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the drug to be transported across the RPE, and therefore knowledge of the barriers to drug delivery is important.

The question of what physiological role this transporter plays when not engaged in the transport of xenobiotics has led to some proposed further actions of P-gp that may or not be true of P-gp in the RPE. For reviews see Johnstone et al. (2000) (78,79). These roles include the transport of phospholipids (80,81) and inhibiting apoptosis (82,83). P-gp has also been associated with modulation of Cl- channel activity and cell volume (84,85). Furthermore, P-gp has been implicated in cholesterol esterification at the plasma membrane (86). P-gp transcription is also up-regulated by cytokines in response to inflammation (87). There is some evidence that statins may be substrates of P-gp or interfere with its activity (88). In vitro studies suggest that simvastatin, atorvastin, and pravastatin inhibit P-gp activity, but pravastatin was ineffective (89).

Inhibition of P-glycoprotein

One method for increasing the effectiveness of chemotherapeutic drugs is to co-administer drugs that inhibit the activity of P-gp. The calcium channel blockers, ver-apamil, and diltiazem inhibited the activity of P-gp in cultures of tumor cells (90). Verapamil acts as a competitive inhibitor for the binding sites of adriamycin, vincris-tine, and colchicine (90). The detection of P-gp in body tissues has exploited the specific transport of certain drugs using fluorescent or radioactively labeled compounds, in conjunction with a specific inhibitor of P-gp. Verapamil is used as a relatively specific inhibitor of P-gp activity and has been used as an effective inhibitor of P-gp in vivo (91) and in vitro in a number of systems (8,92-94). Verapamil acts as a competitive inhibitor of P-gp and is used clinically to reverse the multidrug-resistance profile of P-gp by competing with chemotherapeutic drugs such as vinblastine (90,95-97). Cyclosporine A may also be used to reduce drug resistance associated with P-gp in patients undergoing chemotherapy (98).

The fluorescent probes, such as rhodamine-123 or calcein, are commonly used to determine functional P-gp activity. Rhodamine-123 enters cells passively, but can be extruded from the cytosol by P-gp. By adding a competitive inhibitor with rhodamine-123, it is possible to determine whether P-gp is functional or not. When functional P-gp is present, the cytosolic accumulation of rhodamine-123 is greater on addition of a P-gp inhibitor than in the absence of the inhibitor. Radiolabeled substrates of P-gp can also be used, such as colchicine.

Inhibitors of P-gp are generally classified as belonging to the first-, second-, third-or the current fourth-generation drugs. First-generation drugs, such as verapamil and cyclosporine A, and the second generation analogues of the first generation drugs, such as R-verapamil and cinchonine, have not been completely successful in reversing drug resistance. The first-generation drugs also had adverse side-effects and were used owing to their low affinity for P-gp transport. The second generation analogues, although designed to inhibit P-gp and reduce adverse side-effects, were not successful in clinical trials (47,99,100). Third-generation drugs such as biricodar and R101933 that were designed specifically to inhibit P-gp are currently in clinical trials (101,102).

The fourth-generation compounds, comprising monoclonal antibodies as well as high-affinity hydrophobic polypeptides (reversins), have also been used to demonstrate

Table 2 List of first- and second-generation inhibitors of P-glycoprotein (P-gp) activity

Cyclosporine A FK-506 (fujimycin)

Sirolimus (rapamycin) Lonafarnib (SCH66336) Verapamil

Diltiazem Nifedipine

Carvedilol

Erythromycin

Rifampicin Amitriptyline

Immunosuppressants

Calcium-channel inhibitors

ß-adrenergic agonist Antibiotics

Antidepressant

Foxwell et al. (1989) (110) Pourtier-Manzanedo et al. (1991) (111)

Arceci et al. (1992) (112) Wang et al. (2001) (113) Cornwell et al. (1987) (90) Safa et al. (1987) (114) Jonsson et al. (1999) (115) Hofsli and Nissen-Meyer (1989) (116)

Fardel et al. (1995) (117) Varga et al. (1996) (118)

functional P-gp (103,104). Further developments, following a greater understanding of the structure and function of P-gp, have led to the development of novel synthetic drugs targeted at inhibiting P-gp (105). Phenoxazine derivatives show promise in inhibiting P-gp (106), along with modified reversins, and are reported to show greater P-gp inhibition than cyclosporine A without side-effects (107). Down-regulating MDR1 transcription by inhibiting messenger RNA (mRNA) is also promising, although to date only in vitro studies have been performed (108,109). See Table 2 for a list of known inhibitors of P-gp. Strategies designed around inhibiting MDR1 transcription, or the delivery of drugs directly to tumor cells in liposomes, as well as the development of P-gp specific antibodies and antisense oligonucleotides, should provide better therapeutic outcomes in the future. For a review see Nobili et al. (2006) (99).

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