WO2011009140A1 - Method of assaying 5-fu - Google Patents
Method of assaying 5-fu Download PDFInfo
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- WO2011009140A1 WO2011009140A1 PCT/US2010/042485 US2010042485W WO2011009140A1 WO 2011009140 A1 WO2011009140 A1 WO 2011009140A1 US 2010042485 W US2010042485 W US 2010042485W WO 2011009140 A1 WO2011009140 A1 WO 2011009140A1
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- inactivator
- fluorouracil
- blood
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/94—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving narcotics or drugs or pharmaceuticals, neurotransmitters or associated receptors
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61B—DIAGNOSIS; SURGERY; IDENTIFICATION
- A61B5/00—Measuring for diagnostic purposes; Identification of persons
- A61B5/15—Devices for taking samples of blood
- A61B5/150007—Details
- A61B5/150015—Source of blood
- A61B5/15003—Source of blood for venous or arterial blood
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61B—DIAGNOSIS; SURGERY; IDENTIFICATION
- A61B5/00—Measuring for diagnostic purposes; Identification of persons
- A61B5/15—Devices for taking samples of blood
- A61B5/150007—Details
- A61B5/150206—Construction or design features not otherwise provided for; manufacturing or production; packages; sterilisation of piercing element, piercing device or sampling device
- A61B5/150236—Pistons, i.e. cylindrical bodies that sit inside the syringe barrel, typically with an air tight seal, and slide in the barrel to create a vacuum or to expel blood
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61B—DIAGNOSIS; SURGERY; IDENTIFICATION
- A61B5/00—Measuring for diagnostic purposes; Identification of persons
- A61B5/15—Devices for taking samples of blood
- A61B5/150007—Details
- A61B5/150206—Construction or design features not otherwise provided for; manufacturing or production; packages; sterilisation of piercing element, piercing device or sampling device
- A61B5/150244—Rods for actuating or driving the piston, i.e. the cylindrical body that sits inside the syringe barrel, typically with an air tight seal, and slides in the barrel to create a vacuum or to expel blood
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61B—DIAGNOSIS; SURGERY; IDENTIFICATION
- A61B5/00—Measuring for diagnostic purposes; Identification of persons
- A61B5/15—Devices for taking samples of blood
- A61B5/150007—Details
- A61B5/150351—Caps, stoppers or lids for sealing or closing a blood collection vessel or container, e.g. a test-tube or syringe barrel
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61B—DIAGNOSIS; SURGERY; IDENTIFICATION
- A61B5/00—Measuring for diagnostic purposes; Identification of persons
- A61B5/15—Devices for taking samples of blood
- A61B5/150007—Details
- A61B5/150374—Details of piercing elements or protective means for preventing accidental injuries by such piercing elements
- A61B5/150381—Design of piercing elements
- A61B5/150389—Hollow piercing elements, e.g. canulas, needles, for piercing the skin
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61B—DIAGNOSIS; SURGERY; IDENTIFICATION
- A61B5/00—Measuring for diagnostic purposes; Identification of persons
- A61B5/15—Devices for taking samples of blood
- A61B5/150007—Details
- A61B5/150374—Details of piercing elements or protective means for preventing accidental injuries by such piercing elements
- A61B5/150381—Design of piercing elements
- A61B5/150503—Single-ended needles
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61B—DIAGNOSIS; SURGERY; IDENTIFICATION
- A61B5/00—Measuring for diagnostic purposes; Identification of persons
- A61B5/15—Devices for taking samples of blood
- A61B5/150007—Details
- A61B5/150374—Details of piercing elements or protective means for preventing accidental injuries by such piercing elements
- A61B5/150534—Design of protective means for piercing elements for preventing accidental needle sticks, e.g. shields, caps, protectors, axially extensible sleeves, pivotable protective sleeves
- A61B5/150694—Procedure for removing protection means at the time of piercing
- A61B5/150717—Procedure for removing protection means at the time of piercing manually removed
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61B—DIAGNOSIS; SURGERY; IDENTIFICATION
- A61B5/00—Measuring for diagnostic purposes; Identification of persons
- A61B5/15—Devices for taking samples of blood
- A61B5/150007—Details
- A61B5/150755—Blood sample preparation for further analysis, e.g. by separating blood components or by mixing
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61B—DIAGNOSIS; SURGERY; IDENTIFICATION
- A61B5/00—Measuring for diagnostic purposes; Identification of persons
- A61B5/15—Devices for taking samples of blood
- A61B5/153—Devices specially adapted for taking samples of venous or arterial blood, e.g. with syringes
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61B—DIAGNOSIS; SURGERY; IDENTIFICATION
- A61B5/00—Measuring for diagnostic purposes; Identification of persons
- A61B5/15—Devices for taking samples of blood
- A61B5/153—Devices specially adapted for taking samples of venous or arterial blood, e.g. with syringes
- A61B5/154—Devices using pre-evacuated means
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K31/00—Medicinal preparations containing organic active ingredients
- A61K31/33—Heterocyclic compounds
- A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
- A61K31/495—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with two or more nitrogen atoms as the only ring heteroatoms, e.g. piperazine or tetrazines
- A61K31/505—Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K31/00—Medicinal preparations containing organic active ingredients
- A61K31/33—Heterocyclic compounds
- A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
- A61K31/495—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with two or more nitrogen atoms as the only ring heteroatoms, e.g. piperazine or tetrazines
- A61K31/505—Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim
- A61K31/513—Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim having oxo groups directly attached to the heterocyclic ring, e.g. cytosine
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P35/00—Antineoplastic agents
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/26—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving oxidoreductase
- C12Q1/32—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving oxidoreductase involving dehydrogenase
Definitions
- the invention generally relates to diagnostic testing, and particularly to a composition and diagnostic method for assaying 5 -fluoro uracil in patient samples.
