A flow-through tissue culture system with fast dynamic response

Lankelma, J.; Laurensse, E.; Pinedo, H.M.

doi:10.1016/0003-2697(82)90184-1

Cited by 15 publications

(6 citation statements)

References 4 publications

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“…In a previously described flow-through system [17,28,32], the initial change of the steady-state daunorubicin concentration in the eluent after a change in the intracellular pH was measured. Basically, a monolayer of approximately lo7 cells present on the bottom of a chamber of 500 p1 was perfused with drug-containing medium A or medium C. Medium C was medium A without glucose but supplemented with 10 mM sodium azide, 6.1 mM deoxyglucose and 5% fetal bovine serum that had been dialyzed for 24 h against 1 mM KH,PO,, pH 7.3, using a Visking-30 dialysis tube, 1 :200 VjV (Radiometer, Den Haag, The Netherlands).…”

Section: Methodsmentioning

confidence: 99%

Kinetics of daunorubicin transport by P‐glycoprotein of intact cancer cells

Spoelstra

Westerhoff

Dekker

et al. 1992

European Journal of Biochemistry

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Drug permeation across the plasma membrane of multidrug-resistant cells depends on the kinetics of the P-glycoprotein-mediated pump activity as well as on the passive permeation of the drug. We here demonstrate a method to characterize kinetically the pump in intact cells.To this purpose, we examined the membrane-transport properties of daunorubicin in various sensitive cancer cell lines and in their multidrug resistant (MDR) counterparts. First, we determined the passive permeability coefficient for daunorubicin. Then, using a flow-through system, the drug flux into the cell was measured after inhibition of the P-glycoprotein-mediated efflux pump. Combining the two results allowed us to calculate the intracellular free concentration of the drug. In the steady-state, the pump rate must equal the net rate of passive diffusion of the drug and, therefore, the same experiments gave us the pumping rate of daunorubicin. These experiments were then repeated at various extracellular drug concentrations. By plotting the pumping rate versus the intracellular drug concentration, we then characterized the P-glycoprotein kinetically.Four independent methods were used to measure the passive permeability coefficient for the cell line A2780. Similar values were obtained. Maximal pump rates (Vmax) showed a good correlation with the amount of P-glycoprotein in the cell lines used. We obtained saturation curves for the variation of the pump rates with the intracellular daunorubicin concentrations. These curves were typical for positive cooperativity, which provides evidence that at least two binding sites for daunorubicin are present on the active transport system of daunorubicin. The apparent K,,, values for P-glycoprotein-mediated transport, the intracellular free cytosolic daunorubicin concentrations at half-maximal velocity for the cell lines used, were approximately 1.5 pM. Except for the cell lines with the highest amount of P-glycoprotein, the passive efflux rate of daunorubicin proved to be a substantial part of the total daunorubicin efflux rate for the cell lines used. In cell lines with relatively low levels of P-glycoprotein, passive daunorubicin efflux was even the main route of daunorubicin transport from the cells, determining the intracellular steady-state concentrations of daunorubicin.The cellular pharmacological basis for multidrug resistance (MDR) often appears to be a reduced accumulation of drugs [I -31. One of the mechanisms responsible for MDR is thought to be an energy-dependent transport system for natural-product cytotoxic drugs such as doxorubicin, vinblastine and daunorubicin [l, 21. MDR is often correlated with the overexpression of a 150 -180 kDa membrane protein, called P-glycoprotein [4].Study of the saturability of drug transport may influence the choice of alternative drugs in chemotherapy. In the case of P-glycoprotein-mediated efflux, saturability for a certain drug opens the opportunity for the use of competitive inhibitors. Kinetic studies of drug efflux across the cell membrane are normally ...

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Section: Methodsmentioning

confidence: 99%

Kinetics of daunorubicin transport by P‐glycoprotein of intact cancer cells

Spoelstra

Westerhoff

Dekker

et al. 1992

European Journal of Biochemistry

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“…22 The rat hepatocytes obtained by the incubation at 37°C for 24 h were used for the perfusion experiment in vitro. 23 V max and K m in the present investigation are estimated to be 33.0 g/ mL/min and 2.88 g/mL (‫ס‬mg/L), respectively, in Table III. The unit of V max is given to be g/mL/ min, which is considered to be the maximum velocity per the volume of peripheral compartment (‫ס‬kЈV B ).…”

Section: Discussionmentioning

confidence: 51%

Dose-dependency in local disposition of 5-fluorouracil under non-steady-state condition in rat liver

Higashimori¹,

Yamaoka²,

Nakagawa³

2000

J. Pharm. Sci.

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“…17 Alternatively,thepermeationcoefficient(volume‚time -1 ‚number of cells -1 ) may be calculated when reference is made to the amount of cells instead of surface. 23,24 Octanol/Water Partition CoefficientsThe octanol/water partition coefficient is the ratio of the anthracycline concentration in the octanol phase divided by that in the water () phosphatebuffered saline (PBS)) phase at equilibrium. The ratio was determined at ambient temperature (20-25°C), by adding 750 µL octanol to 750 µL of a 10 µM anthracycline solution prepared in PBS (pH 7.3).…”

Section: Figure 2snormalmentioning

confidence: 99%

“…Until now transepithelial transport has been measured discontinuously in time, mainly by measuring either the amount of substrate (mostly radioactively labeled, or fluorescent) transported from the apical to the basolateral (A to B) side of the cells (or vice versa) or by measuring the TER. [17][18][19][20] By combining knowledge of the study of MDR, 21,22 the use of fluorescence for detection, and the experience in developing in vitro techniques, 23,24 we developed a technique that can be used to study transepithelial transport continuously. The method makes use of commercially available plasticware and a fluorometer, equipment present in most biochemical laboratories.…”

Section: Introductionmentioning

confidence: 99%

In vitro transepithelial drug transport by on-line measurement: Cellular control of paracellular and transcellular transport

Wielinga

Waal

Westerhoff³

et al. 1999

Journal of Pharmaceutical Sciences

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Studies on transcellular transport across epithelial cell layers are performed mostly by discontinuous sampling of the transported compound. This has several drawbacks, e.g., it gives disturbances in volume, it limits the time-resolution, and is often laborious. In this report we introduce a method to measure transepithelial transport of fluorescent compounds continuously. The time-resolution is at the (sub)minute scale, allowing the measurement of the change in transport rate before and after transport modulation. We will describe how we used the method to measure transcellular and paracellular transport. For highly membrane-impermeable compounds, the paracellular transport and the regulation of the tight junctions was studied in wild-type and MDR1 cDNA transfected epithelial canine kidney cells (MDCKII). The effect of the multidrug transporter P-glycoprotein (Pgp) on the transepithelial transport was studied. Addition of the Pgp inhibitor SDZ PSC 833 showed a modulation of the idarubicin (IDA) and daunorubicin (DNR) transport, which was larger during transport from the basolateral to the apical side than in the reverse direction. By modeling the transepithelial transport, we found that in these cells Pgp had more effect on the basolateral to apical transport than vice versa, which can be attributed to a relatively large passive permeation coefficient for the cellular basolateral plasma membrane.

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A flow-through tissue culture system with fast dynamic response

Cited by 15 publications

References 4 publications

Kinetics of daunorubicin transport by P‐glycoprotein of intact cancer cells

Kinetics of daunorubicin transport by P‐glycoprotein of intact cancer cells

Dose-dependency in local disposition of 5-fluorouracil under non-steady-state condition in rat liver

In vitro transepithelial drug transport by on-line measurement: Cellular control of paracellular and transcellular transport

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