The relationship between modulation of MDR and glutathione in MRP-overexpressing human leukemia cells

Multidrug resistance-associated protein (MRP) causes multidrug resistance (MDR) involving the anthracyclines and epipodophyllotoxins. Many studies show modulation of anthracycline levels and cytotoxicity in MRP-overexpressing cells, but there is limited data on the modulation of etoposide levels and cytotoxicity in MRP-overexpressing or in P-glycoprotein-expressing cells. Etoposide accumulation was 50% reduced in both the CEM/E1000 MRP-overexpressing subline and the CEM/VLB100 P-glycoprotein-expressing subline compared to the parental CEM cells, correlating with similar resistance to etoposide (200-fold) of the two sublines. For the CEM/VLB100 subline, the P-glycoprotein inhibitor SDZ PSC 833, but not verapamil, was able to increase etoposide accumulation and cytotoxicity. For the CEM/E1000 subline, neither SDZ PSC 833 nor verapamil had any effect on etoposide accumulation. However, verapamil caused a 4-fold sensitization to etoposide in this subline, along with an 80% decrease in cellular glutathione (P < 0.05). Buthionine sulfoximine (BSO), which depletes glutathione, also caused a 2.5-fold sensitization to etoposide with no effect on accumulation in the CEM/E1000 subline. In contrast, SDZ PSC 833 was able to increase daunorubicin accumulation in the CEM/E1000 subline (P < 0.05), but had no effect on daunorubicin cytotoxicity, or cellular glutathione. These results show that modulation of etoposide cytotoxicity in MRP-overexpressing cells may be through changes in glutathione metabolism rather than changes in accumulation and confirm that changes in drug accumulation are not related to drug resistance in MRP-overexpressing cells.     

Analysis of the interactions of SDZ PSC 833 ([3'-keto-Bmt1]-Val2]-Cyclosporine), a multidrug resistance modulator, with P-glycoprotein

Multidrug resistance (MDR) is considered to be an important impediment to the effective treatment of cancer. P-glycoprotein, the drug efflux pump that mediates this resistance, can be inhibited by a wide variety of pharmacological agents, resulting in the circumvention of the MDR phenotype. SDZ PSC 833 ([3'-keto-Bmt1]-Val2]-cyclosporine), a nonimmunosuppressive cyclosporine D derivative, was identified to be a potent MDR modulator (Gaveriaux et al. J. Cell Pharmacol. 2:225-234; 1991). In this study, the interactions of P-glycoprotein with two cyclosporine derivatives, SDZ PSC 833 and cyclosporine A (CsA, Sandimmune), were analyzed. SDZ PSC 833 enhanced the sensitivity of the MDR cells to anticancer drugs by increasing the accumulation and inhibiting the efflux of cytotoxic agents from resistant cells more efficiently than CsA. The two cyclosporine analogs competed with the labeling of P-glycoprotein by a photoactive cyclosporine derivative. In addition, membrane vesicles derived from resistant cells bound SDZ PSC 833. However, CsA was transported by P-glycoprotein, whereas SDZ PSC 833 was not actively transported. This resulted in a prolonged inhibitory effect by SDZ PSC 833. The studies suggest that the binding of SDZ PSC 833 to P-glycoprotein in the absence of its transport from MDR cells mediated its high potency as an MDR reversing agent. In addition, the comparison of the two cyclosporine analogs indicated that limited chemical modifications of MDR reversing agents can affect their potential to inhibit P-glycoprotein function.     

Reversal of multidrug resistance by SDZ PSC 833, combined with VAD (vincristine, doxorubicin, dexamethasone) in refractory multiple myeloma. A phase I study

