Composition comprising prostacyclin andor analogues thereof for treatment of acute critically ill patients
11714094 · 2023-08-01
Assignee
Inventors
Cpc classification
A61K31/5585
HUMAN NECESSITIES
A61K45/06
HUMAN NECESSITIES
A61K2300/00
HUMAN NECESSITIES
A61K2300/00
HUMAN NECESSITIES
A61K31/5585
HUMAN NECESSITIES
International classification
A61K31/5585
HUMAN NECESSITIES
A61K45/06
HUMAN NECESSITIES
Abstract
Use of prostacyclin or an analogue thereof for treatment of a new medical indication in acute critically ill patients, in particular acute critically ill patients with systemic endothelial damage, a biomarker for identifying individuals that have a new medical indication, and a method for identifying a new medical indication.
Claims
1. A method of diagnosing for an individual diagnosed with an acute respiratory failure due to capillary leakage in the lungs caused by sepsis, if said individual is a candidate for combination treatment of standard care including ventilator therapy for said acute respiratory failure in combination with administration of prostacyclin or an analogue thereof, said method comprising: measuring a baseline concentration of soluble thrombomodulin in blood or plasma of said individual; comparing the level of said baseline concentration of soluble thrombomodulin in blood or plasma of said individual to a threshold level of 2.5 ng/ml; and identifying said individual whose thrombomodulin is greater than 2.5 ng/ml followed by administering the combination treatment of said standard care for said acute respiratory failure in combination with administration of prostacyclin or an analogue thereof to said individual period if said baseline concentration of soluble thrombomodulin in blood or plasma is above said threshold level of at least 2.5 ng/ml.
2. A method according to claim 1, wherein said threshold level is at least 4 ng/ml.
3. A method according to claim 1, wherein said method is a method of diagnosing severe endothelial damage in said individual diagnosed with said acute respiratory failure; wherein said individual is diagnosed with severe endothelial damage in addition to said acute respiratory failure, if said baseline concentration is above said threshold level.
4. A method according to claim 1, wherein said method is a method of diagnosing systemic endotheliopathic syndrome in an individual diagnosed with said acute respiratory failure; wherein said individual is diagnosed with systemic endotheliopathic syndrome when said individual is diagnosed with both said acute respiratory failure and severe endothelial damage.
5. A method of using thrombomodulin as a biological marker in a method for diagnosing for an individual diagnosed with an acute respiratory failure due to capillary leakage in the lungs caused by sepsis, if said individual is a candidate for combination treatment with standard care including ventilator therapy for said acute respiratory failure in combination with administration of prostacyclin or an analogue thereof, said method comprising: measuring a baseline concentration of soluble thrombomodulin in blood or plasma of said individual; comparing the level of said baseline concentration of soluble thrombomodulin in blood or plasma of said individual to a threshold level of 2.5 ng/ml; and identifying said individual whose thrombomodulin is greater than 2.5 ng/ml followed by administering the combination treatment of said standard care for said acute respiratory failure in combination with administration of prostacyclin or an analogue thereof to said individual if said baseline concentration of soluble thrombomodulin in blood or plasma is above said threshold level of at least 2.5 ng/ml.
6. A method according to claim 5, wherein said threshold level is at least 4 ng/ml.
7. A method according to claim 5, wherein said method is a method of diagnosing severe endothelial damage in said individual diagnosed with said acute respiratory failure; wherein said individual is diagnosed with severe endothelial damage in addition to said acute respiratory failure, if said baseline concentration is above said threshold level.
8. A method according to claim 5, wherein said method is a method of diagnosing systemic endotheliopathic syndrome in an individual diagnosed with said acute respiratory failure; wherein said individual is diagnosed with systemic endotheliopathic syndrome when said individual is diagnosed with both said acute respiratory failure and severe endothelial damage.
9. A method according to claim 5, wherein administration of prostacyclin or an analogue thereof to said individual includes administering a dose of 0.5-4 ng/kg/min of prostacyclin or an analogue thereof to said individual continuously for a first time period if said baseline concentration of soluble thrombomodulin in blood or plasma is above said threshold level of at least 2.5 ng/ml.
10. A method of treating an acute respiratory failure in an individual diagnosed with said acute respiratory failure and undergoing standard care including ventilator therapy for said acute respiratory failure and concurrent increase in a measured thrombomodulin level in blood or plasma of said individual, said method comprising: (a) measuring a baseline concentration of soluble thrombomodulin in blood or plasma of said individual; (b) comparing the level of said baseline concentration of soluble thrombomodulin in blood or plasma of said individual to a threshold level of 2.5 ng/ml; (c) identifying said individual whose thrombomodulin is greater than 2.5 ng/ml followed by administering a dose of 0.5-4 ng/kg/min of prostacyclin or an analogue thereof to said individual continuously for a first time period if said baseline concentration of soluble thrombomodulin in blood or plasma is above said threshold level of at least 2.5 ng/ml; (d) measuring at the end of said first time period a concentration of soluble thrombomodulin in blood or plasma of said individual; (e) determining if said concentration of soluble thrombomodulin is lower by at least a decrease of 10% compared to said baseline concentration of thrombomodulin determined prior to initiation of said prostacyclin administration; (f) assessing if a clinical improvement of said acute respiratory failure in said individual has occurred during said first time period; and (g) if both a concentration reduction and a clinical improvement is observed, ceasing prostacyclin administration while continuing said standard care for said acute respiratory failure; or (h) otherwise continue prostacyclin administration and said standard care for a second time period not exceeding said first time period wherein the steps (d) to (h) are repeated until ceasing prostacyclin administration to said individual following step (g).
11. A method according to claim 10, wherein said threshold level is at least 4 ng/ml.
12. A method according to claim 10, wherein said dose is 1-2 ng/kg/min.
13. A method according to claim 10, wherein said analogue of prostacyclin is either Iloprost or Flolan.
14. A method according to claim 10, wherein said first time period is at least 48 hours or at least 72 hours.
15. A method according to claim 10, wherein said second time period is 12 hours or 24 hours.
16. A method according to claim 10, wherein in step (e) said decrease is at least 20%.
17. A method according to claim 10, wherein said prostacyclin administration is initiated immediately or shortly after a completion of step (b) has determined that said baseline concentration of thrombomodulin in blood or plasma is above said threshold level.
18. A method according to claim 10, wherein prostacyclin or an analog thereof is administered as according to step (c) prior to steps (a) and (b) having been completed.
19. A method according to claim 10, wherein said concurrent increase in a measured thrombomodulin level in blood or plasma of said individual is the result of severe endothelial damage.
20. A method according to claim 1, wherein the measurement of thrombomodulin is conducted by a point-of-care (POC) assay.
21. A method according to claim 10 wherein the measurement of thrombomodulin is conducted by a point-of-care (POC) assay.
Description
DESCRIPTION OF DRAWINGS AND TABLES
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DETAILED DESCRIPTION
(36) The aspects of the disclosed embodiments relate to the treatment of acute critically ill patients. Patients that are acute critically ill suffer from e.g. severe trauma including burn injury, severe infection including sepsis and septic shock, acute myocardial infarction, resuscitated cardiac arrest, major surgery and/or other conditions that require organ supportive care/intensive care therapy to survive i.e. ventilator therapy, continuous fluid infusion or blood transfusion, vasopressor therapy etc.
(37) The inventors have discovered that some acute critically ill patients, independent of the type of injurious hit, display evidence of a common unifying medical condition that is associated with increased mortality. In prior research papers, e.g. [Johansson and Ostrowski, 2010], the present inventors have suggested that this common unifying medical condition is a distinct diagnosis which the inventors have now labelled systemic endothelial damage.
(38) Based on their findings in thousands acute critically ill patients, the inventors discovered that a large fraction of these patients presented with systemic endothelial damage that across all patients was the strongest marker for mortality. The combination of acute critical illness and systemic endothelial damage has been suggested by the present inventors to represent a novel disease entity which the inventors now label systemic endotheliopathic syndrome, characterized by impaired vascular barrier function and capillary leakage, bleeding, hypotension, multiple organ failure and death in acute critically ill patients [Johansson and Ostrowski 2010]. A patient is considered to have systemic endotheliopathic syndrome when the patient suffers from acute critical illness AND is diagnosed with systemic endothelial damage.
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(40) The present disclosure demonstrates that systemic endothelial damage can be identified and diagnosed in a simple manner by using the soluble thrombomodulin level in a blood sample as a biological marker for the presence or absence of systemic endothelial damage. Using thrombomodulin as a biological marker, acute critically ill patients presenting with blood thrombomodulin levels above a given threshold value can be rapidly identified as individuals suffering from systemic endothelial damage; thereby in a simple manner differentiating this group of patients from acute critically ill patients that do not suffer from systemic endothelial damage.
