Volume 38, No. 4/2000(April)
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 Int. Journal of Clinical Pharmacology and Therapeutics
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Clinical Pharmacology of P-glycoprotein and related transporters
P-glycoprotein inhibitor erythromycin increases oral bioavailability of talinolol in humans
U.I. Schwarz, T. Gramatté, J. Krappweis, R. Oertel and W. Kirch
Abstract
U.I. Schwarz1, T. Gramatté2, J. Krappweis1, R. Oertel1 and W. Kirch1
1Institute of Clinical Pharmacology, Faculty of Medicine, University of Technology, and 2APOGEPHA Arzneimittel GmbH, Dresden, Germany
Objective: Increased bioavailability of the P-glycoprotein (Pgp) substrates digoxin and cyclosporin due to erythromycin has been observed in vivo. The aim of the present study was to investigate the effect of orally administered erythromycin on the oral bioavailability of the b-blocker talinolol. Talinolol is a suitable model compound for Pgp drug-drug interaction studies due to its Pgp-related active intestinal secretion and lack of any significant metabolism. Methods: In a randomized crossover study, the oral pharmacokinetics of talinolol (50 mg) after a concomitant single oral dose of erythromycin (2 g) or placebo were investigated in 9 healthy men. Concentrations of talinolol were measured in serum and urine by HPLC. Results: The area under the curve of talinolol serum concentrations from 0 to 24 h (AUC(0-24)) and the maximum serum concentrations (Cmax) were significantly increased after administration of erythromycin compared to placebo. tmax values were significantly reduced. The renal clearance (CLR) of talinolol was unchanged after co-administration of erythromycin and there was a small but statistically significant decrease in elimination half-life (t1/2). Serum pharmacokinetics correlate with the results derived from urine concentration measurement. One subject suffered from moderate diarrhea after erythromycin and was excluded from the analysis. Conclusion: We suggest that the increase in oral bioavailability of talinolol after concomitant erythromycin is caused by increased intestinal net absorption due to Pgp inhibition by erythromycin.Correspondence to:
Dr. U.I. Schwarz; Division of Clinical Pharmacology, Departments of Medicine and Pharmacology, Vanderbilt University School of Medicine, 532 Medical Research Building, Nashville, TN 37232-6602, USA
Clinical Pharmacology of P-glycoprotein and related transporters
Affinities at the verapamil binding site of MDR1-encoded P-glycoprotein: drugs and analogs, stereoisomers and metabolites
S. Neuhoff, P. Langguth, C. Dressler, T.B. Andersson, C.G. Regårdh and H. Spahn-Langguth
Abstract
S. Neuhoff1,2, P. Langguth3, C. Dressler1, T.B. Andersson2, C.G. Regårdh2 and H. Spahn-Langguth1
1School of Pharmacy, Martin-Luther-University Halle-Wittenberg, Halle/Saale, 2AstraZeneca R&D, DMPK, Mölndal, Sweden, and 3School of Pharmacy, Johannes Gutenberg-University, Mainz, Germany
Objectives: The multidrug transporter P-glycoprotein (P-gp) appears to play a significant role in drug absorption and disposition. Hence, it is of interest to evaluate structure-affinity relationships for the purpose of making predictions. Methods: The affinity to P-gp of related molecular structures from various groups of drugs was determined using competitive radioligand-binding assays. Structural analogs, stereoisomers and metabolites of verapamil-type calcium antagonists, b-adrenoceptor antagonists as well as omeprazole, omeprazole enantiomers and the sulfone metabolite of omeprazole were investigated. Results: Whereas some stereoselectivity was detected for the high-affinity P-gp substrates verapamil and carvedilol, little or no differences were observed in the case of other b-blockers. One of the 4 labetalol stereoisomers, the R,R-isomer dilevalol, had an IC50 value half that of labetalol. Conclusions: Metabolites of verapamil, gallopamil, carvedilol and omeprazole are characterized by having higher IC50 values (lower P-gp affinity) than the respective parent compounds. Only the acebutolol metabolite, diacetolol, had a P-gp affinity comparable to that of the parent compound.Correspondence to:
Prof. Dr. H. Spahn-Langguth; Institut für Pharmazeutische Chemie, Martin-Luther-Universität, Wolfgang-Langenbeck-Straße 4, D-06120 Halle/ Saale, Germany
Clinical Pharmacology of P-glycoprotein and related transporters
Simultaneous measurement of cellular P-glycoprotein content and function by multiparametric flow-cytometry
M.R. Müller, K. Lennartz, B. Baack, M.M.E. Heim, S. Seeber and M.E. Scheulen
Abstract
M.R. Müller1, K. Lennartz2, B. Baack1, M.M.E. Heim1, S. Seeber1 and M.E. Scheulen1
1Department of Internal Medicine (Cancer Research) and 2Institute of Cell Biology (Cancer Research) (IFZ), West German Cancer Centre Essen, University of Essen Medical School, Essen, Germany
Objective: A multiparametric approach was applied to simultaneously determine expression and function of the drug efflux pump P-glycoprotein (PGP) in multidrug-resistant (MDR) human leukemic lymphoblast cell lines and isolated leukemic blasts using flow-cytometry in a patient with acute myeloid leukemia (AML). Methods: The antigen was measured by staining PGP using the monoclonal antibody 4e3 which does not inhibit the function of PGP. The 4e3 antibody binds to an external epitope of PGP and can therefore be used for staining living cells. Drug transport, mediated by PGP, was determined simultaneously by measuring rhodamine 123 (rho123) efflux. The MDR cell lines, CEM/VLB10-2 and CEM/VBL100 are 10-fold and 270-fold resistant to vinblastine (VBL), respectively, compared to the human PGP-negative parent cell line CEM/ WT and they express different amounts of PGP. Initially, living cells were stained using the 4e3 antibody and a secondary antibody labeled with 7-amino-4-methylcoumarin-3-acetic acid (AMCA). Cells were then incubated for 60 min with rho123 (10 mM) and analyzed for rhodamine and AMCA-derived fluorescence. The decrease in rho123 fluorescence was determined after a further period of 30 min. Results: CEM/VLB100 cells expressed larger amounts of PGP, and rho123 fluorescence after 30 min was 85% lower than the parent cell line. PGP expression and rho123 efflux were also detected in CEM/VLB10-2 cells which display a low degree of resistance, thus reflecting the high sensitivity of this method. PGP-expressing blasts and moderate rho123 efflux were also observed in a specimen derived from a patient with clinically resistant acute myeloid leukemia (AML). Conclusion: A multiparametric approach using flow-cytometry allows the reliable and sensitive measurement of both PGP expression and function simultaneously in single cells.Correspondence to:
Dr. M.R. Müller; Innere Klinik und Poliklinik (Tumorforschung), Universitätsklinikum Essen, Hufelandstraße 55, D-45122 Essen, Germany
Clinical Pharmacology of P-glycoprotein and related transporters
Diverse effects of P-glycoprotein inhibitory agents on human leukemia cells expressing the multidrug resistance protein (MRP)
G. Lehne, L. Mrrkrid, M. den Boer and H.E. Rugstad
Abstract
G. Lehne1,2, L. Mrrkrid3, M. den Boer4 and H.E. Rugstad1
1Department of Clinical Pharmacology, 2Institute for Surgical Research, 3Department of Clinical Chemistry, The National Hospital, Rikshospitalet, University of Oslo, Norway, and 4Department of Pediatrics, Free University Hospital, Amsterdam, The Netherlands