SDZ PSC 833, a non-immunosuppressive cyclosporin analogue reverses multidrug resistance (MDR) in vitro by inhibiting P-glycoprotein (P-gp) mediated drug efflux. We performed a dose escalation study of SDZ PSC 833 combined with VAD chemotherapy in refractory multiple myeloma (MM). Twenty-two MM patients who were refractory to doxorubicin/vincristine/dexamethasone (VADr, n=11) or had failed multiple regimens (n=6) or were melphalan-refractory (MELr, n=5), were treated with one to three cycles of VAD combined with oral SDZ PSC 833, which was administered at escalating dosages starting at 5 mg/kg/day to 15 mg/kg/day for 7 days. The median trough and peak blood levels of SDZ PSC 833 ranged from 461/1134 ng/ml at 5 mg/kg/day to 821/2663 ng/ml at 15 mg/kg, respectively. With addition of SDZ PSC 833 (5 mg/kg) the mean plasma AUC 0-->96 h of doxorubicin as compared with control patients treated with VAD increased from 779 to 1510 ng/ml/h (P=0.0071), while the doxorubicin clearance was reduced from 47.6 to 27.8 l/h/m2 (P=0.0002). The clearance of doxorubicinol was reduced accordingly. Because of the increased plasma AUC, the dose of doxorubicin and vincristine had to be reduced in 13 patients to 50% (n=1) or 75% (n=12). A further dose-escalation of SDZ PSC 833 did not lead to a proportional increase of doxorubicin AUC. Toxicity WHO CTC grade 2 or 3 included hypoplasia (18/22), constipation (10/22), hyponatremia (3/22) and infections (6/22). A partial response or stable disease was achieved in eight and six patients, respectively. In 17 evaluable patients the mean percentage of pretreatment bone marrow plasma cells which expressed P-glycoprotein was 40%. The pretreatment in vitro rhodamin retention in CD38++ myeloma cells was reversible by 2 microM SDZ PSC 833 with 15-98% in 7/9 tested patients. In 4/5 responding patients analyzed before and after treatment with VAD + SDZ PSC 833, a reduction of P-gp + plasma cells was observed. It is concluded, that the blood concentrations of SDZ PSC 833 attained in MM patients increase with dose after oral administration. It can be safely combined with VAD chemotherapy. SDZ PSC 833 diminishes the clearance of doxorubicin, leading to an increase of the plasma AUC of doxorubicin. In addition, it is an effective inhibitor of P-gp mediated efflux of doxorubicin in myeloma tumor cells in vitro. Therefore, a proportional dose-reduction of doxorubicin and vincristine is warranted. Phase II/III studies in refractory MM are in progress to evaluate the therapeutic efficacy of SDZ PSC 833 with VAD.     

Expression, purification, and characterization of a recombinant 5-lipoxygenase from potato tuber

The involvement of mdr1a P-glycoprotein (P-gP) on the tissue distribution of the multidrug resistance-reversing agent SDZ PSC 833 was assessed by use of mdr1a (-/-) mice. The mdr1a (-/-) and wild-type mdr1a (+/+) mice received a 4-h constantrate i.v. infusion (2 micrograms/min) of [14C]SDZ PSC 833. Mice were sacrificed at 0, 0.5, 1, 2 and 4 h during infusion and at 0.5, 1, 3, 8 and 24 h after stopping the infusion. Blood and tissues were analyzed on total (14C) and parental SDZ PSC 833 concentrations. Mdr1a (-/-) mice exhibited increased SDZ PSC 833 accumulation in cerebrum, cerebellum and somewhat in testes and small intestine compared with the wild-type mice. The difference between mdr1a (-/-) and (+/+) brain (cerebrum and cerebellum) penetration depended on SDZ PSC 833 blood concentrations, because this cyclosporin analog apparently governs its own brain penetration by inhibiting the P-glycoprotein pump in mdr1a (+/+) mice. Thus the mdr1a (-/-)/(+/+) ratio of brain concentrations tended to decrease and increase at high and low blood concentrations, respectively. These findings clearly demonstrate the interaction of SDZ PSC 833 with the P-glycoprotein present at the blood-brain barrier. The SDZ PSC 833 distribution in other mdr1a P-glycoprotein-expressed tissues, as well as its metabolism and elimination, was not affected by the mdr1a gene disruption. This suggests that factors other than mdr1a P-gP are involved in the disposition of this multidrug resistance-reversing agent.     

