Modulation Of P-glycoprotein Function Research Articles

Reversion of resistance to immunosuppressive agents in three patients with psoriatic arthritis by cyclosporine A: Modulation of P-glycoprotein function

Secondary resistance may be a major problem in the management of autoimmune diseases. P-glycoprotein (P-gp) over-function has been described as a mechanism of drug resistance in autoimmune patients. P-gp function can in vitro be inhibited by cyclosporine A (CSA) and verapamil; moreover, P-gp reduction by CSA in systemic lupus erythematosus and rheumatoid arthritis has been demonstrated. Here, P-gp function before and after CSA administration in three psoriatic arthritis (PsA) patients, who developed a resistance to MTX/SSA, has been evaluated. P-gp function on patient cells was analyzed by measuring the changes in rhodamine-123 (Rh-123) fluorescence after verapamil incubation. CSA treatment resulted in good clinical outcome that was related with a significant P-gp function reduction at CD3+ and CD8+ levels. In addition to its immunosuppressive activity, CSA results may also be related to MTX/SSA effect restoration through P-gp inhibition. This is the first time that CSA has been demonstrated as being able to revert MTX/SSA resistance in PsA.

Modulation of P-glycoprotein function and reversal of multidrug resistance by (–)-epigallocatechin gallate in human cancer cells

Multidrug resistance (MDR) is a major obstacle in the chemotherapeutic treatment of many human cancers. In this study, the reversal of P-glycoprotein (P-gp) mediated multidrug resistance by (–)-epigallocatechin gallate (EGCG) and its molecular mechanism were investigated. A three-dimensional model of carboxyl-terminal nucleotide binding domain (NBD2) from P-gp was built by homology modeling. The structural model of the complex indicates that EGCG was tightly bound to the ATP-binding site of NBD2. EGCG modulated the function of P-gp and increased the intracellular accumulation of chemotherapeutic agent doxorubicin (DOX) in drug-resistant KB-A1 cells. When KB-A1 cells were exposed to 10 μg/ml DOX combined with 10, 30, 50 μM EGCG for 4 h, the intracellular concentrations of DOX were increased 1.5, 1.9, 2.3 times, respectively compared with DOX alone treatment. In vitro EGCG potentiated the cytotoxicity of DOX to drug-resistant KB-A1 cells. In KB-A1 cell xenograft model, EGCG could also enhance the efficacy of DOX and increased the DOX concentration in the resistant tumors. Thus, these results suggest that EGCG modulated the function of P-gp and reversed P-gp mediated multidrug resistance in human cancer cells.

Modulation of P-glycoprotein function by amlodipine derivatives in brain microvessel endothelial cells of rats1

To investigate whether the amlodipine derivatives, CJX1 and CJX2, have a modulative effect on P-glycoprotein (P-gp) function in rat brain microvessel endothelial cells (RBMEC). Isolated RBMEC were cultured in DMEM/F12 (1:1) medium. The amount of intracellular rhodamine (Rh123) was determined, using a fluorescence spectrophotometer, to evaluate the function of P-gp. The accumulation of Rh123 in RBMEC was potentiated in a concentration-dependent manner after incubation with CJX1 and CJX2 at 1, 2.5, 5, and 10 micromol/L (P<0.01), but no accumulation of Rh123 was observed in human umbilical vein endothelial cells after incubation with CJX1 and CJX2 10 micromol/L (P>0.05). Accumulation of intracellular Rh123 was increased and efflux of intracellular Rh123 was decreased in a time-dependent manner from 0-100 min after CJX1 and CXJ2 at 10 micromol/L treatment. The inhibitory effect of CJX1 and CJX2 on P-gp function was reversible and remained even at 120 min after removal of CJX1 and CJX2 at 2.5 micromol/L from the medium. CJX1 and CJX2 exhibited a potent effect in the inhibition of P-gp function in vitro.

