Uses of an adamts13 composition for treating, improving or preventing vaso-occlusive crisis

Abstract

The disclosure provides compositions and methods for treating, improving, and / or preventing vaso-occlusive crisis (VOC) in an individual suffering from sickle cell disease (SCD). The disclosure also provides compositions and methods for treating, improving, and / or preventing lung injury in an individual suffering from or at risk of suffering from acute lung injury (ALI) and / or acute respiratory distress syndrome (ARDS). The disclosure provides a disintegrin and metalloproteinase with thrombospondin domains type 1, member 13 (ADAMTS13) or a composition comprising ADAMTS13 for treating, improving, and / or preventing VOC, or for treating, improving, and / or preventing lung injury in an individual suffering from or at risk of suffering from ALI and / or ARDS.

Description

1 / 132 DESCRIPTIVE REPORT Uses of an Adamts13 composition to treat, improve, or prevent a vaso-occlusive crisis. Cross-reference to related applications.

[0001] This patent application claims priority under Title 35 of the U.S.C. § 119(e) of Provisional Patent Application No. U.S. 62 / 371,030, filed August 4, 2016, which is incorporated herein by reference in its entirety. FIELD OF THE INVENTION

[0002] The disclosure relates to a method for treating sickle cell disease with a Disintegrin and Metalloproteinase with Thrombospondin domains type 1, member 13 (ADAMTS13). More particularly, the disclosure relates to a method for treating, improving and / or preventing vaso-occlusive crisis (VOC) in an individual with sickle cell disease (SCD) by administering ADAMTS13. The disclosure includes uses of ADAMTS13 and / or compositions comprising ADAMTS13 for the preparation of medicaments for the treatment, improvement and / or prevention of VOC in SCD. The disclosure also relates to a method for treating, improving or preventing lung injury in an individual suffering from or at risk of suffering from acute lung injury (ALI) and / or acute respiratory distress syndrome (ARDS) with ADAMTS13, and uses of ADAMTS13 and / or compositions comprising ADAMTS13 for the preparation of medicaments for the treatment, improvement and / or prevention of ALI and / or ARDS. BACKGROUND OF THE INVENTION

[0003] Sickle cell disease (SCD) is a disorder Petition 870240042003, dated 05 / 17 / 2024, page 17 / 22 Sickle cell anemia (SCD) is a hereditary form of red blood cell disease distributed worldwide, resulting from a point mutation (β3, 6V) in the β-globin chain that leads to the production of a defective form of hemoglobin, hemoglobin S (HbS). Studies of the polymerization kinetics of HbS after deoxygenation have shown that this is a high-order exponential function of hemoglobin concentration, thus highlighting a fundamental role for cellular HbS concentration in sickle cell disease. Pathological studies have shown that dense dehydrated red blood cells have a central function in chronic and acute clinical manifestations of SCD, where intravascular sickling in capillaries, small vessels, and large vessels leads to vaso-occlusion and impaired blood flow with ischemic cell damage in a variety of organs and tissues.

[0004] In patients with DF, increased levels of von Willebrand factor (vWF) and ultralarge vWF multimers have been observed and are associated with acute vaso-occlusive events. The levels of ultralarge vWF multimers are dependent on the activity of Disintegrin and Metalloproteinase with Thrombospondin domains type 1, member 13 (ADAMTS13), which cleaves hyperadherent ultralarge vWF multimers under conditions of high fluid shear stress, playing an important role in maintaining an adequate balance of hemostatic activity and thrombotic risk. ADAMTS13 cleaves vWF between residues Tyr1605 and Met1606, which corresponds to residues 842-843 after cleavage of the pre-pro sequence. It is this ADAMTS13-mediated cleavage of VWF that is largely responsible for modulating the hemostatic activity and multimeric size of VWF. Petition 870200095920, dated 07 / 31 / 2020, page 12 / 151 3 / 132 VWF released through stimulation or circulation in the blood is important in the formation of platelet thrombi because it plays a role with collagen in platelet aggregation and adhesion in subendothelial tissue, including damaged vascular walls. VWF release is accompanied and partially triggered by vascular endothelial activation. Thus, biomarkers of vascular inflammation provide additional information about the risk of vaso-occlusive events.

[0005] Extracellular hemoglobin (ECHb) is increased in patients with DF and inhibits ADAMTS13-mediated VWF proteolysis by binding to the A2 domain of VWF, particularly the ADAMTS13 cleavage site. Thrombospondin 1 (TSP1), which is also increased in patients with DF, binds to the A2 domain of ultralarge VWF multimers and also prevents VWF degradation by ADAMTS13 by competitively inhibiting ADAMTS13 activity.

[0006] Sickle cell disease (SCD) is a congenital, lifelong illness. People with SCD inherit two abnormal pS hemoglobin genes, one from each parent. When a person has two hemoglobin S genes, Hemoglobin SS (Hb SS), the disease is called sickle cell anemia. This is the most common and often the most severe type of SCD. Hemoglobin SC disease and hemoglobin Sp-thalassemia are two other common forms of SCD. In all forms of SCD, at least one of the two abnormal genes causes a person's body to produce hemoglobin S, or sickle cell hemoglobin, in their red blood cells. Hemoglobin is a protein in red blood cells that carries oxygen throughout the body. Sickle cell hemoglobin differs from normal hemoglobin in its propensity to Petition 870200095920, dated 07 / 31 / 2020, page 13 / 151 4 / 132 forming polymers under low oxygen tension conditions, which form rigid rods within the red blood cell, causing it to have a sickle or crescent shape. Sickle-shaped cells are not flexible, which can cause a blockage that slows or stops blood flow and essentially obstructs microcirculation. When this happens, oxygen cannot reach nearby tissues. The lack of tissue oxygen can cause sudden attacks of severe pain, called a vaso-occlusive crisis (VOC), pain crisis, or sickle cell crisis, which results in ischemic injury to the supplied organ and resulting pain. Pain crises constitute the most distinctive clinical feature of SCC and are the leading cause of emergency department visits and hospitalizations for affected patients.

[0007] Vaso-occlusion (VOC) is initiated and sustained by interactions between sickle cells, including plasma constituents, leukocytes, endothelial cells, and sickle reticulocytes, including vanwhamnid fibroblasts (VF). Vaso-occlusion is responsible for a wide variety of clinical complications of sickle cell disease (SCD), including pain syndromes, stroke, leg ulcers, spontaneous abortion, and renal failure. VOC pain is often incompletely treated. Current treatment of VOC includes, among other things, the use of fluids, oxygen, and analgesia, while the incidence of VOC can be reduced with chronic red blood cell (RBC) transfusion, as well as hydroxyurea. Despite advances in pain management, however, physicians are often reluctant to administer appropriate dosages of narcotic analgesics to patients due to concerns about... Petition 870200095920, dated 07 / 31 / 2020, p. 14 / 151 5 / 132 Dependence, tolerance, and side effects. In addition to acute CVO, other chronic and acute complications of DF include kidney disease, splenic infarction, increased risk of bacterial infection, chronic and acute anemia, chest syndrome, stroke, and eye disease.

[0008] Acute pain in patients with sickle cell disease (SCD) is caused by ischemic tissue injury resulting from the occlusion of microvascular beds by sickle cells during an acute crisis. For example, the severe bone pain characteristic of chronic osteoporosis (CVO) is believed to be caused by increased intramedullary pressure, especially within the juxta-articular areas of long bones, secondary to an acute inflammatory response to vascular necrosis of the bone marrow by sickle cells. Pain may also occur due to involvement of the periosteum or periarticular soft tissue of the joints. The effect of unpredictable recurrences of acute crises of chronic pain creates a unique pain syndrome. The severity of sickle cell disease (SCD) varies widely 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 a person with SCD is now around 40-60 years, whereas it was only 14 years about 40 years ago. Currently, however, hematopoietic stem cell transplantation (HSCT) is the only cure for SCD. Unfortunately, most people with SCD are too old for a transplant or do not have a relative who has a good enough genetic match to be a donor for a successful transplant. Thus, there is a need in Petition 870200095920, dated 07 / 31 / 2020, page 15 / 151 6 / 132 Enhanced treatment techniques for DF, including the treatment of vaso-occlusive events in DF, that can reduce symptoms, prevent complications, and improve quality and length of life. SUMMARY OF THE INVENTION

[0009] The disclosure includes a method for treating, improving and / or preventing a vaso-occlusive crisis (VOC) in an individual who has sickle cell disease (SCD), wherein the method comprises administering to the individual in need thereof a therapeutically effective amount of a composition comprising ADAMTS13.

[0010] The disclosure includes a method for treating, improving and / or preventing lung injury in an individual who has acute lung injury (ALI) and / or acute respiratory distress syndrome (ARDS), wherein the method comprises administering to the individual in need thereof a therapeutically effective amount of a composition comprising ADAMTS13.

[0011] The disclosure includes uses of ADAMTS13 and / or compositions comprising ADAMTS13 for the preparation of medicaments. Other related aspects are also provided in the disclosure.

[0012] The disclosure provides a method for treating, improving, and / or preventing CVO in an individual who has DF, wherein the method comprises administering to the individual in need a therapeutically effective amount of a composition comprising ADAMTS13. In some modalities, the individual is treated after the symptoms of a CVO become present. In some modalities, the individual is treated before the symptoms of a crisis of Petition 870200095920, dated 07 / 31 / 2020, page 16 / 151 7 / 132 If CVOs are present, treatment reduces at least one of the following: inflammation, vasoconstriction, or platelet aggregation, or a combination of any of these. In some modalities, treatment results in at least one of the following: improved survival, improved lung function, or reduced organ damage, reduced pulmonary vascular leakage, or a combination of any of these. In some modalities, treatment reduces and / or prevents at least one of the following: impaired blood flow (e.g., ischemia), blood clotting, vascular inflammation, thrombosis, ischemic cell damage, or organ damage, or a combination of any of these. In some modalities, treatment reduces and / or prevents pain or pain severity. In some modalities, treatment reduces the frequency of occurrence of CVOs and / or the duration of CVO episodes.In certain modalities, administration of ADAMTS13 results in reduced expression, level, and / or activation of at least one of VCAM-1, ICAM-1, P-NF-kB / NF-kB ratio, ET-1, TXAS, and HO-1 in an organ. In some modalities, the comparison is with a control individual. In some modalities, the comparison is with measurements taken before treatment.

[0013] In certain modalities, the organs include, but are not limited to, lung, liver, pancreas, skin, retina, prostate, ovary, lymph node, adrenal gland, kidney, heart, gallbladder, or GI tract. In some modalities, the organ tissue includes, but is not limited to, the lungs, liver, spleen, and / or kidneys. In certain modalities, the organ is a lung. In certain modalities, the organ is a kidney. Petition 870200095920, dated 07 / 31 / 2020, page 17 / 151 8 / 132

[0014] In certain modalities, administration of ADAMTS13 results in an increase in at least one of the following blood Hct, Hb, MCV, and MCH levels and / or a reduction in at least one of the following blood MCHC, HDW, LDH, and neutrophil counts compared with the control.

[0015] In some aspects of the revelation, the therapeutically effective amount of ADAMTS13 to treat, improve, or prevent a CVO in an individual who has DF is about 20 to about 6000 international units per kilogram of body weight. In some aspects, the therapeutically effective amount is about 40 to about 4000 international units per kilogram of body weight. In some aspects, the therapeutically effective amount is about 100 to about 3000 international units per kilogram of body weight. In some aspects, the therapeutically effective amount is about 50 to about 500 international units per kilogram of body weight.

[0016] In particular aspects, the therapeutically effective dosage or amount to treat, improve, or prevent a CVO in an individual who has DF is about 10 to about 500 international units per kilogram of body weight. In some aspects, the therapeutically effective dosage or 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. Petition 870200095920, dated 07 / 31 / 2020, p. 18 / 151 9 / 132 body weight. In some respects, the therapeutically effective dosage or amount is about 100 to about 400 international units per kilogram of body weight. In some respects, the therapeutically effective amount is about 100 to about 300 international units per kilogram of body weight. In some respects, the therapeutically effective amount is about 300 to about 500 international units per kilogram of body weight. In some respects, the therapeutically effective dosage or amount is about 200 to about 300 international units per kilogram of body weight. In some respects, the therapeutically effective dosage or 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.

[0017] In additional aspects, the therapeutically effective dosage or amount to treat, improve, or prevent a CVO in an individual who has DF is about 50 to about 1000 international units per kilogram of body weight. In some aspects, the therapeutically effective dosage or amount is about 100 to about 900 international units per kilogram of body weight. In some aspects, the therapeutically effective dosage or amount is about 200 to about 800 international units per kilogram of body weight. In some aspects, the therapeutically effective dosage or amount is about 300 to about 700 international units per kilogram of body weight. In some aspects, the therapeutically effective dosage or amount is about 400 to Petition 870200095920, dated 07 / 31 / 2020, page 19 / 151 10 / 132 approximately 600 international units per kilogram of body weight. In some respects, the therapeutically effective dosage or amount is approximately 500 international units per kilogram of body weight.

[0018] In some embodiments, the composition comprising ADAMTS13, to treat, improve or prevent a CVO in an individual who has DF, is administered as a single bolus injection, monthly, every two weeks, weekly, twice a week, daily, every 12 hours, every eight hours, every six hours, every four hours or every two 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.

[0019] In some aspects of the disclosure, the therapeutically effective amount of the composition comprising ADAMTS13 is administered to the individual within 48 hours of the start of CVO. In some aspects, the therapeutically effective amount of the composition comprising ADAMTS13 is administered to the individual within 24 hours of the start of CVO. In some aspects, the therapeutically effective amount of the composition comprising ADAMTS13 is administered to the individual within 12 hours of the start of CVO. In some aspects, the therapeutically effective amount of the composition comprising ADAMTS13 is administered to the individual within 6 hours of the start of CVO.

[0020] In some aspects of the revelation, the amount Petition 870200095920, dated 07 / 31 / 2020, page 20 / 151 The therapeutically effective amount of the composition comprising ADAMTS13 to prevent CVO is sufficient to maintain an effective level of ADAMTS13 activity in the individual. In some cases, the therapeutically effective amount of the composition comprising ADAMTS13 to prevent CVO is administered monthly, bi-weekly, weekly, or twice weekly to prevent CVO. In some modes, administration is subcutaneous. In some modes, administration is intravenous.

[0021] The disclosure includes the use of a composition comprising ADAMTS13 to treat or prevent a CVO in an individual who has DF. In some embodiments, the disclosure includes a composition comprising ADAMTS13 for use as a medicine for the treatment or prevention of a CVO in an individual who has DF.

[0022] In certain modalities, the methods for treating or preventing CVO comprise (i) administering ADAMTS13 and (ii) assessing whether there has been a change in a parameter or symptom, wherein the parameter is selected from the group consisting of inflammation, vasoconstriction, platelet aggregation, pulmonary function, organ damage (e.g., lung or kidney), pulmonary vascular leakage, blood flow, blood coagulation, vascular inflammation, thrombosis, ischemic cell damage, presence of pain, severity of pain, frequency of CVO occurrence, duration of CVO episodes, VCAM-1, ICAM-1, P-NF-kB / NF-kB ratio, ET-1, TXAS, HO-1, Hct, Hb, MCV, HDW, reticulocyte counts, and neutrophil counts.

[0023] The revelation also provides a method for treating, improving, and / or preventing lung injury in an individual who Petition 870200095920, dated 07 / 31 / 2020, page 21 / 151 12 / 132 suffers or is at risk of suffering from ALI and / or ARDS, the method comprising administering to the individual in need a therapeutically effective amount of a composition comprising ADAMTS13. In some respects, the individual has a condition or a combination of conditions selected from the group consisting of inflammatory pulmonary edema, inflammatory pulmonary infiltrates, impaired oxygenation, and hypoxemia. In some respects, the treatment results in at least one of improved survival, improved lung function, or reduced organ damage, reduced pulmonary vascular leakage, or a combination of any of these. In some respects, the treatment reduces at least one of inflammation, vasoconstriction, or platelet aggregation, or a combination of any of these.In some aspects, the treatment reduces and / or prevents at least one of impaired blood flow (e.g., ischemia), blood clotting, vascular inflammation, thrombosis, ischemic cell damage, or organ damage, or a combination of any of these. In some aspects, the treatment reduces and / or prevents pain or pain severity. In some modalities, the treatment reduces the frequency of occurrence of ALI and / or ARDS and / or the duration of ALI and / or ARDS episodes. In certain modalities, ADAMTS13 administration results in reduced expression, level, and / or activation of at least one of VCAM-1, ICAM-1, PNF-kB / NF-kB ratio, ET-1, TXAS, and HO-1 in an organ. In some modalities, the comparison is with a control individual. In some modalities, the comparison is with measurements taken before treatment. Petition 870200095920, dated 07 / 31 / 2020, page 22 / 151 13 / 132

[0024] In certain modalities, the organs include, but are not limited to, lung, liver, pancreas, skin, retina, prostate, ovary, lymph node, adrenal gland, kidney, heart, gallbladder, or GI tract. In some modalities, the organ tissue includes, but is not limited to, the lungs, liver, spleen, and / or kidneys. In certain modalities, the organ is a lung. In certain modalities, the organ is a kidney.

[0025] In certain modalities, the administration of ADAMTS13 results in a reduction in blood neutrophil counts compared to the control.

[0026] In some aspects of the disclosure, the therapeutically effective amount of ADAMTS13 to treat, improve, or prevent lung injury in an individual suffering from or at risk of suffering from ALI and / or ARDS is approximately 20 to approximately 6000 international units per kilogram of body weight. In some aspects, the therapeutically effective amount is approximately 40 to approximately 4000 international units per kilogram of body weight. In some aspects, the therapeutically effective amount is approximately 100 to approximately 3000 international units per kilogram of body weight. In some aspects, the therapeutically effective amount is approximately 50 to approximately 500 international units per kilogram of body weight.

