Pump P-glycoprotein Research Articles

LY294,002, a specific inhibitor of PI3K/Akt kinase pathway, antagonizes P-glycoprotein-mediated multidrug resistance

The transmembrane transport pump P-glycoprotein (P-gp) causes the efflux of chemotherapeutic agents from cells and is an important system that secures multidrug resistance (MDR) of neoplastic cells. In the present study drug sensitive L1210 and multidrug resistant L1210/VCR mouse leukemic cell lines were used as an experimental model. We found that LY 294,002, a specific inhibitor of PI3K/Akt kinase pathway, reduced the degree of vincristine resistance in L1210/VCR cells significantly and in a concentration-dependent manner. This was accompanied by decrease in IC 50 value to vincristine from 3.195 ± 0.447 to 1.898 ± 0.676 μmol/l for 2 μmol/l, to 0.947 ± 0.419 μmol/l for 4 μmol/l, and to 0.478 ± 0.202 μmol/l for 8 μmol/l LY294,002. The IC 50 value of sensitive cells for vincristine was about 0.010 μmol/l. FACS analysis of the proportion of cells in apoptosis or necrosis by annexin-V apoptosis kit showed the following: (i) vincristine-induced apoptosis in resistant cell to a much lower extent than in sensitive cells; (ii) LY294,002 alone did not induce apoptosis or necrosis in both sensitive and resistant cells; (iii) LY294,002 applied together with vincristine significantly increased the number of apoptotic cells. Transport activity of P-gp in resistant cells was monitored using calcein/AM as substrate and was depressed by LY294,002 in a concentration dependent manner. Significant differences in calcein retention were not observed when cells were preincubated with LY294,002 at different times from 0.5 to 24 h. Sensitive and resistant cells contain similar amounts of uncleaved (i.e., unactivated) caspase-3 but in latter cells the activation of caspase-3 by proteolytic cleavage was decreased. The reversal of vincristine resistance by LY294,002 was associated with marked activation of caspase-3. Western blot analysis revealed that the development of MDR phenotype in L1210/VCR cells was also associated with increased level of Bcl-2 protein. All the above findings point to the possible involvement of PI3K/Akt kinase pathway in modulation of P-gp mediated multidrug resistance in L1210/VCR mouse leukemic cell line. MDR reversal effect of LY294,002 is accompanied with this compound's influence on vincristine-induced apoptosis.

Stimulation of P-Glycoprotein ATPase by Analogues of Tetramethylrosamine: Coupling of Drug Binding at the “R” Site to the ATP Hydrolysis Transition State

The multidrug resistance efflux pump P-glycoprotein (Pgp) couples drug export to ATP binding and hydrolysis. Details regarding drug trajectory, as well as the molecular basis for coupling, remain unknown. Nearly all drugs exported by Pgp have been assayed for competitive behavior with rhodamine123 transport at a canonical "R" drug binding site. Tetramethylrosamine (TMR) displays a relatively high affinity for Pgp when compared to other rhodamines. Here, we present the construction and characterization of a library of compounds based upon the TMR scaffold and use this set to assess the determinants of drug binding to the "R" site of Pgp. This set contained modifications in (1) the number, location, and conformational mobility of hydrogen-bond acceptors; (2) the heteroatom in the xanthylium core; and (3) the size of the substituent in the 9-position of the xanthylium core. Relative specificity for coupling to the distal ATP catalytic site was assessed by ATPase stimulation. We found marked ( approximately 1000-fold) variation in the ATPase specificity constant within the library of TMR analogues. Using established methods involving ADP-Vi trapping by wild-type Pgp and ATP binding by catalytic carboxylate mutant Pgp, these effects can be extended to ATP hydrolysis transition-state stabilization and ATP occlusion at a single site. These data support the idea that drugs trigger the engagement of ATP catalytic site residues necessary for hydrolysis. Further, the nature of the drug binding site and coupling mechanism may be dissected by variation of a drug-like scaffold. These studies may facilitate development of novel competitive inhibitors at the "R" drug site.

