A device for removing albumin and endotoxins from the blood.

Abstract

The present invention relates to the treatment of individuals suffering from cytokine storm syndrome. The present invention also relates to methods for treating such individuals, as well as systems and apparatus for use in such treatment. [Solution] A method for treating an individual having cytokine storm syndrome, (a) A process for selectively removing albumin from the blood of an individual, (b) A process for selectively removing endotoxins from the blood of an individual, and (c) A process in which albumin not derived from the individual is supplied to the individual's blood, if applicable. This includes processing the blood of the said individual, The method wherein step (a) comprises the use of means for dialysis of albumin, wherein the means comprises a membrane having a pore size greater than 50 kDa and less than 100 kDa.

Description

Technical Field

[0001] The present invention relates to the treatment of individuals suffering from cytokine storm syndrome. The present invention relates to methods of treating such individuals and to systems and devices for use in such treatment.

Background Art

[0002] An excessive response of the body's immune system can be fatal. Such an excessive response is referred to as cytokine storm and is characteristic of various pathological conditions such as autoimmune conditions, secondary hemophagocytic lymphohistiocytosis (sHLH), severe viral infections, severe sepsis, postoperative multiple organ failure, multiple organ failure after hepatectomy, and cytokine storm after chemotherapy.

[0003] Cytokines are a diverse group of small proteins secreted by cells for cell - to - cell signaling and communication. Specific cytokines have autocrine, paracrine, and / or endocrine activity and can induce various responses via receptor binding depending on the cytokine and the target cell. Among the many functions of cytokines are the control of cell growth and differentiation, as well as the regulation of angiogenesis and immune and inflammatory responses.

[0004] Cytokine storm syndrome (CSS) is a form of systemic inflammatory response syndrome that can be induced by various factors such as infections and certain drugs. Cytokine storm is associated with a wide variety of infectious and non - infectious diseases. Cytokine storm occurs when a large number of white blood cells are activated, release inflammatory cytokines, and those cytokines further activate a large number of white blood cells. The symptoms of cytokine storm syndrome can include high fever, splenomegaly, excessive bleeding, a decrease in the number of all types of blood cells (red blood cells, white blood cells, and platelets), and potentially multiple organ failure.

[0005] The cytokine storm triggered by influenza infection is often associated with a surge of activated immune cells into the lungs. The resulting lung inflammation and fluid accumulation can lead to respiratory distress and, contaminated by secondary bacterial pneumonia, can often increase patient mortality.

[0006] Human coronaviruses (hCoVs) belong to the virological family Coronaviridae and are enveloped, positive-sense RNA viruses. Generally, they can be classified into low-pathogenic and high-pathogenic CoVs depending on the outcome after infection in humans. High-pathogenic CoVs include severe acute respiratory syndrome coronavirus (SARS-CoV), Middle East respiratory syndrome coronavirus (MERS-CoV), and the newly emerged severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), which causes COVID-19. High-pathogenic CoVs primarily infect the lower respiratory tract, causing fatal pneumonia, and sometimes fatal acute lung injury and acute respiratory distress syndrome (ARDS), resulting in high morbidity and mortality (Channappanavar R, Perlman S 2017).

[0007] Furthermore, recent evidence suggests that some patients with severe COVID-19 also experience cytokine storm syndrome. A recent retrospective multicenter study of 150 confirmed COVID-19 cases in Wuhan, China, identified elevated ferritin and IL-6 as predictors of mortality, suggesting that death may be due to virus-induced hyperinflammation and that respiratory failure due to acute respiratory distress syndrome (ARDS) is the most common cause of death in COVID-19 patients (Ruan Q et al., 2020). A retrospective study focusing on 113 deceased COVID-19 patients showed that older age, male gender, and multiple organ failure were more frequent in deceased patients compared to recovered patients (Chen T et al., 2020). Leukocytosis, lymphopenia, hypoalbuminemia, and elevated CRP levels were also found to be more frequent in deceased patients (Chen T et al., 2020). Other predictors of poor prognosis included a higher Sequential Organ Failure Assessment (SOFA) score and D-dimer levels greater than 1 microgram / L (Zhou F et al., 2020). Regarding the clinical course of critically ill patients, a retrospective study in China reported that 61.5% of 52 critically ill patients died by day 28, with a median time from ICU admission to death of 7 days (Yang X et al., 2020). Age, comorbidities, organ failure, hypoalbuminemia, and marked inflammatory response are independent determinants of mortality in COVID-19 patients.

[0008] Public health interventions such as social isolation aim to prevent viral infection. Active efforts are underway to reduce viral replication using various measures. Treatment options for COVID-19 become limited once patients develop systemic inflammation and begin to experience organ failure. Several methods have been proposed to control systemic inflammation in COVID-19. Corticosteroids are commonly used to suppress the inflammatory state. However, the use of steroids in COVID-19 remains controversial (Russell CD et al., 2020; Shang L et al., 2020). Observational data from SARS-CoV and MERS-CoV suggest increased mortality and secondary infection rates, impaired clearance, and complications of corticosteroid therapy in survivors (Russell CD et al., 2020). Clinical data on the efficacy and safety of steroids in COVID-19 are not publicly available. Another approach being considered is the use of IL-6 antibodies to reduce the severity of inflammation. However, once patients require organ support in the ICU, treatment becomes limited to providing multi-organ support. However, despite the best supportive care, mortality rates remain high, and targeting cytokine storms in COVID-19 patients represents an unmet medical need.

[0009] Therefore, it is necessary to treat individuals with cytokine storm syndrome, or diseases or conditions associated with cytokine storms, in order to reduce the mortality rate of such individuals. [Overview of the Initiative]

[0010] Summary of the Invention This invention relates to diseases or conditions associated with cytokine storms, the treatment of cytokine storm syndrome, or the treatment of patients with severe cytokine storms. This invention addresses key factors that can influence morbidity and mortality in patients with cytokine storm syndrome, namely, structural, functional, and level defects of albumin, as well as elevated endotoxin levels in the blood of such patients. This invention also addresses elevated cytokine levels in the blood of such patients.

[0011] This invention is based on the inventors' finding that patients suffering from cytokine storm syndrome, such as those with COVID-19, showed remarkable similarities in inflammatory responses and associated immunopathology to patients with acute exacerbations of chronic liver failure (ACLF). Therefore, a device that has been shown to address cytokineemia and organ failure characteristic of ACLF will address cytokine storms characteristic of conditions such as severe COVID-19 infection and reduce mortality in these patients.

[0012] Therefore, the present invention provides a method for treating an individual having cytokine storm syndrome, and the said method is (a) A process for selectively removing albumin from the blood of an individual, (b) A process for selectively removing endotoxins from the blood of an individual, and (c) A process in which albumin not derived from the individual is supplied to the individual's blood, if applicable. This includes processing the blood of the said individual, Step (a) comprises the use of means for dialysis of albumin, the means comprising a membrane having a pore size greater than 50 kDa and less than 100 kDa.

[0013] The present invention also provides albumin not derived from the blood of the individual being treated for use in a method for treating cytokine storm syndrome, and the method is (a) A process to remove albumin from the blood of an individual, (b) A process to reduce the level of endotoxins in the blood of an individual, and (c) Depending on the circumstances, the step of introducing albumin that does not originate from the individual being treated into the individual's blood. Includes, Step (a) is performed by dialysis using a means that includes a membrane having a pore size greater than 50 kDa and less than 100 kDa.

[0014] The present invention also provides an apparatus for use in a method for treating an individual having cytokine storm syndrome, and the method is (a) means for selectively removing albumin from the blood of an individual, (b) means for selectively removing endotoxins from the blood of an individual, and (c) Depending on the circumstances, the process may include processing the blood of the individual with an apparatus that includes means for supplying non-individual albumin to the blood of the individual, The means (a) includes means for dialysis of albumin, which includes a membrane having a pore size greater than 50 kDa and less than 100 kDa.

[0015] The present invention also provides an in vitro method for processing blood outside the body by selectively removing albumin and endotoxins from the blood, the blood being from an individual having cytokine storm syndrome, and the method is (a) A step of bringing a solid support that selectively binds to albumin into contact with blood, thereby removing albumin from the blood. (b) A step of bringing a solid support that selectively binds to endotoxins into contact with blood, thereby removing endotoxins from the blood, and (c) Depending on the circumstances, the procedure may include adding albumin to the blood that does not originate from the same individual as the blood.

[0016] The present invention also provides a method for treating an individual having COVID-19 infection, the method being: (a) A process for selectively removing albumin from the blood of an individual, (b) A process for selectively removing endotoxins from the blood of an individual, and (c) optionally, supplying albumin not derived from the individual to the blood of the individual including treating the blood of the individual by wherein the step (a) includes dialysis of albumin by means including a membrane having a pore size exceeding 50 kDa and less than 100 kDa.

