Methods for monitoring methotrexate therapy

1. A method for assessing efficacy of a methotrexate (MTX) dosing regimen in a patient, comprising

(a) determining an intracellular amount of

(i) 5-methyltetrahydrofolate (5CH3THF) polyglutamates (PGs); and

(ii) non-methylated folate PGs

in a biological sample from the patient before or after initiation of MTX treatment or modification of MTX treatment in the patient; and

(b) assessing efficacy of the MTX dosing regimen in the patient based on the amount of 5CH3THF PGs and the amount of non-methylated folate PGs.

2. The method of claim 1 , wherein assessing the efficacy of the MTX dosing regimen comprises one or more of (a) assessing MTX treatment efficacy in the patient; (b) determining an appropriate MTX dosing regimen in the patient; and (c) assessing a propensity for the patient to retain MTX.

3. The method of claim 1 , wherein determining the intracellular amount of 5CH3THF PGs and non-methylated folate PGs is carried out after initiation or after modification of MTX treatment in the patient.

4. The method of claim 1 , wherein the biological sample is treated to convert intracellular folate PGs in the biological sample to monoglutamates (MGs) prior to the determining in step (a).

5. The method of claim 4 , wherein the treating comprises

(a) converting intracellular folate PGs to MGs by use of plasma conjugase; and

(b) deproteinizing the MGs by acid treatment.

6. The method of claim 1 , wherein the non-methylated PGs are selected from the group consisting of tetrahydrofolate (THF) PGs; 5 methylenetetrahydrofolate (5MeTHF) PGs; 5,10 methylenetetrahydrofolate (5,10CH2THF) PGs; dihydrofolate polyglutamates, and 10-formyltetrahydrofolate (10CHOTHF) PGs.

7. The method of claim 1 , wherein the non-methylated PGs are 5,10 methylenetetrahydrofolate (5,10CH2THF) PGs and 10-formyltetrahydrofolate (10CHOTHF) PGs.

8. The method of claim 7 , wherein the biological sample is treated to convert intracellular folate PGs in the biological sample to monoglutamates (MGs) prior to the determining in step (a), and wherein the treating comprises conversion of 5,10CH2THF and 10CHOTHF to 5,10methenyltetrahydrofolate (5,10CH=THF).

9. The method of claim 8 , wherein the determining the intracellular amount of the non-methylated folate PGs comprises separation and detection of 5,10CH=THF and 10CHOTHF.

10. The method of claim 1 , wherein determining the intracellular amount of 5CH3THF PGs and non-methylated folate PGs comprises a method selected from the group consisting of reverse phase high performance liquid chromatography combined with tandem mass spectrometric detection (LC MS/MS), a microbiological assay, a dipstick assay, and liquid chromatography coupled with fluorometric detection.

11. The method of claim 1 , wherein an intracellular 5CH3THF PG level outside a range of between about 100 nmol/L and about 400 nmol/L indicates that the MTX dosage should be adjusted.

12. The method of claims 1 , wherein an intracellular non-methylated folate PG level above about 50 nmol/L indicates that the MTX dosage should be decreased.

13. The method of claim 1 , wherein an intracellular non-methylated folate PG percentage of total folate PG above 40% indicates a need to decrease MTX dosage in the patient.

14. The method of claim 1 , wherein an intracellular 5CH3THF PG level below about 50 nmol/L indicates a likelihood of toxicity in the patient.

15. The method of claim 1 , wherein the patient suffers from an inflammatory disease or cancer.

16. The method of claim 15 , wherein the patient suffers from an autoimmune disorder.

17. The method of claim 1 , wherein the biological sample is selected from the group consisting of whole blood, erythrocytes, reticulocytes, leukocytes, peripheral mononuclear cells, CD4+ cells, CD8+ cells, monocytes, neutrophils, and platelets.

18. The method of claim 1 , wherein assessing the efficacy of the MTX dosing regimen comprises determining an appropriate MTX dosing regimen in the patient.

19. The method of claim 1 , wherein assessing the efficacy of the MTX dosing regimen comprises assessing a propensity for the patient to retain MTX.

20. The method of claim 1 , wherein determining the intracellular amount of 5CH3THF PGs and non-methylated folate PGs is carried out after modification of MTX treatment in the patient.

21. The method of claim 16 , wherein the autoimmune disorder is selected from the group consisting of rheumatoid arthritis, Crohn's disease, ulcerative colitis, and systemic lupus erythematosus.

22. The method of claim 1 , wherein the biological sample comprises whole blood, leukocytes, and/or erythrocytes.