Use of a tryptase in the preparation of a product for assessing the prognosis of the interventional treatment of acute ST-segment elevation myocardial infarction

Abstract

This invention relates to the field of biomarkers. Specifically, this invention provides the application of trypsin-like enzymes in the preparation of products for assessing the prognosis of interventional treatment for acute ST-segment elevation myocardial infarction (STEMI). Furthermore, this invention provides a predictive model capable of assessing the prognosis of interventional treatment for STEMI. The model includes trypsin-like enzyme expression levels, the traditional TIMI risk scoring system, and patient clinical information. It exhibits extremely high predictive accuracy in determining whether MACE will occur after interventional treatment for STEMI, and can be effectively used clinically to identify high-risk groups for poor prognosis in STEMI patients at an early stage, providing strong support for patient treatment decisions and prognostic management.

Description

Technical Field

[0001] This invention relates to the field of biomarkers, and more specifically, to the use of a trypsin-like enzyme in the preparation of a product for evaluating the prognosis of interventional treatment for acute ST-segment elevation myocardial infarction. Background Technology

[0002] Ischemic heart disease is one of the leading causes of death worldwide, with acute ST-segment elevation myocardial infarction (STEMI) being the most severe type. Percutaneous coronary intervention (PPCI) is the most important treatment for STEMI. In STEMI patients, there is a strong inflammatory response during plaque rupture, thrombosis, and reperfusion.

[0003] Multiple cell types are known to participate in inflammatory responses, including macrophages, lymphocytes, dendritic cells, and mast cells (MCs). Cardiac MCs are primarily distributed in the adventitia of coronary arteries and the cardiac interstitium. Following myocardial infarction, MCs are activated through various pathways, producing a variety of inflammatory cytokines (such as tumor necrosis factor-α, interferon-γ, and interleukin-6), histamine, chymotrypsin, trypsin-like proteins, and matrix metalloproteinases, which participate in atherosclerotic plaque rupture, inappropriate ventricular remodeling, and myocardial fibrosis. Trypsin-like proteins, stored in MCs in vesicle form, are the gold standard for evaluating the degree of MC activation. In clinical studies, immunoglobulin E, a MC activator, significantly increases during the acute phase of STEMI. Serum trypsin-like proteins are negatively correlated with ST segment regression rate and left ventricular ejection fraction (LVEF) on electrocardiogram 2 hours after PPCI.

[0004] However, the relationship between acute-phase trypsin and long-term major adverse cardiovascular events (MACE) in STEMI patients remains unclear to date. Summary of the Invention

[0005] To fill the gap in the prior art, the present invention provides the use of a trypsin-like enzyme in the preparation of a product for evaluating the prognosis of interventional treatment for acute ST-segment elevation myocardial infarction (STEMI).

[0006] Firstly, in a first aspect, the present invention provides the use of a trypsin-like enzyme in the preparation of a product for evaluating the prognosis of interventional treatment for acute ST-segment elevation myocardial infarction (STEMI).

[0007] In a second aspect, the present invention provides an application of a prognostic prediction model in the preparation of products for evaluating the prognosis of interventional treatment for acute ST-segment elevation myocardial infarction (STEMI), wherein the prognostic prediction model is composed of trypsin expression levels in patient samples and patient clinical information, including data from the traditional TIMI risk scoring system (age, hypertension, diabetes, angina, systolic blood pressure, heart rate, Killip classification, weight, electrocardiogram, and time of visit), complete revascularization, segmental motion abnormalities, left ventricular ejection fraction, hemoglobin, and global longitudinal strain of the left ventricle.

[0008] In one implementation, the application refers to substituting the expression level of trypsin-like enzymes in patient samples and the patient's clinical information into a nomogram, and obtaining the patient's 1-year, 3-year, and 5-year probability of no MACE through the nomogram.

[0009] In one implementation, the patient sample refers to patient plasma.

[0010] In a preferred embodiment, the plasma is derived from a blood sample taken 6 hours after the patient completes PPCI treatment.