[0017] The present invention also provides a method for treating an individual having multiple organ failure, the method comprising (a) selectively removing albumin from the blood of the individual, (c) selectively removing endotoxin from the blood of the individual, and (c) optionally, supplying albumin not derived from the individual to the blood of the individual including treating the blood of the individual by wherein the step (a) includes dialysis of albumin by means including a membrane having a pore size exceeding 50 kDa and less than 100 kDa. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] [Figure 1A] Configuration of DIALIVE, a device incorporating two filters targeting cytokine storm and albumin function, which are characteristics of acute-on-chronic liver failure (ACLF). [Figure 1B] See Figure 1A. [Figure 2] Study design of a randomized comparative trial of DIALIVE in patients with ACLF. [Figure 3A] Performance indicators of DIALIVE. (A) Significant decrease in human non-mercaptalbumin, (B) increase in human mercaptalbumin, (C) improvement in albumin binding efficiency, and (D) evidence of albumin loss in those treated with the device. (SOC: standard treatment). (E) Change in endotoxin activity assay, which is the second performance indicator. [Figure 3B] See Figure 3A. [Figure 4A]Effects on organ function. (A) Patients treated with DIALIVE showed a significant decrease in CLIF-ACLF score compared to patients treated with standard therapy. (B) Distribution of ACLF grade over time for each treatment. [Figure 4B] See Figure 4A. [Figure 5] The effect of DIALIVE on circulating cytokines. Significant reductions in IL-6, IL-8, and TNFα, and an increase in IL-10, were observed in patients treated with DIALIVE compared to patients treated with standard therapy. [Figure 6] Effect of DIALIVE on circulating markers of cell death. Significant reduction in M30 and M65 fragments of cytokeratin 18 (marker of cell death) in patients treated with DIALIVE and in patients treated with standard therapy. [Figure 7] The inflammatory response and cytokine storm in ACLF are similar to those seen in severe COVID-19. [Figure 8] An example of a typical device. [Modes for carrying out the invention]

[0019] Detailed description of the invention It should be understood that the various applications of the disclosed methods and apparatus may be adapted to specific needs in the art. The terms used herein are intended solely to describe specific embodiments of the invention and should not be considered limiting.

[0020] Throughout this specification, the word “includes” or variations such as “includes” or “includes” is understood to mean the inclusion of the element, integer or process or group of elements, integers or processes shown, and not the exclusion of any other element, integer or process or group of elements, integers or processes.

[0021] Furthermore, the singular form used in this specification and the appended claims includes multiple subjects unless it is clearly contradictory. Therefore, for example, a reference to “syndrome” includes multiple “syndromes.” The terms “cytokine storm syndrome” and “severe cytokine storm” are used interchangeably.

[0022] All publications, patents, and patent applications cited herein, both those mentioned above and those mentioned below, are incorporated herein by reference.

[0023] This invention is based on the unexpected discovery that the removal of endotoxins and the exchange of albumin had a significant effect on the severity of cytokineemia and organ function in patients. Therefore, devices and methods for removing endotoxins and exchanging albumin can suppress cytokine storms characterized by cytokineemia and organ dysfunction. Such devices and methods suppress harmful pro-inflammatory cytokines (e.g., IL-6, IL-8, and TNFα) while increasing the anti-inflammatory cytokine IL-10.

[0024] Accordingly, the present invention relates to the use of a DIALIVE device or a modified form of the device for treating an individual or patient having cytokine storm syndrome or an individual suffering from a disease or condition associated with a cytokine storm. In a preferred embodiment, cytokine storm syndrome results from a viral infection. In a preferred embodiment, the viral infection is COVID-19. The DIALIVE device is described in WO 2008 / 050148 (which is incorporated herein by reference).

[0025] Cytokine storm syndromes include conditions involving unsuppressed inflammatory responses, such as multiple organ failure or dysfunction, Alzheimer's disease and stroke, protein-binding substance poisoning, cholestatic syndrome and severe iron accumulation. Therefore, the methods and apparatus described herein may also be used to treat individuals or patients having multiple organ failure or dysfunction, Alzheimer's disease and stroke, protein-binding substance poisoning, cholestatic syndrome or severe iron accumulation.

[0026] Albumin is the major plasma protein produced in the liver. It plays various roles, including fatty acid transport, metal chelation, drug binding, and antioxidant activity. In liver diseases such as ACLF, its concentration decreases due to reduced synthesis or increased degradation. In patients with liver diseases like ACLF, the proportion of albumin in the bloodstream is structurally abnormal. This albumin profile is consistent with that observed in patients with cytokine storm syndrome, such as those with severe COVID-19 (Figure 7).

[0027] Therefore, in one embodiment, the present invention relates to the removal of albumin from the blood of an individual having cytokine storm syndrome.

[0028] In this context, albumin removal means the removal of structurally normal albumin, and also the removal of any structurally or functionally modified forms of albumin present in the blood of an individual. That is, preferably, means for albumin removal used according to the present invention can remove not only naturally occurring normal albumin, but also albumin that may have abnormal structures or modified albumin. Removal of damaged or abnormal albumin can also be therapeutically useful because damaged albumin has poor functionality and may cause harmful side effects. For example, albumin that can be removed by means for albumin removal has reduced molecular flexibility, reduced fatty acid binding affinity, reduced transport performance, reduced transport efficiency, and / or reduced detoxification capacity compared to normal, unmodified albumin. Means for albumin removal can also remove specific modified forms of albumin, such as ischemic modified albumin (IMA). Such structural and functional modifications can be detected using conventional techniques. In particular, the functionality of albumin can be evaluated using spin-labeling and electron paramagnetic resonance spectroscopy. The presence of IMA can be detected by examining albumin's ability to bind to metal atoms.

[0029] Furthermore, the removal of albumin can detoxify the blood by removing associated albumin-binding toxins. In other words, means of removing albumin can, as a result, also remove toxins in the blood that are bound to albumin.

[0030] Preferably, the means for removing albumin from blood selectively removes albumin; that is, albumin is removed preferentially over other substances in the blood, such as other proteins. Preferably, the amount of albumin removed from the blood is significantly greater than the amount of other blood components removed. For example, more than 99% by weight (i.e., greater than 99% by weight) of the components removed in this embodiment may be albumin. More than 98%, more than 95%, more than 90%, more than 80%, more than 70%, more than 60%, or more than 50% of the components removed in this embodiment may be albumin. In this case, the removal of albumin includes the removal of various modified forms of albumin as described herein.

[0031] The means for removing albumin can be any means that can selectively remove albumin from the blood.

[0032] In one embodiment, albumin is selectively removed using a ligand that binds to albumin. The ligand can be any molecule that binds to albumin. For example, many reactive dyes are known to bind to albumin. The ligand can be an antibody or other affinity ligand that specifically binds to albumin. Typically, a ligand that specifically binds to albumin is a ligand that can selectively remove albumin from blood as described above. For example, the ligand may bind to albumin more strongly than other components of blood. For example, the ligand can be an antibody that specifically binds to human albumin. The ligand can be an antibody that binds to an epitope specific to albumin. The ligand can be a combination of molecules that each bind to albumin, for example, a combination of molecules that bind to different parts of the albumin molecule. The ligand can be a polyclonal antibody or a mixture of antibodies that bind to multiple epitopes on the albumin protein. Such a combination approach can be useful for removing modified forms of albumin because the antibodies can thereby target different parts of the albumin molecule.

[0033] Antibodies can be produced against specific epitopes of albumin molecules. For example, antibodies can be specifically produced against regions that are expected to be structurally similar in both the unmodified and specific modified forms of albumin.

[0034] For the purposes of this invention, the term "antibody" includes albumin-binding fragments unless otherwise specified. Such fragments include Fv, F(ab'), and F(ab')2 fragments, as well as single-chain antibodies. Furthermore, antibodies and their fragments may be chimeric antibodies, CDR-grafted antibodies, or humanized antibodies.

[0035] The antibodies used in this invention can be produced by any suitable method. Means for producing and characterizing antibodies are well known in the art; see, for example, Harlow and Lane (1988) “Antibodies: A Laboratory Manual”, COLD Spring Harbor Laboratory Press, Cold Spring Harbor, NY. For example, antibodies can be produced in a host animal by causing it to produce antibodies against an entire polypeptide or a fragment thereof, such as its antigenic epitope (hereinafter referred to as “immunogen”).

[0036] A method for producing polyclonal antibodies involves immunizing a suitable host animal, such as an experimental animal, with an immunogen and isolating immunoglobulins from the animal's serum. Therefore, it is possible to inoculate an animal with an immunogen, then collect blood from the animal, and purify the IgG fraction.

[0037] Methods for producing monoclonal antibodies involve immortalizing cells that produce the desired antibody. Hybridoma cells can be produced by fusing spleen cells from inoculated experimental animals with tumor cells (Kohler and Milstein (1975) Nature 256, 495-497).

[0038] Immortalized cells that produce the desired antibody can be selected by conventional methods. Hybridomas can be grown in culture or injected intraperitoneally to generate ascites, or into the bloodstream of an allogeneic host or an immunodeficient host. Human antibodies can be produced by in vitro immunization of human lymphocytes, followed by transformation of the lymphocytes with Epstein-Barr virus.

[0039] For the production of both monoclonal and polyclonal antibodies, the experimental animals are appropriately goats, rabbits, rats, or mice. In some cases, the immunogen may be administered as a conjugate, in which the immunogen is bound to a suitable carrier, for example, via a side chain of one amino acid residue. The carrier molecule is typically a physiologically acceptable carrier. The resulting antibodies may be isolated and, if necessary, purified.