Synthesis and structure–activity relationship studies of 4,11-diaminonaphtho[2,3- f]indole-5,10-diones

We describe the synthesis of derivatives of 4,11-diaminonaphtho[2,3- f]indole-5,10-dione and their cytotoxicity for human tumor cells that express major determinants of altered anticancer drug response, the efflux pump P-glycoprotein, and non-functional p53. Nucleophilic substitution of methoxy groups in 4,11-dimethoxynaphtho[2,3- f]indole-5,10-dione with various ethylenediamines yielded the derivatives of 4,11-diaminonaphtho[2,3- f]indole-5,10-dione, the indole containing analogues of the antitumor agent ametantrone. The cytotoxicity of novel compounds for multidrug resistant, P-glycoprotein-expressing tumor cells is highly dependent on the N-substituent at the terminal amino group of the ethylenediamine moiety. Whereas p53 null colon carcinoma cells were less sensitive to the reference drug doxorubicin than their counterparts with wild type p53, the majority of novel naphthoindole derivatives were equally potent for both cell lines, regardless of the p53 status.

What are the mechanisms associated with post-ivermectin serious adverse events?

What are the mechanisms associated with post-ivermectin serious adverse events?

Screening for the P-glycoprotein inhibitory pump activity of plant extracts that are used in Tanzanian traditional medicine

An in vitro screening experiment of 45 methanolic plant extracts was carried out to investigate the inhibition of P-glycoprotein (P-gp) in MCF-7R cells. The investigation was carried out using standard functional assay with rhodamine 6G as the fluorescent probe and reserpine, a known inhibitor of P-glycoprotein pump, was used as a reference drug. The results revealed that out of the 45 plant extracts tested, 3 extracts i.e. Bauhinia thoningii , Clerodendrum myricoides and Rhus natalensis exhibited pronounced activity at the concentration of 100 µg/ml. In comparison to the negative control, B. thoningii , C. myricoides and R. natalensis extracts inhibited the pump by a factor of about 2.5, 4 and 4.3, respectively. The remaining 42 extracts did not show any activity. The reference drug reserpine (20 μM) inhibited the pump by a factor of about 11. These preliminary results indicate that the inhibition of P-gp in MCF-7R cells is through a concerted action of compounds present in the extracts and that if isolated might be effective P-gp pump inhibitors in the treatment of cancer. Keywords : plant extract, P-glycoprotein inhibition; rhodamine 6G; MCF-7R cells East and Central African Journal of Pharmaceutical Sciences Vol. 7 2004: 16-22

Biological Evaluation of Bishydroxymethyl-Substituted Cage Dimeric 1,4-Dihydropyridines as a Novel Class of P-Glycoprotein Modulating Agents in Cancer Cells

A series of N-substituted cage dimeric 1,4-dihydropyridines 3a-e was evaluated as inhibitors of membrane efflux pump P-glycoprotein (P-gp) in multidrug resistant (mdr) cancer cells. Structure-activity relationships (SAR) and cytotoxic properties are discussed. Effective concentrations for overcoming mdr have been demonstrated in competition studies with the P-gp substrate epirubicin.

Enhanced nimodipine bioavailability after oral administration of nimodipine with morin, a flavonoid, in rabbits