Inhibitory effects of a cyclosporin derivative, SDZ PSC 833, on transport of doxorubicin and vinblastine via human P-glycoprotein

The inhibitory effects of SDZ PSC 833 (PSC833), a non-immunosuppressive cyclosporin derivative, on the P-glycoprotein (P-gp)-mediated transport of doxorubicin and vinblastine were compared with those of cyclosporin A (Cs-A). The transcellular transport of the anticancer drugs and PSC833 across a monolayer of LLC-GA5-COL150 cells, which overexpress human P-gp, was measured. Both PSC833 and Cs-A inhibited P-gp-mediated transport of doxorubicin and vinblastine in a concentration-dependent manner and increased the intracellular accumulation of doxorubicin and vinblastine in LLC-GA5-COL150 cells. The values of the 50%-inhibitory concentration (IC50) of PSC833 and Cs-A for doxorubicin transport were 0.29 and 3.66 microM, respectively, and those for vinblastine transport were 1.06 and 5.10 microM, respectively. The IC50 of PSC833 for doxorubicin transport was about 4-fold less than that for vinblastine transport, suggesting that the combination of PSC833 and doxorubicin might be effective. PSC833 itself was not transported by P-gp and had higher lipophilicity than Cs-A. These results indicated that the inhibitory effect of PSC833 on P-gp-mediated transport was 5- to 10-fold more potent than that of Cs-A, and this higher inhibitory effect of PSC833 may be related to the absence of PSC833 transport by P-gp and to the higher lipophilicity of PSC833.     

Changes in doxorubicin distribution and toxicity in mice pretreated with the cyclosporin analogue SDZ PSC 833

SDZ PSC 833 (PSC 833) is a cyclosporin A analogue that is under clinical investigation in combination with doxorubicin (Dx) or other anticancer agents as a type-1 multidrug resistance (MDR-1)-reversing agent. The present study was focused on the effects of PSC 833 on the distribution and toxicity of Dx in non-tumor-bearing CDF1 male mice. Mice were given PSC 833 i.p. at 30 min before i.v. Dx treatment. Dx levels were determined by a high-performance liquid chromatography (HPLC) assay at different times during a 72-h period following Dx treatment in the serum, heart, intestine, liver, kidney, and adrenals of mice. In all tissues, Dx area under the concentration-time curve (AUC) values were much greater in mice receiving 10 mg/kg Dx in combination with 12.5 or 25 mg/kg PSC 833 than in mice receiving Dx alone. The highest increase in Dx concentrations was found in the intestine, liver, kidney, and adrenals. Lower, albeit significant, differences were found in the heart. PSC 833 did not appear to influence either urinary or fecal Dx elimination or Dx metabolism to a great extent. Doses of PSC 833 devoid of any toxicity potentiated the acute and delayed toxicity of Dx dramatically. The mechanism responsible for this enhanced toxicity has not yet been elucidated but is likely to be related to an increased tissue retention of Dx due to inhibition of the P-glycoprotein (Pgp) pump by PSC 833, as has recently been proposed for cyclosporin A.     

Full blockade of intestinal P-glycoprotein and extensive inhibition of blood-brain barrier P-glycoprotein by oral treatment of mice with PSC833

Mice lacking mdr1-type P-glycoproteins (mdr1a/1b [-/-] mice) display large changes in the pharmacokinetics of digoxin and other drugs. Using the kinetics of digoxin in mdr1a/1b (-/-) mice as a model representing a complete block of P-glycoprotein activity, we investigated the activity and specificity of the reversal agent SDZ PSC833 in inhibiting mdr1-type P-glycoproteins in vivo. Oral PSC833 was coadministered with intravenous [3H]digoxin to wild-type and mdr1a/1b (-/-) mice. The direct excretion of [3H]digoxin mediated by P-glycoprotein in the intestinal mucosa of wild-type mice was abolished by administration of PSC833. Hepatobiliary excretion of [3H]digoxin was markedly decreased in both wild-type and mdr1a/1b (-/-) mice by PSC833, the latter effect indicating that in vivo, PSC833 inhibits not only mdr1-type P-glycoproteins, but also other drug transporters. Upon coadministration of PSC833, brain levels of [3H]digoxin in wild-type mice showed a large increase, approaching (but not equaling) the levels found in brains of PSC833-treated mdr1a/1b (-/-) mice. Thus, orally administered PSC833 can inhibit blood-brain barrier P-glycoprotein extensively, and intestinal P-glycoprotein completely. These profound pharmacokinetic effects of PSC833 treatment imply potential risks, but also promising pharmacological applications of the use of effective reversal agents.     

