Influence of multidrug resistance (MDR) proteins at the blood-brain barrier on the transport and brain distribution of enaminone anticonvulsants.

Previous in vitro studies evaluating the permeability of enaminones suggested that their blood-brain barrier (BBB) transport might be influenced by the presence of an efflux mechanism. Therefore, transport mechanisms responsible for these anticonvulsants across the BBB were examined. The transport of enaminones (1 x 10(-4) M) were evaluated over 120 min with verapamil (50 microM) and probenecid (100 microM) using bovine brain microvessel endothelial cells (BBMECs) to assess the role of multidrug resistant (MDR) transport proteins [i.e., P-glycoprotein (Pgp) and MDR protein 1 (MRP1)] on efflux, respectively. Uptake studies in the presence and absence of rhodamine 123 (R123; 3.2 and 5.0 microM) were also performed in a Pgp overexpressing cell line, MCF-7/Adr. Select enaminone esters (12.5 mg/kg) were administered intravenously to mdr 1 a/b (+/+), mdr 1 a/b (-/-) knockout and probenecid pretreated mice (20 +/- 5g). Enaminones and R123 were assayed with validated ultraviolet and fluorescence high-performance liquid chromatography methods, respectively. Verapamil and probenecid significantly ( p>0.05) inhibited the transport of select enaminone esters across BBMECs. Two enaminones caused a statistically significant increase in the uptake of R123 in MCF-7/Adr cells. Concentrations of select enaminones in mdr 1 a/b (-/-) mice brains were significantly higher ( p<0.05) compared with those in mdr 1 a/b (+/+) mice brains; however, no differences were observed in probenecid pretreated animals. Taken together, these results strongly suggest that Pgp may influence enaminone transport at the BBB and hence affect epilepsy treatment with these agents.     

Mdr1 limits CYP3A metabolism in vivo

We determined whether the drug efflux protein P-glycoprotein (Pgp) could influence the extent of CYP3A-mediated metabolism of erythromycin, a widely used model substrate for CYP3A. We compared CYP3A metabolism of erythromycin (a Pgp substrate) using the erythromycin breath test in mice proficient and deficient of mdr1 drug transporters. We first injected mdr1(+/+) mice with [(14)C]N-methyl erythromycin and measured the rate of appearance of (14)CO(2) in the breath as a measure of hepatic CYP3A activity. Animals treated with CYP3A inducers or inhibitor showed accelerated or diminished (14)CO(2) in the breath, respectively. The erythromycin breath test was next administered to mdr1a(-/-) and mdr1a/1b(+/+) and (-/-) mice. These animals had equivalent levels of immunoreactive CYP3A and CYP3A activity as measured by erythromycin N-demethylase activity in liver microsomes. Nevertheless, the rate of (14)CO(2) appearance in the breath showed no relationship with these measurements of CYP3A, but changed proportionally to expression of mdr1. The average breath test (14)CO(2) area under the curves were 1.9- and 1.5-fold greater in mdr1a/1b(-/-) and mdr1a(-/-) mice, respectively, compared with (+/+) mice, and CER(max) was 2-fold greater in mdr1a/1b(-/-) compared with (+/+) mice. We conclude that Pgp, by limiting intracellular substrate availability can be an important determinant of CYP3A metabolism of numerous medications that are substrates for CYP3A and Pgp.     

Caco-2 permeability,P-glycoprotein transport ratios and brain penetration of heterocyclic drugs

In this study the gastrointestinal absorption and P-glycoprotein (Pgp) efflux transport of heterocyclic drugs was investigated with the Caco-2 cell model. Based on the calculation of the physico-chemical properties a good oral absorption was predicted for all the drugs tested in this study which corresponded well with the measured Caco-2 permeabilities (Papp). Generally a high permeability of the tested heterocyclic drugs was measured being in agreement with earlier published human in vivo absorption data. Based on the transport data of domperidone and verapamil it was found that the Pgp efflux transporter was expressed in the Caco-2 cells. Many of the drugs tested were indicated to be potential Pgp efflux substrates. Since Pgp is expressed at the Blood Brain Barrier (BBB) as well, it was expected that CNS penetration will be impaired if a drug is a Pgp substrate. However, no correlation could be found between brain penetration in rats and the Pgp efflux ratio as measured with the Caco-2 cells. From the data it is concluded that Pgp efflux ratio's as determined in in vitro High Throughput Screening (HTS) tests, where the transport conditions are fixed (pH gradient, concentration, etc.), cannot routinely be used to predict a possible limited brain penetration.     