[0040] An antibody or other ligand can be said to "specifically bind" to a protein if it preferentially or with high affinity binds to the protein it is specific to, but substantially does not bind to other proteins or binds to them with low affinity. In other words, an antibody can be said to specifically bind to albumin if it binds to albumin more strongly than it binds to other blood components, such as other proteins in the blood. As stated above, the specificity of binding can be such that it binds not only to unmodified albumin but also to structurally or functionally modified forms of albumin. Preferably, it binds to structurally or functionally modified forms of albumin with the same or substantially the same binding affinity as to unmodified albumin. Preferably, it binds to both modified and unmodified forms of albumin with greater affinity than other blood components, such as other proteins in the blood. Various protocols for competitive binding or immunoradioactive assays to determine the specific binding ability of antibodies are well known in the art (see, for example, Maddox et al., J. Exp. Med. 158, 1211-1226, 1993). Such immunoassays typically involve the formation of a complex between a specific protein and its antibody, and the measurement of complex formation.

[0041] When ligands are used to achieve albumin removal, the ligands may be supplied bound to a solid support. The ligands may be immobilized on such a solid support. A suitable solid support may be in the form of a column through which blood can pass. A suitable solid support may be a porous material such as a membrane, particle bed, or filter that is sufficiently porous to allow blood cells to pass through. Alternatively, a suitable solid support may be a solid substrate over which blood can pass. Preferably, the solid support has a large surface area to maximize the contact area between the solid blood and the ligand bound to the support. The solid support may be in the form of beads that can be filled into a container through which blood can be inserted or passed. The beads preferably have a size sufficient to allow sufficient porosity when filled into a column or filter bed. Various bead materials are known in the art.

[0042] Accordingly, the means for removing albumin according to the present invention may include, or consist of, a solid support on which a ligand binding to albumin is bound or immobilized on its surface. The means for removing albumin may include, or consist of, a container through which blood passes. Accordingly, the container may include an inlet and an outlet. The inlet and outlet are arranged so that the blood passing through the container comes into contact with the solid support described herein. Preferably, the means for removing albumin is designed or selected so as to maximize the contact area between the blood and the solid support. Various such designs are known in the art. For example, the means for removing albumin may be a column or filter bed filled with beads, in which case a ligand for albumin is immobilized on the beads.

[0043] In another embodiment, albumin removal can be achieved by dialysis. Such an approach may also result in the removal of other blood components that are dialyzed along with albumin. Typically, albumin should be the primary component to be removed. This dialysis process can use any albumin dialysis system. Various such systems are known in the art. One such system is the molecular absorbance recirculation system (MARS). An alternative is the common single-pass albumin dialysis (SPAD) system. These systems use a 50 kDa pore membrane to dialyze albumin from the blood. This system is specifically designed to remove albumin-binding toxins from a patient's blood.

[0044] As an alternative to this system, a membrane with larger pores may be used so that albumin derived from the patient's blood is actively exchanged for fresh albumin through dialysis. This allows for the removal of toxic and abnormally shaped albumin in the same process. This also allows for the introduction of new, non-individual-derived albumin into the bloodstream, as will be described in more detail later.

[0045] Studies on modifications to the MARS system to include membranes with larger pores have shown significant improvements in the removal of protein-bound toxins compared to standard 50 kDa pore systems. For example, membranes with pore sizes greater than 50 kDa (i.e., above 50 kDa), greater than 60 kDa, greater than 70 kDa, greater than 80 kDa, greater than 90 kDa, or greater than 100 kDa may be used. Membranes may have pore sizes less than 60 kDa, less than 75 kDa, less than 100 kDa, or less than 150 kDa.

[0046] In a preferred embodiment, the film has a pore diameter greater than 50 kDa and less than 20 kDa. In a preferred embodiment, the film has a pore diameter of at least 50 kDa and less than 120 kDa. In a preferred embodiment, the film has a pore diameter of at least 50 kDa and less than 100 kDa. In a preferred embodiment, the film has a pore diameter greater than 50 kDa and less than 100 kDa.

[0047] In a preferred embodiment, the film has a pore size defined as 10 nm ± 2 standard deviations.

[0048] Albumin removal can also be an alternative to the direct removal of cytokines. Therefore, in one embodiment of the present invention, albumin removal removes cytokines from the blood of an individual.

[0049] Depending on the individual means used to remove albumin, other blood components may be removed along with albumin. In one embodiment, the other components removed along with albumin may be returned to the individual's blood. The returned components can be purified from the albumin mixture removed from the blood, or they can be replaced with fresh equivalent components not derived from the individual.

[0050] Various methods for removing albumin from blood are known in the art. For example, U.S. Patent No. 4,093,612 discloses a reactive dye composition that can be used to remove albumin from a fluid. According to the present invention, such a composition can be used to remove albumin from the blood of an individual. Therefore, this can be a selective albumin capture system based on a compound that specifically binds to albumin. These can be, for example, the reactive dye described in U.S. Patent No. 4,093,612, such as cibacron blue, or other molecules that can bind to albumin, such as an albumin-specific antibody.

[0051] In a preferred embodiment, albumin removal is performed using a septeX filter (Gambro) or a similar filter.

[0052] Accordingly, according to the present invention, albumin is removed from the blood of an individual with liver disease. In one embodiment, this albumin may be replaced with new albumin not derived from the individual. The new albumin is preferably structurally and functionally normal. That is, the new albumin may contain no or substantially any structurally or functionally modified forms of albumin. If the albumin removed from the individual's blood contains one or more modified forms of albumin, the albumin returned to the individual's blood preferably contains less modified albumin than the removed albumin. For example, the albumin returned to the individual's blood may contain less than 50%, less than 30%, less than 20%, less than 10%, less than 5%, or less than 1% of the amount of modified albumin removed from the individual. Preferably, the albumin returned to the individual does not contain modified albumin, or does not contain one or more types of modified albumin removed from the individual, such as ischemic modified albumin (IMA).

[0053] Novel albumin may be human albumin. Novel albumin may originate from another individual, such as an individual without liver disease, without liver failure, or with normal liver function. Novel albumin may be albumin removed from an individual but purified or cleansed to remove toxins and / or modified albumin molecules. Novel albumin typically contains a higher percentage of structurally and functionally normal albumin than albumin removed from an individual. Novel albumin may be pharmaceutical-grade albumin. Novel albumin may be manufactured artificially.

[0054] This new albumin is introduced into the individual's bloodstream to ensure that the individual has an appropriate level of circulating albumin. This can be a direct replacement of the removed albumin; for example, the same or equivalent amount of albumin as removed may be returned to the blood. In this system, the individual's albumin is effectively replaced by the new albumin. Alternatively, by varying the amount of albumin introduced at this stage, the total albumin concentration in the blood can be increased or decreased as needed. Therefore, the amount of albumin introduced into the blood can be greater or less than the amount removed. For example, cytokine storm syndrome can lead to a decrease in circulating albumin levels, which can result in reduced functional capacity. According to the present invention, the amount of new albumin introduced into the patient's blood can be greater than the amount of albumin removed. This can replenish the level of albumin in the individual's circulation. For example, an amount of new albumin can be introduced that raises the total albumin level in the blood to the same or similar level as that seen in individuals without liver disease.

[0055] Fresh albumin can be introduced into an individual's blood simultaneously with the removal of albumin from the individual. For example, albumin replacement can be achieved by dialysis. Alternatively, the steps of albumin removal and albumin return can be performed sequentially or separately. For example, if the present invention is carried out ex vivo, the individual's blood can be passed through means for albumin removal, and then fresh albumin can be added thereto. This can be achieved by different parts of the same apparatus. Alternatively, the addition of fresh albumin can be performed separately. Typically, the addition of fresh albumin is performed after albumin has been removed from the patient's blood.

[0056] Endotoxins have been shown to be components in the blood of patients with ACLF that may be associated with patient prognosis, such as patient susceptibility to infection or organ failure, patient mortality risk, and patient potential response to certain therapies, such as immunosuppression. These prognostic factors have been shown to be related to neutrophil activation in the blood of patients with ACLF, and such activation may be associated with the presence of infectious agents in the plasma of those patients. Therefore, plasma from individuals exhibiting high neutrophil activation may increase the activation level of normal neutrophils.

[0057] Similar effects can be achieved by exposing normal neutrophils to endotoxins, and the removal of endotoxins from the blood of patients exhibiting high neutrophil activation may reduce the level of neutrophil activation in their blood. Therefore, endotoxin removal is considered useful in treating ACLF patients with activated neutrophils. By restoring normal neutrophil function, the ability of these individuals to fight infection may be improved. The inventors have found that the immune profiles of patients with ACLF are remarkably similar to those of patients with cytokine storm syndromes such as COVID-19. Therefore, removing endotoxins from the blood of patients with cytokine storm syndromes should yield similar benefits to those seen in ACLF patients.

[0058] Therefore, the present invention also relates to the removal of endotoxins from the blood of patients with cytokine storm syndrome. The removal of albumin addresses one of the detoxification problems, while the removal of endotoxins addresses another problem, the reduced immune response. By combining these two approaches in a single device or method, a particularly effective treatment of cytokine storm syndrome can be achieved.

[0059] Preferably, the means for removing endotoxins from the blood selectively removes endotoxins; that is, endotoxins are removed preferentially over other substances in the blood. Preferably, the amount of endotoxins removed from the blood is significantly greater than the amount of other blood components removed. For example, more than 99% by weight (i.e., greater than 99% by weight) of the components removed in this embodiment may be endotoxins. More than 98%, more than 95%, more than 90%, more than 80%, more than 70%, more than 60%, or more than 50% of the components removed in this embodiment may be endotoxins.

[0060] The means for removing endotoxins can be any means that can selectively remove endotoxins from the blood.

