Method for determining FXIII levels as a prognostic biomarker in acute myocardial infarction

Abstract

A diagnostic method for determining prognosis of a myocardial infarcted patient, wherein the amounts of FXIII protein are determined on the day of myocardial infarction (t0) and at least on the following three days (t1 to t3), wherein a lowering of FXIII amount on any one of t0 to t3 below a threshold value is indicative of an increased risk of poor prognosis.

Claims

1. A method for predicting prognosis of and treating a myocardial infarcted patient, the method comprising: providing at least four biological samples of the infarcted patient, the at least four samples being collected on the day of myocardial infarction (t0) and at least on the following three days (t1 to t3); determining the amounts of Factor XIII (FXIII) protein in each biological sample; and administering FXIII to said patient if the patient has a lowering of FXIII protein amount in any one of t0 to t3 biological samples below a threshold value which is indicative of a poor prognosis in the absence of said administering or any other intervention/treatment.

2. The method according to claim 1, wherein the method further comprises: providing at least two further biological samples of the infarcted patient, the at least two further samples being collected on at least the fourth and fifth day following myocardial infarction (t4 to t5); determining the amounts of FXIII protein in t4 and t5 biological sample; and administering FXIII to said patient if the patient has a lowering of FXIII protein amount in any one of t4 and t5 biological samples below a threshold value which is indicative of a poor prognosis in the absence of said administering.

3. The method according to claim 1, wherein the threshold value of FXIII protein amount is evaluated as a percentage of the FXIII amount of a positive control.

4. The method according to claim 2, wherein the threshold value of FXIII protein amount for each t0 to t5 biological sample is expressed as percentage of FXIII protein amount of a normal control, such amount being fixed to 100%, wherein the threshold values are: TABLE-US-00006 Threshold sample (FXIII %) t0 88.0 t1 87.7 t2 85.8 t3 75.6 t4 74.9 t5 63.0.

5. The method according to claim 1, wherein the patient biological samples are collected in sample collecting tubes containing at least one anti-proteolytic agent and an anti-coagulating agent.

6. The method according to claim 1, wherein the patient biological samples are whole blood, plasma or serum samples.

7. The method of claim 1 wherein determining the amounts of Factor XIII (FXIII) protein is performed by measuring Factor XIII (FXIII) antigen.

8. A method of treating a myocardial infarcted patient having a poor prognosis in the absence of treatment, the poor prognosis being identifiable by a lower than threshold value of Factor XIII (FXIII) protein amount in at least one of four biological samples from said patient, said four biological samples having been collected daily on the day of myocardial infarction (t0) and the following three days (t1 to t3), said method comprising administering FXIII to said myocardial infarcted patient having a poor prognosis.

9. The method of claim 8 wherein said patient has a lower than threshold value of FXIII protein amount in at least one of six biological samples, said six biological samples having been collected daily on the day of infarction (t0) and the following five days (t1 to t5).

10. The method according to claim 8, wherein the threshold value of FXIII protein amount is a percentage of the FXIII protein amount of a positive control.

11. The method according to claim 9, wherein the threshold value of FXIII protein amount for each t0 to t5 biological sample is expressed as percentage of FXIII protein amount of a normal control, such amount being fixed to 100%, wherein the threshold values are: TABLE-US-00007 Threshold sample (FXIII %) t0 88.0 t1 87.7 t2 85.8 t3 75.6 t4 74.9 t5 63.0.

12. The method according to claim 8, wherein the biological samples were collected in sample collecting tubes containing at least one anti-proteolytic agent and an anti-coagulating agent.

13. The method according to claim 9, wherein the biological samples were collected in sample collecting tubes containing at least one anti-proteolytic agent and an anti-coagulating agent.

