ADAMTS13 for the treatment, amelioration, and / or prevention of vaso-occlusive crises in sickle cell disease, acute lung injury, and / or acute respiratory distress syndrome

Abstract

The present disclosure provides compositions and methods for treating, ameliorating and / or preventing vaso-occlusive crises (VOCs) in subjects with sickle cell disease (SCD). The present disclosure also provides compositions and methods for treating, ameliorating and / or preventing lung injury in subjects with acute lung injury (ALI) and / or acute respiratory distress syndrome (ARDS) or at risk of developing acute lung injury (ALI) and / or acute respiratory distress syndrome (ARDS). The present disclosure provides a disintegrin-like metalloproteinase 13 (ADAMTS13) containing a type I thrombobinding protein motif or a composition comprising ADAMTS13 for treating, ameliorating and / or preventing VOCs, or for treating, ameliorating and / or preventing lung injury in subjects with ALI and / or ARDS or at risk of developing ALI and / or ARDS.

Description

[0001] This application is a divisional application of an invention patent application with an application date of August 4, 2017, application number 201780061626.5 (international application number PCT / US2017 / 045573), and titled “Using ADAMTS13 to treat, improve and / or prevent vaso-occlusive crisis in sickle cell disease, acute lung injury and / or acute respiratory distress syndrome”.

[0002] CROSS-REFERENCE TO RELATED APPLICATIONS

[0003] This patent application claims priority under 35 USC §119(e) to U.S. Provisional Patent Application No. 62 / 371,030, filed on August 4, 2016, which is incorporated herein by reference in its entirety. Technical Field

[0004] The present disclosure relates to methods for treating sickle cell disease with a disintegrin-like metalloproteinase 13 (ADAMTS13) containing a type I platelet-binding protein motif. More specifically, the present disclosure relates to methods for treating, improving and / or preventing vaso-occlusive crises (VOCs) in subjects with sickle cell disease (SCD) by administering ADAMTS13. The present disclosure includes the use of ADAMTS13 and / or a composition comprising ADAMTS13 in the preparation of a medicament for treating, improving and / or preventing VOCs in SCD. The present disclosure also relates to methods for treating, improving or preventing lung injury in subjects with or at risk of acute lung injury (ALI) and / or acute respiratory distress syndrome (ARDS) with ADAMTS13, as well as the use of ADAMTS13 and / or a composition comprising ADAMTS13 in the preparation of a medicament for treating, improving and / or preventing ALI and / or ARDS. Background Art

[0005] Sickle cell disease (SCD) is a worldwide inherited red blood cell disorder caused by a point mutation in the β-globin chain (β s , 6V) results in the production of a defective form of hemoglobin, hemoglobin S (HbS). Studies on the kinetics of HbS polymerization following deoxygenation have shown that it is a high-order exponential function of hemoglobin concentration, thus highlighting the key role of cellular HbS concentration in sickle cells. Pathophysiological studies have shown that dense, dehydrated red blood cells play a significant role in the acute and chronic clinical manifestations of SCD, in which intravascular sickling within capillaries, small vessels, and large blood vessels leads to vascular obstruction and impaired blood flow, with ischemic cellular damage in various organs and tissues.

[0006] In patients with SCD, elevated levels of von Willebrand factor (VWF) and very large VWF multimers are observed and are associated with acute vaso-occlusive events. The level of very large VWF multimers depends on the activity of a disintegrin-like metalloproteinase containing type I thrombobinding protein motifs 13 (ADAMTS 13), which cleaves hyperadhesive very large VWF multimers under conditions of high fluid shear stress and plays an important role in maintaining an appropriate balance between hemostatic activity and thrombotic risk. ADAMTS 13 is expressed at residue Tyr 1605 and the Met 1606 VWF is cleaved between the two residues, which correspond to residues 842-843 after the presequence cleavage. It is this ADAMTS13-mediated VWF cleavage that is primarily responsible for regulating VWF multimer size and hemostatic activity. VWF released by stimulation or blood circulation is important for the formation of platelet thrombi because it acts with collagen on platelet adhesion and aggregation in subendothelial tissue (including damaged vascular walls). The release of VWF is accompanied by and is partly triggered by the activation of the vascular endothelium. Therefore, biomarkers of vascular inflammation provide additional information about the risk of vascular occlusive events.

[0007] Extracellular hemoglobin (ECHb) is increased in SCD patients and inhibits ADAMTS13-mediated VWF proteolysis by binding to the A2 domain of VWF, specifically the ADAMTS13 cleavage site. Thrombospondin-1 (TSP1), which is also increased in SCD patients, binds to the A2 domain of very large VWF multimers and also prevents VWF degradation by ADAMTS13 by competitively inhibiting ADAMTS13 activity.

[0008] SCD is a lifelong congenital disease. People with SCD inherit two abnormal hemoglobin beta sGenes, one from each parent. When a person has two copies of the hemoglobin S gene, known as hemoglobin SS (Hb SS), the condition is called sickle cell anemia. This is the most common and often most severe form of SCD. Hemoglobin SC disease and hemoglobin Sβ thalassemia are two other common forms of SCD. In all forms of SCD, at least one of the two abnormal genes causes the body to produce hemoglobin S or sickle hemoglobin in its red blood cells. Hemoglobin is a protein in red blood cells that transports oxygen throughout the body. Sickle hemoglobin differs from normal hemoglobin in that it tends to form aggregates under conditions of low oxygen tension. These aggregates form rigid rods within red blood cells, giving them a crescent or sickle shape. Sickle cells are inflexible and can cause blockages that slow or stop blood flow and ultimately impede microcirculation. When this occurs, oxygen cannot reach nearby tissues. The lack of tissue oxygen can cause sudden, severe episodes of pain known as vaso-occlusive crises (VOCs), pain crises, or sickle cell crises, leading to ischemic damage to organs and resulting pain. Painful crises are the most distinguishing clinical feature of VOCs in SCD and the leading cause of emergency department visits and hospitalizations in affected patients.

[0009] VOCs are initiated and maintained by interactions between sickle cells (including sickle cell reticulocytes, endothelial cells, leukocytes, and plasma components, including von Willebrand Factor (VWF)). Vaso-occlusive disease leads to various clinical complications of SCD, including pain syndromes, stroke, leg ulcers, spontaneous abortion, and renal insufficiency. The pain of VOCs is often incompletely treated. Current treatments for VOCs include the use of fluids, oxygen, and analgesia, and the incidence of VOCs can be reduced by chronic red blood cell (RBC) transfusions and hydroxyurea. However, despite advances in pain management, physicians are often reluctant to provide patients with adequate doses of narcotic analgesics due to concerns about addiction, tolerance, and side effects. In addition to acute VOCs, other acute and chronic complications of SCD include renal disease, splenic infarction, increased risk of bacterial infection, acute and chronic anemia, chest syndrome, stroke, and ocular disease.

[0010] Acute pain in patients with SCD is caused by ischemic tissue damage during acute crises, which is caused by obstruction of the microvascular bed by sickled red blood cells. For example, the severe bone pain characteristic of VOC is thought to result from increased intramedullary pressure, particularly in the juxtaarticular regions of long bones, secondary to an acute inflammatory response to bone marrow vascular necrosis by sickled red blood cells. Pain may also occur due to involvement of the joint periosteum or periarticular soft tissue. The unpredictable recurrence of acute crises contributes to chronic pain, resulting in a unique pain syndrome.

[0011] The severity of SCD varies from person to person. Advances in the diagnosis and care of SCD have extended the life expectancy of people with SCD. In high-income countries like the United States, the life expectancy of people with SCD is now approximately 40-60 years, compared to only 14 years about 40 years ago. However, currently, hematopoietic stem cell transplantation (HSCT) is the only cure for SCD. Unfortunately, most people with SCD are either too old to undergo a transplant or do not have a relative who is a sufficient genetic match to serve as a donor for a successful transplant. Therefore, there is a need in the art for improved treatments for SCD, including treatments for vaso-occlusive events in SCD, that can alleviate symptoms, prevent complications, and improve the length and quality of life. Summary of the Invention

[0012] The present disclosure includes methods of treating, ameliorating, and / or preventing vaso-occlusive crisis (VOC) in a subject with sickle cell disease (SCD), wherein the method comprises administering to a subject in need thereof a therapeutically effective amount of a composition comprising ADAMTS13.

[0013] The present disclosure includes methods for treating, ameliorating and / or preventing lung injury in a subject with acute lung injury (ALI) and / or acute respiratory distress syndrome (ARDS), wherein the method comprises administering to a subject in need thereof a therapeutically effective amount of a composition comprising ADAMTS13.

[0014] The present disclosure includes the use of ADAMTS13 and / or a composition comprising ADAMTS13 for the preparation of a medicament. Other related aspects are also provided in the present disclosure.

[0015] The present disclosure provides a method for treating, improving and / or preventing VOC in a subject with SCD, the method comprising administering a therapeutically effective amount of a composition comprising ADAMTS13 to a subject in need thereof. In some embodiments, the subject is treated after the presence of VOC symptoms. In some embodiments, the subject is treated before the presence of VOC crisis symptoms. In some embodiments, the treatment reduces at least one of inflammation, vasoconstriction, or platelet aggregation, or any combination thereof. In some embodiments, the treatment results in improved survival, improved lung function, or reduced organ damage, reduced pulmonary vascular leakage, or any combination thereof. In some embodiments, the treatment reduces and / or prevents at least one of impaired blood flow (e.g., local ischemia), blood coagulation, vascular inflammation, thrombosis, ischemic cell damage, or organ damage, or any combination thereof. In some embodiments, the treatment reduces and / or prevents pain or the severity of pain. In some embodiments, the treatment reduces the frequency of VOC occurrences and / or the duration of VOC attacks. In certain embodiments, administration of ADAMTS13 results in a decrease in the expression, level, and / or activation of at least one of VCAM-1, ICAM-1, P-NF-κB / NF-κB ratio, ET-1, TXAS, and HO-1 in an organ. In some embodiments, the comparison is to a control subject. In some embodiments, the comparison is to a measurement before treatment.

[0016] In certain embodiments, organs include but are not limited to lung, liver, pancreas, skin, retina, prostate, ovary, lymph node, adrenal gland, kidney, heart, gallbladder or gastrointestinal tract. In some embodiments, organ tissues include but are not limited to lung, liver, spleen and / or kidney. In certain embodiments, the organ is lung. In certain embodiments, the organ is kidney.

[0017] In certain embodiments, administration of ADAMTS13 results in an increase in the level of at least one of Hct, Hb, MCV, and MCH in the blood, and / or a decrease in at least one of CHCM, HDW, LDH, and neutrophils in the blood, compared to a control.

[0018] In some aspects of the present disclosure, the therapeutically effective amount of ADAMTS13 for treating, ameliorating, or preventing VOCs in a subject with SCD is from about 20 to about 6,000 international units per kilogram of body weight. In some aspects, the therapeutically effective amount is from about 40 to about 4,000 international units per kilogram of body weight. In some aspects, the therapeutically effective amount is from about 100 to about 3,000 international units per kilogram of body weight. In some aspects, the therapeutically effective amount is from about 50 to about 500 international units per kilogram of body weight.

[0019] In certain aspects, the dosage or therapeutically effective amount of VOCs for treating, improving or preventing a subject with SCD is from about 10 to about 500 international units per kilogram of body weight. In some aspects, the dosage or therapeutically effective amount is from about 50 to about 450 international units per kilogram of body weight. In some aspects, the therapeutically effective amount is from about 40 to about 150 international units per kilogram of body weight. In some aspects, the therapeutically effective amount is from about 100 to about 500 international units per kilogram of body weight. In some aspects, the dosage or therapeutically effective amount is from about 100 to about 400 international units per kilogram of body weight. In some aspects, the therapeutically effective amount is from about 100 to about 300 international units per kilogram of body weight. In some aspects, the therapeutically effective amount is from about 300 to about 500 international units per kilogram of body weight. In some aspects, the dosage or therapeutically effective amount is from about 200 to about 300 international units per kilogram of body weight. In some aspects, the dosage or therapeutically effective amount is about 100, about 150, about 200, about 250, about 300, about 350, about 400, about 450, or about 500 International Units per kilogram of body weight.

[0020] In other aspects, the dosage or therapeutically effective amount for treating, ameliorating or preventing VOCs in a subject with SCD is from about 50 to about 1,000 international units per kilogram of body weight. In some aspects, the dosage or therapeutically effective amount is from about 100 to about 900 international units per kilogram of body weight. In some aspects, the dosage or therapeutically effective amount is from about 200 to about 800 international units per kilogram of body weight. In some aspects, the dosage or therapeutically effective amount is from about 300 to about 700 international units per kilogram of body weight. In some aspects, the dosage or therapeutically effective amount is from about 400 to about 600 international units per kilogram of body weight. In some aspects, the dosage or therapeutically effective amount is about 500 international units per kilogram of body weight.

[0021] In some embodiments, the composition comprising ADAMTS13 for treating, improving or preventing VOCs in subjects with SCD is administered monthly, biweekly, weekly, twice a week, daily, every 12 hours, every 8 hours, every 6 hours, every 4 hours or every 2 hours as a single bolus. In some embodiments, the composition comprising ADAMTS13 is administered intravenously or subcutaneously. In some embodiments, the composition comprising ADAMTS13 is administered intravenously. In some embodiments, the composition comprising ADAMTS13 is administered subcutaneously.

[0022] In some aspects of the present disclosure, a therapeutically effective amount of a composition comprising ADAMTS13 is administered to a subject within 48 hours after the onset of a VOC. In some aspects, a therapeutically effective amount of a composition comprising ADAMTS13 is administered to a subject within 24 hours after the onset of a VOC. In some aspects, a therapeutically effective amount of a composition comprising ADAMTS13 is administered to a subject within 12 hours after the onset of a VOC. In some aspects, a therapeutically effective amount of a composition comprising ADAMTS13 is administered to a subject within 6 hours after the onset of a VOC.

[0023] In some aspects of the present disclosure, a therapeutically effective amount of a composition comprising ADAMTS13 for preventing VOCs is sufficient to maintain an effective level of ADAMTS13 activity in a subject. In some aspects, a therapeutically effective amount of a composition comprising ADAMTS13 for preventing VOCs is administered monthly, biweekly, weekly, or twice weekly to prevent VOCs. In some embodiments, administration is subcutaneous. In some aspects, administration is intravenous.

[0024] The present disclosure includes the use of compositions comprising ADAMTS 13 to treat or prevent VOCs in a subject with SCD. In some embodiments, the present disclosure includes compositions comprising ADAMTS 13 for use as a medicament for treating or preventing VOCs in a subject with SCD.

[0025] In certain embodiments, the method of treating or preventing VOC comprises (i) administering ADAMTS13 and (ii) assessing whether a parameter or symptom has changed, wherein the parameter is selected from inflammation, vasoconstriction, platelet aggregation, lung function, organ (such as lung or kidney) damage, pulmonary vascular leakage, blood flow, blood coagulation, vascular inflammation, thrombosis, ischemic cell damage, presence of pain, severity of pain, frequency of VOCs, duration of VOC episodes, VCAM-1, ICAM-1, P-NF-κB / NF-κB ratio, ET-1, TXAS, HO-1, Hct, Hb, MCV, HDW, reticulocyte count, and neutrophil count.

[0026] The present disclosure also provides a method for treating, improving and / or preventing lung injury with ALI and / or ARDS or with a subject suffering from ALI and / or ARDS risk, the method comprising administering a therapeutically effective amount of a composition comprising ADAMTS13 to a subject in need thereof. In some aspects, the subject suffers from a condition or combination of conditions selected from inflammatory pulmonary edema, inflammatory pulmonary infiltration, impaired oxygenation and hypoxemia. In some aspects, treatment results in improved survival, improved lung function or reduced organ damage, reduced pulmonary vascular leakage, or any combination thereof. In some aspects, treatment reduces at least one of inflammation, vasoconstriction or platelet aggregation, or any combination thereof. In some aspects, treatment reduces and / or prevents at least one of impaired blood flow (e.g., ischemia), blood coagulation, vascular inflammation, thrombosis, ischemic cell damage or organ damage, or any combination thereof. In some aspects, treatment reduces and / or prevents pain or the severity of pain. In some embodiments, treatment reduces the frequency of occurrence of ALI and / or ARDS, and / or the duration of ALI and / or ARDS episodes. In certain embodiments, administration of ADAMTS13 results in a decrease in the expression, level, and / or activation of at least one of VCAM-1, ICAM-1, P-NF-κB / NF-κB ratio, ET-1, TXAS, and HO-1 in an organ. In some embodiments, the comparison is to a control subject. In some embodiments, the comparison is to a measurement before treatment.

[0027] In certain embodiments, organs include but are not limited to lung, liver, pancreas, skin, retina, prostate, ovary, lymph node, adrenal gland, kidney, heart, gallbladder or gastrointestinal tract. In some embodiments, organ tissues include but are not limited to lung, liver, spleen and / or kidney. In certain embodiments, the organ is lung. In certain embodiments, the organ is kidney.

[0028] In certain embodiments, administration of ADAMTS 13 results in a decrease in the number of neutrophils in the blood compared to a control.

[0029] In some aspects of the present disclosure, the therapeutically effective amount of ADAMTS 13 for treating, improving or preventing lung injury in a subject with ALI and / or ARDS or a risk of ALI and / or ARDS is from about 20 to about 6,000 international units per kilogram of body weight. In some aspects, the therapeutically effective amount is from about 40 to about 4,000 international units per kilogram of body weight. In some aspects, the therapeutically effective amount is from about 100 to about 3,000 international units per kilogram of body weight. In some aspects, the therapeutically effective amount is from about 50 to about 500 international units per kilogram of body weight.

[0030] In certain aspects, the dosage or therapeutically effective amount for treating, improving or preventing lung injury in a subject with ALI and / or ARDS or a risk of ALI and / or ARDS is about 10 to about 500 international units per kilogram of body weight. In some aspects, the dosage or therapeutically effective amount is about 50 to about 450 international units per kilogram of body weight. In some aspects, the therapeutically effective amount is about 40 to about 150 international units per kilogram of body weight. In some aspects, the therapeutically effective amount is about 100 to about 500 international units per kilogram of body weight. In some aspects, the dosage or therapeutically effective amount is about 100 to about 400 international units per kilogram of body weight. In some aspects, the therapeutically effective amount is about 100 to about 300 international units per kilogram of body weight. In some aspects, the therapeutically effective amount is about 300 to about 500 international units per kilogram of body weight. In some aspects, the dosage or therapeutically effective amount is about 200 to about 300 international units per kilogram of body weight. In some aspects, the dosage or therapeutically effective amount is about 100, about 150, about 200, about 250, about 300, about 350, about 400, about 450, or about 500 International Units per kilogram of body weight.

[0031] In other aspects, the dosage or therapeutically effective amount for treating, improving or preventing lung injury in a subject with ALI and / or ARDS or a risk of ALI and / or ARDS is about 50 to about 1,000 international units per kilogram of body weight. In some aspects, the dosage or therapeutically effective amount is about 100 to about 900 international units per kilogram of body weight. In some aspects, the dosage or therapeutically effective amount is about 200 to about 800 international units per kilogram of body weight. In some aspects, the dosage or therapeutically effective amount is about 300 to about 700 international units per kilogram of body weight. In some aspects, the dosage or therapeutically effective amount is about 400 to about 600 international units per kilogram of body weight. In some aspects, the dosage or therapeutically effective amount is about 500 international units per kilogram of body weight.

[0032] In some embodiments, within 48 hours after detecting inflammatory pulmonary edema, inflammatory pulmonary infiltrates, impaired oxygenation or hypoxia, a therapeutically effective amount of a composition comprising ADAMTS13 for treating, improving and / or preventing lung injury in a subject with ALI and / or ARDS or with a risk of ALI and / or ARDS is administered to the subject. In some embodiments, within 24 hours after detecting inflammatory pulmonary edema, inflammatory pulmonary infiltrates, impaired oxygenation or hypoxia, a therapeutically effective amount of a composition comprising ADAMTS13 is administered to the subject. In some embodiments, within 12 hours after detecting inflammatory pulmonary edema, inflammatory pulmonary infiltrates, impaired oxygenation or hypoxia, a therapeutically effective amount of a composition comprising ADAMTS13 is administered to the subject. In some embodiments, within 6 hours after detecting inflammatory pulmonary edema, inflammatory pulmonary infiltrates, impaired oxygenation or hypoxia, a therapeutically effective amount of a composition comprising ADAMTS13 is administered to the subject.

[0033] In some embodiments, the composition comprising ADAMTS13 is administered as a single bolus injection monthly, biweekly, weekly, twice weekly, daily, every 12 hours, every 8 hours, every 6 hours, every 4 hours, or every 2 hours. In some embodiments, the composition comprising ADAMTS13 is administered intravenously or subcutaneously. In some embodiments, the composition comprising ADAMTS13 is administered intravenously. In some embodiments, the composition comprising ADAMTS13 is administered subcutaneously.

[0034] In various aspects of the disclosure, ADAMTS 13 is recombinant ADAMTS 13. In some aspects, ADAMTS 13 is plasma-derived.

[0035] In various aspects of the present disclosure, the subject is a mammal. In certain aspects, the subject is a human.

[0036] In some aspects, the composition is in a stable aqueous solution ready for administration.

[0037] In some aspects, the therapeutically effective amount of a composition comprising ADAMTS 13 for treating, ameliorating, and / or preventing lung injury is sufficient to maintain effective circulating levels of ADAMTS 13 activity in a subject.

[0038] The present disclosure includes using a composition comprising ADAMTS13 to treat, improve and / or prevent lung injury in a subject suffering from ALI and / or ARDS or having a risk of suffering from ALI and / or ARDS. In some aspects, the subject suffers from ALI. In some aspects, the subject suffers from ARDS.

[0039] The present disclosure also includes a composition containing ADAMTS13 for use as a medicament for treating, ameliorating, or preventing lung injury in a subject suffering from or at risk of suffering from ALI and / or ARDS.

[0040] In certain embodiments, the method of treating or preventing ALI / ARDS comprises (i) administering ADAMTS13 and (ii) assessing whether a parameter or symptom has changed, wherein the parameter is selected from inflammation, vasoconstriction, platelet aggregation, lung function, organ (such as lung or kidney) damage, pulmonary vascular leakage, blood flow, blood coagulation, vascular inflammation, thrombosis, ischemic cell damage, frequency of ALI / ARDS, duration of ALI / ARDS episodes, VCAM-1, ICAM-1, P-NF-κB / NF-κB ratio, ET-1, TXAS, HO-1, Hct, Hb, MCV, HDW, reticulocyte count, and neutrophil count.