Modulation of P-Glycoprotein Function by St John’s Wort Extract and its Major Constituents

Recent data suggest some relevant drug interactions caused by St John's wort extract, which can be explained by interactions with the Cytochrome P450 system or P-Glycoprotein (Pgp). Interaction with Pgp, including activation, inhibition and induction, can lead to altered plasma or brain levels of Pgp substrates. The aim of the present study was to investigate the possible interactions of St John's wort extract and most relevant constituents with the transport activity of Pgp. We characterized the modulatory potencies in two in vitro assays using calcein-AM, first in VLB cells (a human lymphocytic leukemia cell line expressing Pgp) and second in PBCEC cells (porcine brain capillary endothelial cells). The extract, as well as some of the tested constituents modulate the transport by Pgp in the micromolecular range. Quercetin and hyperforin seem to be most potent. These findings suggest the possibility of drug interactions at the level of the gastro-intestinal absorption of drugs. Plasma levels of the constituents of St John's wort are very likely too low to interfere with Pgp at the blood-brain-barrier with the possible exception of quercetin.

Enhanced cerebral uptake of receptor ligands by modulation of P-glycoprotein function in the blood-brain barrier.

Low cerebral uptake of some therapeutic drugs can be enhanced by modulation of P-glycoprotein (P-gp), an ATP-driven drug efflux pump at the blood-brain barrier (BBB). We investigated the possibility of increasing cerebral uptake of the beta-adrenergic ligands S-1'-[(18)F]-fluorocarazolol (FCAR) and [(11)C]-carazolol (CAR) in P-gp knockout mice (mdr1a (-/-)) and by modulation of P-gp with cyclosporin A (CsA) in rats. Specific and nonspecific binding of FCAR in the rat brain were doubled by CsA, while target/nontarget ratios and clearance from plasma (area under curve (AUC)) were not affected. Cerebral uptake of CAR in rats was much lower than FCAR and nonspecific. CsA increased this uptake 5-6-fold, not only due to P-gp modulation in the BBB but also to a 2-fold higher plasma AUC. In the CNS of mdr1a (-/-) mice, uptake of FCAR and CAR was, respectively, 2-fold and 3-fold higher than in mdr1a (+/+) mice. These results indicate that the cerebral uptake of beta-adrenoceptor ligands can be increased by administration of P-gp modulators such as CsA without affecting regional distribution in the brain. P-gp modulation could improve the count statistics in PET studies of the CNS.

Multidrug Resistance Phenotype and Paclitaxel (Taxol®) Sensitivity in Human Renal Carcinoma Cell Lines of Different Histologic Types

We compared the effects of paclitaxel (Taxol®) in human renal cell carcinoma (RCC) of different histologic types. The growth inhibiotory effects of paclitaxel on 34 human RCC cell lines of strictly defined different histologic types were determined by 3–[4,5-dimethylthiazilyl]-2,5-diphenyltetrazoliumbromide (MTT) assays. Paclitaxel-induced morphologic alterations were visualized by light and immunofluorescence and by transmission electron microscopy. The expression and function of P-glyco-protein arid multidrug resistance-associated protein (MRP) were defined by reverse transcriptase polytnercise chain reaction and fluorescence-activated cell sorting (FACS) analysis, respectively. Modulation of P-glycoprotein function was performed by verapamil or Cretnophor EL. A significant (p < 0.05') dose-dependent paclitaxel-induced growth inhibition could be demonstrated in all cell lines, with the effects of paclitaxel dissolved in Cremophor® EL/ethanol (= Taxol®) exceeding the effects of piclitaxel dissolved in dimethyl sulfoxide. The extent of response markedly varied between the different cell lines, although chromophilic RCCs exhibited a more pronounced response to Taxol (IC50: 0.03–0.38 μM) than clear cell RCCs (IC50,: 0.01–36.69 μM). Exposure to paclitaxe/Taxol® induced an increuse of micro-tubule bundles in the clear cell and the chromophobe RCCs but not in the chromophilic RCCs. The expression of the MRP was low in RCC cell lines and was not found to be related to paclitaxel/Taxol® sensitivity. In contrast, the expression level of P-glycoprotein was much more pronounced and showed a positive correlation (p < 0.05) with the response to paditaxel. Reversal of P-glycoprotein function by verapamil or Cremophor® EL enhanced the growth inhibitory effects of paclitaxel and further supported the role of P-glycoprotein for paclitaxel sensitivity of human RCCs. Paclitaxel/Taxol® effktively inhibits proliferation of human RCCs in vitro, irrespective of their histologic types. Moreover, expression and function of P-glycoprotein markedly contribute to paclitaxel responsiveness, although other as yet undefined drug resistance mechanisms are effective in humun RCCs us well.