14. The method according to claim 8, wherein the biological samples are one of whole blood, plasma or serum.

15. The method according to claim 9, wherein the biological samples are one of whole blood, plasma or serum.

Description

BRIEF DESCRIPTION OF THE ANNEXED DRAWINGS

(1) Further features and advantages of the invention will become apparent from the detailed description which follows and which is given purely by way of non limiting examples with reference to the annexed figures of drawings. These drawings are essentially in the form of diagrams showing exemplary delivery profiles of various active agents that may be achieved using compositions as described herein. Specifically:

(2) FIG. 1: Mean and Median values of FXIII levels at the scheduled time in the whole cohort of MI patients analyzed.

(3) FIG. 2A: Mean and Median values of FXIII levels at the scheduled time in those cases who did not experience MACEs during the follow-up.

(4) FIG. 2B: Mean and Median values of FXIII levels at the scheduled time in those cases who experienced or kind of MACE during the follow-up.

(5) FIG. 3A: Mean and Median values of FXIII levels at the scheduled time of patients having a MACE who did not experience AMI (re-infarction) as first end-point in the follow-up after the first acute MI.

(6) FIG. 3B: Mean and Median values of FXIII levels at the scheduled time of patients who experienced AMI (re-infarction) as first end-point in the follow-up after the first acute MI.

(7) FIG. 4A: Mean and Median values of FXIII levels at the scheduled time in those patients who experienced heart failure (HF).

(8) FIG. 4B: Mean and Median values of FXIII levels at the scheduled time in those patients who deceased during the follow-up, t30 was not reported due to the partial and scanty availability of samples (see results).

(9) FIG. 5: Mean and Median values of FXIII levels at the scheduled time in those patients who experienced unstable angina as main symptom during the follow-up.

(10) FIG. 6: ROC curve of FXIII at day 4th (t4*); the associated informative details are reported in Table 1.

DETAILED DESCRIPTION OF THE INVENTION

(11) In the following description, numerous specific details are given to provide a thorough understanding of embodiments. The embodiments can be practiced without one or more of the specific details, or with other methods, components, materials, etc. In other instances, well-known structures, materials, or operations are not shown or described in detail to avoid obscuring aspects of the embodiments.

(12) Reference throughout this specification to one embodiment or an embodiment means that a particular feature, structure, or characteristic described in connection with the embodiment is included in at least one embodiment. Thus, the appearances of the phrases in one embodiment or in an embodiment in various places throughout this specification are not necessarily all referring to the same embodiment. Furthermore, the particular features, structures, or characteristics may be combined in any suitable manner in one or more embodiments.

(13) The headings provided herein are for convenience only and do not interpret the scope or meaning of the embodiments.

(14) The present disclosure concerns the identification of a novel biomarker useful in determining potential recovery of patients who suffered a myocardial infarct.

(15) In an embodiment, the present description provides for a diagnostic kit for evaluating FXIII amounts in a myocardial infarcted patient, having FXIII amounts prognostic value about recovery of the patient, wherein the kit comprises means for detecting the amount of FXIII protein in at least four, preferably six, biological samples and at least four, preferably six, sample collecting tubes suitable to collect the at least four, preferably six, patient biological samples taken on the day of myocardial infarction (t0) and at least on the following three to five days (t1 to t3 or t1 to t5).

(16) A still further embodiment of the instant disclosure concerns a diagnostic method for determining prognosis of a myocardial infarcted patient, wherein the amounts of FXIII protein are determined on the day of myocardial infarction (t0) and at least on the following three days (t1 to t3), wherein a lowering of FXIII amount on any one of t0 to t3 below a threshold value is indicative of a poor prognosis.

(17) In a further embodiment, the threshold value of FXIII protein amount is evaluated as a percentage of a FXIII protein reference value conventionally fixed as 100% (if not differently reported) of a reference sample (normal control), wherein such a reference sample is obtained by standard reference materials like for example reference calibration lyophilized pooled plasma.

(18) As an example, the threshold value (or cut-off value), below which the patient is at risk of a poor prognosis (i.e. heart failure or death), varies according to the time considered (t0-t5). All the thresholds are expressed as percentage of FXIII protein amount of the normal control (reference). The threshold values for t0 to t5 (determined by the ROC analysis), provided appreciable and significant results, i.e. allowed to correctly determine the prognosis of an infarcted patient. Accordingly, in the example below, patients who developed heart failure or those who died after AMI are joined together because of their behaviours (in FXIII consumption) are very similar. Hence, comparing by the ROC procedure the FXIII levels of this MACE+ subgroup with the FXIII levels of all the MACE group, the results were statistically significant.