(41) Furthermore, the present disclosure demonstrates that acute critically ill patients diagnosed with systemic endothelial damage can beneficially receive early treatment with compositions comprising prostacyclin or analogues thereof, such as prostacyclin (PGI2) and prostacyclin (PGX) or analogues thereof such as e.g. iloprost, epoprostenol, epoprostenol Sodium, treprostenil sodium, selexipag or beraprost; thereby significantly reducing development of organ failure, improving long term full recovery and reducing mortality.
(42) A central part of the present invention is a personalized medicine approach to treatment i.e., that only acute critically ill patients suffering from systemic endotheliopathic syndrome should be treated with prostacyclin or analogues whereas acute critically ill patients without systemic endotheliopathic syndrome (with low thrombomodulin) should not be treated with prostacyclin or analogues thereof.
(43) Given the direct effects of prostacyclin and analogs thereof on the endothelium (described in details below), the inventors suggest that infusion of low-dose prostacyclin can be used to treat systemic endotheliopathic syndrome.
(44) Proof-of-concept studies of this intervention and its effect on the endothelium measured by changes in circulating levels of thrombomodulin is described below.
(45) The inventors suggest that prostacyclin analogs should be used for treatment of systemic endotheliopathic syndrome, identified in acute critically ill patients by a quick test measuring the level of circulating thrombomodulin.
(46) In further detail the present invention relates to a method of diagnosing, uses of thrombomodulin as a biological marker, a method of treatment; prostacyclin or an analogue thereof for use in novel treatments and compositions comprising prostacyclin or an analogue thereof for use in novel treatments, all according to the embodiments described in this section in particular, and throughout the present disclosure.
(47) In a first aspect and embodiment there is disclosed a method of diagnosing for an individual diagnosed with an acute critical illness, if the individual is a candidate for combination treatment of standard care for the acute critical illness in combination with administration of prostacyclin or an analogue thereof, the method comprising: measuring a baseline concentration of soluble thrombomodulin in blood or plasma of the individual; determining if the baseline concentration of soluble thrombomodulin in blood or plasma is above a threshold level of at least 2.5 ng/ml; and diagnosing the individual as a candidate for combination treatment if the baseline concentration is above the threshold level.
(48) This first aspect and embodiment of the invention is further detailed in
(49) Generally, the information contained in
(50) The advantage of presenting the information of
(51) As an example, treatment with prostacyclin or an analog thereof according to the disclosure of the present invention can be initiated immediately upon diagnosis of the patient with an acute critical illness, without hesitating to obtain the further diagnose of systemic endothelial damage through measurement of the patient's thrombomodulin levels. This expedites the initiation of the treatment with prostacyclin or an analogue thereof and is possible due to the low risk of an adverse effect from the mentioned treatment.
(52) When initiating rapid treatment as described above, the first measurement for identification of systemic endothelial damage following the disclosure of the present invention, the also becomes the first control measurement, since a patient which is not in need of prostacyclin treatment can be stopped from receiving further treatment once the first test results have been obtained.
EXAMPLES
(53) Studies of the Endothelium in Acute Critically Ill Patients
(54) The inventors have investigated more than 4,400 patients suffering from severe trauma, sepsis, acute myocardial infarction, resuscitated cardiac arrest and major surgery, including 1,750 patients with varying degrees of sepsis. In these studies, the inventors found that a common and critical link may exist between the severity of the injuries; development of coagulopathy and progressive endothelial damage (endotheliopathy) and mortality.
(55) In the above studies, across the different patient groups, the inventors found that endothelial cellular damage, evidenced by high circulating levels of soluble thrombomodulin, was the strongest marker for mortality.
(56) Importantly, across the investigated groups of different patients with acute critical illness, not all patients developed systemic endotheliopathic syndrome and these patients, with low circulating thrombomodulin, had an excellent outcome with high survival rates. This caused the inventors to realize that in some instances systemic endotheliopathic syndrome may result from acute critical illness such as e.g. severe trauma, sepsis, acute myocardial infarction, resuscitated cardiac arrest and major surgery or other types of acute critical illness, but that this outcome is not necessarily given.
(57) Consequently, patients presenting with concurrent acute critical illness AND high circulating thrombomodulin levels above a given critical threshold limit can be diagnosed as presenting with systemic endotheliopathic syndrome.
(58) To the surprise of the inventors they have now further discovered that patients presenting with systemic endotheliopathic syndrome, i.e. presenting with an acute critical illness and high (above a given threshold) thrombomodulin levels in the blood, will benefit from treatment with prostacyclin or analogues thereof with the aim of curing or alleviating the suffered acute critical illness and reducing patient mortality in situations where patients presenting with the same acute critical illness but not presenting with the additional diagnose of systemic endothelial damage as evidence by a high (above a given threshold) thrombomodulin level in the blood, will not.
(59) Many acute critically ill patients are in shock i.e. a medical emergency condition in which the tissue and organs of the body are deprived of oxygen due to inadequate blood flow.
(60) There are four stages of shock: Initial, compensatory, progressive and refractory. In the initial stage, hypoperfusion causes hypoxia which is followed by compensatory physiological mechanisms, including neural (including sympathoadrenal activation) and hormonal mechanisms in an attempt to reverse the condition. If the condition is not reversed it will proceed into the progressive stage where the compensatory mechanisms begin to fail resulting in severely reduced cell, tissue and organ perfusion and a rise in anaerobic metabolism. Eventually this will lead to leakage of fluid and protein into tissues and upon loss of this (intravascular) fluid, the blood concentration and viscosity will increase, causing sludging of the microcirculation. Also, the prolonged vasoconstriction will cause the vital organs to be compromised due to reduced perfusion. In the final refractory stage, the vital organs will have failed and the shock can no longer be reversed.
(61) All stages of shock are associated with systemic endothelial perturbations ranging from reversible activation and damage to irreversible extensive injury. Therefore the present inventors have realized that administration of prostacyclin or an analogue thereof to acute critically ill patients according to the teaching of the present disclosure, will serve through its beneficial effects on the vascular endothelium and its cytoprotective effects, as an anti-shock therapy.
(62) Testing and Treatment
(63) Acute Critically Ill Patients
(64) Patients that are acute critically ill suffer from e.g. severe trauma, including burn injury, severe infection including sepsis and septic shock, acute myocardial infarction, resuscitated cardiac arrest, major surgery and other conditions that happens immediately and require organ supportive care to survive i.e. ventilator therapy, continuous fluid infusion or blood transfusion, vasopressor therapy etc.
(65) When an acute critically ill patient requires pre-hospital emergency care OR is admitted to the Hospital (the Emergency Department, the Trauma Center or other Departments that have the initial contact with the patient) OR when a hospitalized patient becomes acute critically ill in-hospital (in hospital cardiac arrest (INCA), in hospital sudden development of septic shock etc.), a blood sample for measurement of thrombomodulin is drawn as early as possible i.e., during transportation of the patient to the Hospital or within minutes up to hours from Hospital admission or in-hospital recognition of the acute critical illness. At the same time point the patient receives standard care for the acute critical illness i.e. ventilation, fluid therapy, blood transfusion, antibiotics, vasopressor therapy, damage control surgery, specific drugs used for the specific acute critical illness e.g. platelet inhibitors and anticoagulants in cardiac arrest caused by myocardial infarction.
(66) Optimally, the patient blood sample is drawn within the first minutes or hours pre-hospital or within the first minutes or hours after admission/recognition of acute critical illness. However, blood samples taken up to 12 hours of admission/recognition of acute critical illness can still be relevant.
(67) Thrombomodulin Testing
(68) The whole blood sample is immediately spun in a centrifuge (e.g. 3000 rpm for 10 minutes) to separate the plasma phase from the blood cell phase. A small volume of the plasma fraction (e.g. from 10-200 μl) is pipetted onto an immunoassay e.g. an enzyme linked immunosorbent assay (ELISA). Currently available ELISA assays require approximately 1 h incubation with patient sample, hereafter 30 min incubation with detection reagent and hereafter 15 min incubation with substrate solution before the ELISA plate can be read by an ELISA reader, corresponding to a total assay time of less than 2 hours. The thrombomodulin concentration in the patient sample is calculated by comparing the assay output e.g. absorbance in the sample to reference samples with a known concentration of thrombomodulin (a standard curve).
(69) When the concentration of thrombomodulin in the patient plasma sample has been determined, this will either be above or below the predetermined threshold level of at least 2.5 ng/ml, preferably 4 ng/ml. Patients with thrombomodulin values above the threshold level suffer from systemic endotheliopathic syndrome and are candidates for treatment with prostacyclin and analogues thereof. Patients with thrombomodulin values below the threshold level do not have systemic endothelial damage and will not be candidates for treatment with prostacyclin and analogues thereof.
(70) Preferably, the thrombomodulin test is be a quick point-of-care (POC) test based on whole blood input so a drop of whole blood is placed onto e.g. a lateral flow assay (stick) or another immunoassay platform like e.g. cartridges based on microfluidics technology, to display a result within 2-5 minutes reporting whether a patient has a thrombomodulin level above or below a predetermined threshold level.
