Methods for predicting methotrexate polyglutamate levels using pharmacogenetics

Abstract

The present invention provides methods for determining a level of methotrexate polyglutamates (MTXPGs) in an individual undergoing methotrexate (MTX) therapy and for optimizing dose efficacy of MTX therapy in an individual by genotyping the individual at a polymorphic site in at least one folate pathway gene (e.g., a reduced folate carrier (RFC-1) gene, a gamma glutamyl hydrolase (GGH) gene, etc.). Methods are also provided for determining a level of MTXPGs in an individual undergoing MTX therapy and for optimizing dose efficacy of MTX therapy in an individual by generating a pharmacogenetic index based upon the genotype of the individual at a polymorphic site in an RFC-1 gene and/or a GGH gene.

1 . A method for determining a level of methotrexate polyglutamates (MTXPGs) in an individual undergoing methotrexate therapy, said method comprising:
 genotyping said individual at a polymorphic site in at least one folate pathway gene, wherein the presence of a variant allele at said polymorphic site is indicative of said level of MTXPGs in said individual.   
     
     
         2 . The method of  claim 1 , wherein said individual has an inflammatory disease, an autoimmune disease, or cancer. 
     
     
         3 . The method of  claim 2 , wherein said inflammatory disease is rheumatoid arthritis. 
     
     
         4 . The method of  claim 1 , wherein said at least one folate pathway gene is selected from the group consisting of a reduced folate carrier (RFC-1) gene, a gamma glutamyl hydrolase (GGH) gene, and a combination thereof. 
     
     
         5 . The method of  claim 4 , wherein said variant allele in the RFC-1 gene comprises a 80 G to A mutation in the RFC-1 coding region. 
     
     
         6 . The method of  claim 5 , wherein said individual is homozygous for said variant allele. 
     
     
         7 . The method of  claim 6 , wherein said individual has an increased level of MTXPGs relative to wild-type or heterozygous individuals. 
     
     
         8 . The method of  claim 4 , wherein said variant allele in the GGH gene comprises a -401 C to T mutation in the GGH promoter. 
     
     
         9 . The method of  claim 8 , wherein said individual is homozygous for said variant allele. 
     
     
         10 . The method of  claim 9 , wherein said individual has a decreased level of MTXPGs relative to wild-type or heterozygous individuals. 
     
     
         11 . The method of  claim 1 , wherein said level of MTXPGs comprises MTXPG 1-5 . 
     
     
         12 . The method of  claim 1 , wherein said level of MTXPGs comprises MTXPG 3-5 . 
     
     
         13 . The method of  claim 1 , wherein said level of MTXPGs is determined using multivariate linear regression analysis. 
     
     
         14 . The method of  claim 13 , wherein said level of MTXPG 1-5  is determined according to the formula:
   MTXPG 1-5  (nmol/L)=intercept+(β 1 ×Age)+(β 2 ×Dose)+(β 3 ×Route of Administration)+(β 4 ×RFC-1 genotype)+(β 5 ×GGH genotype),   
       wherein
 intercept=−103±16, 
 β 1 =2.47±0.29, 
 β 2 =4.95±0.78, 
 β 3 =16.2±8.0, 
 β 4 =20.8±9.7, 
 β 5 =−21.0±9.6, 
 Route of Administration=0 for oral or 1 for injected, 
 RFC-1 genotype=0 for 80G/G or 80G/A or 1 for 80A/A, and 
 GGH genotype=0 for -401C/C or -401C/T or 1 for -401T/T. 
 
     
     
         15 . The method of  claim 13 , wherein said level of MTXPG 3-5  is determined according to the formula:
   MTXPG 3-5  (nmol/L)=intercept+(β 1 ×Age)+(β 2 ×Dose)+(β 3 ×Route of Administration)+(β 4 ×RFC-1 genotype)+(β 5 ×GGH genotype),   
       wherein
 intercept=−120±24, 
 β 1 =1.53±0.20, 
 β 2 =4.39±0.53, 
 β 3 =15.6±5.4, 
 β 4 =14.8±6.6, 
 β 5 =−15.3±6.5, 
 Route of Administration=0 for oral or 1 for injected, 
 RFC-1 genotype=0 for 80G/G or 80G/A or 1 for 80A/A, and 
 GGH genotype=0 for -401C/C or -401C/T or 1 for -401T/T. 
 
     
     
         16 . The method of  claim 1 , wherein said genotyping is performed on a nucleic acid sample obtained from whole blood. 
     
     
         17 . A method for determining a level of MTXPGs in an individual undergoing methotrexate therapy, said method comprising:
 determining whether said individual is homozygous for a 80 G to A mutation in the RFC-1 coding region, wherein the presence of said homozygous mutation is indicative of an increased level of MTXPGs relative to wild-type or heterozygous individuals.   
     
