Methods to reduce the incidence of thrombosis or thromboembolism

Abstract

The present disclosure relates to methods and compositions for reducing the incidence of thrombosis or thromboembolism in patients identified as being at risk thereof, the methods comprising administering to the patient a therapeutically effective amount of one or more anticoagulants selected from the group consisting of unfractionated heparin, low molecular weight heparin, heparinoid, fondaparinux, idraparinux, and combinations thereof, and antithrombin III (ATIII).

Description

Technical Field

[0001] The present disclosure relates to the field of pharmaceutical products. Certain embodiments herein relate to methods and compositions for reducing the occurrence of thrombosis or thromboembolism in patients identified as being at risk, the method comprising administering to the patient a therapeutically effective amount of one or more anticoagulants, and antithrombin III (ATIII).

Background Art

[0002] Description of Related Art Venous thromboembolism (VTE), a disorder including deep vein thrombosis (DVT) and pulmonary embolism (PE), is a common complication in patients with multiple traumatic injuries. VTE is still one of the most common preventable causes of in-hospital death in this population despite intensive preventive efforts to reduce the risk [1, 2]. Depending greatly on the patient's urgency and degree of monitoring, up to 30% of patients under prophylaxis experience VTE during hospitalization. These events result in aggressive interventions, prolonged hospital stays, and increased healthcare costs [2, ³]. Three months after discharge, the VTE rate in surviving trauma patients still exceeds 10% [4], and 30% of thromboembolic events in the entire trauma population occur after discharge [5]. These are not clinically insignificant events. In fact, Drake et al. recently showed that 10.8% of preventable or potentially preventable deaths after discharge are due to extensive PE [6].

[0003] The exact pathophysiology of VTE in trauma patients is not fully understood, but it is undoubtedly multifactorial and is thought to occur subsequent to a combination of risk factors including abnormal coagulation activation, endothelial dysfunction, persistent inflammation, prolonged immobility, massive blood transfusion, and mechanical ventilation [2]. An increase in post-traumatic thrombin generation, a strong predictor of VTE in trauma patients, has been reported in several studies [7-11]. Therefore, restoring hemostatic homeostasis by limiting thrombin generation after bleeding control has been achieved is critically important for preventing thromboembolic complications in recovering trauma patients.

[0004] To address this, the American Academy of Chest Physicians has established standard treatment recommendations for aggressive VTE prophylaxis in trauma patients, including protocolized anticoagulation.

[12] One of the most commonly used prophylactic anticoagulants is enoxaparin (Lovenox), a low molecular weight heparin that can be monitored in hospital by measuring anti-FXa levels. Enoxaparin acts by enhancing the activity of antithrombin III (AT), a circulating anticoagulant. Enoxaparin binds to AT, inhibiting FXa and thrombin and thus increasing their activity to downmodulate coagulation. Despite increasingly aggressive institutional thromboprophylaxis protocols, 50–70% of trauma patients do not achieve the recommended anti-FXa target range (0.1–0.4 IU / mL), which indicates a change in responsiveness to enoxaparin anticoagulation.[13,14] Such unresponsiveness has been associated with an increased risk of VTE. For example, Malinoski et al. showed an increased incidence of DVT in trauma and surgical patients who did not achieve the recommended anti-FXa range

[15] . When the enoxaparin dose is adjusted in response to low anti-FXa levels, the incidence of VTE remains unchanged, so escalating the heparinoid dose appears to be ineffective

[16] . In the mid-1980s, Sabbagh et al. first introduced the use of fresh frozen plasma (FFP) to reverse heparin resistance in patients undergoing cardiopulmonary bypass

[17] . [Overview of the project] [Problems that the invention aims to solve]

[0005] Therefore, there remains a need to provide compositions and methods of treatment that can help achieve the recommended anti-FXa target range showing a good response to enoxaparin anticoagulation and offer novel treatment strategies for safely improving heparin-based thromboprophylaxis. [Means for solving the problem]

[0006] Surprisingly, the inventors found that ex vivo treatment with ATIII, rather than fresh frozen plasma (FFP), resulted in improved enoxaparin-mediated inhibition of thrombin generation, thereby increasing the effectiveness of enoxaparin-induced prophylactic thrombosis.

[0007] This disclosure provides methods and compositions for reducing the incidence of thrombosis or thromboembolism in patients identified as being at risk. In some embodiments, the method includes administering to a patient a therapeutically effective amount of an anticoagulant selected from the group consisting of unfractionated heparin, low molecular weight heparin, heparinoids, fondaparinux, hydraparinux, and combinations thereof, as well as antithrombin III (ATIII).

[0008] In some embodiments, thrombosis is venous thrombosis. In some embodiments, the thromboembolism is venous thromboembolism (VTE).

[0009] In some embodiments, patients identified as being at risk are selected from a group consisting of patients with physical trauma, perioperative patients, perinatal patients, patients with restricted mobility, cancer patients, and combinations thereof. In some embodiments, the patient is a patient with physical trauma, and the patient has suffered blunt trauma, penetrating trauma, or a combination thereof.