(71) Treatment Initiation
(72) Immediately after the test result is available i.e., from minutes to approximately 2-3 hours after blood sampling pre-hospital or patient admission/recognition of acute critical illness when using the ELISA assay described above or 15-30 min after blood sampling pre-hospital or patient admission/recognition of acute critical illness if using the quick whole blood test or POC assay as described above, the patient starts treatment with an intravenous infusion of prostacyclin or analogues thereof in a dose of 0.5-4 ng/kg/min, preferably 1-2 ng/kg/min.
(73) To administer the treatment through an intravenous infusion system, the prostacyclin or analogues thereof either has to be diluted in an appropriate infusion media (e.g. 0.9% saline) to obtain an appropriate concentration of prostacyclin and analogues thereof in the infusion fluid or the prostacyclin and analogues thereof are available in a ready-to-use appropriate infusion solution.
(74) An appropriate amount of prostacyclin and analogues thereof diluted in the intravenous infusion fluid corresponds to a 24-30 hour weight adjusted dose in a small volume, e.g., 100-500 ml. For example, in a patient with a body weight of 70 kg treated for 24 hours with 1 ng/kg/min prostacyclin and analogues thereof as a 4 ml/hour intravenous infusion, this corresponds to a concentration of prostacyclin and analogues thereof of 105 μg/100 ml in the infusion dilution bag (1 ng/kg/min*70 kg*60 min*25 hours=105,000 ng/100 ml=105 μg/100 ml).
(75) For each 24 hours, a new infusion dilution of prostacyclin and analogues thereof will be prepared and administered (as described above) as continues intravenous infusion to the patient for the following 24 hours.
(76) The treating staff (pre-hospital emergency staff, medical doctors, nurses etc.) has simple instructions for how to prepare an appropriate infusion dilution of the treatment for a patient with a given weight. The instructions should be flexible so the treating staff can either administer the treatment as a fixed volume infusion (with variable concentration of prostacyclin and analogues thereof in the infusion fluid, adjusted to patient weight) or a variable volume infusion (with a fixed concentration of prostacyclin and analogues thereof in the infusion fluid), depending on the type of infusion the treating staff normally use pre-hospital or in their Department/ICU/ward.
(77) During the treatment with prostacyclin and analogues thereof the patient receives normal standard of care i.e., supportive care e.g. ventilation, fluid therapy, blood transfusion, antibiotics, vasopressor therapy, damage control surgery, specific drugs used for the specific acute critical illness etc. The therapy is thus an add-on to the normal standard of care treatment regimen.
(78) Optimally the intravenous infusion with prostacyclin or analogues thereof in the dose should begin as early as possible after the thrombomodulin test results has become available i.e., within 30 min-12 hours after known test results, pre-hospital as well as in-hospital. The time to test should not exceed 6 hours after presentation.
(79) Monitoring Treatment Response and Deciding Length of Treatment
(80) The intravenous infusion with prostacyclin and analogues thereof should continue for a minimum of 72 hours (3 days) up till several weeks.
(81) After the first 3 treatment days, the circulating thrombomodulin level should be measured to reveal whether this has decreased, increased or has remained unchanged compared to the baseline (pre-treatment) value. If the thrombomodulin level has decreased from baseline (e.g. a drop of 10% or preferably by 20% or more) and the patient has improved clinically judged by the attending physician (e.g., reduced need for supportive care), the intravenous infusion with prostacyclin and analogues thereof can be ceased so the patient receives just standard care. If however the thrombomodulin level has increased (>10% increase) or remained unchanged from baseline or the patient has not improved clinically judged by the attending physician (e.g., increased need for supportive care), the intravenous infusion with prostacyclin and analogues should continue for the next 24 hours as an add-on to standard care.
(82) After another 24 hours treatment (total 4 treatment days), the circulating thrombomodulin level should be measured to reveal whether this has decreased, increased or has remained unchanged compared to the baseline (pre-treatment) value. If the thrombomodulin level has decreased from baseline (by the aforementioned >10% drop or, preferably, by the aforementioned >20% drop) and the patient has improved clinically judged by the attending physician (e.g., reduced need for supportive care), the intravenous infusion with prostacyclin and analogues thereof can be ceased. If however the thrombomodulin level has increased (>10% increase) or remained unchanged from baseline or the patient has not improved clinically judged by the attending physician (e.g., increased need for supportive care), the intravenous infusion with prostacyclin and analogues should continue for the next 24 hours as an add-on to standard care.
(83) This loop of thrombomodulin guided prostacyclin treatment continues for up till several weeks, or until the patient has recovered clinically to an extend that allows discharge from ICU or receives an stop-resuscitation-code or dies.
(84) After the last 24 hours treatment (until a total of treatment time of up till several weeks), the intravenous infusion with prostacyclin and analogues thereof is ceased.
(85) During the treatment with prostacyclin and analogues thereof, the patient receives standard of care.
(86) Optimally, thrombomodulin values should be measured on a daily basis to decide if the treatment should continue for another 24 hours or should cease.
Example 1
Evidence of Mechanistic Link
(87) A mechanistic link between systemic endotheliopathic syndrome and mortality in acute critically ill patients was first indicated to the inventors in a study of ˜1,200 septic patients who were followed daily with blood samples from intensive care unit (ICU) admission and the following four days. Here, the inventors observed that patients who had a reduction in their levels of soluble thrombomodulin (a recognized crude marker of endothelial cell damage) over that time had an increased survival rate compared to patients with stable or increasing levels of thrombomodulin as an indicator of continued endothelial damage. Similarly, the inventors found that patients suffering from severe infection (from local infection to septic shock), trauma, acute myocardial infarction or resuscitated cardiac arrest all present with the same type of systemic endothelial damage, and across all patients the inventors found that thrombomodulin is the strongest marker for mortality.
(88) Together this supports the new notion relevant for the present disclosure that various types of acute critically ill patients may develop the same disease, systemic endotheliopathic syndrome, which can be diagnosed by clinical determination of an acute critical illness and high levels of circulating soluble thrombomodulin
(89) Evidence for Medical Use of Prostacyclin and Analogues Thereof for Treatment of Systemic Endotheliopathic Syndrome
(90) The inventors have investigated the anti-thrombotic potential of prostacyclin with functional whole blood hemostatic assays proven to correlate with clinical bleeding conditions and transfusion requirements (thrombelastography (TEG) and impedance aggregometry (Multiplate)) and surprisingly they discovered that low-dose prostacyclin infusion had no measurable anti-thrombotic effects.
(91) The inventors investigated (WO 2013/143548, examples 2 and 3) healthy volunteers and acute critical illness and found that in healthy volunteers, 1-4 ng/kg/min prostacyclin infusion neither influenced clot formation nor platelet aggregation. Furthermore, in acute critically ill patients undergoing major abdominal surgery (Example 6) undergoing PCI after acute myocardial infarction [Holmvang et al 2012, Example 5], 0.5-1 ng/kg/min prostacyclin infusion neither influenced clot formation nor platelet aggregation.
(92) Importantly, the inventors surprisingly found that intra- and post-operative prostacyclin infusion was associated with improved clot formation in patients undergoing major abdominal surgery (Example 6). The finding that low-dose prostacyclin infusion (0.5-1 ng/kg/min) in acute critically ill patients does not compromise hemostasis or induce bleeding or adverse events related to impaired hemostasis is in accordance with several other studies of acute critically ill patients treated for hours-to-days with low-dose (0.5-4 ng/kg/min) prostacyclin infusion: Traumatic brain injury patients (0.5-2 ng/kg/min for up to 72 hours) [Grande et al 2000; Naredi et al 2001], liver transplantation patients (4 ng/kg/min for 6 days and 1 ng/kg/min for 7 days, respectively) [Barthel et al 2012; Neumann et al 1999], CABG patients (2 ng/kg/min for up to 48 hours) [Morgera et al 2002], patients with septic shock (1 ng/kg/min for 24 h and a dose increasing cardiac index 15% (1-2 ng/kg/min) for several hours, respectively) [Kiefer et al 2001; Lehmann et al 2000], patients undergoing surgery for acute lower limp ischemia (0.5-2 ng/kg/min for 6 hours/day for 4-7 days) [de D G et al 2006; de D G et al 2007] and patients undergoing elective open repair of abdominal aortic aneurysm (0.8-1.2 ng/kg/min for 72 h) [Beirne et al 2008].
(93) In conclusion it has now been found that prostacyclin dose-dependently inhibits platelet aggregation but low-dose prostacyclin infusion (up to 4 ng/kg/min) but does not compromise hemostasis or induce bleeding or adverse events related to impaired hemostasis in healthy volunteers and acute critically ill patients.
Example 2
Reevaluation of a Prior Art Study
(94) Healthy Volunteer Study: Safety and Efficacy of 4 ng/kg/min i. v. Flolan® Infusion in Healthy Subjects, Reported in WO 2013/143548.
