Methods for optimizing clinical responsiveness to methotrexate therapy using metabolite profiling and pharmacogenetics

Abstract

The present invention provides methods for optimizing clinical responsiveness to chemotherapy in an individual through genotypic analysis of polymorphisms in at least one gene. The methods of the present invention may further comprise determining the level of at least one long-chain methotrexate polyglutamate (MTXPG) in a sample obtained from the individual. The present invention also provides methods for generating a pharmacogenetic index for predicting clinical responsiveness to chemotherapy in an individual through genotypic analysis of polymorphisms in at least one gene. In addition, the present invention provides methods for optimizing therapeutic efficacy of chemotherapy in an individual by calculating the level of at least one long-chain MTXPG in a sample obtained from the individual.

1 . A method for optimizing clinical responsiveness to anti-folate therapy in a human, said method comprising genotyping said human at a polymorphic site in a thymidylate synthase (TS) gene, wherein the presence of a variant allele at said polymorphic site is indicative of a characteristic clinical responsiveness to said anti-folate therapy. 
     
     
         2 . The method of  claim 1 , further comprising genotyping said human at a polymorphic site in at least one other gene selected from the group consisting of a folate pathway gene, a purine synthesis gene, a cytokine synthesis gene, and a pyrimidine synthesis gene. 
     
     
         3 . The method of  claim 1 , wherein said anti-folate is methotrexate (MTX). 
     
     
         4 . The method of  claim 1 , wherein said human has a disease selected from the group consisting of cancer, an inflammatory disease, and an autoimmune disease. 
     
     
         5 . The method of  claim 1 , wherein said human has rheumatoid arthritis. 
     
     
         6 . The method of  claim 1 , wherein the presence of the variant allele in said TS gene is indicative of superior clinical responsiveness to said anti-folate therapy. 
     
     
         7 . The method of  claim 1 , wherein the variant allele in said TS gene is associated with decreased TS activity or expression. 
     
     
         8 . The method of  claim 1 , wherein the presence of the variant allele in said TS gene is indicative of inferior clinical responsiveness to said anti-folate therapy. 
     
     
         9 . The method of  claim 1 , wherein the variant allele in said TS gene is associated with increased TS activity or expression. 
     
     
         10 . The method of  claim 1 , wherein said polymorphic site is located in a non-coding region of said TS gene. 
     
     
         11 . The method of  claim 10 , wherein said non-coding region is a promoter. 
     
     
         12 . The method of  claim 1 , wherein said variant allele in said TS gene comprises a two 28 base pair tandem repeat in the promoter. 
     
     
         13 . The method of  claim 12 , wherein the presence of said variant allele is indicative of superior clinical responsiveness to said anti-folate therapy. 
     
     
         14 . The method of  claim 12 , wherein the homozygosity of said variant allele is indicative of superior clinical responsiveness to said anti-folate therapy. 
     
     
         15 . The method of  claim 1 , further comprising genotyping said human at a polymorphic site in a reduced folate carrier (RFC-1) gene, an aminoimidazole carboxamide ribonucleotide transformylase (ATIC) gene, or a combination thereof. 
     
     
         16 . The method of  claim 1 , further comprising resolving at least one long-chain methotrexate polyglutamate (MTXPG) in a sample obtained from said human and determining a level of said at least one long-chain MTXPG, wherein the level of said at least one long-chain MTXPG is indicative of a characteristic clinical responsiveness to said anti-folate therapy. 
     
     
         17 . The method of  claim 16 , wherein said at least one long-chain MTXPG is selected from the group consisting of MTXPG 3 , MTXPG 4 , MTXPG 5  (SEQ ID NO: 12), and combinations thereof. 
     
     
         18 . The method of  claim 16 , wherein said at least one long-chain MTXPG is MTXPG 3 . 
     
     
         19 . The method of  claim 18 , wherein the level of MTXPG 3  is predictive of the level of MTXPG 3-5  (SEQ ID NO: 13). 
     
     
         20 . The method of  claim 16 , wherein a level of MTXPG 3  greater than about 60 nmol/L is indicative of superior clinical responsiveness to said anti-folate therapy. 
     
     
         21 . The method of  claim 16 , wherein a level of MTXPG 3  less than about 40 nmol/L is indicative of inferior clinical responsiveness to said anti-folate therapy. 
     
     
         22 . The method of  claim 16 , wherein said sample is red blood cells. 
     
     
         23 . The method of  claim 16 , wherein said resolving comprises chromatography. 
     
     
         24 . The method of  claim 23 , wherein said chromatography is high performance liquid chromatography (HPLC). 
     
     
         25 . The method of  claim 16 , wherein the level of said at least one long-chain MTXPG is determined using fluorimetry, spectrophotometry, or mass spectrometry. 
     
     
         26 . A method for optimizing clinical responsiveness to anti-folate therapy in a human, said method comprising:
 a) genotyping said human at a polymorphic site in a TS gene and, optionally, at least one other gene selected from the group consisting of a folate pathway gene, a purine synthesis gene, a pyrimidine synthesis gene, and a cytokine synthesis gene;   b) identifying the presence or absence of a variant allele at said polymorphic site(s);   c) determining whether said human is wild-type, heterozygous, or homozygous for said variant allele at said polymorphic site(s); and   d) generating a pharmacogenetic index by calculating the sum of said wild-type, heterozygous, and homozygous variant alleles, wherein said pharmacogenetic index is indicative of a characteristic clinical responsiveness to said anti-folate therapy.   
     