- 5-fluorouracil is one of the most important chemotherapeutics in cancer treatment. It is commonly used in various chemotherapy regimens for colorectal, stomach, breast and pancreatic cancers. For example, 5-FU alone or in combination regimens such as FOLFOX and FOFIRI is the standard first line therapy in metastatic colorectal cancer. However, 5-FU is frequently associated with severe adverse effects including myelosuppression, mucositis, dermatitis, diarrhea, cardiac toxicity and even death. These adverse effects are caused at least in part by the current approach to dose determination for individual patients. The current standard of care for dosing-Body Surface Area (BSA)-only takes into account a patient's height and weight.
- BSA dosing-Body Surface Area
- BSA does not account for many of the factors that impact 5-FU metabolism and efficacy such as genotype, age, gender, disease state, drug-drug interactions, organ function, and comorbidities. It is therefore not so surprising that blood concentrations of 5-FU in different patients can vary by more than ten fold despite BSA-based equal dose administration.
- the present invention generally provides a method of assaying 5- fluorouracil (5-FU) in a patient sample.
- a method for assaying 5-FU in a sample from a patient administered 5-FU and optionally other therapeutics.
- a sample is obtained from a patient administered 5-FU.
- the sample is then mixed with an inactivator of dihydropyrimidie dehydrogenase (DPD), and the amount of 5-FU is measured in the sample preferably in admixture with the DPD inactivator.
- DPD dihydropyrimidie dehydrogenase
- the present invention provides a method of treating a patient with 5-FU, comprising the steps of: administering to the patient a first dose of 5-fluorouracil; determining the amount of 5-FU in a sample obtained from the patient administered with 5-FU, wherein said sample is admixed in vitro with an inactivator of DPD, and wherein the amount of 5-FU in said sample is measured preferably in the presence of the inactivator; and administering to the patient a second dose of 5-FU.
- the amount of 5-FU administered in the second dose can be determined based on the measured 5-FU level in the patient sample in reference to a predetermined optimal or target 5-FU AUC.
- the present invention also provides methods of processing a blood sample from a patient undergoing a therapy with 5-FU and optionally other drugs, for a 5-FU assay.
- the method includes the following steps: obtaining a blood sample containing 5-FU from a patient administered 5-FU; isolating plasma from the blood sample; admixing the separated plasma with an inactivator of DPD to form a mixture; and sending the mixture to a laboratory for diagnostic testing for the level of 5-FU in the mixture.
- the method comprises the steps of: obtaining a blood sample containing 5-fluorouracil from a patient administered 5-FU; admixing the blood sample with a DPD inactivator to form a mixture; and isolating plasma containing 5-fluorouracil and the inactivator from the mixture.
- the thus obtained plasma can be sent to a laboratory for assaying 5 -FU.
- the method of processing a blood sample from a patient administered 5-FU entails the following steps: obtaining a blood sample containing 5-FU from the patient; admixing the blood sample with a DPD inactivator to form a mixture; and sending the mixture to a laboratory for assaying 5-FU.
- a composition comprising a sample isolated from a patient administered 5-FU, in admixture with an inactivator of dihydropyrimidie dehydrogenase, wherein said sample is blood or plasma or serum, and the inactivator is at a concentration of at least 850 ng/ml in the composition.
- the present invention provides a kit having in a container: (a) a sample (blood, plasma or serum) isolated from a patient administered 5- FU, in admixture with an inactivator of DPD at a concentration of at least 850 ng/ml; and (b) means for cooling said sample to a temperature below 1O 0 C.
- the sample is not frozen within 5 to 10 hours after separation from said patient.
- the inactivator include, e.g., pyrimidine or pyridine derivatives, particularly uracil analogs such as eniluracil and gimeracil.
- 5-FU can be assayed in a sample by any known techniques, e.g., HPLC, Mass-Spec, or immunoassays using antibodies against 5-FU.
- Figure 1 is a diagram demonstrating an embodiment the sample processing method implemented in a clinic setting
- Figure 2 is a graph showing that eniluracil was effective in an preventing degradation of 5-FU in blood samples
- Figure 3 is a graph showing that 5-vinyluracil, 5-iodouracil, and gimeracil were all effective in stabilizing 5-FU in blood samples;
- Figure 4 is a graph showing that low concentrations of gimeracil in blood is effective in inhibiting DPD-mediated degradation of 5-FU;
- Figure 5 is a graph showing the effect of keeping gimeracil in blood without plasma separation for a longer time
- Figure 6 is a drawing showing an embodiment of a transferring syringe assembly
- Figures 7 A and 7B are drawings showing an embodiment of a transferring syringe assembly
- Figure 8 is a drawing showing an embodiment of a transferring syringe assembly.
- a method for processing patient samples containing 5-FU (e.g., from patients treated with 5-FU or a prodrug of 5-FU) for use in a 5-FU assay.
- the method results in increased stability of 5-fluorouracil in the samples and significantly improved accuracy of 5-FU measurements.
- the present invention also provides a method of assaying 5-FU using a patient sample processed according to the processing method.
- the processing method comprises the steps of obtaining a sample containing 5-FU (e.g., from a patient administered 5-FU or a prodrug of 5-FU);
- the 5-FU-containing patient sample used in the present invention can be from any patient administered 5-FU in whom 5-FU level is to be measured. It can also be from patients administered one or more 5-FU prodrugs such as tegafur, doxifluridine, and capecitabine.
- 5-FU is commonly used in chemotherapy for cancer patients including patients with breast cancer, lung cancer, colorectal cancer, pancreatic cancer or head and neck cancer.
- 5-FU is also used in ophthalmic surgery in glaucoma patients as an anti-scarring agent at the trabeculectomy site.
- 5-FU is used topically as a cream for treating Bowen's disease, solar keratoses, and basal cell carcinomas of the skin.
- the methods of the present invention are particularly
- the patient can be a cancer patient undergoing a treatment regimen comprising 5-FU.
- 5-FU is typically administered along with folic acid or leucovorin.
- 5-FU is often used in combination regimens such as FOLFOX (5-FU plus oxaliplatin and leucovorin) and FOLFIRI (5-FU plus irinotecan and leucovorin).
- Newer drugs such as bevacizumab, panitumumab and cetuximab may also be added to the regimens.
- the present invention is applicable in all such therapies and treatment regimens for assaying 5-FU in samples obtained from patients being treated with such regimens, and to optimizing the amount 5-FU administered to a particular patient.