[0010] In some embodiments, ATIII is administered at a concentration that increases the patient's ATIII level to over approximately 1.0 IU / mL. In some embodiments, ATIII is administered at a concentration that increases the patient's ATIII level to over approximately 1.2 IU / mL. In some embodiments, ATIII is administered at a concentration that increases the patient's ATIII level to over approximately 1.3 IU / mL. In some embodiments, ATIII is administered at a concentration that increases the patient's ATIII level to over approximately 1.4 IU / mL. In some embodiments, ATIII is administered at a concentration that increases the patient's ATIII level to over approximately 1.5 IU / mL. In some embodiments, ATIII is administered at a concentration that increases the patient's ATIII level to a range of approximately 1.5 to approximately 2.5 IU / mL. In some embodiments, ATIII is administered at a concentration that increases the patient's ATIII level to a range of approximately 1.5 to approximately 2.0 IU / mL. In some embodiments, ATIII is administered at a concentration that increases the patient's ATIII level to a range of approximately 1.0 IU / mL to approximately 1.5 IU / mL.

[0011] In some embodiments, ATIII is plasma-derived or recombinant ATIII.

[0012] In some embodiments, the low molecular weight heparin is selected from the group consisting of bemiparin, sertoparin, dalteparin, enoxaparin, nadroparin, parnaparin, reviparin, tinzaparin, combinations thereof, and / or pharmaceutically acceptable salts thereof. In some embodiments, the heparinoid is selected from the group consisting of danaparoid, dermatan sulfate, thulodexide, combinations thereof, and / or pharmaceutically acceptable salts thereof. In some embodiments, the anticoagulant is a low molecular weight heparin, more preferably the low molecular weight heparin is enoxaparin or a pharmaceutically acceptable salt thereof.

[0013] In some embodiments, the compositions of the present invention are administered subcutaneously. In some embodiments, compositions comprising enoxaparin or a pharmaceutically acceptable salt thereof, and ATIII are administered subcutaneously. In some embodiments, the therapeutically effective dose of low molecular weight heparin is about 20 mg to about 180 mg, preferably 20 mg to 40 mg per day. In some embodiments, enoxaparin may be administered in amounts of about 20 mg to about 180 mg per day, or about 20 mg to about 40 mg per day. In some embodiments, the therapeutically effective dose of low molecular weight heparin is about 0.1 to about 2.5 mg / kg. In some embodiments, the therapeutically effective dose of low molecular weight heparin is 0.5 to about 1.5 mg / kg. [Brief explanation of the drawing]

[0014] [Figure 1] This diagram shows the flow of admitted and registered patients in the study of the present invention. PE = pulmonary embolism. [Figure 2] This figure shows antithrombin (AT) levels in trauma patient plasma at baseline and after treatment with fresh frozen plasma (FFP) or AT concentrate. AT is shown as an activity percentage. FFP treatment was 30% by volume. AT was supplemented to final concentrations of 120, 150, 180, or 200%. Data are shown as mean with standard deviation. The dotted line represents the detection limit. * indicates p<0.05 compared to baseline after one-way ANOVA with Bonferroni correction. [Figure 3] This figure shows the levels of anti-FXa and thrombin after enoxaparin treatment. Anti-FXa levels (A) and changes in peak thrombin (B) were measured in trauma patients after treatment of their plasma with enoxaparin, with or without FFP or AT supplementation. The delta peak represents the percentage change in thrombin in treated plasma compared to baseline untreated plasma. Data are shown as mean with standard deviation. * indicates p<0.05 compared to enoxaparin alone after one-way ANOVA with Bonferroni correction. [Figure 4]This figure shows the levels of anti-FXa and thrombin after enoxaparin treatment in patients with PE (pregnancy-induced hypertension) versus those without PE. Anti-FXa levels (A) and changes in peak thrombin (B) were measured in plasma from patients who developed PE (white bars) or did not develop PE (black bars) after enoxaparin treatment with or without FFP or AT supplementation. Data are shown as mean with standard deviation. * indicates p<0.05 after two-way ANOVA comparing "PE" and "no PE" patients with Sidak correction. [Modes for carrying out the invention]

[0015] definition Where used herein, section headings are for structural purposes only and should never be construed as limiting the subject matter described herein. All documents and similar materials cited in this application, including but not limited to patents, patent applications, articles, books, essays, and internet web pages, are expressly incorporated by reference in their entirety for any purpose. If the definitions of terms in the incorporated references appear to differ from the definitions provided in this instruction, the definitions provided in this instruction shall prevail. You will notice that there is an implied “approximately” before the temperature, concentration, time, etc., discussed in this instruction, so that only minor non-substantial deviations are within the scope of this instruction as specified herein.

[0016] In this application, the use of the singular form includes the plural form unless explicitly stated otherwise. Furthermore, the use of "comprise," "comprises," "comprising," "contain," "contains," "containing," "include," "includes," and "including" is not intended to be limiting.

[0017] As used in this specification and claims, the singular forms “a,” “an,” and “the” include multiple references unless the context clearly indicates otherwise.

[0018] As used herein, “about” means a quantity, level, value, number, frequency, percentage, dimension, size, volume, mass, or length that varies by only about 20, 15, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1% relative to the reference quantity, level, value, number, frequency, percentage, dimension, size, volume, mass, or length.

[0019] The term "pharmaceutically acceptable salt" of a given compound refers to a salt that retains the biological efficacy and properties of the given compound and is not biologically or otherwise undesirable. "pharmaceutically acceptable salt" or "physiologically acceptable salt" includes, for example, salts of compounds (or deuterated analogs, stereoisomers, or mixtures of stereoisomers) with inorganic or organic acids. In addition, when the compounds described herein are obtained as acid addition salts, the free base can be obtained by basicizing a solution of the acidic salt. Conversely, when the product is a free base, the addition salt, in particular a pharmaceutically acceptable addition salt, can be produced by dissolving the free base in a suitable organic solvent and treating the solution with an acid, following conventional procedures for preparing acid addition salts from base compounds. Those skilled in the art will be aware of the various synthetic methods that can be used to prepare non-toxic, pharmaceutically acceptable addition salts. pharmaceutically acceptable acid addition salts can be prepared from inorganic and organic acids.