Altered expression of hepatic cytochromes P-450 in mice deficient in one or more mdr1 genes

We hypothesized that the drug efflux protein P-glycoprotein (Pgp), the product of the multidrug resistance gene MDR1, might influence hepatic expression of CYP3A or other cytochromes P-450 (P-450s) because Pgp can transport endogenous regulators of these cytochromes. We began with variants of a CF-1 mouse strain containing a defective mdr1a gene that is inherited in a Mendelian fashion. The amount of CYP3A protein in liver was inversely related to the gene dose of the normal mdr1a allele in these mice. mdr1a knockout mice of either mixed (FVB x 129/Ola) or pure FVB genetic background and housed in Amsterdam display an increased expression of CYP2B and CYP3A proteins. However, because mdr1a ablation causes a compensatory increase in hepatic mdr1b (which can efflux intracellular glucocorticoids), we reasoned that mdr1b might mask the overall effect of mdr1a absence on P-450 gene expression. Targeted inactivation of the mdr1b gene increased P-450 expression, but the effect was modest compared with mdr1a ablation. Mice nullizygous for both mdr1a and mdr1b-type Pgps and kept in Amsterdam had dramatically increased levels of CYP3A protein as well as other P-450s examined and of the electron donor P-450 reductase. Consistent with the protein results, CYP3A catalytic activity measured as midazolam 1'- and 4-hydroxylation in liver microsomes from these knockout mice revealed a rank order of activities with mdr1a/1b > mdr1a > mdr1b > (+/+) mice. In contrast to results in mice housed in Amsterdam, in the genetically identical mdr1a or mdr1a/1b (-/-) male mice housed in the United States, hepatic P-450 expression was unaffected by mdr1 genotype or actually showed a slight decrease in mdr1a (-/-) mice. These results provide a revealing picture of mdr1-type Pgp as an upstream regulator of hepatic P-450 expression, and demonstrate that these pharmacologically relevant phenotypes in knockout mice depend not only on the genetic make-up of the mice but also on the environment.     

Effect of cell differentiation and passage number on the expression of efflux proteins in wild type and vinblastine-induced Caco-2 cell lines.

The mRNA level expression of MDR1, MRP1-6, BCRP and CYP3A4 was determined by quantitative PCR in wild type (Caco-2WT) and vinblastine-treated (Caco-2VBL) Caco-2 cells at different passage levels (32-53). Differentiation increased the mRNA levels of MDR1, BCRP and all the MRPs except MRP4. Corresponding mRNA levels were observed in Caco-2WT and Caco-2VBL, except that the expression of MRD1 was higher in Caco-2VBL than in Caco-2WT cells. CYP3A4 was barely detected in either cell line. MDR1 functionality was studied using rhodamine123 and verapamil as a substrate-inhibitor pair. Corresponding to the observed differences in mRNA levels, MDR1 activity was higher in the Caco-2VBL cells. In Caco-2WT, MDR1 functionality was elevated at low passage numbers (32-35) compared to higher ones (49-53). Verapamil inhibited MDR1 efflux except at higher passage Caco-2WT cells, where no MDR1 activity could be observed. The results support the use of Caco-2VBL cells in MDR1 screening. The functional expression is higher than in Caco-2WT and remains consistent across the studied passages without major differences in mRNA levels of other efflux proteins. As both the passage number and the level of cell differentiation affect the expression profile of efflux proteins, short-term cell growth protocols should be evaluated accordingly.     