[0027] In particular, the therapeutically effective dosage or amount to treat, improve, or prevent lung injury in an individual suffering from or at risk of suffering from ALI and / or ARDS is approximately 10 to approximately 500 international units per kilogram of body weight. In Petition 870200095920, dated 07 / 31 / 2020, p. 23 / 151 14 / 132 In some aspects, the therapeutically effective dosage or quantity is about 50 to about 450 international units per kilogram of body weight. In some aspects, the therapeutically effective quantity is about 40 to about 150 international units per kilogram of body weight. In some aspects, the therapeutically effective quantity is about 100 to about 500 international units per kilogram of body weight. In some aspects, the therapeutically effective dosage or quantity is about 100 to about 400 international units per kilogram of body weight. In some aspects, the therapeutically effective quantity is about 100 to about 300 international units per kilogram of body weight. In some aspects, the therapeutically effective quantity is about 300 to about 500 international units per kilogram of body weight.In some respects, the therapeutically effective dosage or amount is about 200 to about 300 international units per kilogram of body weight. In some respects, the therapeutically effective dosage or 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.

[0028] In additional aspects, the therapeutically effective dosage or amount to treat, improve, or prevent lung injury in an individual suffering from or at risk of suffering from ALI and / or ARDS is approximately 50 to approximately 1000 international units per kilogram of body weight. In some aspects, the therapeutically effective dosage or amount Petition 870200095920, dated 07 / 31 / 2020, p. 24 / 151 The therapeutically effective dosage is approximately 100 to approximately 900 international units per kilogram of body weight. In some respects, the therapeutically effective dosage is approximately 200 to approximately 800 international units per kilogram of body weight. In some respects, the therapeutically effective dosage is approximately 300 to approximately 700 international units per kilogram of body weight. In some respects, the therapeutically effective dosage is approximately 400 to approximately 600 international units per kilogram of body weight. In some respects, the therapeutically effective dosage is approximately 500 international units per kilogram of body weight.

[0029] In some embodiments, the therapeutically effective amount of the composition comprising ADAMTS13, to treat, improve and / or prevent lung injury in an individual suffering from or at risk of suffering from ALI and / or ARDS, is administered to the individual within 48 hours of the detection of inflammatory pulmonary edema, inflammatory pulmonary infiltrates, impaired oxygenation or hypoxemia. In some embodiments, the therapeutically effective amount of the composition comprising ADAMTS13 is administered to the individual within 24 hours of the detection of inflammatory pulmonary edema, inflammatory pulmonary infiltrates, impaired oxygenation or hypoxemia. In some embodiments, the therapeutically effective amount of the composition comprising ADAMTS13 is administered to the individual within 12 hours of the detection of inflammatory pulmonary edema, inflammatory pulmonary infiltrates, impaired oxygenation Petition 870200095920, dated 07 / 31 / 2020, page 25 / 151 16 / 132 impaired or hypoxemia. In some modalities, the therapeutically effective amount of the composition comprising ADAMTS13 is administered to the individual within 6 hours of the detection of inflammatory pulmonary edema, inflammatory pulmonary infiltrates, impaired oxygenation, or hypoxemia.

[0030] In some embodiments, the composition comprising ADAMTS13 is administered as a single bolus injection, monthly, every two weeks, weekly, twice a week, daily, every 12 hours, every eight hours, every six hours, every four hours, or every two 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.

[0031] In several aspects of the revelation, ADAMTS13 is recombinant ADAMTS13. In some aspects, ADAMTS13 is plasma-derived.

[0032] In several aspects of the revelation, the individual is a mammal. In some aspects, the individual is a human being.

[0033] In some aspects, the composition is in a Stable aqueous solution ready for administration.

[0034] In some respects, the therapeutically effective amount of the composition comprising ADAMTS13 to treat, improve and / or prevent lung injury is sufficient to maintain an effective circulating level of ADAMTS13 activity in the individual.

[0035] The revelation includes the use of a composition that Petition 870200095920, dated 07 / 31 / 2020, p. 26 / 151 17 / 132 comprises ADAMTS13 to treat, improve and / or prevent lung injury in an individual who suffers from or is at risk of suffering from ALI and / or ARDS. In some respects, the individual is suffering from ALI. In some respects, the individual is suffering from ARDS.

[0036] The disclosure also includes a composition comprising ADAMTS13 for use as a medicament for the treatment, improvement or prevention of lung injury in an individual who suffers from or is at risk of suffering from ALI and / or ARDS.

[0037] In certain modalities, methods for treating or preventing ALI / ARDS include (i) administering ADAMTS13 and (ii) assessing whether there has been a change in a parameter or symptom, wherein the parameter is selected from the group consisting of inflammation, vasoconstriction, platelet aggregation, pulmonary function, organ damage (e.g., lung or kidney), pulmonary vascular leakage, blood flow, blood coagulation, vascular inflammation, thrombosis, ischemic cell damage, frequency of ALI / ARDS occurrence, duration of ALI / ARDS episodes, VCAM-1, ICAM-1, P-NF-kB / NF-kB ratio, ET-1, TXAS, HO-1, Hct, Hb, MCV, HDW, reticulocyte counts, and neutrophil counts.

[0038] The preceding summary is not intended to define every aspect of the invention, and additional aspects are described in other sections, such as the detailed description below. The entire document is intended to be reported 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 Petition 870200095920, dated 07 / 31 / 2020, p. 27 / 151 18 / 132 found together in the same sentence, paragraph, or section of this document. Other features and advantages of the invention will become apparent from the detailed description that follows. It should be understood, however, that the detailed description and specific examples, while indicating specific embodiments of the invention, are provided for illustrative purposes only, as various changes and modifications in the spirit and scope of the invention will become apparent to those skilled in the art from this detailed description. DESCRIPTION OF THE FIGURES

[0039] Figure 1 is a graph showing that ADAMTS13 protects sickle cell (SC) mice from death related to severe acute CVO. Mice (n=6) were treated with rADAMTS13 (BAX930 / SHP655 (2940 FRETS-U / kg (~3200 IU / kg))) and exposed to 7% oxygen for 10 h followed by 3 h of recovery at 21% oxygen. The survival curves for rADAMTS13-treated SC mice, vehicle-treated (healthy) AA mice, and ADAMTS13-treated AA mice were significantly different (p<0.001) from those of vehicle-treated SC mice. After 13 hours, no animals survived in the vehicle-treated SC mouse group, while 100% of the animals in all three other groups survived.

[0040] Figures 2A-2C: Figure 2A shows that mice (SS) with DF had a significantly higher number of leukocytes and a significantly higher protein content in bronchoalveolar lavage compared to controls, indicating vascular leakage. The Petition 870200095920, dated 07 / 31 / 2020, page 28 / 151 Treatment with rADAMTS 13 (BAX930 / SHP655) considerably reduced this effect, indicating a reduction in systemic inflammation and abnormalities in pulmonary vascular dysfunction. Figure 2B shows that rADAMTS 13 (BAX930 / SHP655) prevented hypoxia-induced NF-κB activation in the lungs of mice with DF and AA, indicating that rADAMTS 13 reduces the hypoxia-triggered pulmonary inflammation process. Figure 2C shows that rADAMTS 13 (BAX930 / SHP655) prevented the activation of several markers of vascular activation and inflammatory vasculopathy in the lungs of mice with DF after exposure to hypoxic conditions.

[0041] Figures 3A-B: Figure 3A shows that rADAMTS13 (BAX930 / SHP655) prevented hypoxia-induced NF-kB activation in the kidneys of mice with DF and AA, as well as in mice with DF under normoxic conditions, indicating that ADAMTS 13 reduces the hypoxia-triggered inflammation process in the kidneys as well as in the lungs.Figure 3B shows that rADAMTS13 (BAX930 / SHP655) prevented the activation of several markers of vascular activation and inflammatory vasculopathy in the kidneys of mice with DF after exposure to hypoxic conditions. DETAILED DESCRIPTION OF THE INVENTION

[0042] The disclosure provides, in several respects, ADAMTS 13 for preventing, improving and / or treating a CVO in DF. Before any embodiment of the disclosure is explained in detail, it should be understood that the invention is not limited, in its application, to the details of construction and component arrangement presented in the following description or illustrated in the figures and examples. The section headings used in this document are for general purposes. Petition 870200095920, dated 07 / 31 / 2020, p. 29 / 151 20 / 132 organizational only and should not be interpreted as limiting the subject matter described. All references cited in this application are expressly incorporated by reference herein for all purposes.

[0043] Disclosure encompasses other modalities and is practiced or performed in various ways. Furthermore, it should be understood that the phraseology and terminology used in this document serve descriptive purposes only and should not be considered limiting. The terms include, comprise, or have, and variations thereof. are considered to encompass the items listed below and additional equivalents thereof, as well as items

[0044] The following abbreviations reveal.

[0045] AA Mice Homozygous for Hemoglobin A (HbA) are used by trans all genes

[0046] ADAMTS A Disintegrin and Metalloproteinase with Thrombospondin Domains

[0047] ADAMTS13 A Disintegrin and Metalloproteinase with Thrombospondin type 1 domains, member 13

[0048] LPA Acute lung injury

[0049] ARDS Acute respiratory distress syndrome

[0050] BAL Bronchoalveolar lavage

[0051] DNA Deoxyribonucleic acid

[0052] ET-1 Endothelin 1

[0053] FRETSU Units FRETS

[0054] GAPDH Glyceraldehyde 3-phosphate dehydrogenase

[0055] HbA Hemoglobin A Petition 870200095920, dated 07 / 31 / 2020, page 30 / 151 21 / 132

[0056] HbS Sickle cell hemoglobin

[0057] HO-1 Heme-oxygenase 1

[0058] H / R Hypoxia / Reoxygenation

[0059] ICAM-1 Intercellular Adhesion Molecule 1

[0060] IU International Units

[0061] kDa Quilodálton

[0062] LDH Lactate dehydrogenase

[0063] NF-kB Nuclear kappa factor B

[0064] P-NF-kB Phosphonuclear kappa factor B

[0065] rADAMTS13 recombinant ADAMTS13

[0066] RBC Red Blood Cell

[0067] RNA Ribonucleic acid

[0068] DF Sickle cell disease

[0069] SS Mouse Transgenic mouse homozygous for HbS

[0070] TXAS Thromboxin synthase

[0071] VCAM-1 Vascular Cell Adhesion Molecule 1

[0072] CVO Vaso-occlusive crisis

[0073] FVW von Willebrand factor

[0074] It is noted here that, as used in this descriptive report and the accompanying claims, the singular forms a, an, and the include plural reference unless the context clearly indicates otherwise. With respect to aspects of the revelation described as a genus, all individual species are considered separate aspects of the revelation. If aspects of the revelation are described as comprising a feature, modalities are also contemplated as consisting of or essentially consisting of the feature.

[0075] As used in this document, the terms a Petition 870200095920, dated 07 / 31 / 2020, page 31 / 151 22 / 132 below have the meanings assigned to them, unless the context specifies otherwise.

[0076] The term sickle cell disease (SCD), as used in this document, describes a group of inherited red blood cell disorders that exist in multiple forms. Some forms of SCD are Hemoglobin SS, Hemoglobin SC, Hemoglobin Sβ0-thalassemia, Hemoglobin Sp+-thalassemia, Hemoglobin SD, and Hemoglobin SE. Although Hemoglobin SC disease and Hemoglobin Sp-thalassemia are two common forms of SCD, disclosure refers to and includes all forms of SCD.

[0077] The term vaso-occlusive crisis (VOC), as used in this document, is a sudden attack of severe pain that can occur without warning. A VOC, also known as a pain crisis or sickle cell crisis, is a common painful complication of SCD in adolescents and adults. A VOC is initiated and sustained by interactions between sickle cells, endothelial cells, and plasma constituents. Vaso-occlusion is responsible for a wide variety of clinical complications of SCD, including pain syndromes, stroke, leg ulcers, miscarriage, and / or renal failure.

[0078] The terms acute lung injury (ALI) and acute respiratory distress syndrome (ARDS) describe clinical syndromes of acute respiratory failure with substantial morbidity and mortality (Johnson et al., J. Aerosol Med. Pulmon. Drug Deliv. 23:243-52, 2010). Both ALI and more severe ARDS represent a spectrum of lung disease characterized by the sudden onset of inflammatory pulmonary edema secondary to a Petition 870200095920, dated 07 / 31 / 2020, page 32 / 151 23 / 132 a myriad of local or systemic insults, including bilateral inflammatory pulmonary infiltrates and impaired oxygenation or hypoxemia (Walkey et al., Clinical Epidemiology 4:159-69, 2012). Although ALI and ARDS are two clinical syndromes of lung disease or injury, the breakthrough refers to and includes the use of ADAMTS13 in the treatment, prevention, or improvement not only of ALI and ARDS, but of all forms of lung injury and lung disease, especially lung disease associated with impaired oxygenation.

[0079] A disintegrin and metalloproteinase with thrombospondin domains type 1, member 13 (ADAMTS13) is also known as von Willebrand factor cleavage protease (VWFPC). The term ADAMTS13 or ADAMTS13 protein, as used herein, includes analogs, variants, derivatives of ADAMTS13 (including chemically modified derivatives) and fragments thereof. In some respects, analogs, variants, derivatives and fragments thereof have increased biological activity compared to ADAMTS13. In various respects, ADAMTS13 is recombinant ADAMTS13 (rADAMTS13) or is blood-derived ADAMTS13, including plasma- and serum-derived ADAMTS13.

[0080] As used in this document, an analogue refers to a polypeptide, for example, ADAMTS13, with substantially similar structure and the same biological activity, although in certain situations to a different degree, as a naturally occurring molecule. Analogues differ in the composition of their amino acid sequences compared to the naturally occurring peptide. Petition 870200095920, dated 07 / 31 / 2020, page 33 / 151 24 / 132 natural from which the analog is derived, 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 one or more internal regions of the naturally occurring peptide sequence, (ii) insertion or addition of one or more amino acids at one or more termini (typically an addition analog) of the polypeptide and / or one or more internal regions (typically an insertion analog) of the naturally occurring peptide sequence, or (iii) substitution of one or more amino acids for other amino acids in the naturally occurring peptide sequence. Substitutions are conservative or non-conservative based on the physicochemical or functional relationship of the amino acid being substituted and the amino acid replacing it.

[0081] Conservatively modified analogs apply to both amino acid and nucleic acid sequences. With respect to particular nucleic acid sequences, conservatively modified nucleic acids refer to those nucleic acids that encode identical or essentially identical amino acid sequences, or where the nucleic acid does not encode an amino acid sequence in essentially identical sequences. Due to the degeneracy of the genetic code, a large number of functionally identical nucleic acids encode any given protein. For example, all codons GCA, GCC, GCG, and GCU encode the amino acid alanine. Thus, at any position where alanine is specified by a codon, the codon can be Petition 870200095920, dated 07 / 31 / 2020, p. 34 / 151 25 / 132 altered to any of the corresponding codons described without altering the encoded polypeptide. These nucleic acid variations are silent variations, which are a kind of conservatively modified analogs. In this document, each nucleic acid sequence encoding a polypeptide also describes every possible silent variation of the nucleic acid. A knowledgeable person will recognize that each codon in a nucleic acid (except AUG, which is commonly the single codon for methionine, and TGG, which is commonly the single codon for tryptophan) can be modified to generate a functionally identical molecule. Consequently, every silent variation of a nucleic acid encoding a polypeptide is implied in each sequence described.

[0082] As with amino acid sequences, a person skilled in the art will recognize that individual substitutions, insertions, deletions, additions, or truncations to a sequence of nucleic acids, peptides, polypeptides, or proteins that alters, adds, or deletes a single amino acid or a small percentage of amino acids in the encoded sequence is a conservatively modified analogue in which the alteration results in the substitution of one amino acid for a chemically similar amino acid. Conservative substitution tables that provide functionally similar amino acids are well known in the art. Such conservatively modified variants are additional to, and do not exclude, polymorphic variants, interspecies homologs, and revealing alleles.

[0083] Each of the following eight groups contains amino acids that are conservative substitutions for the Petition 870200095920, dated 07 / 31 / 2020, p. 35 / 151 26 / 132 other: 1) Alanine (A), Glycine (G); 2) Aspartic acid (D), Glutamic acid (E); 3) Asparagine (N), Glutamine (Q); 4) Arginine (R), Lysine (K); 5) Isoleucine (I), Leucine (L), Methionine (M), Valine (V); 6) Phenylalanine (F), Tyrosine (Y), Tryptophan (W); 7) Serine (S), Threonine (T); and 8) Cysteine ​​(C), Methionine (M) (see, for example, Creighton, Proteins (1984)).

[0084] As used in this document, a variant refers to a polypeptide, protein, or analogue thereof comprising at least one amino acid substitution, deletion, insertion, or modification, provided that the variant retains the biological activity of the native polypeptide. The term variant is, in some respects, used interchangeably with the term mutant.

[0085] As used in this document, an allelic variant refers to any one of two or more polymorphic forms of a gene occupying the same genetic locus. Allelic variations arise naturally through mutation and, in some respects, result in phenotypic polymorphism in populations. In certain respects, genetic mutations are silent (no change in the encoded polypeptide) or, in other respects, encode polypeptides that have altered amino acid sequences. Allelic variants also refer to cDNAs derived from mRNA transcripts of genetic allelic variants, as well as the proteins encoded by them.

[0086] The term derivative refers to polypeptides that Petition 870200095920, dated 07 / 31 / 2020, page 36 / 151 27 / 132 are covalently modified by conjugation with therapeutic or diagnostic agents, identification (e.g., with radionuclides or various enzymes), covalent polymer linkage such as pegylation (derivation with polyethylene glycol), and insertion or substitution by chemical synthesis of non-natural amino acids. In some respects, derivatives are modified to comprise additional chemical fractions that are not normally a part of the molecule. In certain respects, these derivatives are termed chemically modified derivatives. Such fractions, in various respects, modulate the solubility, absorption, and / or biological half-life of the molecule. The fractions, in various other respects, alternatively reduce the toxicity of the molecule and eliminate or attenuate any undesirable side effects of the molecule, etc. Fractions capable of mediating such effects are disclosed in Remington's Pharmaceutical Sciences (1980).Procedures for linking such fragments to a molecule are well known in the art. For example, in some respects, an ADAMTS13 derivative is an ADAMTS13 molecule that has a chemical modification that confers a longer in vivo half-life to the protein. In one embodiment, polypeptides are modified by the addition of a water-soluble polymer known in the art. In a related embodiment, polypeptides are modified by glycosylation, PEGylation, and / or polysialylation.