(95) The study was conducted investigating the influence of 2 hours i.v. Flolan® (epoprostenol sodium, prostacyclin analog) infusion at a dose of 4 ng/kg/min in eight healthy male volunteers.
(96) Endothelial function was evaluated in the study e.g. by measuring plasma levels of endothelial derived biomarkers by commercially available ELISA kits. Biomarkers indicating: Endothelial glycocalyx damage (syndecan-1), endothelial cell damage (thrombomodulin) or necrosis (nucleosomes, HMGB1), endothelial cell activation (PAI-1) and endothelial cell anticoagulation (protein C, antithrombin) were investigated (
(97) Prostacyclin in the administered dose did not change blood pressure or heart rate from baseline values at any time point during the study period. Furthermore, no changes were observed in Multiplate and TEG values comparing baseline and later values, indicating that 4 ng/kg/min i.v. prostacyclin infusion does not influence hemodynamics or hemostasis.
(98) The administered dose of prostacyclin was observed to have an endothelial protective effect evidenced by a marked decrease in the circulating level of thrombomodulin, an effect that appeared to be prolonged continuing for several hours after ceasing the prostacyclin infusion. Also, the circulating level of protein C decreased in the hours after ceasing the prostacyclin infusion, indicating that prostacyclin enhanced activation of protein C. The circulating level of PAI-1, an inhibitor of fibrinolysis shed from the activated endothelium, also declined further indicating that the prostacyclin infusion deactivated the endothelium and enhanced endogenous fibrinolysis. Finally, the circulating level of antithrombin decreased indicating that a higher amount of antithrombin was attached to the endothelial glycocalyx rather than being circulating in its soluble form i.e. providing the endothelium with improved anticoagulant properties.
(99) The finding that the administered dose of prostacyclin was associated with concurrent decreases in circulating thrombomodulin, protein C, PAI-1 and antithrombin in healthy individuals is reported here as a proof-of-concept of the endothelial protective effect of prostacyclin of importance for its capacity to treat systemic endotheliopathic syndrome.
(100) Conclusion:
(101) In this study the inventors found that i.v. infusion of a prostacyclin analog at a dose applied widely clinically (4 ng/kg/min) neither influenced hemodynamics nor whole blood hemostatic competence. The finding that the prostacyclin analog protected the endothelium supports the notion, that administration of low-dose prostacyclin or prostacyclin analogs, in particular iv-administration, is useful for treatment of systemic endotheliopathic syndrome.
Example 3
Reevaluation of a Prior Art Study
(102) Healthy Volunteer Study: Efficacy of 1 ng/kg/min i. v. Ilomedin® Infusion in Healthy Subjects. Reported in WO 2013/143548.
(103) A study was reported investigating the influence of 2 hours i.v. Ilomedin® (iloprost, prostacyclin analog) infusion at a dose of 1 ng/kg/min in eight healthy male volunteers. Blood samples for endothelial function were collected at the following time-points: Before the infusion (0 h), after 30 min infusion (30 min), immediately after ceasing the infusion (2 h) and 2 hours after the end of iloprost infusion.
(104) Endothelial function was evaluated by measuring plasma levels of endothelial derived biomarkers by commercially available ELISA kits. Biomarkers indicating: Endothelial glycocalyx damage (syndecan-1), endothelial cell damage (thrombomodulin) and endothelial cell anticoagulation (protein C) were investigated. Furthermore, we measured plasma concentration of prostacyclin (
(105) The administered dose of iloprost had an endothelial protective effect evidenced by a marked decrease in the circulating level of thrombomodulin, an effect that appeared to be prolonged continuing for several hours after ceasing the iloprost infusion. Also, the circulating levels of protein C and Syndecan-1, the latter a biomarker of glycocalyx damage, decreased during iloprost infusion, indicating that prostacyclin enhanced activation of protein C (resulting in a decline in the non-activated form of protein C) and protected the glycocalyx. Finally, the circulating level of prostacyclin increased approximately 15% during iloprost infusion confirming that systemically detectable increases in prostacyclin were obtained by low-dose 1 ng/kg/min iloprost infusion.
(106) Conclusion:
(107) This study support the notion that an endothelial protective effect of low-dose i.v. infusion with a prostacyclin analog (1 ng/kg/min, iloprost, Ilomedin®) supporting the capacity of administration of low-dose prostacyclin or prostacyclin analogs, in particular iv-administration, for treatment of systemic endotheliopathic syndrome.
Example 4
Revaluation of Prior Art
(108) Patient Study: Safety and Efficacy of Pre-Filter Flolan® Infusion in Critically Ill Patients [Windelov et al 2010]
(109) Furthermore, in a retrospective study of intensive care patients needing continuous renal replacement therapy, the inventors found that patients who received prostacyclin in the dialysis filter had lower 30-day mortality compared to patients who received heparin in the filter as an anticoagulant (21% vs. 39%)
(110) The inventors conducted a retrospective study of critically ill patients treated or not treated with prostacyclin infusion during their intensive care unit (ICU) stay [Windelov et al 2010]. Ninety-four critically ill patients admitted to the ICU for medical or surgical complications, underwent hemofiltration (continuous renal replacement therapy, CRRT) with or without concomitant Flolan® (epoprostenol sodium, prostacyclin analog) treatment. Flolan® was administered at a low dose (5 ng/kg/min) in the filters to prevent these from clotting and consequently, there was only a minor spill-over of Flolan® to the systemic circulation.
(111) The study revealed that the two groups (prostacyclin vs heparin) were comparable with regard to disease severity (APACHE II score) at ICU admission but during their ICU stay (before CRRT), prostacyclin patients appeared more severely ill evidenced by more patients having septic shock, disseminated intravascular coagulation (DIC), severe thrombocytopenia and a lower platelet count at start of hemofiltration (Table 1).
(112) During CRRT with either prostacyclin or heparin, platelet count increased in the prostacyclin patients whereas it declined in the heparin patients, indicating reduced disease severity in the patients treated with prostacyclin (Table 1). Importantly, when comparing mortality, the prostacyclin patients tended to have decreased mortality at 30 days (21% vs. 39%, p=0.12), 90 days (34% vs. 53%, p=0.10) and 365 days (38% vs. 57%, p=0.09).
(113) Conclusion:
(114) The finding of increased platelet count and reduced mortality in CRRT patients receiving prostacyclin in the filters indicate that the minor spill-over of prostacyclin to the systemic circulating influences the endothelium beneficially in acute critically ill patients by limiting consumption of platelets, microvascular occlusion, organ failure and mortality, i.e. hallmarks of systemic endotheliopathic syndrome.
Example 5
Revaluation of Prior Art
(115) Patient Study: Safety and Efficacy of 0.5 ng/kg/min i.v. Ilomedin® Infusion in PCI Patients [Holmvang et al 2012]
(116) The inventors conducted a randomized placebo controlled double blind clinical trial investigating safety and efficacy of 24 hours i.v. Ilomedin® (iloprost, prostacyclin analog) infusion at a dose of 0.5 ng/kg/min compared to placebo in 17 patients with acute myocardial infarction (AMI) undergoing percutanuous coronary intervention (PCI) with stent implantation [Holmvang et al 2012]. Patients randomized to 24 hours active (n=9) or placebo (n=8) therapy were evaluated for hemodynamics, bleeding events and for functional whole blood hemostasis (Multiplate, TEG) and endothelial function in blood sampled before iloprost infusion (baseline), during infusion (1 h, 6 h and 24 h) and 24 hours after ceasing iloprost infusion (48 h).
(117) Bleeding was evaluated by GUSTO criteria (severe, moderate, mild, none) and hemostasis was evaluated by Multiplate and TEG according to the manufactures recommendations. Endothelial function was evaluated by measuring plasma levels of endothelial derived biomarkers by commercially available ELISA kits. Biomarkers indicating: Endothelial glycocalyx damage (syndecan-1), endothelial cell damage (thrombomodulin), endothelial cell activation (sE-selectin, ICAM-1) and anticoagulation (protein C) were investigated.
(118) The study revealed that patients in the two groups (iloprost vs placebo) were comparable with regard to baseline demography and disease severity. Furthermore, no differences in hemodynamics, bleeding events, Multiplate or TEG were observed between groups during or after iloprost infusion.
(119) Importantly, the inventors found that sE-selectin, a biomarker reflecting endothelial activation, decreased significantly from baseline to 48 h in the iloprost patients whereas it increased in the placebo patients (
(120) Conclusion:
(121) AMI PCI patients treated with 24 hours i.v. prostacyclin infusion displayed evidence of prolonged reduction in endothelial activation continuing 24 hours after ceasing the infusion. The prostacyclin infusion did not negatively influence hemodynamics, bleeding events or hemostasis. This study supports the notion that prolonged low-dose i.v. prostacyclin infusion in acute critically ill patients is safe and effective in protecting the endothelium and hence capable of treating systemic endotheliopathic syndrome.