Cyclosporin A and PSC 833 prevent up-regulation of MDR1 expression by anthracyclines in a human multidrug-resistant cell line

We have previously demonstrated that within 24 h of exposure of the CEM/A7R cell line to epirubicin (EPI), MDR1 gene expression is induced. The aim of the current study was to investigate the role of cyclosporin A (CyA) and PSC 833, two biochemical modulators of the classical multidrug-resistant phenotype, in this model. CEM/A7R cells were exposed to EPI in the presence or absence of various concentrations of CyA or PSC 833. MDR1 expression was assessed using Northern blot analysis and quantitated using a phosphorimager. P-glycoprotein (P-gp) expression was analyzed by the determination of MRK16 binding using flow cytometry. P-gp function was measured in an assay of [3H]daunomycin accumulation. The coincubation of CyA or PSC 833 with EPI prevented the increase in MDR1 gene expression induced by EPI alone. This effect of the two modulators was dose dependent. Neither modulator alone had any significant effect on the expression of MDR1. In these experiments, changes in MDR1 expression correlated with changes in P-gp levels (based on MRK16 binding) and P-gp function. Thus, both PSC 833 and CyA appear to prevent the induction of MDR1 gene expression caused by the short-term exposure of CEM/A7R cells to EPI.     

Enhanced oral absorption and decreased elimination of paclitaxel in mice cotreated with cyclosporin A

Recent experiments in mice have demonstrated that the systemic exposure to p.o. administered paclitaxel is significantly enhanced with coadministration of the P-glycoprotein blocker SDZ PSC 833 (J. van Asperen et al, Br. J. Cancer, 76: 1181-1183, 1997). To facilitate further research on the feasibility of a clinically effective oral formulation of paclitaxel, it is important to know whether cotreatment with a commonly applied and commercially available P-glycoprotein blocker, e.g., cyclosporin A, has a similar effect. Here, we present a detailed study about the effects of cyclosporin A on the pharmacokinetics of p.o. and i.v. administered paclitaxel. Female FVB mice received a combined treatment of 5 or 10 mg/kg paclitaxel (either i.v. or p.o.) plus 0, 10, or 50 mg/kg cyclosporin A (p.o.). The plasma concentrations of paclitaxel were determined at several time points after drug administration using high-performance liquid chromatography. Calculated relative to the area under the plasma concentration-time curve of i.v. administered paclitaxel in mice treated without cyclosporin A, the oral bioavailability of paclitaxel increased from 9.3% up to 67% with coadministration of cyclosporin A. The bioavailability in mice cotreated with 10 or 50 mg/kg cyclosporin A appeared to be similar. The effect of cyclosporin A on the systemic exposure to p.o. administered paclitaxel was the result of both a significantly decreased clearance and an increased uptake. A histological examination revealed that the enhanced absorption was not caused by gastrointestinal toxicity. We conclude that cyclosporin A and SDZ PSC 833 are equally effective in increasing the systemic exposure to p.o. administered paclitaxel. These data are promising for the development of a clinically useful oral formulation of this cytostatic drug and indicate that cyclosporin A is a suitable agent for further research of this concept.     

Mechanism of multidrug resistant tumors and chemotherapeutic approaches against the resistant tumors

Research on multidrug resistance (MDR) has spread widely, with the emphasis on the development of therapeutic approaches. This line of research began in the early 1970s. In 1981 and 1982, calcium channel blockers such as verapamil and calmodulin inhibitors were found to enhance the intracellular levels of vincristine (VCR) and adriamycin (ADM) in resistant tumor cells by inhibiting their outward transport and to circumvent MDR in animal experiments. Since these results were noted for verapamil, various other compounds have been investigated to overcome drug resistance. Among these compounds, two compounds were evaluated in our laboratory. The non-immunosuppressive cyclosporin derivative SDZ PSC833 (PSC) has been shown to reverse MDR completely in vitro and in vivo. The second compound is MS-209, a novel quinoline derivative. MS209 completely reversed the resistance against VCR and ADM in vitro. MS209 enhanced the chemotherapeutic effects of VCR and ADM in P388/VCR- and P388/ADM-bearing mice. MS-209 has now started clinical trials in Japan. In addition to these chemical agents, monoclonal antibodies (moAb) against P-glycoprotein such as MRK16 could be useful tools for selective killing of MDR tumor cells. Furthermore another moAb MRK17 can be used against human MDR cells transfected with macrophage-colony stimulating factor (M-CSF) gene. M-CSF can act as an enhancer of antibody dependent cellular cytotoxicity (ADCC) in therapy of human MDR cancer with the anti-P-glycoprotein antibody.     