[0011] In one implementation, the aforementioned product is a reagent kit. Attached Figure Description

[0012] The accompanying drawings are provided to further illustrate the invention and form part of the specification. They are used in conjunction with embodiments of the invention to explain the invention and do not constitute a limitation thereof. In the drawings:

[0013] Figure 1 The difference in endpoint events between the trypsin-eluting and trypsin-depleting groups;

[0014] Figure 2 The KM curves for each endpoint event in the trypsin-eluting and trypsin-depleting groups are shown.

[0015] Figure 3 LASSO regression analysis was performed for major adverse cardiovascular events. The adjustment parameter (λ) was selected using 10-fold cross-validation (A), and the LASSO coefficient curves for the variables were obtained (B).

[0016] Figure 4 To compare the discriminative power of different prediction models and determine the incremental prognostic value of trypsin. (A) Nonograph of Model 5; (B) ROC curves of the two analytical models; (C) Comparison of the discriminative power of the two analytical models. Detailed Implementation

[0017] The preferred embodiments of the present invention will be described below with reference to the accompanying drawings. It should be understood that the preferred embodiments described herein are for illustration and explanation only and are not intended to limit the present invention.

[0018] Example 1: Screening of biomarkers and establishment of diagnostic models

[0019] Research Design and Population

[0020] This is a single-center prospective cohort study that included STEMI patients who underwent PPCI at Peking University Third Hospital between July 2020 and July 2023. Inclusion criteria were: 1) age 18 years and older but under 80 years old, regardless of sex; 2) meeting the diagnostic criteria for STEMI (diagnostic criteria: ischemic chest pain lasting ≥30 min; ST-segment elevation or new-onset left bundle branch block in two or more adjacent leads on ECG; with or without elevated cardiac markers); 3) completion of PPCI and opening of the culprit vessel; 4) consent to participate in this study and signing an informed consent form. Exclusion criteria were: 1) concomitant chronic renal insufficiency with a glomerular filtration rate <30 ml / min / 1.73 m³ / min. 2 2) The patient has an infectious or neoplastic disease; 3) The patient has a coexisting allergic or autoimmune disease; 4) The patient is currently using or plans to use oral or intravenous glucocorticoids long-term. This study followed the Declaration of Helsinki and was approved by the Ethics Committee of Peking University Third Hospital.

[0021] Data collection

[0022] The inpatient electronic medical record system was used to collect general demographic characteristics, including gender, age, past medical history, and medication history, as well as laboratory test results and echocardiogram results. All patients underwent selective coronary angiography, in which two experienced surgeons identified the culprit vessel based on the electrocardiogram and coronary angiography results and performed PPCI treatment. Postoperatively, the clinician determined the use of secondary prevention drugs for coronary artery disease based on the patient's condition.

[0023] Trypsin detection

[0024] Six hours after the patient completes PPCI treatment, blood samples are collected using blood collection tubes containing EDTA anticoagulant and immediately stored at 4°C. Within 30 minutes of blood collection, the sample is centrifuged at 3000 rpm for 10 minutes at 4°C to obtain plasma, which is then frozen at -80°C until analysis. Repeated freeze-thaw cycles are avoided. Trypsin levels in the plasma are measured according to the manufacturer's product instructions (ml060170B, mlbio, Shanghai, China).

[0025] To observe the stability of trypsin assays across different batches, samples from 50 randomly selected patients were tested repeatedly. The consistency of results across different batches was calculated using a single-item randomized model with a consistency criterion. Results showed excellent consistency: inter-observer consistency reached 0.999 (95% CI: 0.999–1.000).

[0026] Follow-up strategy

[0027] Patient follow-up information was obtained through outpatient or inpatient systems. Patients not regularly followed up in the outpatient department were followed up by telephone every six months. The primary endpoint of this study was MACE, including cardiac death, all-cause mortality, readmission due to heart failure, and recurrent coronary events (recurrent myocardial infarction, unplanned PCI, and coronary artery bypass grafting). The secondary endpoint was major adverse cardiovascular and cerebrovascular events (MACCE), including MACE and stroke. All events were independently assessed by two experienced cardiologists unaware of trypsin levels; if the two physicians disagreed, a third senior physician made the final decision.