Example 6
Safety and Efficacy of 1 ng/kg/min i.v
(122) Patient Study: Safety and Efficacy of 1 ng/kg/min i. v. Ilomedin® Infusion in Whipple (Major Abdominal Surgery) Patients
(123) The inventors conducted a randomized placebo controlled double blind clinical trial investigating safety and efficacy of intra- and post-operative i.v. Ilomedin® (iloprost, prostacyclin analog) infusion at a dose of 1 ng/kg/min compared to placebo in 16 patients undergoing whipple surgery at Rigshospitalet, a tertiary level teaching University hospital in Copenhagen, Denmark. The inclusion criteria were adults (age 8 years) undergoing whipple surgery based on surgical indication due to cancer/dysplasia, familial adenomatous polyposis or inflammation/pancreatitis. However, all included patients had cancer as a surgical indication: C. pancreas n=12; C. papilla vateri n=2; C. ductus choledochus n=1; Cholangiocarcinoma n=1. The patients were randomized to receive i.v. infusion with iloprost (1 ng/kg/min, active treatment, n=8) or saline (0.9%, placebo treatment, n=8) at an equal volume during surgery (intra-operative: median ˜5 hours) and 6 hours after surgery (post-operative), yielding a total active/placebo infusion time of ˜11 hours.
(124) Data on demography, co-morbidities, surgical time and 28-day mortality were assessed. Hemodynamics were measured continuously (heart rate (HR), systolic, diastolic and mean arterial pressure (SBT, DBT, MAP)) or at baseline, end of surgery and 6 h and 24 h post-operative (systemic vascular resistance (SVR), stroke volume (SV), central venous pressure (CVP)). The volume and number of units of red blood cells (RBC), plasma and platelet concentrates administered were recorded at the end of surgery, 6 h and 24 h post-operative. Bleeding volume was assessed intra-operative and 6 h and 24 h post-operative. The volume of crystalloids and colloids (only albumin was allowed) was, together with urine output, recorded intra-operative and 6 h and 24 h post-operative.
(125) Blood samples for evaluation of functional whole blood hemostasis (TEG) and endothelial function were collected pre-commencing infusion with study drug (baseline), at the end of surgery (post-op) and 6 h after surgery just before ceasing study drug infusion (6 h). Blood samples for routine biochemistry (hemoglobin, creatinine and lactate) were collected at baseline, post-op and 6 h and 24 h post-operative.
(126) TEG was performed according to the manufactures recommendations. Endothelial function was evaluated by measuring plasma levels of endothelial derived biomarkers by commercially available ELISA kits. Biomarkers indicating: Endothelial glycocalyx damage (syndecan-1), endothelial cell damage (thrombomodulin) and necrosis (nucleosomes) were investigated.
(127) Comparing iloprost and placebo patients, no differences were found with regard to demography, comorbidities, surgical indication or 28-day mortality. Furthermore, the groups received a similar volume of fluids intra- and post-operative and displayed comparable changes in hemodynamics.
(128) However, with regard to transfusion requirements, hemostasis and endothelial function, the inventors surprisingly found major differences between the treatment groups as iloprost patients had reduced RBC transfusion requirements, reduced increases in lactate, improved functional hemostasis and reduced endothelial damage (
(129) Thus, in brief, placebo patients received more intra- and post-operative RBC compared to iloprost patients (median 0 ml (IQR 0-163) vs. 695 ml (IQR 0-969), p=0.083) and a significantly larger volume of RBC from arrival at the post-operative ward and 6 h post-operatively (iloprost patients 0 ml (IQR 0-0) vs. placebo patients 115 ml (IQR 0-296), p=0.027). Furthermore, placebo patients tended to have lower post-operative hemoglobin compared to iloprost patients (median 6.2 mmol/l (IQR 5.9-6.4) vs. 6.7 mmol/l (IQR 6.5-7.7), p=0.058) and post-operatively lactate increased in placebo patients but remained unchanged in iloprost patients indicating that iloprost infusion improved microvascular perfusion. Considering hemostatic competence, placebo patients displayed impaired intra-operative hemostatic competence as TEG maximum clot firmness decreased intra-operatively in placebo patients (p=0.034) whereas it remained stable in iloprost patients (p=NS).
(130) Importantly, the inventors found that iloprost patients displayed evidence of improved endothelial protection and reduced endothelial damage compared to placebo patients (
(131) Conclusion:
(132) Whipple patients treated with iloprost infusion intra- and post-operatively received fewer RBC transfusions, had improved hemostatic capacity, improved microvascular perfusion and evidence of reduced endothelial damage together supporting a capacity of prostacyclin infusion to treat systemic endotheliopathic syndrome.
Example 7
Revaluation of Prior Art
(133) The inventors have measured soluble thrombomodulin levels in blood plasma from 2,118 acute critically ill patients suffering from trauma, myocardial infarction, cardiac arrest, sepsis, severe sepsis and septic shock (Table 2). The blood sample for thrombomodulin measurement was taken immediately upon admission to the hospital or immediately after observation of the occurrence of the acute critical illness.
(134) The inventors discovered that the acute critically ill patients could be stratified into a group with low mortality rate and a group with high mortality rate based on the measured thrombomodulin level. Thus, a high level of thrombomodulin in blood plasma was associated with high 28-day mortality whereas a low thrombomodulin level in blood plasma was associated with low 28-day mortality.
(135) Table 2 details a study of median thrombomodulin levels in plasma/serum (ng/ml) in 2,118 acute critically ill patients suffering from trauma, myocardial infarction, resuscitated cardiac arrest, sepsis, severe sepsis or septic shock, stratified according to gender and 28-day outcome (survival vs. mortality).
(136) The inventors discovered that there was a significant difference in thrombomodulin levels between survivors and non-survivors across all diseases (p<0.0001), but no difference in thrombomodulin between male and female (p=0.114).
(137)
(138) Based on the inventors data, choosing a lower threshold level of thrombomodulin results in increased true-positive rate (sensitivity) and increased false-positive rate (1-specificity) whereas choosing a higher threshold level of thrombomodulin results in reduced true-positive rate (sensitivity) and reduced false-positive rate (1-specificity) (Table 3). Consequently, both a lower and higher threshold level of thrombomodulin will result in a lower Youden Index and hence lower performance of the diagnostic test.
(139) It should be noted that a thrombomodulin level of 4 ng/ml in plasma cannot be expected to be identical to the thrombomodulin level measured in whole blood since the plasma fraction of whole blood is only approximately 55%. (Discussed below based on data comparing the thrombomodulin level in plasma and whole blood).
(140) Conclusion:
(141) Acute critically ill patients with blood or blood plasma thrombomodulin values above 4 ng/ml suffer from systemic endotheliopathic syndrome and will benefit from treatment with prostacyclin or analogs thereof.
(142) Due, however, to the observed benefits of the present invention; the inventors suggest to operatively use a lower threshold level than 4 ng/ml (plasma/serum) as an indicator for treatment with prostacyclin or an analogue thereof according to the present invention. A threshold level corresponding to a plasma/serum level of 2.5 ng/ml is considered satisfactory in the method of the invention or values in between 2.5 ng/ml to 4 ng/ml, such as e.g. 3.0 or 3.5 ng/ml thrombomodulin in plasma/serum.
(143) Selecting e.g. a threshold level of 2.5 ng/ml will result in an almost 91% capture of treatment suited subjects (true positive), however at the cost of a larger number of patients being unnecessarily treated with prostacyclin or an analogue thereof (false positive). Due to the high safety profile and low risk of adverse effects in treatments using the suggested low-dose prostacyclin or analogues thereof, it seems reasonable to proceed in such a manner, in particular since later control according to the invention will limit the unnecessary treatment exposure received by patients not in need thereof.
Example 8
Trauma
(144) Table 4a details results based on 635 trauma patients. It was found that the level of sTM (soluble thrombomodulin) at admission in trauma patients depended on injury severity (increases most in patients with high abbreviated injury severity score (AIS) for abdomen and thorax i.e., severe abdominal and thorax injuries) including shock degree reflecting that the proportion of patients with sTM above 2.5 ng/ml differ between cohorts of severely (84.9%) and moderately (45.2%) injured patients (Table 4a).
(145) Table 4b details results based on 270 trauma patients. It was found that in the first 24 h and 72 h, the level of sTM increases in 64% and 80% of the moderately injured patients, respectively, and from 24 h to 72 h, sTM increases in 61% of the patients. The absolute and proportional change in sTM from admission and 72 h onward is displayed in Table 4b, ranging from a median increase of 0.31-0.72 ng/ml corresponding to 12-34% increases.
(146) It was observed that:
(147) The Level of sTM at Baseline and During Follow-Up is Linked to Complications
(148) Mortality
(149) When comparing the sTM level in survivors and 28d non-survivors, the sTM level at admission (median IQR) (2.95 ng/ml (1.73-4.21) vs. 2.29 ng/ml (1.44-3.38)), at 24 h (3.92 ng/ml (3.7-5.55) vs. 2.94 ng/ml (2.2-4.32)) and 72 h (4.56 ng/ml (3.95-5.85) vs. 3.35 ng/ml (2.67-4.6)) is higher in non-survivors compared to survivors (p=0.047, p=0.016 and p=0.076, respectively).
