Fibrinolytic potential: a test of pleural fluid to predict outcomes and guide dosing in fibrinolytic therapy

Patent metadata

Field	Value
Publication number	US-10175255-B2
Application number	US-201615086623-A
Country	US
Kind code	B2
Filing date	Mar 31, 2016
Priority date	Mar 31, 2015
Publication date	Jan 8, 2019
Grant date	Jan 8, 2019

Abstract

An assay that informs precision-based intrapleural fibrinolytic therapy (IPFT) is disclosed. Provided is a simple assay based on measurement of the Fibrinolytic Potential (FP)—total fibrinolytic activity in pleural fluid when plasminogen activator inhibitors (PAIs) are neutralized and endogenous plasminogen (PLG) is activated. The assay is used to determine FP in baseline pleural fluids of patients undergoing IPFT with agents such as tPA or scuPA to determine the dose and dose intervals for the particular patient. Pleural fluids are also collected after IPFT to confirm that intrapleural fibrinolysis is inhibited and endogenous PLG accumulates in the pleural fluid. Inhibition of intrapleural fibrinolysis in combination with its increase after supplementation of pleural fluid with a plasminogen activator indicates whether or not the subject should be treated with more or higher doses of an IPFT drug.

First claim

What is claimed is:

1. An assay method for measuring a mammalian subject's fibrinolytic potential (FP) in a sample of pleural fluid of the subject, comprising:
(a) incubating said pleural fluid sample with detectably labeled fibrin bound to a solid support,
(b) adding to the sample of (a) an activator of plasminogen (PLG), and
(c) adding to separate solid containers to which said labeled fibrin is bound in varying concentrations of
(i) a fibrinolytic enzyme or
(ii) PLG and said PLG activator
and incubating said containers in parallel with, and under the same condition as said sample, to generate a standard curve of FP arbitrary units (FP A.U.) of fibrinolytic activity from which the sample FP value is determined,

wherein the amount of label released per unit time or during a preset time when compared with the standard curve is a measure of said subject's FP.

2. The method of claim 1 wherein DNA is incorporated into the bound, labeled fibrin.

3. The method of claim 1 wherein the detectably labeled fibrin is labeled with a detectable label selected from the group consisting of:
(a) a fluorescer or fluorogen,
(b) a chromophore or chromogen,
(c) a phosphorescer,
(d) a chemiluminescer,
(e) a bioluminescer, and
(f) a radionuclide.

4. The method of claim 3 wherein the fibrin is detectably labeled with the fluorescer fluorescein.

5. The method of claim 1 wherein the solid support is a polymer selected from the group consisting polystyrene, polypropylene, polyethylene, dextran, nylon, polyacrylamide, polyvinylidene difluoride, natural cellulose, modified cellulose, nitrocellulose, and agarose.

6. The method of claim 5 wherein the solid support is in the form of a multiwell plate to which the labeled fibrin is bound.

7. The method of claim 1 wherein the subject is a human.

8. The method of claim 1 wherein, in step (c), the containers comprise said PLG and PLG activator.

9. The method of claim 1 wherein, in step (c), the containers comprise said fibrinolytic enzyme.

10. The method of claim 9 wherein the fibrinolytic enzyme is plasmin.

11. A method of predicting the severity of a pleural disease or condition and treating it by selecting an effective dose or dosing interval with an intrapleural fibrinolytic therapeutic (IPFT) agent in a mammalian subject suffering from a disease or condition in need of the IPFT agent, which method comprises:
(a) performing the assay method of claim 1 on a pleural fluid sample from the subject to determine the FP of the subject and using the standard curve which is defined over a range of from 0 to 10 FP A.U. of fibrinolytic activity to obtain the subject's FP value, and
(b) considering the subject's clinical condition, to make a clinical decision concerning the state of disease and/or treatment to be selected for the subject,
wherein, depending on the FP A.U. values of the standard curve:
(i) if the subject's FP is between 0 and 0.1 FP A.U., the probability of successful IPFT treatment is low, and a determination that the subject should be treated with an alternative treatment approach is made and the subject treated accordingly; or
(ii) if the subject's FP is between 0.1 FP A.U. and 5 FP A.U., a determination that further doses or a higher dose of the IFPT agent should be administered for effective treatment of the disease or condition is made and the subject treated accordingly; or
(iii) if the subject's FP is equal to or greater than 5 FP A.U., a determination that no further dose nor higher dose of the IPFT agent is required for effective treatment of the disease or condition is made and the subject treated accordingly.