- Patient samples suitable for use in the present invention can be from any part of the body of a patient.
- suitable samples can be tissue or bodily samples, such as tumor tissues, blood, plasma, serum, urine, etc.
- 5 -fluoro uracil is typically administered by intravenous bolus injection or intravenous infusion.
- the administration can be repeated daily, weekly, every four weeks, depending on the choice of treatment regimen.
- Bolus injection can be given over 1 to 10 minutes or over 20 to 60 minutes, and for example, can be repeated for 5 consecutive days, every four week for 6 cycles.
- 5-FU can be administered over several hours, e.g., in 8 hours on a weekly basis.
- Blood samples can be drawn from a patient at any time before, after or during the administration of 5-FU. Often times, measuring blood 5-FU level at a steady state is desirable, in which case blood samples are drawn after a steady state is reached. For example in an eight-hour continuous infusion, a steady state can be reached two hours after the initiation of the infusion.
- blood samples can be collected in any conventional ways of drawing peripheral venous blood.
- a blood sample can be collected into a tube or vial typically used in clinics.
- the amount of blood collected depends on the amount required for a particular 5-FU assay. For example, 1 milliliter to 2 milliliters to 5 milliliters to 10 milliliters can be sufficient for most types of 5-FU assays.
- EDTA or heparin may be mixed with the blood sample to prevent clotting.
- the mixing can be achieved by e.g., using a heparinized tube or an EDTA tube.
- the obtained patient sample containing 5-FU is then admixed with an inactivator of dihydropyrimidine
- DPD dehydrogenase
- the patient sample can be chilled at a temperature of about O 0 C, e.g., for at about 5 minutes or longer before a DPD inactivator is added to the patient sample and mixed together. But preferably, the patient sample is admixed with a DPD inactivator immediately after its separation from the patient's body. The resulting mixture (patient sample in admixture with a DPD inactivator) can then be sent to a laboratory for assaying the amount or level or concentration of 5-FU in the sample.
- the method of processing a patient sample for a 5- FU assay comprises obtaining a blood sample containing 5 -fluoro uracil from a patient administered 5-FU or a 5-FU prodrug, admixing the blood sample with a DPD
- inactivator to form a mixture, and optionally sending the mixture to a laboratory for assaying the amount of 5-FU in the patient sample.
- the method of processing a patient sample for a 5-FU assay comprises obtaining a blood sample containing 5-FU from a patient treated with 5-FU or a 5-FU prodrug, admixing the blood sample with an inactivator of dihydropyrimidie dehydrogenase to form a mixture, isolating plasma containing 5 -FU and the inactivator from the mixture; and optionally sending the plasma to a laboratory for assaying 5-FU level.
- the admixing step takes place immediately after the blood sample is drawn from a patient. That is, after the blood sample is separated from the patient's body, a DPD inactivator should be added to and admixed with the collected blood sample as soon as practical.
- the step of isolating plasma from the blood sample admixed with a DPD inactivator is performed within 4 or 5 hours, more preferably within 1 hour, from the time the blood is drawn.
- the method of processing a patient sample for a 5-FU assay comprises obtaining a blood sample containing 5-FU from a patient treated with 5-FU or a 5-FU prodrug, isolating plasma from the blood sample, admixing the separated plasma with a DPD inactivator to form a mixture, and sending the mixture to a laboratory for assaying 5-FU level.
- Figure 1 demonstrates how the sample processing method can be implemented in a clinic.
- a DPD inactivator syringe (containing DPD inactivator solution) and a transfer device can be prepared as illustrated in Figure 1.
- the cap of a DPD inactivator syringe (see also Figure 6) is unscrewed, the syringe is connected to the BD Transfer Device (see also Figure 7), and the combined unit is set aside until the blood sample has been collected.
- Patient blood can be drawn as shown in Figure 1.
- blood can be drawn into a 6-mL K2-EDTA Vacutainer ® tube while the patient is still being continuously infused with 5-FU at the prescribed infusion rate.
- Blood is drawn from a peripheral vein, not from the infusion port, at least 2 hours after the start of 5-FU continuous infusion. Blood is drawn until the tube is, e.g., at least 3/4 full, and preferably the exact 5-FU dose administered in mg/m 2 and the total infusion duration are recorded.
- a DPD inactivator solution is then immediately dispensed into the blood sample tube as shown in Figure 1. Specifically, the combined syringe-transfer device unit is connected over the K2-EDTA tube and firmly inserted through the tube stopper, after which the syringe plunger is completely depressed. The sample is then gently mixed by inverting the tube at least 3 times. Plasma is separated by centrifugation at 1 ,50Og - 2,00Og for 10 minutes.
- dihydropyrimidine dehydrogenase EC 1.3.1.2
- the term "inactivator of DPD” or paraphrases thereof shall mean any organic or inorganic molecule that inhibits the enzymatic activity of DPD, i.e., its ability to catalyze the reduction of pyrimidine or 5-FU.
- the term “inactivator of DPD” does not encompass 5- FU. Examples of the inactivator include, e.g., pyrimidine derivatives, pyridine derivatives, and babituric acid derivatives.
- the DPD inactivator is a DPD inhibitor, preferably an irreversible DPD inhibitor.
- the DPD inactivator is a uracil derivative, i.e., a uracil compound substituted in the 5- position by a moiety chosen from halogen atom, a C 2 - 4 alkenyl group, a C 2 - 4
- haloalkenyl a C2-6 alkynyl group, a C2-6 haloalkynyl group, a cyano group, a Ci_4 alkyl group and a Ci_4 haloalkyl group.
- the DPD inactivator is a uracil derivative selected from the group consisting of eniluracil, 5-propynyluracil, 5- cyanouracil, 5-bromoethynyluracil, 5-(l-chlorovinyl)uracil, 5-iodouracil, 5- bromovinyluracil, (E)-5-(2-bromovinyl)uracil, 5-hex-l-ynyluracil, 5-vinyluracil, 5- trifluorouracil, 5-bromouracil and 5-(2-bromo-l-chlorovinyl)uracil.