The aim of this study was to investigate the effect of morin on the bioavailability of nimodipine after administering nimodipine (15 mg/kg) orally to rabbits either co-administered or pretreated with morin (2, 10 and 20 mg/kg). The plasma concentrations of nimodipine in the rabbits pretreated with morin were increased significantly (p < 0.05 at 10 mg/kg, p < 0.01 at 20 mg/kg) compared with the control, but the plasma concentrations of nimodipine co-administered with morin were not significant. The areas under the plasma concentration-time curve (AUC) and the peak concentrations (Cmax) of the nimodipine in the rabbits pretreated with morin were significantly higher (p < 0.05 at 10 mg/kg, p < 0.01 at 20 mg/kg), but only the Cmax of nimodipine coadministered with morin 10 mg/kg was increased significantly (p < 0.05). The absolute bioavailability (A.B%) of nimodipine in the rabbits pretreated with morin was significantly (p < 0.05 at 10 mg/kg, p < 0.01 at 20 mg/kg) higher (54.1-65.0%) than the control (36.7%). The increased bioavailability of nimodipine in the rabbits pretreated with morin might have been resulted from the morin, which inhibits the effiux pump P-glycoprotein and the first-pass metabolizing enzyme by cytochrome P-450 3A4 (CYP 3A4).

Tesmilifene may enhance breast cancer chemotherapy by killing a clone of aggressive, multi-drug resistant cells through its action on the p-glycoprotein pump

Tesmilifene is a novel potentiator of chemotherapy which, when added to doxorubicin, achieved an unexpected and very large survival advantage over doxorubicin alone in a randomized trial in advanced breast cancer. This trial was unusual in that the early endpoints (response rate and median progression-free survival) were equivalent in the two arms, despite the ultimate survival difference. These aspects, coupled with the absence of a coherent molecular mechanism of action, and a pending confirmatory trial, have led oncologists to hold judgement on this drug. This paper reacts to this in three ways: firstly, a forensic subgroup analysis is presented with an explanation as to why it strongly supports the veracity of the survival difference; secondly a novel cellular explanation is provided for the decoupling of the early and late (survival) endpoints; finally, a molecular mechanism of action is proposed, for the first time, which reconciles the peculiarities of the trial with the laboratory data and background literature. This hypothesis explains how tesmilifene could meld two of the apparent strengths of the cancer cell (drug resistance pumps, and hypoxia-adapted energetics) into a potent weapon of self-destruction. Tesmilifene is proposed to allow chemotherapy (e.g. anthracycline or taxane) to additionally kill a small but critical population (clone) of aggressive, multi-drug resistant cells, the benefits of which cannot be appreciated until a period of time (about 6-8 months) has elapsed. These cells, present in women with more rapidly relapsing disease, very likely carry an energy-dependent extrusion pump which is paradoxically activated by tesmilifene plus the chemotherapy. The result is that, despite the chemotherapy's remaining extracellular, the cell dies from reactive oxygen species leaking from the electron chain transport in the abnormal mitochondria which characterize cancer. These mitochondria are activated in response to the ATP cost of this pump activation, in these predominantly glycolytic cells.

Multidrug-resistant colonic cancer cell line LoVoDx is efficiently killed by lymphokine-activated killer cells from patients with carcinoma of the colon.

Multidrug-resistant (MDR+) cancer cells have the ability to grow in the presence of cytotoxic concentrations of antineoplastic drugs as a result of possessing the transmembrane drug efflux pump p-glycoprotein. The MDR+ colonic cancer cell line LoVoDx (derived from the drug-sensitive line LoVo) was tested for sensitivity to lymphokine-activated killer (LAK) cell-mediated toxicity. LAK cells were cultured from patients with colonic cancer and from matched controls with benign disorders. LAK cells from patients with cancer were as effective as those from controls in mediating cytotoxicity. The MDR+ cell line was significantly more sensitive to LAK cell-mediated cell killing than its parental drug-sensitive line LoVo (P < 0.05). These results indicate a possible role for adoptive immunotherapy in MDR+ tumours expressing p-glycoprotein.