Modulation of P-glycoprotein function in human lymphocytes and Caco-2 cell monolayers by HIV-1 protease inhibitors.

To determine the effect of the protease inhibitors ritonavir, nelfinavir and indinavir on the P-glycoprotein (P-gp)-mediated transport of saquinavir in Caco-2 cell monolayers. To study the modulation of P-gp function in human lymphocytes by saquinavir, ritonavir, nelfinavir and indinavir. We examined the effect of the protease inhibitors on P-gp function in human lymphocytes by using Rhodamine 123 (Rh 123; a fluorescent substrate of P-gp) by flow cytometry. Efflux of Rh 123 correlates with P-gp function and inhibition of P-gp results in dye retention. Verapamil, a P-gp modulator and inhibitor of active transport at 4 degrees C was used as a positive control. The transport of [14C]saquinavir (1 microM) across Caco-2 cell monolayers was investigated, alone and in the presence of verapamil and ketoconazole (500 microM) and the protease inhibitors at 100 microM. Caco-2 cells are an in vitro model of the intestinal epithelium that is widely used for the study of P-gp function. The transport of saquinavir was determined in both the apical to basolateral (AP-BL) and basolateral to apical (BL-AP) directions. Saquinavir and ritonavir (10 microM) markedly inhibited Rh 123 efflux with an increase in fluorescence intensity similar to that obtained with verapamil. A small but statistically significant increase in fluorescence intensity was observed with nelfinavir; however indinavir did not modulate Rh 123 efflux. In Caco-2 cells the apparent permeability coefficient for BL-AP efflux of saquinavir exceeded that for AP-BL efflux by a factor of 26: this is indicative of an active efflux pump. Known P-gp modulators caused a decrease in BL-AP efflux and an increase in AP-BL transport. The protease inhibitors displayed some P-gp modulation with ritonavir having the most potent effect. We have demonstrated that saquinavir is a substrate for P-gp and that ritonavir, nelfinavir and indinavir modulate P-gp function in both human lymphocytes and Caco-2 cells.

Suppression of multi-drug resistance gene expression in the mouse liver by 1,4-bis[2,(3,5-dichloropyridyloxy)]benzene.

P-glycoproteins encoded by the (multi-drug resistance) mdr genes play a central role in the resistance of tumor cells to a wide range of anti-cancer drugs. Modulation of P-glycoprotein function could therefore provide a means of sensitising tumor cells to chemotherapy. Studies in this context have centred around the use of compounds which antagonise the P-glycoprotein membrane transport system. To investigate the possibility of modulating P-glycoprotein expression at a transcriptional level, we investigated the effects of hormonal factors and cytochrome P450-inducing agents on hepatic expression of murine mdr 1, mdr 2 and mdr 3. Hepatic mdr 2 and mdr 3 expressions were significantly suppressed in hypophysectomised animals, indicating that pituitary hormones activate the hepatic expression of these genes. Many of the foreign compounds and anti-cancer drugs tested did not significantly induce mdr 1, 2 or 3 expression. However, it was of particular interest that a potent cytochrome P450 inducer, 1,4-bis[2-(3,5-dichloropyridyloxy)]benzene, almost completely suppressed hepatic mdr 2 and 3 expressions.

