Modulation Of Drug Transporters Research Articles

Abstract 3250: Microbial metabolite, Urolithin A acts as chemo-sensitizing adjuvant in conventional drug therapies via modulation of drug transporters and EMT markers

Abstract Colon cancer is the third leading cause in cancer related deaths in United States of America. Chemoresistance (drug resistance) of tumors is the primary reason for the failure of chemotherapy and a major cause of mortality in colon cancer. The molecular mechanisms involved in chemoresistance or methods to chemosensitization of cancer cells to chemotherapy remain elusive. Recent studies suggest that gut microbiota and microbial metabolites play a crucial role in the development and progression of colon cancer. Urolithin A (UroA) is a gut microbial metabolite derived from ellagitannin/ellagic acid rich-diets such as pomegranate and berries. Here, we identified that UroA and its novel structural analogue (UAS03) chemosensitize 5FU resistant (5FUR) colon cancer cells to 5FU treatment. Our data suggested that combination of 5FU with UroA or UAS03 significantly reduced cell viability of 5FUR colon cancer cell lines with combination index (CI) less than 1 suggesting their synergism. Moreover, UroA or UAS03 in combination with 5FU significantly inhibited 5FUR colon cancer cell proliferation, migration as well as induced apoptosis. Mechanistically, UroA or UAS03 treatment down regulated drug transporters (MDR1, BCRP and MRP2) leading to decreased efflux of drug (e.g., 5FU) and potentially responsible for chemosensitization. Furthermore, treatment with these compounds also modulated EMT makers (e.g., downregulated Snail and β-catenin; upregulated ZO-1 and E-cadherin) to reduce EMT transition. Treatment of UroA or UAS03 in combination with 5FU significantly reduced the growth of 5FUR-tumor xenografts in NRGS mice. Taken together, utilization of UroA or UAS03 in combination with 5FU treatment may provide better therapeutic options for 5FUR colon cancer alleviation. Citation Format: Sweta Ghosh, Rajbir Singh, Zachary M. Vanwinkle, Haixun Guo, Praveen K. Vemula, Ajay Goel, Bodduluri Haribabu, Venkatakrishna Rao Jala. Microbial metabolite, Urolithin A acts as chemo-sensitizing adjuvant in conventional drug therapies via modulation of drug transporters and EMT markers [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 3250.

Inhibitory Effects of Benzaldehyde, Vanillin, Muscone and Borneol on P-Glycoprotein in Caco-2 Cells and Everted Gut Sac

Aims: In clinical practice, herbal medicines have played an important role in the modulation of drug transporters through the combination of conventional prescription drugs, which necessitates the elucidation of herb-drug interactions. The present study was designed to investigate the inhibitory effects and mechanisms of benzaldehyde, vanillin, muscone, and borneol on P-glycoprotein (P-gp). Methods: The effects of the 4 compounds on the intracellular accumulation of rhodamine-123 (Rho-123) in vinblastine-treated Caco-2 (VB-Caco-2) cells were studied by monitoring fluorescence intensity through a flow cytometry assay, and the effects of these compounds on Rho-123 transport through VB-Caco-2 monolayers and Rho-123 intestinal absorption in the rat everted gut sac were investigated by high-performance liquid chromatography. Moreover, P-gp expression in VB-Caco-2 cells was assessed using flow cytometry and Western blot analysis, and the relative ABCB1 mRNA level was determined by Real-time RT-PCR. Key Findings: The results showed that benzaldehyde, vanillin, muscone, and borneol significantly increased Rho-123 uptake in VB-Caco-2 cells, increased the absorption rate and apparent permeability coefficient of Rho-123 in rat jejunum and ileum, and decreased the efflux ratio of Rho-123 from 6.52 to less than 2 during transport across VB-Caco-2 cell monolayers. In addition, these compounds reduced the protein and ABCB1 mRNA levels of P-gp in VB-Caco-2 cells. Conclusions: These data indicate that benzaldehyde, vanillin, muscone and borneol could effectively reverse multidrug resistance via inhibiting the P-gp function and expression pathway. The data provide fodder for further investigation into the interaction between the 4 compounds and other drugs transported by P-gp.

Endogenous Biomarkers to Assess Drug-Drug Interactions by Drug Transporters and Enzymes.