[0020] The term “treatment” or “to treat” means any treatment of a disease or disorder in a mammal or other subject, including preventing or protecting against a disease or disorder, i.e., preventing the development of clinical symptoms; inhibiting a disease or disorder, i.e., preventing or suppressing the development of clinical symptoms; and / or mitigating a disease or disorder, i.e., causing a reduction in clinical symptoms.

[0021] As used herein, the term "preventing" refers to prophylactic treatment of a patient who needs it. Prophylactic treatment can be achieved by providing a suitable dose of a therapeutic agent to a subject at risk of suffering from a disease, condition, disorder, or impairment, thereby substantially preventing the onset of the disease, condition, disorder, or impairment.

[0022] As used herein, the term "suppressing" refers to prophylactic treatment of a patient who needs it. Prophylactic treatment provides a suitable dose of a therapeutic agent to a subject suffering from the root cause of a disease, condition, disorder, or impairment, but can be achieved by substantially preventing the onset of the symptoms of the disease, condition, disorder, or impairment.

[0023] In human medicine, it will be understood by those skilled in the art that it is not always possible to distinguish between "preventing" and "suppressing" because the final one or more inducing events may be unknown, potential, or the patient is not confirmed until well after the occurrence of one or more events. Thus, as used herein, the term "prevention" is intended to encompass both "preventing" and "suppressing" as defined herein as elements of "treatment".

[0024] The term "therapeutically effective amount" refers to an amount typically delivered as a pharmaceutical composition that is sufficient to effect treatment, as defined herein, when administered to a subject who needs such treatment. On the other hand, the term "composition" may be a parts kit and does not necessarily need to be both active substances co-formulated in a unit dosage form.

[0025] As used herein, the term "critically ill patient" refers to a patient who is admitted to an intensive care unit (ICU) and stays in the ICU for at least 12 hours. In some embodiments, the critically ill patient stays in the ICU for at least 24 hours. In some embodiments, the critically ill patient stays in the ICU for at least 2, 3, or 4 days. In some embodiments, the critically ill patient remains hospitalized after being discharged from the ICU.

[0026] "Severe" or "Severely Ill Patient" 1. Acute decompensated heart failure, New York Heart Association (NYHA) class III or IV. 2. Acute respiratory failure that does not require long-term (≤2 days) respiratory support. 3. Acute infection without septic shock, 4. Acute rheumatic disorders (including acute lower back pain, sciatica, spinal compression, acute arthritis of the leg, or episodes of inflammatory bowel disease) or 5. Cancer It may have one or more states, including but not limited to these.

[0027] Risk factors for venous thromboembolism (VTE) are: a) Age > 75 years old, b) A history of VTE requiring anticoagulant therapy, c) Expected significant immobility ≥ 3 days (Level 1 - bed rest without permission to go to the toilet), d) Obesity (Body Mass Index (BMI) > 30 for men or BMI > 28.6 for women); e) Varicose veins or chronic venous insufficiency, f) lower limb motor paralysis, g) Central venous catheter placement, h) Hormone therapy (anti-androgens, estrogens, or selective estrogen receptor modulators (SERMs)), i) chronic heart failure; j) chronic respiratory failure; k) active collagen vascular disease; l) The acute infectious disease that is causing the current hospitalization, m) Erythrocyte production stimulants, n) inflammatory bowel disease, o) Venous compression (tumor, hematoma or arterial abnormality), p) Nephrotic syndrome, and q) Hereditary or acquired tendency to form thrombi, r) or any combination thereof, including but not limited to these.

[0028] In some embodiments, severely ill patients have any one or more of the above conditions 1-4 and have at least two venous thromboembolism (VTE) risk factors a)-q) as outlined above, or a D-dimer result greater than twice the upper limit of normal.

[0029] As used herein, the term “thrombosis” refers to the formation of a blood clot in a blood vessel that blocks the flow of blood through the circulatory system. In some embodiments, thrombosis is “venous thrombosis,” which is a blood clot that forms in a vein. The term “thromboembolism” refers to the formation of a blood clot in a blood vessel that breaks off and is carried by the bloodstream to block another blood vessel. The clot may block blood vessels in the lungs (pulmonary embolism), brain (stroke), gastrointestinal tract, kidneys, and legs, for example, among others.

[0030] While this disclosure is within the context of certain embodiments and examples, those skilled in the art will understand that this disclosure extends beyond the specifically disclosed embodiments to the use of other alternative embodiments and / or embodiments, as well as obvious variations and equivalents thereof. In addition, while some variations of the embodiments are shown and described in detail, other variations that fall within the scope of this disclosure will be readily apparent to those skilled in the art based on this disclosure.

[0031] It is also intended that various combinations or partial combinations of specific features and aspects of the embodiments may be made and still fall within the scope of the disclosure. It should be understood that various features and aspects of the disclosed embodiments may be combined with or substituted for each other to form various forms or embodiments of the disclosure. Accordingly, it is intended that the scope of the disclosure disclosed herein should not be limited by the specific disclosed embodiments described above.

[0032] However, since various changes and modifications within the spirit and scope of this disclosure will be apparent to those skilled in the art, it should be understood that this description, while illustrating preferred embodiments of this disclosure, is provided merely as examples.