(150) Sepsis
(151) High sTM level at admission tend to be associated with increased risk of sepsis the first 28d (ROC for admission sTM on development of sepsis within 28d is AUC 0.594 (95% Cl 0.487-0.700), p=0.085).
(152) Renal Replacement Therapy (RRT) (Single Organ Failure)
(153) sTM levels at 24 h and 72 h tend to be associated with increased risk of renal replacement therapy (RRT) during the first 28d (ROC for 24 h sTM AUC 0.771 (0.556-0.985), p=0.068 and ROC for 72 h sTM AUC 0.877 (0.789-0.966), p=0.072)) emphasizing that high sTM levels after admission are also linked to organ failure.
(154) The Change in sTM Level from Admission and Onward is Linked to Complications
(155) Bleeding
(156) sTM increases from 24-72 h in more patients who experience bleedings within the first 24 h of admission than in patients who do not bleed the initial 24 h (76% vs. 50% display sTM increases, p=0.063).
(157) Renal Replacement Therapy (RRT) (Single Organ Failure)
(158) sTM increases from 0-24 h, from 0-72 h and from 24-72 h in all (100%) patients who during the first 28 days require RRT due to development of kidney failure (single organ failure). Furthermore, an increase in sTM the initial 24 h from admission (0-24 h) is associated with increased risk of RRT (ROC for sTM change 0-24 h on RRT within 28 d is 0.794 (0.619-0.969), p=0.048). Also, the change in sTM from 0-24 h is positively correlated with RRT days (kidney failure) (both p<0.05) emphasizing that increases in sTM the first 24h from admission is associated with enhanced development of organ failure.
(159) Ventilation (Single Organ Failure)
(160) Also, the change in sTM from 0-24 h is positively correlated with ventilator days (length of ventilator treatment i.e., respiratory failure) emphasizing that increases in sTM the first 24 h from admission is associated with enhanced development of organ failure.
(161) Disease severity scores for trauma were: Injury Severity Score (ISS) [evaluated at admission], Glasgow Coma Scale (GCS) score [evaluated at admission], and Sequential Organ Failure Assessment (SOFA) score [evaluated daily in the ICU].
(162) Together, these findings indicate that early and continued measurement of blood or plasma thrombomodulin levels in trauma patients will allow identification of high-risk patients i.e., patients with systemic endothelial damage and thus patients with the highest thrombomodulin levels or highest increases in thrombomodulin. High-risk patients with high thrombomodulin levels due to systemic endothelial damage will benefit from therapy with low-dose prostacyclin or analogs thereof, which will be safe even in trauma patients due to the low-dose regimen.
Example 9
Cardiac Arrest
(163) Based on n=169 patients resuscitated from cardiac arrest. It was found that the level of sTM at admission in resuscitated cardiac arrest patients depends on the time in shock and the depth of shock (higher sTM with longer time from CA to ROSC and with lower pH and higher lactate), the catecholamine level (higher sTM with higher adrenaline dose administered) and the age of the patient (higher sTM in older patients).
(164) Table 5a details the proportion of OHCA patients with sTM>2.5-5-5 ng/ml at admission, 24 h, 48 h and 72 h. In the first 24 h, 48 h and 72 h, the level of sTM increases in 35%, 52% and 43% of the patients compared to baseline and sTM increases in 77% from 24-48 h, in 38% from 24 h-72 h and in 16% from 48 h-72 h. The median absolute changes from 0-24 h, from 0-48 h and from 0-72 h in all patients are 0.27 ng/ml, 1.08 ng/ml and 0.80 ng/ml, respectively.
(165) Table 5b details the absolute and proportional (median) changes in sTM from admission and 24 h and 72 h onward in 28 day survivors and non-survivors. Importantly, the change in sTM in 28 d survivors compared to non-survivors differs significantly, with the most pronounced difference being that sTM remains unchanged in survivors the first 24 h whereas it increases 16% in non-survivors.
(166) Table 5c details the predictive value of sTM at different time-points for poor cognitive outcome (CPC≥3/mRS≥4) evaluated by ROC analysis. As can be learned from the results continued high sTM levels remain closely linked to high mortality and poor outcome.
(167) It was observed that:
(168) The Level of sTM at Baseline and During Follow-Up is Linked to Complications
(169) Mortality
(170) When comparing the sTM level in 28 d survivors and non-survivors, the sTM levels at admission, 24 h, 48 h and 72 h are all increased in non-survivors compared to survivors: Admission (7.78 ng/ml (6.36-11.41) vs. 6.56 ng/ml (5.17-8.31), p<0.0001), 24 h (9.04 ng/ml (6.28-11.61) vs. 6.6 ng/ml (5.28-9.27), p<0.0001), 48 h (9.96 ng/ml (7.3-12.85) vs. 7.79 ng/ml (6.31-10.52), p<0.006) and 72 h (8.46 ng/ml (5.95-12.16) vs. 7.23 ng/ml (5.46-9.91), p=0.058).
(171) Poor Neurologic Outcome
(172) Also, high sTM level at admission and after 24 h, 48 h and 72 h all predict poor cognitive outcome evaluated by the CPC and mRS scores (Table 5c).
(173) The Change in sTM Level from Admission and Onward is Linked to Complications
(174) The increase in sTM from admission to 24 h, 48 h and 72 h onward all correlate with the time in and depth of shock (time from CA to ROSC, pH and lactate) and the administered adrenaline dose (all p<0.05) emphasizing that the severity of the initial “hit” (shock, catecholamines) drives progressive endothelial damage and hence increases in sTM.
(175) Mortality
(176) The magnitude of the sTM increase from 0-24 h is associated with mortality evidenced by ROC: Higher sTM increase from 0-24 h is associated with increased 28 d mortality with AUC 0.587 (0.484-0.689), p=0.103.
(177) Poor Neurologic Outcome
(178) Also, the magnitude of the sTM increase from 0-24 h is associated with cognitive outcome evidenced by ROC: The predictive value of the sTM increase from 0-24 h is associated with CPC (AIC 0.566 (0.469-0.633), p=0.192) and mRS≥4 (AUC 0.578 (0.480-0.676), p=0.124) indicating that progressive increases in sTM are associated with poor cognitive outcome.
(179) Disease severity scores for cardiac arrest were: Cerebral Performance Category (CPC), and modified Rankin Scale (mRS) [cardiac arrest].
(180) Together, these findings indicate that early and continued measurement of blood or plasma thrombomodulin levels in patients resuscitated from cardiac arrest will allow identification of high-risk patients i.e., patients with systemic endothelial damage and thus patients with the highest thrombomodulin levels or highest increases in thrombomodulin. High-risk patients with high thrombomodulin levels due to systemic endothelial damage will benefit from therapy with low-dose prostacyclin or analogs thereof, which will be safe in patients resuscitated from cardiac arrest due to the low-dose regimen.
Example 10
Myocardial Infarction
(181) Based on n=571 STEMI patients. It was found that the level of sTM at admission in patients with acute myocardial infarction (ST segment elevation myocardial infarction, STEMI) is linked to disease severity and outcome with higher sTM levels in patients with more severe disease (highest levels in patients with shock and ICU requirement).
(182) Table 6 details the proportion of OHCA patients with sTM>2.5-5-5 ng/ml at ICU admission (data from two different cohorts of septic patients).
(183) It was observed that:
(184) The Level of sTM at Admission is Linked to Mortality and Complications
(185) Mortality
(186) When comparing the level of sTM at admission in 28-day and 90-day non-survivors and survivors, the 28-day (3.23 ng/ml (2.23-4.16) vs. 2.09 ng/ml (1.56-2.96), p<0.0001) and 90-day (3.23 ng/ml (2.20-4.16) vs. 2.10 ng/ml (1.56-2.96), p<0.0001) non-survivors have higher sTM levels compared to survivors. sTM at admission correlates inversely with days to death (p=0.045) reflecting that high sTM is associated with faster progression to death. By ROC analyses, admission sTM is a strong predictor of 28-day (AUC 0.718 (0.635-0.801), p<0.0001) and 90-day (AUC 0.713 (0.633-0.792), p<0.0001) all cause mortality and of 28-day (AUC 0.736 (0.648-0.824), p<0.0001) and 90-day (AUC 0.726 (0.639-0.812), p<0.0001) cardiovascular disease mortality.
(187) Disease Severity
(188) Admission sTM correlates positively with Troponin I (a biochemical marker for myocardial injury) (p=0.008) by ROC analysis sTM is associated with shock before PCI (AUC 0.580 (0.495-0.666), p=0.061) and with risk of ICU admission (AUC 0.605 (0.510-0.701), p=0.053). Also, sTM is a strong predictor of 28-day (AUC 0.640 (0.555-0.725), p=0.007) and 90-day (AUC 0.644 (0.564-0.724), p<0.0001) congestive heart failure.
