The subject of the present invention is a method of determining the level of transport activity of the ABCBl protein in a tested mammalian cell and the use of a rhodamine X derivative ester, preferably selected from a group encompassing: X-rhodamine-1 ester and X-rhodamine-5F ester, in the embodiment of such a method. This method can be used particularly in searching for new drugs, particularly antineoplastic drugs.

The ABCBl protein is one of the membrane transporters which regulate the rate of therapeutic substance entry into cells, and is for this reason called the ABCBl multidrug resistance protein. In tumour cells, it is often pathologically overexpressed due to which they become insensitive to chemotherapy. The inhibition of the activity of these proteins through the use of inhibitors is one of the strategies in the design of novel anti-neoplastic drugs.

The physiological presence of the ABCBl protein in tissue barrier cells (i.e. the blood-brain, blood-placenta or blood- testes barriers) can significantly alter the distribution of drugs in an organism, blocking a therapeutic agent from its target site.

An evaluation of the activity of the ABCBl protein in a given material or the evaluation of a candidate substrate on the activity of this protein can be of great clinical significance, i.e. during the selection of effective chemotherapy, or a practical use can be found during the design of novel drugs or the evaluation of drug interactions in vitro.

Many functional tests that evaluate the activity of the ABCBl protein that make use of fluorescence are known. Application US20060263834A1 relates to methods of measuring multidrug resistance of tumour cells using fluorescent markers, amongst others rhodamine 123, that penetrate into a cell and antibodies directed against antigens present on the healthy cell surface as well as antigens of tumour cells. Rhodamine 123 freely penetrates into cells and is accumulated in the mitochondria. In tumour cells, the increased expression of ABC proteins causes the removal of rhodamine 123 to the outside and by the same token reduces its intracellular concentration.

Application WO2011/139634 relates to an assay for measuring the activity of proteins responsible for multidrug resistance, in particular membrane transporters, in the presence and absence of their inhibitors. The degree of penetrance of cell membranes by active ingredients is measured. The markers used in the test are fluorescent compounds and others such as calcein AM, pheophorbide A, chloromethylfluorescein diacetate and other xanthan compounds.

Application WO1/998007 relates to a quantitative assay for evaluating the membrane transport of active ingredients and drug resistance. The disclosed assay makes use of fluorescent markers such as rhodamine and its selected derivatives.

Despite the continued progress in the design of methods for measuring the activity of multidrug resistance proteins, there is still a need for perfected techniques that facilitate more precise measurement. In particular, it is desirable to obtain a method based on the use of a fluorescent marker which would be capable of freely diffusing into cells in a non- fluorescent form, would exhibit fluorescent activity shifted towards the red light band of the visible spectrum only inside of the cell, and at the same time would naturally be sequestered in mitochondria and would be selectively removed from the cell by the ABCBl protein. The subject of the present invention is a method of determining the level of transport activity of the ABCBl protein in a tested cell characterised in that a tested cell is incubated with a solution containing an ester of a rhodamine X derivative, and then the fluorescence of the tested cell is measured, wherein the measured level of fluorescence is a measure of the level of transport activity of the ABCBl protein in a tested cell.

The ester used can be a lower order organic acid C1-C5 ester, for example an acetic, methyl or acetoxymethyl ester.

Preferably the ester of a rhodamine X derivative is an acetoxymethyl ester, particularly selected from a group encompassing: X-rhodamine-1 acetoxymethyl ester and X- rhodamine-5F acetoxymethyl ester.

Preferably, a cell from a line in which the ABCBl protein transport activity is detected is put into contact with a solution containing an acetoxymethyl ester a rhodamine X derivative in the presence of an inhibitor of the ABCBl protein, and then a measurement is made of the resulting fluorescence in the tested cell, wherein the change determined in the level of fluorescence is a measure of the effect of the aforementioned inhibitor on the level of transport activity of the ABCBl protein in a tested cell, wherein preferably the inhibitor of the ABCBl protein is a known inhibitor of this protein, particularly one selected from a group encompassing: verpamil and cyclosporin A.

The next the subject of the present invention is the use of a rhodamine X derivative ester, preferably selected from a group encompassing: X-rhodamine-1 acetoxymethyl ester and X- rhodamine-5F acetoxymethyl ester, to determine the level of transport activity of the ABCBl protein in mammalian cell.

Unexpectedly it was determined that an a lower order organic acid ester, for example acetoxymethyl, of a rhodamine X derivative, particularly X-rhodamine-1 acetoxymethyl ester and X-rhodamine-5F acetoxymethyl ester, diffuses into a cell and is hydrolysed in the cytoplasm into an appropriate fluorescent dye, which is a specific substrate of the ABCB1 protein removed by this protein from the cell. At the same time, unexpectedly, the fluorescence of the rhodamine X derivatives (particularly rhodamine X-rhod-1 and X-rhod-5F) obtained in the cytoplasm is preferably significantly shifted towards red wavelengths (emissions above 600 nm) in relation to other known dyes, which facilitates the precise measurement of ABCB1 protein activity and enables the simultaneous use of other fluorescent substances i.e. in the green band, for example for the simultaneous measurement of other multidrug resistance proteins.