Multidrug resistance (MDR) to cancer chemotherapy is frequently associated with decreased drug accumulation in cancer cells due to drug expulsion by multidrug transporters such as P-glycoprotein (Pgp) and multidrug resistance protein (MRP). The novel resistance modifying agents PSC 833, 280-446, and LY 335979 are primarily targeted at inhibition of Pgp, and their MRP inhibitory potential is largely unknown. Objective: In the present study we addressed the effect of these agents on MRP-derived drug resistance. Materials: Drug-resistant human leukemia cells with Pgp+/MRP– (KG1a/200, K562/150) and Pgp–/MRP+ (HL60/130) phenotypes were maintained in suspension cultures for experimental studies of drug accumulation and drug sensitization by Pgp inhibitors. Methods: Intracellular accumulation of the fluorescent anthracycline daunorubicin was measured by flow cytometry and fluorescence detection. Daunorubicin dose-response curves were generated by non-linear regression of electronically measured cell counts of 72- – 96-h cultures. The half-maximal growth inhibitory dose (GI50) was used as measure of growth inhibition. Results: All MDR phenotypes studied exercised significant resistance to daunorubicin. PSC 833, 280-446 and LY335979 were equal in sensitizing Pgp+/MRP– cells to daunorubicin-induced growth inhibition (p < 0.0001). The Pgp–/MRP+ cells responded to PSC 833 and 280-446 by increased accumulation of daunorubicin (p = 0.0022 and p = 0.0005, respectively) and sensitization to the drug (p = 0.0009 and p = 0.0007, respectively). Conversely, LY335979 did not affect accumulation of daunorubicin in Pgp–/MRP+ cells nor sensitize these cells to daunorubicin. Conclusion: Pgp inhibitory agents have differential effects on MRP-derived drug resistance which could be exploited in treatment of multidrug resistance in cancer patients.Correspondence to:
Dr. G. Lehne; Department of Clinical Pharmacology, Rikshospitalet, The National Hospital of Norway, N-0027 Oslo
Clinical Pharmacology of P-glycoprotein and related transporters
Resistance modulation in CHO cells by R-verapamil and bile salts is associated with physical and chemical changes in the cell membrane
J.H. Dolderer, G. Zimmer, B.G. Woodcock, H. Bockhorn, R. Bickeböller and H. Schuldes
Abstract
J.H. Dolderer1,4, G. Zimmer2, B.G. Woodcock3, H. Bockhorn4, R. Bickeböller1 and H. Schuldes4
1Cellular Research Laboratory of the Urological Clinic, Center of Surgery, 2Membrane Structure Group, Center of Biological Chemistry, 3Institute of Clinical Pharmacology, 4Department of Surgery, Nordwest-Hospital, Johann-Wolfgang-Goethe-University Hospital, Frankfurt/Main and 5Urology Department, St. Katharinen-Hospital, Frechen, Germany
Objectives: Changes in multidrug resistance by resistance modifiers such as R-verapamil cause changes in fluidity of the cell membrane. The extent to which these changes involve structural alterations in membrane lipids has been investigated in CHO cells. Methods: Sensitive (AUXB1) and resistant (CHRC5) chinese hamster ovary cells (CHO) were grown in culture. Incubations were carried out with R-verapamil (0 – 10 mM) or the membrane perturbing agents tauro-cheno-deoxycholate (0 – 1.6 mM, TCDC) and tauro-urso-deoxycholate (0 – 3.5mM, TUDC). Cell membrane fluidity was determined by electron-paramagnetic resonance spectroscopy and membrane lipids by HPLC and TLC. Results: The resistant CHRC5 subline had a higher cell membrane order (lower fluidity, S = 0.7234) in the interface region of the cell membrane than sensitive AUXB1 cells (S = 0.6984) determined using EPR. The MDR-modulator R-verapamil and TCDC, but not TUDC, lowered cell membrane order in a concentration-dependent manner and increased membrane fluidity of the resistant CHRC5 subline. TCDC and R-verapamil were without effect on the cell membrane fluidity of AUXB1 cells. These changes were accompanied by alterations in the fatty acid composition of the plasma membrane. Untreated sensitive AUXB1 cells had higher levels of unsaturated fatty acids than resistant CHRC5 cells. In CHRC5 cells, R-verapamil increased the content of poly-unsaturated fatty acids and TCDC, but not TUDC, increased the content of mono-unsaturated fatty acids. Conclusions: The results demonstrate that resistance modifiers such as verapamil may influence cytostatic drug action by producing structural changes to lipid domains in the plasma membrane.Correspondence to:
Dr. B.G. Woodcock; Institut für Klinische Pharmakologie, Universitätsklinik Frankfurt, Theodor-Stern-Kai 7, D-60590 Frankfurt, Germany
Email: woodcock@em. uni-frankfurt.de
Clinical Pharmacology of P-glycoprotein and related transporters
Cytostatic sensitivity and MDR in bladder carcinoma cells: implications for tumor therapy
H. Schuldes, J.H. Dolderer, C. Schoch, R. Bickeböller and B.G. Woodcock
Abstract
H. Schuldes1, J.H. Dolderer2, C. Schoch2, R. Bickeböller2 and B.G. Woodcock3
1Klinik für Urologie und Kinderurologie, St. Katharinenhospital Frechen, 2Klinik für Urologie und Kinderurologie, 3Institut für Klinische Pharmakologie, Zentrum der Pharmakologie,Johann-Wolfgang-Goethe Universität, Frankfurt am Main, Germany
The clinical success generally seen in chemotherapy of advanced bladder carcinoma is far from optimal. The mechanism of resistance development is unclear and the expression of P-170 glycoprotein is generally low. The aim of this study, carried out in vitro in sensitive and cisplatin-resistant cell lines, was to examine sensitivity modulation using R-verapamil and cell membrane perturbing agents. Cell growth rates and changes in the order of the cell membrane, determined using electron-paramagnetic resonance spectrometry, were recorded. R-verapamil increased the toxic effect of doxorubicin in the cisplatin-resistant cell line which showed the highest membrane order. Linolenic acid had a similar effect and also increased sensitivity to cisplatin and methotrexate. Bile salts (tauro-cheno-deoxycholate,TCDC, and tauro-urso-deoxycholate TUDC), had little effect on cytotoxity. These results indicate that R-verapamil and linolenic acid can act as sensitivity modulators in bladder carcinoma cells and that the action of these agents may involve membrane fluidity changes, a phenomenon noted previously in regard to sensitivity modulation in chinese hamster ovary cell lines.Correspondence to:
PD Dr. H. Schuldes; Abtlg Urologie, St.-Katharinen-Hospital Frechen, Akademisches Lehrkrankenhaus der Universität Köln, Kapellenstraße 1 – 5, D-50226 Frechen, Germany
Clinical Pharmacology of P-glycoprotein and related transporters
Modulation of multidrug resistance in human leukemia cells with mdr1-targeted antisense oligonucleotides using variable treatment schedules
H. Dassow, D. Lassner, H. Remke and R. Preiss
Abstract
H. Dassow1, D. Lassner2, H. Remke2 and R. Preiss1
1Department of Clinical Pharmacology, and 2Department of Clinical Chemistry and Pathological Biochemistry, University of Leipzig, Leipzig, Germany
Objective: The purpose of the current study was to characterize the effect of chimeric AS-ODNs encapsulated with cationic lipids on MDR in human leukemia cells and to determine if this modification of the ODN alone or in combination with the cationic lipid might offer advantages over classical ODN treatment with free unmodulated or phosphorothiolated AS-ODNs. Furthermore, we extended the antisense method to the use of AS-ODNs in the parental drug-sensitive leukemia cells which express mdr1-mRNA at a relative low level and lack P170 expression to evaluate the effectiveness of prophylactic AS-ODN treatment. Methods: The effect of a 4-day AS-ODN treatment in drug-resistant human leukemia cells which exhibit the classic MDR phenotype at a moderate level was examined. Twenty-four hours after the last ODN administration the