Roles for mitochondria in pentamidine susceptibility and resistance in Leishmania donovani

Pentamidine resistant Leishmania donovani was raised in the laboratory by stepwise exposure to increasing drug pressure until a line capable of growth in 8 μM pentamidine (R8) had been selected. An IC 50 value of 40 μM was determined for this line, some 50-fold higher than that recorded for the parental wild-type line. The pentamidine resistant promastigotes were cross-resistant to other toxic diamidine derivatives but not to antimonials or substrates of multidrug resistance pumps. Decreased mitochondrial transmembrane potential was observed in pentamidine resistant promastigotes. A substantial net decrease in accumulation of [ 3H]-pentamidine accompanied the resistance phenotype. Inhibitors of P-glycoprotein pumps, including prochlorperazine and trifluoperazine, did not reverse this decreased drug uptake, which distinguishes the L. donovani resistant line studied here from L. mexicana promastigotes previously studied for pentamidine resistance. Kinetic analysis identified a carrier with an apparent K m value of 6 μM for pentamidine. No significant difference between wild-type and resistant parasites could be detected with respect to this transporter in rapid uptake experiments. However, in longer-term uptake experiments and also using concentrations of pentamidine up to 1 mM, it was demonstrated that wild-type cells, but not resistant cells, could continue to accumulate pentamidine after apparent saturation via the measured transporter had been reached. Agents that diminish the mitochondrial membrane potential inhibited this secondary route. A fluorescent analogue of pentamidine, 2,5-bis-(4-amidophenyl)-3,4-dimethylfuran (DB99), accumulated in the kinetoplast of wild-type but not resistant parasites indicating that uptake of this cationic compound into mitochondria of wild-type cells was more pronounced than in the resistant line. These data together indicate that resistance to pentamidine in L. donovani is associated with alterations to the mitochondria of the parasites, which lead to reduced accumulation of drug.

Efficacy and Safety of Ursodeoxycholic Acid Versus Cholestyramine in Intrahepatic Cholestasis of Pregnancy

Treatment of intrahepatic cholestasis of pregnancy with ursodeoxycholic acid appears promising, but data are limited so far. The aim of this randomized study was to evaluate the efficacy and safety of ursodeoxycholic acid in comparison with cholestyramine. Eighty-four symptomatic patients with intrahepatic cholestasis of pregnancy were randomized to receive either ursodeoxycholic acid, 8-10 mg/kg body weight daily (n = 42), or cholestyramine, 8 g daily (n = 42), for 14 days. The primary end point was a reduction of pruritus by more than 50% after 14 days of treatment as evaluated by a pruritus score. Secondary end points were outcome of pregnancy, reduction of serum aminotransferase activities and serum bile acid levels, and drug safety. Intention-to-treat analysis was applied. Pruritus was more effectively reduced by ursodeoxycholic acid than cholestyramine (66.6% vs 19.0%, respectively; P < .005). Babies were delivered significantly closer to term by patients treated with ursodeoxycholic acid than those treated with cholestyramine (38.7 +/- 1.7 vs 37.4 +/- 1.5 weeks, respectively, P < .05). Serum alanine and aspartate aminotransferase activities were markedly reduced by 78.5% and 73.8%, respectively, after ursodeoxycholic acid, but by only 21.4%, each, after cholestyramine therapy (P < .01 vs ursodeoxycholic acid). Endogenous serum bile acid levels decreased by 59.5% and 19.0%, respectively (P < .02). Ursodeoxycholic acid, but not cholestyramine was free of adverse effects. Ursodeoxycholic acid is safe and more effective than cholestyramine in intrahepatic cholestasis of pregnancy.