(189) Together, these findings indicate that early and continued measurement of blood or plasma thrombomodulin levels in patients with myocardial infarction will allow identification of high-risk patients i.e., patients with systemic endothelial damage and thus patients with the highest thrombomodulin levels or highest increases in thrombomodulin. High-risk patients with high thrombomodulin levels due to systemic endothelial damage will benefit from therapy with low-dose prostacyclin or analogs thereof, which will be safe in myocardial infarction patients due to the low-dose regimen. It is expected that prostacyclin therapy in myocardial infarction patients treated with PCI (percutaneous coronary intervention) will prevent and treat sequelae from the injurious hit such as myocardial infarction with shock, ischemia-reperfusion, catechol-amines etc.
Example 11
Sepsis/Severe Sepsis/Septic Shock
(190) Based on two different cohorts (n=749 and n=184) of patients with sepsis/severe sepsis/septic shock it was observed that the level of sTM at admission in patients with sepsis/severe sepsis/septic shock is closely linked to disease severity with higher sTM levels in patients with higher disease severity scores (APACHE II, SOFA) and hence higher degree of organ dysfunction i.e., higher lactate, lower blood pressure, lower platelet count and hemoglobin, lower urine output, higher BUN and creatinine.
(191) It was observed that
(192) The Level of sTM at Admission is Linked to Mortality and Complications
(193) Mortality
(194) When comparing the level of admission sTM in 7-day, 28-day and 90-day non-survivors and survivors, sTM is increased in the 7-day (9.21 ng/ml (6.74-12.79) vs. 7.35 ng/ml (4.77-10.35), p=0.001), 28-day (9.34 ng/ml (6.86-12.5) vs. 6.81 ng/ml (4.47-9.66), p<0.0001) and 90-day (9.11 ng/ml (6.31-11.92) vs. 6.66 ng/ml (4.61-9.7), p=0.006) non-survivors compared to survivors.
(195) sTM at admission correlates inversely with days to death (p=0.001) reflecting that high sTM is associated with faster progression to death. By Cox proportional hazards models, the admission sTM level is a strong predictor of 7-day, 28-day and 90-day mortality (all p<0.001) and by ROC analysis, sTM is a strong predictor of mortality: 7-day mortality AUC 0.65 (0.56-0.75), p=0.005; 28-day mortality AUC 0.68 (0.60-0.76), p<0.001 and 90-day mortality AUC 0.63 (0.54-0.71), p=0.004.
(196) Disease Severity Scores
(197) sTM at admission correlates strongly positively with SAPS II score (p<0.0001) and SOFA score (p=0.003), and for each 1 ng/ml increase in sTM, SAPS II score increases 1.32 (95% Cl 0.73-1.91) (p<0.001) points and SOFA score increases 0.17 (95% Cl 0.07-0.27) points (p=0.001).
(198) Bleeding and Transfusion Requirements
(199) The sTM level at admission predicts general bleeding in the ICU (ROC AUC 0.598 (0.498-0.678), p=0.059), serious bleeding (requiring >3 RBC) (ROC AUC 0.612 (0.506-0.717), p=0.068) and upper GI bleeding (ROC AUC 0.680 (0.551-0.809), p=0.037). Also, admission sTM predicts need for RBC (ROC AUC 0.605 (0.517-0.693), p=0.018), FFP (ROC AUC 0.582 (0.495-0.669), p=0.067) and platelets (ROC AUC 0.614 (0.523-0.705), p=0.016) during ICU stay.
(200) Renal Replacement Therapy (RRT) (Single Organ Failure)
(201) Admission sTM predicts the 90-day need for dialysis therapy by ROC with AUC 0.688 (0.595-0.782), p<0.001.
(202) Together, these findings indicate that early and continued measurement of blood or plasma thrombomodulin levels in patients with sepsis, severe sepsis, or septic shock will allow identification of high-risk patients i.e., patients with systemic endothelial damage and thus patients with the highest thrombomodulin levels or highest increases in thrombomodulin. High-risk patients with high thrombomodulin levels due to systemic endothelial damage will benefit from therapy with low-dose prostacyclin or analogs thereof, which will be safe in sepsis, severe sepsis, or septic shock patients due to the low-dose regimen.
(203) Disease severity scores for sepsis were: Simplified Acute Physiology Score II (SAPS II) [evaluated at admission], and Sequential Organ Failure Assessment (SOFA) score [evaluated daily in the ICU] [sepsis].
(204) Together, these findings indicate that early and continued measurement of blood or plasma thrombomodulin levels in patients with sepsis/severe sepsis/septic shock will allow identification of high-risk patients i.e., patients with systemic endothelial damage and thus patients with the highest thrombomodulin levels or highest increases in thrombomodulin. High-risk patients with high thrombomodulin levels due to systemic endothelial damage will benefit from therapy with low-dose prostacyclin or analogs thereof, which will be safe in these patients due to the low-dose regimen.
Example 12
Rapid Method of Identifying Patients with Systemic Endotheliopathic Syndrome
(205) The inventors have discovered a new disease entity, systemic endotheliopathic syndrome. Patients with systemic endotheliopathic syndrome suffer from acute critical illness and concurrent evidence of systemic endothelial cell damage identified by increases in circulating levels of a specific marker reflecting endothelial cell damage. Consequently, identification of patients with systemic endotheliopathic syndrome among acute critically ill patients can be done by measuring circulating levels of thrombomodulin, a specific marker of endothelial cell damage with a high sensitivity for diagnosing systemic endotheliopathic syndrome. The fact that patients with systemic endotheliopathic syndrome can be identified by a pre-determined threshold level of circulating thrombomodulin introduces personalized medicine into the invention, which is opposite to the “one size fits all” strategy currently used in medical care. Personalized medicine refers to therapy fitted to the individual patient based on risk stratification, disease phenotype, genetic profile etc.
(206) Accordingly, acute critically ill patients with systemic endotheliopathic syndrome can be rapidly identified (and diagnosed as having that syndrome) using a method that involves measuring thrombomodulin level in a biological sample of the acute critically ill patient, and comparing the measured thrombomodulin level to a predetermined threshold.
(207) Preferably, the thrombomodulin test is a quick point-of-care (POC) test based on whole blood input so a drop of whole blood is placed onto e.g. a lateral flow assay (stick) or another immunoassay platform like e.g. cartridges based on microfluidics technology, to display a result within 2-5 minutes reporting whether a patient has a thrombomodulin level above or below a predetermined threshold level.
(208) It is suggested that thrombomodulin may be detected using a quick POC test such as lateral flow assays (sticks) or other immunoassay platforms like e.g. cartridges based on microfluidics technology, as the time to receive a result would significantly decrease, e.g. assays could be performed en route aboard an ambulance between a site of an accident and a trauma center or immediately upon hospital admission/recognition of acute critical illness. Obtaining a result from a POC test within minutes (instead of hours) would allow rapid allocation to prostacyclin therapy for patients with systemic endotheliopathic syndrome.
(209) As a proof of concept, that soluble thrombomodulin in whole blood is a sufficiently sensitive marker over plasma, the present inventors performed a measurement of soluble thrombomodulin by ELISA in whole blood versus plasma.
(210) Measurement of Soluble Thrombomodulin by ELISA in Whole Blood Compared to Plasma
(211) Background
(212) In the enzyme linked immunosorbent assay (ELISA) method, bound antigen (or antibody) is detected by an antibody linked (primarily or secondarily) to an enzyme whose activity can be determined. The activity of the antibody-linked enzyme serves as a quantitative estimate of the amount of the investigated antigen (or antibody) in the biological specimen.
(213) In thrombomodulin ELISA, patient sample (typically plasma or serum) is added to ELISA wells pre-coated with an antibody directed against thrombomodulin. After a washing step, a detection antibody directed against (another epitope on) thrombomodulin is added (the detection antibody may be primarily or secondarily linked to an enzyme whose activity can be determined). The amount of enzyme activity in each sample is mathematically converted to a concentration measure based on the enzyme activity in samples with known amounts of thrombomodulin (standard curve).
(214) When the thrombomodulin level in acute critically ill patients is used to guide therapy it is pivotal that the thrombomodulin test can be performed rapid. One way to speed up testing is to measure thrombomodulin in whole blood instead of plasma/serum as whole blood requires no blood sample processing (it takes ˜15-20 min to prepare plasma and ˜30-60 min to prepare serum from a whole blood sample).
(215) Since not all molecules can be easily detected in whole blood (whole blood may contain substances that can interfere with specificity of the test), the inventors investigated if thrombomodulin was directly detectable in whole blood when measured by ELISA. By conducting the below described experiments, the inventors surprisingly discovered that thrombomodulin could be precisely and accurately detected in whole blood.
(216) Experiments
(217) Whole blood from 10 healthy human volunteers was sampled (two tubes 4 ml EDTA tubes from each subject). From each person, one tube was spun at 3000 rpm for 10 min to generate plasma and one tube was left unprocessed (whole blood). Immediately after the spinning (plasma), plasma and whole blood was pipetted onto the ELISA plate.
