Methods for direct detection of individual methotrexate metabolites
View Patent ↗The present invention provides a method for determining a level of a methotrexate polyglutamate (MTXPG) in a cellular extract by resolving at least one MTXPG in a cellular extract obtained from a human undergoing methotrexate therapy; and detecting at least one resolved MTXPG, thereby determining a level of the resolved MTXPG. A method of the invention can be useful, for example, for determining a level of MTXPG 3 , MTXPG 4 or MTXPG 5 or for determining a level of each methotrexate polyglutamate species (MTXPG 1 to MTXPG 7 ) present in the cellular extract.
1. A method for determining a level of a methotrexate polyglutamate (MTXPG) in a red blood cell extract, comprising:
(a) resolving at least one MTXPG in a red blood cell extract obtained from a human undergoing methotrexate therapy; and
(b) detecting at least one resolved MTXPG, thereby determining a level of said resolved MTXPG,
wherein detection of said resolved MTXPG occurs without fractionation of said resolved MTXPG from other MTXPG species.
2. The method of claim 1 , comprising determining a level of MTXPG 3 , MTXPG 4 or MTXPG 5 .
3. The method of claim 1 , comprising determining a level of each of MTXPG 1 to MTXPG 7 .
4. The method of claim 1 , which can detect less than 500 nmol of each resolved MTXPG per liter packed red blood cells.
5. The method of claim 1 , which can detect less than 50 nmol of each resolved MTXPG per liter packed red blood cells.
6. The method of claim 1 , wherein said human has an autoimnume disease.
7. The method of claim 6 , wherein said autoimmune disease is arthritis.
8. The method of claim 6 wherein said autoimmune disease is selected from the group consisting of rheumatoid arthritis, polyarthritis, systemic lupus erythematosus, and psoriasis.
9. The method of claim 1 , wherein said human has cancer.
10. The method of claim 1 , wherein step (a) comprises chromatography.
11. The method of claim 10 wherein said chromatography is high performance liquid chromatography (HPLC).
12. The method of claim 1 , wherein step (b) comprises fluorimetry.
13. The method of claim 1 , wherein step (b) comprises spectrophotometry.
14. The method of claim 1 , wherein step (b) comprises mass spectrometry.
15. A method for determining a level of a methotrexate polyglutamate (MTXPG) in a red blood cell extract, comprising:
(a) resolving at least one MTXPG in the red blood cell extract, wherein said MTXPG contains no more than a natural abundance of radioactive isotopes; and
(b) detecting at least one resolved MTXPG, thereby determining a level of said resolved MTXPG,
wherein detection of said resolved MTXPG occurs without fractionation of said resolved MTXPG from other MTXPG species.
16. The method of claim 15 , comprising determining a level of MTXPG 3 , MTXPG 4 or MTXPG 5 .
17. The method of claim 15 , comprising determining a level of each of MTXPG 1 to MTXPG 7 and wherein detection of MTXPG 1 to MTXPG 7 does not require fractionation of the MTXPG 1 to MTXPG 7 species from each other.
18. The method of claim 15 , wherein said red blood cell extract is obtained from a human.
19. The method of claim 15 , which can detect less than 500 nmol of each resolved MTXPG per liter packed red blood cells.
20. The method of claim 15 , which can detect less than 50 nmol of each resolved MTXPG per liter packed red blood cells.
21. The method of claim 15 , wherein said red blood cell extract is from a human undergoing methotrexate therapy.
22. The method of claim 21 , wherein said human has an autoimmune disease.
23. The method of claim 22 , wherein said autoimmune disease is arthritis.
24. The method of claim 22 , wherein said autoimmune disease is selected from the group consisting of rheumatoid arthritis, polyarthritis, systemic lupus erythematosus, and psoriasis.
25. The method of claim 21 , wherein said human has cancer.
26. The method of claim 15 , wherein step (a) comprises chromatography.
27. The method of claim 26 , wherein said chromatography is high performance liquid chromatography (HPLC).
28. The method of claim 15 or 27 , wherein step (b) comprises fluorimetry.
29. The method of claim 15 or 27 , wherein step (b) comprises spectrophotometry.
30. The method of claim 15 or 27 , wherein step (b) comprises mass spectrometry.
31. A method for determining a level of a methotrexate polyglutamate (MTXPG) in a red blood cell extract, comprising:
(a) resolving at least one MTXPG in the red blood cell extract;
(b) irradiating said at least one resolved MTXPG, thereby producing at least one resolved fluorescent MTXPG photolytic product; and
(c) detecting said at least one resolved fluorescent MTXPG photolytic product, thereby determining a level of said MTXPG,
wherein detection of said resolved fluorescent MTXPG photolytic product occurs without fractionation of said resolved fluorescent MTXPG photolytic product from other MTXPG species.
32. The method of claim 31 , comprising determining a level of MTXPG 3 , MTXPG 4 or MTXPG 5 .
33. The method of claim 31 , comprising determining a level of each of MTXPG 1 to MTXPG 7 .
34. The method of claim 31 , wherein step (b) comprises UV irradiating said at least one MTXPG.
35. The method of claim 34 , wherein said UV irradiating utilizes radiation having a wavelength in the range of 225 nm to 275 nm.
