Patent Application: US-24312902-A

Abstract:
disclosed are unique methods for identifying the lowest , yet optimal , aspirin doses for patients . these methods are also characterized as having little to no aspirin - related side - effects . these methods may be used pre - as well as post - thrombotic event , and employs a patient &# 39 ; s urinary thromboxane b 2 metabolic levels , to identify the patient &# 39 ; s platelet activation level . a patient &# 39 ; s urinary thromboxane b 2 metabolic level is then used to calculate and appropriate and individualized treatment effective for utilizing platelet activation . kits for utilizing this technique are also provided . in yet another particular aspect , the invention provides a method for utilizing a random urine sample obtained from a patient to determine whether a patient or particular individual &# 39 ; s current dosage of aspirin is providing an adequate and appropriate level of inhibition of platelet activation levels , as compared to inhibition levels observed in individuals not taking aspirin .

Description:
the following examples are included to demonstrate preferred embodiments of the invention . it should be appreciated by those of skill in the art that the techniques disclosed in the examples which follow represent techniques discovered by the inventor to function well in the practice of the invention , and thus can be considered to constitute preferred modes for its practice . however , those of skill in the art should , in light of the present disclosure , appreciate that many changes can be made in the specific embodiments which are disclosed and still obtain a like or similar result without departing from the spirit and scope of the invention . one may detect and measure in vitro platelet activation using aggregometry , which detects platelet aggregation ; lumiaggregometry , which detects both aggregation and platelet - specific secretions ; or platelet - activation instrumentation such as the dade - behring pfa - 100 platelet function analyzer . plasma , serum , or urine assays of platelet secretions and arachidonic acid pathway metabolites may be employed in the detection of in vitro platelet activation . assays of plasma or serum txb 2 are used extensively in platelet function research laboratories where specimen management is carefully controlled , but routine clinical measurement is hampered by the in vitro instability of platelets . measurement of aspirin - induced platelet suppression has led to some unexpected findings . using the arachidonic acid metabolite 12 - l - 5 , 8 , 10 - heptadecatrienoic acid ( 12 - hht ) as marker , beving et . al . measured aspirin &# 39 ; s suppression of platelet activity at three dosages , 30 , 75 , and 150 mg / day . ( 42 ) his group demonstrated that , after seven days &# 39 ; treatment , discontinuing the higher dosages triggered a rebound phenomenon . this was reflected in significantly elevated 12 - hht levels persisting for up to six weeks after stopping aspirin therapy . further , they demonstrated the rebound to be greater in patients whose baseline levels of 12 - hht were elevated . they concluded that the degree of platelet suppression and ultimate rebound effect could be controlled by determining the pre - aspirin platelet activity via 12 - hht level analysis and by adjusting aspirin dosage accordingly . the rebound effect did not occur in the 30 mg / day dosage arm . buerke et . al . used bleeding times , platelet aggregometry , and serum txb 2 assays to demonstrate effective aspirin dosages in healthy males . ( 43 ) comparing various dosage combinations , they recommended a loading dose of 300 mg aspirin in combination with 40 mg / day as a maintenance dose to achieve optimum suppression of platelets . loading dosages of 40 or 100 mg failed to elicit significant changes within two hours of administration . whole blood or plasma specimens for platelet metabolite assays require special management because the platelets tend to become activated by changes in temperature , exposure to non - biological surfaces , and mild mechanical agitation . aggregometry assays are qualitative and technology - intensive , and bleeding time tests have poor predictive values . thus , it is necessary to locate a metabolite formed in vivo from the products of platelet activation . ( 45 ) hepatocyte 11 - hydroxy thromboxane dehydrogenase acts upon plasma txb 2 to produce 11 - dehydro txb 2 . ( 46 ) the plasma half - life of 11 - dehydro txb 2 is 45 minutes , and plasma levels remain in the nanogram range , as it is rapidly cleared by the kidney . ( 47 ) the urine concentration of 