Drug-Drug Interactions (DDI) by modulation of drug transporters or drug metabolizing enzymes are common in multi-drug therapy. DDI potential of any new drug is assessed by conducting separate clinical studies using relevant probe substrates, which involves additional resource and cost. Recently, several endogenous compounds have been evaluated as substrates of transporters and enzymes that could be assessed as part of early clinical trials along with the assessment of drug pharmacokinetics, pharmacodynamics and safety studies. This enables an early readout on potential DDIs avoiding or minimally delaying the conduct of definitive DDI studies until later in clinical development. This review describes various endogenous biomarkers reported for drug transporters and metabolizing enzymes with their advantages and limitations. Furthermore, the authors describe strategies to adopt while exploring a new endogenous biomarker, and factors to be considered in selection of biomarkers with the current challenges and opportunities.

Inverse association between microRNA-124a and ABCC4 in HepG2 cells treated with antiretroviral drugs

The ATP-binding cassette (ABC) super-family of drug transporters regulates efflux of xenobiotic compounds. The subfamily, multi-drug resistance proteins (MRPs) transports cyclic nucleotides and xenobiotics. Epigenetic modulation of drug transporters is scarcely described. The regulatory role of microRNA (miR)-124a on drug transporter gene ABCC4 was only recently reported.Our study investigated the differential regulation of miR-124a by nucleoside reverse transcriptase inhibitors (NRTIs): Zidovudine (AZT), Stavudine (d4T) and Tenofovir (TFV); at 24 h and 120 h treatments in HepG2 cells. ABCC4 mRNA (qPCR) and ABCC4 protein (western blot) were quantified. Cytotoxicity was evaluated by lactate dehydrogenase (LDH) levels.All NRTIs elevated miR-124a levels at 24 h, with a concomitant decline in ABCC4 mRNA levels (p<0.05). At 120 h, d4T and TFV elevated miR-124a and depleted ABCC4 mRNA levels (p<0.0001), while the inverse was observed with AZT (p<0.005). ABCC4 protein was increased by d4T and TFV at 24h. A significant reduction in protein levels was observed at 120 h in all three treatments (p<0.005). The disjoint in mRNA and protein levels is likely due to ABCC4 being a membrane bound protein. Following prolonged exposure, membrane integrity was compromised as evidenced by increased LDH leakage (p<0.005).We conclude antiretroviral drugs have varying effects on miR-124a and ABCC4.

Transporter modulation by Chinese herbal medicines and its mediated pharmacokinetic herb-drug interactions.

The increasing use of Chinese herbal medicines (CHMs) as complementary therapy and dietary supplement has been greatly raising the concerns about potential herb-drug interactions (HDIs). HDIs may cause the augmented or antagonized effects of prescription drugs, resulting in unexpected clinical outcomes. Therefore, it is of significance to identify or predict potential HDIs, and to delineate the underlying mechanisms. Drug transporters play key roles in transmembrane passage of a large number of drugs, affecting their absorption, distribution and elimination. Modulation of drug transporters has been recognized as one of the main causes of HDIs. In the last decade, a growing number of Chinese medicinal herbs and their derived phytochemicals have been identified to have modulatory effect toward transporter proteins, leading to pharmacokinetic HDIs when concomitantly used with conventional drugs. Some of these transporter-mediated interactions have already shown clinical significance. This review article focuses on two major transporter superfamilies, the solute carrier (SLC) and the ATP-binding cassette (ABC) transporters, to provide the recent advanced knowledge on CHMs and their inherent phytochemicals that interact with these transporters, and their induced pharmacokinetic HDIs from both preclinical and clinical aspects. In addition, the challenges and strategy for studying HDIs are also discussed.

Effect of Multi Drug Resistance Protein 4 (MRP4) Inhibition on the Pharmacokinetics and Pharmacodynamics of Ciprofloxacin in Normal and Rats with LPS-Induced Inflammation.

