A method for assessing aspirin resistance and relative risk of a cardiovascular event in a patient taking aspirin is provided. The concentration of 11-dehydro-thromboxane B2 in a urine sample is measured and compared to a set of standardized quartile concentrations. A concentration of urinary 11-dehydro-thromboxane B2 that falls within the second, third, or fourth quartile is indicative of aspirin resistance and an elevated risk of a recurrent cardiovascular event.

FIELD OF THE INVENTION

The present invention relates to the rapid detection of aspirin resistance as an indicator of the risk of cardiovascular events. Particularly the invention relates to methods and devices for the measurement of suppression of thromboxane generation in response to treatment with aspirin.

BACKGROUND OF THE INVENTION

Cardiovascular disease ranks as a leading cause of mortality and morbidity and represents a significant drain on health resources in many countries.

It is well established that aspirin therapy reduces the risk of a stroke and a first heart attack in healthy individuals, and subsequent heart attacks, strokes, or cardiovascular death in patients with established cardiovascular disease. For example, U.S. Pat. No. 5,240,917 relates to the percutaneous administration of aspirin as an antithrombotic agent.

Studies have shown that aspirin reduces the risk of cardiovascular events by as much as 25% in patients with arterial vascular disease.

Most heart attacks and strokes are caused by blood clots in the heart or brain arteries that form on top of cracked atherosclerotic plaques. These blood clots are predominantly composed of clumped platelets. Aspirin works to prevent blood clot formation at these sites by reducing the ability of the platelets to clump together and form platelet aggregates. Aspirin, also known as acetylsalicylic acid, reduces platelet reactivity because its acetyl group acetylates a key intra-platelet enzyme known as cyclo-oxygenase. Once acetylated, cyclo-oxygenase cannot work to generate thromboxane A2, a substance released from the platelets that serves to activate other platelets and induce them to clump together in aggregates. In order for aspirin to work, therefore, it must reduce thromboxane A2 levels.

Thromboxane A2 has a very short half-life, and is rapidly converted to a stable metabolite called thromboxane B2. Although thromboxane B2 can be measured in blood, the tests can be problematic because platelets can be activated during the collection process. Once activated, the platelets will release thromboxanes that can interfere with the assay. It is therefore preferable to measure thromboxane B2 in the urine.

Even though platelets are an important part of blood clots, rapid technology to measure and predict platelet physiology is lacking. Some accepted laboratory methods include: i) Bleeding Time, a test which is qualitative, not quantitative; ii) Platelet Aggregometry. This test measures the clumping of platelets in response to various stimuli. The test is arduous, time-consuming, and expensive and is not specific for the effects of aspirin on platelet activation. iii) Tests of platelet activation using fluorescent cell sorting techniques. This test can only be done on freshly collected blood and uses size separation to separate platelets from other blood cells and fluorescently-tagged antibodies to identify activated platelets. This test is cumbersome and does not provide aspirin-specific information.

The present invention provides a novel method for assessing platelet function and correlating a readout of that function with the risk of a cardiovascular event.

Aspirin is effective for patients with heart attacks, strokes or peripheral arterial disease or those at risk of these disorders. Aspirin has also been shown to be effective in reducing the incidence of pregnancy-induced hypertension and pre-eclamptic toxicity in women at risk. A role for aspirin in reducing the risk of fatal colon cancer has also been suggested and aspirin may be useful in the treatment of patients with antiphospholipid antibodies, including the lupus anticoagulant. Thus, determining the effectiveness of aspirin treatment in many conditions is an important prognostic factor and may help physicians recommend the most appropriate therapeutic course.

While aspirin is effective in many individuals, approximately 10 to 20% of patients with arterial thrombosis who are treated with aspirin have a recurrent vascular event during long-term follow-up. The failure of these patients to derive a beneficial effect from aspirin is termed "aspirin resistance". There are several possible explanations for aspirin resistance but, whatever the underlying cause, the result is the same. It would obviously be beneficial to be able to identify those patients who are aspirin resistant in order to help physicians determine the advisability of altering the aspirin dose or administering alternative or additional anti-platelet therapies. A need therefore exists for a simple method to accurately determine the response to aspirin and predict the likelihood of onset of a cardiovascular event or other medical condition that would benefit from lowering of thromboxane-A2 levels.

SUMMARY OF THE INVENTION

To be able to identify those people at particular risk of having a recurrent vascular event, so that they can be appropriately treated before a heart attack or stroke occurs, would be of great clinical importance. Former attempts to develop predictive assays, particularly those utilizing blood, have had mixed results. Thus, it is an object of one aspect of the present invention to provide a rapid, non-invasive, reproducible method for determining aspirin resistance. The present invention demonstrates for the first time an association between aspirin resistance, defined as failure of suppression of thromboxane generation, and cardiovascular risk. Determination of the degree of resistance to aspirin is used to predict the risk of a cardiovascular event or other condition that would benefit from lowering thromboxane A2 levels.

The present invention is based on the observation that urinary thromboxane A2 metabolite levels in patients are a surprisingly accurate predictor of recurrent cardiovascular mortality. Thus, determination of metabolite levels in patients may serve to identify those patients at particular risk of developing cardiac ischemia or stroke.

