Method of assaying 5-FU
Inventors
Roa, Benjamin • Colvin, Carrie • Overfield, Michael • McLeod, Howard • Salamone, Salvatore
Assignees
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Publication Number
US-8440442-B2
Publication Date
2013-05-14
Expiration Date
Abstract
The present invention provides methods, compositions, devices and kits for assaying 5-FU.
Core Innovation
The invention relates to diagnostic assay methods for measuring 5-fluorouracil in a whole blood sample from a patient administered 5-fluorouracil or a prodrug thereof. The sample is obtained from the patient and is admixed in vitro with an inactivator of dihydropyrimidine dehydrogenase (DPD) at an amount sufficient to inhibit dihydropyrimidine dehydrogenase activity in the sample, such that there is no more than 10% degradation of 5-fluorouracil within about 0.5 hour after the sample is obtained from the patient.
The disclosed approach further includes processing whole blood for the 5-fluorouracil assay by admixing with a first inactivator of dihydropyrimidine dehydrogenase and isolating plasma containing 5-fluorouracil and the inactivator, or by admixing with a second inactivator to form a second mixture. In each case, the sample is admixed with a dihydropyrimidine dehydrogenase inactivator at an amount sufficient to inhibit dihydropyrimidine dehydrogenase activity, with the same degradation performance target of no more than 10% degradation within about 0.5 hour after obtaining the sample.
The invention specifies that the dihydropyrimidine dehydrogenase inactivator is a pyrimidine or pyridine derivative, including uracil/pyridine derivatives such as eniluracil and gimeracil. Additional described inactivators include 5-vinyluracil and 5-iodouracil, as well as other listed DPD inactivators. The document characterizes assay technologies for measuring 5-fluorouracil, including HPLC/LC-MS and antibody-based immunoassays, including competitive homogenous two-reagent nanoparticle agglutination immunoassay using 5-FU antibodies with low cross-reactivity.
The described system also supports sample transport and cooling using cooled, non-frozen sample preparations and kit components that include a DPD-inactivator transfer syringe and cooled sample preparation. The document further states that extended inhibitor contact before plasma separation can artificially increase measured 5-fluorouracil, and it describes performance constraints such as maintaining no more than 10% degradation within about 0.5 hour, and in an extended constraint, within about 10 hours under room temperature conditions.
Claims Coverage
Independent claims are directed to methods for assaying and processing a whole blood sample to measure 5-fluorouracil while preventing DPD-mediated degradation, using a dihydropyrimidine dehydrogenase inactivator with a defined degradation performance target (no more than 10% degradation within about 0.5 hour). Across the independent claims, two inventive-feature themes are present: in vitro admixing with a DPD inactivator for degradation control, and whole-blood processing that optionally includes plasma isolation with the same degradation target.
In vitro admixing with a dihydropyrimidine dehydrogenase inactivator with a 0.5-hour degradation constraint
A method of assaying 5-fluorouracil in a whole blood sample obtained from a patient administered 5-fluorouracil or a prodrug thereof by admixing the sample in vitro with an inactivator of dihydropyrimidine dehydrogenase after being obtained from the patient, at an amount sufficient to inhibit dihydropyrimidine dehydrogenase activity such that there is no more than 10% degradation of 5-fluorouracil in the sample within about 0.5 hour after the sample is obtained from the patient.
Whole-blood processing with optional plasma isolation using a dihydropyrimidine dehydrogenase inactivator with a 0.5-hour degradation constraint
A method of processing a whole blood sample from a patient treated with a regimen comprising 5-fluorouracil or a prodrug thereof for a 5-fluorouracil assay by obtaining a whole blood sample containing 5-fluorouracil and processing it by either admixing the sample with a first inactivator of dihydropyrimidine dehydrogenase and isolating plasma containing 5-fluorouracil and the inactivator, or admixing the sample with a second inactivator of dihydropyrimidine dehydrogenase to form a second mixture, wherein the sample is admixed with the first or second inactivator at an amount sufficient to inhibit dihydropyrimidine dehydrogenase activity such that there is no more than 10% degradation of 5-fluorouracil within about 0.5 hour after the sample is obtained from the patient.
The independent claims cover in vitro admixing of patient-derived whole blood with a DPD inactivator to inhibit DPD activity and maintain no more than 10% 5-fluorouracil degradation within about 0.5 hour, and processing whole blood for the assay with optional plasma isolation while retaining the same 0.5-hour, 10% degradation constraint.
Stated Advantages
Prevents DPD-mediated degradation of 5-fluorouracil in the sample such that there is no more than 10% degradation within about 0.5 hour after sample obtainment.
Allows 5-fluorouracil to be assayed from a patient whole blood sample administered 5-fluorouracil or a prodrug thereof while controlling degradation performance.
Supports extended stability performance in some conditions, including no more than 10% degradation within about 10 hours under room temperature conditions.
Documented Applications
Diagnostic assay for measuring 5-fluorouracil in patient whole blood/plasma/serum after administration of 5-fluorouracil or a prodrug thereof.
Processing of a whole blood sample for a 5-fluorouracil assay, including isolating plasma containing 5-fluorouracil and the DPD inactivator.
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