Enzymatic Measurement of Imatinib Mesylate
The present invention provides a method for the enzymatic measurement of protein tyrosine kinase inhibitors in biological samples. The present invention also provides assay reagents and packaged kits useful for performing enzymatic measurement of imatinib mesylate and other bcr-abl protein kinase inhibitors in biological samples.
1 . An enzymatic assay for determining the presence of a chemotherapeutic agent selected from the group consisting of imatinib, imatinib salts and chemotherapeutically active metabolites thereof by means of the conversion of NADH to NAD comprising:
a) forming in an aqueous medium a mixture containing a sample suspected of containing said chemotherapeutic agent with an abl protein tyrosine kinase and a polypeptide substrate containing NADH, which substrate is capable of converting NADH to NAD by means of said enzyme, said enzyme being present in an amount effective to convert the NADH in the substrate to NAD;
b) incubating said mixture under conditions which cause said chemotherapeutic agent in the sample to react with said enzyme; and
c) thereafter measuring the amount of the NADH in the substrate which is converted to NAD whereby the presence of the chemotherapeutic agent can be determined.
2 . The enzymatic assay of claim 1 , wherein the sample is a human sample.
3 . The enzymatic assay of claim 2 , wherein said abl kinase enzymes are v-abl protein tyrosine kinase, bcr-abl protein tyrosine kinase or mixtures thereof.
4 . The enzymatic assay of claim 3 , wherein the chemotherapeutic agent is imatinib mesylate.
5 . The enzymatic assay of claim 1 , wherein the protein substrate in addition to NADH contains:
a) adenosine triphosphate;
b) a tyrosine containing polypeptide capable of phosphorolation by tyrosine kinase;
c) phosphoenyl pyruvate;
d) lactate dehyrogenase; and
e) pyruvate kinase.
6 . The enzymatic assay of claim 5 , wherein said abl kinase enzymes are v-abl protein tyrosine kinase, bcr-abl protein tyrosine kinase or mixtures thereof.
7 . The enzymatic assay of claim 6 , wherein said tyrosine containing polypeptide is capable of phosphorolation is bovine serum albumin.
8 . The enzymatic assay of claim 1 , wherein the amount of chemotherapeutic agent, in said sample, is measured by comparing the amount of NADH converted to NAD in the sample with that amount of NADH converted to NAD in one or more samples containing known amounts of said chemotherapeutic agent utilizing the same assay.
9 . The enzymatic assay of claim 8 , wherein said abl kinase enzymes are v-abl protein tyrosine kinase, bcr-abl protein tyrosine kinase or mixtures thereof.
10 . The enzymatic assay of claim 9 , wherein the substrate, in addition to NADH, contains:
a) adenosine triphosphate;
b) a tyrosine containing polypeptide capable of phosphorolation by tyrosine kinase;
c) phosphoenol pyruvate;
d) lactate dehydrogenase; and
e) pyruvate kinase.
11 . The enzymatic assay of claim 10 , wherein said tyrosine containing polypeptide is capable of phosphorolation is bovine serum albumin.
12 . A kit for conducting an enzymatic assay comprising predetermined amounts of the following reagents:
a) a first enzyme component containing abl protein tyrosine kinase;
b) pyruvate kinase;
c) a tyrosine containing polypeptide capable of phosphorolation by tyrosine kinase;
d) lactate dehydrogenase;
e) phosphoenol pyruvate;
f) NADH; and
g) ATP;
wherein the reagents are packaged in at least two separate containers within the kit, with the ATP being in a separate container from the one or more containers containing the first enzyme component.
13 . The kit of claim 12 , wherein said abl protein is v-abl protein tyrosine kinase, bcr-abl protein tyrosine kinase or mixtures thereof.
14 . The kit of claim 13 , wherein the reagents are present in the containers in an aqueous medium containing a buffer and a stabilizer.
15 . The kit of claim 14 , wherein the reagents are present in two separate containers, one of said containers containing ATP, the other container containing reagents b), c), d), e), f) and the first enzyme component of reagent a).
16 . The kit of claim 4 , wherein the reagents are present in three separate containers, one container containing ATP, the second container containing the first enzyme component and the third container containing the reagents of b), c), d), e) and f).
17 . The kit of claim 12 , wherein said tyrosine containing polypeptide capable of phosphorolation is bovine serum albumen.
18 . The kit of claim 12 , wherein said kit is for determining the presence of a chemotherapeutic agent which inhibits abl protein tyrosine kinase
19 . The kit of claim 18 , wherein said abl protein is v-abl protein tyrosine kinase, bcr-abl protein tyrosine kinase or mixtures thereof.
20 . The kit of claim 19 , wherein the kit contains an additional separate container containing a sample with a predetermined amount of the chemotherapeutic agent to be detected.
21 . The kit of claim 19 , wherein the reagents are present in the containers in an aqueous medium containing a buffer and a stabilizer.
22 . The kit of claim 19 , wherein the reagents are present in two separate containers, one of said containers containing ATP, the other container containing reagents b), c), d), e), f), g) and the first enzyme component of reagent a).
23 . The kit of claim 22 , wherein the kit contains an additional separate container containing a sample with a predetermined amount of a chemotherapeutic agent.
24 . The kit of claim 19 , wherein the reagents are present in three separate containers, one container containing ATP, the second container containing the first enzyme component and the third container containing the reagents of b), c), d), e) and f).
25 . The kit of claim 24 , wherein the chemotherapeutic agent is imatinib, imatinib salts and chemotherapeutically active metabolites thereof.
26 . The kit of claim 25 , wherein said tyrosine containing polypeptide capable of phosphorolation is bovine serum albumen.
27 . The kit of claim 25 , wherein the kit contains a separate container containing a sample with a predetermined amount of a chemotherapeutic agent.