[0033] The technical terms used in the descriptions presented herein are not intended to be interpreted in any limited or restrictive manner. Rather, the technical terms are simply used in conjunction with the detailed descriptions of embodiments of the systems, methods, and related components. Furthermore, embodiments may include several novel features, none of which alone constitute their desired characteristics, nor are any of them considered essential for the execution of the embodiments described herein.

[0034] This disclosure provides methods and compositions for reducing the incidence of thrombosis or thromboembolism in patients identified as being at risk. In some embodiments, the method includes administering to a patient a therapeutically effective amount of an anticoagulant selected from the group consisting of unfractionated heparin, low molecular weight heparin, heparinoids, fondaparinux, hydraparinux, and combinations thereof, as well as antithrombin III (ATIII). In some embodiments, the thrombosis is venous thrombosis. In some embodiments, the thromboembolism is venous thromboembolism (VTE).

[0035] In some embodiments, patients identified as being at risk are selected from a group consisting of patients with physical trauma, perioperative patients, perinatal patients, patients with limited mobility, cancer patients, sepsis patients, patients with systemic inflammatory response syndrome, and combinations thereof.

[0036] In some embodiments, the patient is a trauma patient, and the patient has suffered blunt trauma, penetrating trauma, or a combination thereof.

[0037] Surprisingly, the inventors found that anticoagulants and ATIII supplementation, rather than plasma supplementation, increased ATIII levels in patients and improved thrombin production by anticoagulants, such as enoxaparin-mediated inhibition.

[0038] In some embodiments, patients are at risk of thromboembolic complications.

[0039] In some embodiments, patients have moderate or severe motor limitations and other risk factors for VTE.

[0040] In some embodiments, the patient is a critically ill patient admitted to or being treated in an intensive care unit, or a patient at risk of developing venous thromboembolic disease. In some embodiments, the patient suffers from one or more of the following: (a) acute decompensated heart failure, (b) acute respiratory failure, (c) acute infection without septic shock, (d) acute rheumatic disorder, or (e) cancer. In some embodiments, the patient suffers from reduced mobility.

[0041] In some embodiments, acute decompensated heart failure is New York Heart Association (NYHA) class III or IV. In some embodiments, acute respiratory failure does not require long-term (≤2 days) respiratory support. In some embodiments, acute infection does not involve septic shock. In some embodiments, the patient suffers from one or more acute rheumatic disorders (including episodes of acute low back pain, sciatica, spinal compression, acute arthritis of the leg, or inflammatory bowel disease).

[0042] In some embodiments, the patient is at risk of developing venous thromboembolic disease. In other embodiments, the patient suffers from reduced mobility. In yet another embodiment, the thrombosis is venous thrombosis.

[0043] In some cases, thrombosis is a characteristic of an underlying disease or condition. Non-limited examples of such diseases or conditions include acute coronary syndrome, myocardial infarction, unstable angina, refractory angina, occlusive coronary thrombosis occurring after thrombolytic therapy or coronary angioplasty, thromboembolic cerebrovascular syndrome, embolic stroke, thrombotic stroke, thromboembolic stroke, systemic embolism, ischemic stroke, venous thromboembolism, atrial fibrillation, non-valvular atrial fibrillation, atrial flutter, transient ischemic attack, venous thrombosis, deep vein thrombosis, pulmonary embolism, coagulation disorders, disseminated intravascular coagulation, thrombotic thrombocytopenic purpura, thromboangiitis obliterans, and heparin-induced platelet thromboembolism. This includes conditions involving thrombotic disorders associated with hypoplasia, thrombotic complications associated with extracorporeal circulation, thrombotic complications associated with the use of medical devices, thrombotic complications associated with the attachment of prosthetic devices, occlusive coronary thrombosis resulting from thrombolytic therapy or percutaneous transluminal coronary angioplasty, thrombosis in the venous vascular system, disseminated intravascular coagulation disorders, rapid consumption of coagulation factors and systemic coagulation, leading to widespread organ failure, life-threatening thrombus formation occurring throughout the microvessel system, hemorrhagic stroke, renal dialysis, blood oxygenation, and cardiac catheterization.

[0044] In some embodiments, the disease or condition is selected from the group consisting of embolic stroke, thrombotic stroke, venous thrombosis, deep vein thrombosis, acute coronary syndrome, and myocardial infarction.

[0045] In some embodiments, ATIII is administered at a concentration that increases the patient's ATIII level to over approximately 1.2 IU / mL. In some embodiments, ATIII is administered at a concentration that increases the patient's ATIII level to over approximately 1.3 IU / mL. In some embodiments, ATIII is administered at a concentration that increases the patient's ATIII level to over approximately 1.4 IU / mL. In some embodiments, ATIII is administered at a concentration that increases the patient's ATIII level to over approximately 1.5 IU / mL. In some embodiments, ATIII is administered at a concentration that increases the patient's ATIII level in the range of approximately 1.2 to approximately 2.5 IU / mL. In some embodiments, ATIII is administered at a concentration that increases the patient's ATIII level in the range of approximately 1.5 to approximately 2.0 IU / mL.

[0046] In some embodiments, ATIII is plasma-derived or recombinant ATIII.

[0047] Enoxaparin In some embodiments, enoxaparin is the sodium salt of enoxaparin. Enoxaparin sodium is a biomaterial obtained by alkaline depolymerization of heparin benzyl ester derived from porcine intestinal mucosa. Its structure is characterized by a 2-O-sulfo-4-empyranosuronic acid group at the non-reducing end of the chain and a 2-N,6-O-disulfo-D-glucosamine at the reducing end. Approximately 20% (ranging from 15% to 25%) of enoxaparin structures contain a 1,6-anhydro derivative at the reducing end of the polysaccharide chain. The average molecular weight is approximately 4500 daltons. Non-limiting examples of enoxaparin sodium are commercialized under the trademark name Lovenox®. A concentration of Lovenox 100 mg / mL contains 10 mg of enoxaparin sodium per 0.1 mL of water for injection (approximately 1000 IU of anti-factor Xa activity [refer to the WHO First International Low Molecular Weight Heparin Reference Standard]).