Infection and inflammation are known to cause wide variability in disposition of drugs through modulation of drug transporters. However, the effects of inhibition of multidrug resistance protein 4 (MRP4) on pharmacokinetics and pharmacodynamics are poorly understood in normal and inflamed conditions. We hypothesized that inflammation alters the pharmacokinetic parameters of ciprofloxacin; and Pharmacokinetic/Pharmacodynamic indices, such as ratio of peak plasma concentration to minimum inhibitory concentration (C max/MIC) and ratio of area under the plasma drug concentration-time curve to minimum inhibitory concentration (AUC/MIC) of ciprofloxacin will be improved with the co-administration of a MRP4 inhibitor, dipyridamole, in inflammatory conditions. In this study, the role of MRP4 on the pharmacokinetic and pharmacodynamic parameters of ciprofloxacin was investigated by the co-administration of dipyridamole in rats with or without lipopolysaccharide (LPS)-induced inflammation. The pharmacokinetic parameters for ciprofloxacin were calculated by non-compartmental approach. MIC of ciprofloxacin was determined using broth microdilution technique. Induction of inflammation in rats resulted in marked reduction in C max and AUC; and an increase in the volume of distribution (V d/F) and clearance (Cl/F) of ciprofloxacin, compared to normal rats. Co-administration of dipyridamole with ciprofloxacin in inflamed rats resulted in a threefold increase in AUC, a twofold decrease in V d/F and a threefold decrease in Cl/F of ciprofloxacin with significantly prolonged half-life compared to inflamed rats who received ciprofloxacin alone. Co-administration of dipyridamole enhanced AUC/MIC values of ciprofloxacin in both normal and inflamed rats. The results suggest that MRP4 inhibition increases the systemic exposure of ciprofloxacin in both normal and inflammatory conditions.

Inhibitory effects of herbal constituents on P-glycoprotein in vitro and in vivo: Herb–drug interactions mediated via P-gp

Modulation of drug transporters via herbal medicines which have been widely used in combination with conventional prescription drugs may result in herb–drug interactions in clinical practice. The present study was designed to investigate the inhibitory effects of 50 major herbal constituents on P-glycoprotein (P-gp) in vitro and in vivo as well as related inhibitory mechanisms. Among these herbal medicines, four constituents, including emodin, 18β-glycyrrhetic acid (18β-GA), dehydroandrographolide (DAG), and 20(S)-ginsenoside F1 [20(S)-GF1] exhibited significant inhibition (>50%) on P-gp in MDR1-MDCKII and Caco-2 cells. Emodin was the strongest inhibitor of P-gp (IC50=9.42μM), followed by 18β-GA (IC50=21.78μM), 20(S)-GF1 (IC50=76.08μM) and DAG (IC50=77.80μM). P-gp ATPase activity, which was used to evaluate the affinity of substrates to P-gp, was stimulated by emodin and DAG with Km and Vmax values of 48.61, 29.09μM and 71.29, 38.45nmol/min/mg protein, respectively. However, 18β-GA and 20(S)-GF1 exhibited significant inhibition on both basal and verapamil-stimulated P-gp ATPase activities at high concentration. Molecular docking analysis (CDOCKER) further elucidated the mechanism for structure–inhibition relationships of herbal constituents with P-gp. When digoxin was co-administered to male SD rats with emodin or 18β-GA, the AUC0−t and Cmax of digoxin were increased by approximately 51% and 58%, respectively. Furthermore, 18β-GA, DAG, 20(S)-GF1 and Rh1 at 10μM significantly inhibited CYP3A4/5 activity, while emodin activated the metabolism of midazolam in human liver microsomes. In conclusion, four herbal constituents demonstrated inhibition of P-gp to specific extents in vitro and in vivo. Taken together, our findings provided the basis for the reliable assessment of the potential risks of herb–drug interactions in humans.

Abstract 883: Imatinib-induced modulation of gastrointestinal and hepatic membrane transporters in mice.