36. The method of claim 35 , wherein said UV irradiating utilizes radiation having a wavelength of 254 nm.
37. The method of claim 31 , wherein step (a) comprises chromatography.
38. The method of claim 37 , wherein said chromatography is high performance liquid chromatography (HPLC).
39. The method of claim 38 , wherein step (b) is performed by irradiating said at least one MTXPG in a solvent having 0.05% to 1% H 2 O 2 .
40. The method of claim 39 , wherein step (b) is performed by irradiating said at least one MTXPG in a solvent having 0.1% to 0.3% H 2 O 2 .
41. The method of claim 31 , wherein step (b) comprises irradiating said at least one MTXPG for 0.5 to 60 seconds.
42. The method of claim 41 , wherein step (b) comprises irradiating said at least one MTXPG for 0.5 to 15 seconds.
43. The method of claim 31 , wherein step (c) comprises detecting fluorescence upon excitation in the range of 240 nm to 420 nm.
44. The method of claim 43 , wherein step (c) comprises detecting fluorescence upon excitation in the range of 240 nm to 300 nm.
45. The method of claim 44 , wherein step (c) comprises detecting fluorescence upon excitation with UV radiation at 274 nm.
46. The method of claim 43 , wherein step (c) comprises detecting fluorescence upon excitation in the range of 360 nm to 410 nm.
47. The method of claim 31 , wherein step (c) comprises detecting fluorescence at an emission wavelength in the range of 320 nm to 550 nm.
48. The method of claim 47 , wherein step (c) comprises detecting fluorescence at an emission wavelength in the range of 440 nm to 500 nm.
49. The method of claim 48 , wherein step (c) comprises detecting fluorescence at an emission wavelength of 464 nm.
50. The method of claim 49 , wherein step (c) comprises detecting fluorescence upon excitation with UV radiation at 274 nm and at an emission wavelength of 464 nm.
51. The method of claim 31 , wherein said red blood cell extract is obtained from a human.
52. The method of claim 31 , which can detect less than 500 nmol of each resolved MTXPG per liter packed red blood cells.
53. The method of claim 31 , which can detect less than 50 nmol of each resolved MTXPG per liter packed red blood cells.
54. The method of claim 31 , wherein said red blood cell extract is from a human undergoing methotrexate therapy.
55. The method of claim 54 , wherein said methotrexate is low-dose therapy.
56. The method of claim 54 , wherein said human has an autoimmune disease.
57. The method of claim 56 , wherein said autoimmune disease is arthritis.
58. The method of claim 56 , wherein said autoimmune disease is selected from the group consisting of rheumatoid arthritis, polyarthritis, systemic lupus erythematosus, and psoriasis.
59. The method of claim 54 , wherein said human has cancer.
60. A method of optimizing therapeutic efficacy or reducing toxicity associated with a predetermined methotrexate (MTX) dosage administered to a human, comprising:
(a) resolving at least one, methotrexate polyglutamate (MTXPG) in a red blood cell extract obtained from said human;
(b) detecting at least one resolved MTXPG, thereby determining a level of said resolved MTXPG; and
(c) selecting a MTX dosage or a dose of folic acid or a derivative thereof to be subsequently administered to said human based on said level of resolved MTXPG,
wherein detection of said resolved MTXPG occurs without fractionation of said resolved MTXPG from other MTXPG species.
61. The method of claim 60 , comprising determining a level of MTXPG 3 , MTXPG 4 or MTXPG 5 .
62. The method of claim 60 , comprising determining a level of each of MTXPG 1 to MTXPG 7 .
63. The method of claim 60 , wherein step (c) comprises altering said predetermined MTX dosage subsequently administered to said human.
64. The method of claim 63 , wherein step (c) comprises reducing said predetermined MTX dosage subsequently administered to said human.
65. The method of claim 63 , wherein step (c) comprises increasing said predetermined MTX dosage subsequently administered to said human.
66. The method of claim 60 , wherein step (c) comprises altering a dose of folic acid, or a derivative thereof, subsequently administered to said human.
67. The method of claim 60 , which can detect less than 500 nmol of each resolved MTXPG per liter packed red blood cells.
68. The method of claim 60 , which can detect less than 50 nmol of each resolved MTXPG per liter packed red blood cells.
69. The method of claim 60 , wherein said human has an autoimmune disease.
70. The method of claim 69 , wherein said autoimmune disease is arthritis.
71. The method of claim 69 , wherein said autoimmune disease is selected from the group consisting of rheumatoid arthritis, polyarthritis, systemic lupus erythematosus, and psoriasis.
72. The method of claim 60 , wherein said human has cancer.
73. The method of claim 60 , wherein step (a) comprises chromatography.
74. The method of claim 73 , wherein said chromatography is HPLC.
75. The method of claim 60 or 74 , wherein step (b) comprises fluorimetry.
76. The method of claim 60 or 74 , wherein step (b) comprises spectrophotometry.
77. The method of claim 60 or 74 , wherein step (b) comprises mass spectrometry.