(19) In table 1 the threshold values of FXIII protein amount on any of t0 to t5 are provided.

(20) TABLE-US-00001 TABLE 1 FXIII % Sensitivity/ Cut-off Specificity Time (ROC) (%) P AUC CI (95%) t0 88.0 69.23/70.43 <0.0001 0.720 0.663-0.769 t1 87.7 65.45/66.67 <0.0001 0.700 0.638-0.754 t2 85.8 65.31/67.05 <0.0001 0.710 0.644-0.768 t3 75.6 64.44/75.94 <0.0001 0.760 0.709-0.800 t4* 74.9 74.29/67.01 <0.0001 0.740 0.677-0.787 t5 63.0 73.33/76.00 <0.0001 0.770 0.660-0.855 ROC = Receiver Operating Characteristic; AUC = Area Under Curve; CI (95%) = Confidence Interal (95%). The reported thresholds were obtained comparing the FXIII levels of the MACE+ (heart failure and death) with the whole MACE subgroup.

(21) The content of Table 1 indicates that the prognostic value of FXIII is already informative in the earliest times (t0-t3). Anyhow, the monitoring of FXIII in a quite complete way (t0-t5) is recommended, because it is difficult to exactly predict the degree of FXIII protein consumption for any infarcted patient. As an example, the ROC curve at day 4 (t4; FXIII cut-off=74.9%) is provided in FIG. 6.

(22) Today, FXIII assays are performed to determine the amount and/or activity of this enzyme in blood to understand if the enzyme level may be responsible or not for a determined pathological/clinical condition, generally haemorrhage. The test, if performed in acute condition (i.e. haemorrhage), must always be confirmed in a steady-state-condition far from the acute event. The assay is performed as an individual single test, that is sufficient for a definite diagnosis.

(23) Conversely, the instant disclosure provides data about the prognostic value of FXIII levels in post-infarcted patient: monitoring in acute phase the FXIII levels on a time window of four, preferably six, days (i.e. on the day of acute myocardial infarct (t0) and on the following three to five days (t1 to t3 or t0 to t5)) allows to predict the healing potential (the degree to heal) of such a patient, regardless other associated and/or concomitant interfering/confounding factors.

(24) The experimental data herein collected provide evidence that the acute FXIII fall, due to heart injury, virtually happens in quite all myocardial infarction patients but at different extent. This is caused by both coronary thrombus formation and tissue healing/myocardial scar formation. In detail, the present inventor found that a patient undergoing excessive FXIII consuming at the time of myocardial infarct (MI) is more prone to develop severe heart failure or death, whereas less drastic consuming is found in patients relapsing with re-infarction or u-angina.

(25) Having low/not optimal FXIII levels during the critical scar formation phase hampers heart healing giving back not properly tissue repair with chronic future consequences on myocardial performances. In this restricted period of time, the quality of infarct healing determines the fate of the patient for years to come.

(26) Monitoring of FXIII levels during the acute MI enables clinicians to early discover those patients at risk of poor post-MI prognosis giving the possibility to intervene with targeted treatments.

(27) Additionally, FXIII monitoring allows to recognize in advance those patients really in need to receive Possible FXIII infusion therapy (to ameliorate myocardial healing); indiscriminate FXIII infusion is, in fact, responsible of undesirable pro-clotting function before reliable coronary reperfusion after infarction is completely re-established and must be performed only in patients with actual need thereof.

(28) Taken together, these observations indicate FXIII a useful prognostic biomarker with potential prospective in therapeutic handling of a patient after MI.

(29) The present invention describes how different degrees in the fall of FXIII levels allow to predict the risk to experience the major post-MI adverse cardiovascular events.

(30) So monitoring the amount of FXIII drop observed in the acute MI phases allows prognosticating whether such a patient is at risk to develop future accidents.

