Novel sequence variants of multi-drug resistance genes, mdr1 and mrp1, and recombinant cells expressing mrp1 and mdr1 for assessment of drug penetration and disposition

Abstract

Provided are compositions relating to novel MDR1 polymorphisms, including nucleic acids, polypeptides, and recombinant cells, as well as methods for detection of MDR1 polymorphisms in biological samples and elucidation of the influence of MDR1 polymorphisms on MDR1 protein function. Also provided are a rat MRP1 cDNA and protein, stable cell lines expressing the rat MRP1 protein, and methods of assessing drug penetration or disposition in a cell line expressing a recombinant mammalian MRP1 or MDR1 protein, or a homolog thereof.

1 . An isolated nucleic acid molecule comprising a nucleic acid sequence comprising at least 15 contiguous nucleotides of a variant of the nucleotide sequence set forth in SEQ ID NO:5, wherein:
 (i) the nucleic acid sequence comprises the nucleotide at position 163 of SEQ ID NO:5 and said variant encodes T55P;   (ii) the nucleic acid sequence comprises the nucleotide at position 886 of SEQ ID NO:5 and said variant encodes N296H;   (iii) the nucleic acid sequence comprises the nucleotide at position 1199 of SEQ ID NO:5 and said variant encodes S400I;   (iv) the nucleic acid sequence comprises at least one of the nucleotide at position 1292 or position 1293 of SEQ ID NO:5 and said variant encodes C431L;   (v) the nucleic acid sequence comprises the nucleotide at position 2814 of SEQ ID NO:5 and said variant encodes F938L;   (vi) the nucleic acid sequence comprises the nucleotide at position 3258 of SEQ ID NO:5 and said variant encodes F3258F; or   (vii) the nucleic acid sequence comprises the nucleotide at position 3271 of SEQ ID NO:5 and said variant encodes A1091P.   
     
     
         2 . The isolated nucleic acid molecule of  claim 1 , further comprising a complementary second nucleic acid molecule. 
     
     
         3 . The isolated nucleic acid molecule of  claim 1 , wherein the nucleic acid sequence comprises the nucleotide at position 163 of SEQ ID NO:5 and said variant comprises A163C. 
     
     
         4 . The isolated nucleic acid molecule of  claim 1 , wherein the nucleic acid sequence comprises the nucleotide at position 886 of SEQ ID NO:5 and said variant comprises A886C. 
     
     
         5 . The isolated nucleic acid molecule of  claim 1 , wherein the nucleic acid sequence comprises the nucleotide at position 1199 of SEQ ID NO:5 and said variant comprises G1199T. 
     
     
         6 . The isolated nucleic acid molecule of  claim 1 , wherein the nucleic acid sequence comprises the nucleotide at position 1292 of SEQ ID NO:5 and said variant comprises G1292T. 
     
     
         7 . The isolated nucleic acid molecule of  claim 1 , wherein the nucleic acid sequence comprises the nucleotide at position 1293 of SEQ ID NO:5 and said variant comprises T1293G. 
     
     
         8 . The isolated nucleic acid molecule of  claim 1 , wherein the nucleic acid sequence comprises the nucleotide at position 2814 of SEQ ID NO:5 and said variant comprises T2814G. 
     
     
         9 . The isolated nucleic acid molecule of  claim 1 , wherein the nucleic acid sequence comprises the nucleotide at position 3258 of SEQ ID NO:5 and said variant comprises C3258T. 
     
     
         10 . The isolated nucleic acid molecule of  claim 1 , wherein the nucleic acid sequence comprises the nucleotide at position 3271 of SEQ ID NO:5 and said variant comprises G3271C. 
     
     
         11 . The nucleic acid molecule of  claim 1 , wherein the encoded variant MDR1 protein or a fragment thereof comprises MDR1 activity. 
     
     
         12 . An expression vector comprising the nucleic acid sequence of  claim 1 . 
     
     
         13 . An isolated cell line expressing an MDR1 protein which is a variant of the wild-type MDR1 protein having the amino acid sequence set forth in SEQ ID NO:4, said MDR1 variant having at least one of the amino acid substitutions selected from the group consisting of T55P, N296H, S400I, C431L, F938L, and A1091P. 
     
     
         14 . The isolated cell line of  claim 13 , which is a stable recombinant cell line comprising the expression vector of  claim 12 . 
     
     
         15 . The isolated cell line of  claim 13 , which is an epithelial cell line. 
     
     
         16 . The isolated cell line of  claim 13 , which expresses an MDR1 variant having the S4001 amino acid substitution. 
     
     
         17 . A method for evaluating the influence of a MDR1 polymorphism on MDR1 mediated efflux, uptake, or transcellular transport, comprising:
 (a) contacting a first cell with a therapeutic agent, said first cell expressing an MDR1 variant having at least one of the amino acid substitutions selected from the group consisting of T55P, N296H, S400I, C431L, F938L, and A1091P;   (b) contacting a control cell with the therapeutic agent, wherein said control cell expresses the wild type MDR1 protein;   (c) culturing the first cell and control cell;   (d) determining for each of the first cell and control cell at least one of the uptake, efflux, or transcellular transport of the therapeutic agent; and   (e) comparing the level of uptake, efflux, or transcellular transport of the therapeutic agent in the first cell with that of the control cell.   
     
     
         18 . A stably transformed cell line capable of transcellular transport and comprising a non-viral vector encoding and expressing a recombinant mammalian MRP1 or MDR1 protein, or a homolog thereof. 
     
     
         19 . The cell line of  claim 18 , wherein the cell line is a renal or intestinal epithelial cell line. 
     
     
         20 . The cell line of  claim 18 , wherein the epithelial cell line is LLC-PK1, MDCKII, or Caco-2. 
     
     
         21 . A method for evaluating the contribution of an MRP1 or MDR1 protein to the disposition or penetration of an agent, comprising:
 (a) contacting a stably transformed cell line capable of transcellular transport and comprising an expression vector encoding and expressing a recombinant mammalian MRP1 or MDR1 protein, or a homolog thereof, with the agent;   (b) culturing the contacted cell line; and   (c) determining at least one of the uptake, efflux, or transcellular transport of the agent.   
     
     
         22 . A method for evaluating the influence of a MDR1 or MRP1 polymorphism on MDR1- or MRP1-mediated efflux, uptake, or transcellular transport, comprising:
 (a) contacting a first stably transformed cell capable of transcellular transport and comprising an expression vector encoding and expressing a recombinant mammalian allelic variant of a wild-type MRP1 or MDR1 protein, or a homolog thereof, with a therapeutic agent;   (b) contacting a control cell with the therapeutic agent, wherein said control cell expresses the wild type MRP1 or MDR1 protein corresponding to the allelic variant;   (c) culturing the first cell and control cell;   (d) determining for each of the first cell and control cell at least one of the uptake, efflux, or transcellular transport of the therapeutic agent; and   (e) comparing the level of uptake, efflux, and/or transcellular transport of the therapeutic agent in the first cell with that of the control cell.   
     
     
         23 . The method of  claim 22 , wherein the stably transformed cell comprises an expression vector encoding and expressing a recombinant mammalian allelic variant of a wild-type MDR1 protein, or a homolog thereof. 
     
     
         24 . The method of  claim 22 , wherein the stably transformed cell comprises an expression vector encoding and expressing a recombinant mammalian allelic variant of the wild-type MRP1 protein, or a homolog thereof.