[0087] As used in this document, a fragment of a polypeptide refers to any portion of the polypeptide smaller than the full-length polypeptide or protein expression product. Fragments are typically analogous to deletions of the polypeptide. Petition 870200095920, dated 07 / 31 / 2020, page 37 / 151 28 / 132 total length, in which one or more amino acid residues have been removed from the amino terminus and / or the carboxy terminus of the total length polypeptide. Consequently, fragments are a subset of deletion analogs described below.

[0088] The term recombinant or recombinant expression system, when used in reference, for example, to a cell, indicates that the cell has been modified by the introduction of a heterologous protein or nucleic acid or by alteration of a native protein or nucleic acid, or that the cell is derived from a cell so modified. Thus, for example, recombinant cells express genes that are not found in the native (non-recombinant) form of the cell or express native genes that are otherwise abnormally expressed, underexpressed, or not expressed at all. This term also means host cells that stably integrate a recombinant genetic element, or elements, that have a regulatory function in gene expression, for example, promoters or enhancers.Recombinant expression systems, as defined herein, will express endogenous polypeptides or proteins within the cell by inducing regulatory elements linked to the endogenous gene or DNA segment to be expressed. The cells can be prokaryotic or eukaryotic.

[0089] The term recombinant, when used in this document to refer to a polypeptide or protein, means that a polypeptide or protein is derived from recombinant expression systems (e.g., microbial or mammalian). Microbial refers to polypeptides or Petition 870200095920, dated 07 / 31 / 2020, page 38 / 151 29 / 132 recombinant proteins produced in bacterial or fungal expression systems (e.g., yeast). The term recombinant variant refers to any polypeptide different from naturally occurring polypeptides created by amino acid insertions, deletions, and substitutions using recombinant DNA techniques. Guidance in determining which amino acid residues can be substituted, added, or deleted without abolishing activities of interest can be obtained by comparing the sequence of the particular polypeptide with that of homologous peptides and minimizing the number of amino acid sequence changes made in regions of high homology.

[0090] The term agent or compound describes any molecule, for example, a protein or pharmaceutical product, with the ability to affect a biological parameter in the development.

[0091] A control, as used in this document, may refer to an active, positive, negative, or vehicle control. As will be understood by those skilled in the art, controls are used to establish the relevance of experimental results and to provide a comparison for the condition being tested. In certain respects, a control is an individual who does not receive an active prophylactic or therapeutic composition. In certain respects, a control is an individual who does not experience DF, CVO, LPA, and / or SARA, for example, but without limitation to a healthy control or an individual without any symptoms.

[0092] The term reduces the severity, when in Petition 870200095920, dated 07 / 31 / 2020, page 39 / 151 30 / 132 referring to symptoms of DF, CVO in DF and / or LPA / SARA, means that the symptom has a delayed onset, reduced severity, reduced frequency, or causes less harm to the individual. Generally, the severity of a symptom is compared to a control, for example, an individual who does not receive an active prophylactic or therapeutic composition, or compared to the severity of the symptom before administration of the therapeutic product. In any case, it can be said that a composition reduces the severity of a symptom of DF, CVO in DF and / or LPA / SARA if the symptom is reduced by approximately 10%, approximately 15%, approximately 20%, approximately 25%, approximately 30%, approximately 35%, approximately 40%, approximately 50%, approximately 60%, approximately 70%, approximately 80%, approximately 90%, or approximately 100% (that is, essentially eliminated), compared to the symptom control level.In certain aspects, it can be said that a composition reduces the severity of a symptom of DF, CVO in DF and / or LPA / SARA if the symptom is reduced by approximately 10% to approximately 100%, approximately 20% to approximately 90%, approximately 30% to approximately 80%, approximately 40% to approximately 70%, or approximately 50% to approximately 60%, compared to the symptom control level. In certain respects, it can be said that a composition reduces the severity of a symptom of DF, CVO in DF and / or LPA / SARA if the symptom is reduced by approximately 10% to approximately 30%, approximately 20% to approximately 40%, approximately 30% to approximately 50%, approximately 40% to approximately 60%, approximately 50% to approximately 70%, approximately 60% to approximately 80%, approximately 70% to approximately 90%, or approximately % to approximately 100%, compared to the level of symptom control. In some respects, treatment by... Petition 870200095920, dated 07 / 31 / 2020, p. 40 / 151 31 / 132 methods of disclosure reduce the severity of pain and / or other symptoms of CVO in DF and / or LPA / SARA.

[0093] The terms reduced expression, reduced level, and reduced activation when referring to a biomarker of DF, CVO in DF and / or LPA / SARA (e.g., but not limited to VCAM-1, ICAM-1, P-NF-kB / NF-kB ratio, ET-1, TXAS, HO-1, Hct, Hb, MCV, HDW, reticulocyte numbers, and neutrophil numbers) mean that the expression, level, and / or activation of a biomarker has been reduced compared to the control. In any case, it can be said that a composition reduces the expression, level and / or activation of a biomarker of DF, CVO in DF and / or LPA / SARA if the biomarker is reduced by approximately 10%, approximately 15%, approximately 20%, approximately 25%, approximately 30%, approximately 35%, approximately 40%, approximately 50%, approximately 60%, approximately 70%, approximately 80%, approximately 90%, or approximately 100% (i.e., essentially eliminated), compared to the control.In certain aspects, it can be said that a composition reduces the expression, level, and / or activation of DF, CVO in DF, and / or LPA / SARA if the expression, level, and / or activation is reduced by approximately 10% to approximately 100%, approximately 20% to approximately 90%, approximately 30% to approximately 80%, approximately 40% to approximately 70%, or approximately 50% to approximately 60%, compared to the control. In certain aspects, it can be said that a composition reduces the expression, level and / or activation of a biomarker of DF, CVO in DF and / or LPA / SARA if the biomarker is reduced by between approximately 10% to approximately 30%, approximately 20% to approximately 40%, approximately 30% to approximately 50%, approximately 40% to approximately 60%, approximately 50% to approximately 70%, approximately 60% to approximately 80%, approximately 70%. Petition 870200095920, dated 07 / 31 / 2020, p. 41 / 151 32 / 132% to about 90% or about 80% to about 100%, compared to the control.

[0094] The terms increase expression, increase level, and increase activation when referring to a biomarker of DF, CVO in DF, and / or LPA / SARA, mean that the expression, level, and / or activation of a biomarker has been increased compared to the control. In any case, a composition can be said to increase the expression, level, and / or activation of a biomarker of DF, CVO in DF, and / or LPA / SARA if the biomarker is increased by approximately 10%, approximately 15%, approximately 20%, approximately 25%, approximately 30%, approximately %, approximately 40%, approximately 50%, approximately 60%, approximately %, approximately 80%, approximately 90%, or approximately 100% (i.e., essentially eliminated), compared to the control. In certain aspects, it can be said that a composition increases the expression, level, and / or activation of DF, CVO in DF, and / or LPA / SARA if the expression, level, and / or activation is increased by approximately 10% to approximately 100%, approximately 20% to approximately 90%, approximately 30% to approximately 80%, approximately 40% to approximately 70%, or approximately 50% to approximately 60%, compared to the control. In certain aspects, it can be said that a composition increases the expression, level, and / or activation of a biomarker of DF, CVO in DF, and / or LPA / SARA if the biomarker is increased by approximately 10% to approximately 30%, approximately 20% to approximately 40%, approximately 30% to approximately 50%, approximately 40% to approximately 60%, approximately 50% to approximately 70%, approximately 60% to approximately 80%, approximately 70% to approximately 90%, or approximately 80% to approximately 100%, compared to the control.

[0095] Each of the terms effective quantity and Petition 870200095920, dated 07 / 31 / 2020, p. 42 / 151 33 / 132 Therapeutically effective amount refers to the amount of polypeptide, for example, ADAMTS13 polypeptide, or composition used to sustain an observable level of one or more biological activities of ADAMTS13 polypeptide, as presented herein. For example, an effective amount, in some aspects of the development, would be the amount needed to treat or prevent CVO symptoms in DF and / or LPA / SARA.

[0096] An individual is conventionally defined as a non-plant, non-protist living being. In most respects, the individual is an animal. In particular respects, the animal is a mammal. In even more particular respects, the mammal is a human being. In other respects, the mammal is a pet or companion animal, a domesticated farm animal, or a zoo animal. In certain respects, the mammal is a mouse, rat, rabbit, guinea pig, pig, or non-human primate. In other respects, the mammal is a cat, dog, horse, or cow. In various other respects, the mammal is a deer, mouse, chipmunk, squirrel, opossum, or raccoon.

[0097] It is also specifically understood that any numerical value cited in this document includes all values ​​from the lowest to the highest value, that is, all possible combinations of numerical values ​​between the lowest and highest enumerated values ​​should be considered as being expressly stated in this application. For example, if a concentration range is stated as approximately 1% to 50%, it is intended that values ​​such as 2% to 40%, 10% to 30%, or 1% to 3%, etc., should be considered. Petition 870200095920, dated 07 / 31 / 2020, page 43 / 151 34 / 132 expressly listed in this descriptive report. The values ​​listed above are only examples of what is specifically intended.

[0098] Ranges, in various respects, are expressed in this document as about or approximately a particular value and / or at or approximately another particular value. When values ​​are expressed as approximations, using the antecedent about, it will be understood that some amount of variation is included in the range. Such a range may be within an order of magnitude, preferably within 50%, more preferably within 20%, even more preferably within 10%, and with even greater preference, within 5% of a given value or range. The permissible variation encompassed by the term about or approximately depends on the particular system under study, and can be readily verified by a person of ordinary skill in the art. Sickle Cell Disease and Vaso-Occlusion in Sickle Cell Disease

[0099] In some respects, the disclosure includes ADAMTS13 and compositions comprising ADAMTS13 in the treatment, improvement, and / or prevention of CVO in SCD. SCD is a hereditary red blood cell disorder distributed worldwide caused by a point mutation in the β-globin gene that results in pathological HbS synthesis and abnormal HbS polymerization under hypoxic conditions. The two clinical manifestations of SCD are chronic hemolytic anemia and acute CVO, which are the main causes of hospitalization for patients with SCD. Recent studies have reinforced the central role of sickle cell vasculopathy in generating acute events. Petition 870200095920, dated 07 / 31 / 2020, page 44 / 151 35 / 132 related to sickle cell disease 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 CVO, impaired blood flow, vascular inflammation and / or thrombosis with ischemic cell damage.

[0100] The most common clinical manifestation of SCD is CVO. A CVO occurs when the microcirculation is obstructed by sickle cells, causing ischemic damage to the supplied organ and resulting pain. Pain crises constitute the most distinctive clinical feature of SCD and are the main cause of emergency department visits and / or hospitalizations of patients or individuals affected by SCD.

[0101] Approximately half of patients or individuals with SCD who have homozygous HbS disease experience OVC. The frequency of attacks is extremely variable. Some patients or individuals with SCD have up to six or more episodes per year, while others may have episodes only at long intervals or none at all. Each patient or individual typically has a consistent pattern for the frequency of attacks.

[0102] The disclosure includes methods for reducing at least one symptom of CVO including, but not limited to, ischemia and pain (e.g., dactylitis, priapism, abdominal, chest, and joint pain), jaundice, bone infarction, abnormal breathing (e.g., tachypnea and shortness of breath), Petition 870200095920, dated 07 / 31 / 2020, page 45 / 151 36 / 132 Hypoxia, acidosis, hypotension, and / or tachycardia associated with CVO. In certain aspects, CVO can be defined as a condition comprising one or more of these symptoms. Pain crises begin suddenly. The crisis can last from several hours to several days and end as abruptly as it began. The pain can affect any part of the body and frequently involves the abdomen, appendix, chest, back, bones, joints, and soft tissue and may present as dactylitis (both feet and / or hands swollen and painful in children), acute joint necrosis or avascular necrosis, or acute abdominal necrosis. With repeated episodes in the spleen, infarctions and autosplenectomy predisposing to potentially fatal infection are usual. The liver may also infarct and progress to failure over time. Papillary necrosis is a common renal manifestation of CVO, leading to isosthenuria (i.e., inability to concentrate urine).

[0103] Severe deep pain is present in the extremities, involving long bones. Abdominal pain may be severe, resembling acute abdominal pain; this may result from pain originating elsewhere or from infarction of intra-abdominal soft tissue or solid organ. Reactive ileus leads to pain and intestinal distension. The face may also be involved. Pain may be accompanied by fever, malaise, difficulty breathing, painful erections, jaundice, and leukocytosis. Bone pain is frequently due to bone marrow infarction. Certain patterns are predictable, as pain tends to involve bones with the highest bone marrow activity and because bone marrow activity changes with age. During the first 18 Petition 870200095920, dated 07 / 31 / 2020, p. 46 / 151 Between 37 and 132 months of age, the metatarsals and metacarpals may be involved, presenting as dactylitis or hand-foot syndrome. Although the above patterns describe commonly found presentations, any area of ​​the individual's body with a blood supply and sensory nerves can be affected in CVO.

[0104] Frequently, no precipitating cause can be identified in relation to what causes CVO. However, since deoxygenated HbS becomes semi-solid, the most likely physiological trigger for CVO is hypoxemia. This may be due to acute chest syndrome or accompany respiratory complications. Dehydration can also precipitate pain, as acidosis results in an alteration of the oxygen dissociation curve (Bohr effect), causing hemoglobin to desaturate more rapidly. Hemoconcentration is also a common mechanism. Another common trigger for CVO is changes in body temperature, either an increase due to fever or a decrease due to a change in ambient temperature. A decrease in body temperature likely leads to seizures as a result of peripheral vasoconstriction.

[0105] In certain modalities, CVO can be defined as having an increase in peripheral neutrophils compared to a control. In certain modalities, CVO can be defined as an increase in pulmonary vascular leakage (e.g., increased number of leukocytes in a bronchoalveolar lavage (BAL) and / or protein content (protein in BAL (mg / ml)) compared to a control.

[0106] In certain modalities, the increase in levels Petition 870200095920, dated 07 / 31 / 2020, page 47 / 151 38 / 132 vascular activation (e.g., as measured by increased expression, levels, and / or activity of VCAM-1 and / or ICAM-1) in an organ, compared to control, is a marker for CVO. In certain modalities, increased levels of inflammatory vasculopathy (e.g., as measured by increased expression, levels, and / or activity of VCAM-1 and / or ICAM-1) in an organ, compared to control, is a marker for CVO. In certain modalities, increased levels of vascular activation and inflammatory vasculopathy in a tissue, compared to control, is a marker for CVO. In certain modalities, the organ is the lung and / or kidney. In certain modalities, the organ is the kidney.

[0107] In certain modalities, CVO can be defined as increased expression, levels, and / or activation of at least one of NF-κB (where NF-κB activation is measured by P-NF-κB or the P-NF-κB / NF-κB ratio), VCAM-1, and ICAM-1 compared to control. In certain modalities, CVO can be defined as increased expression or level of at least one of endothelin-1 (ET-1), thromboxane synthase (TXAS), and heme oxygenase-1 (HO-1) compared to control. In certain modalities, these increases are seen in lung tissue. In certain modalities, these increases are seen in kidney tissue. In certain modalities, increased expression and / or levels of TXAS, ET-1, and VCAM-1 and NF-κB activation in kidney tissue are markers for CVO.

[0108] In certain modalities, CVO can be defined by hematological parameters. In certain modalities, CVO can be defined as a reduction in Petition 870200095920, dated 07 / 31 / 2020, p. 48 / 151 39 / 132 levels of at least one of Hct, Hb, MCV, and MCH compared to the control. In certain modalities, CVO may be defined as a reduction in the levels of at least two of Hct, Hb, MCV, and MCH compared to the control. In certain modalities, CVO may be defined as a reduction in the levels of at least three of Hct, Hb, MCV, and MCH compared to the control. In certain modalities, CVO may be defined as an increase in the levels of at least one of MCHC, HDW, neutrophil counts, and LDH compared to the control. In certain modalities, CVO may be defined as an increase in the levels of at least two of MCHC, HDW, neutrophil counts, and LDH compared to the control. In certain modalities, CVO may be defined as an increase in the levels of at least three of MCHC, HDW, neutrophil counts, and LDH compared to the control.In certain modalities, CVO can be defined as a reduction in Hct levels compared to the control. In certain modalities, CVO can be defined as a reduction in Hb levels compared to the control. In certain modalities, CVO can be defined as a reduction in MCV compared to the control. In certain modalities, CVO can be defined as a reduction in MCH compared to the control. In certain modalities, CVO can be defined as an increase in MCHC compared to the control. In certain modalities, CVO can be defined as an increase in HDW compared to the control. In certain modalities, CVO can be defined as an increase in neutrophil numbers compared to the control. Petition 870200095920, dated 07 / 31 / 2020, page 49 / 151 40 / 132 control. In certain modalities, CVO can be defined as an increase in LDH compared to the control. In certain modalities, CVO can be defined as a reduction in the levels of at least one of Hct, Hb, MCV, and MCH compared to the control, and / or an increase in the levels of at least one of MCHC, HDW, neutrophil counts, and LDH compared to the control. In certain modalities, CVO can be defined as a reduction in the levels of Hct, Hb, MCV, and MCH compared to the control, and / or an increase in the levels of MCHC, HDW, neutrophil counts, and LDH compared to the control. Models of DF and Methods for Testing the Effectiveness of Prophylaxis or Treatment

[0109] In some embodiments, the disclosure includes studying the effects of a recombinant ADAMTS13 (i.e., BAX930 / SHP655) in a mouse model of sickle cell disease (Tim Townes mouse) during events related to acute sickle cell disease, simulated by exposing sickle cell mice to hypoxia. Studies are conducted under normoxic and hypoxic conditions, in which the efficacy of the prophylaxis or treatment dose(s) in the mouse model (including measurement of overall survival) and biological effects of the BAX930 / SHP655 treatment(s) on lung injury and vascular inflammation are studied after exposing sickle cell mice to hypoxia.

[0110] In some embodiments, a transgenic mouse model with DF is used (Kalish et al., Haematologica 100:870-80, 2015). In some respects, healthy control mice (Hbatm1(HBA)TowHbbtmyHBGyHB^Tow) and mice with DF are used. Petition 870200095920, dated 07 / 31 / 2020, p. 50 / 151 41 / 132 (Hbatmi(HBA)Tow Hbbtm2 (HBGi,HBB*)Tow) are exposed to hypoxia / reoxygenation (H / R) stress (Kalish et al., infra). This H / R stress has been shown to biologically recapitulate the acute CVO and organ damage observed in acute CVO in human DF patients. In some respects, healthy (AA) and DF (SS) mice are subjected to hypoxia (e.g., about 7 or 8% oxygen) for specified periods of time (e.g., about 10 hours) followed by specified periods of time (e.g., 3 hours) of reoxygenation (e.g., about 21% oxygen, ambient air condition) (Kalish et al., infra).