Bile Acid Transport in Sister of P-Glycoprotein (ABCB11) Knockout Mice

In vertebrates, bile flow is essential for movement of water and solutes across liver canalicular membranes. In recent years, the molecular motor of canalicular bile acid secretion has been identified as a member of the ATP binding cassette transporter (ABC) superfamily, known as sister of P-glycoprotein (Spgp) or bile salt export pump (Bsep, ABCB11). In humans, mutations in the BSEP gene are associated with a very low level of bile acid secretion and severe cholestasis. However, as reported previously, because the spgp(-)(/)(-) knockout mice do not express severe cholestasis and have substantial bile acid secretion, we investigated the "alternative transport system" that allows these mice to be physiologically relatively normal. We examined the expression levels of several ABC transporters in spgp(-)(/)(-) mice and found that the level of multidrug resistance Mdr1 (P-glycoprotein) was strikingly increased while those of Mdr2, Mrp2, and Mrp3 were increased to only a moderate extent. We hypothesize that an elevated level of Mdr1 in the spgp(-)(/)(-) knockout mice functions as an alternative pathway to transport bile acids and protects hepatocytes from bile acid-induced cholestasis. In support of this hypothesis, we showed that plasma membrane vesicles isolated from a drug resistant cell line expressing high levels of P-glycoprotein were capable of transporting bile acids, albeit with a 5-fold lower affinity compared to Spgp. This finding is the first direct evidence that P-glycoprotein (Mdr1) is capable of transporting bile acids.

Vincristine resistance development in lymphoblastoid cells under conditions of selective P-glycoprotein inhibition

Most studies looking at the reversal of drug resistance in hematological malignancies have focused on the blockade of P-glycoprotein (Pgp) pump activity, as one of the more important proteins involved in cell resistance to the chemotherapeutic agents used. In model experiments using the IM-9 lymphoblastoid cell line cultured with increasing doses of vincristine (Vcr) in the presence or absence of the Pgp blocker cyclosporin A (CsA) we established two Vcr resistant sublines. By measuring Pgp expression in target cells, and the activity and sensitivity to cytostatics we believe that our results suggest the presence of other mechanisms involved in cell resistance to xenobiotics and that these mechanisms should be considered in the development of clinical trials designed to overcome drug resistance.

Phase II and pharmacological study of oral paclitaxel plus cyclosporine A in anthracycline pretreated metastatic breast cancer

652 Purpose: Paclitaxel is an important chemotherapeutic agent for metastatic breast cancer and often used as second line treatment. Due to its high affinity for the drug efflux pump P-glycoprotein...

Dual Infection with Helicobacter bilis and Helicobacter hepaticus in P-Glycoprotein-Deficient mdr1a−/− Mice Results in Colitis that Progresses to Dysplasia

Patients with inflammatory bowel disease (IBD) are at increased risk for developing high-grade dysplasia and colorectal cancer. Animal IBD models that develop dysplasia and neoplasia may help elucidate the link between inflammation and colorectal cancer. Mdr1a-/- mice lack the membrane efflux pump p-glycoprotein and spontaneously develop IBD that can be modulated by infection with Helicobacter sp: H. bilis accelerates development of colitis while H. hepaticus delays disease. In this study, we determined if H. hepaticus infection could prevent H. bilis-induced colitis. Unexpectedly, a proportion of dual-infected mdr1a-/- mice showed IBD with foci of low- to high-grade dysplasia. A group of dual-infected mdr1a-/- animals were maintained long term (39 weeks) by intermittent feeding of medicated wafers to model chronic and relapsing disease. These mice showed a higher frequency of high-grade crypt dysplasia, including invasive adenocarcinoma, possibly because H. hepaticus, in delaying the development of colitis, allows time for transformation of epithelial cells. Colonic epithelial preparations from co-infected mice showed increased expression of c-myc (5- to 12-fold) and interleukin-1alpha/beta (600-fold) by real-time polymerase chain reaction relative to uninfected wild-type and mdr1a-/- animals. This animal model may have particular relevance to human IBD and colorectal cancer because certain human MDR1 polymorphisms have been linked to ulcerative colitis and increased risk for colorectal cancer.