Inhibitors of mdr1-dependent transport activity delay accumulation of the mdr1 substrate rhodamine 123 in primary rat hepatocyte cultures

P-glycoproteins (P-gps) encoded by mdr1 (multidrug resistance) genes mediate extrusion of numerous lipophilic xeno- and endobiotics through the plasma membrane. Rhodamine 123 (Rh123), a fluorescent dye which is accumulated by mitochondria, is a mdr1 substrate and a well-established tool to study mdr1 transport activity. Inhibitors of mdr1-dependent transport such as verapamil or cyclosporin A have been found to decrease Rh123 efflux from mdr1-expressing cells. Mdr1b gene expression increases with time in primary rat hepatocyte culture. In hepatocytes cultured for 4 days and expressing high levels of P-gp, intracellular Rh123 accumulation was enhanced in the presence of mdr1 inhibitors (cyclosporin A, 8 and 80 microM, verapamil, 8 and 80 microM, or triton X-100, 8 microM). Surprisingly, in hepatocytes expressing low levels of P-gp (after 1 day of culture), time-dependent Rh123 accumulation was not enhanced, but delayed by cyclosporin A, verapamil or triton X-100. In these cells orthovanadate (50 microM), an inhibitor of P-glycoprotein ATPase activity, suppressed Rh123 accumulation, while tetraethylammonium (200 microM), an organic cation transporter (OCT) substrate, had no effect. The paradoxical delay in Rh123 accumulation by verapamil and cyclosporin A occurred eventhough these compounds decreased dye extrusion from Rh123 pre-loaded cells. These observations suggest that a hitherto unknown mechanism which is sensitive to modulators of mdr1-activity contributes to Rh123 uptake or accumulation in primary rat hepatocytes.     

The anthelminthic agent albendazole does not interact with p-glycoprotein.

Albendazole is a clinically important anthelminthic agent known to have variable and low oral bioavailability. The aim of this work was to determine whether albendazole, a CYP3A4 substrate, is also a substrate for the multidrug efflux transporter P-glycoprotein. Both in vitro and in vivo methods were used to assess the role of P-glycoprotein-mediated albendazole transport. In cultured LLC-PK1, L-MDR1, and Caco-2 cells, albendazole was found not to be a P-glycoprotein substrate; the transport across LLC-PK1 and L-MDR1 cells revealed basal to apical versus apical to basal transport to a similar extent. In addition, there was no inhibitory effect of albendazole on digoxin transport in Caco-2 cells, and P-glycoprotein inhibitors (verapamil and quinidine) did not affect transport across Caco-2 cells. The in vivo relevance of P-glycoprotein to albendazole disposition was assessed using mdr1a/1b(-/-) mice after intravenous administration of albendazole (15 mg/kg). A similar pattern of tissue distribution in both P-glycoprotein-deficient and wild-type mice was observed. In conclusion, albendazole is neither a substrate nor an inhibitor of P-glycoprotein. Therefore, interactions between albendazole and P-glycoprotein substrates or inhibitors are unlikely to be clinically important.     

What kinds of substrates show P-glycoprotein-dependent intestinal absorption? Comparison of verapamil with vinblastine

The influence of P-glycoprotein (P-gp) on intestinal absorption of drugs was investigated by comparison of the uptakes of two P-gp substrates, verapamil and vinblastine, using intestinal segments of wild-type and mdr1a/1b gene-deficient (mdr1a/1b(-/-)) mice, and Caco-2 cells. When [(3)H]vinblastine was injected into intestinal segments of wild-type mice, vinblastine was absorbed from duodenum and ileum, but not from jejunum. This difference among intestinal regions could not be explained by segmental differences of mdr1a mRNA expression. In Caco-2 cells, it was found that vinblastine had a high value of efflux/influx ratio (an index of affinity for P-gp) of 12.1, and a low permeability of less than 1 x 10(-6) cm/sec. The corresponding values for verapamil were 4.9 and 10.6 x 10(-6) cm/sec, respectively. After oral administration of [(3)H]vinblastine to mice, the maximum concentration (C(max)) and the area under the plasma concentration time-curve from time 0 to 24 hr (AUC(0-24 hr)) for mdr1a/1b(-/-) mice were 1.5 times greater than those for wild-type mice, while these parameters were not significantly different between the two strains in the case of [(3)H]verapamil. Therefore, P-gp substrates may be classified into at least two types, i.e., verapamil-type, for which the intestinal absorption is unaffected by P-gp, and vinblastine-type, for which the intestinal absorption is influenced by P-gp. Vinblastine-type P-gp substrates, with low permeability and high affinity for P-gp, would be unfavorable candidates for oral drugs.     