(31) The need to have an effective prognostic test is mainly due to the fact that starting from the seventies, large part of efforts in the cardiovascular field have been addressed to diagnostic and surgical (intervention) procedures reaching exceptional results. Due to these effective actions, the rate of patients who survived MI drastically improved and they continue to rise.

(32) The present invention concerns a test able to recognize in advance those patients at increased risk to develop additional cardiovascular accidents in order to approach them with a tailor-made therapeutical treatment.

(33) Nowadays, does not exist a prognostic test or a specific treatment to effectively avert the adverse events secondarily to MI; there exists, in fact, a restricted period of time (1-2 weeks from the establishment of MI) during which it is possible to act in order to potentiate the intrinsic healing power of the heart and thus saving patients from death.

(34) For these reasons, the present disclosure concerns the assessment and monitoring of FXIII levels in the routine laboratory-test-profile for acute myocardial infarction (AMI) detection. The classical marker tests for myocardial infarction are merely passive indicators of heart damage, though very precocious and informative diagnostic tools. Conversely, the complex combined test evaluation herein disclosed results in precious prognostic information very useful for the clinicians. Once verified the diminished FXIII levels it could be possible to act by adjunctive (also therapeutic) treatments. Assessment and monitoring of FXIII levels can also be combined with the classical ischemic markers.

(35) Materials and Methods

(36) Patients and Methods

(37) 350 acute MI patients (whole group; mean age 68.212.95 years; 72.8% men) admitted to the Emergency Department of the University-Hospital of Ferrara were recruited.

(38) Inclusion criteria were: prolonged chest pain occurring at rest accompanied by electrocardiography (ECG) ischemic changes. CK-MB and/or Troponin-I values were greater than the upper reference limit in two separate blood samples.

(39) All noteworthy patient characteristic are reported in Table 2.

(40) TABLE-US-00002 TABLE 2 Total STEMI NSTEMI (n = 350) (n = 252) (n = 98) P Age (y, SD, 68.2 12.95 67.1 13.50 71.05 11.33 <0.05 range) (31-80) (31-80) (38-80) Male (n, %) 255 (72.8) 186 (73.8) 69 (70.4) NS PCI (n, %) 316 (90.3) 235 (93.25) 81 (82.6) <0.05 PCI = percutaneous coronary intervention; STEMI = patients showing ST-segment elevation MI at enrolment; NSTEMI = patients NOT showing ST-segment elevation MI at enrolment.

(41) Blood Samples

(42) Blood was collected in Trisodium Citrate Coagulation tubes at admission (t0) and every 24 h for additional five days (t1-t5) from the acute MI event. Control samples were drawn at least after 30-days (t30) to have basal FXIII levels far from the acute ischemic events.

(43) To exclude, possible, further in vitro enzyme degradation/activation additional comparative samples were drawn in EDTA plus Aprotinin tubes.

(44) Plasma was obtained by blood centrifugation at 2500 g10 min at room temperature, and different aliquots were frozen at 80 C.

(45) In order to verify, in actual fact, how different collection tubes could influence the results, additional MI patients (n=45) were recruited and blood was drawn in duplicate by means of both the classical Trisodium Citrate Coagulation tubes and the EDTA plus Aprotinin tubes. The recruitment scheme, was the same as above described (t0-t5, t30) and the plasma obtained was frozen (80 C.) till the assessment.

(46) FXIII Level Measurements

(47) FXIII antigen levels were assessed by means of a Latex Reagent which is a suspension of uniform size polystyrene latex particles coated with rabbit polyclonal antibodies, highly specific for the A-subunit of FXIII according to the manufacturer's instructions (Instrumentation Laboratory, Milan, Italy). Briefly the method is based on the principle that when a sample (plasma) containing the A-subunit of FXIII is mixed with the Latex reagent included in the kit, the coated latex particles agglutinate. The degree of agglutination is directly proportional to the concentration of FXIII antigen in the sample and is determined by measuring the decrease of transmitted light caused by the aggregates. The test is easy-to-perform, very fast, and routinely practicable.