[0041] The foregoing summary of the invention is not intended to limit every aspect of the present invention, and other aspects are described in other parts, such as the following detailed description. The entire text is intended to be interrelated as a unified disclosure, and it should be understood that all combinations of features described herein can be expected, even if the combination of features is not found in the same sentence, paragraph or part of the text. From the following detailed description, other features and advantages of the present invention will become apparent. However, it should be understood that the detailed description and specific examples, although indicating specific embodiments of the present invention, are only given by way of illustration, because from this detailed description, various changes and modifications within the spirit and scope of the present invention will be apparent to those skilled in the art. BRIEF DESCRIPTION OF THE DRAWINGS

[0042] Figure 1 Figure 2 is a graph showing that ADAMTS13 protects sickle cell mice (SCD) from death associated with severe acute VOCs. Mice (n=6) were treated with rADAMTS13 (BAX930 / SHP655 (2,940 FRETS-U / kg (~3,200 IU / kg))) and exposed to 7% oxygen for 10 hours, followed by a 3-hour recovery period at 21% oxygen. The survival curves of rADAMTS13-treated SCD mice, vehicle-treated AA (healthy) mice, and ADAMTS13-treated AA mice were significantly different from the survival curve of vehicle-treated SCD mice (p<0.001). After 13 hours, no animals survived in the vehicle-treated SCD mouse group, while 100% of the animals survived in all three other groups.

[0043] Figures 2A-2C : Figure 2AThe results showed that SCD (SS) mice had significantly more leukocytes and significantly higher protein levels in bronchoalveolar lavage fluid compared to controls, indicating vascular leakage. Treatment with rADAMTS 13 (BAX930 / SHP655) significantly reduced this effect, indicating reduced systemic inflammation and abnormal pulmonary vascular dysfunction. Figure 2B We show that rADAMTS13 (BAX930 / SHP655) prevents hypoxia-induced NF-κB activation in the lungs of AA and SCD mice, indicating that ADAMTS 13 reduces the lung inflammatory process triggered by hypoxia. Figure 2C showed that rADAMTS 13 (BAX930 / SHP655) prevented the activation of various markers of vascular activation and inflammatory vasculopathy in the lungs of SCD mice exposed to hypoxia.

[0044] Figure 3A -B: Figure 3A We showed that rADAMTS 13 (BAX930 / SHP655) prevented hypoxia-induced NF-κB activation in the kidneys of AA and SCD mice, and prevented hypoxia-induced NF-κB activation in the kidneys of SCD mice under normoxic conditions, indicating that ADAMTS13 reduced the inflammatory process triggered by hypoxia in the kidneys and lungs. Figure 3B We show that rADAMTS13 (BAX930 / SHP655) prevents the activation of various markers of vascular activation and inflammatory vasculopathy in the kidneys of SCD mice exposed to hypoxia. DETAILED DESCRIPTION

[0045] In various aspects, the present disclosure provides ADAMTS 13 for preventing, ameliorating and / or treating VOCs in SCD. Before explaining any embodiment of the present disclosure in detail, it should be understood that the application of the present invention is not limited to the structural details and component arrangements set forth in the following description or shown in the accompanying drawings and examples. The section headings used herein are for organizational purposes only and should not be interpreted as limiting the subject matter described. All references cited in this application are expressly incorporated herein by reference for all purposes.

[0046] The present disclosure encompasses other embodiments and may be practiced or implemented in various ways. Furthermore, it should be understood that the phraseology and terminology used herein are for descriptive purposes and should not be considered restrictive. The terms "include," "comprising," or "having" and variations thereof are intended to include the items listed thereafter and their equivalents as well as additional items.

[0047] The following abbreviations are used throughout.

[0048] AA mice hemoglobin A (HbA) homozygous transgenic mice

[0049] ADAMTS Disintegrin-like metalloproteinase with thrombobinding protein motif

[0050] ADAMTS13 A disintegrin-like metalloproteinase containing type I thrombobinding protein motif 13

[0051] ALI acute lung injury

[0052] ARDS acute respiratory distress syndrome

[0053] BAL bronchoalveolar lavage

[0054] DNA deoxyribonucleic acid

[0055] ET-1 Endothelin 1

[0056] FRETS U FRETS Unit

[0057] GAPDH Glyceraldehyde 3-phosphate dehydrogenase

[0058] HbA hemoglobin A.

[0059] HbS sickle hemoglobin

[0060] HO-1 heme oxygenase 1

[0061] H / R Hypoxia / Reoxygenation

[0062] ICAM-1 intercellular adhesion molecule 1

[0063] IU International Unit

[0064] kDa kilodaltons

[0065] LDH lactate dehydrogenase

[0066] NF-κB Nuclear factor-κB

[0067] P-NF-κB Phospho-nuclear factor-κB

[0068] rADAMTS13 recombinant ADAMTS 13

[0069] RBC Red blood cells

[0070] RNA ribonucleic acid

[0071] SCD sickle cell disease

[0072] SS mice HbS homozygous transgenic mice

[0073] TXAS thromboxane synthase

[0074] VCAM-1 Vascular cell adhesion molecule-1

[0075] VOC vaso-occlusive crisis

[0076] VWF von Willebrand factor

[0077] It should be noted here that, as used in this specification and the appended claims, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise. With respect to aspects of the disclosure described as "genus," all individual species are considered independent aspects of the disclosure. If aspects of the disclosure are described as "comprising" a feature, embodiments are also contemplated as "consisting of" or consisting essentially of that feature.

[0078] As used herein, the following terms have the meanings ascribed to them unless otherwise specified.

[0079] As used herein, the term "sickle cell disease (SCD)" describes a group of inherited red blood cell disorders that exist in various forms. Some forms of SCD are hemoglobin SS, hemoglobin SC, hemoglobin Sβ, 0 Thalassemia, hemoglobin Sβ + Thalassemia, Hemoglobin SD, and Hemoglobin SE. Although hemoglobin SC disease and hemoglobin Sβ thalassemia are two common forms of SCD, the present disclosure relates to and encompasses all forms of SCD.

[0080] As used herein, the term "vaso-occlusive crisis (VOC)" is an episode of sudden, severe pain that can occur without warning. VOC, also known as pain crisis or sickle cell crisis, is a common painful complication of SCD in adolescents and adults. VOC is initiated and sustained by interactions between sickle cells, endothelial cells, and plasma components. Vaso-occlusive disease contributes to various clinical complications of SCD, including pain syndromes, stroke, leg ulcers, spontaneous abortion, and / or renal insufficiency.

[0081] The terms "acute lung injury" (ALI) and "acute respiratory distress syndrome" (ARDS) describe clinical syndromes of acute respiratory failure with significant morbidity and mortality (Johnson et al., J. Aerosol Med. Pulmon. Drug Deliv. 23: 243-52, 2010). Both ALI and the more severe ARDS represent a spectrum of lung diseases characterized by exacerbations of inflammatory pulmonary edema (including bilateral, inflammatory lung infiltrates and impaired oxygenation or hypoxemia) secondary to numerous local or systemic injuries (Walkey et al., Clinical Epidemiology 4: 159-69, 2012). Although ALI and ARDS are two clinical syndromes of lung injury or disease, the present disclosure relates to and includes the use of ADAMTS13 not only in treating, preventing or ameliorating ALI and ARDS, but also in treating, preventing or ameliorating all forms of lung injury and lung disease, particularly lung disease with impaired oxygenation.

[0082] "A disintegrin-like metalloproteinase 13 (ADAMTS13) containing a type I thrombobinding protein motif" is also referred to as von Willebrand factor cleaving protease (VWFCP). As used herein, the terms "ADAMTS13" or "ADAMTS13 protein" include ADAMTS13 analogs, variants, derivatives (including chemically modified derivatives) and fragments thereof. In some aspects, analogs, variants, derivatives and fragments thereof have increased biological activity compared to ADAMTS13. In various aspects, ADAMTS13 is recombinant ADAMTS13 (rADAMTS13) or blood-derived ADAMTS13, including plasma- and serum-derived ADAMTS13.

[0083] As used herein, an "analog" refers to a polypeptide that is substantially similar in structure and has the same biological activity as a naturally occurring molecule (although in some cases to a different extent). Compared to the naturally occurring polypeptide from which the analog is derived, the composition of the amino acid sequence of the analog differs based on one or more mutations involving: (i) deletion of one or more amino acid residues at one or more termini of the polypeptide (including fragments as described above), and / or at one or more internal regions of the naturally occurring polypeptide sequence, (ii) insertion or addition of one or more amino acids at one or more termini of the polypeptide (usually "addition" analogs), and / or at one or more internal regions of the naturally occurring polypeptide sequence (usually "insertion" analogs), or (iii) substitution of one or more amino acids in the naturally occurring polypeptide sequence with other amino acids. Substitutions are conservative or non-conservative based on the physicochemical or functional relevance of the substituted amino acid and the amino acid that replaces it.

[0084] "Conservatively modified analogs" apply to both amino acid and nucleic acid sequences. With respect to a particular nucleic acid sequence, conservatively modified nucleic acids refer to those that encode identical or substantially identical amino acid sequences, or, if the nucleic acid does not encode an amino acid sequence, to substantially identical sequences. Due to the degeneracy of the genetic code, a large number of functionally identical nucleic acids encode any given protein. For example, the codons GCA, GCC, GCG, and GCU all encode the amino acid alanine. Thus, at each position where alanine is specified by a codon, the codon can be changed to any of the corresponding codons described without changing the encoded polypeptide. Such nucleic acid variations are "silent variations," which are a type of conservatively modified analog. Each nucleic acid sequence encoding a polypeptide herein also describes every possible silent variation of the nucleic acid. Those skilled in the art will recognize that every codon in a nucleic acid (except AUG, which is typically the only codon for methionine, and TGG, which is typically the only codon for tryptophan) can be modified to produce functionally identical molecules. Thus, every silent variation of a nucleic acid encoding a polypeptide is implicit in each described sequence.

[0085] With respect to amino acid sequences, the skilled artisan will recognize that a single substitution, insertion, deletion, addition, or truncation of a nucleic acid, peptide, polypeptide, or protein sequence, which alters, adds, or deletes a single amino acid or a small number of amino acids in the encoded sequence, is a "conservatively modified analog," wherein the alteration results in the replacement of an amino acid with a chemically similar amino acid. Conservative substitution tables providing functionally similar amino acids are well known in the art. Such conservatively modified variants are in addition to and do not exclude polymorphic variants, interspecies homologs, and alleles of the present disclosure.

[0086] Each of the following eight groups contains amino acids that are conservatively substituted for each other:

[0087] 1) Alanine (A), Glycine (G);

[0088] 2) Aspartic acid (D), glutamic acid (E);

[0089] 3) Asparagine (N), glutamine (Q);

[0090] 4) Arginine (R), Lysine (K);

[0091] 5) Isoleucine (I), Leucine (L), Methionine (M), Valine (V);

[0092] 6) Phenylalanine (F), tyrosine (Y), tryptophan (W);

[0093] 7) Serine (S), Threonine (T); and

[0094] 8) Cysteine ​​(C), Methionine (M) (see, for example, Creighton, Proteins (1984)).

[0095] As used herein, "variant" refers to a polypeptide, protein, or its analog that comprises at least one amino acid substitution, deletion, insertion, or modification, provided that the variant retains the biological activity of the native polypeptide. In some aspects, the term "variant" can be used interchangeably with the term "mutant."

[0096] As used herein, "allelic variant" refers to any of two or more polymorphic forms of a gene occupying the same genetic locus. Allelic variation occurs naturally through mutation and, in some aspects, results in phenotypic polymorphism within a population. In some aspects, a gene mutation is silent (the encoded polypeptide does not change), or in other aspects, the encoded polypeptide has an altered amino acid sequence. "Allelic variant" also refers to cDNAs derived from mRNA transcripts of genetic allelic variants, and proteins encoded by them.

[0097] The term "derivative" refers to a polypeptide that has been covalently modified by conjugation with a therapeutic or diagnostic agent, labeling (e.g., with a radionuclide or various enzymes), attachment of a covalent polymer (e.g., pegylation (derivatization with polyethylene glycol)), and insertion or substitution of a non-natural amino acid by chemical synthesis. In some aspects, the derivative is modified to include other chemical moieties that are not normally part of the molecule. In some aspects, these derivatives are referred to as chemically modified derivatives. In various aspects, these moieties regulate the solubility, absorption, and / or biological half-life of the molecule. In various other aspects, these moieties optionally reduce the toxicity of the molecule and eliminate or reduce any undesirable side effects of the molecule, etc. Remington's Pharmaceutical Sciences (1980) discloses moieties that can mediate such effects. Methods for coupling these moieties to molecules are well known in the art. For example, in some aspects, an ADAMTS13 derivative is an ADAMTS13 molecule with a chemical modification that imparts a longer in vivo half-life to the protein. In one embodiment, the polypeptide is modified by adding a water-soluble polymer known in the art. In related embodiments, the polypeptide is modified by glycosylation, PEGylation, and / or polysialylation.

[0098] As used herein, a "fragment" of a polypeptide refers to any portion of a polypeptide that is smaller than the full-length polypeptide or protein expression product. Fragments are typically deletion analogs of a full-length polypeptide in which one or more amino acid residues are removed from the amino and / or carboxyl termini of the full-length polypeptide. Thus, a "fragment" is a subset of the deletion analogs described below.

[0099] When used with reference to, for example, a cell, the term "recombinant" or "recombinant expression system" means that the cell has been modified by the introduction of heterologous nucleic acids or proteins or by the modification of native nucleic acids or proteins, or that the cell is derived from a cell so modified. Thus, for example, a recombinant cell expresses genes not found in the native (non-recombinant) form of the cell, or expresses native genes that are abnormally expressed, underexpressed, or not expressed at all. The term also refers to a host cell that has stably integrated recombinant genetic elements, such as promoters or enhancers, that have a regulatory role in gene expression. A recombinant expression system, as defined herein, will express polypeptides or proteins endogenous to the cell when induced by regulatory elements linked to the endogenous DNA segment or gene to be expressed. The cell can be a prokaryotic cell or a eukaryotic cell.

[0100] When used herein to refer to a polypeptide or protein, the term "recombinant" means that the polypeptide or protein is derived from a recombinant (e.g., microbial or mammalian) expression system. "Microbial" refers to a recombinant polypeptide or protein produced in a bacterial or fungal (e.g., yeast) expression system. The term "recombinant variant" refers to any polypeptide that differs from a naturally occurring polypeptide by amino acid insertion, deletion, and substitution produced using recombinant DNA technology. Guidance in determining which amino acid residues can be replaced, added, or deleted without eliminating the activity of interest can be found by comparing the sequence of a particular polypeptide with the sequence of a homologous peptide and minimizing the number of changes in the amino acid sequence in regions of high homology.

[0101] The term "agent" or "compound" describes any molecule, such as a protein or a drug, that has the ability to affect a biological parameter in the present disclosure.

[0102] As used herein, "control" can refer to an active, positive, negative, or vehicle control. As will be appreciated by those skilled in the art, controls are used to establish the relevance of experimental results and to provide a comparison of test conditions. In some aspects, a control is a subject that does not receive an active prophylactic or therapeutic composition. In some aspects, a control is a subject that has not experienced SCD, VOC, ALI, and / or ARDS, such as, but not limited to, a healthy control or a subject without any symptoms.

[0103] When referring to the symptoms of SCD, VOCs in SCD and / or ALI / ARDS, the term "reducing severity" means that the symptom has a delayed onset, reduced severity, reduced frequency, or causes less damage to the subject. Typically, the severity of the symptom is compared to a control (e.g., a subject who has not received an active preventive or therapeutic composition) or compared to the severity of the symptom before the therapeutic agent is administered. In this case, if the symptom is reduced by about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90% or about 100% (i.e., substantially eliminated) compared to the control level of the symptom, it can be said that the composition reduces the severity of SCD symptoms, VOCs in SCD and / or ALI / ARDS. In some aspects, compared to the control level of symptoms, if the symptoms reduce by about 10% to about 100%, about 20% to about 90%, about 30% to about 80%, about 40% to about 70% or about 50% to about 60%, then it can be said that the composition reduces the severity of SCD symptoms, VOCs in SCD and / or ALI / ARDS. In some aspects, compared to the control level of symptoms, if the symptoms reduce by about 10% to about 30%, about 20% to about 40%, about 30% to about 50%, about 40% to about 60%, about 50% to about 70%, about 60% to about 80%, about 70% to about 90% or about 80% to about 100%, then it can be said that the composition reduces the severity of SCD symptoms, VOCs in SCD and / or ALI / ARDS. In some aspects, the treatment by the method of the present disclosure reduces the severity of pain and / or other symptoms in VOCs and / or ALI / ARDS in SCD.

[0104] When referring to biomarkers of SCD, VOCs in SCD, and / or ALI / ARDS (e.g., but not limited to, VCAM-1, ICAM-1, P-NF-κB / NF-κB ratio, ET-1, TXAS, HO-1, Hct, Hb, MCV, HDW, reticulocyte count, and neutrophil count), the terms "reduce expression," "reduce levels," and "reduce activation" mean that the expression, level, and / or activation of the biomarker has been reduced compared to a control. In this case, a composition can be said to reduce the expression, level, and / or activation of a biomarker of SCD, VOCs in SCD, and / or ALI / ARDS if the biomarker is reduced by about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, or about 100% (i.e., substantially eliminated) compared to a control. In certain aspects, a composition is said to reduce expression, level, and / or activation of SCD, VOCs in SCD, and / or ALI / ARDS if the expression, level, and / or activation is reduced by about 10% to about 100%, about 20% to about 90%, about 30% to about 80%, about 40% to about 70%, or about 50% to about 60%, as compared to a control. In certain aspects, a composition is said to reduce expression, level, and / or activation of a biomarker for SCD, VOCs in SCD, and / or ALI / ARDS if the biomarker is reduced by about 10% to about 30%, about 20% to about 40%, about 30% to about 50%, about 40% to about 60%, about 50% to about 70%, about 60% to about 80%, about 70% to about 90%, or about 80% to about 100%, as compared to a control.

[0105] When referring to a biomarker for SCD, VOCs in SCD, and / or ALI / ARDS, the terms "increase expression," "increase levels," and "increase activation" mean that the expression, level, and / or activation of the biomarker has been increased compared to a control. In this case, a composition can be said to increase the expression, level, and / or activation of a biomarker for SCD, VOCs in SCD, and / or ALI / ARDS if the biomarker is increased by about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, or about 100% (i.e., substantially eliminated) compared to a control. In certain aspects, a composition is said to increase expression, level, and / or activation of SCD, VOCs in SCD, and / or ALI / ARDS if the expression, level, and / or activation is increased by about 10% to about 100%, about 20% to about 90%, about 30% to about 80%, about 40% to about 70%, or about 50% to about 60%, as compared to a control. In certain aspects, a composition is said to increase expression, level, and / or activation of a biomarker for SCD, VOCs in SCD, and / or ALI / ARDS if the biomarker is increased by about 10% to about 30%, about 20% to about 40%, about 30% to about 50%, about 40% to about 60%, about 50% to about 70%, about 60% to about 80%, about 70% to about 90%, or about 80% to about 100%, as compared to a control.

[0106] The terms "effective amount" and "therapeutically effective amount" each refer to an amount of a polypeptide, such as an ADAMTS13 polypeptide, or a composition that supports observable levels of one or more biological activities of an ADAMTS13 polypeptide, as described herein. For example, in some aspects of the present disclosure, an effective amount is the amount necessary to treat or prevent VOCs in SCD and / or symptoms in ALI / ARDS.

[0107] "Subject" is given its conventional meaning of non-plant, non-protist. In most aspects, the subject is an animal. In particular aspects, the animal is a mammal. In more specific aspects, the mammal is a human. In other aspects, the mammal is a pet or companion animal, a domesticated farm animal, or a zoo animal. In certain aspects, the mammal is a mouse, rat, rabbit, guinea pig, pig, or non-human primate. In other aspects, the mammal is a cat, dog, horse, or cow. In various other aspects, the mammal is a deer, mouse, chipmunk, squirrel, opossum, or raccoon.

[0108] It is also specifically understood that any numerical value cited herein includes all values ​​from the lower limit to the upper limit, that is, all possible combinations of values ​​between the lowest value and the highest value enumerated should be considered to be explicitly stated in this application. For example, if a concentration range is expressed as about 1% to 50%, it means that values ​​such as 2% to 40%, 10% to 30%, or 1% to 3% are explicitly enumerated in this specification. The values ​​listed above are only examples.

[0109] In various aspects, ranges are expressed herein as "about" or "approximately" one particular value and / or "about" or "approximately" another particular value. When values ​​are expressed as approximate values ​​by using the antecedent "about," it is understood that some amount of variation is included within the range. Such ranges can be within an order of magnitude, preferably within 50%, more preferably within 20%, still more preferably within 10%, and even more preferably within 5% of a given value or range. The permissible variation encompassed by the term "about" or "approximately" depends on the particular system under consideration and is readily understood by one of ordinary skill in the art.

[0110] Sickle cell disease and vascular obstruction in sickle cell disease

[0111] In some aspects, the present disclosure includes ADAMTS13 and compositions comprising ADAMTS13 for use in treating, ameliorating, and / or preventing VOCs in SCD. SCD is a worldwide inherited red blood cell disorder caused by a point mutation in the β-globin gene, which leads to the synthesis of pathological HbS and abnormal HbS polymerization under hypoxic conditions. The two main clinical manifestations of SCD are chronic hemolytic anemia and acute VOCs, which are the main reasons for hospitalization of SCD patients. Recent studies have emphasized the important role of sickle vascular lesions in the development of sickle cell-related acute events and chronic organ complications (Sparkenbaugh et al., Br. J. Haematol 162: 3-14, 2013; De Franceschi et al., Semin. Thromb. Hemost. 226-36, 2011; and Hebbel et al., Cardiovasc. Hematol. Disord. Drug Targets, 9: 271-92, 2009). The pathophysiology of these complications is based on intravascular sickling in capillaries and small vessels, leading to VOC, impaired blood flow, vascular inflammation, and / or thrombosis with ischemic cellular damage.

[0112] The most common clinical manifestation of SCD is vena cava (VOC). VOC occurs when microcirculation is blocked by sickled red blood cells, leading to ischemic damage to organs and resulting pain. Pain crises are the most prominent clinical feature of SCD and the leading cause of emergency department visits and / or hospitalizations in affected SCD subjects or patients.

[0113] Approximately half of SCD subjects or patients with homozygous HbS disease experience VOCs. The frequency of crises is extremely variable. Some SCD subjects or patients have up to six or more crises per year, while others may have crises only at long intervals or not at all. Each subject or patient generally has a consistent pattern of crisis frequency.