[0061] In one embodiment, endotoxins may be selectively removed using ligands that bind to endotoxins. The ligand can be any molecule that binds to endotoxins. For example, anti-endotoxin antibodies, LPS-binding proteins, polymyxin B, polyethyleneimine, arginine ligands, and various peptides are known to bind to endotoxins. The ligand can be an antibody or other affinity ligand that specifically binds to endotoxins. For example, the ligand can be an antibody that specifically binds to endotoxins. Typically, a ligand that specifically binds to endotoxins is a ligand that can selectively remove endotoxins from the blood as described above. For example, the ligand may bind to endotoxins more strongly than other components of the blood. The ligand can be an antibody that binds to an endotoxin-specific epitope. The ligand can be a combination of molecules that each bind to an endotoxin, for example, a combination of molecules that bind to different parts of an endotoxin molecule or different endotoxins. The ligand can be a polyclonal antibody or a mixture of antibodies that bind to multiple epitopes on an endotoxin protein or to various endotoxins.

[0062] Antibodies can be produced against specific epitopes of endotoxin molecules. Suitable antibody types can be any type of antibody, as described above with respect to albumin, such as antibody fragments.

[0063] Antibodies that bind to endotoxins can be produced by any means, for example, as described above with respect to albumin-binding antibodies. The obtained antibodies can be isolated and, if necessary, purified.

[0064] An antibody or other ligand can be said to "specifically bind" to a protein if it preferentially or with high affinity binds to the specific protein, but substantially does not bind to other proteins or binds with low affinity. That is, it binds to endotoxins more strongly than it binds to other blood components, such as other proteins in the blood. The specificity of the binding may be such that it binds to various forms of endotoxins. Preferably, it binds to various forms of endotoxins with greater affinity than other blood components.

[0065] When ligands are used to achieve endotoxin removal, the ligands may be supplied bound to a solid support. The ligands may be immobilized on such a solid support. Suitable solid supports are as described above with respect to albumin-binding ligands.

[0066] Accordingly, the means for removing endotoxins according to the present invention may include, or consist of, a solid support on which a ligand capable of binding to endotoxins is bound or immobilized on its surface. The means for removing endotoxins may include, or consist of, a container through which blood passes. Accordingly, the container may include an inlet and an outlet. The inlet and outlet are arranged so that the blood passing through the container comes into contact with the solid support described herein. Preferably, the means is designed or selected so as to maximize the contact area between the blood and the solid support. Various such designs are known in the art. For example, the means may be a column or filter bed filled with beads, in which case a ligand for albumin is immobilized on the beads.

[0067] In a preferred embodiment, endotoxin removal is carried out using an oXiris filter (Baxter).

[0068] In another embodiment, it is possible to reduce endotoxin levels by administering drugs to an individual rather than physically removing endotoxins from the blood. For example, endotoxins in the blood may be functionally neutralized rather than removed. Various methods for neutralizing endotoxins are known in the art. This may include administering drugs to an individual that can selectively remove or neutralize the activity of endotoxins. This may depend on the host immune system assisting in the removal of endotoxins. For example, a suitable drug may bind to the endotoxin, allowing the individual's immune system to remove the endotoxin-drug complex from the blood. Various drugs for reducing circulating endotoxin levels are known, such as anti-endotoxin antibodies, albumin and LPS-binding proteins, and LPS-neutralizing CD14 antibodies.

[0069] Depending on the specific means used to remove endotoxins, other blood components may be removed along with the endotoxins. For example, some methods for removing endotoxins may also remove other toxins from the blood. This may be beneficial to the patient. Some methods for removing endotoxins may also remove other blood components that are desirable to be retained in the blood. In this case, the blood components removed along with the endotoxins may be returned to the individual's blood. The returned components can be purified from the removed endotoxin mixture or replaced with fresh equivalent components not derived from the individual.

[0070] Various approaches for removing endotoxins from samples have been described in the art. For example, EP-A-0 129 786 describes the use of polymyxin B covalently immobilized on polystyrene fibers for removing endotoxins from blood. Falkenhagen et al. (Artificial Organs (1996) 20:420) describe the removal of endotoxins from plasma using polyethyleneimine-coated beads. WO 01 / 23413 describes highly dispersible oligopeptides used for selective removal of endotoxins from blood or plasma. US 5,476,715 describes a material for removing endotoxins from samples, comprising a porous carrier composed of polymers of acrylic acid and methacrylic acid with specific particle sizes and spacings. Staubach et al. (Transfusion and Apheresis Science (2003) 29: 93-98) describe an apparatus for endotoxin adsorption based on immobilized albumin. Thus, there are numerous available methods that can be used to remove endotoxins from samples. Any of these methods may be used or adapted for use according to the present invention. A person skilled in the art will be able to select an appropriate method and conditions for such use.

[0071] The apparatus or method of the present invention is preferably effective in achieving a significant reduction in endotoxin levels in circulating blood. For example, the apparatus or method may result in a reduction of at least 25%, at least 50%, at least 70%, at least 80%, at least 90%, at least 95%, at least 99%, or more in endotoxin levels in the blood of an individual.

[0072] Furthermore, the two aforementioned aspects of the present invention are related. Albumin can bind to endotoxins in the blood. Therefore, removal of albumin may also result in the removal of some endotoxins bound to albumin. Moreover, by increasing the level of normal albumin in an individual, it is possible to make blood albumin capable of binding to circulating endotoxins, thereby reducing the level of free endotoxins in the blood. However, the amount of endotoxin reduction achieved by this method is relatively small, and therefore, the present invention preferably utilizes separate means for (a) removing albumin and (b) reducing endotoxin levels. This effect of albumin removal and / or substitution can complement other means for reducing endotoxins and help to "clear" any remaining endotoxins in the blood of an individual.

[0073] Based on these findings, the inventors have developed novel methods and apparatus for use in the treatment of individuals with cytokine storm syndrome. These include combinations of components that act to remove both albumin and endotoxins from the blood of individuals with liver disease.

[0074] In particular, the inventors have developed a device for use in the treatment of individuals with cytokine storm syndrome. The device includes means for selectively removing albumin from the individual's blood (including means for albumin dialysis, which includes a membrane having a pore size greater than 50 kDa and less than 100 kDa) and means for selectively removing endotoxins from the individual's blood. Preferably, these are two separate means, each aimed at achieving one of these results. The device may also include means for supplying new albumin, not derived from the individual, into the individual's blood.

[0075] Furthermore, the apparatus may also include means for removing cytokines from the individual's blood. The means for removing cytokines from the blood may be the same as, or different from, the means for removing albumin and endotoxins from the individual's blood.

[0076] Therefore, the use of such devices results in the removal of albumin from the individual's blood, a reduction in the level of endotoxins in the individual's blood, and, if applicable, the introduction of new albumin into the individual's blood. This is therapeutically useful in various ways. Removing endogenous albumin from the individual's blood can result in the removal of undesirable toxins bound to albumin, and it is also possible to remove abnormally modified forms of albumin from the blood. Such modified forms often exhibit reduced function. To replace the removed albumin with fresh albumin, the introduction of new albumin may be performed if appropriate. Preferably, the fresh albumin does not contain toxins bound to the albumin molecule, and preferably, the fresh albumin is in an unmodified form. The addition of fresh albumin to the blood may be performed to compensate for the reduced endogenous albumin levels resulting from cytokine storm syndrome. Finally, removing endotoxins from the blood of an individual with cytokine storm syndrome can help reduce the activation level of neutrophils in the individual's blood. This reduction in neutrophil activation can lead to a lower risk of infection, organ failure, and death, and may improve an individual's response to immunosuppressive therapy and steroid or antibiotic treatment.

[0077] Therefore, the method and apparatus of the present invention provide a targeted approach for the treatment of cytokine storm syndrome, addressing multiple factors associated with such disease and providing benefits to the individual in multiple ways.

[0078] Accordingly, in one embodiment, the present invention relates to a device for use in the treatment of an individual having cytokine storm syndrome. The device may include several components, which may be used in combination or separately. The device of the present invention includes, or is essentially composed of, means for selectively removing albumin from the blood of an individual, and means for selectively removing endotoxins from the blood of an individual. The device of the present invention may include means for removing cytokines from the blood of an individual.

[0079] Any means described herein may be used to remove albumin, endotoxins, or cytokines. In one embodiment, a single component of the apparatus may be used for the removal of both albumin and endotoxins. Typically, such a component of the apparatus includes both means for removing albumin and means for removing endotoxins. For example, if both albumin and endotoxins are removed by binding to specific ligands, one or more ligands specific to albumin and one or more ligands specific to endotoxins may be used together in the same component of the apparatus. For example, the apparatus may include a single container, such as a column or filter bed, containing a solid support on which such two ligands are immobilized. The ligands may be immobilized on different supports or on the same support. Thus, albumin and endotoxins may be removed simultaneously from the blood of an individual.

[0080] Alternatively, the device may include separate means for removing albumin and endotoxins. The device may include multiple means capable of removing albumin and / or multiple means capable of removing albumin. In one embodiment, a single component of the device may be used to remove both albumin and cytokines. In one embodiment, a single component of the device may be used to remove both endotoxins and cytokines. In one embodiment, a single component of the device may be available to remove both albumin and cytokines, and another component of the device may be available to remove both endotoxins and cytokines. For example, a septeX filter may remove cytokines in addition to albumin. An oXiris filter may remove cytokines in addition to endotoxins.

[0081] The device may further include other components. For example, the device may include means for removing cytokines from an individual's blood. The device may include means for supplying new albumin, i.e., albumin not derived from the individual, to the blood. The device may be for ex vivo or in vitro use. For example, the device may be designed so that blood from an individual passes through it and achieves albumin removal and endotoxin removal before returning to the individual's body.