[0111] In several respects, DF models and controls are subjected to normoxia or hypoxia conditions. In normoxia experiments, healthy control (AA) and DF (SS) mice receive a single intravenous administration of rADAMTS13 (e.g., 2940 FRETS-U / kg (~3200 IU / kg)) or buffer (vehicle) at a fixed volume (e.g., 10 ml / kg) and are subjected to normoxic conditions (e.g., approximately 21% oxygen, room air condition). The animals are studied for varying periods of time after treatment with ADAMTS13 or vehicle and exposure to normoxia or hypoxia. Blood is collected, and a complete blood count (CBC) is measured. A CBC is a blood test used to assess general health and detect a wide range of disorders, including, among other things, anemia. Several other indicators, including but not limited to hematology, coagulation parameters, inflammatory biomarkers, vasculopathy, and histopathology are measured. Petition 870200095920, dated 07 / 31 / 2020, p. 51 / 151 42 / 132

[0112] By way of example, hypoxia experiments are performed in which healthy (AA) and DF (SS) control mice receive a single intravenous administration of ADAMTS13 (e.g., 500 IU / kg, 1000 IU / kg, or 3200 IU / kg) or vehicle at a fixed volume (e.g., 10 ml / kg). In certain embodiments, the dose administered to a human subject is approximately 10% of that administered to a rodent subject (e.g., mouse). In certain embodiments, the dose administered to a human subject is approximately 9% of that administered to a rodent subject (e.g., mouse). In certain embodiments, the dose administered to a human subject is approximately 8% of that administered to a rodent subject (e.g., mouse). In certain embodiments, the dose administered to a human subject is approximately 7% of that administered to a rodent subject (e.g., mouse).In certain embodiments, the dose administered to a human individual is less than about 10%, for example, about 7% to about 10%, of that administered to a rodent individual (e.g., mouse).

[0113] 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 a period of reoxygenation time (e.g., about 3 hours) to simulate VOC events related to DF. In some respects, the same parameters as detailed for normoxic studies are evaluated. Petition 870200095920, dated 07 / 31 / 2020, page 52 / 151 43 / 132

[0114] In further illustrative aspects, hypoxia experiments are performed, in which healthy control mice (AA) and mice with DF (SS) are exposed to hypoxia (e.g., about 8% oxygen, or more) for a period of time (e.g., about 10 hours) followed by a period of reoxygenation time (e.g., about 3 hours) to simulate VOC events related to DF. Then, at various subsequent time points including, but not limited to, immediately after, or approximately 1, 3, 6, 12, 24, 36, 48, or 72 hours after the experimentally induced vaso-occlusive event, mice receive a single intravenous administration of ADAMTS13 (e.g., 500 IU / kg, 1000 IU / kg, or 3200 IU / kg) or vehicle at a fixed volume (e.g., 10 ml / kg), or multiple injections at 12- or 24-hour intervals. In some respects, the same parameters as detailed for normoxic studies are evaluated.

[0115] In several respects, any target tissue is examined for the efficacy of treatment with ADAMTS13 in in vitro or in vivo models and / or under CVO conditions. In some respects, organ tissue includes, but is not limited to, lung, liver, pancreas, skin, retina, prostate, ovary, lymph node, adrenal gland, kidney, heart, gallbladder, or GI tract. In some respects, organ tissue includes, but is not limited to, the lungs, liver, spleen, and / or kidneys.

[0116] For example, in some aspects, target tissues are collected to examine the effects of ADAMTS13 under normoxia or hypoxia conditions. The tissues are frozen and / or formalin-fixed. The frozen tissues are used to Petition 870200095920, dated 07 / 31 / 2020, page 53 / 151 44 / 132 Immunoblot analysis with specific antibodies against nuclear factor kappa B (NF-κB), endothelin-1 (ET-1), heme oxygenase 1 (HO-1), intracellular 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).

[0117] In some modalities, markers of vasoconstriction, platelet aggregation, inflammation, oxidative stress, antioxidant response, and / or tissue damage are measured to determine treatment efficacy. In some aspects, nuclear factor kappa B is measured in its normal (NF-κB) and activated (P-NF-κB) forms. NF-κB is a transcription factor that has been described to coordinate the inflammatory and antioxidant response. The ratio between the activated and normal forms is evaluated. In some aspects, ET-1 is measured. ET-1 is a potent vasoconstrictor that is 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 the inducible rate-limiting enzyme in heme catabolism and can attenuate the severity of vaso-occlusive and hemolytic crisis outcomes by acting as a vasoprotective antioxidant.In some aspects, ICAM-1 is measured. ICAM-1 is continuously present in low concentrations on 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 CVO by promoting leukocyte adhesion. In some aspects, TXAS is measured. TXAS is a... Petition 870200095920, dated 07 / 31 / 2020, p. 54 / 151 45 / 132 endoplasmic reticulum membrane protein that catalyzes the conversion of prostaglandin H2 to thromboxane A2. TXAS is a potent vasoconstrictor and inducer of platelet aggregation. Thus, 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 therefore may contribute to vaso-occlusive events. In some aspects, inflammatory cellular infiltrates are measured in organ tissue.

[0118] By way of example, immunoblot analyses with antibodies specific for NF-kB, ET-1, HO-1, ICAM-1, TXAS, and VCAM-1 are performed to measure the expression of these enzymes in cells and tissues of development models or individuals to determine treatment efficacy. By way of example, the expression of NF-kB, ET-1, HO-1, ICAM-1, TXAS, and / or VCAM-1 is measured in organ tissue from mice with DF and AA treated with vehicle or ADAMTS13. 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 GI tract. In certain embodiments, the organ is lung, liver, spleen, and / or kidney.

[0119] In certain modalities, the administration of ADAMTS13 results in reduced levels of vascular activation and / or inflammatory vasculopathy in an organ compared Petition 870200095920, dated 07 / 31 / 2020, page 55 / 151 46 / 132 with the control. In certain modalities, the organ is the lung. In certain modalities, the organ is the kidney.

[0120] In certain embodiments, administration of ADAMTS13 results in a reduction in the expression, level, and / or activation of at least one of VCAM-1, ICAM-1, NF-kB (where the reduction in NF-kB activation is measured by P-NF-kB or the P-NF-kB / NF-kB ratio), ET-1, TXAS, and HO-1 compared to the control. In certain embodiments, administration of ADAMTS13 results in a reduction in the expression, level, and / or activation of at least two of VCAM-1, ICAM-1, NF-kB, ET-1, TXAS, and HO-1 compared to the control. In certain embodiments, administration of ADAMTS13 results in a reduction in the expression, level, and / or activation of at least three of VCAM-1, ICAM-1, NF-kB, ET-1, TXAS, and HO-1 compared to the control. In certain modalities, administration of ADAMTS13 results in a reduction in the expression, level, and / or activation of at least four of VCAM-1, ICAM-1, NF-kB, ET-1, TXAS, and HO-1 compared to the control.In certain modalities, ADAMTS13 administration results in reduced expression, level, and / or activation of at least five of VCAM-1, ICAM-1, NF-κB, ET-1, TXAS, and HO-1 compared to the control. In certain modalities, ADAMTS13 administration results in reduced expression, level, and / or activation of VCAM-1, ICAM-1, NF-κB, ET-1, TXAS, and HO-1 compared to the control. In certain modalities, ADAMTS13 administration results in reduced expression, level, and / or activation of VCAM-1 compared to the control. In certain modalities, ADAMTS13 administration results in reduced expression, level, and / or activation of... Petition 870200095920, dated 07 / 31 / 2020, page 56 / 151 47 / 132 ICAM-1 compared to control. In certain modalities, ADAMTS13 administration results in reduced expression, level, and / or activation of VCAM-1 and ICAM-1 compared to control. In certain modalities, ADAMTS13 administration results in reduced expression and / or level of ET-1 compared to control. In certain modalities, ADAMTS13 administration results in reduced expression and / or level of TXAS compared to control. In certain modalities, ADAMTS13 administration results in reduced expression and / or level of HO-1 compared to control. In certain modalities, ADAMTS13 administration results in a reduction in the P-NF-kB / NF-kB ratio compared to control. In certain modalities, the administration of ADAMTS13 results in a reduction of at least one of the following: P-NF-kB / NF-kB ratio, ET-1 expression and / or level, TXAS expression and / or level, and HO-1 expression and / or level compared to the control.In certain modalities, administration of ADAMTS13 results in a reduction in the P-NF-kB / NF-kB ratio, ET-1 expression and / or level, TXAS expression and / or level, and HO-1 expression and / or level compared to the control. In certain modalities, the organ is the lung. In certain modalities, the organ is the kidney.

[0121] In further exemplary aspects, the measurement of these markers is performed after the animal models have been subjected to hypoxia and reoxygenation (H / R) conditions as described in this document. In further exemplary aspects, the measurement of these markers is performed after the individuals have experienced Petition 870200095920, dated 07 / 31 / 2020, page 57 / 151 48 / 132 CVO.

[0122] In some modalities, blood flow is measured as an indicator of treatment effectiveness. In some modalities, blood flow is measured, but not limited to, ultrasound, PET, fMRI, MRI, laser Doppler, electromagnetic blood flow meter, or a wearable device.

[0123] In some modalities, the reduction or prevention of thrombosis is a measure of treatment efficacy. In some modalities, the presence of thrombosis is measured, but not limited to, histopathological examination, ultrasound, D-dimer test, venography, MRI, or CT / CAT scan. In some aspects, thrombus formation is determined in the organ tissue.

[0124] In some modalities, the reduction or prevention of pulmonary vascular leak (i.e., lung damage and leakage) is a measure of treatment efficacy. In some modalities, measurements or parameters (total protein and leukocyte count) of bronchoalveolar lavage (BAL) are measured as markers of pulmonary vascular leak (to determine the extent of lung damage and treatment efficacy (e.g., treatment with ADAMTS13)). Pulmonary leak can result in an increase in leukocyte and protein content in the BAL. BAL fluids are collected, and the cell count is recovered by centrifugation and counted by microcytometry as previously reported (Kalish et al., Haematologica 100:870-80, 2015, incorporated herein by reference in its entirety for all purposes). In some modalities, the reduction or prevention Petition 870200095920, dated 07 / 31 / 2020, page 58 / 151 A 49 / 132 increase in peripheral neutrophils is a measure of treatment efficacy. The percentage of neutrophils is determined by cytopsin centrifugation, and the supernatant fluids are used to determine the total protein content (Kalish et al., supra).

[0125] In some modalities, the improvement in lung function is measured as an indicator of treatment effectiveness. Lung function can be measured, but is not limited to, a peak flow test, a reversibility test and spirometry, a lung volume test, a gas transfer test, a respiratory muscle test, an exhaled carbon monoxide test, or an exhaled nitric oxide test.

[0126] In some modalities, hematological parameters are measured to determine treatment efficacy (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 erythrocyte viability; hemoglobin (Hb), mean corpuscular hemoglobin (MCH), and hemoglobin concentration (MCHC) as indicators of oxygen-binding capacity; red cell distribution heterogeneity (HDW) as an indicator of the presence of dense red cells; reticulocyte count as an indicator of anemia status; and neutrophil count as an indicator of systemic inflammatory status.

[0127] In certain modalities, ADAMTS13 administration improves the reduction of blood levels of at least one of Hct, Hb, MCV, and MCH compared to Petition 870200095920, dated 07 / 31 / 2020, p. 59 / 151 50 / 132 control. In certain modalities, ADAMTS13 administration improves the reduction of at least two of the following blood Hct, Hb, MCV, and MCH levels compared to the control. In certain modalities, ADAMTS13 administration improves the reduction of at least three of the following blood Hct, Hb, MCV, and MCH levels compared to the control. In certain modalities, ADAMTS13 administration improves the reduction of blood Hct, Hb, MCV, and MCH levels compared to the control. In certain modalities, ADAMTS13 administration improves the increase of at least one of the following MCHC, HDW, LDH, and neutrophil count compared to the control. In certain modalities, ADAMTS13 administration improves the increase of at least two of the following MCHC, HDW, LDH, and neutrophil count compared to the control.In certain modalities, ADAMTS13 administration improves the increase in at least three of MCHC, HDW, LDH, and neutrophil count compared to control. In certain modalities, ADAMTS13 administration improves the increase in MCHC, HDW, LDH, and neutrophil count compared to control. In certain modalities, ADAMTS13 improves the reduction in Hct, Hb, MCV, and MCH levels and improves the increase in MCHC, HDW, LDH, and neutrophil levels compared to control.

[0128] In certain embodiments, administration of ADAMTS13 results in an increase in the levels of at least one of Hct, Hb, MCV, and MCH in the blood compared to the control. In certain embodiments, administration of ADAMTS13 results in an increase in the levels of at least two of Hct, Hb, MCV, and MCH in the blood compared to Petition 870200095920, dated 07 / 31 / 2020, page 60 / 151 51 / 132 the control. In certain modalities, administration of ADAMTS13 results in an increase in the levels of at least three of Hct, Hb, MCV, and MCH in the blood compared to the control. In certain modalities, administration of ADAMTS13 results in an increase in the levels of Hct, Hb, MCV, and MCH in the blood compared to the control. In certain modalities, administration of ADAMTS13 results in a reduction in at least one of MCHC, HDW, LDH, and neutrophil count compared to the control. In certain modalities, administration of ADAMTS13 results in a reduction in at least two of MCHC, HDW, LDH, and neutrophil count compared to the control. In certain modalities, administration of ADAMTS13 results in a reduction in at least three of MCHC, HDW, LDH, and neutrophil count compared to the control.In certain modalities, ADAMTS13 administration results in a reduction in MCHC, HDW, LDH, and neutrophil count compared to the control. In certain modalities, ADAMTS13 results in an increase in Hct, Hb, MCV, and MCH levels and a reduction in MCHC, HDW, LDH, and neutrophil count compared to the control.

[0129] In some modalities, methods for measuring VWF levels and ultralarge VWF multimers are used. In patients with DF, increased levels of VWF factor and ultralarge VWF multimers have been observed and are associated with acute vaso-occlusive events. Increased levels of circulating VWF multimers are dependent on the activity of ADAMTS13, which cleaves hyperadherent VWF under high fluid shear stress conditions, playing an important role in maintaining a Petition 870200095920, dated 07 / 31 / 2020, page 61 / 151 52 / 132 adequate balance of hemostatic activity and thrombotic risk. More specifically, ADAMTS13 cleaves VWF between amino acid residues Tyr1605 and Met1606, which correspond to amino acid residues 842-843 after cleavage of the pre-pro sequence. It is this ADAMTS13-mediated cleavage that is largely responsible for the size of VWF multimers, which correlates with primary hemostatic activity. Measurement methods for VWF and ultralarge VWF multimers, including various types of immunoblot analyses with specific antibodies against VWF, are performed to measure VWF expression or level. Additionally, other known VWF measurement methods are included in various aspects of the research.

[0130] In some respects, efficacy is measured by reduction of organ damage compared to control or reference measurements. In some modalities, organ damage is measured by radiological imaging such as, but not limited to, CT / CAT scan, ultrasound, X-ray, MRI, and nuclear medicine. In some modalities, organ damage is measured by a change in several biomarkers including, but not limited to, blood urea nitrogen (BUN), creatinine, BUN / creatinine ratio, troponin, neuron-specific enolase (NSE). In some modalities, tissue changes are measured by histopathological examination.

[0131] A person of ordinary skill in the art is able to select an appropriate measurement of any biomarker disclosed herein associated with the organ (defined above) and / or body fluid to be measured. Body fluids include, but are not limited to, blood (including blood plasma and blood serum), lymph, Petition 870200095920, dated 07 / 31 / 2020, p. 62 / 151 53 / 132 cerebrospinal fluid, lactation products (e.g., milk), amniotic fluids, urine, saliva, perspiration, tears, menstruation, feces and including fractions thereof.

[0132] In some respects, effectiveness is measured by assessing the individual’s Quality of Life (e.g., using the Adult Sickle Cell Quality-of-Life Measurement Information System (ASCQ-Me) as reported by Treadwell et al., Clin. J. Pain 30(10):902-915 (2016)).The ASCQ-Me focuses around seven topics: emotional impact (five-question survey related to emotional distress (e.g., hopelessness, loneliness, depression, and worry); severity and frequency of pain episodes (number of episodes, time since the last episode; severity of pain in the last attack on a scale of 1-10); how long the attack lasted, how much the attack impacted your life); impact of pain (questions about frequency and severity and how much it impacted activities); sickle cell disease medical history checklist; impact on sleep (how easy it was to fall asleep, how often you can't fall asleep); impact on social functioning (trust in others, how health impacted activities); and impact on stiffness (stiff joints causing insomnia, daytime movement, movement while awake).

[0133] In several respects, the effectiveness of prophylaxis and / or treatment is determined by measuring the severity of pain (e.g., as measured by a pain rating scale), pain relief, perceived need for medication, satisfaction with treatment, and the frequency of occurrence of Petition 870200095920, dated 07 / 31 / 2020, p. 63 / 151 54 / 132 CVO, the duration of the CVO episode, the extent and / or duration of hospitalization, costs associated with the hospital stay, and / or the duration of pain medication requirements (e.g., IV opioids).

[0134] In certain aspects, pain severity is measured using the McGill / Melzack Pain Questionnaire (Melzack et al., Pain set 1975; 1(3):277-99), in which the individual selects one or more words that best describe their pain. In certain aspects, pain severity is measured using the Visual Analogue Scale (VAS). The VAS is a 10 cm unshaded line, anchored at one end as no pain and at the other end as the worst possible pain. Patients are instructed to mark their pain level on the line between the two anchors. VAS ratings are calculated by measuring the distance, in centimeters, between the no-pain anchor and the patient's mark indicating their pain level, resulting in a pain severity rating ranging from 0 mm to 10 cm. In certain aspects, pain severity is measured using the Numerical Rating Scale (NRS). The NRS is an 11-point scale anchored between no pain and worst possible pain.Patients are instructed to report their current pain level on a scale of 0 to 10, where 0 means no pain and 10 means the worst possible pain.