Abstract Drug-induced changes in the expression or activity of ADME genes may critically affect the pharmacokinetics (PK) and the efficacy of chemotherapy as well as the incidence and severity of unwanted side effects. Drug uptake and efflux transporters are likely to be involved in the intestinal absorption, distribution and hepatic excretion of imatinib, an oral tyrosine kinase inhibitor approved for the treatment of CML and GIST. Previously, we demonstrated that imatinib is a substrate drug of both ABCB1 and ABCG2. Furthermore, we showed that prolonged imatinib exposure in Caco-2 cell cultures leads to increased expression of these drug efflux transporters. Evidently, some GIST patients chronically treated with imatinib show a time-dependent decrease in imatinib plasma levels. Altered transporter activity in the intestine and/or liver affecting the PK of imatinib may (partially) account for the observed PK resistance. We hypothesized that acquired PK resistance by decreased absorption and/or increased clearance of imatinib may be attributable to induction of intestinal and hepatic transport proteins. So, we investigated in mice whether imatinib-induced modulation of drug transporters occurs over time upon prolonged daily use of imatinib. To mimic the clinical situation we chronically treated C57/Bl6 mice, either with imatinib or with a solute control (H2O) for 4, 8 or 12 weeks. Imatinib (100 mg/kg once daily) was administered to the mice via oral gavage (∼300 μl / mice). We collected all organs involved in the absorption and elimination of imatinib including the liver and the complete GI-tract (esophagus, stomach, small and large intestine and caecum) and subsequently isolated total RNA and protein from these tissues. We performed mRNA and miRNA profiling using mouse genome 430 PM (Affymetrix) and LNA-modified oligonucleotide arrays (Exiqon), respectively, and examined the expression of &amp;gt;80 transporter genes using the RT2 Profiler PCR array system (SABiosciences). Prolonged imatinib treatment resulted in the modulation of various intestinal and hepatic drug transporters and clearly changed the mRNA and miRNA profiles of ADME-involved organs. Apart from the imatinib-associated transporters Abcb1a/b, Abcg2, Slc22a1 and Slco1b2, indicating proof of concept, our search identified various other potential imatinib transporter which are currently tested in uptake experiments using transporter-transfected HEK293 cells. In summary, we showed that continuous exposure of mice to daily dosages of imatinib markedly affects the expression of drug transporters and changes the mRNA and miRNA expression profiles in organs involved in uptake, distribution and elimination of imatinib. These imatinib-induced changes may critically affect the PK, efficacy and adverse events of imatinib chemotherapy. Citation Format: Herman Burger, Alexander T. den Dekker, Antonius WM Boersma, Peter de Bruijn, Jaap Verweij, Ron HJ Mathijssen, Erik AC Wiemer. Imatinib-induced modulation of gastrointestinal and hepatic membrane transporters in mice. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 883. doi:10.1158/1538-7445.AM2013-883

Induction of Drug Transporters Alters Disposition of Risperidone - A Study in Mice.

Pharmacokinetic interactions, e.g. modulation of drug transporters like P-glycoprotein at the blood-brain barrier, can be a reason for treatment non-response. This study focuses on the influence of induction of drug transporters on the disposition of the antipsychotic drugs risperidone and 9-hydroxyrisperidone. Brain and serum concentrations of risperidone and its active metabolite 9-hydroxyrisperidone, which are known P-glycoprotein substrates, were measured after drug transporter induction with rifampicin, dexamethasone or 5-pregnene-3beta-ol-20-on-16alpha-carbonitrile using high performance liquid chromatography. Disposition of risperidone and 9-hydroxyrisperidone was dramatically decreased in mouse brain and serum after drug transporter induction. The metabolism of risperidone was also affected.

The accumulation and metabolism of zidovudine in 3T3‐F442A pre‐adipocytes

Cultured pre-adipocytes accumulate and metabolize zidovudine (ZDV), but its mode of accumulation into these cells is unclear. We investigated the mode of accumulation of [(3)H]-ZDV, and the impact of changes in external pH and modulators of drug transporters on its accumulation and metabolism. The initial rate and steady-state accumulation of [(3)H]-ZDV were measured in 3T3-F442A cells. P-glycoprotein (P-gp) expression was detected by Western blotting. External pH was varied, and modulators of intracellular pH and drug transporters were used to study the mode of accumulation of ZDV. Phosphorylated ZDV metabolites were detected by high-performance liquid chromatography. Intracellular accumulation of ZDV was rapid, reaching equilibrium within 20 min; nigericin increased accumulation by 1.9-fold, but this did not alter the generation of ZDV mono-, di- and triphosphate. The accumulation and metabolism were pH dependent, being maximal at pH 7.4 and least at pH 5.1. Monensin, carbonyl cyanide p-trifluoromethoxy) phenyl hydrazone, brefeldin A, bafilomycin A1 and concanamycin A increased accumulation; 2-deoxyglucose, dipyridamole, thymidine and tetraphenylphosphonium inhibited accumulation. The accumulation was saturable; the derived K(d) and capacity of binding were 250 nmol per 10(6) cells and 265 nM respectively. 3T3-F442A cells express P-gp; inhibitors of P-gp (XR9576 and verapamil), P-gp/BCRP (GF120918), multidrug resistance protein (MRP) (MK571) and MRP/OATP (probenecid) increased the accumulation of ZDV. Saquinavir, ritonavir, amprenavir and lopinavir increased accumulation. The accumulation of ZDV in 3T3-F442A cells was rapid, energy dependent, saturable and pH sensitive. Western blot analysis showed that 3T3-F442A cells express P-gp, and direct inhibition assays suggest that ZDV is a substrate of P-gp and MRP.