cells were analyzed for mdr1-mRNA (quantitative RT-PCR) and P170 expression (FCM), for R123 accumulation/efflux capacity (FCM) and for sensitivity to vincristine (MTT). In the parental drug-sensitive CCRF-CEM cells the mdr1-mRNA expression was assessed 24, 48 and 72 h after AS-ODN treatment administered as free phosphorothioate or conjugated with DMRIE-C. Results: Cationic lipids produced a clear increase in cellular ODN uptake but also caused an increase in variability of uptake rates (30% vs. 10% variability after free phosphorothioates). Both AS-ODNs inhibit P170 expression whereby the antisense effect of the chimeric ODN seems to be stronger compared to the phosphorothioate (30% vs. 22% MRK16 staining). Consistent with the inhibition of P170 expression, an increased sensitivity to vincristine was observed. In parental drug-sensitive cells, AS-ODN treatment caused nearly complete inhibition of mdr1-mRNA expression (5% of control). Conclusion: The data demonstrate that it is nearly impossible to achieve a complete reversal of the MDR phenotype in drug-resistant cells using AS-ODNs. A more promising approach seems to be the prophylactic treatment with AS-ODNs.Correspondence to:
Dr. H. Dassow; Abteilung Klinische Pharmakologie, Universität Leipzig, Haertelstraße 16-18, D-04107 Leipzig 653, Germany
Clinical Pharmacology of P-glycoprotein and related transporters
Induction of apoptosis by idarubicin: how important is the plasma peak?
F. Gieseler, M. Clark, K. Stiebeling, M. Puschmann and S. Valsamas
Abstract
F. Gieseler, M. Clark, K. Stiebeling, M. Puschmann and S. Valsamas
Schwerpunkt Hämatologie/Onkologie, Klinik für Allgemeine Innere Medizin, Christian-Albrechts-Universität, Kiel, Germany
Objectives: It is still not clear whether a high plasma peak or a prolonged plasma presence of the drug is optimal for chemotherapy with anthracyclines. A high plasma peak seems to correlate with the liberation of oxygen radicals and cumulative delayed cardiotoxicity, and therefore, should be avoided. On the other hand, its role in attaining the desired therapeutic effect, i.e. induction of apoptosis in tumor cells, has not been clearly elucidated. Methods: Idarubicin is the only anthracycline that can be applied orally. We measured the DNA-binding of idarubicin and idarubicinol and the induced apoptosis in the human promyelocytic HL-60 leukemia cell line. Various pharmacokinetic profiles, with and without clinically relevant peak concentrations, were simulated in vitro. Results: The concentration necessary for maximal DNA-binding and subsequent induction of apoptosis was 1.5 mg/ml for 20 minutes which is well above the plasma concentration achievable in therapy. A plateau of apoptosis was observed after 90 minutes of incubation; a prolongation above 90 minutes did not increase the rate of apoptosis. We simulated a bolus application and a continuous infusion using two different pharmacokinetic profiles of idarubicin with comparable AUCs (area under the time curve). After 48 hours of total incubation, the viability of HL-60 cells was 56.88% with profile 1 (50 ng/ml idarubicin for 2 hours) and 83.00% with profile 2 (4.25 ng/ml for 24 hours). Conclusions: Although these in vitro experiments are not directly applicable to the clinical situation, they do indicate that a prolongation of the application time up to at least 90 minutes, either by continuous infusion or by oral application, may be acceptable as a method of increasing apoptosis. On the other hand, the plasma peak seems to be an important factor for the induction of apoptosis. Further studies are in progress to define the minimal plasma peak necessary to induce a maximum of apoptosis.Correspondence to:
Prof. Dr. F. Gieseler; Klinik für Allgemeine Innere Medizin Schlittenhelmstraße 12, D-24105 Kiel, Germany