[0135] In certain respects, pain relief can be measured as a global assessment of how a patient's pain may have changed since the last assessment (i.e., current assessment minus previous assessment), as used to anchor changes observed on the NRS and VAS scales. Patients reported pain relief in response to Petition 870200095920, dated 07 / 31 / 2020, page 64 / 151 55 / 132 question: Compared to the last time you rated your pain, how much has your pain changed? Patients could respond that their pain was much worse, a little worse, the same, a little better, or much better.

[0136] In certain aspects, the need for medication may be reported by the patient or by the healthcare professional.

[0137] In certain aspects, satisfaction with treatment can be reported by the patient. The report can be on a scale of not at all, somewhat satisfied (happy), very satisfied (happy), or I don't know. Acute Lung Injury and Acute Respiratory Failure Syndrome

[0138] In some embodiments, the disclosure includes ADAMTS13, compositions comprising ADAMTS13, and methods of using ADAMTS13 in the treatment, improvement, and / or prevention of acute lung injury (ALI) and acute respiratory distress syndrome (ARDS), including ventilator-associated lung injury resulting from ALI. The pathogenesis of ALI / ARDS is explained by injury to the vascular endothelium and alveolar epithelium. Phase III clinical trials by NHLBI SARA Network resulted in improved survival and a reduction in the duration of mechanical ventilation with a protective lung ventilation strategy and fluid-conserving protocol. However, there is a strong unmet medical need for additional treatments because there are no existing specific pharmacological therapies for ALI / ARDS. Therefore, the use of ADAMTS13 in the treatment of ALI / ARDS represents an advance in the treatment of ALI / ARDS. Petition 870200095920, dated 07 / 31 / 2020, p. 65 / 151 56 / 132

[0139] In some respects, APL is an acute inflammatory disorder that causes disruption of the pulmonary epithelial and endothelial barriers. Cellular features of APL include loss of alveolar-capillary membrane integrity, excessive transepithelial migration of neutrophils, and release of proinflammatory cytotoxic mediators. Several studies have documented increased VWF release and upregulation of intracellular adhesion molecule 1 (ICAM-1) following endothelial damage (Johnson, supra). Transepithelial migration of neutrophils is an important feature of APL because neutrophils are the primary perpetrators of inflammation. Prolonged neutrophil activation contributes to basement membrane destruction and increased permeability of the alveolar-capillary barrier (Johnson, supra).

[0140] ARDS, in some aspects, includes acute-onset tachypnea, hypoxemia, diffuse pulmonary infiltrates, and loss of pulmonary compliance characterized by high short-term mortality in adults (Walkey, supra). Therapeutic strategies for ARDS focus on treating the underlying etiology and providing supportive care that reduces the progression of lung injury. Most patients with ARDS develop respiratory failure severe enough to require mechanical ventilatory support. Mechanical ventilation can cause additional lung damage termed ventilator-associated lung injury (VALI) due to the combined mechanical forces of overdistension and cyclic recruitment. VALI produces biotrauma due to the systemic release of inflammatory cytokines. Currently, the main objective Petition 870200095920, dated 07 / 31 / 2020, page 66 / 151 57 / 132 for the management of SARA is the reduction of VALI. (Walkey, supra).

[0141] ADAMTS13 significantly reduced markers of lung injury and vascular dysfunction in normal mice. More specifically, the revelation shows that dosing normal (control) mice with recombinant ADAMTS13 under hypoxic conditions resulted in reduced pulmonary expression of several protein markers of lung injury and vascular dysfunction, indicating that ADAMTS13 may be used in the treatment or improvement of lung damage resulting from acute lung injury characterized by the sudden onset of pulmonary edema (including inflammatory pulmonary edema) secondary to a myriad of systemic or local insults, including bilateral inflammatory pulmonary infiltrates and impaired oxygenation or hypoxemia.

[0142] In certain modalities, ALI and / or ARDS may be defined by one or more of, but not limited to, ischemia, abnormal breathing (e.g., tachypnea and shortness of breath), non-cardiogenic pulmonary edema, pulmonary infiltrates, reduced oxygenation, and reduced ventilation associated with ALI / ARDS. The disclosure includes methods to reduce ALI / ARDS symptoms including, but not limited to, at least one of ischemia, abnormal breathing (e.g., tachypnea and shortness of breath), non-cardiogenic pulmonary edema, pulmonary infiltrates, reduced oxygenation, reduced ventilation, and combinations thereof associated with ALI / ARDS.

[0143] In certain modalities, LPA and / or SARA can be defined as having an increase in neutrophils. Petition 870200095920, dated 07 / 31 / 2020, page 67 / 151 58 / 132 peripheral compared to a control. In certain modalities, ALI and / or ARDS can be defined as an increase in pulmonary vascular leakage (e.g., increased leukocyte count in bronchoalveolar lavage (BAL) and / or protein content (protein in BAL (mg / ml)) compared to a control.

[0144] In certain modalities, increased levels of vascular activation in an organ, compared to the control, is a marker for ALI and / or ARDS. In certain modalities, increased levels of inflammatory vasculopathy in an organ, compared to the control, is a marker for ALI and / or ARDS. In certain modalities, increased levels of vascular activation and inflammatory vasculopathy in a tissue, compared to the control, is a marker for ALI and / or ARDS. In certain modalities, the organ is the lung and / or kidney.

[0145] In certain modalities, LPA and / or SARA can be defined as increased expression, levels, and / or activation of at least one of NF-κB (where NF-κB activation is measured by P-NF-κB or the P-NF-κB / NF-κB ratio), VCAM-1, and / or ICAM-1 compared to control. In certain modalities, LPA and / or SARA can be defined as increased expression or level of at least one of endothelin-1 (ET-1), thromboxane synthase (TXAS), and heme oxygenase-1 (HO-1) compared to control. In certain modalities, these increases are seen in lung tissue. In certain modalities, these increases are seen in kidney tissue. In certain modalities, increased expression and / or levels of TXAS and Petition 870200095920, dated 07 / 31 / 2020, page 68 / 151 59 / 132 ET-1 and NF-kB activation in renal tissue are markers for ALI and / or ARDS.

[0146] In certain modalities, LPA and / or SARA can be defined by hematological parameters. In certain modalities, LPA and / or SARA can be defined as an increase in neutrophil counts compared to the control. In certain modalities, LPA and / or SARA can be defined as an increase in neutrophil counts compared to the control.

[0147] In certain modalities, LPA and / or SARA may 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, for example, Tzouvelekis et al., Respiratory Research 2005, 6:62), which is incorporated herein in its entirety. LPA / SARA Models and Methods for Testing the Effectiveness of Prophylaxis or Treatment

[0148] Animal models for 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 some cases, ALI in humans is histopathologically characterized by neutrophilic alveolitis, endothelial and alveolar epithelial injury, hyaline membrane formation, and microvascular thrombi. In some respects, animal models of experimental lung injury can be used to investigate the mechanisms Petition 870200095920, dated 07 / 31 / 2020, p. 69 / 151 60 / 132 of ALL. For example, risk factors for ARDS, such as sepsis, lipid embolism secondary to bone fracture, acid aspiration, ischemia-reperfusion of distal or pulmonary 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 intrapulmonary inflammatory response begins before the onset of clinically defined 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 autopsy specimens or lung biopsies.

[0149] 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 respects, these conditions can be modeled in mice using the gram-negative bacterial endotoxin LPS, which can be administered directly to the lungs via intratracheal injection or inhalation, or given intraperitoneally or intravenously to elicit a systemic inflammatory response. Mice treated with intratracheal LPS have a robust and acute inflammatory cell influx to the lung with resolution within 48 hours. LPS activates systemic inflammation and is associated Petition 870200095920, dated 07 / 31 / 2020, page 70 / 151 61 / 132 to a mild lung injury. This injury can be increased with repeated injections of LPS or implantation of an LPS pump in the peritoneal cavity to release LPS continuously for hours, or even days. Another commonly used model of lung injury is hyperoxia, in which mice breathe a high partial pressure of oxygen that is highly toxic to the alveolar epithelium and causes extensive alveolar epithelial injury with only a modest amount of inflammation. An additional commonly studied model is ventilator-induced lung injury, which correlates excellently with human ventilator-induced lung injury; however, in the absence of an additional stimulus or extremely high tidal volumes, this model does not induce substantial lung injury in mice.A recent study showed a modest degree of pulmonary inflammation, vascular leakage, and alveolar coagulation activation with tidal volumes of 15 ml / kg compared to low tidal volumes of 7.5 ml / kg. Higher tidal volumes (up to 35 ml / kg) are required for more severe injury. A recent comprehensive review of animal models of ALI by MatuteBello et al. (Am. J. Physiol. Lung Cell Mol. Physiol. 295:L379-L399, 2008), also incorporated herein by reference for all purposes, discusses each model in great detail.

[0150] In certain respects, the methods and composition disclosed may improve symptoms such as, but not limited to, ischemia, abnormal breathing (e.g., tachypnea and shortness of breath), non-cardiogenic pulmonary edema, pulmonary infiltrates (e.g., measured by Petition 870200095920, dated 07 / 31 / 2020, page 71 / 151 62 / 132 chest x-ray), reduced oxygenation (e.g., measured by pulse oximetry [SpO2] or arterial blood gas [PaO2]) and reduced ventilation (e.g., measured by expired CO2 or arterial blood gas [PaCO2], a reduction on days with mechanical ventilation or an increase on days without mechanical ventilation) associated with ALI / ARDS.

[0151] In many respects, the effectiveness of prophylaxis and / or treatment is determined by measuring survival, extent and / or frequency of hospitalization, extent and / or duration of ICU admissions and / or costs associated with hospital stay.

[0152] In many respects, the effectiveness of prophylaxis 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 (CAM), and airway complications); gastrointestinal complications (e.g., bleeding (ulcer, lesions), dysmotility, pneumoperitoneum, and bacterial translocation); cardiac complications (e.g., abnormal heart rhythms and myocardial dysfunction); renal complications (acute renal failure and positive fluid balance); mechanical complications (e.g., vascular injury, pneumothorax (pulmonary artery catheter placement), tracheal injury / stenosis (resulting from intubation and / or endotracheal tube irritation); nutritional complications (e.g., malnutrition (catabolic state), electrolyte deficiency); and general complications (e.g., muscle weakness and tolerance to...). Petition 870200095920, dated 07 / 31 / 2020, page 72 / 151 63 / 132 exercise).

[0153] In certain modalities, the measurement of treatment efficacy, improvement and prevention is the same as those revealed above for CVO.

[0154] In certain modalities, the organs include, but are not limited to, lung, liver, pancreas, skin, retina, prostate, ovary, lymph node, adrenal gland, kidney, heart, gallbladder, or GI tract. In certain modalities, the organ is lung, liver, spleen, and / or kidney.

[0155] In certain modalities, administration of ADAMTS13 results in reduced levels of vascular activation and / or inflammatory vasculopathy in an organ compared to the control. In certain modalities, the organ is the lung. In certain modalities, the organ is the kidney.

[0156] In certain embodiments, administration of ADAMTS13 results in a reduction in the expression, level, and / or activation of at least one of ICAM-1, NF-kB (where the reduction in NF-kB activation is measured by P-NF-kB or the P-NF-kB / NF-kB ratio), ET-1, TXAS, and HO-1 compared to the control. In certain embodiments, administration of ADAMTS13 results in a reduction in the expression, level, and / or activation of at least two of ICAM-1, NF-kB, ET-1, TXAS, and HO-1 compared to the control. In certain embodiments, administration of ADAMTS13 results in a reduction in the expression, level, and / or activation of at least three of ICAM-1, NF-kB, ET-1, TXAS, and HO-1 compared to the control. In certain modalities, administration of ADAMTS13 results in a reduction in the expression, level, and / or activation of at least four of ICAM-1, NFkB, ET-1, TXAS, and HO-1 compared to the control. Petition 870200095920, dated 07 / 31 / 2020, page 73 / 151 64 / 132 In certain modalities, ADAMTS13 administration results in a reduction in the expression, level, and / or activation of ICAM-1, NF-kB, ET-1, TXAS, and HO-1 compared to the control. In certain modalities, ADAMTS13 administration results in a reduction in the expression, level, and / or activation of ICAM-1 compared to the control. In certain modalities, ADAMTS13 administration results in a reduction in the expression and / or level of ET-1 compared to the control. In certain modalities, ADAMTS13 administration results in a reduction in the expression and / or level of TXAS compared to the control. In certain modalities, ADAMTS13 administration results in a reduction in the expression and / or level of HO-1 compared to the control. In certain modalities, ADAMTS13 administration results in a reduction in the P-NF-kB / NF-kB ratio compared to the control.In certain embodiments, the administration of ADAMTS13 results in a reduction of at least one of the following: P-NF-kB / NF-kB ratio, ET-1 expression and / or level, TXAS expression and / or level, and HO-1 expression and / or level compared to the control. In certain embodiments, the administration of ADAMTS13 results in a reduction of the P-NF-kB / NF-kB ratio, ET-1 expression and / or level, TXAS expression and / or level, and HO-1 expression and / or level compared to the control.

[0157] In certain modalities, administration of ADAMTS13 results in improved increase in blood neutrophil count compared to control.

[0158] In certain modalities, administration of ADAMTS13 results in a reduction in at least one of the following serum biomarkers compared with Petition 870200095920, dated 07 / 31 / 2020, p. 74 / 151 65 / 132 control: surfactant-associated protein (SP)-A, SP-B, SPD, KL-6 / MUC1, IL-1, IL-2, IL-3, IL-6, IL-8, IL-10, IL-15, TNF«, adhesion molecules (e.g., L E, M4 e Ferlectin ADAMTS13

[0159] In some aspects, the disclosure includes ADAMTS13 (also known as A13) and compositions comprising ADAMTS13 in the treatment and prevention of DF. In particular aspects, the disclosure includes ADAMTS13 and compositions comprising ADAMTS13 in the treatment and prevention of CVO in DF. The protease ADAMTS13 is approximately 180 kDa to 200 kDa glycosylated protein produced predominantly by the liver. ADAMTS13 is a plasma metalloprotease that cleaves VWF multimers and downregulates their 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-related DIC.

[0160] All forms of ADAMTS13 known in the art are contemplated for use in the methods and applications of the discovery. Mature ADAMTS13 has a calculated molecular mass of about 145 kDa, while ADAMTS13 derived from purified plasma has an apparent molecular mass of about 180 kDa to 200 kDa, likely due to post-translational modifications consisting of consensus sequences present for 10 potential N-glycosylation sites and several O-glycosylation sites and one C-mannosylation site. Petition 870200095920, dated 07 / 31 / 2020, p. 75 / 151 66 / 132 in the TSP1 repetitions.

[0161] As used in this document, ADAMTS13 refers to a metalloprotease of the ADAMTS family (a disintegrin and metalloproteinase with type 1 thrombospondin domains) that cleaves FVW between residues Tyr1605 and Met1606. In the context of disclosure, ADAMTS13, A13, or an ADAMTS13 protein encompasses any ADAMTS13 protein, for example, ADAMTS13 from a mammal such as a primate, human (NP620594), monkey, rabbit, pig, bovine (XP610784), rodent, mouse (NP001001322), rat (XP342396), hamster, gerbil, canine, feline, frog (NP001083331), chicken (XP415435) and biologically active derivatives thereof. As used in this document, ADAMTS13, A13, or ADAMTS13 protein refers to recombinant, natural, or plasma-derived ADAMTS13 protein. Variant and mutant ADAMTS13 proteins that have activity are also included, as they are functional fragments and fusion proteins of ADAMTS13 proteins.In some respects, an ADAMTS13 protein additionally comprises an identifier that facilitates purification, detection, or both. The ADAMTS13 protein of the discovery, in some respects, is additionally modified with an additional therapeutic fraction or a fraction suitable for in vitro or in vivo imaging.

[0162] The ADAMTS13 protein includes any protein or polypeptide with ADAMTS13 activity, particularly the ability to cleave the peptide bond between the Tyr-842 and Met-843 residues of VWF. Human ADAMTS13 proteins include, without limitation, polypeptides comprising the amino acid sequence with GenBank NP accession number Petition 870200095920, dated 07 / 31 / 2020, p. 76 / 151 67 / 132 620594 (NM139025.3) or a processed polypeptide thereof, for example, a polypeptide in which the signaling peptide (amino acids 1 to 29) and / or propeptide (amino acids 30-74) has been removed. In certain respects, an ADAMTS13 protein refers to a polypeptide comprising an amino acid sequence that is highly similar to that of NP 620596 (ADAMTS13 isoform 2, preproprotein) or amino acids 75 to 1371 of P 620594 (ADAMTS13 isoform 2, mature polypeptide). In yet another embodiment, ADAMTS13 proteins include polypeptides comprising an amino acid sequence highly similar to that of NP 620595 (ADAMTS13 isoform 3, preproprotein) or amino acids 75 to 1340 of NP 620595 (ADAMTS13 isoform 1, mature polypeptide). In certain respects, an ADAMTS13 protein includes natural variants with FVW cleavage activity and artificial constructs with FVW cleavage activity.In certain respects, ADAMTS13 encompasses 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 covered by the development formulations, some of which include mutations selected from R7W, V88M, H96D, R102C, R193W, T196I, H234Q, A250V, R268P, W390C, R398H, Q448E, Q456H, P457L, P475S, C508Y, R528G, P618A, R625H, 1673F, R692C, A732V, E740K, A900V, S903L, C908Y, C951G, G982R, C1024G, A1033T, R1095W, R1123C, C1213Y, T12261, G1239V, and R1336W. Additionally, ADAMTS13 proteins include natural and recombinant proteins that have been mutated, for example, by one or more mutations. Petition 870200095920, dated 07 / 31 / 2020, p. 77 / 151 68 / 132 are conservative in a non-essential amino acid. Preferably, the amino acids essential for the enzymatic activity of ADAMTS13 will not be mutated. These include, for example, residues known or presumed to be essential for metal binding such as residues 83, 173, 224, 228, 234, 281, and 284, and residues observed in the enzyme's active site, for example, residue 225. Similarly, in the context of discovery, ADAMTS13 proteins include alternative isoforms, for example, isoforms lacking amino acids 275 to 305 and / or 1135 to 1190 of the full-length human protein.