[0114] The present disclosure includes methods for reducing at least one symptom of VOC, including, but not limited to, ischemia and pain associated with VOC (e.g., dactylitis, priapism, abdomen, chest, and joints), jaundice, bone infarction, respiratory abnormalities (e.g., tachypnea and shortness of breath), hypoxia, acidosis, hypotension, and / or tachycardia. In certain aspects, VOC can be defined as a condition that includes one or more of these symptoms. The pain crisis begins suddenly. The crisis may last from several hours to several days and end as suddenly as it began. The pain can affect any part of the body, commonly involving the abdomen, appendages, chest, back, bones, joints, and soft tissues, and may manifest as dactylitis (bilateral pain and swelling of the hands and / or feet in children), acute joint necrosis or avascular necrosis, or acute abdomen. With repeated attacks in the spleen, infarction and autologous splenectomy, which predispose to life-threatening infections, often occur. The liver may also become infarcted over time and progress to failure. Papillary necrosis is a common renal manifestation of VOC, resulting in isotonic urine (i.e., inability to concentrate urine).

[0115] Severe, deep pain occurs in the extremities, including the long bones. Abdominal pain may be severe and resemble an acute abdomen; it may result from pain arising from other sites or from infarction of solid organs or soft tissues within the abdominal cavity. Reactive ileus causes intestinal distension and pain. The face may also be involved. The pain may be accompanied by fever, malaise, dyspnea, painful erections, jaundice, and leukocytosis. Bone pain is usually caused by bone marrow infarction. Certain patterns are predictable because pain tends to involve bones with the most bone marrow activity and because bone marrow activity varies with age. In the first 18 months of life, the metatarsal and metacarpal bones may be involved, presenting as dactylitis or hand-foot syndrome. Although the above patterns describe common presentations, any area of ​​the subject's body with a blood supply and sensory nerves may be affected by VOCs.

[0116] Often, the cause of VOCs cannot be determined. However, the most likely physiological trigger for VOCs is hypoxia, as deoxygenated HbS becomes semisolid. This may be due to acute chest syndrome or concomitant respiratory complications. Dehydration can also cause pain, as acidosis causes a shift in the oxygen dissociation curve (Bohr effect), making hemoglobin more susceptible to desaturation. Hemoconcentration is also a common mechanism. Another common trigger for VOCs is a change in body temperature, either an increase due to fever or a decrease due to a change in ambient temperature. A decrease in body temperature can lead to a crisis due to peripheral vasoconstriction.

[0117] In certain embodiments, VOC can be defined as having an increase in peripheral neutrophils compared to a control. In certain embodiments, VOC can be defined as an increase in pulmonary vascular leakage (e.g., an increase in the number of white blood cells and / or protein content in bronchoalveolar lavage (BAL) (BAL protein (mg / mL)) compared to a control).

[0118] In certain embodiments, an increase in the level of vascular activation in an organ (e.g., as measured by an increase in the expression, level, and / or activation of VCAM-1 and / or ICAM-1) compared to a control is a marker for VOCs. In certain embodiments, an increase in the level of inflammatory vasculopathy in an organ (e.g., as measured by an increase in the expression, level, and / or activation of VCAM-1 and / or ICAM-1) compared to a control is a marker for VOCs. In certain embodiments, increased levels of vascular activation and inflammatory vasculopathy in a tissue compared to a control are markers for VOCs. In certain embodiments, the organ is the lung and / or kidney. In certain embodiments, the organ is the kidney.

[0119] In certain embodiments, VOCs can be defined as an increase in the expression, level, and / or activation of at least one of NF-κB (wherein NF-κB activation is measured by P-NF-κB or the ratio of P-NF-κB / NF-κB), VCAM-1, and ICAM-1, compared to a control. In certain embodiments, VOCs can be defined as an increase in the expression or level of at least one of endothelin-1 (ET-1), thromboxane synthase (TXAS), and heme oxygenase-1 (HO-1), compared to a control. In certain embodiments, these increases are observed in lung tissue. In certain embodiments, these increases are observed in kidney tissue. In certain embodiments, increased expression and / or levels of TXAS, ET-1, and VCAM-1, as well as increased activation of NF-κB in kidney tissue, are markers of VOCs.

[0120] In certain embodiments, VOC can be defined by hematological parameters. In certain embodiments, VOC can be defined as a decrease in the level of at least one of Hct, Hb, MCV, and MCH compared to a control. In certain embodiments, VOC can be defined as a decrease in the level of at least two of Hct, Hb, MCV, and MCH compared to a control. In certain embodiments, VOC can be defined as a decrease in the level of at least three of Hct, Hb, MCV, and MCH compared to a control. In certain embodiments, VOC can be defined as an increase in the level of at least one of CHCM, HDW, neutrophil count, and LDH compared to a control. In certain embodiments, VOC can be defined as an increase in the level of at least two of CHCM, HDW, neutrophil count, and LDH compared to a control. In certain embodiments, VOC can be defined as an increase in the level of at least three of CHCM, HDW, neutrophil count, and LDH compared to a control. In certain embodiments, VOC can be defined as a decrease in the level of Hct compared to a control. In certain embodiments, VOC can be defined as a decrease in the level of Hb compared to a control. In certain embodiments, VOC can be defined as a decrease in the level of MCV compared to a control. In certain embodiments, VOC can be defined as a decrease in MCH compared to a control. In certain embodiments, VOC can be defined as an increase in CHCM compared to a control. In certain embodiments, VOC can be defined as an increase in HDW compared to a control. In certain embodiments, VOC can be defined as an increase in neutrophil count compared to a control. In certain embodiments, VOC can be defined as an increase in LDH compared to a control. In certain embodiments, VOC can be defined as a decrease in the level of at least one of Hct, Hb, MCV, and MCH compared to a control, and / or an increase in the level of at least one of CHCM, HDW, neutrophil count, and LDH compared to a control. In certain embodiments, VOC can be defined as a decrease in the level of Hct, Hb, MCV, and MCH compared to a control, and / or an increase in the level of CHCM, HDW, neutrophil count, and LDH compared to a control.

[0121] SCD models and methods for testing the effectiveness of prevention or treatment

[0122] In some embodiments, the present disclosure includes studying the effects of recombinant ADAMTS13 (i.e., BAX930 / SHP655) in a SCD mouse model (Tim Townes mice) during acute SCD-related events (mimicked by exposing SCD mice to hypoxia). The studies were conducted under normoxic and hypoxic conditions, wherein the efficacy of prophylactic or therapeutic doses in the mouse model (including measuring overall survival) and the biological effects of BAX930 / SHP655 treatment on lung injury and vascular inflammation were studied after exposing sickle cell disease mice to hypoxia.

[0123] In some embodiments, a transgenic mouse model of SCD is used (Kalish et al., Haematologica 100:870-80, 2015). In some aspects, healthy controls (Hba tm1(HBA)Tow Hbb tm3(HBG1,HBB)Tow ) and SCD(Hba tm1(HBA) Tow Hbb tm2(HBG1,HBB*)Tow ) mice were exposed to hypoxia / reoxygenation (H / R) stress (Kalish et al., below). This H / R stress has been shown to biologically recapitulate the acute VOC and organ damage observed in acute VOC in human SCD patients. In some aspects, healthy (AA) and SCD (SS) mice were subjected to hypoxia (e.g., about 7 or 8% oxygen) for a period of time (e.g., about 10 hours) and then reoxygenated (e.g., about 21% oxygen, room air conditions) for a period of time (e.g., 3 hours) (Kalish et al., below).

[0124] In various aspects, the models of SCD and control are subjected to normoxic or hypoxic conditions. In normoxic experiments, healthy controls (AA) and SCD (SS) mice receive a single intravenous administration of rADAMTS13 (e.g., 2,940FRETS-U / kg (~3,200IU / kg)) or buffer (vehicle) in a fixed volume (e.g., 10mL / kg) and are subjected to normoxic conditions (e.g., about 21% oxygen, room air conditions). After being treated with ADAMTS13 or vehicle and exposed to normoxia or hypoxia, the animals are studied for different time periods. Blood is collected and a complete blood count (CBC) is measured. CBC is a blood test used to assess overall health and detect various diseases, including anemia. Various other endpoints are measured, including but not limited to hematology, coagulation parameters, biomarkers of inflammation, vascular lesions, and tissue pathology.

[0125] In exemplary aspects, hypoxia experiments are carried out, wherein healthy controls (AA) and SCD (SS) mice receive a single intravenous administration of ADAMTS13 (e.g., 500 IU / kg, 1,000 IU / kg or 3,200 IU / kg) or a vehicle with a fixed volume (e.g., 10 mL / kg). In certain embodiments, the dosage applied to human subjects is about 10% of the dosage applied to rodent (e.g., mouse) subjects. In certain embodiments, the dosage applied to human subjects is about 9% of the dosage applied to rodent (e.g., mouse) subjects. In certain embodiments, the dosage applied to human subjects is about 8% of the dosage applied to rodent (e.g., mouse) subjects. In certain embodiments, the dosage applied to human subjects is about 7% of the dosage applied to rodent (e.g., mouse) subjects. In certain embodiments, the dosage applied to human subjects is less than about 10% of the dosage applied to rodent (e.g., mouse) subjects, for example, about 7% to about 10%.

[0126] After injection (e.g., about 1-3 hours after injection), mice are exposed to hypoxia (e.g., about 7% or 8% oxygen) for a period of time (e.g., about 10 hours) followed by reoxygenation for a period of time (e.g., about 3 hours) to simulate SCD-related VOC events. In some aspects, the same parameters as detailed in the normoxic studies are assessed.

[0127] In other exemplary aspects, hypoxia experiments are performed in which healthy control (AA) and SCD (SS) mice are exposed to hypoxia (e.g., about 8% oxygen or higher) for a period of time (e.g., about 10 hours) followed by reoxygenation for a period of time (e.g., about 3 hours) to simulate SCD-related VOC events. Then, at various time points thereafter, including but not limited to, immediately or about 1, 3, 6, 12, 24, 36, 48, or 72 hours after the onset of experimentally induced vascular occlusion, mice receive a single intravenous administration of ADAMTS13 (e.g., 500 IU / kg, 1,000 IU / kg, or 3,200 IU / kg) or vehicle in a fixed volume (e.g., 10 mL / kg), or multiple injections at 12 or 24 intervals. In some aspects, the same parameters as detailed in the normoxic study are evaluated.

[0128] In various aspects, any target tissue is examined for effectiveness of treatment with ADAMTS13 in an in vitro or in vivo model and / or under VOC conditions. In some aspects, organs include, but are not limited to, lung, liver, pancreas, skin, retina, prostate, ovary, lymph node, adrenal gland, kidney, heart, gallbladder, or gastrointestinal tract. In some embodiments, organ tissues include, but are not limited to, lung, liver, spleen, and / or kidney.

[0129] For example, in some aspects, target tissues are collected to examine the effects of ADAMTS13 under normoxic or hypoxic conditions. The tissues are frozen and / or fixed in formalin. Immunoblot analysis is performed on frozen tissues using specific antibodies against nuclear factor-κB (NF-κB), endothelin-1 (ET-1), heme oxygenase 1 (HO-1), intercellular adhesion molecule-1 (ICAM-1), thromboxane synthase (TXAS), and vascular cell adhesion molecule-1 (VCAM-1). Fixed organs are used for standard pathology (H&E staining).

[0130] In some embodiments, markers of vasoconstriction, platelet aggregation, inflammation, oxidative stress, antioxidant response and / or tissue damage are measured to determine the effectiveness of the treatment. In some aspects, the normal (NF-κB) and activated (P-NF-κB) forms of nuclear factor κB are measured. NF-κB is a transcription factor that has been described to coordinate inflammatory and antioxidant responses. The ratio between the activated and normal forms is assessed. In some aspects, ET-1 is measured. ET-1 is a potent vasoconstrictor produced by vascular endothelial cells. ET-1 plays a role in several pathophysiological processes, including cardiovascular hypertrophy, pulmonary hypertension and chronic renal failure. In some aspects, HO-1 is measured. HO-1 is an inducible rate-limiting enzyme in heme catabolism that can reduce the severity of vascular obstruction and the consequences of hemolytic crisis, acting as a vascular protective antioxidant. In some aspects, ICAM-1 is measured. ICAM-1 is continuously present in low concentrations in the membranes of leukocytes and endothelial cells. Although ICAM-1 does not appear to be involved in sickle cell adhesion to the vascular endothelium, ICAM-1 may exacerbate VOCs by promoting leukocyte adhesion. In some aspects, TXAS is measured. TXAS is an endoplasmic reticulum membrane protein that catalyzes the conversion of prostaglandin H2 to thromboxane A2. TXAS is a potent vasoconstrictor and inducer of platelet aggregation. Therefore, TXAS is a potent inducer of vasoconstriction and platelet aggregation. TXAS plays a role in several pathophysiological processes, including hemostasis, cardiovascular disease, and stroke. In some aspects, VCAM-1 is measured. VCAM-1 mediates the adhesion of lymphocytes and other blood cells to the vascular endothelium and may therefore contribute to vaso-occlusive events. In some aspects, inflammatory cell infiltration is measured in organ tissues.

[0131] In exemplary aspects, immunoblot analysis is performed using specific antibodies against NF-κB, ET-1, HO-1, ICAM-1, TXAS, and VCAM-1 to measure the expression of these enzymes in the cell and tissue models or subjects of the present disclosure to determine the effectiveness of the treatment. In exemplary aspects, the expression of NF-κB, ET-1, HO-1, ICAM-1, TXAS, and / or VCAM-1 is measured in organ tissues of AA and SCD mice treated with vehicle or ADAMTS13. In certain embodiments, the organs include, but are not limited to, the lung, liver, pancreas, skin, retina, prostate, ovary, lymph node, adrenal gland, kidney, heart, gallbladder, or gastrointestinal tract. In certain embodiments, the organ is the lung, liver, spleen, and / or kidney.

[0132] In certain embodiments, administration of ADAMTS13 results in a decrease in the level of vascular activation and / or inflammatory vasculopathy in an organ compared to a control. In certain embodiments, the organ is a lung. In certain embodiments, the organ is a kidney.

[0133] In certain embodiments, administration of ADAMTS13 results in a decrease in the expression, level, and / or activation of at least one of VCAM-1, ICAM-1, NF-κB (wherein NF-κB activation is reduced as measured by P-NF-κB or the ratio of P-NF-κB / NF-κB), ET-1, TXAS, and HO-1, compared to a control. In certain embodiments, administration of ADAMTS13 results in a decrease in the expression, level, and / or activation of at least two of VCAM-1, ICAM-1, NF-κB, ET-1, TXAS, and HO-1, compared to a control. In certain embodiments, administration of ADAMTS13 results in a decrease in the expression, level, and / or activation of at least three of VCAM-1, ICAM-1, NF-κB, ET-1, TXAS, and HO-1, compared to a control. In certain embodiments, administration of ADAMTS13 results in a decrease in the expression, level, and / or activation of at least four of VCAM-1, ICAM-1, NF-κB, ET-1, TXAS, and HO-1, compared to a control. In certain embodiments, administration of ADAMTS13 results in a decrease in the expression, level, and / or activation of at least five of VCAM-1, ICAM-1, NF-κB, ET-1, TXAS, and HO-1, compared to a control. In certain embodiments, administration of ADAMTS13 results in a decrease in the expression, level, and / or activation of VCAM-1, ICAM-1, NF-κB, ET-1, TXAS, and HO-1, compared to a control. In certain embodiments, administration of ADAMTS13 results in a decrease in the expression, level, and / or activation of VCAM-1, compared to a control. In certain embodiments, administration of ADAMTS13 results in a decrease in the expression, level, and / or activation of ICAM-1, compared to a control. In certain embodiments, administration of ADAMTS13 results in a decrease in the expression, level, and / or activation of VCAM-1, compared to a control. In certain embodiments, administration of ADAMTS13 results in a decrease in the expression, level, and / or activation of VCAM-1 and ICAM-1, compared to a control. In certain embodiments, administration of ADAMTS13 results in a decrease in the expression and / or level of ET-1, compared to a control. In certain embodiments, administration of ADAMTS 13 results in a decrease in the expression and / or level of TXAS compared to a control. In certain embodiments, administration of ADAMTS 13 results in a decrease in the expression and / or level of HO-1 compared to a control. In certain embodiments, administration of ADAMTS 13 results in a decrease in the ratio of β-NF-κB / NF-κB compared to a control. In certain embodiments, administration of ADAMTS 13 results in a decrease in at least one of the ratio of β-NF-κB / NF-κB, expression and / or level of ET-1, expression and / or level of TXAS, and expression and / or level of HO-1 compared to a control.In certain embodiments, administration of ADAMTS13 results in a decrease in the P-NF-κB / NF-κB ratio, ET-1 expression and / or levels, TXAS expression and / or levels, and HO-1 expression and / or levels compared to a control. In certain embodiments, the organ is the lung. In certain embodiments, the organ is the kidney.

[0134] In further exemplary aspects, the measurement of these markers is performed after the animal model is subjected to hypoxia and reoxygenation (H / R) conditions as described herein. In further exemplary aspects, the measurement of these markers is performed after the subject experiences a VOC.

[0135] In some embodiments, blood flow is measured as an indicator of treatment effectiveness. In some embodiments, blood flow is measured by, but not limited to, ultrasound, PET, fMRI, NMR, laser Doppler, electromagnetic blood flowmetry, or a wearable device.

[0136] In some embodiments, reducing or preventing thrombosis is a measure of therapeutic effectiveness. In some embodiments, the presence of thrombosis is measured by, but not limited to, histopathological examination, ultrasound, D-dimer testing, venography, MRI, or CT / CAT scanning. In some aspects, thrombosis is measured in organ tissues.

[0137] In some embodiments, reducing or preventing pulmonary vascular leakage (i.e., lung leakage and damage) is a measure of therapeutic effectiveness. In some embodiments, bronchoalveolar lavage (BAL) measurements or parameters (total protein and leukocyte content) are measured as markers of pulmonary vascular leakage (to determine the extent of lung damage and the effectiveness of treatment (e.g., treatment with ADAMTS13)). Pulmonary leakage can lead to an increase in protein and / or leukocyte content in BAL. BAL fluid is collected and the cell contents are recovered by centrifugation and counted by micrometry as previously reported (Kalish et al., Haematologica 100:870-80, 2015, incorporated herein by reference in its entirety and for all purposes). In some embodiments, reducing or preventing an increase in peripheral neutrophils is a measure of therapeutic effectiveness. The percentage of neutrophils is determined by cytospin centrifugation, and the supernatant is used to determine the total protein content (Kalish et al., supra).

[0138] In some embodiments, improvement in lung function is measured as an indicator of treatment effectiveness. Lung function can be measured by, but is not limited to, peak flow testing, spirometry and reversibility testing, lung capacity testing, gas transfer testing, respiratory muscle testing, exhaled carbon mononucleoside testing, or exhaled nitric oxide testing.

[0139] In some embodiments, hematological parameters are measured to determine the effectiveness of treatment (e.g., treatment with ADAMTS13). The following hematological parameters are determined: lactate dehydrogenase (LDH) as a general marker of cell damage; hematocrit (Hct) and mean corpuscular volume (MCV) as a measure of red blood cell viability; hemoglobin (Hb), mean corpuscular hemoglobin (MCH) and cell hemoglobin concentration (CHCM) as indicators of oxygen binding capacity; heterogeneity of red blood cell distribution (HDW) as an indicator of the presence of dense red blood cells; reticulocyte count as an indicator of anemic state; and neutrophil count as an indicator of systemic inflammatory state.

[0140] In certain embodiments, compared with the control, the administration of ADAMTS13 improves the reduction of at least one of the levels of Hct, Hb, MCV and MCH in the blood. In certain embodiments, compared with the control, the administration of ADAMTS13 improves the reduction of at least two of the levels of Hct, Hb, MCV and MCH in the blood. In certain embodiments, compared with the control, the administration of ADAMTS13 improves the reduction of at least three of the levels of Hct, Hb, MCV and MCH in the blood. In certain embodiments, compared with the control, the administration of ADAMTS13 improves the reduction of Hct, Hb, MCV and MCH levels in the blood. In certain embodiments, compared with the control, the administration of ADAMTS13 improves the increase of at least one of CHCM, HDW, LDH and neutrophil counts. In certain embodiments, compared with the control, the administration of ADAMTS13 improves the increase of at least two of CHCM, HDW, LDH and neutrophil counts. In certain embodiments, administration of ADAMTS13 improves increases in at least three of CHCM, HDW, LDH, and neutrophil counts compared to a control. In certain embodiments, administration of ADAMTS13 improves increases in CHCM, HDW, LDH, and neutrophil counts compared to a control. In certain embodiments, ADAMTS13 improves decreases in Hct, Hb, MCV, and MCH levels and improves increases in CHCM, HDW, LDH, and neutrophil levels compared to a control.

[0141] In certain embodiments, compared with the control, the administration of ADAMTS13 results in an increase in the level of at least one of Hct, Hb, MCV and MCH in the blood. In certain embodiments, compared with the control, the administration of ADAMTS13 results in an increase in the level of at least two of Hct, Hb, MCV and MCH in the blood. In certain embodiments, compared with the control, the administration of ADAMTS13 results in an increase in the level of at least three of Hct, Hb, MCV and MCH in the blood. In certain embodiments, compared with the control, the administration of ADAMTS13 results in an increase in the level of Hct, Hb, MCV and MCH in the blood. In certain embodiments, compared with the control, the administration of ADAMTS13 results in a decrease in at least one of CHCM, HDW, LDH and neutrophil counts. In certain embodiments, compared with the control, the administration of ADAMTS13 results in a decrease in at least two of CHCM, HDW, LDH and neutrophil counts. In certain embodiments, compared with the control, the administration of ADAMTS13 results in a decrease in at least three of CHCM, HDW, LDH and neutrophil counts. In certain embodiments, administration of ADAMTS13 results in a decrease in CHCM, HDW, LDH, and neutrophil counts compared to a control. In certain embodiments, ADAMTS13 results in an increase in Hct, Hb, MCV, and MCH levels and a decrease in CHCM, HDW, LDH, and neutrophil levels compared to a control.