[0082] Two possible devices are illustrated in Figure 8 (quoted from WO 2008 / 050148). It will be apparent from the discussion herein and from the two devices illustrated in Figure 8 that numerous different components can be used in various combinations to achieve the desired effect. For example, the device of the present invention can also remove cytokines.

[0083] The components of the device initially illustrated in Figure 8 are as follows: 1. A means of capturing albumin. An albumin trap removes albumin from the patient's blood. 2. Means for performing albumin dialysis to remove toxins. For example, using a large-pore membrane (over 50 kDa) to enable albumin exchange allows for improved toxin removal by enabling interaction between the "cleaning" filter and the patient's albumin. This also allows for the removal of the patient's albumin because it is exchanged with the albumin dialysate through the filter. 3. A filter for purifying albumin. 4. Components of endotoxin removal. 5. Infusion of new albumin.

[0084] The components of the second example device in Figure 8 are as follows: 1. Bacterial lipopolysaccharide trap. This is a method for removing LPS / endotoxins from a patient's blood. 2. Means for performing albumin dialysis to remove toxins. For example, as described with respect to the apparatus first illustrated in Figure 8. 3. A filter for purifying albumin. 4. Albumin replacement means. For example, a diffusion gradient for removing a patient's albumin and replacing it with fresh albumin. 5. Infusion of new albumin.

[0085] The present invention also relates to a method for treating cytokine storm syndrome using the apparatus of the present invention. The present invention also relates to a method for treating a disease or condition associated with a cytokine storm using the apparatus of the present invention. For example, it is possible to remove albumin and endotoxins from the blood by bringing blood from an individual into contact with the apparatus of the present invention, and optionally supplying albumin not derived from the individual. In a preferred embodiment, cytokines are also removed from the individual's blood. This method can be carried out ex vivo or in vitro, and the blood can then be returned to the individual.

[0086] The present invention relates to a method for treating an individual having cytokine storm syndrome. The present invention relates to a method for treating an individual having a disease or condition associated with a cytokine storm. The method comprises, or essentially consists of, the following steps: (a) removing albumin from the individual's blood, and (b) reducing the level of endotoxins in the individual's blood. This can be achieved by any method or means described herein. The method can be carried out ex vivo. Step (a) can be achieved using dialysis. Step (a) can be achieved using a ligand that can specifically bind to albumin. Step (b) can be achieved by directly removing endotoxins from the blood. Step (b) can be achieved using a ligand that specifically binds to endotoxins. Step (b) can be achieved by administering to the individual a therapeutically effective amount of an agent that can reduce the level of endotoxins in the blood. Any combination of the methods of steps (a) and (b) described herein can be used. Optionally, the method may further include the step of introducing non-individual albumin into the individual's blood. Depending on the circumstances, the method may further include a step of removing cytokines from the blood of an individual.

[0087] The present invention also relates to a method for processing blood extracorporeally by selectively removing albumin and endotoxins from the blood of an individual having cytokine storm syndrome. The present invention also relates to a method for processing blood extracorporeally by selectively removing albumin and endotoxins from the blood of an individual having a disease or condition associated with a cytokine storm. This method is achievable by any suitable means described herein and may include an additional step of adding non-individual albumin to the blood. The blood thus processed can be returned to the individual for therapeutic purposes or used for another purpose. For example, the blood may be processed in this manner before transfusion to a different individual.

[0088] The methods and apparatus described herein may be used to treat cytokine storm syndrome. Cytokine storm syndrome, or cytokine storm, is characteristic of, or may be associated with, diseases or conditions such as autoimmune conditions, secondary hemophagocytic lymphohistiocytosis (sHLH), viral infection or severe viral infection, severe sepsis, postoperative multiple organ failure, post-hepatectomy multiple organ failure, and cytokine storm after chemotherapy. In preferred embodiments, the cytokine storm syndrome to be treated is a viral infection. In preferred embodiments, the viral infection is influenza or CoVD-19.

[0089] Cytokine storm syndromes include conditions involving unsuppressed inflammatory responses, such as multiple organ failure or dysfunction, Alzheimer's disease and stroke, protein-binding substance poisoning, cholestatic syndrome and severe iron accumulation. Therefore, the methods and apparatus described herein may also be used to treat individuals or patients having multiple organ failure or dysfunction, Alzheimer's disease and stroke, protein-binding substance poisoning, cholestatic syndrome or severe iron accumulation. In preferred embodiments, the cytokine storm syndrome to be treated is multiple organ failure or dysfunction.

[0090] Multiple organ failure or dysfunction may be defined as failure of at least two organ systems resulting from physiological damage that cannot maintain homeostasis without medical intervention. Commonly affected organ systems include the central nervous system, respiratory system, renal or urinary system, cardiovascular system, coagulation system, gastrointestinal system, musculoskeletal system, and hepatobiliary system. As defined herein, multiple organ failure may be considered to include failure of at least the central nervous system and respiratory system, or the central nervous system and the renal or urinary system. As defined herein, multiple organ failure may be considered to include failure of at least the brain and lungs, or the brain and kidneys.

[0091] When drugs are administered to an individual to reduce endotoxin levels, the drugs can be administered in various dosage forms. For example, drugs can be administered orally as tablets, lozenges, aqueous or oily suspensions, dispersible powders, or granules. Drugs can also be administered parenterally by subcutaneous, intravenous, intramuscular, intrasternal, percutaneous, or infusion techniques. Drugs can also be administered in the form of suppositories. A physician may determine the necessary route of administration for each individual patient.

[0092] The formulation of a drug depends on factors such as the exact nature of the drug and whether it is intended for pharmaceutical or veterinary use. Drugs used to treat liver disease may be formulated for simultaneous, separate, or sequential use.

[0093] The drug is typically formulated for administration in the present invention using a pharmaceutically acceptable carrier or diluent. The pharmaceutically acceptable carrier or diluent may be, for example, an isotonic solution. For example, a solid oral form may contain, together with the active compound, a diluent such as lactose, dextrose, saccharose, cellulose, corn starch, or potato starch; a lubricant such as silica, talc, stearic acid, magnesium stearate, or calcium stearate and / or polyethylene glycol; a binder such as starch, gum arabic, gelatin, methylcellulose, carboxymethylcellulose, or polyvinylpyrrolidone; a de-agglomerating agent such as starch, alginic acid, arginate, or sodium starch glycolate; a foaming mixture; a dye; a sweetener; a wetting agent such as lecithin, polysorbate, or lauryl sulfate; and generally, pharmacologically inert and non-toxic substances used in pharmaceutical formulations. Such pharmaceutical formulations may be produced by known methods, such as mixing, granulation, tableting, sugar coating, or film coating processes.

[0094] Dispersions for oral administration may be syrups, emulsions, or suspensions. Syrups may contain, for example, saccharose, or saccharose in the presence of glycerin and / or mannitol and / or sorbitol as a carrier.

[0095] Suspensions and emulsions may contain, for example, natural rubber, agar, sodium alginate, pectin, methylcellulose, carboxymethylcellulose, or polyvinyl alcohol as a carrier. Suspensions or solutions for intramuscular injection may contain, together with the active compound, a pharmaceutically acceptable carrier, such as sterile water, olive oil, ethyl oleate, glycol, such as propylene glycol, and optionally an appropriate amount of lidocaine hydrochloride.

[0096] Solutions for intravenous administration or infusion may contain, for example, sterile water as a carrier, or preferably, they may be in the form of sterile, aqueous, isotonic salt solutions.

[0097] The dosage of a drug can be determined according to various parameters, in particular, the substance used; the age, weight, and condition of the patient being treated; the route of administration; and the required regimen.

[0098] In this case as well, the physician may determine the necessary route and dosage for each individual patient. A typical daily dose is approximately 0.1 to 50 mg per kg of body weight, depending on the activity of the specific inhibitor, the age, weight and condition of the individual being treated, the type and severity of the degeneration, and the frequency and route of administration. Preferably, the daily dose level is 5 mg to 2 g.

[0099] Examples Example 1: The DIALIVE extracorporeal liver support device is repurposed to target a severe cytokine storm for the treatment of severe COVID-19 infection (TREAT-CoV).

[0100] DIALIVE is a novel "liver dialysis device" developed and optimized by the ALIVER Consortium. The DIALIVE device is based on the understanding that (i) the function of albumin, a circulating protein involved in detoxification, is irreversibly reduced, and (ii) endotoxemia contributes to an increased risk of infection in liver failure. DIALIVE removes and replaces albumin and removes endotoxins and is TRL5. In animal models of liver failure, DIALIVE has been shown to be easy to use, safe, reduce endotoxemia, improve albumin and immune function, and extend survival time.