[0028] Statistical methods

[0029] Based on the preliminary trial results, 51 patients were included and divided into an exposed group and a non-exposed group, with the median TPS of 1.62 ng / ml as the cutoff. After one year of follow-up, 3 patients (12.00%) in the non-exposed group (25 cases) experienced MACE, and 6 patients (23.08%) in the exposed group (26 cases) experienced MACE. Using PASS to calculate the sample size, with β=0.10 and α=0.05, a sample size of 492 patients was required. Assuming a loss to follow-up rate of 10%, a total sample size of 541 patients was needed.

[0030] For normally distributed continuous variables, expressed as mean ± standard deviation, the independent samples t-test was used for comparisons between two groups. For non-normally distributed continuous variables, expressed as quartiles, the rank-sum test was used for comparisons between two groups. For categorical variables, expressed as number of cases and percentages, the chi-square test was used for comparisons. The relationship between trypsin and traditional cardiovascular risk factors was assessed by plotting restriction cubes (RCS). Groups were divided into elevated and decreased trypsin groups according to the optimal cutoff value for trypsin. Survival analysis was performed using the Kaplan-Meier method, survival curves were plotted for the two groups, and log-rank comparisons were used for comparisons between groups. LASSO regression was used to screen variables for inclusion in the Cox multivariate analysis. Potential confounding factors were corrected for by Cox multivariate regression to determine the risk stratification effect of trypsin on MACE, calculating HR values ​​and providing 95% CI. The predictive role of trypsin in the prognosis of STEMI patients was assessed in different subgroups according to general demographic data.

[0031] result

[0032] 1. Population characteristics

[0033] A total of 823 STEMI patients who visited Peking University Third Hospital between July 2020 and July 2023 were screened. 42 patients were excluded due to comorbidities, and 31 patients were excluded due to insufficient follow-up (less than 8 months), leaving 750 patients for the final analysis. Of these, 514 patients were randomly assigned to the screening set and 236 to the validation set. Among the 514 screening patients, the average age was 59.27 ± 13.26 years, with 87 females (16.93%) and 199 patients (38.72%) having anterior wall myocardial infarction (Table 1). The median follow-up time was 13.28 (10.47, 37.61) months. A total of 85 cases of MACE (16.54%) and 89 cases of MACCE (17.32%) occurred. Among them, 12 cases of cardiac death (2.33%), 22 cases of all-cause death (4.28%), 17 cases of rehospitalization due to HF (3.31%), 51 cases of recurrent coronary events (9.92%), and 6 cases of stroke (1.17%).

[0034] Table 1. Clinical characteristics of acute ST-segment elevation myocardial infarction treated with direct coronary intervention.

[0035]

[0036]

[0037] The optimal cutoff value for MACE was determined to be 2.20 ng / ml using the ROC curve of trypsin-mediated MACE. This was used to detect whether the trypsin concentration in patient samples was higher or lower than the optimal cutoff value, and patients were divided into an elevated trypsin group and a decreased trypsin group. In the elevated trypsin group, the proportion requiring device assistance (intra-aortic balloon pump and extracorporeal membrane oxygenation), the proportion of anterior wall myocardial infarction and Killip grade II or above, the TIMI risk score for myocardial infarction, and the peak value of creatine kinase isoenzymes were all higher than in the decreased trypsin group. Hemoglobin, LVEF, and left ventricular strain were lower in the elevated trypsin group. There were no statistically significant differences between the two groups in age, sex, comorbidities, and secondary prevention medications for coronary artery disease (Table 1). Patients in the elevated trypsin group had significantly higher MACE, a composite endpoint of rehospitalization due to heart failure and cardiac death, and a significantly higher incidence of recurrent coronary events than those in the decreased trypsin group. Figure 1 ).

[0038] 2. The relationship between trypsin-like enzymes and the prognosis of STEMI patients

[0039] Further survival analysis using the Kaplan-Meier method was performed on the two groups. Patients in the elevated and decreased trypsin levels showed differences in MACE (HR 2.60 (1.68, 4.01), P <0.001), cardiac death and rehospitalization due to heart failure (HR 3.02 (1.44-6.33), P=0.002), and recurrent coronary events (HR 2.31 (1.31, 4.10), P=0.003). Figure 2 ).