Silybin and Silymarin - New and Emerging Applications in Medicine

This review critically surveys the literature published mainly within this millennium on the new and emerging applications of silybin (pure, chemically defined substance) and silymarin (flavonoid complex from Silybum marianum - milk thistle seeds). These compounds used so far mostly as hepatoprotectants were shown to have other interesting activities, e.g. anticancer and canceroprotective and also hypocholesterolemic activity. These effects were demonstrated in a large variety of illnesses of different organs, e.g. prostate, lungs, CNS, kidneys, pancreas and also in the skin protection. Besides the cytoprotective activity of silybin mediated by its antioxidative and radical-scavenging properties also new functions based on the specific receptor interaction were discovered. These were studied on the molecular level and modulation of various cell-signaling pathways with silybin was disclosed--e.g. NF-kappaB, inhibition of EGFR-MAPK/ERK1/2 signaling, activity upon Rb and E2F proteins, IGF-receptor signaling. Proapoptotic activity of silybin in pre- and/or cancerogenic cells and anti-angiogenic activity of silybin are other important findings that bring silymarin preparations closer to respective application in the cancer treatment. Discovery of the inhibition and modulation of drug transporters, P-glycoproteins, estrogenic receptors, nuclear receptors by silybin and some of its new derivatives contribute further to the better understanding of silybin activity on the molecular level. Silymarin application in veterinary medicine is reviewed as well. Recent works using optically pure silybin diastereomers clearly indicate extreme importance of the use of optically active silybin namely in the receptor studies. Significance of silymarin and its components in the medicine is clearly indicated by an exponential growth of publications on this topic--over 800 papers in the last 5 years.

P-glycoprotein: Pharmacological relevance

P-glycoprotein (P-gp) is a 170 kDa membrane-bound protein, an energy-dependent efflux transporter driven by ATP hydrolysis. It is responsible for multidrug resistance of many drugs. Physiologically, it is involved in limiting the harmful exposure of toxins, drugs, and xenobiotics to the body by extruding them out of cells. It is increasingly recognized to play an important modulating role in the pharmacokinetic properties of many clinically important therapeutic agents and because of its importance in pharmacokinetics, its screening has to be incorporated into the drug discovery process. The modulation of drug transporters through inhibition or induction by various drugs or herbs can lead to significant drug-drug or drug-herb interactions by affecting various pharmacokinetic parameters of the drug. In addition, genetic polymorphism of P-gp has also been reported, which may affect drug disposition, produce variable drug effects, and may change disease risk susceptibility. As drug interactions and genetic polymorphism are important factors to be considered during drug development, P-gp may have an impact on drug development in future.

Silybin and silymarin--new effects and applications.

This article aims to review critically literature published mainly within this millennium on the new and emerging applications of silymarin, the polyphenolic fraction from the seeds of Silybum marianum and its main component silybin. Silymarin and silybin used so far mostly as hepatoprotectants were shown to have other interesting activities as e.g., anticancer and canceroprotective. These activities were demonstrated in a large variety of illnesses of different organs as e.g., prostate, lungs, CNS, kidneys, pancreas and others. Besides the cytoprotective activity of silybin mediated by its antioxidative and radical-scavenging properties also new activities based on the specific receptor interaction were discovered--e.g., inhibition and modulation of drug transporters, P-glycoproteins, estrogenic receptors, nuclear receptors and some others. New derivatives of silybin open new ways to its therapeutic applications. Pharmacology dealing with optically pure silybin diastereomers may suggest new mechanisms of its action.

Modulation of drug transport by selected flavonoids: Involvement of P-gp and OCT?