- a uracil derivative selected from the group consisting of eniluracil, 5-propynyluracil, 5- cyanouracil, 5-bromoethynyluracil, 5-(l-chlorovinyl)uracil, 5-iodouracil, 5- bromovinyluracil, (
- the DPD inactivator used is chosen from derivatives of barbituric acid, derivatives of 2,4-dihydroxypyridine and derivatives of 2,6-dihydroxpyridine.
- the DPD inactivator is a pyridine derivative.
- the DPD inactivator used is gimeracil (5-chloro-2,4-dihydroxypyridine) or 3-cyano-2,6- dihy droxypyridine .
- a single DPD inactivator or a combination of two or more DPD inactivators can be admixed with a single patient sample.
- the one or more DPD inactivators can be used at a total amount to reach a final concentration in the admixture sufficient to substantially inhibit the activity DPD in the patient sample (e.g., urine, whole blood, serum or plasma) and to prevent any significant or meaningful 5-FU degradation in the sample, under normal conditions of sample handling and storage and for a sufficient time to allow assays for 5-FU to be completed.
- the total final concentration of any DPD inactivator(s) in the patient sample can range from about 1 ng/ml to about 60 ⁇ g/ml or more.
- one or more DPD inactivators are added and admixed in a patient sample at an amount sufficient to inhibit DPD activity in a patient sample such that there is no more than 10% degradation of 5-FU in the patient sample within about 0.5 hour after the sample is obtained from the patient and placed under room temperature (about 25 0 C), preferably no more than 10% degradation of 5-FU in the patient sample within about 10 hours after the sample is obtained and placed under room temperature.
- the amount of DPD inactivator used can vary depending on the inhibition activity of a particular DPD inactivator and the amount of DPD in a particular sample. Skilled artisans can readily determine an optimal amount of any particular DPD inactivator by routine experimentation, as demonstrated in the examples below.
- the samples processed by the method of the present invention are particularly suitable for use in assays for 5-FU level, as 5-FU degradation in the samples after they are drawn but before assaying is minimized. Accordingly, the present invention further provides a method for assaying 5-FU in a patient sample from a patient administered 5-FU or a prodrug thereof.
- the method generally includes at least the steps of providing a processed patient sample obtained from a patient administered 5-FU or a prodrug thereof, as described above, wherein the sample is admixed in vitro with an inactivator of the enzyme dihydropyrimidie dehydrogenase (DPD), and measuring the amount of 5-FU in the sample. In some embodiments, the amount of 5-FU in the sample is measured in the presence of the DPD inactivator.
- DPD dihydropyrimidie dehydrogenase
- the method of assaying 5-FU comprises obtaining a blood sample containing 5-fluorouracil from a patient administered 5-FU, admixing the blood sample with a DPD inactivator to form a mixture, and measuring the amount of 5-FU in the mixture.
- the method of assaying 5-FU comprises obtaining a blood sample containing 5-FU from a patient treated with 5-FU, admixing the blood sample with a DPD inactivator to form a mixture, isolating plasma containing 5-FU and the inactivator from the mixture, and measuring the amount of 5-FU in the plasma.
- the step of isolating plasma from the blood sample admixed with a DPD inactivator is performed within 4 or 5 hours, more preferably within 1 hour, from the time the blood is drawn.
- the method of assaying 5-FU comprises obtaining a blood sample containing 5-FU from a patient treated with 5-FU, isolating plasma from the blood sample, admixing the separated plasma with a DPD inactivator to form a mixture, and measuring the amount of 5-FU in the mixture.
- the method of assaying 5-FU of the present invention includes measuring 5-FU in a patient sample containing 5-FU obtained from a patient administered 5-FU or a prodrug thereof using an immunoassay, wherein the patient sample is admixed in vitro with a DPD inactivator after being obtained from the patient. That is, the measuring step comprises contacting the patient sample containing 5-FU with an antibody immunore active with 5-FU and determining the binding of 5-FU to the antibody.
- 5-FU antibody is known in the art and is disclosed in, e.g., U.S. Patent No. 7,205, 116.
- the patient sample can be blood, serum, plasma, urine, etc., and preferably a plasma sample prepared by the sample processing methods described above.
- the step of determining the binding of 5-FU to a 5-FU antibody is performed in the presence of a DPD inactivator as described above, e.g., 5-iodouracil, 5-vinyluracil, eniluracil or gimeracil.
- a DPD inactivator e.g., 5-iodouracil, 5-vinyluracil, eniluracil or gimeracil.
- 5-FU was measured using an antibody-based agglutination assay on the Olympus AU680 Analyzer using an antibody disclosed in U.S. Patent No. 7,205, 116.
- a first reagent comprises a plurality of 5-FU molecules conjugated to a polymer or substrate
- a second reagent comprises a plurality of 5-FU antibodies attached to a solid support (e.g., a nanoparticle or bead).
- a solid support e.g., a nanoparticle or bead
- the concentration of 5-FU in a sample is measured based on the absorbance of a sample, which is determined by the amount of aggregation by first and second reagents.
- the instrument is calibrated with a plurality of reference samples with different known concentrations of 5-FU, and a standard curve is generated (Absorbance vs. 5-FU concentration). Absorbance values from individual samples are compared against this curve to determine concentration.
- the step of measuring 5-FU in the assaying method of the present invention comprises contacting a first reagent having a plurality of 5-FU molecules conjugated to a polymer or substrate, and a second reagent having a plurality of 5-FU antibodies attached to a solid support, in the presence of a patient sample containing 5-FU, and determining the aggregation of first and second reagents.
- the patient sample can be blood, serum, plasma, urine, etc., and preferably a plasma sample prepared by the sample processing methods described above.
- the step of determining the binding of 5-FU to a 5-FU antibody is performed in the presence of a DPD inactivator as described above, e.g., 5-iodouracil, 5- vinyluracil, eniluracil or gimeracil.
- a DPD inactivator e.g., 5-iodouracil, 5- vinyluracil, eniluracil or gimeracil.
- the present invention also provides a composition
- a composition comprising (1) an antibody selectively immunoreactive with 5 -FU and (2) a DPD inactivator.
