US 11,982,675 B2
Method of assessing ABC transporter activity using fluorescent dye accumulation assay
Edit Szabó, Budapest (HU); Dóra Kovács-Türk, Budapest (HU); Ágnes Telbisz, Budapest (HU); Nóra Kucsma, Budapest (HU); Tamás Horváth, Budapest (HU); Gergely Szakács, Budapest (HU); László Homolya, Telki (HU); Balázs Sarkadi, Budapest (HU); and Várady György, Budapest (HU)
Assigned to CELLPHARMA KFT., Budapest (HU); and TERMÉSZETTUDOMÁNYI KUTATÓKÖZPONT, Budapest (HU)
Appl. No. 16/759,422
Filed by CELLPHARMA KFT., Budapest (HU); and TERMÉSZETTUDOMÁNYI KUTATÓKÖZPONT, Budapest (HU)
PCT Filed Oct. 25, 2018, PCT No. PCT/HU2018/050046
§ 371(c)(1), (2) Date Apr. 27, 2020,
PCT Pub. No. WO2019/081957, PCT Pub. Date May 2, 2019.
Claims priority of application No. P1700432 (HU), filed on Oct. 25, 2017.
Prior Publication US 2020/0284797 A1, Sep. 10, 2020
Int. Cl. C07K 14/705 (2006.01); C12P 17/06 (2006.01); G01N 21/64 (2006.01); G01N 33/50 (2006.01); G01N 33/58 (2006.01); G01N 33/60 (2006.01)
CPC G01N 33/582 (2013.01) [C07K 14/705 (2013.01); C12P 17/06 (2013.01); G01N 21/64 (2013.01); G01N 33/5035 (2013.01); G01N 33/60 (2013.01); G01N 2800/52 (2013.01); G01N 2800/7028 (2013.01)] 23 Claims
 
1. A method of assessing adenosine triphosphate-binding cassette (ABC) transporter activity of an ABC multidrug transporter that transports hydrophobic or amphipathic heterocycles, comprising:
(a) exposing a population of biological specimen cells and a population of negative control cells to a fluorescein derivative ester compound of Formula Ia to load the biological specimen cells and the negative control cells with the ester compound in a loading medium, wherein the ester compound is hydrolyzed to a corresponding fluorescein derivative hydroxy compound by cellular esterases inside both cells, wherein at least the hydroxy compound is fluorescent;

OG Complex Work Unit Chemistry
and wherein
R1 and R2 are independently hydrogen, halogen or pseudohalogen,
R3 and R4 are independently methyl, ethyl or propyl,
L is a linker having 2 to 5 chain atoms selected from the group consisting of C and N, the linker forming a conjugated pi electron system with both pi electrons of the fluorescein moiety and of the PH EN moiety, or the L is selected from the group consisting of aminocarbonyl (carboxamide), urea, thiourea, alkenyl, C2 alkenylamine, C4 alkenylamine and C3 alkenylamide,
PHEN is a phenantrene derivative comprising 1, 2 or 3 ring nitrogens in any of positions 1 to 8 of the phenantrene skeleton wherein L is covalently bound to PHEN in a position corresponding to positions 9 or 10 of the phenantrene skeleton, and
(b) assessing the level of the fluorescein derivative ester compound or the corresponding hydroxy compound or both, accumulating in the specimen cells to obtain a level of the fluorescein derivative compound in the specimen cells and in the population of negative control cells to obtain a negative control level of the fluorescein derivative compound, by assessing the level of fluorescence in the cells,
(c) comparing the level of the fluorescein derivative compound in the specimen cells with a negative control level obtained from the negative control cells, and
(d) wherein a lower level of the fluorescein derivative compound in the specimen cells compared to the negative control level assesses the level of ABC transporter activity in the biological specimen.