P-gp inhibitors

An additional example of a bioavailability-predicted absorption plot is shown for a series of calcium antagonists (Fig. 19.8). Again there is considerable scatter in the data, and the four compounds - felodipine, nisoldipine, diltiazem, and verapamil -are predicted to be much better absorbed than was actually observed. Some of these compounds are known to undergo rapid first-pass metabolic clearance, and are also P-gp inhibitors or substrates (diltiazem and felodipine are P-gp substrates nicardipine and nitrendipine are P-gp inhibitors [25] verapamil is a P-gp inhibitor), and this might contribute to the scatter obtained in the graph. 

In the transport assays, the permeability of a compound in both absorption and secretion directions is measured using polarized epithelial cells that constitutively express high levels of P-gp (e.g. Caco-2) or have been transfected with the gene for a specific P-gp (e.g. MDR1-transfected MDCK or LLC-PK1 cells). Since P-gp is expressed on the apical membrane, ratios ofbasolateral-to-apical (B —> A) permeability versus apical-to-basolateral (A —> B) permeability greater than 1 may indicate an active efflux transport process. Bidirectional permeability measurements can also be performed in the presence of a specific P-gp inhibitor. Thus, apical-to-basolateral permeability increases and basolateral-to-apical permeability decreases such that... 

Accumulation/efflux studies can be performed on different cell systems or membrane vesicle preparations. In the accumulation assays, uptake of a probe over time, typically either fluorescent (e.g. calcein-AM (CAM) [25-27]) or radiolabeled, into the cell or membrane vesicles is measured in the presence or absence of a known P-gp inhibitor. As P-gp transports substrates out of the cells, the inhibition of the protein would result in an increase in the amount of the probe in the cell. Accumulation studies in cells that overexpress P-gp can be compared to those obtained in the parental cell line that does not have as high a level of P-gp expression. The probe in the absence of inhibitors shows lower accumulation in P-gp expressing cells than in P-gp deficient cells. Similarly, probe accumulation is increased under conditions where P-gp is inhibited such that the difference in accumulation in P-gp deficient and overexpressing cells, respectively, becomes smaller. Accumulation assays poorly distinguish substrates and inhibitors of P-gp and, as far as transport assays are concerned, are also influenced by a passive diffusion property of molecules [20]. In contrast to transport assays, both accumulation (i.e. calcein-AM assay) and ATPase assays tend to fail in the identification ofrelatively low permeable compounds as P-gp active compounds [20]. 

P-gp inhibitor 16 inhibitors of Test sets in [44] The model produced Binding to the ve- Vinblastine-in- Pharmacophore con- [45]... 

Figure 16.7 (I) A graphical representation ofthe most important 3D pharmacophoric GRIND features forthe astemizole inhibitor as a reference compound [54]. (II) Similar pharmacophoric features found forthree potent P-gp inhibitors [55] as an example, these features are reported for compound 3a. The colored areas around the molecule are the GRID MIFs calculated with the DRY (yellow) and N1 (blue) probes.

Figure 16.8 Structures of three potent P-gp inhibitors taken from the literature [55].

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. 

Less direct evidence for active transport mechanisms includes the high degree of absorption of the P-gp substrate losartan in the isolated perfused rat lung, which indicated an absence of any absorption-retarding effect of P-gp [139], Evidence to the contrary includes an increase in the uptake of idaru-bicin when administered to the IPL via the perfusate in the presence of P-gp inhibitors [70],... 

Some P-gp inhibitors have been tested in clinical trials (e.g., GF120918, PSC 833) [88, 89], Shortly before birth, it is often desirable to expose the fetus to anti-HIV medications to prevent HIV transmission from the mother to the fetus during delivery. Preperfusion with P-gp inhibitors increased fetal penetration of the protease inhibitor saquinavir in in vitro placental models, and it has been hypothesized that P-gp may be responsible for limiting fetal exposure to HIV protease inhibitors, methadone, anthracyclines, and taxanes [90-93],... 