[0048] Antithrombin III Antithrombin (AT) is an alpha-2 glycoprotein with a molecular weight of 58,000 that is normally present in human plasma at a concentration of approximately 12.5 mg / dL and is the primary plasma inhibitor of thrombin. Inactivation of thrombin by AT occurs through the formation of a covalent bond involving the interaction of the active serine of thrombin and the arginine reaction site of AT, resulting in an inactive 1:1 stoichiometric complex between the two. AT can also inactivate other components of the coagulation cascade, including factors IXa, Xa, XIa, and XIIa, as well as plasmin. A non-limiting example of AT is commercialized under the trademark name Thrombate III® (Grifols Therapeutics LLC, USA).

[0049] The compositions of this invention may be in the form of orally administered tablets, capsules, lozenges, elixirs, suppositories, sterile solutions or suspensions, or injectable doses, or may be incorporated into molded articles. The method of administration will vary depending on the subject and depend on factors such as the type of mammal being treated, its sex, weight, diet, concomitant medications, overall clinical condition, the specific compounds and / or salts employed, the specific uses for which these compounds and / or salts are employed, and other factors recognized by those skilled in the medical field.

[0050] The compositions of the present invention may be prepared for storage or administration by mixing an active agent of desired purity with a physiologically acceptable carrier, excipient, stabilizer, etc., and may be provided in sustained-release or time-release formulations. Carriers or diluents acceptable for therapeutic use are well known in the pharmaceutical field.

[0051] In some embodiments, the dosage formulation used for therapeutic administration is sterile. Sterility is readily achieved by filtration through a sterile membrane, such as a 0.2-micron membrane, or by other conventional methods. The formulation is typically stored in lyophilized form or as an aqueous solution. The pH of the preparations of this invention is typically 3 to 11, or 5 to 9, or 7 to 8. The route of administration may be by injection, such as intravenous (bolus and / or infusion), subcutaneous, or intramuscular, or by colon, rectal, nasal, or abdominal cavity. Other dosage forms may be used, such as suppositories, implantable pellets or small cylinders, aerosols, oral formulations (e.g., tablets, capsules, and lozenges), and topical formulations, such as ointments, drops, and patches. The sterile membrane may be incorporated into molded articles such as implants, which may employ inert materials such as biodegradable polymers or synthetic silicones, e.g., Silastic, silicone rubber, or other commercially available polymers. Preferably, the compositions of the present invention are administered subcutaneously. [Examples]

[0052] The present invention is further defined by reference to the following examples. It will be apparent to those skilled in the art that many variations of both the composition and the method can be performed without departing from the scope of the invention.

[0053] The study was a single-center, retrospective cohort analysis of prospectively collected data from Memorial Hermann Hospital, Level-1 Trauma Center, and the University of Texas Health Science Center, Houston, TX. Prior approval from the Institutional Review Board (HSC-GEN-12-0059) was obtained for studies including adult patients (≥16 years) admitted between October 2012 and October 2016 who met the criteria for activation of the highest level trauma team and who provided informed consent from the patient or legally authorized representative within 72 hours of admission. A waiver of consent was obtained if the patient was discharged or died within 24 hours. Patients were excluded if they were <16 years of age, pregnant, a prisoner, had burns covering more than 20% of their total surface area, were taking pre-admission anticoagulants, died within 24 hours, or did not provide informed consent. A prospective power analysis estimated that 50 patients per group (with vs. without thromboembolic events) would be sufficient to detect a 20% difference in thrombin generation, using an alpha of 0.05 and a power of 0.90%.

[0054] Upon admission to the emergency department, patient characteristics, vital signs, standard clinical laboratory values, mechanism and severity of injury, and pre-admission fluid and / or blood product administration were collected. Outcomes (PE, days free from hospitalization, days free from mechanical ventilation, days free from intensive care unit (ICU), mortality), and 24-hour blood product administration and fluid administration were collected from patient records. A matched group design was used to compare patients with and without PE based on age, sex, mechanism and severity of injury. After identifying all PE patients, non-PE patients admitted within this timeframe with similar age, sex, and injury mechanism and severity to the PE group were selected. The Mann-Whitney U test was performed to compare these features between the PE and non-PE groups. If differences approaching p=0.06 were identified, new non-PE patients were selected and the comparison was repeated until the groups were adequately matched.

[0055] PE classification Since the facility routinely monitors for PE symptoms without screening for DVT, VTE was defined as the onset of PE in this study. Even when patients presented with mild symptoms associated with PE, such as cough or shortness of breath, or when an asymptomatic pulmonary embolism was incidentally identified during the examination of other signs, a standard contrast-enhanced computed tomography (CTA) of the chest was performed to confirm the diagnosis. All diagnoses of PE were clinically confirmed using radiological criteria, and chest images derived from CTA were examined by the staff radiologist for contrast-filled defects in the pulmonary vascular system. PE diagnoses were then classified as central (involving the left and / or right main pulmonary artery or one or more lobar arteries) or segmental / subsegmental (involving segmental and / or subsegmental arteries) based on the location of the filling defect.