[0142] In some embodiments, a method for measuring the levels of VWF and very large VWF multimers is used. In SCD patients, increased levels of VWF and very large VWF multimers have been observed and are associated with acute vascular occlusive events. The increase in circulating VWF multimer levels depends on the activity of ADAMTS13, which cuts super-sticky, very large VWF under conditions of high fluid shear stress, and plays an important role in maintaining an appropriate balance between hemostatic activity and thrombotic risk. More specifically, ADAMTS 13 is expressed at the amino acid residue Tyr 1605 and the Met 1606 VWF is cleaved between the pro-sequence and the pro-sequence, corresponding to amino acid residues 842-843. It is this ADAMTS13-mediated cleavage that is primarily responsible for regulating VWF multimer size, which is associated with primary hemostatic activity. Methods for measuring VWF and very large VWF multimers include various types of immunoblot analysis using specific antibodies against VWF to measure VWF expression or levels. In addition, other known methods for measuring VWF are encompassed by various aspects of the present disclosure.

[0143] In some aspects, effectiveness is measured by a reduction in organ damage compared to a control or baseline measurement. In some embodiments, organ damage is measured by radiographic imaging, such as, but not limited to, CT / CAT scans, ultrasound, X-rays, MRI, and nuclear medicine. In some embodiments, organ damage is measured by changes in various biomarkers, including but not limited to blood urea nitrogen (BUN), creatinine, the ratio of BUN / creatinine, troponin, neuron-specific enolase (NSE). In some embodiments, tissue changes are measured by histopathological examination.

[0144] One of ordinary skill in the art will be able to select appropriate measurements of any biomarker disclosed herein that are associated with the organ to be measured (as defined above) and / or body fluid. Body fluids include, but are not limited to, blood (including plasma and serum), lymph, cerebrospinal fluid, breast milk products (e.g., milk), amniotic fluid, urine, saliva, sweat, tears, menstruation, feces, and fractions thereof.

[0145] In some aspects, effectiveness is measured by assessing the subject's quality of life (e.g., using the Adult Sickle Cell Quality of Life Measurement Information System (ASCQ-Me) reported by Treadwell et al., Clin. J. Pain 30(10):902-915 (2016). The ASCQ-Me is structured around seven themes: emotional impact (a five-question survey related to emotional distress (e.g., hopelessness, loneliness, depression, and worry); pain attack frequency and severity (number of attacks, time since last attack; severity of pain during last attack on a 1-10 scale); how long attacks lasted, and how much impact did attacks have on your life); pain impact (asking about frequency and severity and how it affects activities); sickle cell disease history inventory; sleep impact (ease of falling asleep, how often you can't sleep); social functioning impact (dependence on others, impact on healthy activities); and stiffness impact (stiff joints causing insomnia, daytime movement, movement while awake).

[0146] In various aspects, the effectiveness of prevention and / or treatment is determined by measuring pain severity (e.g., as measured by a pain rating scale), pain relief, perceived medication needs, treatment satisfaction, frequency of VOCs, duration of VOCs, length and / or duration of hospital stays, costs associated with hospital stays, and / or duration of need for analgesics (e.g., narcotics),

[0147] In some aspects, pain severity is measured using the McGill / Melzack Pain Questionnaire (Melzack et al., Pain 1975, Sep;1(3):277-99), in which subjects select one or more words that best describe their pain. In some aspects, pain severity is measured using a visual analog scale (VAS). The VAS is a 10 cm unshaded line with one end fixed as "no pain" and the other end as "worst possible pain." Patients are instructed to mark their pain level between the two ends of the line. The VAS score is calculated by measuring the distance (in centimeters) between the "no pain" end and the patient's mark indicating the level of pain, resulting in a pain severity score ranging from 0 mm to 10 cm. In some aspects, pain severity is measured using a numeric rating scale (NRS). The NRS is an 11-point scale with endpoints of "no pain" and "worst possible pain." Patients are instructed to report their current pain level on a scale of 0 to 10, with 0 being no pain and 10 being the worst possible pain.

[0148] In some aspects, pain relief can be measured as an overall assessment of how the patient's pain has changed since the last assessment (e.g., current assessment minus previous assessment), such as used to anchor changes recorded on NRS and VAS scales. Patients report pain relief by answering the following question: "Compared to the last time you marked your pain, please tell us how much your pain has changed." Patients can answer that their pain is "worse," "somewhat worse," "the same," "a little better," or "much better."

[0149] In some aspects, the need for medication can be patient or healthcare worker reported.

[0150] In some aspects, treatment satisfaction can be patient-reported, with responses ranging from “not at all,” “somewhat satisfied (happy),” “very satisfied (happy),” or “don’t know.”

[0151] Acute lung injury and acute respiratory distress syndrome

[0152] In some embodiments, the present disclosure includes: ADAMTS13, compositions comprising ADAMTS13, and methods of using ADAMTS13 to treat, improve and / or prevent acute lung injury (ALI) and acute respiratory distress syndrome (ARDS), including ventilator-associated lung injury caused thereby. The pathogenesis of ALI / ARDS can be explained by damage to the vascular endothelium and alveolar epithelium. The Phase III clinical trial of the NHLBI ARDS Network improved survival and reduced the duration of mechanical ventilation through lung protective ventilation strategies and fluid conservative regimens. However, since there is no specific pharmacological therapy for ALI / ARDS, there is a strong unmet medical need for other treatments. Therefore, the use of ADAMTS13 in the treatment of ALI / ARDS represents a breakthrough in the treatment of ALI / ARDS.

[0153] In some aspects, ALI is an acute inflammatory condition that causes damage to the pulmonary endothelium and epithelial barrier. The cellular characteristics of ALI include loss of integrity of the alveolar-capillary membrane, excessive transepithelial neutrophil migration, and the release of proinflammatory cytotoxic mediators. Some studies have confirmed that the release of VWF increases after endothelial damage and the upregulation of intracellular adhesion molecule-1 (ICAM-1) (Johnson, supra). Transepithelial neutrophil migration is an important feature of ALI because neutrophils are the main perpetrators of inflammation. The prolonged activation of neutrophils helps to destroy the basement membrane and increase the permeability of the alveolar-capillary barrier. (Johnson, supra).

[0154] In certain aspects, ARDS includes acute onset tachypnea, hypoxemia, diffuse pulmonary infiltrates, and loss of lung compliance, characterized by a high short-term mortality rate in adults (Walkey, supra). The treatment strategy for ARDS focuses on treating the underlying cause and providing supportive care to reduce the progression of lung damage. Most ARDS patients develop severe respiratory failure and require mechanical ventilation support. Mechanical ventilation can cause further lung damage due to the combined mechanical forces of overexpansion and circulatory recruitment, known as ventilator-associated lung injury (VALI). VALI produces "biological trauma" due to the systemic release of inflammatory cytokines. Currently, the main goal of ARDS management is to reduce VALI. (Walkey, supra).

[0155] ADAMTS13 significantly reduced markers of lung injury and vascular dysfunction in normal mice. More specifically, the present disclosure shows that administering recombinant ADAMTS13 to normal (control) mice under hypoxic conditions resulted in reduced lung expression of various protein markers of lung injury and vascular dysfunction, suggesting that ADAMTS13 can be used to treat or improve lung injury caused by acute lung injury, which is characterized by the sudden onset of pulmonary edema (including inflammatory pulmonary edema) secondary to numerous local or systemic injuries (including bilateral, inflammatory lung infiltrates and impaired oxygenation or hypoxemia).

[0156] In certain embodiments, ALI and / or ARDS may be defined by one or more of, but not limited to, ischemia associated with ALI / ARDS, abnormal breathing (e.g., tachypnea and shortness of breath), non-cardiogenic pulmonary edema, pulmonary infiltrates, decreased oxygenation, and decreased ventilation. The present disclosure includes methods for alleviating ALI / ARDS symptoms, including but not limited to at least one of ischemia associated with ALI / ARDS, abnormal breathing (e.g., tachypnea and shortness of breath), non-cardiogenic pulmonary edema, pulmonary infiltrates, decreased oxygenation, and decreased ventilation, and combinations thereof.

[0157] In certain embodiments, ALI and / or ARDS can be defined as having an increase in peripheral neutrophils compared to a control. In certain embodiments, ALI and / or ARDS can be defined as an increase in pulmonary vascular leakage compared to a control (e.g., an increase in the number of white blood cells and / or protein content in bronchoalveolar lavage (BAL) (BAL protein (mg / mL)).

[0158] In certain embodiments, an increase in the level of vascular activation in an organ compared to a control is a marker for ALI and / or ARDS. In certain embodiments, an increase in the level of inflammatory vasculopathy in an organ compared to a control is a marker for ALI and / or ARDS. In certain embodiments, an increase in the level of vascular activation and inflammatory vasculopathy in a tissue compared to a control is a marker for ALI and / or ARDS. In certain embodiments, the organ is the lung and / or kidney.

[0159] In certain embodiments, ALI and / or ARDS can be defined as an increase in the expression, level, and / or activation of at least one of NF-κB (wherein NF-κB activation is measured by P-NF-κB or the ratio of P-NF-κB / NF-κB), VCAM-1, and ICAM-1, as compared to a control. In certain embodiments, ALI and / or ARDS can be defined as an increase in the expression or level of at least one of endothelin-1 (ET-1), thromboxane synthase (TXAS), and heme oxygenase-1 (HO-1), as compared to a control. In certain embodiments, these increases are observed in lung tissue. In certain embodiments, these increases are observed in renal tissue. In certain embodiments, increased expression and / or levels of TXAS and ET-1 and increased activation of NF-κB in renal tissue are markers of ALI and / or ARDS.

[0160] In certain embodiments, ALI and / or ARDS can be defined by hematological parameters. In certain embodiments, ALI and / or ARDS can be defined as an increase in neutrophil count compared to a control. In certain embodiments, ALI and / or ARDS can be defined as an increase in neutrophil count compared to a control.

[0161] In certain embodiments, ALI and / or ARDS can also be defined by an increase in at least one of the following serum biomarkers: surfactant-associated protein (SP)-A, SP-B, SP-D, KL-6 / MUC1, IL-1, IL-2, IL-3, IL-6, IL-8, IL-10, IL-15, TNFα, adhesion molecules (e.g., E, L-selectin), MMP-9, LTB4, and ferritin. See, e.g., Tzouvelekis et al., Respiratory Research 2005, 6:62, which is incorporated herein in its entirety.

[0162] Models of ALI / ARDS and methods for testing the effectiveness of prevention or treatment

[0163] Animal models of ALI are described in Matute-Bello et al., Am. J. Physiol. Lung Cell Mol. Physiol. 295(3): L379-99, 2008, which is incorporated by reference in its entirety for all purposes. In certain instances, ALI in humans is histopathologically characterized by neutrophilic alveolitis, alveolar epithelial and endothelial damage, hyaline membrane formation, and microvascular thrombosis. In some aspects, animal models of experimental lung injury can be used to study the mechanisms of ALI. For example, risk factors for ARDS, such as sepsis, lipid embolism secondary to fractures, acid aspiration, ischemia-reperfusion of the lungs or distal vascular beds, and other clinical risks, can be reproduced. In certain aspects, animal models of ALI reproduce the mechanisms and consequences of ALI, including the physiological and pathological changes that occur. In humans, the inflammatory response in the lung begins before the onset of clinically confirmed ALI and is most intense approximately 3 days after the onset of ALI and / or ARDS. The acute inflammatory phase is followed by a chronic fibroproliferative phase. For example, pulmonary function tests may show restriction consistent with parenchymal fibrosis seen in lung biopsy or autopsy specimens.

[0164] Animal models for ARDS are described in Bastarche et al., Dis. Model. Mech. 2(5-8):218-23, 2009, which is incorporated by reference in its entirety for all purposes. For example, in humans, pneumonia and sepsis are the two most common predisposing conditions for the development of ARDS. In some aspects, these conditions can be modeled in mice using the Gram-negative bacterial endotoxin LPS, which can be administered directly to the lungs by intratracheal injection or inhalation, or administered intraperitoneally or intravenously to induce a systemic inflammatory response. Mice treated with intratracheal LPS have an acute and potent influx of inflammatory cells into the lungs that resolves within 48 hours. Intraperitoneal LPS activates systemic inflammation and is associated with mild lung damage. This damage can be increased by repeated injections of LPS or by implanting an LPS pump in the peritoneal cavity to continuously release LPS for hours or even days. Another commonly used model of lung injury is hyperoxia, in which mice breathe high oxygen partial pressures, which are highly toxic to the alveolar epithelium and cause extensive alveolar epithelial damage with only moderate inflammation. Another frequently studied model is ventilator-induced lung injury, which correlates well with human ventilator-induced lung injury; however, this model does not cause substantial lung injury in mice without additional stimulation or extreme tidal volumes. A recent study showed that a tidal volume of 15 ml / kg resulted in moderate lung inflammation, vascular leakage, and alveolar coagulation activation compared to a low tidal volume of 7.5 ml / kg. To obtain more severe injury, higher tidal volumes (up to 35 ml / kg) are required. Matute-Bello et al. recently conducted a comprehensive review of ALI animal models, discussing each model in great detail (Am. J. Physiol. Lung Cell Mol. Physiol. 295: L379-L399, 2008), which is also incorporated herein by reference for all purposes.

[0165] In certain aspects, the disclosed methods and compositions can improve symptoms such as, but not limited to, ischemia associated with ALI / ARDS, abnormal breathing (e.g., tachypnea and shortness of breath), non-cardiogenic pulmonary edema, pulmonary infiltrates (e.g., as measured by chest radiography), decreased oxygenation (e.g., as measured by pulse oximetry [SpO2] or arterial blood gases [PaO2]), and decreased ventilation (e.g., as measured by end-tidal CO2 or arterial blood gases [PaCO2], decreased ventilator-free days or increased ventilator-free days).

[0166] In various aspects, the effectiveness of prevention and / or treatment is determined by measuring survival, length and / or frequency of hospitalizations, length and / or duration of ICU admissions, and / or costs associated with hospitalizations.

[0167] In various aspects, the effectiveness of prevention and / or treatment is determined by measuring a reduction in the number and / or severity of complications associated with ALI and / or ARDS. Complications may include, but are not limited to: pulmonary complications (e.g., barotrauma, volutrauma, pulmonary embolism, pulmonary fibrosis, ventilator-associated pneumonia (CAP), and airway complications); gastrointestinal complications (e.g., bleeding (ulcers, lesions), motility disorders, pneumoperitoneum, and bacterial translocation); cardiac complications (e.g., abnormal heart rhythms and myocardial dysfunction); renal (acute renal failure and aggressive fluid balance); mechanical complications (e.g., vascular injury, pneumothorax (due to placement of a pulmonary artery catheter), tracheal injury / stenosis (as a result of intubation and / or endotracheal tube stimulation); nutritional complications (e.g., malnutrition (catabolic state), electrolyte deficiencies); and general complications (e.g., muscle weakness and exercise tolerance).

[0168] In certain embodiments, the measures of effectiveness of treatment, amelioration, and prevention are the same as those disclosed above for VOCs.

[0169] In certain embodiments, organs include but are not limited to lung, liver, pancreas, skin, retina, prostate, ovary, lymph node, adrenal gland, kidney, heart, gall bladder or gastrointestinal tract.In certain embodiments, the organ is lung, liver, spleen and / or kidney.

[0170] In certain embodiments, administration of ADAMTS13 results in a reduction in the level of vascular activation and / or inflammatory vasculopathy in an organ compared to a control. In certain embodiments, the organ is the lung. In certain embodiments, the organ is the kidney.

[0171] In certain embodiments, administration of ADAMTS13 results in a decrease in the expression, level, and / or activation of at least one of ICAM-1, NF-κB (wherein the activation of NF-κB is reduced as measured by P-NF-κB or the ratio of P-NF-κB / NF-κB), ET-1, TXAS, and HO-1, as compared to a control. In certain embodiments, administration of ADAMTS13 results in a decrease in the expression, level, and / or activation of at least two of ICAM-1, NF-κB, ET-1, TXAS, and HO-1, as compared to a control. In certain embodiments, administration of ADAMTS13 results in a decrease in the expression, level, and / or activation of at least three of ICAM-1, NF-κB, ET-1, TXAS, and HO-1, as compared to a control. In certain embodiments, administration of ADAMTS13 results in a decrease in the expression, level, and / or activation of at least four of ICAM-1, NF-κB, ET-1, TXAS, and HO-1, as compared to a control. In certain embodiments, administration of ADAMTS13 results in a decrease in the expression, level, and / or activation of ICAM-1, NF-κB, ET-1, TXAS, and HO-1 compared to a control. In certain embodiments, administration of ADAMTS13 results in a decrease in the expression, level, and / or activation of ICAM-1 compared to a control. In certain embodiments, administration of ADAMTS13 results in a decrease in the expression and / or level of ET-1 compared to a control. In certain embodiments, administration of ADAMTS13 results in a decrease in the expression and / or level of TXAS compared to a control. In certain embodiments, administration of ADAMTS13 results in a decrease in the expression and / or level of HO-1 compared to a control. In certain embodiments, administration of ADAMTS13 results in a decrease in the ratio of p-NF-κB / NF-κB compared to a control. In certain embodiments, administration of ADAMTS13 results in a decrease in at least one of the ratio of p-NF-κB / NF-κB, ET-1 expression and / or level, TXAS expression and / or level, and HO-1 expression and / or level compared to a control. In certain embodiments, administration of ADAMTS13 results in a decrease in the P-NF-κB / NF-κB ratio, ET-1 expression and / or levels, TXAS expression and / or levels, and HO-1 expression and / or levels compared to a control.

[0172] In certain embodiments, administration of ADAMTS 13 results in an improvement in the increase in neutrophil numbers in the blood compared to a control.

[0173] In certain embodiments, administration of ADAMTS13 results in a decrease in at least one of the following serum biomarkers compared to a control: surfactant-associated protein (SP)-A, SP-B, SP-D, KL-6 / MUC1, IL-1, IL-2, IL-3, IL-6, IL-8, IL-10, IL-15, TNFα, adhesion molecules (e.g., E, L-selectin), MMP-9, LTB4, and ferritin.

[0174] ADAMTS13

[0175] In some aspects, the present disclosure includes: ADAMTS13 (also known as "A13") and compositions comprising ADAMTS13 for treating and preventing SCD. In specific aspects, the present disclosure includes: ADAMTS13 and compositions comprising ADAMTS13 for treating and preventing VOCs in SCD. ADAMTS13 protease is a glycosylated protein of approximately 180 kDa to 200 kDa produced primarily by the liver. ADAMTS13 is a plasma metalloprotease that cleaves VWF multimers and downregulates its activity in platelet aggregation. To date, ADAMTS13 has been associated with coagulation disorders such as hereditary thrombotic thrombocytopenic purpura (TTP), acquired TTP, cerebral infarction, myocardial infarction, ischemia / reperfusion injury, deep vein thrombosis, and disseminated intravascular coagulation (DIC), such as sepsis-associated DIC.

[0176] All forms of ADAMTS13 known in the art are contemplated for use in the methods and uses of the present disclosure. Mature ADAMTS13 has a calculated molecular weight of approximately 145 kDa, while purified plasma-derived ADAMTS13 has an apparent molecular weight of approximately 180 kDa to 200 kDa, likely due to post-translational modifications including 10 potential N-glycosylation sites in the existing consensus sequence, as well as several O-glycosylation sites and one C-mannosylation site in the TSP1 repeat sequence.

[0177] As used herein, "ADAMTS13" refers to a metalloprotease of the ADAMTS (a disintegrin-like metalloprotease containing a type I platelet-binding protein motif) family that has a Tyr 1605 and the Met 1606In the context of the present disclosure, "ADAMTS 13", "A13" or "ADAMTS 13 protein" includes any ADAMTS 13 protein, such as ADAMTS 13 from mammals and biologically active derivatives thereof, such as primates, humans (NP620594), monkeys, rabbits, pigs, cattle (XP610784), rodents, mice (NP001001322), rats (XP342396), hamsters, gerbils, dogs, cats, frogs (NP001083331), chickens (XP415435). As used herein, "ADAMTS 13", "A13" or "ADAMTS 13 protein" refers to recombinant, natural or plasma-derived ADAMTS 13 proteins. Also included are active mutant and variant ADAMTS 13 proteins, as well as functional fragments and fusion proteins of ADAMTS 13 proteins. In some aspects, the ADAMTS 13 protein further comprises a tag that facilitates purification, detection, or both. In some aspects, the ADAMTS 13 protein of the present invention is further modified with an additional therapeutic moiety or a moiety suitable for in vitro or in vivo imaging.

[0178] ADAMTS 13 proteins include any protein or polypeptide with ADAMTS 13 activity (particularly the ability to cut the peptide bond between residues Tyr-842 and Met-843 of VWF). Human ADAMTS13 proteins include, but are not limited to, polypeptides (NM 139025.3) or processed polypeptides thereof comprising the amino acid sequence of GenBank accession number NP 620594, such as polypeptides wherein signal peptide (amino acids 1 to 29) and / or propeptide (amino acids 30 to 74) have been removed. In some aspects, ADAMTS13 proteins refer to polypeptides comprising an amino acid sequence highly similar to NP 620596 (ADAMTS13 isoform 2, preproprotein) or an amino acid sequence highly similar to amino acids 75 to 1371 of P_620594 (ADAMTS13 isoform 2, mature polypeptide). In another embodiment, ADAMTS13 protein includes a polypeptide containing an amino acid sequence highly similar to NP 620595 (ADAMTS13 isoform 3, preproprotein) or an amino acid sequence highly similar to amino acids 75 to 1340 of NP_620595 (ADAMTS13 isoform 1, mature polypeptide). In some aspects, ADAMTS13 protein includes natural variants with VWF cleavage activity and artificial constructs with VWF cleavage activity. In some aspects, ADAMTS13 includes any natural variants, alternative sequences, isoforms or mutant proteins that retain some basal activity. Many natural variants of human ADAMTS13 are known in the art and are encompassed by the formulations disclosed herein, some of which include those selected from R7W, V88M, H96D, R102C, R193W, T196I, H234Q, A250V, R268P, W390C, R398H, Q448E, Q456H, P457L, P475S, C508Y, Mutations of R528G, P618A, R625H, 1673F, R692C, A732V, E740K, A900V, S903L, C908Y, C951G, G982R, C1024G, A1033T, R1095W, R1095W, Rl123C, C1213Y, T1226I, G1239V, and R1336W.

[0179] In addition, ADAMTS13 protein includes natural and recombinant proteins that have been mutated, for example, by one or more conservative mutations of non-essential amino acids. Preferably, the amino acids essential for the enzymatic activity of ADAMTS13 are not mutated. These include, for example, residues known or speculated to be essential for metal binding, such as residues 83, 173, 224, 228, 234, 281 and 284, and residues found in the active site of the enzyme, such as residue 225. Similarly, in the context of the present disclosure, ADAMTS13 protein includes alternative isoforms, for example, isoforms lacking amino acids 275 to 305 and / or 1135 to 1190 of the full-length human protein.