In contrast to P-gp and the MRP proteins, the breast cancer resistance protein (BCRP) contains six transmembrane domains and only one ATP-binding domain. It was first cloned from the breast cancer cell line MCF-7 selected in doxombicin, in the presence of the P-gp inhibitor verapamil. It is found in many human tissues, such as the placenta, small intestine, colon, and liver [133], It is localized to the apical membrane of epithelial cells of the small intestine and colon and to the bile canalicular membrane in the liver and is involved in reducing intestinal uptake, increasing hepatobiliary excretion, etc., leading to diminished oral bioavailability. cDNA sequences identical to BCRP and named MXR and ABCP, respectively, were independently isolated from human colon carcinoma cells and human placenta [134], BCRP requires... 

Absorption - Absorption of tipranavir in humans is limited. Tipranavir is a P-glycoprotein (P-gp) substrate, a weak P-gp inhibitor, and appears to be a potent P-gp inducer as well. Steady state is attained in most subjects after 7 to 10 days of dosing. 

Pancreatic cholesterol esterase (3.1.1.3.) aids in transporting cholesterol to the enterocyte. By utilizing a selective and potent cholesterol esterase inhibitor 6-chloro-3-(l-ethyl-2-cyclohexyl)-2-pyrone, the absorption of cholesterol in hamsters could be reduced [71]. Wadkins et al. [72] synthesized novel sulfonamide derivatives, which demonstrated greater than 200-fold selectivity for human intestinal carboxylesterase compared with the human liver carboxylesterase hCEl, and none of them was an inhibitor of human acetylcholinesterase or butyrylcholinester-ase. Maybe these agents can serve as lead compounds for the development of effective, selective carboxylesterase inhibitors for clinical applications. Also the potent P-gp inhibitor verapamil [73] as well as S,S,S-tributylphosphortrithionate (DEF) [74] may exhibit carboxylesterase inhibitory properties. Various other inhibitors of human esterases are listed in Table 5.6. 

While several P-gp inhibitors have entered clinical trials, the development of specific MRP inhibitors is still in its infancy. Similar to P-gp, various MRP inhibitors are already known— among others MK-571 [98-100], probenecid [101], ritonavir [102], leukotriene C4 [103], gemfibrozil [104], pyrrolpyrimidines [105], sulindac [106], and certain isothiocyanates [107], but their potential as auxiliary agents for oral drug delivery has to be further investigated. 

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. 

Paclitaxel is an antitumor drug, with a low oral bioavailability (6%) [96] due to poor water solubility, hydrophobicity (log P = 4), and its high affinity to P-gp and CYP3A4. A novel SMEDDS of paclitaxel increased the bioavailability by up to 1.5-fold in comparison to the commercial micellar solution taxol. Concomitant intake of cyclosporine A, a known P-gp inhibitor, further increased paclitaxel bioavailability (1.8-fold in comparison to taxol) [97]. 

Recent studies on multidrug reversal in mouse lymphoma and MDR/COLO 320 cells have shown that phenothiazine derivatives, namely perphenazine (2) and prochlorperazine dimaleate (4), effectively inhibited rhodamine efflux [171]. Other phenothiazine derivatives such as promethazine (1), ox-omemazine (20), methotrimeprazine maleate (18), triflupromazine (11), and trimeprazine (17) differently modulated intracellular rhodamine accumulation in these resistant cells. The effect of some substitution in the phenothiazine ring was studied in mouse lymphoma cells expressing P-gp [172], The 3,7,8-trihydroxy- and 7,8-dihydroxychlorpromazine derivatives were effective P-gp inhibitors, whereas 7,8-diacetoxy-, 7,8-dimethoxy-, 7-semicarbazone-, and 5-oxo-chlorpromazine derivatives exerted only a moderate effect. 

In contrast, data from a recent conference presentation by Choc and Robinson [100], indicated that cyclosporin metabolite ratios were similar after oral administration of either Sandimmun or the Neoral formulation, suggesting minimal changes in cyclosporin first-pass metabolism after Neoral administration. Furthermore, Neoral/Sandimmun area under the curve (AUC) ratios were similar (approximately 2) after cyclosporin administration in either the absence or presence of a CYP3A/P-gp inhibitor, whereas an AUC ratio of approximately 1 would have been expected if bioavailability was limited by metabolism or antitransport. However, these data have not yet been published, and the relative contributions of enhanced absorption or reduced metabolism in the CY bioavailability enhancement provided by Sandimmun Neoral are yet to be fully defined. 

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