[0163] In some respects, ADAMTS13 proteins are additionally modified, for example, by post-translational modifications (e.g., glycosylation at one or more amino acids selected from human residues 142, 146, 552, 579, 614, 667, 707, 828, 1235, 1354, or any other natural modification site subjected to genetic engineering) or by ex vivo chemical or enzymatic modification, including without limitation, glycosylation, water-soluble polymer modification (e.g., PEGylation, sialylation, HEsylation, etc.), identification and the like.

[0164] In some respects, the ADAMTS13 protein is human ADAMTS13 or a biologically active fragment or derivative 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.

[0165] In certain respects, recombinant ADAMTS13 may be BAX930 / SHP655. In certain respects, the Petition 870200095920, dated 07 / 31 / 2020, p. 78 / 151 69 / 132 ADAMTS13 protein includes any protein or polypeptide with ADAMTS13 activity, particularly the ability to cleave the peptide bond between Tyr-842 and Met-843 residues of VWF with 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 with BAX930 / SHP655.

[0166] Recombinant proteolytically active ADAMTS13 can be prepared by expression in mammalian cell cultures, as described in Plaimauer et al, (2002, Blood. 15; 100(10):3626-32) and US 2005 / 0266528, the disclosure of which is incorporated herein by reference in its entirety for all purposes. Methods for the expression of recombinant ADAMTS13 in cell culture are disclosed in Plaimauer B, Scheiflinger F. (Semin Hematol. Jan 2004;41(l):24-33) and US 2011 / 0086413, the disclosure of which is incorporated herein by reference in its entirety for all purposes. See also document WO2012 / 006594, incorporated by reference in its entirety for all purposes, regarding methods of producing recombinant ADAMTS13 in cell culture.

[0167] 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. Such methods include, in some respects, enriching ADAMTS13 protein by chromatographic contact of the sample with hydroxyapatite in Petition 870200095920, dated 07 / 31 / 2020, page 79 / 151 70 / 132 conditions that allow ADAMTS13 protein to appear in the eluate or supernatant from hydroxyapatite. The methods may further comprise tandem chromatography with a mixed-mode hydrophobic / cation exchange interaction resin that binds to the ADAMTS13 protein. Optional additional steps involve ultrafiltration / diafiltration, anion exchange chromatography, cation exchange chromatography, and viral inactivation. In some aspects, such methods include the inactivation of viral contaminants in protein samples, wherein the protein is immobilized on a support. Also provided herein, in some aspects, are ADAMTS13 compositions prepared according to the methods described in U.S. Patent No. 8,945,895. Administration and Compositions of ADAMTS13

[0168] In aspects of the disclosure, ADAMTS13 is administered to an individual in need thereof. To administer the ADAMTS13 described herein to an individual, ADAMTS13 is, in some respects, formulated in a composition comprising one or more pharmaceutically acceptable carriers.

[0169] The term pharmaceutically acceptable, as used in conjunction with compositions described herein, refers to molecular entities and other ingredients of those compositions that are physiologically tolerable and typically do not produce undesirable reactions when administered to a mammal (e.g., a human). Preferably, the term pharmaceutically acceptable means approved by a federal or state government regulatory agency or listed in Petition 870200095920, dated 07 / 31 / 2020, page 80 / 151 71 / 132 US Pharmacopoeia or other generally recognized pharmacopoeia for use in mammals and, more particularly, in humans. Pharmaceutically acceptable carriers include any and all clinically useful solvents, dispersing media, coatings, antibacterial and antifungal agents, absorption delay agents, and isotonic and similar substances. In some respects, the composition forms solvates with water or common organic solvents. Such solvates are also included.

[0170] In some aspects, the disclosure provides stabilized formulations of plasma-derived ADAMTS13 proteins and recombinant ADAMTS13 (rADAMTS13) as described in U.S. Patent Application Publication No. 2011 / 0229455 (now U.S. Patent Application No. 8,623,352) and / or U.S. Patent Application Publication No. 2014 / 0271611, both of which are incorporated herein by reference for all purposes. In some embodiments, the formulations provided herein retain significant ADAMTS13 activity when stored for extended periods of time. In some embodiments, the disclosure formulations reduce or retard dimerization, oligomerization, and / or aggregation of an ADAMTS13 protein.

[0171] In some respects, the disclosure provides ADAMTS13 formulations comprising a therapeutically effective amount or dose of an ADAMTS13 protein, a subphysiological or physiological concentration of a pharmaceutically acceptable salt, a stabilizing concentration of one or more sugars and / or sugar alcohols, a nonionic surfactant, a buffering agent that Petition 870200095920, dated 07 / 31 / 2020, p. 81 / 151 72 / 132 provides a neutral pH for the formulation and, optionally, a calcium and / or zinc salt. Generally, the stabilized ADAMTS13 formulations provided herein are suitable for pharmaceutical administration. In some respects, the ADAMTS13 protein is human ADAMTS13 or a biologically active fragment or derivative 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.

[0172] In some respects, ADAMTS13 formulations are lyophilized or liquid formulations. In other embodiments, a lyophilized formulation is lyophilized from a 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 formulations provided herein, the ADAMTS13 protein is human or recombinant human ADAMTS13, or a biologically active fragment or derivative 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.

[0173] The composition of the revelation is, in several aspects, administered orally, topically, transdermally, Petition 870200095920, dated 07 / 31 / 2020, page 82 / 151 73 / 132 parenteral, by inhalation spray, vaginal, rectal or intracranial injection. The term parenteral, as used herein, includes subcutaneous, intravenous, intramuscular, intrasternal injection or infusion techniques. In some embodiments, administration is subcutaneous. Administration by intravenous, intradermal, intramuscular, intramammary, intraperitoneal, intrathecal, retrobulbar, intrapulmonary injection and / or surgical implantation at a particular site is also contemplated. In some embodiments, administration is intravenous. Generally, compositions are essentially free of pyrogens, as well as other impurities that may be harmful to the recipient.

[0174] The formulation of the pharmaceutical composition or compound will vary according to the route of administration selected (e.g., solution or emulsion). An appropriate composition comprising the composition to be administered is prepared in a physiologically acceptable carrier or vehicle. For solutions or emulsions, suitable carriers include, for example, aqueous or alcoholic / aqueous solutions, emulsions or suspensions, including brine and buffered media. Parenteral vehicles, in some respects, include sodium chloride solution, Ringer's dextrose, dextrose and sodium chloride, Ringer's lactate, or fixed oils. Intravenous vehicles, in certain respects, include various additives, preservatives, or fluid, nutrient, or electrolyte replenishers.

[0175] Pharmaceutical compositions or compounds useful in the development of compounds and methods containing ADAMTS13 Petition 870200095920, dated 07 / 31 / 2020, page 83 / 151 74 / 132 as an active ingredient contains, in various respects, pharmaceutically acceptable additives or carriers depending on the route of administration. Examples of such carriers or additives include water, a pharmaceutically acceptable organic solvent, collagen, polyvinyl alcohol, polyvinylpyrrolidone, a carboxyvinyl polymer, sodium carboxymethylcellulose, sodium polyacrylic acid, sodium alginate, water-soluble dextran, sodium carboxymethyl starch, pectin, methyl cellulose, ethyl cellulose, xanthan gum, gum arabic, casein, gelatin, agar, diglycerin, glycerin, propylene glycol, polyethylene glycol, petrolatum, paraffin, stearyl alcohol, stearic acid, human serum albumin (HSA), mannitol, sorbitol, lactose, a pharmaceutically acceptable surfactant and the like. The additives used are chosen from, but not limited to, those mentioned above, or combinations thereof, as appropriate, depending on the dosage form.

[0176] A variety of aqueous carriers, for example, water, buffered water, 0.4% brine, 0.3% glycine, or aqueous suspensions contain, in various respects, the active compound in addition mixtures with excipients suitable for the production of aqueous suspensions. Such excipients are suspending agents, for example, sodium carboxymethylcellulose, methylcellulose, hydroxypropylmethylcellulose, sodium alginate, polyvinylpyrrolidone, tragacanth gum and acacia gum; dispersing or wetting agents, in some cases, are a naturally occurring phosphatide, for example, lecithin, or condensation products of an alkylene oxide with acids. Petition 870200095920, dated 07 / 31 / 2020, page 84 / 151 75 / 132 fatty acids, for example, polyoxyethylene stearate, or condensation products of ethylene oxide with long-chain aliphatic alcohols, for example, heptadecaethyl-eneoxycetanol, or condensation products of ethylene oxide with partial esters derived from fatty acids and a hexitol such as polyoxyethylene sorbitol monooleate, or condensation products of ethylene oxide with partial esters derived from fatty acids and hexitol anhydrides, for example, polyethylene sorbitan monooleate. The aqueous suspensions, in some aspects, contain one or more preservatives, for example, ethyl, or n-propyl, p-hydroxybenzoate.

[0177] In some respects, 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 employed. It is observed by those skilled in the art that lyophilization and reconstitution leads to varying degrees of protein activity loss and that usage levels are often adjusted to compensate.

[0178] Granules and dispersible powders suitable for the preparation of an aqueous suspension by the addition of water provide the active compound in a mixture by addition with a dispersing or wetting agent, a suspending agent and one or more preservatives. Suitable dispersing or wetting agents and suspending agents are exemplified by those already mentioned above.

[0179] In some embodiments, the ADAMTS13 formulations provided in this document may include Petition 870200095920, dated 07 / 31 / 2020, p. 85 / 151 76 / 132 additionally 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.

[0180] In some embodiments, the ADAMTS13 formulations provided herein will have a tonicity in a range described 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.

[0181] In some respects, the disclosure provides ADAMTS13 formulations comprising the exemplary formulations described in Section III (Compositions and Formulations of ADAMTS13) of U.S. Patent Application Publication No. 2011 / 0229455. The methods of production of ADAMTS13 and compositions thereof as described in U.S. Patent Application Publication No. 2011 / 0229455 and / or U.S. Patent Application Publication No. 2014 / 0271611 are incorporated herein by reference in their entirety for all purposes. Additionally, actual methods for preparing parenterally administrable formulations and compositions are known or are evident to those skilled in the art and are described in more detail, for example, in Remington's Pharmaceutical Science, 15th ed., Mack Publishing Company, Petition 870200095920, dated 07 / 31 / 2020, page 86 / 151 77 / 132 Easton, Pa. (1980).

[0182] In several aspects, the pharmaceutical compositions are in the form of dispersions, sterile aqueous injectable oil suspensions, or sterile powders for the extemporaneous preparation of sterile injectable dispersions or solutions. The suspension, in some aspects, is formulated according to known technique, using those suitable dispersing or wetting agents and suspending agents mentioned above. The sterile injectable preparation, in certain aspects, is a sterile injectable suspension or solution in a parenterally acceptable non-toxic solvent or diluent, for example, as a solution in 1,3-butanediol. In some embodiments, the carrier is a dispersing medium or solvent or dispersion containing, for example, water, ethanol, polyol (e.g., glycerol, propylene glycol and liquid polyethylene glycol and the like), suitable mixtures thereof, vegetable oils, Ringer's solution, and isotonic sodium chloride solution.Furthermore, sterile fixed oils are conventionally employed as a solvent or suspension medium. For this purpose, any mild fixed oil is used, in various forms, including synthetic mono- or diglycerides. Additionally, fatty acids such as oleic acid are useful in the preparation of injectable products.

[0183] In all cases, the form must be sterile and must be fluid to the point where easy syringeability exists. Adequate fluidity is maintained, for example, by the use of a coating such as lecithin, by maintaining the necessary particle size in the case of Petition 870200095920, dated 07 / 31 / 2020, page 87 / 151 78 / 132 dispersion and by the use of surfactants. It must be stable under manufacturing and storage conditions and must be protected against the contaminating action of microorganisms such as bacteria and fungi. Prevention of microbial action is achieved by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, sorbic acid, thimerosal and the like. In many cases, it will be desirable to include isotonic agents, for example, sugars or sodium chloride. In certain aspects, prolonged absorption of injectable compositions is achieved by the use of absorption-delaying agents in the compositions, for example, aluminum monostearate and gelatin.

[0184] Compositions useful for administration are, in certain respects, formulated with absorption or incorporation enhancers or to increase their effectiveness. Such enhancers include, for example, salicylate, glycocholate / linoleate, glycolate, aprotinin, bacitracin, SDS, caprate and the like. See, for example, Fix (J. Pharm. Sci., 85:1282-1285, 1996) and Oliyai et al. (Ann. Rev. Pharmacol. Toxicol., 32:521-544, 1993), each of which is incorporated herein by reference in its entirety and for all purposes.

[0185] Furthermore, the hydrophilic and hydrophobic properties of the compositions used in the development compositions and methods are well balanced, thus enhancing their usefulness for in vitro and especially in vivo applications, whereas other compositions lacking this balance are of substantially lesser use. Specifically, the compositions in the development have an appropriate degree of solubility in aqueous media, which Petition 870200095920, dated 07 / 31 / 2020, p. 88 / 151 79 / 132 allows for absorption and bioavailability in the body, while also having a degree of lipid solubility, which allows the compounds to cross the cell membrane to a putative site of action.

[0186] In particular aspects, ADAMTS13 is supplied in a pharmaceutically acceptable (i.e., sterile and non-toxic) solid, semi-solid or liquid diluent that serves as a pharmaceutical medium, excipient or vehicle. Any diluent known in the art is used. Exemplary diluents include, but are not limited to, polyoxyethylene sorbitan monolaurate, magnesium stearate, methyl propyl hydroxybenzoate, talc, alginates, starches, lactose, sucrose, dextrose, sorbitol, mannitol, acacia gum, calcium phosphate, mineral oil, cocoa butter and theobroma oil.

[0187] The composition is packaged in forms convenient for delivery. The composition is contained within a capsule, tablet, sachet, pill, gelatin, paper or other container. These delivery forms are preferred when compatible with the delivery of the composition into the recipient organism and, particularly, when the composition is being delivered in unit dose form. Dosage units are packaged, for example, in bottles, tablets, capsules, suppositories or sachets.

[0188] The disclosure includes methods for treating, improving and / or preventing CVO in DF in an individual, including administering an effective amount of ADAMTS13 or a composition of ADAMTS13 as described herein. The composition is introduced into the individual to be treated by any conventional method as described. Petition 870200095920, dated 07 / 31 / 2020, page 89 / 151 80 / 132 in the present document in detail above. In certain respects, the composition is administered in a single dose or in a plurality of doses over a period of time (as described in more detail below).

[0189] In some modalities, the composition comprising ADAMTS13 is administered to the individual within approximately 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 start of the CVO. In some modalities, the composition comprising ADAMTS13 is administered to the individual within approximately 1-2 hours, approximately 1-5 hours, approximately 1-10 hours, approximately 1-12 hours, approximately 1-24 hours, approximately 1-36 hours, approximately 1-48 hours, approximately 1-60 hours, approximately 1-72 hours, approximately 1-84 hours, approximately 1-96 hours, approximately 1-108 hours, or approximately 1-120 hours after the start of the CVO.In some modalities, the composition comprising ADAMTS13 is administered to the individual within approximately 2-5 hours, approximately 5-10 hours, approximately 10-20 hours, approximately 20-40 hours, approximately 30-60 hours, approximately 40-80 hours, approximately 50-100 hours, or approximately 60-120 hours after the start of the CVO. In some modalities, the composition is administered within 1 week of the CVO. In some modalities, the composition is administered daily after the CVO. In some modalities, the composition is administered weekly after the CVO. In some modalities, the composition is administered every day. In some modalities, the composition is administered every other day. In some modalities, the composition is administered every... Petition 870200095920, dated 07 / 31 / 2020, pp. 90 / 151 81 / 132 three days. In some modalities, the composition is administered twice a week. In some modalities, the composition is administered until clinical manifestations (e.g., symptoms and / or biomarkers) resolve. In some modalities, the composition is administered up to one day after clinical manifestations resolve. In some modalities, the composition is administered for at least two days after clinical manifestations resolve. In some modalities, the composition is administered for at least three days after clinical manifestations resolve. In some modalities, the composition is administered for at least one week after clinical manifestations resolve.

[0190] In some aspects, the composition comprising ADAMTS13 is administered to the individual to prevent the onset of CVO. In this preventive treatment, ADAMTS13 is administered as a single bolus injection or in multiple doses to maintain a circulating level of ADAMTS13 effective in preventing the onset of CVO. In such aspects, the composition comprising ADAMTS13 is administered monthly, every two weeks, weekly, twice a week, every other day, or daily. In particular aspects, the injection is administered subcutaneously. In other aspects, the injection is administered intravenously.

[0191] In some embodiments, the composition comprising ADAMTS13 is administered to the individual prior to the onset of CVO to prevent CVO. In such aspects of the development, the composition is administered in a therapeutically effective amount or dose sufficient to maintain a Petition 870200095920, dated 07 / 31 / 2020, pp. 91 / 151 82 / 132 effective activity level of ADAMTS13 in the individual or in the individual's blood.

[0192] The disclosure includes methods for treating, improving, or preventing ALI or ARDS in an individual, including administering an effective amount of ADAMTS13 or a composition of ADAMTS13 as described herein. The composition is introduced into the individual to be treated by any conventional method as described herein in detail above. In certain respects, the composition is administered in a single dose or in a plurality of doses over a period of time (as described in more detail below).

