P-glycoprotein Inhibitor Cyclosporine Research Articles

Clinical Drug-Drug Interaction Studies to Evaluate the Effects of a P-Glycoprotein Inhibitor, CYP3A Inhibitors, and a CYP3A Inducer on the Pharmacokinetics of Naldemedine in Healthy Subjects.

BackgroundNaldemedine is a peripherally acting μ-opioid receptor antagonist that is indicated to treat opioid-induced constipation.ObjectivesTo assess the potential for drug-drug interactions between a single oral dose of naldemedine and the oral P-glycoprotein inhibitor cyclosporine, cytochrome P450 (CYP) 3A inhibitors itraconazole and fluconazole, and CYP3A inducer rifampin.MethodsThree Phase 1, open-label studies were conducted in healthy subjects. In the P-glycoprotein inhibitor study, subjects received naldemedine 0.4 mg alone or coadministered with cyclosporine 600 mg. In the CYP3A inhibitors study, subjects in separate cohorts received naldemedine 0.2 mg alone or with itraconazole or fluconazole. In the CYP3A inducer study, subjects received naldemedine 0.2 mg alone or with rifampin 600 mg. Geometric mean ratios and 90 % confidence intervals were used to evaluate the effects of coadministered drugs on naldemedine maximum plasma concentration (Cmax) and the area under the concentration-time curve (AUC). Safety assessments included occurrence of adverse events (AEs), laboratory parameters, vital signs, and electrocardiography results.ResultsA total of 56 subjects were enrolled (n = 14 in each cohort). Cyclosporine increased naldemedine AUC0–inf 1.78-fold and Cmax 1.45-fold. Itraconazole and fluconazole increased naldemedine AUC0–inf 2.91-fold and 1.90-fold, and Cmax 1.12-fold and 1.38-fold, respectively. Rifampin decreased naldemedine AUC0–inf by 83% and Cmax by 38%. Across studies, AEs were generally mild. Laboratory, vital sign, or electrocardiogram assessments produced no clinically significant findings.ConclusionsCoadministration of naldemedine with a P-glycoprotein inhibitor or a strong/moderate CYP3A inhibitor increases naldemedine exposure; coadministration with a strong CYP3A inducer decreases its exposure. Coadministration of naldemedine with cyclosporine, itraconazole, fluconazole, or rifampin was generally safe and well tolerated.

Development of Positron Emission Tomography Radiotracers for the GABA Transporter 1.

In vivo positron emission tomography (PET) imaging of the γ-aminobutyric acid (GABA) receptor complex has been accomplished using radiolabeled benzodiazepine derivatives, but development of specific presynaptic radioligands targeting the neuronal membrane GABA transporter type 1 (GAT-1) has been less successful. The availability of new structure-activity studies of GAT-1 inhibitors and the introduction of a GAT-1 inhibitor (tiagabine, Gabatril) into clinical use prompted us to reinvestigate the syntheses of PET ligands for this transporter. Initial synthesis and rodent PET studies of N-[11C]methylnipecotic acid confirmed the low brain uptake of that small and polar molecule. The common design approach to improve blood-brain barrier permeability of GAT-1 inhibitors is the attachment of a large lipophilic substituent. We selected an unsymmetrical bis-aromatic residue attached to the ring nitrogen by a vinyl ether spacer from a series recently reported by Wanner and coworkers. Nucleophilic aromatic substitution of an aryl chloride precursor with [18F]fluoride was used to prepare the desired candidate radiotracer ( R, E/ Z)-1-(2-((4-fluoro-2-(4-[18F]fluorobenzoyl)styryl)oxy)ethyl)piperidine-3-carboxylic acid (( R, E/ Z)-[18F]10). PET studies in rats showed no brain uptake, which was not altered by pretreatment of animals with the P-glycoprotein inhibitor cyclosporine A, indicating efflux by Pgp was not responsible. Subsequent PET imaging studies of ( R, E/ Z)-[18F]10 in rhesus monkey brain showed very low brain uptake. Finally, to test if the free carboxylic acid group was the likely cause of poor brain uptake, PET studies were done using the ethyl ester derivative of ( R, E/ Z)-[18F]10. Rapid and significant monkey brain uptake of the ester was observed, followed by a slow washout over 90 min. The blood-brain barrier permeability of the ester supports a hypothesis that the free acid function limits brain uptake of nipecotic acid-based GAT-1 radioligands, and future radiotracer efforts should investigate the use of carboxylic acid bioisosteres.