(48) Follow-up (FU) and Statistics

(49) The primary endpoint was a composite of cardiovascular death, re-infarction, heart failure or unstable angina (u-angina) at one-year. Major adverse cardiac events (MACEs) were retrospectively analyzed as single variable or combined. Continuous data were presented as meansSD, with the significance of differences judged by t-test. Categorical variables were summarized in terms of number and percentages with the significance of differences judged by Chi-Square test. The recognition of the FXIII threshold(s) at any period of time considered (t0-t5) was obtained by means of the Receiver Operating Characteristic (ROC) analysis. Probability was considered significant at a level of P<0.05.

(50) Results

(51) FXIII was tested at the recruitment (t0) and every 24 h for additional five days (t1-t5) from the acute MI event. Control samples were drawn at least after 30-days (t30) to have basal FXIII levels far from the acute ischemic event.

(52) Table 3 shows the results of FXIII monitoring in acute MI patients obtained using standard sample collecting tubes (indicated Trisodium Citrate Coagulation tubes) and sample collecting tubes containing at least one anti-proteolytic agent and at least one anti-coagulating agent (indicated EDTA plus Aprotinin tubes).

(53) Though slightly lower values resulted from the EDTA plus Aprotinin tubes, no significant differences were obtained by comparing the two different vials in which blood was collected. Anyhow, making use of tubes containing at least one anti-proteolytic agent and at least one anti-coagulating agent is recommended in those situations in which a not so rapid assessment of the test can be possible (delay4 hours from the time in which blood is taken).

(54) The anti-proteolytic agent can be selected among aprotinin, heparin, antithrombin, hirudin, EDTA (Ethylenediaminetetraacetic acid), EGTA (Ethylene glycol-bis(-aminoethyl ether) tetraacetic acid), Leupeptin, iodoacetamide, APMSF ((4-Amidino-phenyl)methane-sulfonyl fluoride), 4-(2-Aminoethyl)-benzenesulfonyl-flouride hydrochloride, a2-Macroglobulin.

(55) The use of collecting tubes containing at least one anti-proteolytic agent allows, in fact, to determine the actual FXIII protein amount in the infarcted patient avoiding further consumption of FXIII protein contained in the collecting tube because of the presence of the anti-proteolytic(s) completely blocks any further (in vitro) enzyme activation. This precaution warrants a more reliable FXIII detection, better reflecting that particular in vivo status.

(56) TABLE-US-00003 TABLE 3 t0 t1 t2 t3 t4 t5 t30 Trisodium Citrate Coagulation tubes Mean 99.48 97.68 94.72 90.72 85.24 76.51 110.15 Me- 98.4 95.30 94.20 88.70 83.20 74.05 108.20 dian SD 30.57 28.06 29.12 30.30 24.93 27.02 20.34 min 40.60 39.90 32.80 29.5 20.90 20.00 60.00 max 186.40 181.10 179.80 169.10 136.20 138.25 147.90 EDTA plus Aprotinin tubes Mean 98.34 96.87 93.61 89.16 83.08 75.45 107.34 Me- 94.40 95.15 92.30 87.90 72.65 74.00 105.90 dian SD 31.87 32.34 34.40 31.25 28.72 29.19 27.37 Min 41.05 38.75 34.70 30.5 22.05 21.90 61.25 Max 193.90 190.90 182.50 178.20 139.60 135.25 152.70 P 0.92 0.32 0.30 0.35 0.28 0.84 0.80

(57) FIG. 1 shows the mean and median levels of FXIII during the assessment period in the whole cohort.

(58) FIGS. 2 to 5 show the FXIII levels in different subgroups of patients during the six days (t0-t5) and at the final check (t30).

(59) Globally, a significant FXIII level drop was clearly observed at t4-t5 as both mean and median regardless the patients experienced or not major adverse cardiac events (MACE) (FIGS. 2B and 2A, respectively).