- the composition comprises a patient sample (blood, serum, plasma, urine, etc.) containing 5-FU and obtained from a patient treated with 5-FU or a prodrug thereof, in admixture with (1) an antibody selectively
- the antibody is not substantially immunoreactive with the DPD inactivator, and at an amount in the composition sufficient to bind substantially all 5-FU molecules in the composition.
- the antibody in the composition has less than 25%, preferably less than 15%, cross-reactivity with the DPD inactivator. Examples of the antibody include those disclosed in U.S. Patent No. 7,205, 116.
- Suitable DPD inactivators are as described above, including, but not limited to, e.g., gimeracil, 5-iodouracil, 5-vinyluracil, and eniluracil.
- the amount of such DPD inactivators to be included in the composition is also as described above.
- a DPD inactivator is included in the composition at an amount of at least 600 ng/ml or at least 850 ng/ml.
- the composition comprises (1) a plurality of antibodies attached to a solid support and selectively immunoreactive with 5-FU; (2) a plurality of 5-FU molecules conjugated to a polymer or substrate; and (3) a DPD inactivator.
- the antibodies in the composition have less than 25%, preferably less than 15%, cross-reactivity with the DPD inactivator.
- the composition further includes a patient sample (blood, serum, plasma, urine, etc.) containing 5-FU and obtained from a patient treated with 5-FU or a prodrug thereof.
- the present invention also provides a composition
- a composition comprising a sample isolated from a patient administered 5-FU or a prodrug thereof, in admixture with a DPD inactivator.
- a DPD inactivator is gimeracil.
- the sample is blood or plasma or serum, and the inactivator is at a concentration of at least 600, 850, 1000, 1200, 2000 or 4000 ng/ml in the composition.
- the present invention provides a kit having in a container: a sample (blood, plasma or serum) isolated from a patient administered 5-FU or a prodrug thereof, in admixture with an inactivator of DPD at a concentration of at least 600 ng/ml, 850 ng/ml, 1000 ng/ml, 1200 ng/ml, 2000 ng/ml or 4000 ng/ml.
- the kit further includes means for cooling the sample to a temperature below 1O 0 C.
- cooling packs such as PolarPack ® (Tegrant) and ThermoSafe ® can be used. Regular ice, or dry ice can also be used.
- An assay kit comprising in a compartmentalized container, (1) a plurality of antibodies attached to a solid support and selectively immunoreactive with 5-FU; (2) a plurality of 5-FU molecules conjugated to a polymer or substrate; and (3) a DPD inactivator.
- the antibodies in the composition have less than 25%, preferably less than 15%, cross-reactivity with the DPD inactivator.
- the kit optionally also contains in a separate compartment 5-FU for use as a reference material. The kit would make it possible for a convenient and easy to use method of determining 5-FU level in any given patient sample.
- the patient sample in admixture with a DPD inactivator is not frozen within 1 hour, 2 hours, 3 hours, 4 hours, or within 5 to 48 hours after separation from a patient.
- the method of assaying 5-FU is particularly useful in optimizing treatment with 5-FU or a prodrug of 5-FU wherein blood 5-FU level is determined after one dose of the drug and used as a reference to adjust/determine the follow-on dose of the drug.
- a method of treating a patient with 5-FU or a 5-FU prodrug is also provided.
- the method comprises the steps of (1) administering to a patient in need of treatment a first dose of 5-FU or a 5-FU prodrug; (2) determining the amount of 5-FU in a sample obtained from the patient, wherein the sample is admixed in vitro with an inactivator of dihydropyrimidie dehydrogenase; and (3) administering to the patient a second dose of 5-FU or a prodrug thereof.
- the amount of 5-FU or the prodrug administered in the second dose can be determined based on the measured 5-FU level in the patient sample in reference to a predetermined optimal or target 5-FU AUC.
- the second dose can then be administered in an amount to provide a blood 5-FU concentration within the target concentration.
- the step of determining the amount of 5-FU in a sample obtained from the patient may comprise assaying 5 -FU in a patient sample according to the assaying method of the present invention as described above, which incorporates the sample processing method of the present invention also described above.
- a physician may also order such an assay from a diagnostic lab and determining a 5 -FU level from the assay report sent back from the lab.
- the treatment method of the present invention is used to optimize 5-FU dosing, and comprises the steps of: (1) administering to a patient a first dose of 5-FU, which can be at an amount determined by, e.g., BSA (body surface area), (2) determining the amount of 5-FU in a sample obtained from the patient, wherein the sample is admixed in vitro with an inactivator of DPD, and (3) based on the amount of 5-FU measured in step (2), administering to the patient a second dose of 5-FU in an amount calculated or expected to provide a blood/plasma 5-FU AUC within the range of 20 to 25 mg»h/L.
- a first dose of 5-FU which can be at an amount determined by, e.g., BSA (body surface area)
- BSA body surface area
- the assaying method and various embodiments thereof have been described above and are
- the determining step of the treatment method comprises obtaining a blood sample containing 5-FU from a patient treated with 5-FU, admixing the blood sample in vitro with a DPD inactivator to form a mixture, isolating plasma containing 5-FU and the inactivator from the mixture, and measuring the amount of 5-FU in the plasma.
- the present invention provides the use of an inactivator of DPD for manufacturing an assay kit for measuring 5-FU in a patient sample for guiding the treatment of the patient with 5-FU or a prodrug thereof, wherein the treatment comprises the steps of (1) administering to a patient in need of treatment a first dose of 5-FU or a 5-FU prodrug; (2) determining the amount of 5-FU in a sample obtained from the patient, wherein the sample is admixed in vitro with an inactivator of DPD; and (3) administering to the patient a second dose of 5-FU or a prodrug thereof, in an amount determined based on the amount of 5-FU determined in step (2), and calculated or expected to result in a blood/plasma 5-FU AUC within a predetermined reference range, e.g., the range of 20 to 25 mg»h/L.
- the components of the assay kit are as described above or those below.
- the present invention also provides transferring devices for
- transferring a DPD inactivator to, and admixing the DPD inactivator with, a patient blood sample encompasses the transferring syringe assemblies described below with or without a DPD inactivator therein.