Prochloraz and nonylphenol diethoxylate inhibit an mdr1-like activity in vitro, but do not alter hepatic levels of P-glycoprotein in trout exposed in vivo.

P-glycoproteins (P-gps) encoded by multidrug resistance 1 (mdr1) genes are ATP-dependent transporters located in the cytoplasmic membrane which mediate the efflux of a broad spectrum of hydrophobic compounds from the cell. The tissue distribution of P-gps suggests their role in the organismal defense against xenobiotics by effecting xenobiotic excretion and reducing xenobiotic uptake. In the present work, the interaction of P-gp(s) in the liver and in primary cultured hepatocytes of rainbow trout with two model pollutants was studied - the imidazole fungicide prochloraz and the alkylphenolic surfactant nonylphenol diethoxylate (NP2EO). Using a monoclonal antibody (mAB C219) directed against a conserved P-gp epitope, an immunoreactive protein of 160 kDa was detected in immunoblots of liver extracts from control trout. In sections of control trout livers, immunohistochemistry with the mAB C219 resulted in specific staining of bile canaliculi. In juvenile trout exposed for 7 days to sublethal concentrations of prochloraz (0.027 microM; 0.27 microM) or NP2EO (0.32 microM; 1.30 microM), no changes in levels of hepatic P-gp(s) were found in immunoblot and immunochemical investigations. The efflux of the fluorescent mdr 1 substrate rhodamine 123 (Rh123) from cultured isolated trout hepatocytes was partly inhibited by verapamil and vinblastine, compounds known to interfere with mdr 1-dependent transport. This demonstrates the presence of a mdr1-like mechanism in trout liver which is probably involved in the biliary excretion of hydrophobic xenobiotics. Non-cytotoxic concentrations of prochloraz and NP2EO were tested for effects on the efflux of Rh123 from trout hepatocytes. Prochloraz was a potent inhibitor of the mdr1-like mechanism, being effective at 0.3 microM and above. NP2EO inhibited Rh123 efflux only at the highest concentration tested (31.6 microM). The accumulation and elimination of 14C-prochloraz by cultured trout hepatocytes was not affected by mdr 1-type substrates (Rh123, vinblastine) and a mdr 1 inhibitor (verapamil). This shows that prochloraz is, despite its inhibitory potency, not a substrate of the mdr1-like mechanism in trout liver. The inhibition by prochloraz and NP2EO of the md r1-like mechanism in trout hepatocytes suggests that water pollutants can interfere with P-gp-function in fish and thus may impair the organismal defense against xenobiotics.     

Suppressive effect of tobacco smoke extracts on oral P-glycoprotein function and its impact in smoke-induced insult to oral epidermal cells

P-glycoprotein (Pgp) participates in the export of numerous toxins, drugs, and physiological compounds. To examine the involvement of Pgp in smoke-induced oral cell insult, the effects of extracts of the mainstream tobacco smoke (TS) on Pgp were studied in an oral epidermal carcinoma cell line, OECM-1. TS was first extracted with cyclohexane (CTS) and the residues were further extracted with isopropanol (ITS). For comparison, cells were exposed to CTS and ITS at the concentrations according to their relative extraction yield. ITS but not CTS decreased the efflux of a Pgp substrate, rhodamine (Rh) 123, in a concentration- and time-dependent manner. The efflux was also decreased by co-exposure to CTS and ITS. However, immunoblot analysis revealed that the protein level of Pgp was not affected by ITS. Naphthalene, mainly detected in the ITS, decreased Rh 123 efflux. However, the efflux activity was not affected by benzo(a)pyrene and nicotine, which were present in the CTS and both extracts, respectively. Co-exposure to CTS in combination with ITS, naphthalene, or verapamil enhanced cell insult compared to single exposure. These results demonstrated that smoke and its constituent, naphthalene, diminished Pgp-mediated efflux. The reduction in Pgp function could be a stimulatory factor of TS-induced oral cell insult.     