11 - dehydro txb 2 , however , is plentiful and , as platelets appear to be its only source , proportionally reflects platelet activity within the previous twelve hours . ( 46 ) urine levels of 11 - dehydro txb 2 are frequently elevated in atherosclerosis , the chronic phase following stroke , transient ischemic attack , intracerebral hemorrhage , and atrial fibrillation . ( 48 ) further , 11 - dehydro txb 2 levels are typically decreased in aspirin therapy , even in cases of atherosclerosis , myocardial infarction , and atrial fibrillation . assays for 11 - dehydro txb 2 have intra - and interassay cvs of ≦ 10 % and do not cross - react with txb 2 , 2 , 3 - dinor txb 2 , nor other cyclooxygenase metabolites . ( 49 ). 11 - dehydro txb 2 in randomly collected urine from aspirin and non - aspirin donors urine 11 - dehydro txb 2 was assayed in random urine specimens to : establish a reference range in non - aspirin users . determine whether aspirin therapy is related to levels below the reference range . detect aspirin resistance in individuals taking aspirin therapy . 11 - dehydro txb 2 reference range and results in aspirin treatment random urine specimens were collected from 65 individuals who had avoided aspirin for at least two weeks and from 45 individuals who were taking 81 or 325 mg / day by prescription . each specimen was assayed for 11 - dehydro - txb 2 . specimens were assayed at two dilutions using acetylcholinesterase - linked enzyme immunoassay ; the results were tested for parallelism and averaged . urine creatinine was assayed using the jaffe picrate reaction . to normalize for urinary output , results were expressed in pg 11 - dehydro txb 2 / mg creatinine . results of all populations were compared using the student t - test . within - day variation is 30 % and day to day variation 20 %. in a crossover study , twenty - four healthy individuals who had avoided aspirin for a minimum of ten days collected random baseline urine specimens . they then took one 81 or 325 mg tablet at 8 : 00 am and collected a second urine specimen 24 hours after the aspirin was taken . following a 2 - week washout period dosages were reversed and another baseline and 24 - hours post - aspirin specimen was collected . individuals taking 81 to 325 mg / day aspirin exhibit significantly lower levels of 11 - dehydro - txb 2 than those not taking aspirin . see table 1 . a decision point of 1000 pg 11 - dehydro - txb 2 / mg creatinine yields both false negative and false positive rates of 10 %, the best achievable combination , as shown in table 2 . aspirin effect is ruled in at a decision point of 800 pg / mg or less and ruled out of 1000 pg / mg . fig3 is a histogram that illustrates the clear separation between the non - aspirin and aspirin population . of the aspirin users , six individuals ( 13 %) bad results above the decision point and two exceeded the aspirin effect rule - out point of 1000 . these six may be aspirin - resistant , and are being observed closely for potential thrombotic events . initiation of aspirin therapy causes mean reduction of 68 % for 81 mg / day and 76 % for 325 mg / day as illustrated in fig4 and 5 . there was no significant difference between the 81 mg / day and 325 mg / day suppression levels . an immunoassay has been investigated here that measures the effect of aspirin on platelet function . a stable metabolite of the platelet activation process , 11 - dehydro - txb 2 , can be measured in random urine , bypassing the need for ex vivo platelet function . utilizing this assay we have been able to show a significant difference between individuals taking 81 or 325 mg of aspirin and individuals not taking aspirin . values equal to or less than 800 pg 11 - dehydro - txb 2 / mg creatinine indicates that aspirin usage is sufficiently inhibiting cox - 1 activity . aspirin users with levels over 1000 pg / mg appear to be resistant and not achieving optimal platelet inhibition , as demonstrated in fig3 . utilizing these decision point criteria , we define 13 % of individuals as potential non - responders , coinciding with other published reports . the present findings demonstrate that the range of non - aspirin results is broad and appears bimodally distributed . the higher peak represents increased platelet activation . from the crossover study , seven individuals ( 30 %) had a less than 50 % response to either 81 mg or 325 mg of aspirin . of these , three demonstrated less 11 - dehydro txb 2 excretion at 81 mg than at 325 mg . however , one individual ( subject 24 ) did not demonstrate 50 % reduction to 81 mg or 325 mg of aspirin suggesting aspirin resistance . the inhibitory contribution of dietary and lifestyle habits should always be taken into consideration when interpreting data and may have contributed to these results . further , urine metabolite interference , exercise , and hormonal variation may be responsible for a 20 - 30 % within - day and day - to - day variation aspirin antithrombotic therapy is long - term therapy . the urine 11 - dehydro txb 2 essay is a readily available means for evaluating individual response to aspirin . it is useful for detecting aspirin resistance and for prescribing and monitoring alternate therapies . the assay is also useful in monitoring patient compliance and determining the lowest aspirin dose that produces effective cox - 1 inhibition while avoiding aspirin &# 39 ; s unpleasant , sometimes dangerous side effects . using the 11 - dehydro txb 2 assay as the measure of platelet activity , we recommend clinical trials that 1 ) further compare biologic response to aspirin &# 39 ; s affect on clinical outcomes , 2 ) evaluate whether dosage adjustment according to cox - 1 inhibition will reduce thrombotic events while limiting untoward side effects , and 3 ) determine whether the influence of inflammatory process , reflected by abnormal c - reactive protein levels , are associated with aspirin resistance . aspirin has been proven to prevent secondary arterial thrombosis in a variety of conditions , and may be used as defined here to prevent primary heart attacks and strokes to those who are at risk . proper monitoring of aspirin therapy will lead to more accurate dosing , prevention of side effects , and the management of aspirin resistance . all of the compositions and methods disclosed and claimed herein can be made and executed without undue experimentation in light of the present disclosure . while the compositions and methods of this invention have been described in terms of preferred embodiments , it will be apparent to those of skill in the art that variations may be applied to the composition , methods and in the steps or in the sequence of steps of the method described herein without departing from the concept , spirit and scope of the invention . more specifically , it will be apparent that certain agents , which are both chemically and physiologically , related may be substituted for the agents described herein while the same or similar results would be achieved . all such similar substitutes and modifications apparent to those skilled in the art are deemed to be within the spirit , scope and concept of the invention as defined by the appended claims . the concept of utilizing 11 - dehydrothromboxane measurements to determine individuals in whom aspirin or other platelet modifying compounds is not having a significant platelet inhibiting effect may have several additional applications . the antigen antibody assay currently performed by conventional elisa technique could also be performed using a point of care instrument . the test method would have to be modified such that the reaction could take place and the end point be determined within the confines of the point of care instrument . additionally , it may be possible to adapt the assay for patient self testing utilizing “ dip stick ” technology similar to that used for glucose . urinary thromboxane b 2 metabolite monitors the anti - platelet action of aspirin concern for side effects and laboratory evidence for aspirin “ resistance ” in up to 20 % of individuals make it essential to establish a simple , effective laboratory monitoring system for aspirin &# 39 ; s antithrombotic efficacy . the present example demonstrates the utility of the invention for the purpose in human patients . 11 - dehydrothromboxane b 2 , ( 11 - de - h - txb 2 ), a stable metabolite of platelet activation was measured in random urine specimens from 65 people who were not taking aspirin and 45 who were taking at least 81 mg / day . the means for non - aspirin users was 2080 pg 11 - de - h - txb 2 / mg creatinine , and for aspirin users was 523 , p .& lt ; 0 . 0000 . 1 . receiver - operating characteristic analysis yields an optimum decision point of 1000 pg 11 - de - h - txb 2 in random urine may be used to document aspirin &# 39 ; s platelet - suppressive effects and to identify individuals who may not achieve antithrombotic benefits . the following references , to the extent that they provide exemplary procedural or other details supplementary to those set forth herein , are specifically incorporated herein by reference . 1 . juul - moller s ., edvardsson n ., jahnmatz b ., et al ., lancet 1992 ; 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