Cyclosporine-inhibitable cerebral drug transport does not influence clinical methadone pharmacodynamics.

Interindividual variability and drug interaction studies suggest that blood-brain barrier drug transporters mediate human methadone brain biodistribution. In vitro and animal studies suggest that methadone is a substrate for the efflux transporter P-glycoprotein, and that P-glycoprotein-mediated transport influences brain access and pharmacologic effect. This investigation tested whether methadone is a transporter substrate in humans [corrected]. Healthy volunteers received oral (N=16) or IV (N=12) methadone in different crossover protocols after nothing (control) or the validated P-glycoprotein inhibitor cyclosporine (4.5 mg/kg orally twice daily for 4 days, or 5 mg/kg IV over 2 h). Plasma and urine methadone and metabolite concentrations were measured by mass spectrometry. Methadone effects were measured by miosis and thermal analgesia (maximally tolerated temperature and verbal analog scale rating of discreet temperatures). Cyclosporine marginally but significantly decreased methadone plasma concentrations and apparent oral clearance, but had no effect on methadone renal clearance or on hepatic N-demethylation. Cyclosporine had no effect on miosis or on R-methadone concentration-miosis relationships after either oral or IV methadone. Peak miosis was similar in controls and cyclosporine-treated subjects after oral methadone (1.4±0.4 and 1.3±0.5 mm/mg, respectively) and IV methadone (3.1±1.0 and 3.2±0.8 mm, respectively). Methadone increased maximally tolerated temperature, but analgesia testing was confounded by cyclosporine-related pain. Cyclosporine did not affect methadone pharmacodynamics. This result does not support a role for cyclosporine-inhibitable transporters mediating methadone brain access and biodistribution.

The P-glycoprotein inhibitor cyclosporin A differentially influences behavioural and neurochemical responses to the antidepressant escitalopram

Recent studies have raised the possibility that P-glycoprotein (P-gp) inhibition may represent a putative augmentation strategy for treatment with certain antidepressants. Indeed, we have previously shown that administration of the P-gp inhibitor verapamil increased the brain distribution and behavioural effects of the antidepressant escitalopram. The aim of the current study was to investigate if similar effects occur with another P-gp inhibitor, cyclosporin A (CsA). CsA pre-treatment exacerbated the severity of behaviours in an escitalopram-induced mouse model of serotonin syndrome, a potentially life-threatening adverse drug reaction associated with serotonergic drugs. P-gp inhibition by CsA enhanced the brain distribution of escitalopram by 70–80%. Serotonin (5-HT) turnover in the prefrontal cortex was reduced by escitalopram, and this effect was augmented by CsA. However, CsA pre-treatment did not augment the effect of escitalopram in the tail suspension test (TST) of antidepressant-like activity. Microdialysis experiments revealed that pre-treatment with CsA failed to augment, but blunted, the increase in extracellular 5-HT in response to escitalopram administration. This blunting effect may contribute to the lack of augmentation in the TST. Taken together, the present studies demonstrate that co-administration of CsA and escitalopram produces differential effects depending on the behavioural and neurochemical assays employed. Thus, the results highlight the need for further studies involving more selective pharmacological tools to specifically evaluate the impact of P-gp inhibition on behavioural responses to antidepressants which are subject to efflux by P-gp.

Cyclosporine-inhibitable Blood–Brain Barrier Drug Transport Influences Clinical Morphine Pharmacodynamics