Enhanced diltiazem bioavailability after oral administration of diltiazem with quercetin to rabbits

The aim of this study was to investigate the effect of quercetin on the bioavailability of diltiazem after administering diltiazem (15 mg/kg) orally to rabbits either co-administered or pretreated with quercetin (2, 10, 20 mg/kg). The plasma concentrations of diltiazem in the rabbits pretreated with quercetin were increased significantly ( p < 0.05, at 2 mg/kg; p < 0.01, at 10 and 20 mg/kg) compared with the control, but the plasma concentrations of diltiazem co-administered with quercetin were not significant. The areas under the plasma concentration-time curve (AUC) and the peak concentrations ( C max) of the diltiazem in the rabbits pretreated with quercetin were significantly higher ( p < 0.05, at 2 mg/kg; p < 0.01, at 10 and 20 mg/kg) than the control. The absolute bioavailability (AB%) of diltiazem in the rabbits pretreated with quercetin was significantly ( p < 0.05 at 2 mg/kg, p < 0.01 at 10 and 20 mg/kg) higher (9.10–12.81%) than the control (4.64%). AUC, AB% and C max of diltiazem co-administered with quercetin were higher than the control, but these were not significant. The bioavailibility of diltiazem in the rabbits pretreated with quercetin is increased significantly compared with the control, but not in the rabbits co-administered with quercetin. The increased bioavailability of diltiazem in the rabbits pretreated with quercetin might have been resulted result from the quercetin, which inhibits the efflux pump P-glycoprotein and the first-pass metabolizing enzyme CYP 3A4.

ABCB1 Genotype of the Donor but Not of the Recipient Is a Major Risk Factor for Cyclosporine-Related Nephrotoxicity after Renal Transplantation

Cyclosporine (CsA) nephrotoxicity is a severe complication in organ transplantation because it leads to impaired renal function and chronic allograft nephropathy, which is a major predictor of graft loss. Animal models and in vivo studies indicate that the transmembrane efflux pump P-glycoprotein contributes substantially to CsA nephrotoxicity. It was hypothesized that the TT genotype at the ABCB1 3435C-->T polymorphism, which is associated with decreased expression of P-glycoprotein in renal tissue, is a risk factor for developing CsA nephrotoxicity. In a case-control study, 18 of 97 patients developed CsA nephrotoxicity and showed complete recovery of renal function in all cases when switched to a calcineurin inhibitor-free regimen. Both recipients and donors were genotyped for ABCB1 polymorphisms at the positions 3435C-->T and 2677G-->T/A. For controlling for population stratification, two additional polymorphisms, CYP2D6*4 and CYP3A5*3, with intermediate allelic frequencies were studied. The P-glycoprotein low expressor genotype 3435TT only of renal organ donors but not of the recipients was overrepresented in patients with CsA nephrotoxicity as compared with patients without toxicity (chi2 = 10.5; P = 0.005). CsA dosage, trough levels, and the concentration per dose ratio were not different between the patient groups. In a multivariate model that included several other nongenetic covariates, only the donor's ABCB1 3435TT genotype was strongly associated with CsA nephrotoxicity (odds ratio, 13.4; 95% confidence interval, 1.2 to 148; P = 0.034). A dominant role of the donor's ABCB1 genotype was identified for development of CsA nephrotoxicity. This suggests that P-glycoprotein is an important factor in CsA nephrotoxicity.

Effects of miltefosine on membrane permeability and accumulation of [ 99mTc]-hexakis-2-methoxyisobutyl isonitrile, 2-[ 18F]fluoro-2-deoxy- d-glucose, daunorubucin and rhodamine123 in multidrug-resistant and sensitive cells