[0180] In some aspects, the ADAMTS13 protein is further modified, for example, by post-translational modification (e.g., glycosylation of one or more amino acids selected from human residues 142, 146, 552, 579, 614, 667, 707, 828, 1235, 1354, or any other natural or engineered modification site), or by ex vivo chemical or enzymatic modification, including but not limited to glycosylation, modification by water-soluble polymers (e.g., PEGylation, sialylation, HESylation, etc.), labeling, etc.

[0181] In some aspects, the ADAMTS 13 protein is human ADAMTS 13 or a biologically active derivative or fragment thereof, as described in US Patent Application Publication No. 2011 / 0229455 and / or US Patent Application Publication No. 2014 / 0271611, each of which is herein incorporated by reference in its entirety for all purposes.

[0182] In certain aspects, the recombinant ADAMTS13 can be BAX930 / SHP655. In certain aspects, the ADAMTS13 protein includes any protein or polypeptide having ADAMTS13 activity (particularly the ability to cleave the peptide bond between residues Tyr-842 and Met-843 of VWF) that has at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence homology to BAX930 / SHP655.

[0183] Proteolytically active recombinant ADAMTS13 can be prepared by expression in mammalian cell culture, as described in Plaimauer et al. (2002, Blood. 15; 100(10): 3626-32) and US 2005 / 0266528, the disclosures of which are incorporated herein by reference in their entirety for all purposes. Methods for expressing recombinant ADAMTS13 in cell culture are disclosed in Plaimauer B, Scheiflinger F. (Semin Hematol. 2004 Jan; 41(1): 24-33 and US 2011 / 0086413, the disclosures of which are incorporated herein by reference in their entirety for all purposes. See also WO 2012 / 006594 for methods of preparing recombinant ADAMTS13 in cell culture, which is incorporated herein by reference in its entirety for all purposes.

[0184] Methods for purifying ADAMTS13 protein from a sample are described in U.S. Patent No. 8,945,895, which is incorporated herein by reference for all purposes. In some aspects, these methods include enriching the ADAMTS13 protein by contacting the sample with hydroxyapatite for chromatography under conditions allowing the ADAMTS13 protein to appear in the eluent or supernatant of hydroxyapatite. The method may further include tandem chromatography, which has a mixed mode cation exchange / hydrophobic interaction resin that binds the ADAMTS13 protein. Additional optional steps include ultrafiltration / diafiltration, anion exchange chromatography, cation exchange chromatography, and viral inactivation. In some aspects, such methods include inactivating viral contaminants in the protein sample, wherein the protein is fixed on a support. In some aspects, also provided herein is a composition of ADAMTS13 prepared according to the method described in U.S. Patent No. 8,945,895.

[0185] ADAMTS13 Composition and Administration

[0186] In aspects of the present disclosure, ADAMTS 13 is administered to a subject in need thereof. In some aspects, to administer the ADAMTS 13 described herein to a subject, ADAMTS 13 is formulated in a composition comprising one or more pharmaceutically acceptable carriers.

[0187] The term "pharmaceutically acceptable" used in conjunction with the compositions described herein refers to the molecules and other ingredients of these compositions that are physiologically acceptable and generally do not produce adverse reactions when administered to mammals (e.g., humans). Preferably, the term "pharmaceutically acceptable" means approved by a regulatory agency of the federal or state government or listed in the U.S. Pharmacopoeia or other generally recognized pharmacopoeia for use in mammals, more particularly humans. "Pharmaceutically acceptable carriers" include any and all clinically useful solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like. In some aspects, the composition forms a solvate with water or a common organic solvent. These solvates are also included.

[0188] In some aspects, the present disclosure provides stable formulations of plasma-derived ADAMTS13 and recombinant ADAMTS13 (rADAMTS13) proteins, as described in U.S. Patent Application Publication 2011 / 0229455 (now U.S. Patent No. 8,623,352) and / or U.S. Patent Application Publication 2014 / 0271611, both of which are incorporated herein by reference for all purposes. In some embodiments, the formulations provided herein retain significant ADAMTS13 activity upon long-term storage. In some embodiments, the formulations disclosed herein reduce or prevent dimerization, oligomerization, and / or aggregation of ADAMTS13 proteins.

[0189] In some aspects, the present disclosure provides a preparation of ADAMTS13, which comprises a therapeutically effective amount or dosage of an ADAMTS13 protein, a pharmaceutically acceptable salt at a subphysiological to physiological concentration, a stable concentration of one or more sugars and / or sugar alcohols, a nonionic surfactant, a buffer providing a neutral pH for the preparation, and optional calcium salts and / or zinc salts. Generally, the stable ADAMTS13 preparations provided herein are suitable for drug administration. In certain aspects, the ADAMTS13 protein is human ADAMTS13 or a biologically active derivative or fragment thereof, as described in U.S. Patent Application Publication No. 2011 / 0229455 and / or U.S. Patent Application Publication No. 2014 / 0271611, both of which are incorporated herein by reference in their entirety for all purposes.

[0190] In some aspects, the ADAMTS 13 formulation is a liquid or lyophilized formulation. In other embodiments, the lyophilized formulation is lyophilized by liquid formulation, as described in U.S. Patent Application Publication No. 2011 / 0229455 and / or U.S. Patent Application Publication No. 2014 / 0271611, each of which is incorporated herein by reference in its entirety and for all purposes. In certain embodiments of the formulation provided herein, the ADAMTS 13 protein is human ADAMTS 13 or recombinant human ADAMTS 13, or a biologically active derivative or fragment thereof, as described in U.S. Patent Application Publication No. 2011 / 0229455 and / or U.S. Patent Application Publication No. 2014 / 0271611, each of which is incorporated herein by reference in its entirety and for all purposes.

[0191] In various aspects, the compositions of the present invention are administered orally, topically, transdermally, parenterally, by inhalation spray, vaginally, rectally or by intracranial injection. The term parenteral as used herein includes subcutaneous injection, intravenous, intramuscular, intracisternal injection or infusion techniques. In some embodiments, administration is subcutaneous. It is also contemplated that administration is by intravenous, intradermal, intramuscular, intramammary, intraperitoneal, intrathecal, retrobulbar, intrapulmonary injection and / or in a specific location surgical implantation. In some embodiments, administration is intravenous. Typically, the compositions are substantially free of pyrogens and other impurities that may be harmful to the recipient.

[0192] The formula of composition or pharmaceutical composition will change according to selected route of administration (such as solution or emulsion).In physiologically acceptable carrier (vehicle) or carrier (carrier), prepare the suitable composition comprising composition to be administered.For solution or emulsion, suitable carrier comprises such as aqueous solution or alcohol / aqueous solution, emulsion or suspension, comprises saline and buffer medium.In some aspects, parenteral carrier comprises sodium chloride solution, Ringer's glucose, dextrose and sodium chloride, lactated Ringer's or fixed oil.In some aspects, intravenous carrier comprises various additives, preservative or fluid, nutrient or electrolyte supplement.

[0193] In various aspects, compositions or pharmaceutical compositions containing ADAMTS 13 as an active ingredient that can be used in the compounds and methods of the present disclosure contain: pharmaceutically acceptable carriers or additives, depending on the route of administration. Examples of these carriers or additives include: water, pharmaceutically acceptable organic solvents, collagen, polyvinyl alcohol, polyvinyl pyrrolidone, carboxyvinyl polymers, sodium carboxymethylcellulose, sodium polyacrylate, sodium alginate, water-soluble dextran, sodium carboxymethyl starch, pectin, methylcellulose, ethylcellulose, xanthan gum, gum arabic, casein, gelatin, agar, diglycerol, glycerol, propylene glycol, polyethylene glycol, petrolatum, paraffin, stearyl alcohol, stearic acid, human serum albumin (HSA), mannitol, sorbitol, lactose, pharmaceutically acceptable surfactants, etc. The additives used are selected from but not limited to the above or a combination thereof according to the dosage form, as appropriate.

[0194] In various aspects, various aqueous carriers, such as water, buffered water, 0.4% saline, 0.3% glycine, or aqueous suspensions contain the active compound in admixture with excipients suitable for the preparation of aqueous suspensions. These excipients are suspending agents, such as sodium carboxymethylcellulose, methylcellulose, hydroxypropylmethylcellulose, sodium alginate, polyvinylpyrrolidone, gum tragacanth, and gum arabic; in some cases, the dispersing or wetting agent is a naturally occurring phospholipid, such as lecithin, or condensation products of alkylene oxides with fatty acids, such as polyoxyethylene stearate, or condensation products of alkylene oxide with long-chain aliphatic alcohols, such as heptadecaethyl-eneoxycetanol, or condensation products of alkylene oxide with partial esters derived from fatty acids and hexitol (e.g., polyoxyethylene sorbitol monooleate), or condensation products of alkylene oxide with partial esters derived from fatty acids and hexitol anhydrides (e.g., polyethylene sorbitan monooleate). In some aspects, aqueous suspensions contain one or more preservatives, for example, ethyl or n-propyl p-hydroxybenzoate.

[0195] In some aspects, ADAMTS13 or ADAMTS13 compositions are lyophilized for storage and reconstituted in a suitable carrier prior to use. Any suitable lyophilization and reconstitution techniques known in the art are used. Those skilled in the art will appreciate that lyophilization and reconstitution result in varying degrees of protein activity loss, and typically use levels are adjusted to compensate.

[0196] Dispersible powders and granules suitable for preparation of an aqueous suspension by the addition of water provide the active compound in admixture with a dispersant or wetting agent, suspending agent and one or more preservatives. Suitable dispersants or wetting agents and suspending agents are exemplified by those already mentioned above.

[0197] In some embodiments, the ADAMTS13 formulations provided herein may further comprise one or more pharmaceutically acceptable excipients, carriers, and / or diluents, as described in U.S. Patent Application Publication No. 2011 / 0229455 and / or U.S. Patent Application Publication No. 2014 / 0271611, each of which is incorporated herein by reference in its entirety and for all purposes.

[0198] In some embodiments, the ADAMTS 13 formulations provided herein will have a tonicity within the ranges described in US Patent Application Publication No. 2011 / 0229455 and / or US Patent Application Publication No. 2014 / 0271611 (each of which is herein incorporated by reference in its entirety and for all purposes).

[0199] In some aspects, the present disclosure provides formulations of ADAMTS13 comprising exemplary formulations described in Section III ("ADAMTS13 compositions and formulations") of U.S. Patent Application Publication 2011 / 0229455. The ADAMTS13 production methods and compositions thereof as described in U.S. Patent Application Publication 2011 / 0229455 and / or U.S. Patent Application Publication 2014 / 0271611 are incorporated herein by reference in their entirety for all purposes. In addition, actual methods for preparing parenteral formulations and compositions are known or apparent to those skilled in the art and are described in more detail in, for example, Remington's Pharmaceutical Science, 15th edition, Mack Publishing Company, Easton, Pennsylvania, (1980).

[0200] In various aspects, the pharmaceutical composition is in the form of sterile water for injection, oily suspension, dispersion or sterile powder for the temporary preparation of sterile injectable solutions or dispersions. In some aspects, the suspension is prepared according to known techniques using those suitable dispersants or wetting agents and suspending agents mentioned above. In some aspects, the sterile injectable preparation is a sterile injectable solution or suspension in a non-toxic parenteral acceptable diluent or solvent, such as a solution in 1,3-butanediol. In some embodiments, the carrier is a solvent or dispersion medium containing, for example, water, ethanol, a polyol (e.g., glycerol, propylene glycol and liquid polyethylene glycol, etc.), a suitable mixture thereof, a vegetable oil, Ringer's solution and an isotonic sodium chloride solution. In addition, sterile fixed oils are generally used as solvents or suspension media. For this purpose, any mild fixed oil is used in various aspects, including synthetic monoglycerides or diglycerides. In addition, fatty acids such as oleic acid can be used to prepare injections.

[0201] In all cases, the form must be sterile and must be fluid to the extent that it is easy to inject. For example, by using a coating such as lecithin, by maintaining the required particle size in the case of dispersion and by using a surfactant, appropriate fluidity is maintained. It must be stable under the conditions of manufacture and storage and must prevent the contaminating effects of microorganisms such as bacteria and fungi. The effects of microorganisms can be prevented by various antibacterial and antifungal agents, such as parabens, chlorobutanol, phenol, sorbic acid, thimerosal, etc. In many cases, it is desirable to include an isotonic agent, such as sugar or sodium chloride. In some aspects, the absorption of the injectable composition can be prolonged by using an agent that delays absorption in the composition, such as aluminum monostearate and gelatin.

[0202] In certain aspects, the compositions for administration are formulated with an uptake or absorption enhancer to increase their efficacy. These enhancers include, for example, salicylates, glycocholate / linoleate, glycinates, aprotinin, bacitracin, SDS, decanoate, and the like. See, for example, Fix (J. Pharm. Sci., 85: 1282-1285, 1996) and Oliya et al. (Ann. Rev. Pharmacol. Toxicol, 32: 521-544, 1993), the entire contents of which are incorporated herein by reference and for all purposes.

[0203] Furthermore, the hydrophilic and hydrophobic properties of the compositions used in the compositions and methods of the present disclosure are well balanced, thereby enhancing their utility in in vitro and, in particular, in vivo applications, whereas other compositions lacking such a balance are much less practical. Specifically, the compositions of the present disclosure have appropriate solubility in aqueous media, which allows for in vivo absorption and bioavailability, while also having solubility in lipids, which allows the compounds to cross cell membranes to the putative site of action.

[0204] In particular aspects, ADAMTS13 is provided in a pharmaceutically acceptable (i.e., sterile and nontoxic) liquid, semisolid or solid diluent as a pharmaceutical carrier, excipient or medium. Any diluent known in the art is used. Exemplary diluents include, but are not limited to, polyoxyethylene sorbitan monolaurate, magnesium stearate, methylhydroxybenzoate and propylhydroxybenzoate, talc, alginate, starch, lactose, sucrose, dextrose, sorbitol, mannitol, gum arabic, calcium phosphate, mineral oil, cocoa butter, and cocoa butter.

[0205] The composition is packaged in a form that facilitates delivery. The composition is enclosed in a capsule, caplet, pouch, cachet, gelatin, paper, or other container. These delivery forms are preferred when compatible with delivery of the composition to a recipient organism, particularly when the composition is delivered in unit dosage form. The dosage unit is packaged, for example, in a vial, tablet, capsule, suppository, or cachet.

[0206] The present disclosure includes methods for treating, improving and / or preventing VOCs in a subject's SCD, comprising administering an effective amount of ADAMTS13 or an ADAMTS13 composition as described herein. The composition is introduced into the subject to be treated by any conventional method described in detail herein. In some aspects, the composition is administered in a single dose or multiple doses over a period of time (as described in more detail below).

[0207] In some embodiments, the composition comprising ADAMTS 13 is administered to a subject within about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 60, 72, 84, 96, 108, or 120 hours after the onset of a VOC. In some embodiments, within about 1-2 hours, about 1-5 hours, about 1-10 hours, about 1-12 hours, about 1-24 hours, about 1-36 hours, about 1-48 hours, about 1-60 hours, about 1-72 hours, about 1-84 hours, about 1-96 hours, about 1-108 hours, or about 1-120 hours after the onset of VOC, a composition comprising ADAMTS13 is administered to a subject. In some embodiments, within about 2-5 hours, about 5-10 hours, about 10-20 hours, about 20-40 hours, about 30-60 hours, about 40-80 hours, about 50-100 hours, or about 60-120 hours after the onset of VOC, a composition comprising ADAMTS13 is administered to a subject. In some embodiments, the composition is administered within 1 week of VOC. In some embodiments, the composition is administered every day after VOC. In some embodiments, the composition is administered weekly after VOC. In some embodiments, the composition is administered every day. In some embodiments, the composition is administered every other day. In some embodiments, the composition is administered every three days. In some embodiments, the composition is administered twice a week. In some embodiments, the composition is administered until the clinical manifestations (e.g., symptoms and / or biomarkers) subside. In some embodiments, the composition is administered until one day after the clinical manifestations subside. In some embodiments, the composition is administered for at least two days after the clinical manifestations subside. In some embodiments, the composition is administered for at least three days after the clinical manifestations subside. In some embodiments, the composition is administered for at least one week after the clinical manifestations subside.

[0208] In some aspects, a composition comprising ADAMTS13 is administered to a subject to prevent the onset of VOCs. In this preventive treatment, ADAMTS13 is administered as a single bolus or in multiple doses to maintain circulating levels of ADAMTS13 that effectively prevent the onset of VOCs. In these aspects, a composition comprising ADAMTS13 is administered monthly, biweekly, weekly, twice a week, every other day, or daily. In certain aspects, injection is administered subcutaneously. In other aspects, injection is administered intravenously.

[0209] In some embodiments, a composition comprising ADAMTS13 is administered to a subject prior to the onset of a VOC to prevent VOC. In these aspects of the disclosure, the composition is administered in a therapeutically effective amount or dose sufficient to maintain an effective level of ADAMTS13 activity in the subject or in the subject's blood.

[0210] The present disclosure includes methods for treating, improving or preventing ALI or ARDS in a subject, comprising administering an effective amount of ADAMTS13 or ADAMTS13 compositions as described herein. The composition is introduced into the subject to be treated by any conventional method described in detail herein. In some aspects, the composition is administered in a single dose or multiple doses over a period of time (as described in more detail below).

[0211] In some embodiments, the composition comprising ADAMTS 13 is administered to a subject within 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 60, 72, 84, 96, 108, or 120 hours after the onset of ALI or ARDS. In some embodiments, within about 1-2 hours, about 1-5 hours, about 1-10 hours, about 1-12 hours, about 1-24 hours, about 1-36 hours, about 1-48 hours, about 1-60 hours, about 1-72 hours, about 1-84 hours, about 1-96 hours, about 1-108 hours or about 1-120 hours after the onset of ALI or ARDS, a composition comprising ADAMTS13 is administered to a subject. In some embodiments, within about 2-5 hours, about 5-10 hours, about 10-20 hours, about 20-40 hours, about 30-60 hours, about 40-80 hours, about 50-100 hours or about 60-120 hours after the onset of ALI or ARDS, a composition comprising ADAMTS13 is administered to a subject. In some embodiments, the composition is administered within 4 hours, 8 hours, 12 hours, 1 day, 2 days, 3 days, 4 days, 5 days, or 6 days after the onset or diagnosis of ALI or ARDS. In some embodiments, the composition is administered within 1 week after the onset or diagnosis of ALI or ARDS. In some embodiments, the composition is administered daily after the onset or diagnosis of ALI or ARDS. In some embodiments, the composition is administered weekly after the onset or diagnosis of ALI or ARDS. In some embodiments, the composition is administered daily. In some embodiments, the composition is administered every other day. In some embodiments, the composition is administered every three days. In some embodiments, the composition is administered twice a week. In some embodiments, the composition is administered until clinical manifestations resolve. In some embodiments, the composition is administered until one day after clinical manifestations resolve. In some embodiments, the composition is administered for at least two days after clinical manifestations resolve. In some embodiments, the composition is administered for at least three days after clinical manifestations resolve. In some embodiments, the composition is administered for at least one week after clinical manifestations resolve.

[0212] In some aspects, a composition comprising ADAMTS13 is administered to a subject to prevent the onset of ALI or ARDS. In this preventive treatment, ADAMTS13 is administered in a single bolus or multiple doses to maintain circulating levels of ADAMTS13 that effectively prevent the onset of ALI or ARDS. In these aspects, a composition comprising ADAMTS13 is administered monthly, biweekly, weekly, twice a week, every other day, or daily. In certain aspects, injection is administered subcutaneously. In other aspects, injection is administered intravenously.

[0213] In some embodiments, a composition comprising ADAMTS13 is administered to a subject prior to the onset of ALI or ARDS to prevent ALI or ARDS. In these aspects of the disclosure, the composition is administered in a therapeutically effective amount or dose sufficient to maintain an effective level of ADAMTS13 activity in the subject or in the subject's blood.

[0214] Administration of ADAMTS13 compositions / treatment methods

[0215] In various aspects, the effective dosage of ADAMTS 13 or ADAMTS 13 composition to be administered varies depending on a variety of factors that alter the effect of the drug, such as the age, condition, weight, sex and diet of the subject, the severity of any infection, the time of administration, the mode of administration, and other clinical factors, including the severity of VOCs of SCD.

[0216] In some aspects, the preparation or composition of the present invention is administered by an initial bolus injection, followed by a boost delivery after a period of time. In some aspects, the preparation of the present invention is administered by an initial bolus injection followed by a continuous infusion to maintain therapeutic circulating levels of ADAMTS 13. In specific aspects, the ADAMTS 13 or ADAMTS 13 composition of the present invention is administered over an extended period of time. In some aspects, ADAMTS 13 or ADAMTS 13 composition is delivered in a rapid treatment regimen to alleviate the acute symptoms of VOC. In some aspects, ADAMTS 13 or ADAMTS 13 composition is delivered in an extended and varied treatment regimen to prevent the occurrence of VOC. As another example, the composition or preparation of the present invention is administered in a disposable dose. One of ordinary skill in the art will readily optimize the effective dose and administration regimen, as determined by good medical practice and the clinical condition of the individual subject. The frequency of administration depends on the pharmacokinetic parameters of the agent, the route of administration, and the condition of the subject.

[0217] Pharmaceutical formulations are determined by those skilled in the art according to route of administration and desired dosage. See, for example, Remington's Pharmaceutical Sciences, 18th edition (1990, Mack Publishing Co., Easton, Pennsylvania, 18042) 1435-1712 pages, the disclosure of which is incorporated herein by reference for all purposes. In some cases, such formulations affect the physical state, stability, in vivo release rate and in vivo clearance rate of the administered composition. According to route of administration (in particular aspects, according to body weight, body surface area or organ size), a suitable dosage is calculated. In some aspects, by using an established assay for determining blood level dosage in combination with appropriate dose-response data, a suitable dosage is determined. In some aspects, individual antibody titers are measured to determine the optimal dose and administration regimen. Final dosage regimen will be determined by the attending physician or doctor, considering the various factors that change the effect of the pharmaceutical composition, for example, the specific activity of the composition, the reactivity of the subject, the age, condition, body weight, sex and diet of the subject, the severity of any infection or malignant condition, administration time and other clinical factors, including the severity of pain or VOC.