[0056] Sample collection Patient samples were collected upon admission, prior to clinical administration of enoxaparin. Along with blood collection for standard hospital clinical testing, an additional 20 mL of blood was obtained for research purposes. The blood was transferred to a Vacutainer tube containing 3.2% citrate and centrifuged at 3,200 rpm for 20 minutes at room temperature after 30 minutes to obtain platelet-poor plasma. The plasma samples were then divided equally and stored at -80°C until analysis. Plasma was also collected from 15 healthy, consenting volunteers under a separate protocol (HSC-MS-09-0314). Volunteers were excluded if they were taking anticoagulants or antiplatelet agents, were pregnant, or had a history of cardiovascular disease. Healthy subjects had a median age of 31 (30, 35), were 50% male, and 63% were Caucasian. Fresh frozen plasma (FFP) from five donors was purchased from the Gulf Coast Regional Blood Center. The pool of five donors was used for all ex vivo experiments.

[0057] plasma analysis Citrate-oxidized plasma samples were thawed immediately before use. First, the ex vivo administration of enoxaparin was optimized using plasma from healthy, intact volunteers. The ex vivo dose of enoxaparin required to achieve 50% inhibition of thrombin generation was found to be 0.13 IU / mL, similar to that previously identified in the literature [22-24]. Considering that the inventors mixed a 0.13 IU / mL dose of enoxaparin into the plasma, the inventors' target anti-FXa level in response to enoxaparin was defined as 0.13 IU / mL. The response to enoxaparin was compared between healthy, intact volunteers and trauma patients by treating all plasma samples ex vivo with 0.13 IU / mL of enoxaparin. Trauma patient samples were supplemented with AT using 30 vol% FFP doses or AT concentrates in the form of THROMBATE III® provided by Grifols, up to final concentrations of 80%, 120%, 150%, 180%, and 200% based on baseline values. This was done to improve the patient plasma response to enoxaparin to levels observed in healthy controls. As in our previous studies, we selected 30 vol% FFP doses to model the effect of transfusing 6 units of plasma

[25] . Calculations were based on the activity (IU) (approximately 500 IU / 10 mL) provided by the manufacturer for a given lot of THROMBATE III® and the patient's baseline AT activity level. AT supplementation was confirmed by measuring AT activity using ACL-TOP. Thrombin generation and anti-FXa levels were measured to determine the response to enoxaparin and AT supplementation. Plasma from healthy volunteers served as an intact control for normal AT, anti-FXa, and thrombin production levels before and after enoxaparin treatment.

[0058] AT activity and anti-FXa levels were determined by colorimetric analysis using an ACL-TOP Coagulation Analyzer (Instrumentation Laboratory, Bedford MA). Thrombinogenesis was measured using a calibrated automated thrombogram (CAT) (Thermo Fisher Scientific, Waltham MA) as previously described [8]. Relevant parameters obtained from CAT analysis include lag time (time to the start of thrombinogenesis; minutes), peak (maximum thrombin production; nM), time to peak [ttpeak] (time to reach the peak; minutes), endogenous thrombin activity [ETP] (total amount of thrombin produced over time; nM), and rate (rate of thrombinogenesis determined by the slope of the thrombinogenesis curve; nM / min).

[0059] statistics All analyses were performed using GraphPad Prism 6 (La Jolla, CA). For multiple comparisons, one-way or two-way ANOVA tests were performed using Bonferroni or Sidak corrections, respectively. Mann-Whitney tests were performed where instructed. Data were considered significant if p<0.05.

[0060] result A consort diagram describing the patient cohort enrollment is provided in Figure 1. During the study period, 6,089 patients underwent the highest level of trauma at the inventors' facility.

[0061] Of these, 3,797 met the inclusion criteria for the inventors' study. Plasma samples were available from the inventors' biorepository for 2,613 of these patients. A total of 55 patients with PE were identified during the study period. Of these patients, 3 were excluded due to pre-admission anticoagulant use, and 7 were excluded due to death from unsalvageable traumatic brain injury. This left 100 patients for complete analysis (46 without PE and 54 with PE). The median time to PE diagnosis was 9 days (4, 14) of hospitalization. Of these patients who developed PE, 68.5% were symptomatic and 31.5% were asymptomatic. Most PEs were located in segmental or subsegmental arteries (67%), with the remainder located centrally (33%). Approximately 85% of PE patients received thrombosis-related interventions, including thrombectomy, tPA infusion, inferior vena cava (IVC) filter placement, unfractionated heparin infusion, or another anticoagulant. Only 8 patients (15%) were maintained with enoxaparin alone. Patient characteristics, injury type / severity, and outcomes are summarized in Table 1. There were no significant differences in characteristics or injury type and severity between patients who developed PE and those who did not. However, patients who developed PE received significantly more pre-admission crystalloids and units of blood products during transport and the first 24 hours of hospitalization (all p<0.05). Furthermore, patients who developed PE had significantly fewer ventilator-free days, ICU-free days, and hospital-free days (all p<0.01), but the overall in-hospital mortality rate was the same (8.5% vs. 9.3%; p>0.05). Finally, although no difference in pelvic fracture rates was observed, patients who developed PE had a significant increase in lower limb fracture rates (28% vs. 11%; p<0.05).

[0062] [Table 1]

[0063] However, the presence of such fractures did not affect the relevant outcomes of this study, such as AT levels with or without AT supplementation, AT deficiency rates, thrombin generation, or responsiveness to enoxaparin.

[0064] Effects of enoxaparin on healthy plasma; effects of enoxaparin on plasma from trauma patients. As in previous reports (7), baseline thrombin generation was significantly elevated in trauma patients compared to healthy donors, as evidenced by reduced lag time, increased peak activity, decreased time to peak, and increased thrombin generation rate (Table 2).