[0040] 3. Correlation between trypsin-like proteins and MACE risk

[0041] In univariate Cox regression analysis, the following factors significantly influenced the outcome: Killip grade (HR 2.65, 95% CI: (1.70 - 4.14), P < 0.001), multivessel disease (HR 2.02, 95% CI: (1.21 - 3.37), P = 0.007), complete revascularization (HR 0.29, 95% CI: (0.18 - 0.46), P < 0.001), hemoglobin (HR 0.98, 95% CI: (0.97 - 0.99), P < 0.001), trypsin inhibitors (HR 1.98, 95% CI: (1.70 - 2.34), P < 0.001), peak CKMB > 300 U / L (HR 2.33, 95% CI: (1.47 - 3.70), P < 0.001), and left ventricular ejection fraction (HR 1.00). The following factors were predictors of MACE: 0.94, 95% CI: (0.92 - 0.95), P<0.001; segmental motion abnormalities (HR 2.42, 95% CI: (1.04 - 5.63), P=0.039); and the absolute value of left ventricular longitudinal strain (HR 0.79, 95% CI: (0.74 - 0.85), P=0.002).

[0042] To reduce the risk of overfitting, the variables in Table 1 were included in LASSO regression to screen for COX multivariate regression variables. A 10-fold forward crossover validation factor was used, and the λ value with the smallest mean squared error was selected. Multivessel disease, complete revascularization, left ventricular ejection fraction, segmental motion abnormalities, hemoglobin, absolute value of left ventricular longitudinal strain, and trypsin were included in the COX multivariate analysis. Figure 3 Multivariate analysis suggested that trypsin-like enzymes (HR 1.56 (1.29 - 1.88), P<0.001) were an independent risk factor for long-term MACE after PPCI in STEMI patients (Table 2).

[0043] Table 2. Cox regression analysis of major adverse cardiovascular events and major adverse cardiovascular and cerebrovascular events

[0044]

[0045] 4. Cox regression to control confounding variables

[0046] According to the rough model of univariate Cox regression analysis, elevated trypsin levels were a risk factor for poor prognosis in STEMI patients (HR 1.98 (1.70 - 2.34), P < 0.001). Further analysis by adding demographic characteristics and comorbidities to Model 2, and adding multivessel disease, complete revascularization, door-balloon time, hemoglobin, left ventricular ejection fraction, left ventricular longitudinal strain, and coronary artery disease drug treatment to Model 3, showed that trypsin levels still had a significant impact on the prognosis of STEMI patients (HR 1.96 (1.57 - 2.4589), P < 0.001). Using the optimal cutoff value for trypsin levels as a dichotomous variable, it still had significant prognostic value. (Table 3)

[0047] Table 3: Cox regression model of trypsin on major adverse cardiovascular events

[0048]

[0049] Note: The variables in each model are as follows:

[0050] Model 1: Trypsin

[0051] Model 2: Adjusted for gender, age, smoking, hypertension, diabetes, and hyperlipidemia.

[0052] Model 3: Based on Model 2, adjustments were made for multivessel disease, complete revascularization, door-to-balloon time, hemoglobin, left ventricular ejection fraction, left ventricular longitudinal strain, and β-blocker, angiotensin-converting enzyme inhibitor, and angiotensin receptor antagonist.

[0053] Based on the optimal cutoff value of trypsin, the groups were divided into an elevated group and a decreased group.