The blood-brain barrier is richly populated by active influx and efflux transporters influencing brain drug concentrations. Morphine, a drug with delayed clinical onset, is a substrate for the efflux transporter P-glycoprotein in vitro and in animals. This investigation tested whether morphine is a transporter substrate in humans. Fourteen healthy volunteers received morphine (0.1 mg/kg, 1-h IV infusion) in a crossover study without (control) or with the infusion of validated P-glycoprotein inhibitor cyclosporine (5 mg/kg, 2-h infusion). Plasma and urine morphine and morphine glucuronide metabolite concentrations were measured by mass spectrometry. Morphine effects were measured by miosis and analgesia. Cyclosporine minimally altered morphine disposition, increasing the area under the plasma morphine concentration versus time curve to 100 ± 21 versus 85 ± 24 ng/ml·h (P < 0.05) without changing maximum plasma concentration. Cyclosporine enhanced (3.2 ± 0.9 vs. 2.5 ± 1.0 mm peak) and prolonged miosis, and increased the area under the miosis-time curve (18 ± 9 vs. 11 ± 5 mm·h), plasma effect-site transfer rate constant (k(e0), median 0.27 vs. 0.17 h(-1)), and maximum calculated effect-site morphine concentration (11.5 ± 3.7 vs. 7.6 ± 2.9 ng/ml; all P < 0.05). Analgesia testing was confounded by cyclosporine-related pain. Morphine is a transporter substrate at the human blood-brain barrier. Results suggest a role for P-glycoprotein or other efflux transporters in brain morphine access, although the magnitude of the effect is small, and unlikely to be a major determinant of morphine clinical effects. Efflux may explain some variability in clinical morphine effects.

Transport and metabolism of some cationic ubiquinone antioxidants (MitoQn) in Caco-2 cell monolayers

MitoQn are mitochondria-targeted antioxidants with structures linking a triphenyphosphonium cation to an ubiquinone moiety by a linear n-carbon alkyl chain. The antioxidant efficacy of MitoQn has been shown to be optimum when n = 10 but little is known about the relative transport and metabolism of these homologues. The present study examined the absorptive and secretory transport and metabolism of MitoQn (n = 3, 5 and 10) in Caco-2 cell monolayers. During absorptive transport in the apical-to-basolateral (AB) direction, intracellular accumulation was found to be proportional to lipophilicity but permeation (PappAB) was not, being high for MitoQ3 and low for MitoQ5 and MitoQ10. Secretory transport was greater than absorptive transport with efflux ratios (PappBA/PappAB) for n = 3, 5 and 10 of 2.3, 24.9 and 4.0, respectively. In the presence of the P-glycoprotein inhibitor cyclosporine A (CsA) 30 microM, PappAB values for n = 3, 5 and 10 were increased by 12, 195% and 30%, respectively whereas PappBA values were decreased by 81%, 61% and 68% respectively. In the presence of protein (4% bovine serum albumin) on the B side, PappAB of MitoQ10 (log P 3.44) increased 9-fold whereas PappAB of MitoQ5 (log P 1.14) remained unchanged, both with no change in permeability to the paracellular probe, mannitol. During transport, metabolism to the corresponding reduced ubiquinol species and their sulfate and glucuronide conjugates was detected by liquid chromatography tandem mass spectrometry. In conclusion, the permeation of these cationic ubiquinone antioxidants in Caco-2 cell monolayers depends on a balance between lipophilicity, transporter affinity, protein binding and affinity for phase 2 metabolizing enzymes.

PET-Evaluated Transport of [11C]Hydroxyurea Across the Rat Blood-Brain Barrier - Lack of Influence of Cyclosporin and Probenecid

The transport of hydroxyurea, a ribonucleoside reductase inhibitor, over biological membranes is slow and it has therefore been suggested that the substance could interact with an active efflux transporter. The transport of [(11)C]hydroxyurea into the rat brain was therefore studied after administration of the multidrug resistance protein inhibitor probenecid (50 and 150 mg/kg), the P-glycoprotein inhibitor cyclosporin A (25 mg/kg), hydroxyurea (50, 150 and 450 mg/kg) and mannitol (25%). None of the intervention drugs affected the brain uptake of [(11)C]hydroxyurea. The brain-to-plasma concentration ratios (K(p)), with or without intervention drug, were in the range 0.12-0.25 after 60 min of [(11)C]hydroxyurea infusion. [(11)C]Verapamil, a P-glycoprotein substrate with low brain penetration, was used to study the ability of hydroxyurea to inhibit P-glycoprotein. Administration of hydroxyurea (150 and 450 mg/kg) did not increase brain concentrations of [(11)C]verapamil. It is therefore unlikely that hydroxyurea is a substrate for or an inhibitor of P-glycoprotein or a substrate for a probenecid sensitive transport system. The low brain concentrations may instead be the result of slow uptake due to the hydrophilic nature of hydroxyurea.