[0193] In some modalities, the composition comprising ADAMTS13 is administered to the individual 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 modalities, the composition comprising ADAMTS13 is administered to the individual within approximately 1-2 hours, approximately 1-5 hours, approximately 1-10 hours, approximately 1-12 hours, approximately 1-24 hours, approximately 1-36 hours, approximately 1-48 hours, approximately 1-60 hours, approximately 1-72 hours, approximately 1-84 hours, approximately 1-96 hours, approximately 1-108 hours, or approximately 1-120 hours after the onset of ALI or ARDS. In some modalities, the composition comprising ADAMTS13 is administered to the individual within approximately 2-5 hours, approximately 5-10 hours, approximately 10-20 hours, approximately 20-40 hours, approximately 30-60 hours, approximately 40-80 hours, approximately 50-100 hours, or approximately 60-120 hours. Petition 870200095920, dated 07 / 31 / 2020, page 92 / 151 83 / 132 hours after the onset of ALI or ARDS. In some modalities, the composition is administered within 4 hours, within 8 hours, within 12 hours, within 1 day, within 2 days, within 3 days, within 4 days, within 5 days, within 6 days after the onset or diagnosis of ALI or ARDS. In some modalities, the composition is administered within 1 week after the onset or diagnosis of ALI or ARDS. In some modalities, the composition is administered daily after the onset or diagnosis of ALI or ARDS. In some modalities, the composition is administered weekly after the onset or diagnosis of ALI or ARDS. In some modalities, the composition is administered every day. In some modalities, the composition is administered every other day. In some modalities, the composition is administered every three days. In some modalities, the composition is administered twice a week.In some modalities, the composition is administered until the clinical manifestations resolve. In some modalities, the composition is administered up to one day after the clinical manifestations resolve. In some modalities, the composition is administered for at least two days after the clinical manifestations resolve. In some modalities, the composition is administered for at least three days after the clinical manifestations resolve. In some modalities, the composition is administered for at least one week after the clinical manifestations resolve.

[0194] In some respects, the composition comprising ADAMTS13 is administered to the individual to prevent the onset of ALI or ARDS. In this preventive treatment, ADAMTS13 is Petition 870200095920, dated 07 / 31 / 2020, page 93 / 151 84 / 132 administered as a single bolus injection or in multiple doses to maintain an effective circulating level of ADAMTS13 to prevent the onset of ALI or ARDS. In such aspects, the composition comprising ADAMTS13 is administered monthly, every other week, weekly, twice a week, every other day, or daily. In particular aspects, the injection is administered subcutaneously. In other aspects, the injection is administered intravenously.

[0195] In some embodiments, the composition comprising ADAMTS13 is administered to the individual prior to the onset of ALI or ARDS to prevent ALI or ARDS. In such aspects of the development, the composition is administered in a therapeutically effective amount or dose sufficient to maintain an effective level of ADAMTS13 activity in the individual or in the individual's blood. Dosage of ADAMTS13 Compositions / Treatment Methods

[0196] In many respects, the effective dosage of ADAMTS13 or an ADAMTS13 composition to be administered varies depending on multiple factors that modify drug action, for example, the individual's age, condition, body weight, sex and diet, the severity of any infection, time of administration, mode of administration, and other clinical factors, including the severity of the CVO of DF.

[0197] In some respects, the development formulations or compositions are administered by an initial bolus followed by booster delivery after a period of time has elapsed. In certain respects, development formulations are administered by an initial bolus followed by a Petition 870200095920, dated 07 / 31 / 2020, page 94 / 151 85 / 132 continuous infusion to maintain therapeutic circulating levels of ADAMTS13. In particular instances, ADAMTS13 or an ADAMTS13 composition is administered over extended periods of time. In some instances, ADAMTS13 or an ADAMTS13 composition is delivered in a rapid treatment regimen to alleviate acute CVO symptoms. In some instances, ADAMTS13 or an ADAMTS13 composition is delivered in a prolonged and varied treatment regimen to prevent the occurrence of CVO. As another example, the development composition or formulation is administered as a single dose. Those skilled in the art quickly optimize effective dosages and administration regimens as determined by good medical practice and the clinical condition of the individual subject. Dosing frequency depends on the pharmacokinetic parameters of the agents, the route of administration, and the individual's condition.

[0198] The pharmaceutical formulation is determined by someone skilled in the art depending on the route of administration and desired dosage. See, for example, Remington's Pharmaceutical Sciences, 18th Ed. (1990, Mack Publishing Co., Easton, PA 18042) pages 1435-1712, the disclosure of which is incorporated herein by reference for all purposes. Such formulations, in some cases, influence the physical state, stability, in vivo release rate, and in vivo removal rate of the administered composition. Depending on the route of administration, an appropriate dose is calculated, in particular aspects, according to body weight, body surface area, or organ size. In some Petition 870200095920, dated 07 / 31 / 2020, page 95 / 151 In certain aspects, appropriate dosages are determined through the use of established assays for determining blood level dosages in conjunction with adequate dose-response data. In certain aspects, an individual's antibody titer is measured to determine the ideal dosage and administration regimens. The final dosage regimen will be determined by the attending physician or doctor, considering various factors that modify the action of pharmaceutical compositions, for example, the specific activity of the composition, the individual's responsiveness, the individual's age, condition, body weight, sex and diet, the severity of any infection or malignancy, time of administration and other clinical factors, including the severity of pain or CVO.

[0199] In certain respects, ADAMTS13 or the ADAMTS13 composition comprises any dose of ADAMTS13 sufficient to elicit a response in the individual. In some modalities, the dose of ADAMTS13 is sufficient to treat CVO. In some modalities, the dose of ADAMTS13 is sufficient to prevent CVO. In some modalities, the dose of ADAMTS13 is sufficient to treat LPA. In some modalities, the dose of ADAMTS13 is sufficient to prevent LPA. In some modalities, the dose of ADAMTS13 is sufficient to treat ARDS. In some modalities, the dose of ADAMTS13 is sufficient to prevent ARDS. The effective amount of ADAMTS13 or the ADAMTS13 composition to be used therapeutically will depend, for example, on the therapeutic goals and context. A person skilled in the art will find that the appropriate dosage levels for treatment or prevention will vary accordingly. Petition 870200095920, dated 07 / 31 / 2020, p. 96 / 151 87 / 132 depending, in part, on the molecule delivered, the indication for which ADAMTS13 or ADAMTS13 composition is being used, the route of administration, and the size (body weight, body surface area, or organ size) and condition (age and general health) of the patient. Consequently, the clinician, in some cases, titrates the dosage and modifies the route of administration to obtain the optimal therapeutic effect.

[0200] Dosage, unless specifically stated otherwise, is given in international units. As discussed in the document below, the use of international units (IU) is the 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 are equivalent to about 18.22 FRETS U of ADAMTS13.

[0201] A typical dosage, in various respects, ranges from about 10 international units per kilogram of body weight to about 10,000 international units per kilogram of body weight. In some respects, a therapeutically effective dosage or amount of ADAMTS13 is up to about 10,000 international units per kilogram of body weight or more, depending on the factors mentioned above. In other respects, the dosage may range from about 20 to about 6,000 international units per kilogram of body weight. In some respects, the therapeutically effective dosage or amount of ADAMTS13 is from about 40 to about 4,000 units. Petition 870200095920, dated 07 / 31 / 2020, page 97 / 151 88 / 132 international units per kilogram of body weight. In some respects, the therapeutically effective dosage or quantity is approximately 100 to approximately 3000 international units per kilogram of body weight.

[0202] In particular aspects, the therapeutically effective dosage or quantity is from about 10 to about 500 international units per kilogram of body weight. In some aspects, the therapeutically effective dosage or quantity is from about 50 to about 450 international units per kilogram of body weight. In some aspects, the therapeutically effective quantity is from about 40 to about 100 international units per kilogram of body weight. In some aspects, the therapeutically effective quantity is from about 40 to about 150 international units per kilogram of body weight. In some aspects, the therapeutically effective dosage or quantity is from about 100 to about 500 international units per kilogram of body weight. In some aspects, the therapeutically effective dosage or quantity is from about 100 to about 400 international units per kilogram of body weight.In some respects, the therapeutically effective dosage or quantity is approximately 100 to approximately 300 international units per kilogram of body weight. In some respects, the therapeutically effective dosage or quantity is approximately 300 to approximately 500 international units per kilogram of body weight. In some respects, the therapeutically effective dosage or quantity is approximately 200 to approximately 300 international units per kilogram of body weight. In some respects, the therapeutically effective dosage or quantity is... Petition 870200095920, dated 07 / 31 / 2020, pp. 98 / 151 89 / 132 of approximately 100, approximately 150, approximately 200, approximately 250, approximately 300, approximately 350, approximately 400, approximately 450, or approximately 500 international units per kilogram of body weight.

[0203] In additional aspects, the therapeutically effective dosage or quantity is about 50 to about 1000 international units per kilogram of body weight. In some aspects, the therapeutically effective dosage or quantity is about 100 to about 900 international units per kilogram of body weight. In some aspects, the therapeutically effective dosage or quantity is about 200 to about 800 international units per kilogram of body weight. In some aspects, the therapeutically effective dosage or quantity is about 300 to about 700 international units per kilogram of body weight. In some aspects, the therapeutically effective dosage or quantity is about 400 to about 600 international units per kilogram of body weight. In some aspects, the therapeutically effective dosage or quantity is about 500 international units per kilogram of body weight.

[0204] In some respects, the therapeutically effective dosage or amount is approximately 10 international units per kilogram of body weight, approximately 20 international units per kilogram of body weight, approximately 30 international units per kilogram of body weight, approximately 40 international units per kilogram of body weight, approximately 50 international units per kilogram of body weight, approximately 60 international units per kilogram of body weight, Petition 870200095920, dated 07 / 31 / 2020, page 99 / 151 90 / 132 approximately 70 international units per kilogram of body weight, approximately 80 international units per kilogram of body weight, approximately 90 international units per kilogram of body weight, approximately 100 international units per kilogram of body weight, approximately 120 international units per kilogram of body weight, approximately 140 international units per kilogram of body weight, approximately 150 international units per kilogram of body weight, approximately 160 international units per kilogram of body weight, approximately 180 international units per kilogram of body weight, approximately 200 international units per kilogram of body weight, approximately 220 international units per kilogram of body weight, approximately 240 international units per kilogram of body weight, approximately 250 international units per kilogram of body weight, approximately 260 international units per kilogram of body weight,approximately 280 international units per kilogram of body weight, approximately 300 international units per kilogram of body weight, approximately 350 international units per kilogram of body weight, approximately 400 international units per kilogram of body weight, approximately 450 international units per kilogram of body weight, approximately 500 international units per kilogram of body weight, approximately 550 international units per kilogram of body weight, approximately 600 international units per kilogram of body weight, approximately 650 international units per, Petition 870200095920, dated 07 / 31 / 2020, pages 100 / 151 91 / 132 kilograms of body weight, approximately 700 international units per kilogram of body weight, approximately 750 international units per kilogram of body weight, approximately 800 international units per kilogram of body weight, approximately 850 international units per kilogram of body weight, approximately 900 international units per kilogram of body weight, approximately 950 international units per kilogram of body weight, approximately 1000 international units per kilogram of body weight, approximately 1100 international units per kilogram of body weight, approximately 1100 international units per kilogram of body weight, approximately 1200 international units per kilogram of body weight, approximately 1300 international units per kilogram of body weight, approximately 1400 international units per kilogram of body weight, approximately 1500 international units per kilogram of body weight,approximately 1600 international units per kilogram of body weight, approximately 1800 international units per kilogram of body weight, approximately 2000 international units per kilogram of body weight, approximately 2500 international units per kilogram of body weight, approximately 3000 international units per kilogram of body weight, approximately 3500 international units per kilogram of body weight, approximately 4000 international units per kilogram of body weight, approximately 4500 international units per kilogram of body weight, approximately 5000 international units per kilogram of body weight, approximately, Petition 870200095920, dated 07 / 31 / 2020, pages 101 / 151 92 / 132 5500 international units per kilogram of body weight, approximately 6000 international units per kilogram of body weight, approximately 6500 international units per kilogram of body weight, approximately 7000 international units per kilogram of body weight, approximately 7500 international units per kilogram of body weight, approximately 8000 international units per kilogram of body weight, approximately 8500 international units per kilogram of body weight, approximately 9000 international units per kilogram of body weight, approximately 9500 international units per kilogram of body weight, and approximately 10000 international units per kilogram of body weight.

[0205] As used in this document, one ADAMTS13 activity unit or one activity unit is defined as the amount of activity in 1 ml of pooled normal human plasma, regardless of the assay being used. As provided above, however, the new standard for measuring or quantifying ADAMTS13 is international units (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 are equivalent to approximately 18.22 FRETS U of ADAMTS13. Thus, the change to the new standard results in an approximate 8.9% change in the conversion from FRETS U to IU.

[0206] In some respects, Fluorescence Resonance Energy Transfer (FRETS) assays are used to measure ADAMTS13 activity. FRETS requires Petition 870200095920, dated 07 / 31 / 2020, pp. 102 / 151 93 / 132 two interaction partners, one of which is identified with a donor fluorophore and the other with an acceptor fluorophore. FRETS assays for ADAMTS13 involve a chemically modified fragment of the A2 domain of VWF that spans the ADAMTS13 cleavage site. This is rapidly cleaved by normal plasma, but not by ADAMTS13-deficient plasma. This cleavage is blocked by EDTA, and therefore samples for this assay must be collected in tubes containing citrate as an anticoagulant, not EDTA. One unit of FRETS-VWF73 activity of ADAMTS13 is the amount of activity required to cleave the same amount of FRETS-VWF73 substrate (Kokame et al., Br J. Haematol. April 2005; 129 (1): 93-100, incorporated herein by reference) as is cleaved by one ml of pooled normal human plasma.

[0207] In some respects, additional activity assays are used to measure ADAMTS13 activity. For example, direct ADAMTS13 activity assays can be performed to detect the cleavage of full-length VWF molecules or VWF fragments using agarose-SDS gel electrophoresis, and indirect detection of ADAMTS13 activity can be achieved with collagen binding assays. Direct assays, including the FRETS assay as described in this document, involve detecting cleavage products of a full-length VWF molecule or a VWF fragment encompassing the ADAMTS13 cleavage site. Using agarose-SDS gel electrophoresis and Western blotting, purified VWF is incubated with plasma for 24 hours. VWF cleavage by ADAMTS13 occurs, leading to a reduction in multimer sizes. Petition 870200095920, dated 07 / 31 / 2020, pp. 103 / 151 94 / 132 The reduction is visualized by agarose gel electrophoresis followed by Western blotting with a peroxidase-conjugated anti-VWF antibody. The ADAMTS13 activity concentration in the test sample can be established by reference to a series of diluted normal plasma samples. SDS-PAGE and Western blotting can also be performed, which involves visualizing dimeric VWF fragments after SDS-PAGE and Western blotting. This assay is technically easier than agarose gel electrophoresis-SDS and appears to be a very sensitive method for measuring ADAMTS13 activity levels.

[0208] In some respects, indirect assays involve the detection of cleavage products from a full-length VWF molecule or a VWF fragment encompassing the 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 the plasma test sample in the presence of BaC12 and 1.5 M urea, which denatures VWF. VWF is cleaved by ADAMTS13, and residual VWF is measured by its binding to Type III collagen. Bound VWF is quantified using an ELISA assay with a conjugated anti-VWF antibody. Another indirect assay is the ristocetin-induced aggregation assay. This is similar to the collagen binding assay above, but residual VWF is measured by ristocetin-induced platelet aggregation using a platelet aggregometer. Another indirect assay is a functional ELISA.In this assay, a recombinant VWF fragment is immobilized on an ELISA plate using an antibody for identification of the VWF. The VWF fragment encodes the domain. Petition 870200095920, dated 07 / 31 / 2020, pp. 104 / 151 95 / 132 A2 and the ADAMTS13 cleavage site in Tyrl605-Metl606 are identified with S-transferase [GST]-histidine [GST-VWF73His]. Plasma is added to the immobilized GST-VWF7 3-His fragment, and cleavage of the immobilized fragment occurs at the ADAMTS13 cleavage site. The residual cleaved VWF fragment is measured using a second monoclonal antibody that recognizes only the cleaved VWF fragment and NOT the interacting fragment. The activity of ADAMTS13 is therefore inversely proportional to the residual substrate concentration.

[0209] In certain embodiments, ADAMTS13 is supplied or administered at a therapeutically effective concentration between about 0.05 mg / ml and about 10 mg / ml in the final formulation. In other embodiments, ADAMTS13 is present at a concentration between about 0.1 mg / ml and about 10 mg / ml. In still other embodiments, ADAMTS13 is present at a concentration between about 0.1 mg / ml and about 5 mg / ml. In another embodiment, ADAMTS13 is present at a concentration between about 0.1 mg / ml and about 2 mg / ml. In still other embodiments, ADAMTS13 may be present at about 0.01 mg / ml, or at 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, mg / ml, 0.8 mg / ml, 1.3 mg / ml, 1.8 mg / ml, 3.5 mg / ml, 6.0 0.3 mg / ml, 0 mg / ml, 0.9 mg / ml, 1.4 mg / ml, 1.9 mg / ml, 4.0 mg / ml, 6.5 .4 mg / ml, 0 mg / ml, 1.0 mg / ml, 1.5 mg / ml, 2.0 mg / ml, 4.5 mg / ml, 7.0 mg / ml, 0.6 mg / ml, 1.1 mg / ml, 1.6 mg / ml, 2.5 mg / ml, 5.0 mg / ml, 7.5 mg / ml, 0.7 mg / ml, 1.2 mg / ml, 1.7 mg / ml, 3.0 mg / ml, 5.5 mg / ml, 8.0 mg / ml, 8.5 mg / ml, 9.0 mg / ml, 9.5 mg / ml, 10.0 mg / ml or a Petition 870200095920, dated 07 / 31 / 2020, pp. 105 / 151 96 / 132 higher concentration.

[0210] In some embodiments, the concentration of a relatively pure ADAMTS13 formulation can be determined by spectroscopy (i.e., total protein measured at A280) or other mass determination (e.g., Bradford assay, silver staining, weight of a lyophilized powder, etc.). In other embodiments, the ADAMTS13 concentration can be determined by an ADAMTS13 ELISA assay (e.g., mg / ml of antigen).

[0211] In some respects, the concentration of ADAMTS13 in a development formulation is expressed as an enzymatic activity level. For example, in some embodiments, an ADAMTS13 formulation contains between about 10 FRETS-VWF73 activity units and about 10,000 FRETS-VWF73 activity units or other suitable ADAMTS13 enzymatic unit (IU). In other embodiments, the formulation may contain between approximately 20 FRETS-VWF73 activity units (Ufv73) and approximately 8000 FRETS-VWF73 activity units, or between approximately 30 Ufv73 and approximately 6000 Ufv73, or between approximately 40 Ufv73 and approximately 4000 Ufv73, or between approximately 50 Ufv73 and approximately 3000 Ufv73, or between approximately 75 Ufv73 and approximately 2500 Ufv73, or between approximately 100 Ufv73 and approximately 2000 Ufv73, or between approximately 200 Ufv73 and approximately 1500 Ufv73, or between approximately other ranges therein.