[0065] [Table 2]

[0066] Trauma patients also showed significantly lower anti-FXa levels after enoxaparin treatment compared to healthy controls. In response to enoxaparin treatment, all healthy donors achieved a targeted anti-FXa response of 0.13 IU / mL compared to 59% of trauma patients (p<0.01). Compared to healthy controls, trauma patients showed a significant reduction in inhibition of thrombin production after enoxaparin treatment, as evidenced by reduced peak thrombin inhibition, reduced endogenous thrombin activity (ETP) inhibition, and reduced prolongation of thrombin production rate.

[0067] Enoxaparin supplementation with FFP or AT To determine whether supplementation with FFP or AT increases AT levels and improves the response to enoxaparin, trauma patient plasma was treated ex vivo with FFP (30% by volume) or AT (final concentration of 120–200%) in addition to enoxaparin (0.13 IU / mL) (Figure 2). FFP had a baseline AT level of 98 (96, 102). Treatment with FFP had no effect on AT levels, but AT supplementation significantly increased baseline AT levels (limit of detection = 180%). Treatment with FFP did not improve anti-FXa levels compared to enoxaparin alone, but AT-supplemented enoxaparin increased anti-FXa levels in a dose-dependent manner (Figure 3A: all doses p<0.05).

[0068] FFP treatment improved the treatment response rate from 59% to 80%, but AT supplementation up to 120% resulted in 92% of trauma patient plasma reaching therapeutic anti-FXa levels. This increased to 100% after increasing AT supplementation to over 150%. In addition, while determining whether FFP or AT supplementation could improve enoxaparin's ability to inhibit thrombin generation, we found that both FFP and AT could significantly increase peak thrombin inhibition compared to enoxaparin alone (all p<0.05). Maximum inhibition was observed at AT levels of 150–200% (Figure 3B).

[0069] The effect of enoxaparin in plasma from patients with and without PE. Baseline AT activity was similar between groups with no difference in the incidence of AT deficiency (Table 3). However, when comparing thrombin generation, patients who developed PE had significantly higher peak (13%) and faster rates (24%) of thrombin generation at baseline compared with patients without PE. Furthermore, patients who developed PE had a significantly reduced response to enoxaparin ex vivo, as measured by both anti-FXa levels and changes in thrombin generation. Anti-FXa levels in response to enoxaparin were significantly higher in plasma from patients without PE compared with patients who developed PE (0.14 vs. 0.13 IU / mL; p<0.05). Ex vivo treatment with enoxaparin resulted in a median decrease of 30.6% in peak thrombin in patients without PE, compared to only a 20.7% decrease in peak thrombin in patients with PE (p<0.01).

[0070] [Table 3]

[0071] Enoxaparin replacement with AT in plasma from PE patients. To determine the effect of AT supplementation on the ex vivo response to enoxaparin, plasma from both PE and non-PE patients was treated with enoxaparin and AT, and changes in anti-FXa and thrombin inhibition were observed. AT significantly increased anti-FXa levels and decreased peak thrombin generation at all doses in plasma from all patients tested. Enoxaparin alone resulted in lower anti-FXa levels (Figure 4A) and reduced peak thrombin inhibition (Figure 4B) in PE patient plasma compared to non-PE patients; however, these differences in the enoxaparin response were no longer apparent when AT supplementation was ≥120%.

[0072] Consideration Despite aggressive protocolized thromboprophylaxis, the persistence of VTE in trauma patient populations is a significant health management concern requiring new, evidence-based treatment strategies. AT concentrate is an FDA-approved, clinically available therapeutic agent used to treat congenital AT deficiency. To date, there is little data demonstrating the potential role of AT supplementation in improving VTE prophylaxis in trauma patients. Here, we demonstrate that plasma samples from severely injured patients, particularly those who developed PE, show reduced sensitivity to enoxaparin, and that AT supplementation improves enoxaparin-mediated thrombin inhibition ex vivo.

[0073] Enoxaparin and other low molecular weight heparin molecules are the most commonly used prophylactic agents in recovering critically ill patients. Enoxaparin increases both anti-FXa levels and thrombin AT inhibition, making it an essential cofactor in preventing VTE. AT deficiency is generally defined by hematological criteria as activity <80% with normal levels ranging from 80 to 120%. According to this definition, hospital-induced AT deficiency occurred in approximately 20% of trauma patients enrolled in this study, which is similar to rates reported elsewhere [3]. While the majority of patients had AT levels within the normal limits, only half of all patient plasma samples achieved a target anti-FXa level of 0.13 IU / mL after treatment with enoxaparin.

[0074] The inventors have previously shown that trauma patients exhibit significantly elevated thrombinogenesis, a known risk factor for VTE, compared to healthy subjects [7] [8]. Indeed, a significant elevation was detected in this study as well. In situations of such excessive and prolonged thrombinogenesis, circulating levels of AT may not be sufficient to promote prophylactic anticoagulation with enoxaparin. Our findings suggest that hyperphysiological levels of ATIII may be required to compete with such high levels of thrombinogenesis. Here, we show that supplementation of AT up to 120% in trauma patient plasma increased enoxaparin efficacy from 59% to 92%, and that increasing AT to over 120% optimized the enoxaparin response in terms of both anti-FXa levels and inhibition of thrombinogenesis. Maximum efficacy of 100% was achieved when AT levels were increased to 150%. These findings are consistent with other reports in the literature. Animal models of sepsis have shown that high doses of AT (approximately 160%) are most beneficial for alleviating hypercoagulation, reducing inflammation, and protecting against organ damage

[26] . Maintaining such high levels of ATIII activity may not be feasible for all trauma patients, but it may be appropriate to screen patients for potential responsiveness after routine assessment of their anti-FXa levels. Patients who do not achieve therapeutic range may be the best candidates for ATIII therapy. This may also offer an effective and safe alternative to risky interventions for patients with segmental or subsegmental thrombi.