- transferring syringe assemblies 600 and 600' are particularly useful in storing and shipping a DPD inactivator solution to a clinic or testing lab, and transferring the stored DPD inactivator solution to a patient sample.
- Transferring syringe assembly 600 (600 and 600') includes a barrel 610 defining a chamber of the transferring syringe, which may be prefilled with a DPD inactivator solution 620 (e.g., a solution of gimeracil or eniuracil in a DMSO or methanol solvent).
- a DPD inactivator solution 620 e.g., a solution of gimeracil or eniuracil in a DMSO or methanol solvent.
- Barrel 610 has a tip 630 extending from a distal end.
- the tip 630 can be operably connected to a needle at the time of use.
- transferring syringe assembly further includes a tip shield 640 attached to the tip or extending from the distal end of barrel 610. Tip shield 640 extends longitudely over tip 630.
- the transferring syringe assembly 600 includes a tip cap 650 that can be operably connected to tip 630 or tip shield 640.
- tip shield 640 and tip cap 650 have mating grooves and are interlocked together.
- tip shield 640 can have an inward groove and tip cap 650 has an outward groove, or vise versa, such that tip cap 650 is tightly screwed upon and interlocked with tip shield 640 so as to seal the tip 630 of the barrel chamber.
- a plunger 660 is slidably inserted inside the chamber of barrel 610.
- Transferring syringe assembly 600' is the same as transferring syringe assembly 600 except a needle 670 is attached to the syringe tip 630.
- the needle is preferably shielded with a needle cap.
- the needle can be used to puncture another container (vial or tube) containing a patient sample to deliver the DPD inactivator solution into the patient sample container by sliding or pushing plunger 660.
- Figure 7 illustrates another transferring syringe assembly 700, which is same as transferring syringe assembly 600 and 600' except that the tip cap 650 of the transferring syringe assembly 600 is replaced with needle cover 710, or that a needle cover 710' is mounted onto transferring syringe assembly 600'.
- needle cover 710 it includes a generally tubular body 730 coaxially disposed about the needle 740, and having a tubular wall extending along needle 740 and beyond the sharpened tip of the needle.
- the needle cover 710' is same as needle cover 710 except for the lack of the needle 740.
- the needle covers can be operably connected and mounted onto the transferring syringe assembly 600 or 600' forming the transferring syringe assembly 700.
- tip shield 640 of transferring syringe assembly 600 can have an inward groove for interlocking the luer adaptor 720 of needle 740.
- tip shield 640 of transferring syringe assembly 600' can have an inward groove for interlocking the luer adaptor 720' such that needle cover 710' is mounted onto transferring syringe assembly 600' shielding needle 670.
- 720 or 720' can be a connector coaxially mounted about the needle 740.
- the transferring syringe assembly 700 can be used to conveniently and safely transfer a DPD inactivator solution into a patient sample tube as shown in Figure 8.
- the transferring syringe assembly 800 in Figure 8 includes the transferring syringe assembly 700 and a tube 810 containing a patient sample (e.g., blood) wherein the tube 810 is inserted into the needle cover 710 or 710' and needle 740 or 670 has been inserted into the sample tube 810 through the puncturable cap 812 of the tube.
- the plunger 660 can be pushed thereby delivering the DPD inactivator solution into the sample tube, if the the DPD inactivator solution is contained in the transferring syringe assembly 700.
- the present invention provides a kit including, one or more the transferring syringe assemblies described above.
- the kit may or may not include a DPD inactivator in any form (e.g., in solution). If included, the DPD inactivator can be stored in a transferring syringe assembly, or in a separate compartment, and can be at an amount sufficient to inhibit DPD enzymatic activity in a patient blood sample and to prevent 5-FU degradation in the sample.
- the kit may further include one or more of the following items: disposable pipettes, blood drawing tubes, cryovial tubes for receiving and storing plasma samples, transportation tubes, bags with absorbent materials for transporting a patient sample, etc.
- the kit may further include instruction sheets explaining to healthcare professionals how to obtain and process patient samples for the purposes of assaying 5-FU in the samples, which are all described in detail above.
- the kit may include an antibody selectively immunoreactive with 5-FU.
- This kit optionally also contains in a separate compartment 5-FU for use as a reference material.
- the kit comprises in a container, (1) a transferring syringe assembly as illustrated in Figure 6 or Figure 7, (2) a DPD inactivator, and (3) an antibody selectively immunoreactive with 5-FU.
- the kit comprises in a container, (1) a transferring syringe assembly illustrated in Figure 6 or Figure 7, (2) a DPD inactivator, (3) a plurality of antibodies attached to a solid support and selectively immunoreactive with 5-FU; (4) a plurality of 5-FU molecules
- the antibodies in the kit have less than 25%, preferably less than 15%, cross-reactivity with the DPD inactivator.
- the kit optionally also contains in a separate compartment 5-FU for use as a reference material.
- the DPD inactivator is in a solution contained in the barrel of the transferring syringe assembly.
- 5-FU was measured using an antibody-based agglutination assay on the Olympus AU680 Analyzer using an antibody disclosed in U.S. Patent No. 7,205, 116.
- concentration of 5-FU in a sample is determined based on the absorbance of a sample, which is determined by the amount of aggregation by the nanoparticles.
- the system requires two reagents, which when mixed in the absence of free 5-FU will form large aggregates of nanoparticles and scatter light to result in a higher absorption value.
- the agglutination reaction becomes somewhat inhibited and scattering of light is less, causing a lower absorption value.
- the instrument is calibrated twice monthly, and a curve is generated (Absorbance vs. 5 -FU concentration). Absorbance values from individual samples are compared against this curve to determine concentration.
- agglutination assay 95 ⁇ l of a buffer containing 5- FU-labeled amino-dextran is pipetted to a cuvette, followed by 7 ⁇ L of a filtered plasma sample. The sample and reagent are mixed and then incubated for about 3.5 minutes. 95 ⁇ l of 5-FU antibody-conjugated nanoparticles is pipetted into the cuvette and mixed. The complete reaction incubates for about 6.2 minutes. A series of 27 measurements at 600 nm are taken at regular intervals by the AU680 analyzer throughout this process, and 5-FU concentration is calculated using the measurements from two specific time points.