P-glycoprotein efflux pump expression and activity in Calu-3 cells

The purpose of this work was to determine if the sub-bronchial epithelial cell model, Calu-3, expresses the functionally active P-glycoprotein (Pgp) efflux pump. Calu-3 cells express lower levels of Pgp than both Caco-2 and A549 cells as determined by Western Blot analysis. In Calu-3 cells, accumulation of the Pgp substrates rhodamine 123 (Rh123) and calcein acetoxymethyl ester (calcein-AM) was increased in the presence of the specific Pgp inhibitors cyclosporin A (CsA), vinblastine, and taxol. Significant inhibition of Pgp activity was not observed until after 2 h in both cell lines. The organic anion/multidrug resistance associated protein-1 (MRP1) inhibitors, probenecid and indomethacin, did not affect Rh123 accumulation, whereas an increase in calcein accumulation was observed by both agents. The metabolic inhibitor sodium azide decreased the efflux of Rh123 out of Calu-3 cells to the same degree as CsA, supporting inhibition of an active, efflux pathway. The basolateral-to-apical transport of Rh123 was significantly higher than that in the reverse direction, indicating a secretory pathway of efflux that was inhibited 25-fold by CsA. Basolateral-to-apical transport of Rh123 was inhibited slightly with both MRP1 inhibitors; however, no significant effect of Rh123 net secretion was observed. Mixed inhibitor studies demonstrated that Rh123 efflux was mainly Pgp mediated. These results support an energy-dependent Pgp efflux pump pathway that is sensitive to inhibition with CsA in Calu-3 cells.     

Interaction of triazolam and ketoconazole in P-glycoprotein-deficient mice.

The role of P-glycoprotein (P-gp) on the distribution of the benzodiazepine triazolam (TRZ) and the azole antifungal agent ketoconazole (KET), and on the TRZ-KET interaction, was studied using mdr1a(-) or mdr1a/b(-/-) mice (P-gp-deficient mice) and matched controls. TRZ and KET also were studied in Caco-2 cells in Transwell culture. After single i.p. injections of TRZ or KET in separate groups of control mice, brain concentrations of TRZ exceeded those in serum [brain/serum area under the concentration curve (AUC) ratio, 5.0], whereas brain/serum AUC ratios for KET were approximately 0.5. On the basis of single time points, brain concentrations of TRZ, or brain/serum ratios, were similar in P-gp-deficient animals compared with controls, whereas P-gp-deficient animals had significantly higher KET brain concentrations and brain/serum ratios. Coadministration of KET with TRZ increased TRZ concentrations in serum, liver, and brain, both in controls and in P-gp-deficient animals, probably attributable to impairment by KET of CYP3A-mediated clearance of TRZ. However, KET did not increase brain/serum ratios of TRZ in either group. In Caco-2 cells, basal-to-apical flux of TRZ was higher than apical-to-basal flux. However, verapamil (100 microM) did not alter flux in either direction. KET inhibited basal-to-apical transport of rho-damine-123, with a 50% inhibitory concentration of 2.7 microM. Thus, TRZ does not appear to undergo measurable blood-brain barrier efflux transport by P-gp in this animal model. KET impairs clearance of TRZ but does not increase tissue uptake. However, KET itself may be a substrate for efflux transport at the blood-brain barrier.     

Modulation by LY335979 of P-glycoprotein function in multidrug-resistant cell lines and human natural killer cells

Resistance to chemotherapy by some human tumors may be due to overexpression of membrane-associated transport proteins. The best characterized of these is the multidrug resistance (MDR) transporter, P-glycoprotein (Pgp). The aim of this study was to measure the inhibitory effects of a potent new MDR modulator, (2R)-anti-5-(3-[4-(10,11-difluoromethanodibenzo-suber-5-yl) piperazin-1-yl]-2-hydroxypropoxy)quinoline trihydrochloride (LY335979), in the drug-resistant cell line HL60/VCR and in normal, human CD56(+) lymphocytes. We used flow cytometric methods to detect the accumulation of rhodamine 123 and daunorubicin, fluorescent MDR substrates, in these cells. Our results indicate that LY335979 was 500-1500 times more potent than cyclosporin A or verapamil in restoring Pgp substrate accumulation in the MDR cell line HL60/VCR. Moreover, LY335979 could effectively block Pgp function on isolated CD56(+) lymphocytes (IC(50) = 1.2 nM) or CD56(+) lymphocytes in whole blood (IC(50) = 174 nM). We conclude that LY335979 is among the most potent Pgp inhibitors described and that it maintains significant potency in whole-human blood. These latter findings are important for establishing the dosing regimens of LY335979 for future clinical studies.     