(218) From each subject, the following duplicates of plasma/whole blood was investigated for its thrombomodulin content: I) Plasma diluted 1:2 with assay buffer (golden standard and recommended for the ELISA) II) Whole blood undiluted III) Whole blood diluted 1:2 with assay buffer IV) Whole blood diluted 1:4 with assay buffer
Besides using whole blood as sample material, the ELISA was conducted according to the manufacturer's recommendations.
(219) The hematocrit values from the healthy volunteers was calculated from an expected normal hemoglobin concentration of 8.3-10.5 mmol/l in male (mean 9.4 mmol/l) and 7.3-9.5 mmol/l in female (mean 8.4 mmol/l) corresponding to hematocrit values of 42% in female and 48% in male volunteers (meaning that the plasma content in whole blood was 58% in female and 52% in male).
(220) Results
(221) Table 8 displays the measured thrombomodulin levels in 1:2 diluted plasma (golden standard) and in different dilutions of whole blood.
(222) In plasma, the mean thrombomodulin concentration was 1.92 (SD 0.12) ng/ml in the 10 healthy volunteers, with a low intra-assay variation between duplicates (CV %, corresponding to high precision) of 6.9%.
(223) In undiluted whole blood, the mean concentration was 0.78 (SD 0.06) ng/ml and after correcting for hematocrit (hct) it was 1.38 (SD 0.30) ng/ml, corresponding to a recovery of 71.5% (SD 5.9%) compared to plasma. Notably, the precision of the test in undiluted whole blood was extremely high with mean CV % between duplicates only being 8.8% (Table 8). Also, the linear correlation between plasma and undiluted whole blood levels of thrombomodulin revealed a strong Pearson product moment correlation, r=0.93, yielding a high coefficient of determination, r.sup.2=0.87 (
(224) In 1:2 diluted whole blood, the mean concentration was 1.23 (SD 0.33) ng/ml and after correcting for hematocrit (hct) it was 2.16 (SD 0.59) ng/ml, corresponding to a recovery of 110.9% (SD 15.8%) compared to plasma. The precision of the test in 1:2 diluted whole blood was extremely high with mean CV % between duplicates only being 10.3% (Table 8). The linear correlation between plasma and undiluted whole blood levels of thrombomodulin revealed a strong Pearson product moment correlation, r=0.91, yielding a high coefficient of determination, r.sup.2=0.83 (
(225) In 1:4 diluted whole blood, the mean concentration was 1.04 (SD 0.51) ng/ml and after correcting for hematocrit (hct) it was 1.83 (SD 0.90) ng/ml, corresponding to a recovery of 90.19% (SD 33.4%) compared to plasma. The precision of the test in 1:4 diluted whole blood was acceptable with mean CV % between duplicates being 21.0% (Table 8). The linear correlation between plasma and undiluted whole blood levels of thrombomodulin revealed a strong Pearson product moment correlation, r=0.93, yielding a high coefficient of determination, r.sup.2=0.86 (
(226) Conclusion
(227) This experiment demonstrated that thrombomodulin was easily detectable in whole blood. This emphasizes that thrombomodulin has biochemical features that allows thrombomodulin antibodies to detect this molecule also in whole blood i.e., a significantly more complex biological sample matrix than plasma. The precision of the thrombomodulin measurement in undiluted whole blood was extremely high and 1:2 dilution of the whole blood retained precision and improved accuracy.
(228) Based on the results of this experiment, the inventors infer that thrombomodulin can be easily measured in a whole blood based quick assay like e.g. a dipstick. This may allow rapid treatment stratification of acute critically ill patients when this is based on their thrombomodulin levels.
(229) Qualified Diagnosis of Systemic Endotheliopathy by Adding Measurement of Other Biomarkers i.e. Syndecan-1, Adrenaline and Vascular Endothelial Growth Factor (VEGF) in Some Patients
(230) The diagnosis of systemic endothelial damage i.e. systemic endotheliopathic syndrome (SES) in acute critically ill patients may in some patients be further qualified besides that obtained by measuring thrombomodulin levels pre-hospital or at hospital admission/recognition of acute critical illness. Thus, adding a pre-hospital or at hospital admission measurement of syndecan-1 (a biomarker reflecting endothelial glycocalyx damage) and/or adrenaline (a endogenous stress biomarker directly drivers endothelial damage) will aid the diagnosis of “systemic endotheliopathic syndrome”.
(231) Patients, in particular trauma patients, with thrombomodulin levels just above a given cut-off but with concurrent syndecan-1 and adrenaline levels above their given cut-offs, suffer from severe systemic endotheliopathic syndrome. These patients, with borderline increased thrombomodulin but high syndecan-1 and adrenaline levels, are thus at higher-than-expected (based on thrombomodulin) risk of disease progression and organ failure and excess mortality, and these patients will benefit from prostacyclin therapy as described above. The prostacyclin therapy should be initiated earliest possible and monitored daily by thrombomodulin and clinical disease scores, as described above.
(232) Thus, similar to thrombomodulin, the measurement of syndecan-1 and adrenaline as additional biomarkers that can diagnose and qualify the severity of systemic endotheliopathy i.e. systemic endotheliopathic syndrome. Preferably, all biomarkers (thrombomodulin, syndecan-1, adrenaline) are measured either individually or simultaneously by a quick POC assay either pre-hospital or immediately upon hospital admission/recognition of the acute critical illness. Applying a quick POC assay that reveal results within a few minutes, enables fast diagnosis of systemic endotheliopathic syndrome and hereby a fast risk stratification and initiation of prostacyclin therapy. Prompt initiation of prostacyclin therapy will minimize further progression of the endotheliopathy in the early phase of the acute critical illness and will thus enable fast reversal and treatment of the endotheliopathy.
(233) Evidence from Trauma
(234) In 635 trauma patients, high syndecan-1 and adrenaline levels were strong and independent predictors of mortality.
(235)
(236)
(237) When using the above cut-offs for syndecan-1 (40 ng/ml) and adrenaline (225 pg/ml), the following hazards ratios (HR) for 28-day mortality were revealed: Plasma syndecan-1 above 40 ng/ml: HR 2.16 (95% CI 1.43-3.24), Wald 14, p<0.0001 Plasma adrenaline above 225 pg/ml: HR 2.47 (95% CI 1.61-3.80), Wald=17, p<0.0001
(238) The power of these cut-off can be illustrated by the proportion of patients dying within 1-28 days when stratified according to syndecan-1 or adrenaline level ad admission.
(239) 1-Day Mortality:
(240) Patients with syndecan-1 levels>40 ng/ml at admission have a 4-fold higher <24 h mortality compared to patients with syndecan-1 levels<40 ng/ml (10.4% vs. 2.7%, p<0.0001). Patients with adrenaline levels>225 pg/ml have 4-fold higher <24 h mortality compared to patients with adrenaline levels<225 pg/ml (8.5% vs. 2.1%, p<0.0001).
(241) Especially for early mortality (<24 h) risk, adding syndecan-1 and adrenaline measurements and their cut-off levels to that of thrombomodulin, tends to improve the diagnosis of traumatic endotheliopathy and thus risk stratification allowing early treatment initiation (syndecan-1 adding significant value compared to thrombomodulin: p=0.153 and adrenaline adding significant value compared to thrombomodulin: p=0.085). The strength for early mortality prediction emphasizes the need for fast diagnosis of systemic endotheliopathic syndrome by POC assays allowing a fast initiation of prostacyclin therapy.
(242) 28-Day Mortality:
(243) Patients with syndecan-1 levels>40 ng/ml at admission have a 2-fold higher 28-day mortality compared to patients with syndecan-1 levels<40 ng/ml (26% vs. 14%, p<0.0001). Patients with adrenaline levels>225 pg/ml have more than 2-fold higher 28-day mortality compared to patients with adrenaline levels<225 pg/ml (25% vs. 11%, p<0.0001)
(244) When applying other cut-offs than 40 ng/ml for syndecan-1 and 225 pg/ml for adrenaline, the sensitivities and 1-specificities for predicting mortality are obtained (data from 635 trauma patients), as shown in Table 9 and 10, respectively.
(245) Finally, in addition to thrombomodulin and/or syndecan-1 and/or adrenaline, measurements of the vascular endothelial growth factor (VEGF) level in patients suffering from acute critical illness can diagnose patients with systemic endotheliopathic syndrome whom have excessive loss of vascular integrity i.e., excessive loss of fluids out of the intra vascular compartment. Risk stratification and prostacyclin therapy based on cut-off levels of VEGF measured pre-hospital or at admission/recognition of acute critical illness represents yet another tool to personalize care for acute critically ill patients.
(246) Thus, a patient is considered to have systemic endotheliopathic syndrome (also denoted systemic endotheliopathy or simply endotheliopathy) when the patient suffers from acute critical illness AND is diagnosed with systemic endothelial damage.