In a preferable embodiment the non-fluorescent AM ester of a given rhodamine X penetrates into the tested mammalian cells through the membrane via diffusion. In the cytoplasm it is hydrolysed to the fluorescent anionic form of the dye, which preferentially accumulates in the mitochondria. Over time (in the order of minutes) the overall fluorescence of the cell increases, which can be measured using standard biological fluorometric techniques, such as flow cytometry or fluorescence microscopy, including confocal microscopy. Cells with an increased expression of the ABCB1 protein are capable of ejecting the ester dyes beyond the cell membrane, whereby their fluorescence increases much slower by several orders of magnitude in relation to control cells.

When using the method according to the present invention to test ABCB1 protein inhibitors, the inhibition of ABCB1 activity by a specific inhibitor/ other substrate of the protein (i.e. verpamil or cyclosporin A) causes an increase of intracellular fluorescence in cells overexpressing it, which may be comparable to the level of intracellular fluorescence observed in cells with a low ABCB1 protein expression. By the same token, the increase in the level of cell fluorescence, preferably in a cell overexpressing ABCBl, incubated with an AM ester of X-rhod-1 or X-rhod-5F at an appropriate concentration in the presence of an appropriate dose of a specific an inhibitor in comparison to the level of fluorescence in a cell of the same type incubated only with an appropriate AM ester is a measure of the decrease of ABCBl protein activity due to the inhibitor. For this reason, the increase of the level of intracellular fluorescence observed due to the tested inhibitor of may be a measure of the activity of the tested inhibitor of the ABCBl protein, and thus of its potential therapeutic utility.

Analogously, by replacing the inhibitor with a tested drug, it is possible to evaluate the ability of the ABCBl protein to transport said drug by observing the accumulation of the selected rhodamine X as dependent on the drug's concentration.

An analogous test may be used to determine the sensitivity of the tested cells, i.e. of tumour cells isolated from a patient, to the reduction of their multidrug resistance by known inhibitors of the ABCBl protein. An inhibitor thus selected may be then used to increase the effectiveness of chemotherapy in a given patient.

Example 1. Measurement of the transport activity of the ABCBl protein

Cells of the line MDCKII (canine kidney epithelial cell line derived by Madin and Darby, ATCC CRL-2936™) and the line MDR-MDCKI I (sub-line of MDCKII stably transfected with an expression construct encoding the human ABCBl gene, source: SOLVO Biotechnology, http : //www . solvobiotech . com/products/categories/mdckii-cell- lines) are trypsinized and suspended in a culture medium previously brought to 37 °C (here: DMEM with an addition of 10% v/v foetal bovine serum) to a final density of lxl0 ⁶ /cm ³ . The lines MDCKII and derivatives were purchased from SOLVO Biotechnology (Segedyn, Hungary) . The cell suspension is divided into an appropriate number of measurement tubes, whereafter each tube is supplemented with an appropriate volume of the initial solution of the ester i.e. acetoxymethyl rhodamine X-l at a concentration of 200 μπιοΐ/dm ³ in DMSO, to a final concentration 1 μπιοΐ/dm ³ in the tubes. This is vortexed, incubated for 40 minutes at a temperature of 37°C, whereafter measurements are made using fluorescence parameters appropriate for each marker, i.e. excitation with an argon laser bandwith of 488 nm and an emission filter of 600/10 nm. When testing the effect of inhibitors on dye transport, an appropriate volume of the given inhibitor is loaded into the cytometry tubes, the fluorescent marker is added to a final concentration of 1 μπιοΐ/dm ³ , the cell suspension is added, whereafter this is vortexed and measured immediately. Inhibitory are used at the following concentrations: verpamil - 25 μπιοΐ/dm ³ , K0143 - 10 μπιοΐ/dm ³ , MK571 - 10 μπιοΐ/dm ³ , cyclosporin A - 1 μπιοΐ/dm ³ , fumitremorgin C - 10 μπιοΐ/dm ³ .

Example 2. Evaluation of the effect of selected inhibitors of ABC proteins on X-rhodamine accumulation

Simultaneously, to confirm the role of the ABCB1 protein in the transport of the evaluated rhodamines in a test according to the present invention, we used known selective ABC protein inhibitors. The tests were performed in the presence of an inhibitor, proceeding substantially as in Example 1. In the basal MDCKII line we observed only a relatively small (though statistically significant) increase in the accumulation of X-Rhod-1 following the use of verpamil, cyclosporin and K0143. In the same cells, in the case of X- Rhod-5F, we observed a significant effect on the accumulation only in the case of cyclosporin A (Fig. 1 and Fig. 2) .

Figure 1 represents the effect of the selected compounds on the accumulation of X-Rhod-1 in the cell line MDCKII where: wer 10 - verpamil 10 μπιοΐ/dm ³ , wer 25 - verpamil 25 μπιοΐ/dm ³ , K0143 10 μπιοΐ/dm ³ , MK571 10 μπιοΐ/dm ³ , Csp A - cyclosporin A 10 μπιοΐ/dni ³ , and Fum C - fumitremorgin C 10 μmol/dm ³ . Average ± SD, n=3, measurement of fluorescence in the PE-Texas Red channel. The statistical analysis was performed using ANOVA.