- Olympus analyzer AU680 200 ⁇ L, spin for 15 minutes at 12,500 rpm. Measurements were taken immediately after plasma collection and 24 hours after the initial reading. Plasma was stored at room temperature. Figure 2 shows that eniluracil was effective in preventing degradation of 5-FU in blood samples.
- Tubes were mixed by inversion. Blood tubes incubated at room temperature for 45 minutes. At the appropriate times, blood tubes were centrifuged at 2000xg for 10 minutes at room temperature to separate plasma. Plasma was aspirated immediately following centrifugation, and dispensed into new conical tubes. Plasma samples were run under standard conditions for Olympus AU680 analyzer (200 ⁇ L, spin for 15 minutes at 12,500 rpm). Plasma was stored at room temperature.
- Figure 3 shows that 5-vinyluracil, 5-iodouracil, and gimeracil were all effective in stabilizing 5- FU in blood samples.
- One volume of blood was spiked with a calculated volume of stock 5-FU (at 200 ng/ ⁇ L) for a final concentration of 500 ng/mL, and the other volume of blood was spiked with a calculated volume of stock 5-FU (at 200 ng/ ⁇ L) for a final concentration of 1500 ng/mL.
- Three sets of eight tubes each were prepared to aliquot the spiked blood into.
- a negative control was used that was inoculated with 300 ⁇ L of 30% methanol/water instead of gimeracil.
- the low concentration 5-FU blood was aliquotted, 6 mL each tube.
- the high concentration 5- FU blood was aliquotted to the remaining four tubes per set, as well as to the negative control tube, 6 mL each.
- the eight tubes were then inoculated with either 300 ⁇ L of 0.12 mg/mL gimeracil in 30% MeOH/water or 300 ⁇ L of 0.04 mg/mL gimeracil.
- Blood tubes were inverted to mix the gimeracil, and then sat at room temperature for 45 minutes. Blood tubes were centrifuged at 2000xg for 10 minutes and room temperature. Plasma was aspirated immediately after centrifugation and dispensed into clean cryovials. The WBC/buffy coat was added to designated tubes of plasma, and vortexed to evenly mix.
- Plasma samples were run under standard conditions for the AU680 analyzer (200 ⁇ L, spin for 15 minutes at 12,500 rpm). Measurements were taken immediately after adding WBC/buffy coat ("initial"), as well as 24 hours (“24-hour”), after addition of WBC/buffy coat. For the 24-hour measurement, plasma was stored at room temperature for the first 24 hours of the experiment to simulate overnight ambient shipping. Table 1 below summarizes the results.
- methanol was added to the blood instead of gimeracil, in order to serve as a negative control.
- the blood tubes sat at room temperature for 45 minutes after the addition of the gimeracil or methanol.
- the blood tubes were centrifuged at 2000xg for 10 minutes at room temperature to separate plasma. Plasma was aspirated immediately after centrifugation, and the plasma was dispensed into clean tubes. Plasma samples were run using standard conditions for Olympus AU680 analyzer (200 ⁇ L, spin for 15 minutes at 12,500 rpm). Measurements of 5-FU concentration were taken after plasma collection ("initial") and 24 hours ("24hr") thereafter. For the 24hr measurement, plasma was stored at room temperature during the 24 hours.
- Table 2 summarizes the results. As shown in Table 2 and Figure 4, low concentrations of gimeracil in blood is also effective at inhibiting DPD-mediated degradation of 5-FU. The overall percent difference in 5-FU concentration between the samples containing 200 ng/mL and 600 ng/mL gimeracil is less than 1%.
- methanol only is added to the control blood tube.
- Blood tubes sat at room temperature for 45 minutes, 24 hours, or 48 hours before plasma separation. At the appropriate times, the blood tubes were centrifuged at 2000xg for lOminutes at room temperature for plasma separation. The plasma was aspirated immediately after centrifugation and dispensed into clean cryovials.
- 5-FU assays the plasma samples were run under standard conditions for the Olympus AU680 analyzer, as described above (200 ⁇ L, spin for 15 minutes at 12,500 rpm). Measurements of 5-FU
- Sample collection consists of venous blood drawn at the clinician site between at least 2 hours after initiation of 5-FU treatment and before the end of drug infusion to ensure steady state levels (the average tm for 5-FU is ⁇ 13 minutes).
- the OnDose Sample Stabilizer is injected and mixed into the blood sample immediately following the blood draw, and the blood sample can then remain at RT for up to 4 hours.
- the sample is then centrifuged (1500-200Og, for lOmin). Approximately half of the plasma is transferred to a plasma transport tube, labeled with a barcode, and shipped overnight at ambient temperature to Myriad Genetic Laboratories, Inc. for analysis.
- the OnDose assay is a competitive homogenous two-reagent nanoparticle agglutination immunoassay.
- the first reagent contains 5-FU conjugate with the second reagent consisting of 5-FU-directed, antibody-conjugated nanoparticles.
- the amount of free 5-FU in the plasma inhibits the aggregation of the two assay reagents.
- the amount of light absorbance at certain wavelengths from nanoparticle agglutination depends on the amount of drug in the plasma. This absorbance is compared to a standardized calibration curve for quantitation.
- the quantitative target range as expressed by Area Under the Curve (AUC), based on Gamelin E, et al, J Clin Oncol. 26(13):2099-105 (2008), is calculated from the measured concentration of 5-FU and the infusion duration provided by the clinician on the test request form.