In one aspect of the invention, a method for assessing aspirin resistance in a patient is provided. The method comprises determining the concentration of a metabolite of thromboxane A2 in a sample of body fluid from the patient. The method preferably further comprises the step of comparing the concentration of metabolite in the sample to a predetermined set of concentration quartiles to determine within which quartile the sample falls and determining aspirin resistance based on the quartile of the sample. A concentration of metabolite within the second, third or fourth quartile is indicative of an increased risk of a cardiovascular event.

In another aspect, a method for assessing the risk of a cardiovascular event in a patient is provided. The method comprises determining the level of thromboxane B2 or another thromboxane A2 metabolite in a body fluid, preferably urine. In a preferred embodiment, the method comprises an immunoassay in which a body fluid sample from the patient is contacted with an antibody that specifically binds to a metabolite of thromboxane-A2. The formation of immune complexes is then detected to determine the level of antigen in the sample and the sample level thus obtained is compared to control levels to determine a relative risk factor.

In another aspect, there is provided a method of screening a patient for risk of having a cardiovascular event which comprises contacting a body fluid sample from the patient with an antibody which specifically binds to a thromboxane-A2 metabolite, determining the degree of immune complex formation by immunoassay, and assessing the patient's risk of a cardiovascular event upon the basis of immune complex formation.

In a preferred embodiment, the patient has arterial vascular disease and the method is used to predict the risk of a recurrent vascular event.

In a further preferred embodiment, the metabolite that is measured is thromboxane-B2 metabolite, preferably 11-dehydro thromboxane B2.

In a further aspect, a urine level of this metabolite of greater than 15 ng/mmol creatinine is indicative of risk of a cardiovascular event, more preferably a urine level greater than 21.9 ng/mmol creatinine is indicative of risk of a cardiovascular event and most preferably a urine level greater than 33.8 ng/mmol creatinine is indicative of risk of a cardiovascular event.

The present invention also provides a kit for assessing aspirin resistance. The kit typically comprises (a) an antibody that specifically binds to a thromboxane A2 metabolite, and (b) a labeled sample of the metabolite.

In another aspect of the invention, a device for detecting 11-dehydro thromboxane B2 in a test sample obtained from a mammal is provided. The device comprises an immobilized moiety that specifically binds to 11-dehydro thromboxane B2 and means for visually determining if the level of 11-dehydro thromboxane exceeds a predetermined threshold amount. The moiety that specifically binds 11-dehydro thromboxane B2 is preferably an antibody, an antibody fragment, a single chain antibody or an antigen-binding domain of an antibody. The binding moiety is immobilized on a solid support selected from the group consisting of glass, polystyrene, nylon, cellulose acetate, nitrocellulose and other polymers. The device may be in the format of a dipstick.

In yet another aspect of the invention a method of predicting increased risk of an increased risk for a recurrent cardiovascular event is provided. The method comprises: a) measuring the concentration of 11-dehydro thromboxane B2 in a test urine sample; b) comparing the concentration of the test sample to the quartile concentration of a series of reference samples; c) determining which quartile concentration the test sample falls within; and d) predicting the risk based on the corresponding quartile concentration.

In a particularly preferred embodiment, an immunoassay device for detecting the presence of an analyte is provided. The device comprises a strip that comprises a reagent that specifically binds to the analyte to be tested. The reagent is preferably distributed in patches to detect different amounts of the analyte.

In another embodiment, an immunoassay device is provided which comprises two strips that are reversibly attached. One strip comprises an absorbent material capable of absorbing a predetermined volume of urine and the second strip comprises patches having different amounts of a moiety that specifically binds the analytes to be tested.

In yet another embodiment, an immunoassay device is provided comprising a first strip having an antibody moiety which specifically binds the analyte to be determined and a second strip containing at least one standardized concentration of the analyte to be determined, wherein upon addition of a test sample, analyte in the test sample competes with analyte on the second strip for binding by the antibody moiety.

The methods and devices of the present invention can prospectively identify patients who are relatively resistant to anti-thrombotic doses of aspirin and who may benefit from higher doses of aspirin or additional or alternative therapies that can either block thromboxane production or activity or inhibit another pathway of platelet activation.
 

Claim 1 of 8 Claims

1. A method for assessing aspirin resistance in a patient, said method comprising determining the concentration of 11-dehydro thromboxane B2 in a sample of body fluid from the patient; comparing the concentration of 11-dehydro thromboxane B2 in the sample to a predetermined set of concentration quartiles comprising a first quartile, a second quartile, a third quartile and a fourth quartile, wherein the first quartile comprises concentrations less than 15.1 ng/mmol creatinine, the second quartile comprises concentrations between 15.1 ng/mmol creatinine and 21.8 ng/mmol creatinine, the third quartile comprises concentrations between 21.9 ng/mmol creatinine and 33.7 ng/mmol creatinine, and the fourth quartile comprises concentrations greater than 33.8 ng/mmol creatinine; and determining within which quartile the sample concentration falls; wherein a concentration of 11-dehydro thromboxane B2 within the second, third or fourth quartile is indicative of aspirin resistance and resistance increases with each increasing quartile.

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