mediated efflux of compounds from the endothelial cells that comprise the BBB. Several examples have demonstrated the usefulness of this system to study polarized efflux via P-gp. For example, the influence of P-gp expressed in brain capillary endothelial cells on the transport of cyclosporin A (388,389), vincristine (381), protease inhibitors (amprenavir, saquinavir, and indinavir) (245,390), rhodamine 123 (211,383), opioid peptides (211,391,392), and the (1-blocking agent bunitrolol (393) have all been determined using this system. 

Yumoto R, Murakami T, Nakamoto Y, et al. Transport of rhodamine 123, a P-glycoprotein substrate, across rat intestine and Caco-2 cell monolayers in the presence of cytochrome P-450 3A-related compounds. J Pharmacol Exp Ther 1999 289(1) 149-155. 

The polysorbates used most regarding efflux pump inhibition are polyoxyethylene sorbitan monolaurates (Tween 20), polyoxyethylene sorbitan monopalmi-tates (Tween 40) and polyoxyethylene sorbitan monooleates (Tween 80). Various studies demonstrate the ability of polysorbates to inhibit efflux pumps. In transport experiments across intestinal mucosa, the efflux ratio (basolateral to apical drug transport/apical to basolateral drug transport) of rhodamine 123 was reduced in the presence of Tween 80 (Shono et al. 2004). In another study, Zhang et al. demonstrated enhanced absorption of the P-glycoprotein substrate digoxin in rats in the presence of Tween 80 (Zhang et al. 2003). 

Recently an effect of PEG glyceryl fatty acid esters such as PEG-8 glyceryl caprylate/caprate (commercially known as Labrasol) on the transport of a P-glycoprotein substrate has been reported. In in situ absorption studies, Labrasol (0.1 % (v/v)) significantly enhanced the intestinal absorption of rhodamine... 

Some studies reported that polyoxyethylene (POE) stearates (under the brand name Myrj) and alkyl-polyethyleneoxide (PEO) surfactants (under the brand name Brij) can inhibit efflux pumps. The oral bioavailability of the P-gp substrate cyclosporine A administered in a solid dispersion of polyoxyethylene 40 stearate (Myrj 52) was in the same range as the oral bioavailability of the commercial product Sandimmune Neoral (Liu et al. 2006). In a study by Lo, it has been shown that apical to basolateral epirubicin transport across Caco-2 cells was enhanced in the presence of polyoxyethylene 40 stearate and the basolateral to apical transport was decreased. These results indicate that polyoxyethylene stearates effect efflux pumps (Lo 2003). Similar results were gained when using polyoxyethylene laurylether (Brij 30). In another study, tablets based on polyoxyethylene 40 stearate containing the P-gp substrate rhodamine 123 increased the oral bioavailability in rats by about 2.4-fold (Foger et al. 2006a). 

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