In vitro efficacy of known P-glycoprotein modulators compared to droloxifene E and Z: studies on a human T-cell leukemia cell line and their resistant variants

P-glycoprotein(P-gp)- related resistance is one of the major obstacles in treating leukemia patients. Therefore, it is of clinical interest to find new potential modulators and compare their P-gp-modulating efficacy. The present analysis investigated the influence of P-gp modulators, such as verapamil, tamoxifen, droloxifene E, droloxifene Z, SDZ PSC 833 (PSC 833) and dexniguldipine in a leukemic T-cell line (CCRF-CEM) and its P-gp-resistant counterparts (CCRF-CEM/ACT400 and CCRF-CEM/VCR1000). P-gp expression was assessed with an immunocytological technique using the monoclonal antibody 4E3.16. It was characterized as the percentage of P-gp positive cells and also expressed as a D value by using the Kolmogorov Smirnov statistic. The efficacy of P-gp modulators was determined with the rhodamine-123 accumulation test and the MTT test. An in vitro modulator concentration between 0.1 microM and 3 microM was determined, where no genuine antiproliferative effect was apparent. The modulators PSC 833 and dexniguldipine were the significant (p相似文献   

Quality control of multidrug resistance assays in adult acute leukemia: correlation between assays for P-glycoprotein expression and activity

We have compared multiple assays for the P-glycoprotein (Pgp/MDR1) phenotype in fresh and thawed adult acute leukemia to validate and quantitate measures for the expression and function of Pgp. The results are related to the Pgp-expressing KB8 and KB8-5 call lines. The most sensitive assay was the measurement of modulation of the rhodamine 123 (R123) fluorescence by 2 micromol/L PSC833, followed by the modulation of the probe calcein-AM. We also found a good intralaboratory and interlaboratory correlation between the values of the R123/PSC833 assay for fresh as well as thawed samples. In addition, the affects of PSC833 on 3H-daunorubicin (DNR) accumulation, DNR fluorescence, and 3H-vincristine accumulation were very similar. The correlation between the DNR/PSC833 and R123/PSC833 test was r = .86 (N = 51). The modulation of drug accumulation by 8 micromol/L verapamil was the some as the PSC833 effect for DNR (117%, N = 21), but was higher for vincristine in every single case (161% v 121%, N = 22; P< .001), indicating additional verapamil effects, not related to Pgp. The correlation of the staining of viable cells for Pgp with the monoclonal antibody MRK16 was r = .77 (N = 52) for the R123/PSC833 functional test and r = .84 (N = 50) for the DNR/PSC833 test. From these results it could be calculated that a maximal increase of the mean DNR accumulation of about 50% can be achieved by blocking Pgp pump activity with PSC833 in leukemic blast samples with the highest mean Pgp expression. Subpopulations of blast calls with higher Pgp activity are likely to be present. Their relevance has to be studied further. The methods outlined here allow the reliable, quantitative monitoring of the Pgp/MDR1 phenotype in leukemias in multicentered, clinical Pgp modulation studies.     

Treatment of refractory and relapsed acute myelogenous leukemia with combination chemotherapy plus the multidrug resistance modulator PSC 833 (Valspodar)

A potential mechanism of chemotherapy resistance in acute myeloid leukemia (AML) is the multidrug resistance (MDR-1) gene product P-glycoprotein (P-gp), which is often overexpressed in myeloblasts from refractory or relapsed AML. In a multicenter phase II clinical trial, 37 patients with these poor risk forms of AML were treated with PSC 833 (Valspodar; Novartis Pharmaceutical Corporation, East Hanover, NJ), a potent inhibitor of the MDR-1 efflux pump, plus mitoxantrone, etoposide, and cytarabine (PSC-MEC). Pharmacokinetic (PK) interactions of etoposide and mitoxantrone with PSC were anticipated, measured in comparison with historical controls without PSC, and showed a 57% decrease in etoposide clearance (P =.001) and a 1.8-fold longer beta half-life for mitoxantrone in plasma (P <.05). The doses of mitoxantrone and etoposide were substantially reduced to compensate for these interactions and clinical toxicity and in Cohort II were well tolerated at dose levels of 4 mg/m2 mitoxantrone, 40 mg/m2 etoposide, and 1 g/m2 C daily for 5 days. Overall, postchemotherapy marrow hypoplasia was achieved in 33 patients. Twelve patients (32%) achieved complete remission, four achieved partial remission, and 21 failed therapy. The PK observations correlated with enhanced toxicity. The probability of an infectious early death was 36% (4 of 11) in patients with high PK parameters for either drug versus 5% (1 of 20) in those with lower PK parameters (P =.04). P-gp function was assessed in 19 patients using rhodamine-123 efflux and its inhibition by PSC. The median percentage of blasts expressing P-gp was increased (49%) for leukemic cells with PSC-inhibitable rhodamine efflux compared with 17% in cases lacking PSC-inhibitable efflux (P =.004). PSC-MEC was relatively well tolerated in these patients with poor-risk AML, and had encouraging antileukemic effects. The Eastern Cooperative Oncology Group is currently testing this regimen versus standard MEC chemotherapy in a phase III trial, E2995, in a similar patient population.     