     
         27 . The method of  claim 26 , wherein said pharmacogenetic index is indicative of superior clinical responsiveness to said anti-folate therapy. 
     
     
         28 . The method of  claim 26 , wherein said pharmacogenetic index is indicative of inferior clinical responsiveness to said anti-folate therapy. 
     
     
         29 . The method of  claim 26 , wherein said at least one other gene is selected from the group consisting of an RFC-1 gene, an ATIC gene, and a combination thereof. 
     
     
         30 . The method of  claim 29 , wherein said pharmacogenetic index is generated by calculating the sum of:
 (i) heterozygous or homozygous variant alleles for said TS gene; and   (ii) heterozygous and homozygous variant alleles for said ATIC gene; and/or   (iii) homozygous variant alleles for said RFC-1 gene.   
     
     
         31 . A method for optimizing clinical responsiveness to anti-folate therapy in a human, said method comprising:
 a) genotyping said human at a polymorphic site in a TS gene and, optionally, at least one other gene selected from the group consisting of a folate pathway gene, a purine synthesis gene, a pyrimidine synthesis gene, and a cytokine synthesis gene;   b) identifying the presence or absence of a variant allele at said polymorphic site(s);   c) if present, determining whether said variant allele is homozygous at said polymorphic site(s); and   d) generating a pharmacogenetic index by calculating the sum of said homozygous variant alleles; wherein said pharmacogenetic index is indicative of a characteristic clinical responsiveness to said anti-folate therapy.   
     
     
         32 . The method of  claim 31 , wherein said pharmacogenetic index is indicative of superior clinical responsiveness to said anti-folate therapy. 
     
     
         33 . The method of  claim 31 , wherein said pharmacogenetic index is indicative of inferior clinical responsiveness to said anti-folate therapy. 
     
     
         34 . The method of  claim 31 , wherein said at least one other gene is selected from the group consisting of an RFC-1 gene, an ATIC gene, and a combination thereof. 
     
     
         35 . A combination of tests for the purpose of predicting whether a human afflicted with, or at risk of developing, rheumatoid arthritis will be responsive to anti-folate therapy, comprising a first test for the presence of a polymorphism in a TS gene in combination with a second test for the presence of a polymorphism in a gene selected from the group consisting of RFC-1 and ATIC. 
     
     
         36 . The combination of  claim 35 , comprising a first test for the presence of a polymorphism in a TS gene in combination with a second test for the presence of a polymorphism in an RFC-1 gene and a third test for the presence of a polymorphism in a ATIC gene. 
     
     
         37 . The combination of  claim 35 , wherein said polymorphism in a TS gene comprises a two 28 base pair tandem repeat in the promoter. 
     
     
         38 . The combination of  claim 35 , wherein said anti-folate is methotrexate. 
     
     
         39 . A method of providing useful information for evaluating whether a human afflicted with, or at risk of developing, rheumatoid arthritis will be responsive to anti-folate therapy, comprising:
 detecting the presence or absence in said human of a first polymorphism in a TS gene, and   providing a result of said first polymorphism detection to an entity that evaluates the result and provides an evaluation of whether said human will be responsive to anti-folate therapy.   
     
     
         40 . The method of  claim 39 , further comprising:
 detecting the presence or absence in said human of a second polymorphism in a gene selected from the group consisting of RFC-1 and ATIC, and   providing results of said first and second polymorphism detection to an entity that evaluates the results and provides an evaluation of whether said human will be responsive to anti-folate therapy.   
     
     
         41 . The method of  claim 39 , further comprising:
 detecting the presence or absence in said human of a second polymorphism in an RFC-1 gene;   detecting the presence or absence in said human of a third polymorphism in an ATIC gene; and   providing results of said first, second, and third polymorphism detection to an entity that evaluates the results and provides an evaluation of whether said human will be responsive to anti-folate therapy.   
     
     
         42 . The method of  claim 39 , wherein said first polymorphism is a two 28 base pair tandem repeat in the promoter. 
     
     
         43 . The method of  claim 39 , wherein said anti-folate is methotrexate. 
     
     
         44 . A collection of results for the purpose of predicting whether a human afflicted with, or at risk of developing, rheumatoid arthritis will be responsive to anti-folate therapy comprising:
 (i) information about the presence or absence of a TS gene polymorphism in said human in combination with   (ii) information about the presence or absence of a second polymorphism in said human, wherein said second polymorphism is in a gene selected from the group consisting of RFC1 and ATIC.   
     
     
         45 . The collection of  claim 44 , comprising:
 (i) information about the presence or absence of a TS gene polymorphism in said human in combination with   (ii) information about the presence or absence of an RFC-1 gene polymorphism in said human, and   (iii) information about the presence of absence of an ATIC gene polymorphism in said human.   
     
     
         46 . The collection of  claim 44 , wherein said TS gene polymorphism is a two 28 base pair tandem repeat in the promoter. 
     
     
         47 . The collection of  claim 44 , wherein said anti-folate is methotrexate.