[0101] Human coronaviruses (hCoVs) belong to the virological family Coronaviridae and are enveloped, positive-sense RNA viruses. Generally, they can be classified into low-pathogenic and high-pathogenic CoVs depending on the outcome after infection in humans. High-pathogenic CoVs include severe acute respiratory syndrome coronavirus (SARS-CoV), Middle East respiratory syndrome coronavirus (MERS-CoV), and the newly emerged severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) that causes COVID-19. High-pathogenic CoVs primarily infect the lower respiratory tract, causing fatal pneumonia, and sometimes fatal acute lung injury and acute respiratory distress syndrome (ARDS), resulting in high morbidity and mortality (Channappanavar R, Perlman S 2017). Although the emergence of COVID-19 only began a few months ago, it has already caused more deaths than SARS and MERS combined (Mahase E 2020). According to the latest report from the World Health Organization (March 27, 2020), the overall mortality rate worldwide is approximately 4.5%. However, mortality rates vary by country, with 10% in Italy, 8% in Iran, 7% in Spain, 5% in the UK, 1% in the US, 0.6% in Germany, and 0.4% in Australia (Coronavirus disease 2019 (COVID-19) - Situation Report). In China, the mortality rate is estimated at 2.3% based on a recent series of 72,314 cases (The epidemiological characteristics of an outbreak of 2019 novel coronavirus diseases (COVID-19) in China, 2020). Results from the limited reporting of the case series have shown that age and pre-existing comorbidities are associated with a poor prognosis for COVID-19.

[0102] Age and comorbidities are the most important independent prognostic factors for mortality. In China, most deaths occurred in patients over 60 years of age and / or with comorbidities (cardiovascular disease 10.5%, diabetes 7.3%, chronic respiratory disease 6.3%, hypertension 6%, and cancer 5.6%) (The epidemiological characteristics of an outbreak of 2019 novel coronavirus diseases (COVID-19) in China, 2020). In the United States, mortality rates also varied by age group, with the highest rates for those 85 years and older (10%–27%), followed by those 65–84 years (3%–11%), 55–64 years (1%–3%), 20–54 years (less than 1%), and under 19 years (no deaths) (Severe Outcomes Among Patients with Coronavirus Disease 2019 (COVID-19) - United States, February 12–March 16, 2020). The most common cause of death in COVID-19 patients is respiratory failure due to ARDS (Ruan Q et al., 2020). A retrospective study focusing on 113 deceased COVID-19 patients showed that older age, male gender, and multiple organ failure were more frequent in deceased patients compared to recovered patients (Chen T et al., 2020). Leukocytosis, lymphopenia, low albumin levels, and elevated CRP levels were also found to be more frequent in deceased patients (Chen T et al., 2020). Other predictors of poor prognosis included a higher Sequential Organ Failure Assessment (SOFA) score and D-dimer levels greater than 1 microgram / L (Zhou F et al., 2020). Regarding the clinical course of critically ill patients, a retrospective study in China reported that 61.5% of 52 critically ill patients died by day 28, and the median time from ICU admission to death was 7 days (Yang X et al., 2020). In summary, age, comorbidities, organ failure, hypoalbuminemia, and marked inflammatory responses are independent determinants of mortality.Strategies that affect organ failure, particularly respiratory failure, could alter the mortality rate of COVID-19 patients admitted to the ICU.

[0103] Disease progression is often associated with rapid viral replication, massive inflammatory cell infiltration, and elevated pro-inflammatory cytokine / chemokine responses. Previous studies on SARS-CoV and MERS-CoV have shown that hCoV infection of airway and / or alveolar epithelial cells, rapid viral replication, delayed IFN response, and accumulation of monocyte-macrophages and neutrophils can all contribute to cytokine storms and worsening immunopathology. Excessive inflammatory responses can result in apoptosis and vascular leakage of epithelial and endothelial cells, suboptimal T cell responses, accumulation of selectively activated macrophages, and alterations in tissue homeostasis and acute respiratory distress syndrome (Channappanavar R, Perlman S 2017). Systemic inflammation appears to be associated with the severity of hCoV infection. For example, compared to individuals with uncomplicated viral infections, severe SARS-CoV or MERS-CoV patients showed higher serum levels of pro-inflammatory cytokines (IFN-γ, IL-1, IL-6, IL-12, and TGFβ) and chemokines (CCL2, CXCL10, CXCL9, and IL-8), as well as lower levels of anti-inflammatory cytokines (IL-10) (Channappanavar R, Perlman S 2017). Similar systemic inflammation is also found in COVID-19 patients. Severe COVID-19 cases show higher neutrophil-lymphocyte ratios (NLR), higher levels of tumor necrosis factor α, IL-2R, IL-6, IL-8, and IL-10 (Qin C et al., 2020). SARS-CoV-2 can bind to Toll-like receptors (TLRs), potentially triggering inflammasome activation and the release of pro-IL-1β (a mediator of pneumonia, fever, and fibrosis) (Conti P et al., 2020). Therapies targeting severe cytokineemia are a potential treatment for patients with COVID-19 infection. Novel therapeutic strategies that can mitigate cytokine storms are likely to alleviate organ immunopathological states and improve survival rates in COVID-19 patients.

[0104] Rationale for repurposing DIALIVE, a liver support device that targets inflammation and immunopathological conditions in liver failure, for the treatment of severely ill COVID-19 patients.

[0105] Acute exacerbation of chronic liver failure (ACLF) is a newly defined syndrome that occurs in patients with cirrhosis whose condition rapidly deteriorates due to triggering events such as infection. Patients present with organ failure, and depending on the severity of organ failure, 20-90% of these patients die. From a pathophysiological perspective, patients present with marked systemic inflammation and a large cytokine storm.

[0106] While treatment options for ACLF are currently limited, a novel strategy involves the use of an extracorporeal liver support device called DIALIVE, which targets severe cytokine storms using two novel filters that enable albumin exchange and the removal of damage-related and pathogen-related molecular patterns. DIALIVE has just completed a randomized controlled clinical trial in ACLF patients and has been shown to be safe and effective in reducing the cytokine storms characteristic of ACLF and improving organ function. The configuration of the DIALIVE device is shown in Figure 1.

[0107] Example 2: Randomized controlled clinical trial of DIALIVE in ACLF patients The program to transfer preclinical data on DIALIVE to ACLF patients was funded by a €6.4 million grant from the EU H2020 program. This study was conducted at 11 hospitals across Europe. While a full analysis is currently underway, preliminary results are presented below. A summary of the study design is shown in Figure 2.

[0108] Concise results This study recruited 30 ACLF patients.

[0109] Safety: Safety data is evaluated by the Data Safety Monitoring Board, which verifies the safety of the device.

[0110] Performance: Two key performance indicators for DIALIVE were evidence that it could remove endotoxins and albumin and improve albumin function (see Figure 3).

[0111] Impact on organ function: The primary efficacy endpoint evaluated in this trial was the change in the CLIF-ACLF score (a score from 0 to 100, combining the CLIF-organ failure score, age, and white blood cell count). The CLIF organ failure score is a modified version of the SOFA score and is widely used in septic patients in the ICU. See Figure 4.

[0112] Effects of DIALIVE on circulating markers and cytokines of cell death: Markers of cell death and associated cytokines in randomized patients. Cytokine data from the first 14 patients in this study are shown in Figure 5. Figure 6 shows a significant decrease in cytokeratin 18 (marker of cell death) M30 and M65 fragments in patients treated with DIALIVE and patients treated with standard care.

[0113] Summary of the effects of DIALIVE in ACLF patients The available data indicate that treatment with DIALIVE in ACLF patients with multiple organ failure is safe and that the device functions appropriately as specified. Patients treated with DIALIVE showed significant improvements in organ function, likely due to favorable regulation of inflammation, as indicated by reduced systemic inflammation, endotoxemia, markers of cell death, and changes in cytokine levels.

[0114] Similarities between cytokine storms in ACLF patients and cytokine storms in severe COVID-19 patients. Existing data suggest significant similarities in inflammatory responses and associated immunopathology between ACLF patients and patients with ACLF. After reviewing this data with new data related to cytokine storm syndrome, the inventors investigated the immune response in ACLF patients in comparison to that of patients with severe COVID-19. The inventors analyzed data from Qin et al., 2020 and Claria et al., 2016, and, taking this analysis into consideration, created Figure 7, which illustrates the significant similarities between cytokine storms in ACLF and those in patients with severe COVID-19.

[0115] This data reveals the unexpected finding that endotoxin removal and albumin exchange have a significant impact on the severity of cytokineemia in patients, as well as on organ function. Therefore, devices like DIALIVE, which remove endotoxins and exchange albumin, may also prevent cytokine storms characterized by cytokineemia and organ dysfunction. Such devices inhibit damage from pro-inflammatory cytokines (e.g., IL6, IL8, and TNFa) while increasing the anti-inflammatory cytokine IL10.

[0116] This data indicates similarities between cytokine storms causing multiple organ failure in ACLF and in patients with severe COVID-19 infection. DIALIVE is safe and addresses the cytokineemia and organ failure characteristic of ACLF. Therefore, DIALIVE will address the cytokine storms characteristic of severe COVID-19 infection and reduce mortality in these patients.

[0117] Example 3 - Additional data from the DIALIVE clinical trial This study was based on a safety set of 32 patients (17 DIALIVE, 15 SOC) for safety analysis, and a subset of 30 patients (15 DIALIVE, 15 SOC) in a modified safety set for biomarker and efficacy analysis. Outcome analyses, including safety, biomarkers, and efficacy, were limited to 28 days, with the exception of 90-day mortality for patients in the subsets.

[0118] Measurement of endotoxins and albumin Endotoxin measurements supported the performance and effectiveness of the appropriate equipment. Regarding mean EAA (Endotoxin Activity Assay), a decreasing trend was observed in the DIALIVE group, most pronounced on day 5. Pre-planned analysis of target and acceptable values ​​was most important when the CV% was less than 15%. The optimistic target (40%) was not achieved, but the acceptable value of 20% was achieved. Acceptable values ​​were met in both groups on day 3 (pre-treatment), with the best difference observed on day 5 (pre-treatment), showing a clinically significant advantage in the DIALIVE group (80%) versus SOC (36.4%) on day 5. A significant decrease in endotoxin activity, measured using horseshoe crab hemocyte extract (LAL) assay, was observed in DIALIVE patients, but no difference in LBP was observed between the groups. This data supports the hypothesis that DIALIVE effectively removes circulating endotoxins.