[0075] Despite similar AT levels at admission, patients who later developed PE had a significantly reduced response to enoxaparin compared to those who did not, and this deficit was corrected by AT supplementation. This finding suggests that while other mechanisms of enoxaparin resistance not evaluated here may exist, modifying AT levels is still an effective intervention to increase plasma enoxaparin sensitivity.

[0076] FFP has been used in the past to treat heparin resistance [17, 28], but we found here that it has no significant effect, which is consistent with previous studies

[18] . Even at 30 vol% equivalent to approximately 6 units or about 20 ml / kg in a 70 kg adult, FFP had no effect on AT levels or on the anti-FXa response to enoxaparin. However, FFP was able to significantly reduce thrombin production more than enoxaparin alone. This is likely due to endogenous AT, in addition to other circulating anticoagulants present in balanced FFP that affect thrombin production, such as tissue factor pathway inhibitors, protein C, protein S, and thrombomodulin. Therefore, as previously shown, FFP administration may function to mitigate thrombin production by diluting endogenously hypercoagulable plasma with balanced plasma

[25] , but our data suggest that it has only a very slight direct effect on improving PE prevention.

[0077] In summary, trauma is associated with decreased AT levels, increased thrombin generation, and reduced responsiveness to enoxaparin. In patients who developed PE, thrombin generation was higher and the response to enoxaparin was lower, and ex vivo treatment with AT rather than FFP resulted in improved enoxaparin-mediated inhibition of thrombin generation.

[0078] Abbreviation AIS, Simplified Injury Scale; ANOVA, Analysis of Variance; AT, Antithrombin III; CAT, Calibrated Automated Thrombogram; CTA, Computed Tomography Angiography; DVT, Deep Vein Thrombosis; ETP, Endogenous Thrombin Activity; FFP, Fresh Frozen Plasma; FXa, Coagulation Factor Xa; GCS, Glasgow Coma Scale; ICU, Intensive Care Unit; ISS, Injury Severity Score; Kg, Kilogram; PE, Pulmonary Embolism; RBC, Red Blood Cells; RPM, Turns Per Minute; ttPeak, Time to Peak; VTE, Venous Thromboembolism; w-RTS, Weighted Modified Trauma Score. (References) TIFF0007854662000004.tif239168TIFF0007854662000005.tif239168TIFF0007854662000006.tif231168TIFF0007854662000007.tif98168

Claims

1. A composition for reducing the occurrence of thrombosis or thromboembolism in patients identified as being at risk, comprising an anticoagulant selected from the group consisting of unfractionated heparin, low molecular weight heparin, heparinoids, fondaparinux, hydraparinux, and combinations thereof, and antithrombin (ATIII), wherein ATIII is administered at a concentration that increases the patient's ATIII level to greater than 1.2 IU / mL, and the patients identified as being at risk are patients with blunt physical trauma.

2. The composition according to claim 1, wherein the thrombosis is venous thrombosis.

3. The composition according to claim 1 or 2, wherein the thromboembolism is venous thromboembolism (VTE).

4. The composition according to claim 1, wherein ATIII is administered at a concentration that increases the patient's ATIII level to more than 1.3 IU / mL.

5. The composition according to claim 1, wherein ATIII is administered at a concentration that increases the patient's ATIII level to more than 1.4 IU / mL.

6. The composition according to claim 1, wherein ATIII is administered at a concentration that increases the patient's ATIII level to more than 1.5 IU / mL.

7. The composition according to claim 1, wherein ATIII is administered at a concentration that increases the patient's ATIII level to a range of 1.5 IU / mL to 2.5 IU / mL.

8. The composition according to claim 1, wherein ATIII is administered at a concentration that increases the patient's ATIII level to a range of 1.5 IU / mL to 2.0 IU / mL.

9. The composition according to claim 1, wherein ATIII is derived from plasma or is recombinant.

10. The composition according to claim 1, wherein the low molecular weight heparin is selected from the group consisting of bemiparin, sertoparin, dalteparin, enoxaparin, nadroparin, parnaparin, reviparin, tinzaparin, combinations thereof, and pharmaceutically acceptable salts thereof.

11. The composition according to claim 1, wherein the heparinoid is selected from the group consisting of danaparoids, dermatan sulfate, throdoxides, combinations thereof, and pharmaceutically acceptable salts thereof.

12. The composition according to claim 1, wherein the anticoagulant is low molecular weight heparin.

13. The composition according to claim 12, wherein the low molecular weight heparin is enoxaparin or a pharmaceutically acceptable salt thereof.

14. The composition according to claim 1, wherein the therapeutically effective dose of low molecular weight heparin is 20 mg to 180 mg per day.

15. The composition according to claim 14, wherein the therapeutically effective amount of low molecular weight heparin is 20 mg to 40 mg per day.

16. The composition according to claim 1, wherein the therapeutically effective dose of low molecular weight heparin is 0.1 to 2.5 mg / kg.

17. The composition according to claim 16, wherein the therapeutically effective amount of low molecular weight heparin is 0.5 to 1.5 mg / kg.

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