[0218] In certain aspects, ADAMTS13 or an ADAMTS13 composition comprises any dose of ADAMTS13 sufficient to elicit a subject's response. In some embodiments, the dose of ADAMTS13 is sufficient to treat VOCs. In some embodiments, the dose of ADAMTS13 is sufficient to prevent VOCs. In some embodiments, the dose of ADAMTS13 is sufficient to treat ALI. In some embodiments, the dose of ADAMTS13 is sufficient to prevent ALI. In some embodiments, the dose of ADAMTS13 is sufficient to treat ARDS. In some embodiments, the dose of ADAMTS13 is sufficient to prevent ARDS. The effective amount of ADAMTS13 or an ADAMTS13 composition used therapeutically will depend on, for example, the therapeutic context and purpose. Those skilled in the art will understand that the appropriate dosage level for treatment or prevention therefore depends in part on the delivered molecule, the indication for the use of ADAMTS13 or an ADAMTS13 composition, the route of administration, and the size (weight, body surface or organ size) and condition (age and general health) of the patient. Therefore, in some cases, clinicians titrate the dose and change the route of administration to obtain the best therapeutic effect.

[0219] Unless otherwise indicated, doses are given in International Units. As discussed below, the use of International Units (IU) is a new standard for measuring ADAMTS13 activity. Until recently, FRETS units (or FRETS test units) were the standard for measuring ADAMTS13 activity. 20 FRETS units (FRETS U) are equivalent to approximately 21.78 IU. In other words, 20 IU of ADAMTS13 is equivalent to approximately 18.22 FRETS U of ADAMTS13.

[0220] In various aspects, typical dosage range is about 10 international units per kilogram of body weight to about 10,000 international units per kilogram of body weight. In some aspects, depending on the above factors, the dosage or therapeutically effective amount of ADAMTS13 is up to more than about 10,000 international units per kilogram of body weight. In other aspects, the dosage can be about 20 to about 6,000 international units per kilogram of body weight. In some aspects, the dosage or therapeutically effective amount of ADAMTS13 is about 40 to about 4,000 international units per kilogram of body weight. In some aspects, the dosage or therapeutically effective amount is about 100 to about 3,000 international units per kilogram of body weight.

[0221] In certain aspects, the dosage or therapeutically effective dose is about 10 to about 500 international units per kilogram of body weight. In some aspects, the dosage or therapeutically effective dose is about 50 to about 450 international units per kilogram of body weight. In some aspects, the therapeutically effective dose is about 40 to about 100 international units per kilogram of body weight. In some aspects, the therapeutically effective dose is about 40 to about 150 international units per kilogram of body weight. In some aspects, the dosage or therapeutically effective dose is about 100 to about 500 international units per kilogram of body weight. In some aspects, the dosage or therapeutically effective dose is about 100 to about 400 international units per kilogram of body weight. In some aspects, the dosage or therapeutically effective dose is about 100 to about 300 international units per kilogram of body weight. In some aspects, the dosage or therapeutically effective dose is about 300 to about 500 international units per kilogram of body weight. In some aspects, the dosage or therapeutically effective dose is about 200 to about 300 international units per kilogram of body weight. In some aspects, the dosage or therapeutically effective amount is about 100, about 150, about 200, about 250, about 300, about 350, about 400, about 450, or about 500 International Units per kilogram of body weight.

[0222] In further aspects, dosage or therapeutically effective dose is about 50 to about 1,000 international units per kilogram of body weight. In some aspects, dosage or therapeutically effective dose is about 100 to about 900 international units per kilogram of body weight. In some aspects, dosage or therapeutically effective dose is about 200 to about 800 international units per kilogram of body weight. In some aspects, dosage or therapeutically effective dose is about 300 to about 700 international units per kilogram of body weight. In some aspects, dosage or therapeutically effective dose is about 400 to about 600 international units per kilogram of body weight. In some aspects, dosage or therapeutically effective dose is about 500 international units per kilogram of body weight.

[0223] In some aspects, the dosage or therapeutically effective amount is about 10 IU / kg body weight, about 20 IU / kg body weight, about 30 IU / kg body weight, about 40 IU / kg body weight, about 50 IU / kg body weight, about 60 IU / kg body weight, about 70 IU / kg body weight, about 80 IU / kg body weight, about 90 IU / kg body weight, about 100 IU / kg body weight, about 120 IU / kg body weight, about 140 IU / kg body weight, about 150 IU / kg body weight, about 160 IU / kg body weight, about 180 IU / kg body weight, about 200 IU / kg body weight, or about 300 IU / kg body weight. , about 220 international units per kilogram of body weight, about 240 international units per kilogram of body weight, about 250 international units per kilogram of body weight, about 260 international units per kilogram of body weight, about 280 international units per kilogram of body weight, about 300 international units per kilogram of body weight, about 350 international units per kilogram of body weight, about 400 international units per kilogram of body weight, about 450 international units per kilogram of body weight, about 500 international units per kilogram of body weight, about 550 international units per kilogram of body weight, about 600 international units per kilogram of body weight, about 650 international units per kilogram of body weight, about 700 international units per kilogram of body weight, about 750 international units per kilogram of body weight, about 800 international units per kilogram of body weight, and about 1000 international units per kilogram of body weight. About 850 IUs per kilogram of body weight, about 900 IUs per kilogram of body weight, about 950 IUs per kilogram of body weight, about 1,000 IUs per kilogram of body weight, about 1,100 IUs per kilogram of body weight, about 1,100 IUs per kilogram of body weight, about 1,200 IUs per kilogram of body weight, about 1,300 IUs per kilogram of body weight, about 1,400 IUs per kilogram of body weight, about 1,500 IUs per kilogram of body weight, about 1,600 IUs per kilogram of body weight, about 1,800 IUs per kilogram of body weight, about 2,000 IUs per kilogram of body weight, about 2,500 IUs per kilogram of body weight, about 3,000 IUs per kilogram of body weight International Units per kilogram of body weight, about 3,500 international units per kilogram of body weight, about 4,000 international units per kilogram of body weight, about 4,500 international units per kilogram of body weight, about 5,000 international units per kilogram of body weight, about 5,500 international units per kilogram of body weight, about 6,000 international units per kilogram of body weight, about 6,500 international units per kilogram of body weight, about 7,000 international units per kilogram of body weight, about 7,500 international units per kilogram of body weight, about 8,000 international units per kilogram of body weight, about 8,500 international units per kilogram of body weight, about 9,000 international units per kilogram of body weight, about 9,500 international units per kilogram of body weight, and about 10,000 international units per kilogram of body weight.

[0224] As used herein, "one unit of ADAMTS13 activity" or "one activity unit" is defined as the amount of activity in 1 mL of combined normal human plasma, regardless of the assay used. However, as described above, the new standard for measuring or dosing ADAMTS13 is the International Unit (IU). 20 FRETS test units or 20 FRETS units (FRETS U) are equivalent to approximately 21.78 IU. In other words, 20 IU of ADAMTS13 is equivalent to approximately 18.22 FRETS U of ADAMTS13. Therefore, changing to the new standard results in an approximate shift of 8.9% in the conversion of FRETS U to IU.

[0225] In some aspects, a fluorescence resonance energy transfer (FRETS) assay is used to measure ADAMTS13 activity. FRETS requires two interacting partners, one of which is labeled with a donor fluorophore and the other with an acceptor fluorophore. The FRETS assay for ADAMTS13 involves a chemically modified fragment of the A2 domain of VWF that spans the ADAMTS13 cleavage site. This is readily cleaved by normal plasma but not by ADAMTS13-deficient plasma. This cleavage is blocked by EDTA, so samples for this assay must be collected into tubes containing citrate rather than EDTA as an anticoagulant. One unit of ADAMTS13 FRETS-VWF73 activity is the amount of activity required to cleave the same amount of FRETS-VWF73 substrate (Kokame et al., Br J. Haematol. 2005 Apr; 129(1): 93-100, incorporated herein by reference) that is present in 1 mL of pooled normal human plasma.

[0226] In some aspects, the activity of ADAMTS13 is measured using another activity assay. For example, direct ADAMTS13 activity assays can be performed using SDS agarose gel electrophoresis to detect the cleavage of full-length VWF molecules or VWF fragments, or ADAMTS13 activity can be indirectly detected using collagen binding assays. Direct assays as described herein, including FRETS assays, involve detecting the cleavage of products of full-length VWF molecules or VWF fragments containing ADAMTS13 cleavage sites. Purified VWF is incubated with plasma for 24 hours using SDS agarose gel electrophoresis and Western blotting. The cleavage of VWF by ADAMTS13 results in a reduction in the size of the multimers. This reduction is visualized by agarose gel electrophoresis followed by Western blotting with a peroxidase-conjugated anti-VWF antibody. The concentration of ADAMTS13 activity in the test sample can be determined by reference to a series of diluted normal plasma samples. SDS-PAGE and Western blotting can also be performed, which involves visualization of dimeric VWF fragments after SDS PAGE and Western blotting. This assay is technically easier than SDS agarose gel electrophoresis and appears to be a very sensitive method for measuring ADAMTS13 activity levels.

[0227] In some aspects, indirect assays involve detecting the cleavage of a product of a full-length VWF molecule or VWF fragment comprising an ADAMTS13 cleavage site in the A2 domain of VWF. Such assays include collagen binding assays, in which normal plasma or purified VWF is incubated with a test plasma sample in the presence of BaCl2 and 1.5M urea to denature VWF. VWF is cleaved by ADAMTS13, and the residual VWF is measured by its binding to type III collagen. Utilizing an ELISA assay, bound VWF is quantified using a conjugated anti-VWF antibody. Another indirect assay is a ristocetin-induced aggregation assay. This is similar to the collagen binding assay above, but the residual VWF is measured by ristocetin-induced platelet aggregation using a platelet aggregometer. Another indirect assay is a functional ELISA. In this assay, recombinant VWF fragments are fixed on an ELISA plate using antibodies directed against a tag on VWF. The VWF fragment encodes the A2 domain and the ADAMTS13 cleavage site at Tyr1605-Met1606 and is tagged with S-transferase [GST]-histidine [GST-VWF73-His]. Plasma is added to the immobilized GST-VWF73-His fragment, and cleavage of the immobilized fragment occurs at the ADAMTS13 cleavage site. Residual cleaved VWF fragments are measured using a secondary monoclonal antibody that recognizes only the cleaved VWF fragment and not the interacting fragment. Therefore, ADAMTS13 activity is inversely proportional to the residual substrate concentration.

[0228] In certain embodiments, ADAMTS13 is provided or administered at a therapeutically effective concentration of about 0.05 mg / mL to about 10 mg / mL in the final formulation. In other embodiments, ADAMTS13 is present at a concentration of about 0.1 mg / mL to about 10 mg / mL. In other embodiments, ADAMTS13 is present at a concentration of about 0.1 mg / mL to about 5 mg / mL. In another embodiment, ADAMTS13 is present at a concentration of about 0.1 mg / mL to about 2 mg / mL. In other embodiments, ADAMTS13 can be administered at about 0.01 mg / mL, or about 0.02 mg / mL, 0.03 mg / mL, 0.04 mg / mL, 0.05 mg / mL, 0.06 mg / mL, 0.07 mg / mL, 0.08 mg / mL, 0.09 mg / mL, 0.1 mg / mL, 0.2 mg / mL, 0.3 mg / mL, 0.4 mg / mL, 0.5 mg / mL, 0.6 mg / mL, 0.7 mg / mL, 0.8 mg / mL, 0.9 mg / mL, 1.0 mg / mL, 1.1 mg / mL, 1.2 mg / mL, 1.3 mg / mL, 1.6 mg / mL, 1.7 mg / mL, 1.8 mg / mL, 1.9 mg / mL, 2.0 mg / mL, 2.1 mg / mL, 2.2 mg / mL, 2.3 mg / mL, 2.4 mg / mL, 2.5 mg / mL, 2.6 mg / mL, 2.7 mg / mL, 2.8 mg / mL, .3mg / mL, 1.4mg / mL, 1.5mg / mL, 1.6mg / mL, 1.7mg / mL, 1.8mg / mL, 1.9mg / mL, 2.0mg / mL, 2.5mg / mL, 3.0mg / mL, 3.5mg / mL, 4.0mg / mL, 4.5m g / mL, 5.0mg / mL, 5.5mg / mL, 6.0mg / mL, 6.5mg / mL, 7.0mg / mL, 7.5mg / mL, 8.0mg / mL, 8.5mg / mL, 9.0mg / mL, 9.5mg / mL, 10.0mg / mL or higher.

[0229] In some embodiments, the concentration of a relatively pure ADAMTS13 preparation can be determined spectroscopically (i.e., total protein measured at A280) or other bulk determinations (e.g., Bradford assay, silver staining, weight of lyophilized powder, etc.). In other embodiments, the concentration of ADAMTS13 can be determined by an ADAMTS13 ELISA assay (e.g., mg / mL antigen).

[0230] In some aspects, the concentration of ADAMTS13 in the formulations of the present disclosure is expressed as an enzymatic activity level. For example, in some embodiments, the ADAMTS13 formulation contains about 10 units of FRETS-VWF73 activity to about 10,000 units of FRETS-VWF73 activity or other suitable ADAMTS13 enzymatic units (IU). In other embodiments, the formulation may contain about 20 units of FRETS-VWF73 (IU).FV73 ) activity to about 8,000 units of FRETS-VWF73 activity, or about 30 U FV73 to about 6,000U FV73 , or about 40U FV73 to about 4,000U FV73 , or about 50U FV73 to about 3,000U FV73 , or about 75U FV73 About 2,500U FV73 Between, or about 100U FV73 to about 2,000U FV73 Between, or about 200U FV73 About 1,500U FV73 between, or between about other ranges therein.

[0231] In some embodiments, ADAMTS 13 is administered at about 10 U per kilogram of body weight. FV73 Up to 10,000 U per kilogram of body weight FV73 In one embodiment, ADAMTS 13 is provided or administered at a dose of about 20 U per kilogram of body weight. FV73 Up to about 8,000 U per kilogram of body weight FV73 In one embodiment, ADAMTS 13 is administered at a dose of about 30 U per kilogram of body weight. FV73 Up to about 6,000 U per kilogram of body weight FV73 In one embodiment, ADAMTS 13 is administered at a dose of about 40 U per kilogram of body weight. FV73 Up to approximately 4,000 U per kilogram of body weight FV73 In one embodiment, ADAMTS 13 is administered at a dose of about 100 U per kilogram of body weight. FV73 Up to 3,000 U per kilogram of body weight FV73 In one embodiment, ADAMTS 13 is administered at a dose of about 200 U per kilogram of body weight. FV73 Up to approximately 2,000 U per kilogram of body weight FV73 In other embodiments, ADAMTS 13 is administered at a dose of about 10 U per kilogram of body weight. FV73, per kilogram of body weight about 20, 30, 40, 50, 60, 70, 80, 90, 100, 150, 200, 250, 300, 350, 400, 450, 500, 600, 700, 800, 900, 1,000, 1100, 1,200, 1,300, 1,400, 1,500, 1,600, 1,700, 1,800, 1,900, 2,000, 2,100, 2,200, 2,300, 2,40 0, 2,500, 2,600, 2,700, 2,800, 2,900, 3,000, 3,100, 3,200, 3,300, 3,400, 3,500, 3,600, 3,700, 3,800, 3,900, 4,000, 4,500, 5,000, 5,500, 6,000, 6,500, 7,000, 7,500, 8,000, 8,500, 9,000, 9,500, or 10,000 U FV73 The dosage may be administered at a dosage level of, or at a dosage level or a dosage range intermediate thereto.

[0232] In some aspects, the ADAMTS 13 preparations provided herein contain from about 20 to about 10,000 U FV73In some embodiments, the formulation contains about 10 units of FRETS-VWF73 activity, or about 20, 30, 40, 50, 60, 70, 80, 90, 100, 150, 200, 250, 300, 350, 400, 450, 500, 600, 700, 800, 900, 1,000, 1,100, 1,200, 1,300, 1,400, 1,500, 1,600, 1,700, 1,800, 1,900, 0, 2,000, 2,100, 2,200, 2,300, 2,400, 2,500, 2,600, 2,700, 2,800, 2,900, 3,000, 3,100, 3,200, 3,300, 3,400, 3,500, 3,600, 3,700, 3,800, 3,900, 4,000, 4,100, 4,200, 4,300, 4,400, 4,500, 4,600, 4,700 , 4,800, 4,900, 5,000, 5,100, 5,200, 5,300, 5,400, 5,500, 5,600, 5,700, 5,800, 5,900, 6,000, 6,100, 6,200, 6,300, 6,400, 6,500, 6,600, 6,700, 6,800, 6,900, 7,000, 7,100, 7,200, 7,300, 7,400, 7,500, 7,600, 7,700, 7,800, 7,900, 8,000, 8,100, 8,200, 8,300, 8,400, 8,500, 8,600, 8,700, 8,800, 8,900, 9,000, 9,100, 9,200, 9,300, 9,400, 9,500, 9,600, 9,700, 9,800, 9,900, 10,000 or more units of FRETS-VWF73 activity.

[0233] In some aspects, the concentration of ADAMTS 13 can be expressed as enzyme activity per unit volume, for example, ADAMTS 13 enzyme units / mL (IU / mL). For example, in some embodiments, the ADAMTS 13 preparation contains about 10 IU / mL to about 10,000 IU / mL. In some other embodiments, the preparation contains about 20 IU / mL to about 10,000 IU / mL, or about 20 IU / mL to about 8,000 IU / mL, or about 30 IU / mL to about 6,000 IU / mL, or about 40 IU / mL to about 4,000 IU / mL, or about 50 IU / mL to about 3,000 IU / mL, or about 75 IU / mL to about 2,500 IU / mL, or about 100 IU / mL to about 2,000 IU / mL, or about 200 IU / mL to about 1,500 IU / mL, or in about other ranges therein. In some embodiments, the ADAMTS13 preparations provided herein contain about 150 IU / mL to about 600 IU / mL. In another embodiment, the ADAMTS13 preparations provided herein contain about 100 IU / mL to about 1,000 IU / mL. In some embodiments, the ADAMTS13 preparations provided herein contain about 100 IU / mL to about 800 IU / mL. In some embodiments, the ADAMTS13 preparations provided herein contain about 100 IU / mL to about 600 IU / mL. In some embodiments, the ADAMTS13 preparations provided herein contain about 100 IU / mL to about 500 IU / mL. In some embodiments, the ADAMTS13 preparations provided herein contain about 100 IU / mL to about 400 IU / mL. In some embodiments, the ADAMTS13 preparations provided herein contain about 100 IU / mL to about 300 IU / mL. In some embodiments, the ADAMTS13 preparations provided herein contain about 100 IU / mL to about 200 IU / mL. In some embodiments, the ADAMTS13 preparations provided herein contain about 300 IU / mL to about 500 IU / mL. In some embodiments, the ADAMTS13 preparations provided herein contain about 100 IU / mL. In some embodiments, the ADAMTS13 preparations provided herein contain about 300 IU / mL.In various embodiments, the formulation contains about 10 IU / mL, or about 20, 30, 40, 50, 60, 70, 80, 90, 100, 150, 200, 250, 300, 350, 400, 450, 500, 600, 700, 800, 900, 1,000, 1,100, 1,200, 1,300, 1,400, 1,500, 1,600, 1,700, 1,800, 1,900, 2,000 0, 2,100, 2,200, 2,300, 2,400, 2,500, 2,600, 2,700, 2,800, 2,900, 3,000, 3,100, 3,200, 3,300, 3,400, 3,500, 3,600, 3,700, 3,800, 3,900, 4,000, 4,100, 4,200, 4,300, 4,400, 4,500, 4,600, 4,700, 4,800, 4,900, 5,000, 5,100, 5,200, 5,300, 5,400, 5,500, 5,600, 5,700, 5,800, 5,900, 6,000, 6,100, 6,200, 6,300, 6,400, 6,500, 6,600, 6,700, 6,800, 6,900, 7,000, 7,100, 7,200, 7,300, 7,400, 7, 500, 7,600, 7,700, 7,800, 7,900, 8,000, 8,100, 8,200, 8,300, 8,400, 8,500, 8,600, 8,700, 8,800, 8,900, 9,000, 9,100, 9,200, 9,300, 9,400, 9,500, 9,600, 9,700, 9,800, 9,900, 10,000 IU / mL or above.

[0234] In some embodiments, the ADAMTS13 formulations provided herein may further comprise one or more pharmaceutically acceptable excipients, carriers, and / or diluents, as described in U.S. Patent Application Publication No. 2011 / 0229455 and / or U.S. Patent Application Publication No. 2014 / 0271611, each of which is incorporated herein by reference in its entirety and for all purposes. In addition, in one embodiment, the ADAMTS13 formulations provided herein will have a tension within the range described in U.S. Patent Application Publication No. 2011 / 0229455 and / or U.S. Patent Application Publication No. 2014 / 0271611, each of which is incorporated herein by reference in its entirety and for all purposes.

[0235] The frequency of administration depends on the pharmacokinetic parameters of the ADAMTS13 molecule in the formulation used. Typically, the clinician will administer the composition until the dosage for achieving the desired effect is reached. Therefore, in various aspects, the composition is administered as a single dose, or as two or more doses (which may comprise the same amount of desired molecules or comprise different amounts of desired molecules) administered over time, or as a continuous infusion by an implant device or catheter. In some aspects, the composition comprising ADAMTS13 is administered monthly, every two weeks, weekly, twice a week, every other day, every day, every 12 hours, every 8 hours, every 6 hours, every 4 hours, or every 2 hours with a single bolus. In terms of prevention or preventive treatment of the present disclosure, ADAMTS13 is administered in multiple doses to maintain the circulating level of ADAMTS13, to effectively prevent the onset of VOC, ALI or ARDS. In these aspects, the composition comprising ADAMTS13 is administered monthly, every two weeks, weekly, twice a week, every other day or every day. In particular aspects, injection (e.g., WO2014151968, incorporated herein by reference in its entirety for all purposes) is administered subcutaneously. In other aspects, intravenous injection is used. Suitable dosage and administration time are further improved routinely by those of ordinary skill in the art and are within the scope of their routine tasks. Suitable dosage is usually determined by using conventionally obtained appropriate dose-response data.

[0236] Kits containing ADAMTS13

[0237] As an additional aspect, the present disclosure includes kits comprising one or more pharmaceutical formulations for administering ADAMTS 13 or an ADAMTS 13 composition to a subject, packaged in a manner that facilitates administration to a subject.

[0238] In a specific embodiment, the present disclosure includes a test kit for producing a single-dose administration unit. In another embodiment, the present disclosure includes a test kit for providing a multi-dose administration unit. In various aspects, the test kit each comprises a first container with a dry protein and a second container with an aqueous formulation. Also included within the scope of the present disclosure are test kits comprising single-chamber and multi-chamber prefilled syringes (e.g., liquid syringes and lyophilizing syringes).