[0119] Albumin measurements supported evidence of the device's performance and effectiveness. For most measurements, consistent improvement and benefits were observed in the DIALIVE group, most of which were statistically significant. The most effective treatment outcomes were observed for HMA%, HMA / HNA ratio, HNA1%, HNA2%, HNA-2 / HMA ratio, and IMAR, where clinically and statistically significant improvements were observed in the DIALIVE group on days 5 and 10, although only minimal changes were observed for SOC. Pre-planned modeling of absolute change from baseline identified key benefits of DIALIVE. The treatment (Rx) covariate (over all days) was significant for seven measurements, while the results on day 5 were significant for six measurements, and the results on day 10 were significant for the same seven measurements as the treatment covariate. All five measurements based on HMA and HNA were significant for treatment on days 5 and 10, respectively.

[0120] Organ function Analysis of improvements in individual organ function (CLIF organ failure components: brain, kidney, CVS, lung, liver, and coagulation) identified clear and significant advantages of DIALIVE compared to SOC. Improvements in brain subscores were observed in the DIALIVE group (p<0.001), but not in the SOC group (73.3% vs. 20%). This was statistically significant between the groups (two-sided Fisher exact test, p=0.0092). Regarding liver subscores, statistically significant improvements were observed in the DIALIVE group (p=0.045) (53.3% vs. 33.3%). Significant deterioration in lung subscores was observed in the SOC group (p=0.025), but not in the DIALIVE group. Taken together, this resulted in statistically significant reductions in CLIF-C organ failure scores on days 5 and 10.

[0121] [Table 1]

[0122] [Table 2]

[0123] Dissipation of ACLF (reaching ACLF Grade 0) DIALIVE demonstrated an average superiority over 9 days, with a reduced time to ACLF resolution (ACLF grade = 0) (two-sided t-test p = 0.044). A significantly larger proportion of patients reached ACLF grade 0 (33.3% vs. 66.7%), demonstrating the superiority of DIALIVE (two-sided log-rank p = 0.0357; two-sided t-test p = 0.044), reaching ACLF grade 0 2.8 times faster, which was close to statistical significance (two-sided Wald test p = 0.059) (Figure 4B and Table 3). These statistically significant superiorities correspond to clinically meaningful superiority. Over the first 28 days, DIALIVE patients achieved a total of 245 days of ACLF grade 0, compared to 110 days for SOC patients.

[0124] [Table 3]

[0125] Inflammatory markers In cytokine measurements, the most significant effects were observed with TNF-α and IL-8. For TNF-α, a therapeutic effect was observed in the borderline (two-sided p=.094) and on day 10 in the borderline (two-sided p=0.053). For IL-8, an overall therapeutic effect (two-sided p=.008) and a day-1 effect (two-sided p=0.019) were observed, and in DIALIVE, significant superiority was observed on both day 5 (two-sided p=0.044) and day 10 (two-sided p=0.006).

[0126] Regarding NGAL, a significant decrease was observed in the DIALIVE group between day 5 and day 10 (two-sided paired t-test, p=0.048), but urinary NGAL remained unchanged. No significant changes were observed in IL1b, IL-6, IL-7, IL-18, CXCL1, CX3CL1, sCD63, CCL5 / RANTES, and CCL2 / MCP.

[0127] Damage-associated molecular patterns (DAMPs) Regarding cytokeratin 18 (M30 CK18 U / L), a significant overall therapeutic effect (two-sided p=0.002), a borderline 1-day effect (two-sided p=0.055), superiority of borderline DIALIVE on day 5 (two-sided p=0.081), and a significant superiority of DIALIVE on day 10 (two-sided p=0.005) were observed.

[0128] Regarding cytokeratin 18 (M65 K18 U / L), a significant overall therapeutic effect (two-sided, p=0.028) and a significant superiority over DIALIVE on day 10 (two-sided, p=0.029) were observed.

[0129] Regarding the CK:K18 ratio, significant superiority was observed in the treatment of borderline cases due to the 1-day interaction (two-sided, p=0.064), and significant superiority was observed in DIALIVE on day 5 (two-sided, p=0.039).

[0130] Regarding receptor-interacting serine / threonine-protein kinase 3 (RIPK3), an overall borderline therapeutic effect was observed (two-sided, p=0.094), and a significant superiority of DIALIVE was observed on day 5 (two-sided, p=0.030).

[0131] For each of the three DAMP measurements, significant or borderline therapeutic effects demonstrating the superiority of DIALIVE were observed on days 5 and 10. The cytokeratin 18 and M30 CK18 U / L markers showed overall significant therapeutic effects (two-sided p=0.002), borderline 1-day effects (two-sided p=0.055), borderline superiority of DIALIVE on day 5 (two-sided p=0.081), and significant superiority of DIALIVE on day 10 (two-sided p=0.005) (reflecting a reduction in apoptotic cell death in DIALIVE-treated patients). The observed decrease in M65 K18 suggests a reduction in non-apoptotic cell death in DIALIVE-treated patients. For RIPK3, overall borderline therapeutic effects (two-sided p=0.094) and significant superiority of DIALIVE on day 5 (two-sided p=0.030) were observed. The decreasing trend in RIPK3 suggests a reduction in necroptosis.

[0132] Endothelial markers Among the four endothelial markers—E-selectin, ICAM-1, VCAM-1, and coagulation factor VIII—coagulation factor VIII best supported the beneficial effects of DIALIVE. Overall, coagulation factor VIII supported significant therapeutic effects (two-sided, p=.009) and borderline treatment due to circadian rhythm interaction (two-sided, p=0.059). Significant superiority for DIALIVE was observed on day 5 (two-sided, p=0.002). No significant trends were observed for E-selectin, ICAM-1, and VCAM-1.

[0133] signal transduction markers Regarding the IL-1β / IL-8 response ratio, a significant overall therapeutic effect (two-sided, p=<0.001) and a nearly significant therapeutic effect due to the 1-day interaction (two-sided, p=0.065) were observed. Significant superiority of DIALIVE was observed on both day 5 (two-sided, p<0.001) and day 10 (two-sided, p=0.002).

[0134] Regarding the TLR4 response rate, a significant overall therapeutic effect (two-sided, p=0.003) was observed, and significant superiority of DIALIVE was observed on both day 5 (two-sided, p=0.005) and day 10 (two-sided, p=0.03).

[0135] Both signaling markers support the efficacy of DIALIVE. Regarding the IL-1β / IL-8 response ratio, a significant overall therapeutic effect (two-sided, p=<0.001) and a significant therapeutic effect due to the 24-day interaction (two-sided, p=0.019) were observed, demonstrating significant superiority of DIALIVE on both day 5 (two-sided, p<0.001) and day 10 (two-sided, p=0.002). Similarly, regarding the TLR4 response rate, a significant overall therapeutic effect (two-sided, p=0.003) was observed, demonstrating significant superiority of DIALIVE on both day 5 (two-sided, p=0.005) and day 10 (two-sided, p=0.03).

[0136] summary The effectiveness of DIALIVE was demonstrated based on the percentage of patients who reached ACLF Grade 0 and the time it took to reach ACLF Grade 0. 66.7% of the DIALIVE group reached ACLF 0 by day 28, while 33.3% of the SOC group reached ACLF 0 by day 28. Regarding the time to achieve ACLF grade, DIALIVE showed statistically significant superiority compared to SOC (two-sided log-rank p=0.0357). Regarding the number of days with ACLF grade 0, DIALIVE showed superiority of an average of 9 days compared to SOC (two-sided t-test, p=0.044). Further efficacy has been demonstrated in terms of improving individual organ function, CLIF-OF score, and MELD score. CLIF OF score: A significant overall therapeutic effect was observed (bilateral p=0.025), and significant superiority of DIALIVE was observed on both day 10 (bilateral p=0.021) and day 14 (bilateral p=0.001). Brain subscores improved in 73.3% of the DIALIVE group, while SOC improved in 20% (p<0.001). Liver subscores improved in 53.3% of the DIALIVE group, compared to 33.3% of the SOC group (p=0.045). Lung subscores worsened in the SOC group (p=0.025), but this was not observed in the DIALIVE group. MELD and MELD-Na scores were significantly improved in the DIALIVE group compared to the SOC group. Compared to SOC, several biomarkers were significantly improved in patients treated with DIALIVE. Regarding endothelial function markers, a significant difference was observed in the treatment group for Factor VIII on both day 5 and day 10, and a significant difference was observed in the treatment group for ADMA on day 10. Regarding the systemic inflammation marker IL-8, a significant difference in the treatment group demonstrating the superiority of DIALIVE was observed, along with a corresponding decrease in TNF-α and NGAL. Regarding cell death markers, significant therapeutic differences demonstrating the superiority of DIALIVE were observed for both cytokeratin 18 and RIPK3. Cellular signaling: Significant differences were observed between the treatment groups on both day 5 and day 10 regarding the IL-1β / IL-8 response ratio and TLR4 response rate.