(60) Interestingly, the combined endpoint analysis showed that the FXIII fall was significantly stronger considering those patients who experienced major adverse cardiac events (MACE) (MACE+; n=101; 28.8%) when compared with the rest of patients free of major post-MI events (MACE; n=249; 71.1%).

(61) It's noteworthy the observation that MACE+ patients presented with significantly lower FXIII levels at t0 and at any time considered as both mean and median. The data of FXIII levels in the t0 to t5 time window for MACE and MACE+ patients are provided in Tables 4 and 5, respectively.

(62) TABLE-US-00004 TABLE 4 MACE (n = 249) t0 t1 t2 t3 t4 t5 t30 Mean 103.73 102.10 101.31 98.02 89.34 83.37 109.53 Me- 102.20 100.53 97.80 94.05 85.20 91.00 108.35 dian SD 28.77 29.17 31.55 29.74 25.58 23.53 24.95 min 46.30 42.60 42.40 40.60 38.30 37.00 54.80 max 236.80 201.20 198.90 193.90 149.60 136.60 172.70

(63) TABLE-US-00005 TABLE 5 MACE+ (n = 101) t0 t1 t2 t3 t4 t5 t30 Mean 88.69 86.22 82.68 76.28 71.00 65.07 91.94 Me- 81.00 81.00 78.00 72.00 66.00 60.55 88.95 dian SD 29.41 29.87 29.71 29.10 29.01 31.12 24.28 min 35 32.8 31.5 30.5 20.9 18 54 max 167.2 160.5 158.4 151 148 136.6 159

(64) Interestingly, the degree of the FXIII lowering became even stronger in the MACE+ subgroup (FIG. 3A) when those who experienced acute myocardial infarction (AMI) as first end-point in the follow-up were excluded. Indeed, in these latter (AMI), the FXIII drop mean behaviour strongly diverged from the other groups considered, being apparently not affected by a sharp fall of FXIII (FIG. 3B).

(65) Conversely to what happened in the previous described patients (heart failure or dead subgroups), here those who developed AMI, diverge from the other patients being paradoxically less FXIII-consuming even than the MACE group. For this reason we did not compute by ROC analyses this kind of results, but strongly underline their importance suggesting they should be taken in consideration and defer their final interpretation.

(66) To note, these patients (AMI) presented with higher mean FXIII level at t0, and never went below a mean value of 94.0% (t4).

(67) According to the literature on the role of FXIII in recovering of tissue damages, that is low FXIII levels might be responsible for anomalous post-MI heart/wall remodelling causing heart failure or even death (due to inefficient/poor myocardial scar formation at the injury site), the present inventor specifically analyzed how FXIII levels were in those patients who experienced post-MI heart failure (HF) or death.

(68) HF patients showed awful FXIII mean reduction and their trend was characterized by a sharper slope. Though they reached very low mean FXIII levels (FIG. 4A), they showed appreciable and normal recovery of FXIII at t30. Conversely, those patients who died due to cardiovascular complications related to the acute experienced MI (FIG. 4B), presented at t0 with slightly lower levels and reached t4-5 with mean and median FXIII values resembling those of the HF subgroup. Unfortunately, t30 was not collected because of large part of these patients died before/or around that time or were definitely not available due to concomitant treatments or complications.

(69) Finally, those patients who experienced u-angina as the prevailing symptom during the follow-up (FIG. 5), though they showed a slow rate in FXIII decreasing, did not show at t30 the classical recover of FXIII levels. This could mean they continue to spend/consume FXIII also far from the acute infarction event, possibly due to the chronic relapsing in angina attacks.

(70) Similarly to what we did in the previous less FXIII-consuming AMI patients, also data from anginous cases were not computed by ROC analyses. We strongly underline as well, their importance suggesting they should be taken into consideration and defer their final interpretation.

(71) Summarizing, the test/procedure presented here gives very informative results as a prognostic indicator of those MACEs related to excessive FXIII consumption (HF or death). Conversely, though noteworthy, the low FXIII consumption observed in anginous and AMI patients deserve future and definite investigations, ascribing anyhow to them a strong potential clinical relevance.

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