Evidence for Modulation of P-glycoprotein-Mediated Efflux by Methoxypolyethylene Glycol-block-Polycaprolactone Amphiphilic Diblock Copolymers

PURPOSE: The purpose of this study was to investigate the pathways involved in rhodamine 123 (R-123) accumulation enhancement in Caco-2 cells with a low molecular-weight methoxypolyethylene glycol-block-polycaprolactone (MePEG-b-PCL) diblock copolymer. METHODS: R-123 accumulation by Caco-2 cells with MePEG17-b-PCL5 was measured in the presence of endocytosis inhibitors or under ATP depletion conditions. Directional flux studies were conducted with cell monolayers on Transwell plates. RESULTS: Endocytosis inhibitors had no effect on reducing R-123 accumulation with MePEG17-b-PCL5. The apical to basolateral (AP-->BL) flux of R-123 with MePEG17-b-PCL5 or verapamil was similar to R-123 alone. However the BL-->AP flux was significantly decreased with MePEG17-b-PCL5 and verapamil. The efflux ratio for R-123 flux was 3.2 and was reduced to 1.06 with MePEG17-b-PCL5 confirming the inhibition of P-glycoprotein (P-gp)-mediated efflux. R-123 accumulation at the conclusion of each of the flux experiments was similar for MePEG17-b-PCL5 and verapamil in the BL-->AP direction. The AP-->BL direction demonstrated a 2-fold increase for verapamil and a 5-fold increase with MePEG17-b-PCL5. This difference in R-123 accumulation was possibly due to the diblock enhancing passive membrane diffusion of R-123. CONCLUSIONS: MePEG17-b-PCL5 diblock reduced R-123 efflux through inhibition of P-gp efflux, and unimers may interact with cell membranes, increasing permeability and enhancing R-123 influx through increased transmembrane diffusion.     

Contribution of mdr1b-type P-glycoprotein to okadaic acid resistance in rat pituitary GH3 cells

Okadaic acid as well as other, structurally different, inhibitors of serine/threonine phosphatases 1 and 2A induce apoptosis in pituitary GH3 cells. Incubation with stepwise raised concentrations of okadaic acid resulted in the isolation of cells that were increasingly less sensitive to the cytotoxic effect of this agent. After about 18 months cells were selected that survived at 300 nM okadaic acid, which is about 30 times the initially lethal concentration. This study revealed that a major pharmacokinetic mechanism underlying cell survival was the development of a P-glycoprotein-mediated multidrug resistance (MDR) phenotype. The increase in mRNA levels of the mdr1b P-glycoprotein isoform correlated with the extent of drug resistance. Functional assays revealed that increasing drug resistance was paralleled by a decreased accumulation of rhodamine 123, a fluorescent dye which is a substrate of mdr1-mediated efflux activity. Resistance could be abolished by structurally different chemosensitizers of P-glycoprotein function like verapamil and reserpine but not by the leukotriene receptor antagonist MK571 which is a modulator of the multidrug resistance-associated protein (MRP). Okadaic acid resistance included cross-resistance to other cytotoxic agents that are substrates of mdr1-type P-glycoproteins, like doxorubicin and actinomycin D, but not to non-substrates of mdr1, e.g. cytosine arabinoside. Thus, functional as well as biochemical features support the conclusion that okadaic acid is a substrate of the mdr1-mediated efflux activity in rat pituitary GH3 cells. Maintenance of resistance after withdrawal of okadaic acid as well as metaphase spreads of 100 nM okadaic acid-resistant cells suggested a stable MDR genotype without indications for the occurrence of extrachromosomal amplifications, e.g. double minute chromosomes.     