- AUC Area Under the Curve
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Priority Applications (8)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
EP20180417.6A EP3792634A3 (en) | 2009-07-17 | 2010-07-19 | Method of assaying 5-fu |
AU2010273964A AU2010273964A1 (en) | 2009-07-17 | 2010-07-19 | Method of assaying 5-FU |
IN1379DEN2012 IN2012DN01379A (en) | 2009-07-17 | 2010-07-19 | |
CA2768488A CA2768488A1 (en) | 2009-07-17 | 2010-07-19 | Method of assaying 5-fu |
CN2010800415495A CN102648412A (en) | 2009-07-17 | 2010-07-19 | Method of assaying 5-FU |
JP2012520842A JP5863047B2 (en) | 2009-07-17 | 2010-07-19 | Method for assaying 5-FU |
EP10800680A EP2454589A4 (en) | 2009-07-17 | 2010-07-19 | Method of assaying 5-fu |
BR112012000873-0A BR112012000873A2 (en) | 2009-07-17 | 2010-07-19 | method of dosing 5-fluorouracil in a sample of a patient treated with f-fu or a prodrug thereof, method of treating a patient with 5-fluorouracil or a prodrug thereof, method of processing a blood sample of a patient treated with a regimen comprising 5-fluorouracil or a prodrug thereof for a 5-fluorouracil test, method of processing a blood sample from a patient treated with a regimen comprising 5-fluorouracil or a prodrug thereof, composition , transfer kit, test kit, syringe set |
Applications Claiming Priority (4)
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US22658909P | 2009-07-17 | 2009-07-17 | |
US61/226,589 | 2009-07-17 | ||
US32424110P | 2010-04-14 | 2010-04-14 | |
US61/324,241 | 2010-04-14 |
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PCT/US2010/042485 WO2011009140A1 (en) | 2009-07-17 | 2010-07-19 | Method of assaying 5-fu |
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US (1) | US8440442B2 (en) |
EP (2) | EP2454589A4 (en) |
JP (1) | JP5863047B2 (en) |
CN (1) | CN102648412A (en) |
AU (1) | AU2010273964A1 (en) |
BR (1) | BR112012000873A2 (en) |
CA (1) | CA2768488A1 (en) |
IN (1) | IN2012DN01379A (en) |
WO (1) | WO2011009140A1 (en) |
Cited By (1)
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JP2015522801A (en) * | 2012-05-15 | 2015-08-06 | ウェルスタット ダイアグノスティクス,エルエルシー | Clinical diagnostic system |
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EP2715359A4 (en) * | 2011-05-30 | 2015-01-21 | String Therapeutics Inc | Methods and compositions for therapeutic drug monitoring and dosing by point-of-care pharmacokinetic profiling |
CA2857645A1 (en) * | 2011-11-30 | 2013-06-06 | Wellstat Diagnostics, Llc | Assays, antibodies, immunogens and compositions related to 5-fu |
US10809264B2 (en) * | 2014-05-29 | 2020-10-20 | Siemens Healthcare Diagnostics Inc. | Rare molecule detection |
WO2018232031A1 (en) * | 2017-06-14 | 2018-12-20 | Epiphany La | Undergarment padding |
Citations (3)
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US7205116B2 (en) * | 2005-02-08 | 2007-04-17 | Salamone Salvatore J | 5-Fluoro-uracil immunoassay |
US20070134251A1 (en) * | 2004-08-06 | 2007-06-14 | Genentech, Inc. | Assays and methods using biomarkers |
US20070225245A1 (en) * | 1998-09-29 | 2007-09-27 | Buchsbaum Donald J | Viral vector driven mutant bacterial cytosine deaminase gene and uses thereof |
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ME01335B (en) * | 2004-04-29 | 2013-12-20 | Taiho Pharmaceutical Co Ltd | Method for reducing gastrointestinal toxicity due to the administration of tegafur |
MX2007006646A (en) * | 2004-12-03 | 2007-11-16 | Adherex Technologies Inc | Methods for administering dpd inhibitors in combination with 5-fu and 5-fu prodrugs. |
CN101333558A (en) * | 2007-06-29 | 2008-12-31 | 上海裕隆生物科技有限公司 | Kit for detecting 5 fluorouracil medicine insensitive gene chip |
-
2010
- 2010-07-19 CN CN2010800415495A patent/CN102648412A/en active Pending
- 2010-07-19 JP JP2012520842A patent/JP5863047B2/en active Active
- 2010-07-19 BR BR112012000873-0A patent/BR112012000873A2/en not_active IP Right Cessation
- 2010-07-19 AU AU2010273964A patent/AU2010273964A1/en not_active Abandoned
- 2010-07-19 IN IN1379DEN2012 patent/IN2012DN01379A/en unknown
- 2010-07-19 WO PCT/US2010/042485 patent/WO2011009140A1/en active Application Filing
- 2010-07-19 CA CA2768488A patent/CA2768488A1/en not_active Abandoned
- 2010-07-19 EP EP10800680A patent/EP2454589A4/en not_active Ceased
- 2010-07-19 US US12/839,336 patent/US8440442B2/en active Active
- 2010-07-19 EP EP20180417.6A patent/EP3792634A3/en active Pending
Patent Citations (3)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US20070225245A1 (en) * | 1998-09-29 | 2007-09-27 | Buchsbaum Donald J | Viral vector driven mutant bacterial cytosine deaminase gene and uses thereof |
US20070134251A1 (en) * | 2004-08-06 | 2007-06-14 | Genentech, Inc. | Assays and methods using biomarkers |
US7205116B2 (en) * | 2005-02-08 | 2007-04-17 | Salamone Salvatore J | 5-Fluoro-uracil immunoassay |
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See also references of EP2454589A4 * |
Cited By (1)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
JP2015522801A (en) * | 2012-05-15 | 2015-08-06 | ウェルスタット ダイアグノスティクス,エルエルシー | Clinical diagnostic system |
Also Published As
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US20110034488A1 (en) | 2011-02-10 |
EP2454589A1 (en) | 2012-05-23 |
CA2768488A1 (en) | 2011-01-20 |
AU2010273964A1 (en) | 2012-02-02 |
CN102648412A (en) | 2012-08-22 |
BR112012000873A2 (en) | 2019-11-05 |
JP5863047B2 (en) | 2016-02-16 |
JP2012533739A (en) | 2012-12-27 |
IN2012DN01379A (en) | 2015-06-05 |
EP3792634A3 (en) | 2021-06-02 |
EP3792634A2 (en) | 2021-03-17 |
US8440442B2 (en) | 2013-05-14 |
EP2454589A4 (en) | 2012-09-05 |
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