4.5 Applications of the calcein-AM efflux assay
4.5.2 Characterization of p-glycoprotein substrates and modulators
Ineffectivity of tumor chemotherapy is often caused by the multidrug resistance of malig-nant cells and by the localization of multidrug transporters such as p-gp at several barriers inside the body. Various substances are developed which are not transported by p-gp but modulate the p-gp mediated efflux of p-gp substrates. The efficacy of these modulators was studied in p-gp overexpressing Kb-V1/VBL cells using the calcein-AM efflux assay.
Cells were preincubated with the test compound for 15 min, and the assay was carried out subsequently according to the procedure described in chapter 4.3. If the test compound interacts with p-gp as a modulator, the intracellular fluorescence intensity increases
be-84 Establishment and application of a calcein-AM efflux assay cause p-gp transport of calcein-AM is blocked and calcein is formed and accumulates in the cells (see Fig. 4.2 c). As a representative of 1st generation modulators, R-verapamil (Fig. 4.9 a) and of 2nd generation modulators, valspodar (Fig. 4.9 b), were examined.
Both are well known as p-gp modulators in the literature (Holland et al. 2003).
N
Figure 4.9: Structures of the 1st generation p-gp modulator verapamil (a) and the 2nd genera-tion p-gp modulator valspodar (b)
Increasing concentrations of either verapamil or valspodar led to an elevated intracel-lular calcein fluorescence (Fig. 4.10 a and Fig. 4.11 a). This is explained by the increasing inhibition of the p-gp activity mediated by the two p-gp modulators. Concentration-response profiles were acquired by incubation with various concentrations of the respec-tive modulator. For that purpose, the geometric means of the obtained histograms were plotted against the modulator concentrations (Fig. 4.10 b and Fig. 4.11 b) and the IC50
values were determined. An IC50 value of 248 µM was calculated using the Hill equation for the p-gp modulation by verapamil. In case of valspodar an IC50 value of 1.63µM was determined. The intensity shift caused by the inhibition of the p-gp mediated efflux is dif-ferent for the two modulators. In the case of valspodar the fluorescence intensity abruptly increased at valspodar concentrations higher than 0.3µM. However, when verapamil was used to inhibit p-gp, the fluorescence intensity increased gradually (for further investiga-tions see chapter 4.5.3). Moreover, the p-gp modulation by verapamil was not maximal as the fluorescence intensity was still increasing at 300 µM of verapamil. Verapamil con-centrations higher than 300µM were not used due to solubility problems. Therefore, the half maximum inhibitory concentration of 248 µM represents only an estimation.
4.5 Applications of the calcein-AM efflux assay 85
Figure 4.10: Modulation of p-gp activity by increasing concentrations of R-verapamil. Fluores-cence intensity increased with increasing R-verapamil concentrations (a). Plotting of geometric means against the R-verapamil concentration leads to concentration response curves (b).
Figure 4.11: Modulation of p-gp activity by increasing concentrations of valspodar. Fluores-cence intensity increased with increasing valspodar concentrations (a). Plotting of geometric means against the valspodar concentration leads to concentration response curves. The calculated IC50 value is 1.63 µM. (b).
86 Establishment and application of a calcein-AM efflux assay In addition to a complete inhibition of the p-gp activity, it is important to regard the limited solubility of the test compounds in the aqueous assay system to calculate precise IC50 values. The results of the p-gp modulation by the immunosuppressant rapamycin in the calcein-AM efflux assay are shown in Fig. 4.12. A correlation between the shift of the fluorescence intensity in channel FL1-H and the added rapamycin could not be established.
The fluorescence intensities decreased at high amounts of rapamycin. Considering the solubility of rapamycin in water (Fig. 4.13), it becomes obvious that rapamycin is only partially soluble in aqueous systems at concentrations higher than 30 µM. In literature
100 101 102 103 104
Figure 4.12: Modulation of p-gp activity by increasing amounts of rapamycin. There is no cor-relation between the shift of the fluorescence intensity and the added rapamycin.
0
Figure 4.13: Solubility of rapamycin in loading buffer determined by turbidimetric analysis at λ600nm. Rapamycin is not soluble in aqueous solutions in concentrations higher than 30 µM.
4.5 Applications of the calcein-AM efflux assay 87 the solubility of rapamycin in water is even given as low as 0.3 µM. Hence, conclusions about the exact concentration of the drug responsible for the p-gp modulation in the calcein-AM efflux assay cannot be drawn.
It becomes clear that accurate results in the calcein-AM efflux assay can only be ob-tained for those substances that are sufficiently soluble in aqueous systems. Furthermore, false negative results are achieved if the affinity of a test compound for p-gp is lower com-pared to the p-gp affinity of calcein-AM (Homolya et al. 1993). In that case calcein-AM is the preferred substrate of p-gp and is being effluxed by p-gp.
In summary, the distinction between p-gp substrates and inhibitors is difficult. Using the calcein-AM efflux assay, the potency of p-gp modulation can be assessed. However, only interactions of p-gp substrates with a higher affinity to p-gp compared to calcein-AM can be detected.
4.5.3 Investigation of transport mechanisms by time-resolved mea-surements
The 1st generation modulator verapamil and the 2nd generation modulator valspodar significantly differ regarding the steepness of the corresponding concentration-response curves (Fig. 4.10 b and Fig. 4.11 b). This could be a hint to different inhibition mechanisms of the p-gp mediated efflux and was investigated by means of time-resolved measurements based on the calcein-AM efflux assay during the incubation period. Flow cytometry offers the possibility to observe changes in the fluorescence over an arbitrary period of time.
In Fig. 4.14 the results of the time resolved measurements are shown. An increase in fluorescence intensity during the incubation period was observed as expected.
Calcein-AM was added after 30 s, and the experiment was aborted after 7.5 min when the fluorescence intensity of the control cells (Kb-V1 wildtype cells) was nearly constant.
The diagrams show the measurements with the most pronounced changes of the slope of the concentration response curves. Immediately after calcein-AM addition, fluorescence rises irrespective of the modulator. The higher the modulator concentration, the faster is the increase in calcein fluorescence. Fluorescence intensities in channel FL1-H reached after 7.5 min correspond to the results depicted in Fig. 4.10 and Fig. 4.11. In conclusion,
88 Establishment and application of a calcein-AM efflux assay
Figure 4.14: Time-resolved measurements of calcein fluorescence during incubation with dif-ferent concentrations of verapamil (VP, a) and valspodar (PSC, b)
with respect to the explanation of the different slopes of the concentrations response curves no additional information could be obtained by this approach. Calculations of typical kinetic parameters (i.e. Michaelis constant Km and maximum velocity vmax) based on the fluorescence intensity increase were impossible as well. Therefore, insight into the kinetic processes could not be gained. It is difficult to assess the transport procedures with respect to kinetic aspects, as the formation of calcein is controlled by two subsequent steps. First, calcein-AM has to be incorporated into the cells and in the second step the ester bonds have to be cleaved to form calcein. The first step is the more interesting one when characterizing the p-gp transport process. However, the separation of the two processes for a calculation is difficult and additional assays have to be carried out to clarify the transport processes.