In vivo effects of cyclosporin A and ketoconazole on the pharmacokinetics of representative substrates for P-glycoprotein and cytochrome P450 (CYP) 3A in rats

In this study, the in vivo effects of cyclosporin A (CsA) and ketoconazole (KCZ), which are used as inhibitors of P-glycoprotein (Pgp) and cytochrome P450 (CYP) 3A, respectively, on the pharmacokinetics of rhodamine 123 (Rho123), nelfinavir (NFV) and erythromycin (EM) were evaluated in rats. The biliary excretion clearance (Clbile) of a known Pgp substrate, Rho123, after intravenous pretreatment with CsA or KCZ (0-20 mg/kg after i.v. administration) showed maximum reduction by 85.6 or 54.1%, respectively, suggesting that the inhibitory potency of KCZ is about half that of Pgp in the liver. Without pretreatment with CsA or KCZ, the clearance ratio of Clbile relative to the total body clearances of Rho123, NFV and EM was 10.5, 0.07 and 31.1%, respectively. After CsA pretreatment, these ratios decreased markedly in a manner dependent on the dose of CsA, while after CZ pretreatment the clearance ratios of NFV and EM increased significantly in a manner dependent on the dose of KCZ. However, in the liver, the contribution of Pgp to the changes in the pharmacokinetic parameters of Rho123, NFV and EM after intravenous administration was much less than that of CYP3A. The portal levels of Rho123 and EM but not NFV after intra-loop administration in the presence of 10 microM CsA in the jejunal loop increased significantly, while in the presence of 25 microM KCZ in the jejunal loop, the portal levels of those substrates showed no notable change as compared to the control levels. In conclusion, KCZ had dual potency to inhibit CYP3A and Pgp, and its inhibitory potency for Pgp was half that of CsA in the rat liver. In addition, metabolism via CYP3A contributed more significantly to the clearance of these substrates that did excretion via Pgp in the liver. In the small intestine, the contribution of Pgp is a more important factor in determining the oral bioavailability of EM than metabolism via CYP enzymes. The elimination of NFV is mainly dependent on liver metabolism via CYP3A, and the Pgp efflux mechanism in the liver and intestine did not contribute as importantly to the oral bioavailability of NFV under in vivo conditions, although NFV has been demonstrated to be a substrate of Pgp under in vitro conditions.     

Resistance to the Translation Initiation Inhibitor Silvestrol is Mediated by ABCB1/P-Glycoprotein Overexpression in Acute Lymphoblastic Leukemia Cells

Protein synthesis is a powerful therapeutic target in leukemias and other cancers, but few pharmacologically viable agents are available that affect this process directly. The plant-derived agent silvestrol specifically inhibits translation initiation by interfering with eIF4A/mRNA assembly with eIF4F. Silvestrol has potent in vitro and in vivo activity in multiple cancer models including acute lymphoblastic leukemia (ALL) and is under pre-clinical development by the US National Cancer Institute, but no information is available about potential mechanisms of resistance. In a separate report, we showed that intraperitoneal silvestrol is approximately 100% bioavailable systemically, although oral doses were only 1% bioavailable despite an apparent lack of metabolism. To explore mechanisms of silvestrol resistance and the possible role of efflux transporters in silvestrol disposition, we characterized multi-drug resistance transporter expression and function in a silvestrol-resistant ALL cell line generated via culture of the 697 ALL cell line in gradually increasing silvestrol concentrations. This resistant cell line, 697-R, shows significant upregulation of ABCB1 mRNA and P-glycoprotein (Pgp) as well as cross-resistance to known Pgp substrates vincristine and romidepsin. Furthermore, 697-R cells readily efflux the fluorescent Pgp substrate rhodamine 123. This effect is prevented by Pgp inhibitors verapamil and cyclosporin A, as well as siRNA to ABCB1, with concomitant re-sensitization to silvestrol. Together, these data indicate that silvestrol is a substrate of Pgp, a potential obstacle that must be considered in the development of silvestrol for oral delivery or targeting to tumors protected by Pgp overexpression.