Cytokine modulation of P-glycoprotein function in human lymphocytes

Cytokine modulation of P-glycoprotein function in human lymphocytes

Clinical trials of modulation of multidrug resistance. Pharmacokinetic and pharmacodynamic considerations.

A growing body of evidence indicates that expression of the mdr1 gene, which encodes the multidrug transporter, P-glycoprotein, contributes to chemotherapeutic resistance of human cancers. Expression of this protein in normal tissues such as the biliary tract, intestines, and renal tubules suggests a role in the excretion of toxins. Modulation of P-glycoprotein function in normal tissues may lead to decreased excretion of drugs and enhanced toxicities. A clinical trial of etoposide with escalating doses of cyclosporine (CsA) as a modulator of multidrug resistance was performed. CsA was delivered as a 2-hour loading dose followed by a 60-hour intravenous infusion, together with etoposide administered as a short infusion daily for 3 days. Patients received one or more courses of etoposide alone before the combined therapy to establish their clinical resistance to etoposide and to study etoposide pharmacokinetics without and then with CsA. Plasma and urinary etoposide was measured by high-performance liquid chromatography and plasma CsA by a nonspecific immunoassay. Conclusions from the initial phase I trial with the use of CsA as a modulator of etoposide are: (1) Serum CsA steady-state levels of up to 4800 ng/ml (4 microM) could be achieved with acceptable toxicity. (2) Toxicities caused by the combined treatment included increased nausea and vomiting, increased myelosuppression, and hyperbilirubinemia, consistent with modulation of P-glycoprotein function in the blood-brain barrier, hematopoietic stem cell, and biliary tract. Renal toxicity was uncommon, but severe in two patients with steady-state plasma CsA levels above 6000 ng/ml. (3) CsA administration had a marked effect on the pharmacokinetics of etoposide, with a doubling of the area under the concentration-time curve as a result of both decreased renal and nonrenal clearance, necessitating a 50% dose reduction in patients with normal renal function and hepatic function. (4) The recommended dose of CsA is a 6-7 mg/kg loading dose administered as a 2-hour intravenous infusion followed by a continuous infusion of 18-21 mg/kg/day for 60 hours with adjustments in the infusion rate to maintain steady-state serum levels of 3000-4800 ng/ml (2.5-4.0 M). We are performing additional phase I trials combining CsA with single-agent doxorubicin and taxol, and the CsA analog PSC-833 with various multidrug-resistant-related cytotoxins.

Differential modulation of P-glycoprotein transport by protein kinase inhibition

Previous studies of P-glycoprotein have demonstrated that its function can be modulated by phosphorylation. In the present study, inhibition of protein kinase C with calphostin C or stauroporine or prolonged treatment with the phorbol ester TPA decreased phosphorylation of P-glycoprotein, and impaired transport of vinblastine. Calphostin C also inhibited transport of actinomycin D, vincristine, rhodamine, and azidopine in SW620 Ad300 multidrug-resistant human colon carcinoma cells. Photoaffinity labeling of P-glycoprotein with azidopine was decreased by calphostin C, suggesting that dephosphorylation alters the affinity of P-glycoprotein for its substrates. Impaired transport of rhodamine in normal T lymphocytes treated with staurosporine demonstrates that modulation of P-glycoprotein function is not limited to cells selected for drug resistance in vitro. Transport of P-glycoprotein antagonists in SW620 Ad300 cells was also affected by calphostin C. Cyclosporin A transport decreased, while verapamil transport increased. Cyclosporin A in calphostin C-treated cells resulted in additive P-glycoprotein antagonism, while no additive effect could be demonstrated with verapamil, suggesting that the increase in verapamil transport makes it a poorer P-glycoprotein antagonist. These studies suggest that transport by P-glycoprotein is a dynamic process which can be modulated by phosphorylation, and that antagonists may block P-glycoprotein differently in different phosphorylation states.