Modulation of the tumor disposition of vinca alkaloids by PSC 833 in vitro and in vivo

PSC 833, a nonimmunosuppressive cyclosporin, is able to inhibit the efflux of antitumor drugs mediated by P-glycoprotein (P-gp). The purpose of the present study is to compare the effect of PSC 833 on the tumor disposition of [3H]vincristine ([3H]VCR) and [3H]vinblastine ([3H]VBL) in in vitro and in vivo experiments from a pharmacokinetic point of view. In in vitro experiments, the effect of PSC 833 was investigated on the cellular uptake of [3H]VCR and [3H]VBL by HCT-15 and COLO 205, human colorectal tumor cell lines with extensive and minimal expression of P-gp, respectively. PSC 833 (2 microM) increased the cellular uptake of [3H]VCR and [3H]VBL by HCT-15 cells, but not that by COLO 205 cells, 8- and 6-fold, respectively, without affecting the initial influx rates. In addition, 2 microM PSC 833 reduced the efflux of [3H]VCR from HCT-15 cells to a level comparable with that from COLO 205 cells. Furthermore, the effect of PSC 833 on the tumor disposition of intravenously administered [3H]VCR and [3H]VBL was studied in tumor inoculated mice. Infusion of PSC 833 (10 microg/hr/mouse) increased the HCT-15 tumor disposition of [3H]VBL and [3H]VCR in vivo to a level comparable with that observed in vitro. These findings demonstrate that PSC 833 enhances the tumor disposition of vinca alkaloids by inhibition of P-gp-mediated efflux not only in vitro but also in vivo in a solid tumor model.     

pH-dependent growth retardation of Escherichia coli caused by overproduction of Na+/H+ antiporter

PSC 833, a nonimmunosuppressive cyclosporin, is a potent inhibitor of the efflux of antitumor drugs mediated by P-glycoprotein and thus has been introduced in clinical trials as an agent to overcome multidrug resistance. The purpose of this study was to evaluate the dose-dependent pharmacokinetics of PSC 833 and its effects on the biliary excretion of endogenous substrates in rats. The major elimination route for PSC 833 is metabolism, followed by excretion into bile. The biliary clearance of PSC 833 was reduced in a dose-dependent manner, whereas no urinary excretion of PSC 833 was detectable. The tissue/blood concentration ratios for PSC 833 in the liver, kidney, intestine, and spleen were reduced in a dose-dependent manner, suggesting the presence of a saturable uptake process and/or saturable binding in these tissues. The dose-dependent increase in the tissue/blood concentration ratio in the brain suggests the presence of efflux transporters on the blood-brain barrier. PSC 833 reduced the bile flow rate by decreasing the biliary excretion of bile acids and reduced and oxidized glutathione, in a dose-dependent manner. The mechanism for the dose-dependent disposition of PSC 833 and its effects on the biliary excretion of endogenous substrates could be related to interactions with transporters.     

