CELLS AND METHODS FOR EVALUATING THE ACTIVITY OF ORGANIC ANION TRANSPORTING POLYPEPTIDE 2A1 (OATP2A1)
Disclosed herein are cells overexpressing a human organic anion transporting polypeptide 2A1 (OATP2A1) and methods of measuring OATP2A1-mediated cellular uptake of a compound, methods of determining if a compound modulates OATP2A1-mediated activity, methods for determining if a cell expresses OATP2A1, as well as kits for carrying out the aforementioned methods.
1 . A cell overexpressing a human organic anion transporting polypeptide 2A1 (OATP2A1), wherein the cell expresses at least 100 times more OATP2A1 mRNA compared to a control cell.
2 . (canceled)
3 . The cell of claim 1 , wherein the cell expresses at least 1000 times more OATP2A1 mRNA compared to the control cell.
4 . The cell of claim 1 , wherein the cell is transfected with an OATP2A1 nucleic acid.
5 . The cell of claim 4 , wherein the cell is stably transfected with the OATP2A1 nucleic acid.
6 . The cell of claim 1 , wherein the cell is a human embryonic kidney 293 (HEK293) cell.
7 . The cell of claim 1 , wherein the cell can be cultured for at least 20 passages without a significant reduction in OATP2A1 expression.
8 . The cell of claim 1 , wherein the cell further comprises a seminaphthorhodafluor (SNARF) substrate or a 6-carboxyfluorescein (6-CF) substrate.
9 . The cell of claim 8 , wherein the SNARF substrate is a 5, 6-Carboxy-SNARF.
10 . The cell of claim 9 , wherein the 5, 6-Carboxy-SNARF is bound to the OATP2A1.
11 . The cell of claim 1 , wherein the cell has an OATP2A1-mediated activity that is at least 20 times greater than an OATP2A1-mediated activity of a control cell.
12 . The cell of claim 1 , wherein the cell has an OATP2A1-mediated uptake of a SNARF substrate that is at least 100 times greater than an OATP2A1-mediated uptake of a SNARF substrate in a control cell.
13 . The cell of claim 11 , wherein the OATP2A1 transporter-mediated activity or uptake is measured at a physiological pH ranging from about 6.5 to about 8.
14 .- 15 . (canceled)
16 . A method of measuring an organic anion transporting polypeptide 2A1 (OATP2A1)-mediated cellular uptake of a compound, the method comprising:
a) contacting a cell that overexpresses the OATP2A1 with the compound; and,
b) measuring the amount of the compound within the cell, wherein the amount of compound within the cell is indicative of the OATP2A1-mediated cellular uptake of the compound.
17 . A method of determining if a compound inhibits an OATP2A1 or modulates an OATP2A1 mediated activity, the method comprising:
a) contacting a cell that overexpresses the OATP2A1 with a fluorescent substrate;
b) contacting a cell that overexpresses the OATP2A1 with the compound and the fluorescent substrate; and
c) measuring an amount of the fluorescent substrate in the cell from step (a) and measuring an amount of the fluorescent substrate in the cell from step (b), wherein a decreased amount or a changed amount of the fluorescent substrate in the cell from step (b) indicates that the compound inhibits the OATP2A1, or that the compound modulates the OATP2A1-mediated activity.
18 .- 19 . (canceled)
20 . The method of claim 17 , wherein an increase in the amount of the fluorescent substrate in the cell from step (b) indicates that the compound increases the OATP2A1-mediated activity and a decrease in the amount of the fluorescent substrate in the cell from step (b) indicates that the compound decreases the OATP2A1-mediated activity.
21 . The method of claim 17 , wherein the fluorescent substrate comprises a seminaphthorhodafluor (SNARF) substrate, a 5, 6-Carboxy-SNARF substrate or a 6-carboxyfluorescein (6-CF) substrate.
22 .- 23 . (canceled)
24 . A method of determining if a cell of interest expresses OATP2A1, the method comprising:
a) contacting the cell of interest with a 5, 6-Carboxy-SNARF substrate;
b) contacting a control cell that does not express OATP2A1 with the 5, 6-Carboxy-SNARF substrate; and
c) comparing an amount of the 5, 6-Carboxy-SNARF substrate within the cell of interest to an amount of the 5, 6-Carboxy-SNARF substrate within the control cell, wherein an increased amount of 5, 6-Carboxy-SNARF substrate in the cell of interest compared to the control cell indicates that the cell of interest expresses the OATP2A1.
25 . The method of claim 16 , wherein the cell overexpresses at least 100 times more OATP2A1 mRNA compared to a control cell.
26 . The method of claim 16 , wherein the cell is extracted from a mammalian tissue or human tissue.
27 . The method of claim 16 , wherein the compound comprises a new chemical entity (NCE), a nonsteroidal anti-inflammatory drug (NSAID), a modulator of eicosanoid receptor, a modulator of prostanoid receptor, or a modulator of prostaglandin transporter (PGT).
28 .- 30 . (canceled)