Assessing neuroprotective P-glycoprotein activity at the blood-brain barrier in killifish (Fundulus heteroclitus) using behavioural profiles

The neuroprotective role of P-glycoprotein, a multixenobiotic resistance transporter (ABCB1/MDR1), in the blood-brain barrier in fish was examined using behavioural toxicological assays. P-glycoprotein acts as cellular efflux pump to prevent substrates from accumulating in the brain, including environmental contaminants such as ivermectin, a common aquaculture pesticide and mammalian anti-parasitic drug. The behavioural toxicological assays were developed to determine the neuropathological effect of ivermectin in killifish (Fundulus heteroclitus). P-glycoprotein function and thus blood-brain barrier integrity can be compromised by chemosensitizers that inhibit transport activity. Fish treated with ivermectin and the P-glycoprotein inhibitor cyclosporin A were significantly more sensitive and succumbed more rapidly to tilting, lethargy, slowing of pectoral-fin movement and loss of haptic-reactivity compared to fish treated with ivermectin-only. P-glycoprotein inhibition is associated with significantly earlier onset and increased mortality in ivermectin-exposed fish. Our results suggest that P-glycoprotein confers resistance against ivermectin-induced behavioural neuropathology and mortality in fish. This assay provides us with a non-invasive tool to study P-glycoprotein function in the blood-brain barrier and evaluate the behavioural effects of potential environmental neurotoxins.

Genetic variants of the P-glycoprotein gene Abcb1b modulate opioid-induced hyperalgesia, tolerance and dependence

Opioid-induced hyperalgesia (OIH) is a state of paradoxically increased nociceptive sensitivity seen in both humans and rodents following the resolution of the acute opioid antinociceptive effects or during periods of chronic opioid administration. Using the power of genetic analysis, we hoped to discover novel mechanisms modulating this trait. The degree of opioid-induced hyperalgesia displayed in response to a thermal stimulus applied to the hind paw was measured in 16 strains of inbred mice after 4 days of morphine administration. The degree of thermal sensitization was then used in a recently developed in silico haplotypic mapping algorithm along with a haplotypic map constructed from a database containing 209,000 single nucleotide polymorphisms. Analysis of the data resulted in the identification of several haplotype blocks strongly associated with the thermal opioid-induced hyperalgesia trait. The most strongly associated block was located within the Abcb1b P-glycoprotein drug transporter gene. Experiments using the P-glycoprotein inhibitor cyclosporine A and P-glycoprotein null mutant mice supported the hypothesis that a functional association exists between P-glycoprotein transporters and opioid-induced hyperalgesia. The observation of a correlation between morphine brain concentrations and the development of opioid-induced hyperalgesia was consistent with this hypothesis as well. In addition, P-glycoprotein gene deletion and pharmacological inhibition altered morphine ED50, tolerance and physical dependence. We conclude that the use of haplotypic mapping to identify novel mechanisms controlling complex traits is a viable approach. Variants of the Abcb1b gene may explain some portion of the interstrain differences in OIH and perhaps other consequences of chronic opioid administration.

Influence of P-glycoprotein inhibition on the distribution of the tricyclic antidepressant nortriptyline over the blood-brain barrier

The distribution of the antidepressant drug nortriptyline (NT) and its main metabolite E-10-hydroxy-nortriptyline (E-10-OH-NT) across the blood-brain barrier was considered in relation to inhibition of the multidrug transporter P-glycoprotein (P-gp). Rats received NT in doses of 25 mg/kg orally, 10 mg/kg i.p. or 25 mg/kg i.p. Half the rats were treated with the P-glycoprotein inhibitor cyclosporine A (CsA) (200 mg/kg) 2 h prior to NT administration, and the other half served as a control group. NT and the metabolite were extracted from brain and serum by liquid-liquid extraction and analysed by HPLC with UV-detection. The brain to serum ratio of NT was increased in the CsA treated groups (22.3-26.8) compared with the control groups (16.5-22.7), the difference being statistically significant in two of the three experiments (p<0.05). Increased brain-serum ratios were also found for E-10-OH-NT, but the differences were not statistically significant. These results suggest that inhibition of P-gp by CsA increases the accumulation of NT in the brain. Administration of the antipsychotic drug risperidone (0.5 mg/kg s.c.), which is a P-gp substrate, instead of CsA did not exert any measurable influence on the blood-brain ratio of NT concentrations. In conclusion, the results show that drug-drug interaction at P-gp may influence the intracerebral NT concentration, but apparently, a major inhibition of P-gp is necessary to attain a measurable effect.