Human hepatoma cells rich in P-glycoprotein display enhanced in vitro invasive properties compared to P-glycoprotein-poor hepatoma cells

Whether the phenotypes of drug resistance and metastatic activity in cancer are dependent on each other or not is controversial. We compared in vitro invasive properties of human hepatoma cells resistant to epirubicin and rich in P-glycoprotein (Pgp) (HB8065/R) with the parental epirubicin-sensitive, Pgp-poor cells (HB8065/S). The HB8065/R cells displayed elevated capacity to migrate in a transwell chamber assay (three- to fourfold compared to the HB8065/S cells), both in the absence and presence of a reconstituted basement membrane extract (Matrigel). In the presence of the P-gp inhibitor PSC 833 (1.5 micrograms/ml) the capacity of the HB8065/R cells to cross Matrigel-coated filters was attenuated by approximately 25%. Compared to the HB8065/S cells, the resistant cell line expressed higher level of plasminogen activator inhibitor (PAI)-1 mRNA (approximately threefold), which was reflected by a approximately fivefold increase in secreted PAI-1 immunoactivity (approximately 50 ng/10(6) HB8065/R cells). Furthermore, treatment with PSC 833 was associated with upregulation of PAI-1 mRNA (approximately 3.5-fold) and immunoactivity (approximately twofold) in the HB8065/R cells. Level of tissue inhibitor of metalloproteinases (TIMP)-1 was also significantly increased in the HB8065/R cells compared to the HB8065/S cells, whereas both cell lines showed low constitutive expression of TIMP-2. Levels of TIMPs were not altered by PSC 833. These data suggest that overexpression of Pgp in these hepatoma cells may covariate with the phenotypes of both enhanced in vitro invasiveness and high PAI-1 expression, whether randomly acquired or not.     

Drug binding to P-glycoprotein is inhibited in normal tissues following SDZ-PSC 833 treatment

Rats were treated with daily injections of SDZ-PSC 833 (PSC) to study the interaction of this potent modulator of multidrug resistance (MDR) with P-glycoprotein (P-gp) expressed in normal tissues. After 2 days of treatment, the level of P-gp expression, detected by Western blot analysis, was not modified in renal brush border membranes (BBMs) and brain capillaries. However, the amount of P-gp detected with the photoaffinity probe [125I]-arylazidoprazosin (IAAP) was decreased in both tissues, suggesting that the drug binding properties of P-gp were altered by PSC treatment. This effect was further characterized by treating rats with PSC for 10 days. Following these treatments, the amount of immunodetected P-gp was increased in renal BBMs and brain capillaries. However, no increase in P-gp expression was observed in photolabeling experiments, suggesting that induced P-gp was not functional. In vitro experiments performed with renal BBMs showed that the inhibition of P-gp photolabeling by cyclosporin A (CsA), verapamil and vinblastine could be reversed by performing washing steps to remove these drugs before incubating the samples with IAAP. However, the inhibition mediated by PSC was less reversible since photolabeling of P-gp remained inhibited following the washing steps. Pre-incubation of intact CHRC5 cells with PSC, CsA and verapamil also inhibited P-gp photolabeling and increased rhodamine 123 accumulation. For PSC pre-treated samples, these effects were not completely reversed following washing, but were abolished for CsA and Ver pre-treated samples. Our results suggest that PSC could block P-gp function by a different mechanism from that of CsA and verapamil, involving modification of the drug binding sites.     

Role of P-glycoprotein in the blood-brain transport of colchicine and vinblastine

Classically, drug penetration through the blood-brain barrier depends on the lipid solubility of the substance, except for some highly lipophilic drugs, like colchicine and vinblastine, both substrates of P-glycoprotein, a drug efflux pump present at the luminal surface of the brain capillary endothelial cells. Colchicine and vinblastine uptake into the brain was studied in the rat using the in situ brain perfusion technique and two inhibitors of P-glycoprotein, verapamil and SDZ PSC-833. When rats were pretreated with PSC-833 (10 mg/kg, intravenous bolus), colchicine and vinblastine uptake was enhanced 8.42- and 9.08-fold, respectively, in all the gray areas of the rat brain studied. The mean colchicine distribution volume was increased from 0.67 +/- 0.41 to 5.64 +/- 0.70 microliters/g and vinblastine distribution volume from 2.74 +/- 1.15 to 24.88 +/- 4.03 microliters/g. When rats were pretreated with verapamil (1 mg/kg, intravenous bolus), colchicine distribution volume was increased 3.70-fold. The increase in colchicine and vinblastine did not differ between the eight brain gray areas. PSC-833 and verapamil pretreatment had no influence on the distribution volume of either drug in the choroid plexus. Nevertheless, distribution volumes remained small, considering the highly lipophilic nature of the substances. We suggest that P-glycoprotein is either only partially inhibited (difficulty of fully saturating P-glycoprotein, especially under in vivo conditions) or not the only barrier to these two drugs.