[0239] In another embodiment, such kit includes a pharmaceutical preparation as described herein (e.g., a composition comprising a therapeutic protein such as ADAMTS13), which is packaged in a container (e.g., a sealed bottle or tube), wherein a label is attached to or contained in the container, and the label describes the use of the compound or composition when implementing the method. In one embodiment, the pharmaceutical preparation is packaged in a container so that the amount of head space in the container (e.g., the amount of air between the liquid preparation and the top of the container) is very small. Preferably, the amount of head space is negligible (i.e., almost absent).

[0240] In some aspects, the pharmaceutical formulation or composition comprises a stabilizer. The term "stabilizer" refers to a substance or excipient that protects the composition from adverse conditions (such as those that occur during heating or freezing) and / or prolongs the stability or shelf life of the composition or pharmaceutical composition in a stable state. Examples of stabilizers include, but are not limited to, sugars such as sucrose, lactose, and mannose; sugar alcohols such as mannitol; amino acids such as glycine or glutamic acid; and proteins such as human serum albumin or gelatin.

[0241] In some aspects, the pharmaceutical preparation or composition comprises an antimicrobial preservative. The term "antimicrobial preservative" refers to any substance added to the composition that inhibits the growth of microorganisms that may be introduced when a multi-dose bottle (when using such a container) is repeatedly punctured. Examples of antimicrobial preservatives include, but are not limited to, substances such as thimerosal, 2-phenoxyethanol, benzethonium chloride, and phenol.

[0242] In one aspect, the kit comprises: a first container having a therapeutic protein or protein composition, and a second container having a physiologically acceptable reconstitution solution for the composition. In one aspect, the pharmaceutical preparation is packaged in unit dosage form. The kit optionally also includes a device suitable for administering the pharmaceutical preparation according to a specific route of administration. In some aspects, the kit includes a label describing the use of the pharmaceutical preparation.

[0243] The entire document is intended to be linked as a unified disclosure, and it should be understood that all combinations of features described herein are contemplated, even if the combination of features is not found in the same sentence, paragraph, or portion of text. The present disclosure also includes, for example, all embodiments that are narrower in scope than the variations specifically mentioned above.

[0244] All publications, patents, and patent applications cited in this specification are herein incorporated by reference to the same extent as if each individual publication or patent application was specifically and individually indicated to be incorporated by reference in its entirety to the extent not inconsistent with this disclosure.

[0245] Other embodiments

[0246] In certain embodiments, a method for treating or preventing vaso-occlusive crisis (VOC) in a subject with sickle cell disease (SCD) is provided. A therapeutically effective amount of a composition comprising ADAMTS13 is administered to the subject. The subject can be a human patient with SCD, or an animal with SCD. ADAMTS13 can be in a recombinant form. ADAMTS13 can be part of a preparation suitable for intravenous injection. ADAMTS13 can be part of a preparation suitable for subcutaneous injection. In rodents, the dosage of ADAMTS13 can be 2,500 IU / kg to 4,000 IU / kg, 2,800 IU / kg to 3,800 IU / kg, 3,000 IU / kg to 3,400 IU / kg, about 3,200 IU / kg or 3,200 IU / kg. In human patients, the dose of ADAMTS13 can be 40 IU / kg to 100 IU / kg, 100 IU / kg to 300 IU / kg, 120 IU / kg to 240 IU / kg, 150 IU / kg to 200 IU / kg, or 300 IU / kg to 500 IU / kg. ADAMTS13 can be administered intravenously before, during, or after acute VOC in a human or animal patient with SCD. ADAMTS13 can be administered subcutaneously before, during, or after acute VOC in a human or animal patient with SCD. The treatment can effectively protect subjects (e.g., human patients or animals with SCD) from morbidity and mortality associated with VOC or hypoxia.

[0247] In certain embodiments, a method for treating or preventing acute lung injury (ALI) or acute respiratory distress syndrome (ARDS) is provided. A therapeutically effective amount of a composition comprising ADAMTS13 is administered to a subject. ADAMTS13 can be in a recombinant form. ADAMTS13 can be part of a preparation suitable for intravenous injection. ADAMTS13 can be part of a preparation suitable for subcutaneous injection. In rodents, the dosage of ADAMTS13 can be 2,500 IU / kg to 4,000 IU / kg, 2,800 IU / kg to 3,800 IU / kg, 3,000 IU / kg to 3,400 IU / kg, about 3,200 IU / kg or 3,200 IU / kg. In human patients, the dosage of ADAMTS13 can be 40 IU / kg to 100 IU / kg, 100 IU / kg to 300 IU / kg, 120 IU / kg to 240 IU / kg, 150 IU / kg to 200 IU / kg, or 300 IU / kg to 500 IU / kg. ADAMTS13 can be administered intravenously before, during, or after ALI or ARDS in human or animal patients. ADAMTS13 can be administered subcutaneously before, during, or after ALI or ARDS in human or animal patients. The treatment can effectively protect subjects (e.g., human patients with ALI and / or ARDS) from morbidity and mortality.

[0248] In another embodiment, a method for treating, improving or preventing (a) VOC in a subject with SCD or (b) lung injury in a subject with acute lung injury (ALI) and / or acute respiratory distress syndrome (ARDS) or a risk of acute lung injury (ALI) and / or acute respiratory distress syndrome (ARDS) is provided. A therapeutically effective amount of ADAMTS13 is administered to the subject. Lung injury or vascular inflammation may be secondary to or caused by hypoxia. Lung injury or vascular inflammation may be secondary to or caused by reoxygenation stress. During hypoxia or reoxygenation stress, the oxygen level may be about 7%, about 8%, about 9%, about 10%, 7-10% or 7-9%. The subject may be a human patient with SCD, a human patient experiencing VOC, a human patient with ALI, a human patient with ARDS, an animal with SCD, an animal experiencing VOC, an animal with ALI, an animal with ARDS, or an animal with HbA homozygotes. ADAMTS13 may be part of a formulation suitable for intravenous injection. ADAMTS13 can be part of a preparation suitable for subcutaneous injection. In rodents, the dosage of ADAMTS13 can be 2,500 IU / kg to 4,000 IU / kg, 2,800 IU / kg to 3,800 IU / kg, 3,000 IU / kg to 3,400 IU / kg, about 3,200 IU / kg or 3,200 IU / kg. In human patients, the dosage of ADAMTS13 can be 40 IU / kg to 100 IU / kg, 100 IU / kg to 300 IU / kg, 120 IU / kg to 240 IU / kg, 150 IU / kg to 200 IU / kg, or 300 IU / kg to 500 IU / kg. Before, during, and after treatment, one or more of the BAL protein content and BAL white blood cell count of the subject can be monitored once or repeatedly. Administration of ADAMTS13 can effectively reduce BAL protein content by at least 35%, at least 36%, at least 37%, at least 38%, at least 39%, at least 40%, 30-45%, 33-43%, 34-42%, 35-41% or 36-40% compared to a control (e.g., an untreated subject). Administration of ADAMTS13 can effectively reduce BAL white blood cell count by at least 35%, at least 36%, at least 37%, at least 38%, at least 39%, at least 40%, 30-45%, 33-43%, 34-42%, 35-41% or 36-40% compared to a control (e.g., an untreated subject).

[0249] In at least the above embodiments, administration of ADAMTS13 is effective to prevent activation or increased expression or levels of at least one of VCAM-1 and ICAM-1, and / or to reduce expression of at least one of ET-1, TXAS, and HO-1. Figure 2B、 2C Administration of ADAMTS13 can be effective in reducing the expression or level of TXAS or ET-1 (as measured by densitometry) by at least 65%, at least 68%, at least 71%, at least 74%, at least 77%, at least 80%, 65-80%, 70-80%, or 70-75% compared to a control (e.g., an untreated subject). Administration of ADAMTS13 can be effective in reducing the expression, level, or activity of ICAM-1 (as measured by densitometry) by at least 53%, at least 56%, at least 59%, at least 62%, at least 65%, 53-65%, 55-62%, or 57-60% compared to a control (e.g., an untreated subject). Administration of ADAMTS13 can be effective in reducing the expression or level of HO-1 (as measured by densitometry) by at least 46%, at least 47%, at least 48%, at least 49%, 46-49%, 47-49%, or 46-48% compared to a control (e.g., an untreated subject). Administration of ADAMTS13 can effectively reduce the ratio of P-NF-κB / NF-κB (as measured by densitometry) by at least 63%, at least 67%, at least 71%, at least 75%, at least 79%, at least 83%, 63-83%, 67-79% or 71-75% compared to a control (e.g., an untreated subject). In subjects with SCD or experiencing VOC, administration of ADAMTS13 can effectively reduce the expression, level or activity of VCAM-1 (as measured by densitometry) by at least 40%, at least 42%, at least 44%, at least 46%, at least 48%, at least 50%, 40-50%, 42-48% or 44-46% compared to a control (e.g., an untreated subject). In certain embodiments, biomarkers (e.g., VCAM-1, ICAM-1, P-NF-κB, NF-κB, ET-1, TXAS and HO-1) are measured in the lung. In certain embodiments, biomarkers (e.g., VCAM-1, ICAM-1, P-NF-κB, NF-κB, ET-1, TXAS, and HO-1) are measured in the kidney.

[0250] In at least the above embodiments, with respect to treating lung injury or vascular inflammation associated with SCD, VOC, ALI and / or ARDS, administration of ADAMTS13 is effective in preventing activation or increased expression or levels of at least one of VCAM-1 and ICAM-1, and / or reducing expression or levels of at least one of ET-1, TXAS and HO-1. Figure 2B and 2CAdministration of ADAMTS13 can be effective in reducing the expression or level of TXAS or ET-1 (as measured by densitometry) by at least 65%, at least 68%, at least 71%, at least 74%, at least 77%, at least 80%, 65-80%, 70-80%, or 70-75% compared to a control (e.g., an untreated subject). Administration of ADAMTS13 can be effective in reducing the expression, level, or activity of ICAM-1 (as measured by densitometry) by at least 53%, at least 56%, at least 59%, at least 62%, at least 65%, 53-65%, 55-62%, or 57-60% compared to a control (e.g., an untreated subject). Administration of ADAMTS13 can be effective in reducing the expression or level of HO-1 (as measured by densitometry) by at least 46%, at least 47%, at least 48%, at least 49%, 46-49%, 47-49%, or 46-48% compared to a control (e.g., an untreated subject). Administration of ADAMTS13 can effectively reduce the ratio of P-NF-κB / NF-κB (as measured by densitometry) by at least 63%, at least 67%, at least 71%, at least 75%, at least 79%, at least 83%, 63-83%, 67-79% or 71-75% compared to a control (e.g., an untreated subject). In subjects with SCD and / or experiencing VOC, administration of ADAMTS13 can effectively reduce the expression, level or activity of VCAM-1 (as measured by densitometry) by at least 40%, at least 42%, at least 44%, at least 46%, at least 48%, at least 50%, 40-50%, 42-48% or 44-46% compared to a control (e.g., an untreated subject). In certain embodiments, biomarkers (e.g., VCAM-1, ICAM-1, P-NF-κB, NF-κB, ET-1, TXAS and HO-1) are measured in the lung.

[0251] In at least the above embodiments, with respect to treating renal injury or vascular inflammation associated with SCD, VOC, ALI and / or ARDS, administration of ADAMTS13 can effectively prevent activation and / or increased expression levels of VCAM-1, reduce the ratio of P-NF-κB / NF-κB and / or reduce the expression or level of at least one of ET-1 or TXAS. Figure 3A and 3BAdministration of ADAMTS13 can effectively reduce the expression or level of TXAS (as measured by densitometry) by at least 70%, at least 73%, at least 76%, at least 78%, at least 80%, at least 82%, 70-82%, 73-80% or 76-78% compared to a control (e.g., an untreated subject). Administration of ADAMTS13 can effectively reduce the ratio of P-NF-κB / NF-κB (as measured by densitometry) by at least 68%, at least 70%, at least 72%, at least 75%, at least 78%, 68-78%, 70-75% or 72-75% compared to a control (e.g., an untreated subject). Administration of ADAMTS13 can effectively reduce the expression or level or activity of VCAM-1 in subjects with SCD or experiencing VOC (as measured by densitometry) by at least 58%, at least 60%, at least 62%, at least 64%, at least 67%, 58-67%, 60-64% or 60-62% compared to a control (e.g., an untreated subject). In certain embodiments, biomarkers (e.g., VCAM-1, P-NF-κB, NF-κB, ET-1, TXAS and HO-1) are measured in the kidney.

[0252] In at least the above embodiments, a blood sample can be collected from a subject, for example, to monitor the treatment of SCD, VOC, ALI and / or ARDS by measuring one or more of the following hematocrit values: hematocrit (Hct) % and mean corpuscular volume (MCV), as indicators of red blood cell viability; hemoglobin (Hb), mean corpuscular hemoglobin (MCH) and mean corpuscular hemoglobin concentration (CHCM), as indicators of oxygen binding capacity; heterogeneity of red blood cell distribution (HDW), as an indicator of the presence of dense red blood cells; reticulocyte count (Retics), as an indicator of anemic state; neutrophil count, as an indicator of systemic inflammatory state; and / or lactate dehydrogenase (LDH), as a general marker of cell damage. Administration of ADAMTS13 can effectively reduce CHCM by at least 5%, at least 5.5%, at least 6%, at least 6.5% or at least 7% compared to a control (e.g., an untreated subject). Compared to a control (e.g., an untreated subject), administration of ADAMTS13 can effectively increase the network by at least 5%, at least 7%, at least 9%, at least 11% or at least 13%. Compared to a control (e.g., an untreated subject), administration of ADAMTS13 can effectively reduce neutrophils (cells / microliter) by at least 30%, at least 35%, at least 40%, at least 45% or at least 50%. Compared to a control (e.g., an untreated subject), administration of ADAMTS13 can effectively reduce LDH (cells / microliter) by at least 5%, at least 10%, at least 15%, at least 20%, at least 25% or at least 30%.

[0253] In at least the above embodiments, ADAMTS13 is administered to subjects suffering from SCD or experiencing VOCs, and administration of ADAMTS13 is effective in changing the levels of Hct%, Hb, MCV, MCH and / or HDW. Compared to a control (e.g., an untreated subject), administration of ADAMTS13 can effectively increase Hct% in subjects suffering from SCD or experiencing VOCs by at least 60%, at least 65%, at least 70%, at least 75%, at least 80% or at least 85%. Compared to a control (e.g., an untreated subject), administration of ADAMTS13 can effectively increase Hb in subjects suffering from SCD or experiencing VOCs by at least 18%, at least 20%, at least 22%, at least 24% or at least 26%. Compared to a control (e.g., an untreated subject), administration of ADAMTS13 can effectively increase MCV in subjects suffering from SCD or experiencing VOCs by at least 18%, at least 20%, at least 22%, at least 24% or at least 26%. Administration of ADAMTS13 can effectively increase MCH in subjects with SCD or experiencing VOCs by at least 5%, at least 5.5%, at least 6%, at least 6.5%, or at least 7%, compared to a control (e.g., an untreated subject). Administration of ADAMTS13 can effectively reduce HDW in subjects with SCD or experiencing VOCs by at least 12%, at least 14%, at least 16%, at least 18%, or at least 20%, compared to a control (e.g., an untreated subject).

[0254] In at least the above embodiments, administration of ADAMTS13 in subjects with SCD experiencing VOCs can reduce or prevent SCD-associated tissue damage. In certain embodiments, the tissue damage is caused by hypoxia. In certain embodiments, the tissue damage is caused by reoxygenation. In certain embodiments, the tissue is lung tissue. In certain embodiments, the tissue is renal tissue. In certain embodiments, administration of ADAMTS13 reduces inflammatory cell infiltration and / or thrombosis in the tissue compared to a control. In certain embodiments, administration of ADAMTS13 reduces inflammatory cell infiltration in lung tissue compared to a control. In some embodiments, administration of ADAMTS13 reduces inflammatory cell infiltration in lung tissue by at least 20%, at least 30%, at least 40%, at least 50%, or at least 60%. In some embodiments, administration of ADAMTS13 reduces thrombosis in lung tissue compared to a control. In certain embodiments, administration of ADAMTS13 reduces pulmonary thrombosis by at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 75%, at least 80%, or at least 85%. In some embodiments, compared with a control, the administration of ADAMTS13 reduces inflammatory cell infiltration in renal tissue. In certain embodiments, administration of ADAMTS13 reduces renal inflammatory cell infiltration by at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99% or at least 100%. In certain embodiments, compared with a control, administration of ADAMTS13 reduces thrombosis in lung tissue. In certain embodiments, administration of ADAMTS13 reduces pulmonary thrombosis by at least 20%, at least 30%, at least 40%, at least 50% or at least 60%.

[0255] In at least the above embodiments, administration of ADAMTS13 in subjects with ALI and / or ARDS can reduce or prevent tissue damage associated with ALI and / or ARDS. In certain embodiments, the tissue damage is caused by hypoxia. In certain embodiments, the tissue damage is caused by reoxygenation. In certain embodiments, the tissue is lung tissue. In certain embodiments, the tissue is renal tissue. In certain embodiments, administration of ADAMTS13 reduces inflammatory cell infiltration and / or thrombosis in the tissue compared to a control. In certain embodiments, administration of ADAMTS13 reduces inflammatory cell infiltration in lung tissue compared to a control. In some embodiments, administration of ADAMTS13 reduces inflammatory cell infiltration in lung tissue by at least 20%, at least 30%, at least 40%, at least 50%, or at least 60%. In some embodiments, administration of ADAMTS13 reduces thrombosis in lung tissue compared to a control. In certain embodiments, administration of ADAMTS13 reduces pulmonary thrombosis by at least 20%, at least 30%, at least 40%, at least 50%, or at least 60%. In some embodiments, administration of ADAMTS13 reduces thrombosis in lung tissue compared to a control. In certain embodiments, administration of ADAMTS13 reduces pulmonary thrombosis by at least 20%, at least 30%, at least 40%, or at least 50%. In some embodiments, administration of ADAMTS13 reduces inflammatory cell infiltration in renal tissue compared to a control. In certain embodiments, administration of ADAMTS13 reduces renal inflammatory cell infiltration by at least 20%, at least 30%, at least 40%, at least 50%, or at least 60%. In certain embodiments, administration of ADAMTS13 reduces thrombosis in lung tissue compared to a control. In certain embodiments, administration of ADAMTS13 reduces pulmonary thrombosis by at least 20%, at least 30%, or at least 40%.

[0256] It should be understood that the examples and embodiments described herein are for illustrative purposes only and that various modifications or changes in light thereof will be suggested to those skilled in the art and are to be included within the spirit and purview of this application and the scope of the appended claims.

[0257] Example

[0258] Other aspects and details of the invention will be apparent from the following examples, which are intended to be illustrative and not restrictive.

[0259] Example 1:

[0260] ADAMTS13 prevents mortality in SCD mice exposed to lethal hypoxia-induced VOCs

[0261] Because acute sickle cell events are triggered by low oxygen (hypoxia), this example was performed to evaluate the effect of ADAMTS13 on survival in a SCD model subjected to hypoxia. The purpose of this example was to determine whether recombinant ADAMTS13 (rADAMTS13 (BAX930 / SHP655)) could protect humanized SCD mice exposed to lethal hypoxia-induced VOCs. It has been previously shown that exposure of SCD mice to very severe, life-threatening hypoxia / reoxygenation stress can be used to evaluate the effectiveness of novel therapeutic approaches for survival in SCD mice (Sabaa et al., JCI 118: 1924, 2008).

[0262] 4-6 weeks old healthy controls (Hba tm1(HBA)Tow Hbb tm3(HBG1,HBB)Tow ) mice (ie, AA) and SCD (Hba tm1(HBA) Tow Hbb tm2(HBG1,HBB*)Tow ) mice (humanized mouse models for sickle cell disease (i.e., SCD or SS mice)). Healthy (AA) and sickle cell disease mice (SCD or SS) were treated intravenously (iv) with vehicle or rADAMTS13 at a dose of 2,940 FRETS-U / kg (~3,200 IU / kg) 1 hour before severe hypoxia / reoxygenation stress (at approximately 7% oxygen for 10 hours), followed by reoxygenation with approximately 21% oxygen for 3 hours, which has previously been shown to biologically recapitulate the organ damage observed in human SCD patients in acute VOCs. See a similar protocol reported by Kalish et al. (supra). More specifically, four groups (n=6) of AA and SCD mice were treated with vehicle or ADAMTS13 (BAX930 / SHP655) (2,940 FRETS-U / kg (~3,200 IU / kg)) and exposed to conditions of hypoxic stress.

[0263] Compared with vehicle-treated SCD mice, treatment with recombinant ADAMTS 13 completely protected SCD mice from death (at 10 h of hypoxia, the mortality rate of rADAMTS13-treated SCD mice was 0%, while that of vehicle-treated SCD mice was 83.3%; at 3 h of reoxygenation after 10 h of hypoxia, the mortality rate of rADAMTS 13-treated SCD mice was 0%, while that of vehicle-treated SCD mice was 100%; P < 0.001) ( Figure 1 No differences in mouse survival were observed in healthy mice treated with vehicle or rADAMTS 13.

[0264] Data show that ADAMTS13 has a protective effect, including increased survival, in a SCD model following exposure to hypoxic stress.

[0265] Example 2:

[0266] ADAMTS13 reduces lung abnormalities induced by hypoxia / reoxygenation stress

[0267] The purpose of this example was to evaluate the effect of ADAMTS13 on lung injury and vascular inflammation induced by hypoxia / reoxygenation (H / R) stress.

[0268] Healthy controls (Hba tm1(HBA)Tow Hbb tm3(HBG1,HBB)Tow ) and SCD(Hba tm1(HBA)Tow Hbb tm2(HBG1,HBB*)Tow ) mice were exposed to hypoxia / reoxygenation (H / R) stress, which has previously been shown to biologically recapitulate acute VOC in human SCD patients and the organ damage observed in acute VOC. Specifically, six experimental groups were used - (1) AA untreated normoxia; (2) SS untreated normoxia; (3) AA vehicle plus H / R; (4) AA ADAMTS13 (BAX930 / SHP655) plus H / R; (5) SS vehicle plus H / R; and (6) SS ADAMTS13 (BAX930 / SHP655) plus H / R. In this experiment, H / R conditions were 8% oxygen for 10 hours, followed by a 3-hour recovery at approximately 21% oxygen, a protocol that is generally not lethal for SCD mice (Kalish et al., Haematologica 100:870-80, 2015).