Miltefosine is a phospholipid analog that exhibits antineoplastic activity against breast cancer metastases, but its mechanism of action remains uncertain. The aim of this study was to investigate the transport mechanism for the removal of miltefosine and [ 99mTc]-hexakis-2-methoxyisobutyl isonitrile ( 99mTc-MIBI) from multidrug-resistant cells. The P-glycoprotein pump function, cell viability, and 99mTc-MIBI and 2-[ 18F]fluoro-2-deoxy- d-glucose ( 18FDG) uptakes were measured in NIH 3T3 (3T3) and NIH 3T3MDR1 G185 (3T3MDR1) mouse fibroblasts and human lymphoid B JY cells. Miltefosine treatment increased the permeability and fluidity of these tumor cells in a concentration-dependent manner. The multidrug-sensitive cells were 3–4 times more sensitive to miltefosine than the multidrug-resistant ones. The extent of 99mTc-MIBI accumulation in the P-glycoprotein-expressing cells increased in the presence of miltefosine, whereas the rhodamine123 and daunorubicin uptakes of the cells did not change significantly. In the 3T3MDR1 cells verapamil reinstated the rhodamine123 and daunorubicin accumulation, but not the 99mTc-MIBI uptake. Cyclosporin A reinstated the uptakes of 99mTc-MIBI, daunorubicin and rhodamine123 by the 3T3MDR1 cells. In a concentration-dependent manner miltefosine decreased the extents of 99mTc-MIBI, rhodamine123, daunorubicin and 18FDG accumulation in the JY and 3T3 cells. Our findings indicate a common transport mechanism for 99mTc-MIBI and miltefosine, which is distinct from that for rhodamine123 and daunorubicin in MDR cells.

Clinical pharmacogenetics of immunosuppressive drugs in organ transplantation

Organ transplantation has become an important additional option for patients with organ failure. Immunosuppressive drugs showing a very narrow therapeutic window have to be administered. Different transporters and metabolic pathways are responsible for absorption and metabolism of these drugs; for instance, the P-glycoprotein (P-gp) pump regulates the absorption of a drug, and its metabolism is catalyzed by cytochrome P450s (CYPs). As the phenotypes of P-gp or the CYPs are predetermined by their genotypes, genetic testing prior to drug therapy may help to predict the drug doses required. This review describes polymorphisms of the genes coding for P-gp and CYPs, and focuses on the compounds cyclosporin and tacrolimus. It is hoped that this information might help to judge the value of pharmacogenetic testing prior to immunosuppressive therapy in solid organ transplantation.

Characterisation of (R/S)-propafenone and its metabolites as substrates and inhibitors of P-glycoprotein

Digoxin is a drug with a narrow therapeutic index, which is a substrate of the ATP-dependent efflux pump P-glycoprotein. Increased or decreased digoxin plasma concentrations occur in humans due to the inhibition or induction of this drug transporter in organs with excretory function such as small intestine, liver and kidney. It is well known that serum concentrations of digoxin increase considerably in humans if propafenone is given simultaneously. However, it has not been investigated in detail whether propafenone and its metabolites are substrates and/or inhibitors of human P-glycoprotein. The aim of this study, therefore, was to investigate the P-glycoprotein-mediated transport and inhibition properties of propafenone and its major metabolites 5-hydroxypropafenone and N-desalkylpropafenone in Caco-2 cell monolayers. Inhibition of P-glycoprotein-mediated transport by propafenone and its metabolites was determined using digoxin as a P-glycoprotein substrate. No polarised transport was observed for propafenone and N-desalkylpropafenone in Caco-2 cell monolayers. However, 5-hydroxypropafenone translocation was significantly greater from basal-to-apical compared with apical-to-basal (P(app) basal-apical vs. P(app) apical-basal, 10.21+/-2.63 x 10(-6) vs. 4.34+/-1.84 x 10(-6) cm/s; P<0.01). Moreover, propafenone, 5-hydroxypropafenone and N-desalkylpropafenone inhibited P-glycoprotein-mediated digoxin transport with IC(50) values of 6.8, 19.9, and 21.3 microM, respectively. In summary, whereas propafenone and N-desalkylpropafenone are not substrates of P-glycoprotein, 5-hydroxypropafenone is translocated by human P-glycoprotein across cell monolayers. In addition, propafenone and its two major metabolites 5-hydroxypropafenone and N-desalkylpropafenone are inhibitors of human P-glycoprotein and therefore contribute to the digoxin-propafenone interaction observed in humans.