209. P-glycoprotein limits activation of the glucocorticoid receptor in placental BeWo cells

Inadequate placental growth and function are key determinants of fetal growth retardation. Glucocorticoids potently inhibit fetal and placental growth via activation of the glucocorticoid receptor (GR). Placental and fetal glucocorticoid exposure is minimised by the ‘placental glucocorticoid barrier’, which consists primarily of placental 11β-hydroxysteroid dehydrogenase type 2 (11β-HSD2) converting maternal glucocorticoids to inactive metabolites. Recent studies in the rodent brain show that P-glycoprotein (P-gp) is also an important physiological regulator of glucocorticoid access to the GR in target cells. Therefore, we hypothesised that placental P-gp may serve to exclude maternal glucocorticoids from the placenta and fetus, and thereby augment the barrier. We have used the placental choriocarcinoma cell line BeWo, and MDR-BeWo, a daughter cell line virally transduced with P-gp, to assess whether P-gp regulates access of dexamethasone to the GR. Quantitative PCR analysis showed that MDR-BeWo cells express ~10-fold higher levels of P-gp mRNA than BeWo cells. Syncytialisation of BeWo and MDR-BeWo cells with 20μM forskolin also increased P-gp mRNA by ~7-fold in each cell line. The elevated P-gp expression in MDR-BeWo cells resulted in a reduced activation of the GR with 1μM dexamethasone by ~50% (P&lt;0.001) in comparison to BeWo cells. Accordingly, dexamethasone-induced apoptosis was reduced in MDR-BeWo cells, as indicated by a lack of induction of cleaved caspase 3 protein. Additionally, the P-glycoprotein inhibitor cyclosporin A (10μM) did not increase the level of dexamethasone-induced GR activation in the low P-gp expressing BeWo cells, but potentiated GR activation by ~2-fold in the MDR-BeWo cells, to a level comparable to that in BeWo cells. These data support the hypothesis that P-glycoprotein contributes to the placental glucocorticoid barrier. Thus, 11β-HSD2 and P-glycoprotein are likely to act in unison to reduce fetal and placental exposure to maternal glucocorticoids and minimise their growth inhibitory actions.

The effects of the cyclosporin A, a P-glycoprotein inhibitor, on the pharmacokinetics of baicalein in the rat: a microdialysis study.

1. Baicalein is a bioactive flavonoid isolated from the root of Scutellaria baicalensis Georgi, a medicinal herb that has been used since ancient times to treat bacterial infections. As little is known concerning its pharmacokinetics, this study focussed on its pharmacokinetics as well as the possible roles of the multidrug transporter P-glycoprotein on its distribution and disposition. 2. Three microdialysis probes were simultaneously inserted into the jugular vein, the hippocampus and the bile duct of male Sprague-Dawley rats for sampling in biological fluids following the administration of baicalein (10, 30 and 60 mg kg(-1)) through the femoral vein. The P-glycoprotein inhibitor cyclosporin A was used to help delineate its roles. 3. The study design consisted of two groups of six rats in parallel: control rats which received baicalein alone and the cyclosporin A treated-group in which the rats were injected cyclosporin A, a P-glycoprotein inhibitor, 10 min prior to baicalein administration. 4. Cyclosporin A treatment resulted in a significant increase in elimination half-life, mean residence time and area under the concentration versus time curve (AUC) of unbound baicalein in the brain. However, AUC in the bile was decreased. 5. The decline of baicalein in the hippocampus, blood and bile suggested that there was rapid exchange and equilibration between the peripheral compartment and the central nervous system. In addition, the results indicated that baicalein was able to penetrate the blood-brain barrier as well as undergoing hepatobiliary excretion. 6. Although no direct transport studies were undertaken and multiple factors may affect BBB penetration and hepatobiliary excretion, strong association of the involvement of P-glycoprotein in these processes is indicated.