Whereas Figure 2 represents the effect of the selected compounds on the accumulation of X-Rhod-5F in the cell line MDCKII; wer 10- verpamil 10 μπιοΐ/dm ³ , Wer 25- verpamil 25 μπιοΐ/dm ³ , KO 143 10 μπιοΐ/dm ³ , MK571 10 μπιοΐ/dm ³ , Csp A- cyclosporin A 10 μπιοΐ/dm ³ , Fum C- fumitremorgin C 10 μπιοΐ/dm ³ . Average ± SD, n=3, measurement of fluorescence in the PE-Texas Red channel. The statistical analysis was performed using ANOVA.

In the line MDCKII-BCRP, transformed with the human ABCG2 gene, we observed no significant effect of the inhibitors used on the accumulation of any of the two evaluated X rhodamines (Fig. 3 and Fig. 4) .

Figure 3 represents the effect of the selected compounds on the accumulation of X-Rhod-1 in the cell line MDCKII-BCRP, wer 10 - verpamil 10 μπιοΐ/dm ³ , wer 25 - verpamil 25 μπιοΐ/dm ³ , K0143 10 μπιοΐ/dm ³ , MK571 10 μπιοΐ/dm ³ , Csp A - cyclosporin A 10 μπιοΐ/dm ³ , Fum C- fumitremorgin C 10 μπιοΐ/dm ³ . Average ± SD, n=3, measurement of fluorescence in the PE-Texas Red channel. The statistical analysis was performed using ANOVA.

Figure 4 represents the effect of the selected compounds on the accumulation of X-Rhod-5F in the cell line MDCKII-BCRP; wer 10 - verpamil 10 μπιοΐ/dm ³ , wer 25 - verpamil 25 μπιοΐ/dm ³ , K0143 10 μπιοΐ/dm ³ , MK571 10 μπιοΐ/dm ³ , Csp A - cyclosporin A 10 μπιοΐ/dm ³ , Fum C- fumitremorgin C 10 μπιοΐ/dm ³ . Average ± SD, n=3, measurement of fluorescence in the PE-Texas Red channel-A. The statistical analysis was performed using ANOVA.

However, we observed the clear effect of the specific

ABCB1 inhibitors on the increased accumulation of X rhodamine derivatives in the line MDCKII-MDR. Verpamil, in a dose dependent manner, enhanced the accumulation of the tested dyes in the line by as much as 650% of the control for X-Rhod-1 and 450% of the control for X-Rhod-5F. We also observed a clear effect of cyclosporin A on the increased accumulation of rhodamine X in the line MDCKI I-MDR : 650% of the control for X- Rhod-1 and 700% of the control for X-Rhod-5F. We did not observe a statistically significant effect of ABCG2 inhibitors (K0143, fumitremorgin C) nor ABCC1 inhibitors (MK571) on the transport of any of the evaluated compounds (Fig. 5 and Fig. 6) .

Figure 5 represents the effect of the selected compounds on the accumulation of X-Rhod-1 in the cell line MDCKI I-MDR; wer 10- verpamil 10 μπιοΐ/dm ³ , wer 25- verpamil 25 μπιοΐ/dm ³ , KO 143 10 μπιοΐ/dm ³ , MK571 10 μπιοΐ/dm ³ , Csp A- cyclosporin A 10 μπιοΐ/dm ³ , Fum C- fumitremorgin C 10 μπιοΐ/dm ³ . Average ± SD, n=3, measurement of fluorescence in the PE-Texas Red channel. The statistical analysis was performed using ANOVA.

Figure 6 represents the effect of the selected compounds on the accumulation of X-Rhod-5F in the cell line MDCKI I-MDR; wer 10- verpamil 10 μπιοΐ/dm ³ , wer 25- verpamil 25 μπιοΐ/dm ³ , KO 143 10 μπιοΐ/dm ³ , MK571 10 μπιοΐ/dm ³ , Csp A- cyclosporin A 10 μπιοΐ/dm ³ , Fum C- fumitremorgin C 10 μπιοΐ/dm ³ . Average ± SD, n=3, measurement of fluorescence in the PE-Texas Red channel. The statistical analysis was performed using ANOVA.

An analogous test may be performed using the method according to the present invention in order to determine the sensitivity of a tested cell, i.e. of tumour cells isolated from a patient, on overcoming their multidrug resistance by known inhibitors of the ABCB1 protein.

Confirmation of the localization of rhodamine X in mitochondria

We evaluated the localization of the evaluated rhodamine X derivatives in the cell line MDCKII and MDCKI I-MDR using confocal microscopy. Rhodamine 123, with a mitochondrial localization known from literature, was used as a positive control. We observed that the evaluated X rhodamines selectively stain oblong, granular structures present throughout the volume of the cytoplasm of living cells, similar to rhodamine 123. It is to be assumed that these are mitochondria .