[0269] Pulmonary vascular leakage was assessed in mice by measuring protein content and leukocyte count (total leukocytes measured in cells per microliter) in bronchoalveolar lavage (BAL) fluid under normoxic and H / R conditions.

[0270] Pulmonary vascular leakage was examined by measuring protein content and white blood cell counts (i.e., cell number) in bronchoalveolar lavage (BAL) fluid. As shown in Table 1, under normoxic conditions, increased BAL protein levels and white blood cell numbers were detected in SCD mice compared with healthy mice, indicating the accumulation of proteins and inflammatory cells in the alveolar space. Interestingly, in response to H / R, BAL protein and white blood cell counts were significantly increased in both SCD and AA mice.

[0271] Table 1: Results of lung leakage experiments performed in AA and SCD mice under normoxic and hypoxic conditions.

[0272]

[0273] AA: Hb homozygous control mice or healthy mice; SCD: HbS homozygous mice or sickle cell mice; and BAL: bronchoalveolar lavage; *P < 0.05 compared with vehicle-treated mice; °P < 0.05 compared with AA mice.

[0274] The data showed that SCD mice had a significant increase in peripheral neutrophils (cells / μL) compared to AA mice; however, treatment with ADAMTS 13 significantly reduced neutrophil counts. The data also showed that SCD mice had a greater number of leukocytes (total leukocytes in bronchoalveolar lavage (BAL) (cells / μL)) and a higher leukocyte protein content (BAL protein in bronchoalveolar lavage (mg / mL)) compared to controls, indicating that SCD mice had vascular leakage. Treatment with ADAMTS 13 significantly reduced this effect ( Figure 2A and Table 1 ), indicating that ADAMTS 13 reduced systemic inflammation and decreased abnormalities in pulmonary vascular dysfunction

[0275] These data indicate that ADAMTS 13 prevents hypoxia-induced inflammatory vasculopathy and abnormal pulmonary vascular leakage in the lungs of SCD mice during acute vaso-occlusive crisis. Furthermore, ADAMTS 13 significantly reduced BAL protein content and leukocyte counts in SCD and AA mice compared with vehicle-treated controls, suggesting that ADAMTS 13 has a protective effect on the lungs under hypoxic conditions.

[0276] Example 3:

[0277] ADAMTS 13 reduces hypoxia / reoxygenation stress-induced pulmonary vascular activation

[0278] To investigate the effects of ADAMTS 13 on lung injury and vascular inflammation, additional experiments were performed using the same six experimental groups as described in Example 2, namely, (1) AA untreated normoxia; (2) SS untreated normoxia; (3) AA vehicle plus H / R; (4) AA ADAMTS13 (BAX930 / SHP655) plus H / R; (5) SS vehicle plus H / R; and (6) SS ADAMTS13 (BAX930 / SHP655) plus H / R. In this example, animals were administered vehicle or ADAMTS13 as described in Example 2, then exposed to 8% oxygen for 10 hours, followed by a 3-hour recovery at 21% oxygen. Additional controls (AA and SCD) were also subjected to normoxia without vehicle or ADAMTS13.

[0279] Immunoblot analysis was performed using specific antibodies against various markers of inflammation, vasoconstriction, and platelet aggregation (i.e., nuclear factor kappa B (NF-κB), endothelin-1 (ET-1), heme oxygenase 1 (HO-1), intercellular adhesion molecule 1 (ICAM-1), thromboxane synthase (TXAS), and vascular cell adhesion molecule 1 (VCAM-1)) to measure the expression of these proteins in the lungs of healthy control (AA) and SCD mice treated with vehicle or rADAMTS13 after exposure to hypoxia (e.g., H / R) or normoxic conditions.

[0280] The data from this example showed that ADAMTS13 prevented hypoxia-induced NF-κB activation in the lung tissues of AA and SCD mice, indicating that ADAMTS13 reduced the lung inflammation process caused by hypoxia ( Figure 2B In the lungs of hypoxic SCD mice, ADAMTS13 prevented the activation of VCAM-1 and ICAM-1 (markers of vascular activation and inflammatory vasculopathy) and reduced the expression of endothelin-1 (ET-1), thromboxane synthase (TXAS), and heme oxygenase-1 (HO-1). Figure 2C ).

[0281] Table 2 reports the optical density values ​​obtained by immunoblot analysis using specific antibodies against nuclear factor kappa B (NF-κB) and its activated form (P-NF-κB), endothelin 1 (ET-1), heme oxygenase 1 (HO-1), intercellular adhesion molecule 1 (ICAM-1), thromboxane synthase (TXAS), and vascular cell adhesion molecule 1 (VCAM-1) in the lungs of healthy control (AA) and sickle cell (SCD) mice treated with vehicle or rADAMTS13 and exposed to normoxia or hypoxia / reoxygenation stress.

[0282] As shown in Table 2, under normoxic conditions, all measured protein markers (except ICAM-1) showed increased protein expression in SCD mice compared to AA mice. Under hypoxic conditions, the expression of all measured markers was further increased in both healthy controls and SCD mice. However, ADAMTS13 (i.e., BAX930 / SHP655) treatment had a protective effect in both AA and SCD mice, as demonstrated by lower levels of all tested markers of inflammation, vasoconstriction, and platelet aggregation.

[0283] Table 2

[0284]

[0285] AA: Hb homozygous control mice or healthy mice; SCD: HbS homozygous mice or sickle cell mice; TXAS: thromboxane synthase; ET-1: endothelin-1; VCAM-1: vascular cell adhesion molecule-1; ICAM-1: intercellular adhesion molecule-1; HO-1: heme oxygenase-1; p-NF-κB: phospho-nuclear factor-κB; and NF-κB: nuclear factor-κB. *P < 0.05 compared with vehicle-treated mice; °P < 0.05 compared with AA mice.

[0286] Recombinant ADAMTS 13 significantly reduced the expression level of each protein marker tested in SCD mice (i.e., compared to vehicle-treated SCD mice) (Table 2). In addition, recombinant ADAMTS 13 reduced the lung expression of all tested protein markers (except VCAM-1) in healthy control (AA) mice (i.e., compared to vehicle-treated control mice).

[0287] These data show that during acute vaso-occlusive crisis, ADAMTS 13 prevented inflammatory vasculopathy and abnormal pulmonary vascular leakage caused by hypoxia in the lungs of SCD mice. In addition, ADAMTS 13 prevented the increased expression of potent regulators of vascular tension (such as ET-1 and TXAS) caused by hypoxia, both of which are indicated as factors leading to vascular dysfunction in SCD during acute events. In addition, the data also show that ADAMTS 13 has a protective effect on the lung tissue of healthy animals under hypoxic conditions. Therefore, the data show that ADAMTS13 reduces vascular activation and inflammatory responses associated with hypoxic stress in the lungs of SCD and healthy mice.

[0288] Example 4:

[0289] ADAMTS13 reduces renal vascular activation induced by hypoxia / reoxygenation stress

[0290] To investigate the effects of ADAMTS13 on injury and vascular inflammation in the kidney, additional experiments were performed using the same six experimental groups as described in Example 2, namely, (1) AA untreated normoxia; (2) SS untreated normoxia; (3) AA vehicle plus H / R; (4) AA ADAMTS13 (BAX930 / SHP655) plus H / R; (5) SS vehicle plus H / R; and (6) SS ADAMTS13 (BAX930 / SHP655) plus H / R. In this example, animals were administered vehicle or ADAMTS13 as described in Examples 2 and 3, then exposed to 8% oxygen for 10 hours, followed by a 3-hour recovery at approximately 21% oxygen, which has previously been shown to biologically recapitulate acute VOCs in human SCD patients and the organ damage observed in acute VOCs. Additional controls (AA and SCD) were also subjected to normoxic conditions without vehicle or ADAMTS13.

[0291] Immunoblot analysis was performed with specific antibodies against NF-κB and its activated form (P-NF-κB), as well as ET-1, TXAS, and VCAM-1, to measure the expression of these proteins in the kidneys of AA and SCD mice treated with vehicle or rADAMTS13.

[0292] Table 3 reports the optical density values ​​obtained by immunoblotting analysis using specific antibodies against nuclear factor kappa B (NF-κB) and its activated form (P-NF-κB), endothelin 1 (ET-1), thromboxane synthase (TXAS), and vascular cell adhesion molecule 1 (VCAM-1) in the kidneys of healthy control (AA) and sickle cell (SCD) mice treated with vehicle or rADAMTS13 and exposed to normoxia or hypoxia (hypoxia / reoxygenation stress). As can be seen in Table 3, under normoxia, the levels of all protein markers were higher in SCD mice than in AA mice. Under hypoxia, the expression levels of all protein markers (except VCAM-1) were further increased in both SCD and AA mice.

[0293] Table 3

[0294]

[0295] AA: Hb homozygous control mice or healthy mice; SCD: HbS homozygous mice or sickle cell mice; TXAS: thromboxane synthase; ET-1: endothelin 1; VCAM-1: vascular cell adhesion molecule 1; p-NF-κB: phospho-nuclear factor κB; and NF-κB: nuclear factor κB. *P < 0.05 compared with vehicle-treated mice; °P < 0.05 compared with AA mice.

[0296] The data from this example show that ADAMTS13 prevents NF-κB activation induced by hypoxia in the kidneys of AA and SCD mice, and prevents NF-κB activation in SCD mice under normoxic conditions (Tables 3 and Figure 3A ). In SCD mice exposed to hypoxia, the expression of VCAM-1, ET-1, and TXAS was increased. ADAMTS13 prevented the hypoxia-induced increase in the expression of VCAM-1 and TXAS in the kidneys of both mouse strains and the increase in ET-1 levels in the kidneys of AA mice (Tables 3 and Figure 3B ).

[0297] These data suggest that ADAMTS13 prevents the hypoxia-induced increase in expression of potent regulators of vascular tone during acute events and / or prevents the increased expression of factors that contribute to the vascular dysfunction described in SCD. The data demonstrate that ADAMTS13 reduces vascular activation and inflammatory responses associated with hypoxic stress in the kidneys of both SCD and healthy mice. The examples demonstrate that rADAMTS13 can reduce acute sickle cell-associated events in the kidney, such as vasoconstriction and inflammatory vasculopathy.

[0298] Example 5:

[0299] ADAMTS 13 improves abnormalities of various hematological parameters induced by hypoxia / reoxygenation stress

[0300] To investigate the effects of ADAMTS 13 on various hematological parameters, additional experiments were performed using the same six experimental groups as described in Example 2, namely, (1) AA untreated normoxia; (2) SS untreated normoxia; (3) AA vehicle plus H / R; (4) AA ADAMTS13 (BAX930 / SHP655) plus H / R; (5) SS vehicle plus H / R; and (6) SS ADAMTS13 (BAX930 / SHP655) plus H / R. In this example, animals were administered vehicle or ADAMTS13 as described in Examples 2-4 and then exposed to normoxia or H / R (8% oxygen for 10 hours, followed by a 3-hour recovery at approximately 21% oxygen).

[0301] The following hematologic parameters were determined: hematocrit (Hct) % and mean corpuscular volume (MCV) as indicators of erythrocyte viability; hemoglobin (Hb), mean corpuscular hemoglobin (MCH), and mean cellular hemoglobin concentration (CHCM) as indicators of oxygen-binding capacity; heterogeneity of red cell distribution (HDW) as an indicator of the presence of densely packed erythrocytes; reticulocyte count as an indicator of anemic status; neutrophil count as an indicator of systemic inflammatory status; and lactate dehydrogenase (LDH) as a general marker of cellular damage.

[0302] Hematocrit is the ratio of red blood cell volume to total blood volume. MCV is the mean volume of an RBC. Hemoglobin is the protein responsible for transporting oxygen in the blood, and MCH is the average amount of hemoglobin per RBC in a blood sample; CHCM reflects the hemoglobin content within intact red blood cells. Hemoglobin distribution width (HDW) is a measure of the heterogeneity of red blood cell hemoglobin concentration. Reticulocytes are newly produced, relatively immature red blood cells; the reticulocyte count indicates whether adequate red blood cell production is occurring in the bone marrow. Within minutes of trauma, neutrophils are recruited to the site of injury; therefore, neutrophils are a hallmark of acute inflammation, and the neutrophil count indicates the inflammatory state.

[0303] Table 4 shows the hematological parameters of healthy control (AA) and sickle cell (SCD) mice under normoxic conditions and after treatment with ADAMTS13 (i.e., BAX930 / SHP655) or vehicle and exposure to hypoxia / reoxygenation stress. As shown in Table 4, under normoxic conditions, Hct and Hb levels were lower in SCD mice compared to control (AA) mice, while MCV and HDW levels were higher, and the number of reticulocytes and neutrophils was also higher. In healthy control mice, hypoxic conditions increased the number of reticulocytes and neutrophils. Administration of ADAMTS13 to control mice improved the large increase in the number of neutrophils, indicating a decrease in inflammation. In SCD mice, hypoxic conditions reduced Hct, Hb, MCV, and MCH, and increased CHCM, HDW, and neutrophil counts. Administration of ADAMTS13 to SCD mice improved the reduction in Hct, Hb, MCV, and MCH, and improved the increase in CHCM, HDW, and neutrophil counts.

[0304] Table 4

[0305]

[0306] AA: Hb homozygous control mice or healthy mice; SCD: HbS homozygous mice or sickle cell mice; Hct: hematocrit; Hb: hemoglobin; MCV: mean corpuscular volume; MCH: mean corpuscular hemoglobin; CHCM: cellular hemoglobin concentration; HDW: heterogeneity of red blood cell distribution; Retics: reticulocytes; and LDH: lactate dehydrogenase. *P < 0.002 compared with vehicle-treated mice; °P < 0.005 compared with AA mice.

[0307] Example 6:

[0308] ADAMTS13 improves abnormalities of various histopathological parameters induced by hypoxia / reoxygenation stress

[0309] To investigate the effects of ADAMTS 13 on various histopathological parameters, additional experiments were performed using four experimental groups: (1) AA vehicle plus H / R; (2) AA ADAMTS 13 (BAX930 / SHP655) plus H / R; (3) SS vehicle plus H / R; and (4) SS ADAMTS 13 (BAX930 / SHP655) plus H / R. In this example, animals were administered vehicle or ADAMTS 13 and then exposed to H / R conditions (10 hours in 8% oxygen, followed by a 3-hour recovery at approximately 21% oxygen).

[0310] Lungs and kidneys were collected after 3 hours of reoxygenation. Lung and kidney pathology was analyzed and the presence of inflammatory cell infiltration and thrombus was determined.

[0311] Histological analysis showed that H / R stress caused severe SCD-related tissue damage in both the lungs and kidneys of SCD mice. In the lungs, H / R caused inflammatory cell infiltration and thrombosis in all SCD mice (Table 5). In AA mice, H / R caused moderate inflammatory cell infiltration and some thrombosis in a few mice. In SCD mice, ADAMTS13 (BAX930 / SHP655) reduced inflammatory cell infiltration and thrombosis compared to vehicle-treated SCD mice. In AA mice, ADAMTS13 (BAX930 / SHP655) reduced cellular inflammatory infiltration in the lungs.

[0312] In the kidneys, H / R induced inflammatory cell infiltration and thrombi in all SCD mice. In AA mice, H / R induced limited inflammatory cell infiltration in a minority of mice, with virtually no thrombus formation. ADAMTS13 (BAX930 / SHP655) reduced inflammatory cell infiltration in the kidneys of SCD mice exposed to H / R and also affected thrombus formation. In AA mice, ADAMTS13 (BAX930 / SHP655) reduced cellular inflammatory infiltration and had no effect on thrombus formation (Table 5).

[0313] Table 5

[0314]

[0315] H / R: hypoxia / reoxygenation stress; the number of thrombi present per field of view at 250× magnification is given; the presence of inflammatory cell infiltration per field of view at 250× magnification (lung tissue 250×, kidney 400×): +1 to 10 cells per field of view of magnification; +10 to 50 cells per field of view of magnification; the number of animals with findings is listed in parentheses.

[0316] Example 7:

[0317] ADAMTS13 reduces organ damage in subjects with hypoxemia and at risk of developing ARDS

[0318] ADAMTS13 has been shown to reduce end-organ damage in a mouse model of severe hypoxia (8% oxygen for 10 hours followed by 3 hours of recovery at approximately 21% oxygen). Because severe hypoxemic injury is a contributing factor to the pathophysiology observed in patients with acute lung injury (ALI) and acute respiratory distress syndrome (ARDS) (ALI / ARDS), it is hypothesized that administering ADAMTS13 to patients at risk for or who have already developed ALI and / or ARDS (ALI / ARDS) may prevent, treat, or ameliorate the disease process and lead to improved outcomes, such as survival, long-term lung function, and avoidance of other end-organ damage.

[0319] LPS is administered to mice, either directly into the lungs by intratracheal injection or inhalation, or intraperitoneally or intravenously to induce a systemic inflammatory response. Mice treated with intratracheal LPS experience an acute and robust influx of inflammatory cells into the lungs, which resolves within 48 hours. Intraperitoneal LPS activates systemic inflammation and is associated with mild lung injury. This injury can be augmented by repeated LPS injections or by implanting an intraperitoneal LPS pump to continuously release LPS for hours or even days.

[0320] ADAMTS13 was administered at doses of approximately 50, 100, 200, 500, 1,000, 2,000, and 3,000 IU / kg body weight before and 12, 24, 48, 72, and 96 hours after LPS treatment. ADAMTS13 doses were administered subcutaneously or intravenously daily or every 12 hours until the subjects were sacrificed to examine inflammatory responses and organ damage in the lungs.

[0321] ADAMTS13 treatment reduced inflammatory responses, including the influx of inflammatory cells into the lungs, as measured by a decrease in the number of neutrophils, macrophages, monocytes, mast cells, eosinophils, and / or basophils present in the lungs of ADAMTS13-treated mice. ADAMTS13 treatment also reduced organ damage, as measured by blood urea nitrogen (BUN), creatinine, BUN / creatinine ratio, troponin, and neuron-specific enolase (NSE).

[0322] The present invention has been described in terms of specific embodiments that have been discovered or proposed, and the embodiments include specific modes for implementing the present invention. Various modifications and variations of the described present invention will be apparent to those skilled in the art without departing from the scope and spirit of the present invention. Although the present invention has been described in conjunction with specific embodiments, it should be understood that the claimed invention should not be unduly limited to these specific embodiments. In fact, various modifications of the modes for implementing the present invention that are apparent to those skilled in the relevant art are intended to fall within the scope of the appended claims.

Claims

1. Use of a composition containing a disintegrin-like metalloproteinase 13 containing a type I thrombobinding protein motif (ADAMTS13) in the preparation of a medicament for treating, ameliorating or preventing lung injury in a subject suffering from acute lung injury (ALI) and / or acute respiratory distress syndrome (ARDS) or at risk of suffering from acute lung injury (ALI) and / or acute respiratory distress syndrome (ARDS), wherein: The ADAMTS13 is in a therapeutically effective amount.

2. The use according to claim 1, wherein The subject suffers from a condition or combination of conditions selected from inflammatory pulmonary edema, inflammatory pulmonary infiltrates, impaired oxygenation, and hypoxemia.

3. The use according to claim 1, wherein: ADAMTS13 results in at least one of improved survival, improved lung function, reduced organ damage, reduced pulmonary vascular leakage, or any combination thereof compared to a control or no treatment.

4. The use according to any one of claims 1 to 3, wherein ADAMTS13 reduces at least one of inflammation, vasoconstriction, platelet aggregation, or any combination thereof, compared to a control or no treatment.

5. The use according to any one of claims 1 to 3, wherein ADAMTS13 reduces and / or prevents at least one of impaired blood flow, blood coagulation, vascular inflammation, thrombosis, ischemic cell damage, or organ damage, or any combination thereof, compared to a control or no treatment.

6. The use according to any one of claims 1 to 3, wherein ADAMTS13 reduces and / or prevents pain or the severity of pain compared to a control or no treatment.

7. The use according to any one of claims 1 to 3, wherein ADAMTS13 reduces the frequency of ALI and / or ARDS and / or the duration of ALI and / or ARDS episodes compared to no treatment.

8. The use according to any one of claims 1 to 3, wherein ADAMTS13 reduces the expression, level and / or activation of at least one of VCAM-1, ICAM-1, P-NF-κB / NF-κB ratio, ET-1, TXAS and HO-1 in an organ compared to a control or no treatment.

9. The use according to any one of claims 1 to 3, wherein ADAMTS13 increases the level of at least one of Hct, Hb, MCV, and MCH in the blood compared to a control or no treatment.

10. The use according to any one of claims 1 to 3, wherein ADAMTS13 reduces the level of at least one of CHCM, HDW, LDH, and neutrophil count in the blood compared to a control or no treatment.

11. The use according to any one of claims 1 to 3, wherein A therapeutically effective amount of ADAMTS13 is 20 to 6,000 international units per kilogram of body weight.

12. The use according to any one of claims 1 to 3, wherein A therapeutically effective amount of ADAMTS13 is 40 to 4,000 international units per kilogram of body weight.

13. The use according to any one of claims 1 to 3, wherein A therapeutically effective amount of ADAMTS13 is 100 to 3,000 international units per kilogram of body weight.

14. The use according to any one of claims 1 to 3, wherein A therapeutically effective amount of ADAMTS13 is 50 to 500 international units per kilogram of body weight.

15. The use according to any one of claims 1 to 3, wherein The composition comprising ADAMTS 13 is administered as a single bolus injection monthly, biweekly, weekly, twice weekly, every other day, daily, every 12 hours, every 8 hours, every 6 hours, every 4 hours, or every 2 hours.

16. The use according to any one of claims 1 to 3, wherein The composition comprising ADAMTS13 is administered intravenously or subcutaneously.

17. The use according to any one of claims 1 to 3, wherein ADAMTS13 is recombinant ADAMTS13.

18. The use according to any one of claims 1 to 3, wherein ADAMTS13 is of plasma origin.

19. The use according to any one of claims 1 to 3, wherein The subject is a mammal.

20. The use according to any one of claims 1 to 3, wherein The subjects are humans.

21. The use according to any one of claims 1 to 3, wherein The composition is in a stable aqueous solution ready for administration.

22. The use according to any one of claims 1 to 3, wherein A therapeutically effective amount of a composition comprising ADAMTS13 is administered to the subject within 48 hours after detection of inflammatory pulmonary edema, inflammatory pulmonary infiltrates, impaired oxygenation, or hypoxemia.

23. The use according to any one of claims 1 to 3, wherein The therapeutically effective amount of the composition comprising ADAMTS13 for treating, ameliorating and / or preventing lung injury is sufficient to maintain effective circulating levels of ADAMTS13 activity in a subject.

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