Assessment of systemic toxicity in children receiving chemotherapy with cyclosporine for sarcoma

Background Overexpression of P-glycoprotein in malignant tumors has been associated with poor responses to chemotherapy. It appears biologically plausible that addition of the P-glycoprotein inhibitor cyclosporine (CsA) to standard chemotherapy may improve the outcome. The protective functions of P-glycoprotein in healthy tissues, however, have not been fully elucidated. Addition of CsA may lead to increased systemic chemotherapy toxicity, so we compared the rate and severity of chemotherapy-associated systemic toxicity in the presence and absence of CsA. Procedure Standard chemotherapy consisted of etoposide/ifosfamide (VP16/IFOS) cycles, alternating with vincristine/dactinomycin/cyclophosphamide (VAC) cycles. CsA was given at a median dose of 20 mg/kg with unaltered doses of the antineoplastic drugs. The analysis of toxicity was performed by comparing clinically significant toxicity events recorded during and after chemotherapy cycles with and without CsA. Results Toxicity-related hospital admissions occurred after 93% of VAC cycles with CsA compared to 40% of the cycles without CsA (P < 0.0001); 29% of VP16/IFOS cycles with CsA led to admissions vs. 12% with non-CsA cycles (P = 0.04). Infections or fever and neutropenia were the main reasons for these admissions. Thirty-seven percent of the VAC cycles with CsA were complicated by culture-proved sepsis, which did not occur in cycles without CsA (P < 0.0001). Requirements for blood and platelet transfusions were greatly increased after VAC cycles with CsA compared to VAC cycles without CsA. Conclusions The chemosensitizer CsA increases the systemic toxicity of VAC chemotherapy in patients with sarcomas. Future trials of chemotherapy with chemosensitizers will have to take into account a potential increase in systemic toxicity. Careful monitoring of chemotherapy-related toxicity becomes mandatory in such studies. Med. Pediatr. Oncol. 34:242–249, 2000. © 2000 Wiley-Liss, Inc.

Detection of P-glycoprotein in the nuclear envelope of multidrug resistant cells.

P-glycoprotein is a plasma membrane efflux pump which is responsible for multidrug resistance of many cancer cell lines. A number of studies have demonstrated the presence of P-glycoprotein molecules, besides on the plasma membrane, also in intracellular sites, such as the Golgi apparatus and the nucleus. In this study, the presence and function of P-glycoprotein in the nuclear membranes of human breast cancer cells (MCF-7 WT) and their multidrug resistant variants (MCF-7 DX) were investigated. Electron and confocal microscopy immunolabelling experiments demonstrated the presence of P-glycoprotein molecules in the nuclear membranes of MCF-7 DX cells. Moreover, the labelling pattern was strongly dependent on pH values of the incubation buffer. At physiological pH (7.2), a strong labelling was detected in the cytoplasm and the nuclear matrix in both sensitive and resistant MCF-7 cells. By raising the pH to 8.0, the P-glycoprotein molecules were easily detected in the cytoplasm (transport vesicles and Golgi apparatus), plasma and nuclear membranes exclusively in MCF-7 DX cells. Furthermore, drug uptake and efflux studies, performed by flow cytometry on isolated nuclei in the presence of the P-glycoprotein inhibitor cyclosporin A, suggested the presence of a functional P-glycoprotein in the nuclear membrane, but not in the nuclear matrix, of drug resistant cells. Therefore, P-glycoprotein in the nuclear envelope seems to represent a further defense mechanism developed by resistant cells against antineoplastic agents.

Rhodamine 123 accumulates extensively in the isolated perfused rat kidney and is secreted by the organic cation system

Rhodamine 123 has been shown to be a substrate for P-glycoprotein in multidrug resistant cells. In the present investigation the disposition of rhodamine 123 was studied in the isolated perfused rat kidney. After exposing the kidneys to perfusate concentrations ranging from 10 to 1000 ng/ml, the renal clearance was 4–1 times the clearance by glomerular filtration, respectively, indicating active and saturable secretion of rhodamine 123. The rate-limiting step in secretion was found to be membrane passage from cell to tubular lumen. Suprisingly, renal clearance was not influenced by the P-glycoprotein inhibitors cyclosporin A or digoxin. However, pretreatment of the kidneys with verapamil and quinidine (inhibitors of both P-glycoprotein and organic cation transport) or cimetidine (organic cation transport inhibitor) resulted in a significantly reduced rhodamine 123 clearance, indicating that the renal organic cation carrier may be involved in active secretion. Rhodamine 123 accumulated extensively in the isolated perfused rat kidney; tissue concentrations of 270–360 times the perfusate concentration were determined. Similar accumulation ratios at different perfusate concentrations were found, suggesting that the compound enters the tubular cells by (facilitated) diffusion. In conclusion, rhodamine 123 accumulated extensively in the isolated perfused rat kidney and active renal secretion appears to be preferentially mediated by the organic cation carrier and not by P-glycoprotein.