     
         18 . A method for determining a level of MTXPGs in an individual undergoing methotrexate therapy, said method comprising:
 determining whether said individual is homozygous for a -401 C to T mutation in the GGH promoter, wherein the presence of said homozygous mutation is indicative of a decreased level of MTXPGs relative to wild-type or heterozygous individuals.   
     
     
         19 . A method for optimizing dose efficacy of methotrexate therapy in an individual, said method comprising:
 a) genotyping said individual at a polymorphic site in at least one folate pathway gene to determine a level of MTXPGs in said individual; and   b) recommending a subsequent dose of methotrexate or an analog thereof based upon said level of MTXPGs in said individual.   
     
     
         20 . The method of  claim 19 , wherein said individual has an inflammatory disease, an autoimmune disease, or cancer. 
     
     
         21 . The method of  claim 20 , wherein said inflammatory disease is rheumatoid arthritis. 
     
     
         22 . The method of  claim 19 , wherein said at least one folate pathway gene is selected from the group consisting of a reduced folate carrier (RFC-1) gene, a gamma glutamyl hydrolase (GGH) gene, and a combination thereof. 
     
     
         23 . The method of  claim 22 , wherein said variant allele in the RFC-1 gene comprises a 80 G to A mutation in the RFC-1 coding region. 
     
     
         24 . The method of  claim 22 , wherein said individual is homozygous for said variant allele. 
     
     
         25 . The method of  claim 24 , wherein said individual has an increased level of MTXPGs relative to wild-type or heterozygous individuals. 
     
     
         26 . The method of  claim 25 , wherein said subsequent dose of methotrexate or an analog thereof is maintained or decreased. 
     
     
         27 . The method of  claim 22 , wherein said variant allele in the GGH gene comprises a -401 C to T mutation in the GGH promoter. 
     
     
         28 . The method of  claim 27 , wherein said individual is homozygous for said variant allele. 
     
     
         29 . The method of  claim 28 , wherein said individual has a decreased level of MTXPGs relative to wild-type or heterozygous individuals. 
     
     
         30 . The method of  claim 29 , wherein said subsequent dose of methotrexate or an analog thereof is increased. 
     
     
         31 . A method for determining a level of methotrexate polyglutamates (MTXPGs) in an individual undergoing methotrexate therapy, said method comprising:
 a) determining whether said individual has a variant allele in at least one gene selected from the group consisting of an RFC-1 gene, a GGH gene, and a combination thereof; and   b) generating a pharmacogenetic index based upon the presence or absence of said variant allele,   
       wherein said pharmacogenetic index is indicative of said level of MTXPGs in said individual. 
     
     
         32 . The method of  claim 31 , wherein said level of MTXPGs comprises MTXPG 3-5 . 
     
     
         33 . The method of  claim 31 , wherein said pharmacogenetic index is indicative of an increase in the level of MTXPGs relative to wild-type individuals. 
     
     
         34 . The method of  claim 31 , wherein said pharmacogenetic index is indicative of a decrease in the level of MTXPGs relative to wild-type individuals. 
     
     
         35 . The method of  claim 31 , wherein said pharmacogenetic index is indicative of a probability that the level of MTXPGs is above a median level. 
     
     
         36 . The method of  claim 35 , wherein said median level is about 56 nmol/L. 
     
     
         37 . A method for determining a level of MTXPGs in an individual undergoing methotrexate therapy, said method comprising:
 a) genotyping said individual at a polymorphic site in an RFC-1 gene and a GGH gene;   b) identifying the presence or absence of a variant allele at said polymorphic site;   c) determining whether said individual is wild-type, heterozygous, or homozygous for said variant allele;   d) assigning to said RFC-1 or GGH gene a value of 0 when said individual is wild-type or heterozygous for said variant allele or a value of 1 when the individual is homozygous for said variant allele; and   e) generating a pharmacogenetic index by subtracting the value assigned to said GGH gene from the value assigned to said RFC-1 gene,   
       wherein said pharmacogenetic index is indicative of said level of MTXPGs in said individual. 
     
     
         38 . A method for optimizing dose efficacy of methotrexate therapy in an individual, said method comprising:
 a) determining whether said individual has a variant allele in at least one gene selected from the group consisting of an RFC-1 gene, a GGH gene, and a combination thereof;   b) generating a pharmacogenetic index based upon the presence or absence of said variant allele; and   c) recommending a subsequent dose of methotrexate or an analog thereof based upon said pharmacogenetic index.   
     
     
         39 . The method of  claim 38 , wherein said pharmacogenetic index is indicative of an increase in the level of MTXPGs relative to wild-type individuals. 
     
     
         40 . The method of  claim 39 , wherein said subsequent dose of methotrexate or an analog thereof is maintained or decreased. 
     
     
         41 . The method of  claim 38 , wherein said pharmacogenetic index is indicative of a decrease in the level of MTXPGs relative to wild-type individuals. 
     
     
         42 . The method of  claim 41 , wherein said subsequent dose of methotrexate or an analog thereof is increased.