[0137] References Channappanavar R, Perlman S: Pathogenic human coronavirus infections: causes and consequences of cytokine storm and immunopathology. Semin Immunopathol 2017, 39(5):529-539. Chen T, Wu D, Chen H, Yan W, Yang D, Chen G, Ma K, Xu D, Yu H, Wang H: Clinical characteristics of 113 deceased patients with coronary artery disease 2019: a retrospective study. BMJ 2020. Claria, J.ら, (2016), Systemic inflammation in decompensated cirrhosis: Characterization and role in acute‐on‐chronic liver failure. Hepatology, 64:1249-1264. Conti P, Ronconi G, Caraffa A, Gallenga CE, Ross R, Frydas I, Kritas SK: Induction of pro-inflammatory cytokines (IL-1 and IL-6) and lung inflammation by coronavirus-19 (COVI-19 or SARS-CoV-2): anti-inflammatory strategies. Journal of Biological Regulators and Homeostatic Agents 2020, 34(2). Coronavirus disease 2019 (COVID-19)-Situation Report - 67. WHO report. Falkenhagenら (Artificial Organs (1996) 20:420) HarlowおよびLane (1988) “Antibodies: A Laboratory Manual”, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY. Kohler and Milstein (1975) Nature 256, 495-497). Maddoxら, J. Exp. Med. 158, 1211-1226, 1993. Mahase E: Coronavirus covid-19 has killed more people than SARS and MERS combined, despite lower case fatality rate. BMJ 2020, 368:m641. Moreau, Richardら. Acute-on-Chronic Liver Failure Is a Distinct Syndrome That Develops in Patients With Acute Decompensation of Cirrhosis. Gastroenterology , Volume 144 , Issue 7 , 1426 -1437.e9 Qin C, Zhou L, Hu Z, Zhang S, Yang S, Tao Y, Xie C, Ma K, Shang K, Wang Wら: Dysregulation of immune response in patients with COVID-19 in Wuhan, China. Clin Infect Dis 2020. Ruan Qら. Clinical predictors of mortality due to COVID-19 based on an analysis of data of 150 patients from Wuhan, China. Intensive Care Med. 2020; (published online March 3 2020) Russell CD, Millar JE, Baillie JK: Clinical evidence does not support corticosteroid treatment for 2019-nCoV lung injury. Lancet 2020, 395(10223):473-475. Severe Outcomes Among Patients with Coronavirus Disease 2019 (COVID-19) - United States, February 12-March 16, 2020. MMWR Morb Mortal Wkly Rep 2020, 69(12):343-346. Shang L, Zhao J, Hu Y, Du R, Cao B: On the use of corticosteroids for 2019-nCoV pneumonia. Lancet 2020, 395(10225):683-684. Staubachら (Transfusion and Apheresis Science (2003) 29: 93-98) [The epidemiological characteristics of an outbreak of 2019 novel coronavirus diseases (COVID-19) in China]. Zhonghua Liu Xing Bing Xue Za Zhi 2020, 41(2):145-151. Yang X, Yu Y, Xu J, Shu H, Xia Ja, Liu H, Wu Y, Zhang L, Yu Z, Fang Mら: Clinical course and outcomes of critically ill patients with SARS-CoV-2 pneumonia in Wuhan, China: a single-centered, retrospective, observational study. The Lancet Respiratory Medicine 2020. Zhou F, Yu T, Du R, Fan G, Liu Y, Liu Z, Xiang J, Wang Y, Song B, Gu Xら: Clinical course and risk factors for mortality of adult inpatients with COVID-19 in Wuhan, China: a retrospective cohort study. The Lancet 2020, 395(10229):1054-1062.

Claims

1. A method for treating individuals with cytokine storm syndrome, The method described above is (a) A process for selectively removing albumin from the blood of an individual, (b) A process for selectively removing endotoxins from the blood of an individual, and (c) A process of supplying albumin not derived from the individual to the individual's blood, if applicable. This includes processing the blood of the said individual, The method wherein step (a) comprises the use of means for dialysis of albumin, wherein the means comprises a membrane having a pore size greater than 50 kDa and less than 100 kDa.

2. The method according to claim 1, wherein steps (a) and (b) include different means for removing albumin and for reducing endotoxin levels.

3. The method is (d) Process to remove cytokines from the blood of an individual. The method according to claim 1 or 2, including the method described in claim 1 or 2.

4. The method is (e) A process to remove toxins bound to albumin from the blood of an individual. A method according to any one of claims 1 to 3, including the method described in any one of claims 1 to 3.

5. The method according to any one of claims 1 to 4, wherein step (a) includes the use of a solid support capable of selectively binding to albumin.

6. The method according to any one of claims 1 to 5, wherein step (b) includes the use of a solid support capable of selectively binding to an endotoxin.

7. The method according to any one of claims 1 to 6, wherein the albumin in portion (c) is pharmaceutical-grade albumin.

8. The method according to any one of claims 1 to 7, performed using ExVivo.

9. The method according to any one of claims 1 to 8, wherein other components removed along with albumin are returned to the blood of the individual.

10. Albumin not derived from the blood of the individual being treated, for use in the treatment of cytokine storm syndrome, The method described above is (a) A process to remove albumin from the blood of an individual, (b) A process to reduce the level of endotoxins in the blood of an individual, and (c) Depending on the circumstances, the step of introducing albumin that does not originate from the individual being treated into the individual's blood. Includes, The albumin, wherein step (a) is performed by dialysis using means including a membrane having a pore size greater than 50 kDa and less than 100 kDa.

11. Albumin for use according to claim 10, wherein steps (a) and (b) of the method include different means for removing albumin and for reducing endotoxin levels.

12. Albumin for use according to claim 10 or claim 11, wherein steps (a) and (c) are performed by dialysis.

13. Albumin for use according to any one of claims 10 to 12, wherein step (b) comprises removing endotoxins from the blood of an individual, or (ii) step (b) comprises administering to an individual a therapeutically effective amount of a drug capable of reducing the level of endotoxins in the blood, and optionally the method further comprises removing cytokines from the blood of the individual.

14. Albumin for use according to any one of claims 10 to 13, wherein the albumin of step (c) is pharmaceutical grade albumin.

15. Albumin for use according to any one of claims 10 to 14, wherein the method is carried out using the apparatus according to any one of claims 1 to 9.

16. A device for use in a treatment method for individuals with cytokine storm syndrome, The method described above is (a) means for selectively removing albumin from the blood of an individual, (b) means for selectively removing endotoxins from the blood of an individual, and (c) Means for supplying non-individual albumin to the blood of an individual, if applicable. This includes processing the blood of the individual using an apparatus that includes, The apparatus, wherein means (a) includes means for dialysis of albumin, wherein means (a) includes a membrane having a pore size greater than 50 kDa and less than 100 kDa.

17. The apparatus for use according to claim 16, wherein means (a) and (b) are separate components of the apparatus.

18. The device is (d) means for removing cytokines from the blood of an individual, and / or (e) Means for removing albumin-bound toxins from the blood of an individual. Apparatus for use according to claim 16 or claim 17, further comprising:

19. (i) means (a) includes a solid support capable of selectively binding to albumin, and / or (ii) The apparatus for use according to any one of claims 16 to 18, wherein the means (b) comprises a solid support capable of selectively binding to an endotoxin.

20. Apparatus for use according to any one of claims 16 to 19, wherein the albumin in portion (c) is pharmaceutical-grade albumin.

21. An in vitro method for processing blood outside the body by selectively removing albumin and endotoxins from the blood, The blood sample is from an individual with cytokine storm syndrome. The method described above is (a) A step of bringing a solid support that selectively binds to albumin into contact with blood, thereby removing albumin from the blood. (b) A step of bringing a solid support that selectively binds to endotoxins into contact with blood, thereby removing endotoxins from the blood, and (c) Depending on the circumstances, the step of adding albumin to the blood that does not originate from the same individual as the blood. The method, including the method described above.

22. (i) Steps (a) and (b) of the method include different means for removing albumin and for removing endotoxins, and / or (ii) The method according to claim 22, wherein the solid support of (a) comprises an antibody that specifically binds to albumin.

23. A method, apparatus for use, albumin for use, or in vitro method according to any one of claims 1 to 22, wherein cytokine storm syndrome is associated with an autoimmune condition, secondary hemophagocytic lymphohistiocytosis (sHLH), viral infection, severe sepsis, postoperative multiple organ failure, post-hepatectomy multiple organ failure, or post-chemotherapy cytokine storm.

24. The method, apparatus for use, albumin for use, or in vitro method according to claim 23, wherein the viral infection is influenza infection or COVID-19 infection.

25. A method for treating an individual infected with COVID-19, The method described above is (a) A process for selectively removing albumin from the blood of an individual, (b) A process for selectively removing endotoxins from the blood of an individual, and (C) A process in which, if applicable, albumin not derived from the individual is supplied to the individual's blood. This includes processing the blood of the aforementioned individual, The method wherein step (a) includes dialysis of albumin by means of a membrane having a pore size greater than 50 kDa and less than 100 kDa.

26. A method for treating individuals with multiple organ failure. The method described above is (a) A process for selectively removing albumin from the blood of an individual, (b) A process for selectively removing endotoxins from the blood of an individual, and (c) A process of supplying albumin not derived from the individual to the individual's blood, if applicable. This includes processing the blood of the aforementioned individual, The method wherein step (a) comprises the use of means for dialysis of albumin, wherein the means comprises a membrane having a pore size greater than 50 kDa and less than 100 kDa.

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