Flavonoids, as a common component of daily nutrition, are a possible source of interference with absorption processes, due to modulation of transporting proteins. In this study, the influence of selected flavonoids (quercetin, isoquercitrin, spiraeoside, rutin, kaempferol, naringenin, naringin, and kaempferol) on the transport of the P-gp substrate [ 3H]talinolol across Caco-2 cell monolayers was investigated. To elucidate the mechanism behind the interaction observed in this system the potency of the flavonoids to replace [ 3H]talinolol from its P-gp binding site as well as their activity to inhibit OCT2-mediated [ 14C]TEA uptake into LLC-PK 1 cells were measured, as P-gp and OCT have been shown to be present in Caco-2 cells. Six of the investigated flavonoids reduced the secretory flux of talinolol across Caco-2 cells (IC 50-values: hesperetin < quercetin < kaempferol ≪ spiraeoside < isoquercitrin < naringin). But none of the selected flavonoids was able to replace [ 3H]talinolol from its binding to P-gp. However, the investigated flavonoids did show potency to inhibit OCT-mediated transport (IC 50-values: quercetin < kaempferol ≪ naringenin < isoquercitrin < spiraeoside ≪ rutin < hesperetin < naringin). The present in vitro results demonstrate that flavonoids bear the ability to interfere with secretory intestinal transport processes. This might be due to an interaction with P-gp, but apparently not via competition at the talinolol binding site of P-gp. Another mode of interaction may be the inhibition of members of the OCT-family, which is located at the basolateral membrane of intestinal epithelial cells.

Efflux Transporters and their Clinical Relevance

It is increasingly recognized that efflux transporters play an important role, not only in chemo protection e.g. multi-drug resistance, but also in the absorption, distribution, and elimination of drugs. The modulation of drug transporters through inhibition or induction can lead to significant drug-drug interactions by affecting intestinal absorption, renal secretion, and biliary excretion, thereby changing the systemic or target tissue exposure of the drug. Few clinically significant drug interactions that affect efficacy and safety are due to a single mechanism and there is considerable overlap of substrates, inhibitors, and inducers of efflux transporters and drug metabolizing enzymes, such as CYP3A. As well, genetic polymorphisms of efflux transporters have been correlated with human disease and variability of drug exposure. Accordingly, this review will discuss drug interactions and suitable probe substrates, as well as, the clinical relevance of the variability and modulation of efflux transporters and the exploitation of substrates as diagnostic tools. An update is given on inhibitors, which clinically reverse drug resistance and minimize the risk of metabolic interactions.

Chapter 2 Rapid Determination of Cellular Resistance-Related Drug Efflux in Tumor Cells

Laser flow cytometry (FCM) offers a unique tool for monitoring of fluorescent antitumor drug retention and its modulation in tumor cells. Besides its rapidity, the laser FCM method can identify heterogeneity of drug retention as well as allow for sorting of subpopulations for further biochemical or morphological characterization. Cellular resistance to some of the clinically important anthracyclines has been suggested to be due to rapid drug efflux. Thus, drugs that block anthracycline efflux and thereby enhance retention can reduce cellular resistance to anthracyclines. Laser FCM is used to monitor anthracycline fluorescence in tumor cells and to monitor the effect of drug efflux blocking agents on drug retention. This method is also used to monitor anthracycline retention and its modulation by phenothiazines and amphotericin-B in P388 and doxorubicin-resistant cells. Some data show that the effect of efflux blockers on doxorubicin retention is cell-cycle proliferation related, and often one can identify subpopulations based on their differential response to efflux blockers. From these studies it follows that modulation of drug transport and thereby cellular resistance by phenothiazines or calcium channel blockers may not be uniform in a population but may be selective and confined to only certain types of cells and subpopulations.

An in vitro approach to study cellular kinetics of drugs

We adapted different existing techniques in order to optimize the methodology for studying kinetic interactions between drugs and cells in vitro. Using the polymorphonuclear leukocyte as a target cell, we measured the binding of various ligands and intracellular drug concentrations. We also studied pharmacological modulation of drug transport under normal and inflammatory conditions. Our approach allows reproducible measurements on ligands with low affinity for association sites on polymorphonuclear leukocytes. We present data for various nonsteroidal antiinflammatory drugs and other ligands to validate our methodological approach. On the basis of the results thus obtained, we proposed a tentative model to fit data and concepts of drug-cell interactions.