[0054] Example 2: Performance evaluation of the trypsin-like predictive model

[0055] To investigate the value of trypsin in predicting incremental MACE, the traditional TIMI risk scoring system (age, hypertension, diabetes, angina, systolic blood pressure, heart rate, Killip classification, weight, ECG, and time to visit) was used as Model 4. Variables with multivariate Cox regression p < 0.05 (complete revascularization, segmental motion abnormalities, left ventricular ejection fraction, hemoglobin, trypsin, and global left ventricular longitudinal strain) were further added to construct Model 5, and a nomogram was plotted (the values ​​of TIMI risk score, complete revascularization, segmental motion abnormalities, left ventricular ejection fraction, hemoglobin, trypsin, and global left ventricular longitudinal strain were all obtained within 24 hours post-surgery). The score of each variable in Model 5 was calculated in the corresponding first row of the nomogram. The scores of each variable were summed, and the probability of MACE-free status at 1, 3, and 5 years could be calculated based on the total score. Figure 4 A). The results showed that, compared with model 4, model 5, which included trypsin-like enzymes, had a higher ability to distinguish MACE events (AUC: 0.877 vs. 0.628, p < 0.001). The sensitivity and specificity of model 5 were 83.2% and 88.5%, respectively. Figure 4 (B and C).

[0056] To demonstrate that trypsin also has the same predictive value in MACCE, MACCE was used instead of MACE in sensitivity analysis. In the trypsin-elevated group, the incidence of MACCE was still significantly higher than in the trypsin-decreased group. Figure 1 Kaplan–Meier survival curve analysis indicated a difference in prognosis between the two groups (MACCE 2.77 (1.81–4.23), P<0.001). Figure 2 In the multivariate regression model, trypsin remained an important predictor of MACCE (HR 2.07 (1.52-2.83), P<0.001) (Table 2).

[0057] Example 3: Clinical application of trypsin-like predictive models

[0058] To further validate the predictive accuracy of Model 5, we used a validation set of 236 patients randomly selected from those with previously collected plasma samples. Among the 236 patients, the age was 60.18 ± 11.75 years, 43 were female (18.22%), and the median follow-up time was 21.45 (10.47, 32.61) months.

[0059] Model 5 and its nomogram were used to predict the probability of MACE-free outcomes in the validation set of patients at 1, 3, and 5 years, including cardiac death, all-cause mortality, readmission due to heart failure, and recurrent coronary events (recurrent myocardial infarction, unplanned PCI, and coronary artery bypass grafting). Patient information was collected through clinical monitoring and follow-up. Two experienced cardiologists, unaware of trypsin levels, independently determined whether each event constituted a target event. If the two physicians disagreed, a third senior physician made the final decision.

[0060] In the validation set, 36 patients (15.25%) experienced MACE. Of these, 15 occurred within one year, with 12 (80.00%) matching the predictions of Model 5. 224 patients did not experience MACE, with 185 (83.71%) matching the predictions of Model 5. Within three years, 33 patients experienced MACE, with 26 (78.79%) matching the predictions of Model 5. 260 patients did not experience MACE, with 207 (79.61%) matching the predictions of Model 5. The results demonstrate that Model 5 has extremely high predictive value for the occurrence of MACE after PPCI in STEMI patients.

[0061] Obviously, those skilled in the art can make various modifications and variations to this invention without departing from its spirit and scope. Therefore, if these modifications and variations fall within the scope of the claims of this invention and their equivalents, this invention also intends to include these modifications and variations.

Claims

1. The application of a prognostic prediction model in the development of a product for assessing the probability of MACE (major adverse eventuality) after interventional treatment of acute ST-segment elevation myocardial infarction (STEMI), wherein, The prognostic prediction model consists of trypsin expression levels in patient samples and patient clinical information, including data from the traditional TIMI risk scoring system (age, hypertension, diabetes, angina, systolic blood pressure, heart rate, Killip classification, weight, electrocardiogram, and time of visit), complete revascularization, segmental motion abnormalities, left ventricular ejection fraction, hemoglobin, and global longitudinal strain of the left ventricle.

2. The application as described in claim 1, characterized in that, The application refers to inputting the expression level of trypsin-like enzymes in patient samples and the patient's clinical information into a nomogram, and obtaining the patient's 1-year, 3-year, and 5-year probability of no MACE through the nomogram.

3. The application as described in claim 2, characterized in that, The patient sample refers to the patient's plasma.

4. The application as described in claim 3, characterized in that, The plasma was obtained from a blood sample taken from the patient 6 hours after completing PPCI treatment.

5. The application as described in claim 1, characterized in that, The product in question is a reagent kit.

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