12. The method of claim 11 wherein the subject is a human.

13. The method of claim 12 wherein the disease or condition is empyema, complicated parapneumonic pleural effusion, loculation occurring in association with malignant pleural effusion, loculation associated with collagen vascular disease, or loculation associated with chronic organized hemothorax.

14. The method of claim 13 wherein the IPFT agent is an activator of endogenous plasminogen selected from the group consisting of tissue plasminogen activator (tPA) and urokinase plasminogen activator (uPA).

15. The method of claim 11 wherein DNA is incorporated into the fibrin that is bound to said solid support.

16. The method of claim 11 wherein the fibrin is detectably labeled with fluorescein.

17. The method of claim 11 wherein the solid support is in the form of a multiwell plate to which the labeled fibrin is bound.

18. The method of claim 11 wherein, in step (c) of the assay method, the containers comprise said PLG and PLG activator.

19. The method of claim 11 wherein, in step (c), of the assay method, the containers comprise said fibrinolytic enzyme.

20. A kit for measuring the fibrinolytic potential (FP) of a mammalian subject in a sample of pleural fluid of the subject, the kit comprising:
(a) detectably labeled fibrin, bound to a solid support;
(b) an activator of plasminogen (PLG);
(c) reagents for measuring release of detectable label from said labeled, bound fibrin as a measure of fibrinolysis of the labeled fibrin wherein the amount of label released per unit time or during a preset time interval is a measure of said subject's FP,
(d) separate containers to be incubated in parallel with, and under the same condition as the sample, said containers comprising varying concentrations of
(i) a fibrinolytic enzyme, or
(ii) PLG and said PLG activator,
for generating a standard curve; and,

(e) instructions for using the kit to determine said FP.

21. The kit of claim 20 further comprising DNA that is incorporated into the fibrin that is bound to said solid support.

22. The kit of claim 20 wherein the detectably labeled fibrin is labeled with a detectable label selected from the group consisting of:
(a) a fluorescer or fluorogen,
(b) a chromophore or chromogen,
(c) a phosphorescer,
(d) a chemiluminescer,
(e) a bioluminescer, and
(f) a radionuclide.

23. The kit of claim 22 wherein the fibrin is detectably labeled with the fluorescer fluorescein.

24. The kit of claim 20 wherein the solid support is a polymer selected from the group consisting of polystyrene, polypropylene, polyethylene, dextran, nylon, polyacrylamide, polyvinylidene difluoride, natural cellulose, modified cellulose, nitrocellulose, and agarose.

25. The kit of claim 24 wherein the solid support is in the form of a multiwell plate to which the labeled fibrin is bound.

26. The kit of claim 20 wherein, in part (d), the containers comprise said PLG and PLG activator.

27. The kit of claim 20 wherein, in part (d), the containers comprise said fibrinolytic enzyme.

28. The kit of claim 27 wherein the fibrinolytic enzyme is plasmin.

29. The kit of claim 20 for, a method comprising
(A) predicting the severity of a pleural disease or condition in the subject based on the subject's determined FP and/or
(B) selecting an effective dose of, or dosing interval for, an intrapleural fibrinolytic therapeutic (IPFT) agent in said mammalian subject that is suffering from a disease or condition in need of treatment with the IPFT agent,

which kit comprises
(a) instructions for predicting said severity of said pleural disease or condition and for selection of said effective doses or dosing interval base

Assignees

• Univ Texas

Inventors

• Komissarov Andrey A
• Florova Galina
• Idell Steven

Classifications

• G01N33/86 Primary

  involving blood coagulating time {or factors, or their receptors} · CPC title