[0212] In some embodiments, ADAMTS13 is supplied or administered at a dose of approximately 10 Wv73 / kg body weight to 10,000 UFV73 / kg body weight. In one embodiment, ADAMTS13 is administered at a dose of approximately 20 UFV73 / kg body weight to approximately 8,000 Wv73 / kg body weight. Petition 870200095920, dated 07 / 31 / 2020, pp. 106 / 151 97 / 132 body weight. In one embodiment, ADAMTS13 is administered at a dose of approximately 30 UFV73 / kg of body weight to approximately 6000 UFV73 / kg of body weight. In one embodiment, ADAMTS13 is administered at a dose of approximately 40 UFV73 / kg of body weight to approximately 4000 UFV73 / kg of body weight. In one embodiment, ADAMTS13 is administered at a dose of approximately 100 UFV73 / kg of body weight to approximately 3000 UFV73 / kg of body weight. In one embodiment, ADAMTS13 is administered at a dose of approximately 200 UFV73 / kg of body weight to approximately 2000 UFV73 / kg of body weight. In other modalities, ADAMTS13 is administered at approximately 10 UFV73 / kg of body weight, approximately 20, 30, 40, 50, 60, 70, 80, 90, 100, 150, 200, 250, 300, 350, 400, 450, 500, 600, 700, 800, 900, 1000, 1100, 1200, 1300, 1400, 1500, 1600, 1700, 1800, 1900, 2000, 2100, 2200, 2300, 2400, 2500, 2600, 2700, 2800, 2900, 3000, 3100, 3200, 3300, 3400, 3500, 3600, 3700, 3800, 3900, 4000, 4500, 5000, 5500, 6000, 6500, 7000, 7500, 8000, 8500, 9000, 9500 or 10000 UFV73 / kg of body weight, or at an intermediate dose or dose range thereof.

[0213] In some respects, an ADAMTS13 formulation provided in this document contains between about 20 and about 10000 Ufv73. In some embodiments, the formulation contains about 10 FRETS-VWF73 activity units, or about 20, 30, 40, 50, 60, 70, 80, 90, 100, 150, 200, 250, 300, 350, 400, 450, 500, 600, 700, 800, 900, 1000, 1100, 1200, 1300, 1400, 1500, 1600, 1700, 1800, 1900, 2000, 2100, 2200, 2300, 2400, 2500, 2600, 2700, 2800, 2900, 3000, 3100, 3200, 3300, 3400, 3500, 3600, 3700, 3800, 3900, 4000, 4100, 4200, 4300, 4400, 4500, 4600, 4700, 4800, 4900, 5000, 5100, 5200, 5300, 5400, 5500, 5600, 5700, 5800, 5900, 6000, Petition 870200095920, dated 07 / 31 / 2020, pp. 107 / 151 98 / 132 6100, 6200, 6300, 6400, 6500, 6600, 6700, 6800, 6900, 7000, 7100, 7200, 7300, 7400, 7500, 7600, 7700, 7800, 7900, 8000, 8100, 8200, 8300, 8400, 8500, 8600, 8700, 8800, 8900, 9000, 9100, 9200, 9300, 9400, 9500, 9600, 9700, 9800, 9900, 10000 or more activity units of FRETS-VWF73.

[0214] In some respects, the concentration of ADAMTS13 can be expressed as enzymatic activity per unit volume, for example, enzymatic units of ADAMTS13 per ml (IU / ml). For example, in some embodiments, an ADAMTS13 formulation contains between about 10 IU / ml and about 10,000 IU / ml. In some other embodiments, the formulation contains between about 20 IU / ml and about 10,000 IU / ml, or between about 20 IU / ml and about 8,000 IU / ml, or between about 30 IU / ml and about 6,000 IU / ml, or between about 40 IU / ml and about 4,000 IU / ml, or between about 50 IU / ml and about 3,000 IU / ml, or between about 75 IU / ml and about 2,500 IU / ml, or between about 100 IU / ml and about 2,000 IU / ml, or between about 200 IU / ml and about 1,500 IU / ml, or within other ranges therein. In some embodiments, an ADAMTS13 formulation provided in this document contains between approximately 150 IU / ml and approximately 600 IU / ml.In another embodiment, an ADAMTS13 formulation provided in this document contains between about 100 IU / ml and about 1000 IU / ml. In some embodiments, an ADAMTS13 formulation provided in this document contains between about 100 IU / ml and about 800 IU / ml. In some embodiments, an ADAMTS13 formulation provided in this document contains between about 100 IU / ml and about 600 IU / ml. In some embodiments, an ADAMTS13 formulation provided in this document contains between... Petition 870200095920, dated 07 / 31 / 2020, pp. 108 / 151 99 / 132 approximately 100 IU / ml and approximately 500 IU / ml. In some embodiments, an ADAMTS13 formulation provided in this document contains between approximately 100 IU / ml and approximately 400 IU / ml. In some embodiments, an ADAMTS13 formulation provided in this document contains between approximately 100 IU / ml and approximately 300 IU / ml. In some embodiments, an ADAMTS13 formulation provided in this document contains between approximately 100 IU / ml and approximately 200 IU / ml. In some embodiments, an ADAMTS13 formulation provided in this document contains between approximately 300 IU / ml and approximately 500 IU / ml. In some embodiments, an ADAMTS13 formulation provided in this document contains approximately 100 IU / ml. In some embodiments, an ADAMTS13 formulation provided in this document contains approximately 300 IU / ml. In several embodiments, the formulation contains approximately of 10 IU / ml, or approximately 20, 30, 40, 50, 60, 70, 80, 90, 100, 150, 200, 250, 300, 350, 400, 450, 500, 600, 700, 800, 900, 1000, 1100, 1200, 1300, 1400, 1500, 1600, 1700, 1800, 1900, 2000, 2100, 2200, 2300, 2400, 2500, 2600, 2700, 2800, 2900, 3000, 3100, 3200, 3300, 3400, 3500, 3600, 3700, 3800, 3900, 4000, 4100, 4200, 4300, 4400, 4500, 4600, 4700, 4800, 4900, 5000, 5100, 5200, 5300, 5400, 5500, 5600, 5700, 5800, 5900, 6000, 6100, 6200, 6300, 6400, 6500, 6600, 6700, 6800, 6900, 7000, 7100, 7200, 7300, 7400, 7500, 7600, 7700, 7800, 7900, 8000, 8100, 8200, 8300, 8400, 8500, 8600, 8700, 8800, 8900, 9900, 9000, 10000, 9100, 9200, 9300, 9400, or more IU / ml. 9500, 9600, 9700, 9800,

[0215] In some embodiments, the ADAMTS13 formulations provided in this document may additionally comprise one or more excipients, carriers and / or Petition 870200095920, dated 07 / 31 / 2020, pp. 109 / 151 100 / 132 pharmaceutically acceptable 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 by reference in its entirety for all purposes. Furthermore, in one embodiment, the ADAMTS13 formulations provided herein will have a tonicity in a range described 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 by reference in its entirety and for all purposes.

[0216] The dosing frequency will depend on the pharmacokinetic parameters of an ADAMTS13 molecule in the formulation used. Typically, a clinician will administer the composition until a dosage is reached that achieves the desired effect. Therefore, in several aspects, the composition is administered as a single dose, or as two or more doses (which may or may not contain the same amount of the desired molecule) over time, or as a continuous infusion via a catheter or implantable device. In some aspects, the composition comprising ADAMTS13 is administered as a single bolus injection, monthly, every two weeks, weekly, twice a week, every two days, daily, every 12 hours, every eight hours, every six hours, every four hours, or every two hours. In aspects of preventive or prophylactic treatment of the development, ADAMTS13 is administered in multiple doses to maintain a circulating level of ADAMTS13 effective in preventing the onset of Petition 870200095920, dated 07 / 31 / 2020, pp. 110 / 151 101 / 132 CVO, LPA, or SARA. In such cases, the composition comprising ADAMTS13 is administered monthly, every two weeks, weekly, twice a week, every other day, or daily. In particular cases, the injection is administered subcutaneously (e.g., WO2014151968, incorporated herein by reference in its entirety for all purposes). In other cases, the injection is administered intravenously. Further refinement of the appropriate dosage administered and the timing of administration is routinely performed by those of ordinary skill in the art and is within the scope of tasks routinely performed by them. Appropriate dosages are frequently determined through the use of appropriate dose-response data that are routinely obtained. Kits that include ADAMTS13

[0217] As an additional aspect, the disclosure includes kits comprising one or more pharmaceutical formulations for administration of ADAMTS13 or a composition of ADAMTS13 to an individual, packaged in a manner that facilitates its use for administration to the individual.

[0218] In one specific embodiment, the disclosure includes kits for producing a single-dose administration unit. In another embodiment, the disclosure includes kits for providing multiple-dose administration units. The kits, in various aspects, each contain a first container having a dry protein and a second container having an aqueous formulation. Also included in the scope of this disclosure are kits containing pre-filled single-chamber and multiple-chamber syringes. Petition 870200095920, dated 07 / 31 / 2020, pp. 111 / 151 102 / 132 chambers (e.g., liquid-filled syringes and lyosyringe-type syringes).

[0219] In another embodiment, such a kit includes a pharmaceutical formulation described in this document (for example, a composition comprising a therapeutic protein, for example, ADAMTS13), packaged in a container such as a sealed vial or bottle, with a label affixed to the container or included in the package describing the use of the compound or composition in the practice of the method. In one embodiment, the pharmaceutical formulation is packaged in the container so that the amount of headspace in the container (for example, the amount of air between the liquid formulation and the top of the container) is very small. Preferably, the amount of headspace is negligible (i.e., almost non-existent).

[0220] In some respects, 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 gelatin or human serum albumin.

[0221] In some respects, the pharmaceutical formulation or composition comprises an antimicrobial preservative. The term antimicrobial preservative refers to any Petition 870200095920, dated 07 / 31 / 2020, pp. 112 / 151 103 / 132 A substance added to the composition that inhibits the growth of microorganisms that may be introduced by repeated puncture of multi-dose vials, should such containers be used. Examples of antimicrobial preservatives include, but are not limited to, substances such as thimerosal, 2-phenoxyethanol, benzethonium chloride, and phenol.

[0222] In one aspect, the kit contains a first container that has a therapeutic protein or protein composition, and a second container that has a physiologically acceptable reconstitution solution for the composition. In one aspect, the pharmaceutical formulation is packaged in a unit dosage form. The kit may optionally include a suitable device for administering the pharmaceutical formulation according to a specific route of administration. In some aspects, the kit contains a label describing the use of the pharmaceutical formulations.

[0223] This entire document is intended to be reported 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 together in the same sentence, paragraph, or section of this document. The disclosure also includes, for example, all disclosure modalities with a scope that is in any way more restricted than the variations specifically mentioned above.

[0224] All publications, patents and patent applications cited in this descriptive report are incorporated herein by reference. Petition 870200095920, dated 07 / 31 / 2020, pp. 113 / 151 104 / 132 as if each individual patent application or publication were individually and specifically indicated as being incorporated by reference in its entirety to the extent that it is not inconsistent with the disclosure. Additional options

[0225] In certain embodiments, a method is provided to treat or prevent vaso-occlusive crisis (VOC) in an individual suffering from sickle cell disease (SCD). A therapeutically effective amount of a composition comprising ADAMTS13 is administered to the individual. The individual may be a human patient with SCD, or an animal with SCD. ADAMTS13 may be in a recombinant form. ADAMTS13 may be part of a formulation suitable for intravenous injection. ADAMTS13 may be part of a formulation suitable for subcutaneous injection. The dosage of ADAMTS13 may be 2500 IU / kg to 4000 IU / kg, 2800 IU / kg to 3800 IU / kg, 3000 IU / kg to 3400 IU / kg, approximately 3200 IU / kg, or 3200 IU / kg in a rodent. The dosage of ADAMTS13 can be from 40 IU / kg to 100 IU / kg, from 100 IU / kg to 300 IU / kg, from 120 IU / kg to 240 IU / kg, from 150 IU / kg to 200 IU / kg, or from 300 IU / kg to 500 IU / kg in a human patient.ADAMTS13 can be administered intravenously before, during, or after an acute CVO in a human or animal patient with DF. ADAMTS13 can also be administered subcutaneously before, during, or after an acute CVO in a human or animal patient with DF. Treatment may be effective in protecting the individual, for example, a human or animal patient with DF, from morbidity and mortality associated with CVO or hypoxia. Petition 870200095920, dated 07 / 31 / 2020, pp. 114 / 151 105 / 132

[0226] In certain embodiments, a method is provided for treating or preventing acute lung injury (ALI) or acute respiratory distress syndrome (ARDS). A therapeutically effective amount of a composition comprising ADAMTS13 is administered to the individual. ADAMTS13 may be in recombinant form. ADAMTS13 may be part of a formulation suitable for intravenous injection. ADAMTS13 may be part of a formulation suitable for subcutaneous injection. The dosage of ADAMTS13 may be 2500 IU / kg to 4000 IU / kg, 2800 IU / kg to 3800 IU / kg, 3000 IU / kg to 3400 IU / kg, approximately 3200 IU / kg, or 3200 IU / kg in a rodent. 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 in a human patient. ADAMTS13 can be administered intravenously at a time before, during, or after an ALI or ARDS in a human or animal patient.ADAMTS13 can be administered subcutaneously at a time before, during, or after an ALI or ARDS in a human or animal patient. Treatment may be effective in protecting the individual, for example, a human patient with ALI and / or ARDS, from morbidity and mortality.

[0227] In another embodiment, a method is provided to treat, improve or prevent (a) CVO in an individual who has DF or (b) lung injury in an individual who suffers from or is at risk of suffering from acute lung injury (ALI) and / or acute respiratory distress syndrome (ARDS). A therapeutically effective amount of ADAMTS13 is administered to the individual. Lung injury or inflammation Petition 870200095920, dated 07 / 31 / 2020, pages 115 / 151 106 / 132 Vascular lung injury may be secondary to, or induced by, hypoxia. Lung injury or vascular inflammation may be secondary to, or induced by, reoxygenation stress. During hypoxic or reoxygenation stress, the oxygen level may be approximately 7%, approximately 8%, approximately 9%, approximately 10%, 7-10%, or 7-9%. The individual may be a human patient with SCD, a human patient experiencing a CVO, a human patient with ALI, a human patient with ARDS, an animal with SCD, an animal experiencing CVO, an animal with ALI, an animal with ARDS, or an animal homozygous for HbA. ADAMTS13 may be part of a formulation suitable for intravenous injection. ADAMTS13 may be part of a formulation suitable for subcutaneous injection. The dosage of ADAMTS13 can be 2500 IU / kg to 4000 IU / kg, 2800 IU / kg to 3800 IU / kg, 3000 IU / kg to 3400 IU / kg, approximately 3200 IU / kg, or 3200 IU / kg in a rodent.The dosage of ADAMTS13 may 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 in a human patient. The individual may be monitored for one or more of the following: BAL protein content and BAL leukocyte count at one or more time points before, during, and after treatment. Administration of ADAMTS13 may be effective in reducing the protein content of BAL 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., untreated individual). Administration of ADAMTS13 may be effective in reducing the leukocyte count in BAL by at least 35%, at least 36%, at least 37%, at least 38%, at least 39%. Petition 870200095920, dated 07 / 31 / 2020, pp. 116 / 151 107 / 132%, at least 40%, 30-45%, 33-43%, 34-42%, 35-41%, or 36-40%, compared to a control (e.g., untreated individual).

[0228] At least in the above modalities, ADAMTS13 administration may be effective in preventing activation or increased expression or level of at least one of VCAM1 and ICAM-1 and / or in reducing the expression of at least one of ET-1, TXAS, and HO-1. See Figures 2B, 2C, and 3B. ADAMTS13 administration may be effective in reducing the expression or level of TXAS or ET-1, as measured by densitometry, for example, 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 with a control (e.g., untreated individual). Administration of ADAMTS13 may be effective in reducing the expression, level, or activity of ICAM-1, as measured by densitometry, for example, 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., untreated individual).Administration of ADAMTS13 may be effective in reducing the expression or level of HO-1, as measured by densitometry, for example, 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., untreated individual). Administration of ADAMTS13 may be effective in reducing the P-NF-kB / NF-kB ratio, as measured by densitometry, for example, 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%. Petition 870200095920, dated 07 / 31 / 2020, pp. 117 / 151 108 / 132 compared to a control (e.g., untreated individual). In an individual with DF or experiencing CVO, ADAMTS13 administration may be effective in reducing VCAM-1 expression, level, or activity, as measured by densitometry, for example, 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., untreated individual). In certain modalities, the biomarker (e.g., VCAM-1, ICAM-1, P-NF-kB, NF-kB, ET-1, TXAS, and HO-1) is measured in the lung. In certain modalities, the biomarker (e.g., VCAM-1, ICAM-1, P-NF-kB, NF-kB, ET-1, TXAS, and HO-1) is measured in the kidney.

[0229] At least in the above modalities, with respect to the treatment of lung injury or vascular inflammation associated with DF, CVO, LPA and / or ARDS, the administration of ADAMTS13 may be effective in preventing the activation or increased expression or level of at least one of VCAM1 and ICAM-1 and / or in reducing the expression or level of at least one of ET-1, TXAS and HO-1. See Figures 2B and 2C. The administration of ADAMTS13 may be effective in reducing the expression or level of TXAS or ET-1, as measured by densitometry, for example, 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 with a control (e.g., untreated individual). Administration of ADAMTS13 may be effective in reducing the expression, level, or activity of ICAM-1, as measured by densitometry, for example, by at least 53%, at least 56%, at least 59%, at least 62%. Petition 870200095920, dated 07 / 31 / 2020, pp. 118 / 151 109 / 132%, at least 65%, 53-65%, 55-62%, or 57-60%, compared to a control (e.g., untreated individual). Administration of ADAMTS13 may be effective in reducing HO-1 expression or level, as measured by densitometry, for example, 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., untreated individual). Administration of ADAMTS13 may be effective in reducing the P-NF-kB / NF-kB ratio, as measured by densitometry, for example, 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., untreated individual).In an individual with DF and / or experiencing a CVO, administration of ADAMTS13 may be effective in reducing the expression, or level or activity of VCAM-1, as measured by densitometry, for example, 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., untreated individual). In certain modalities, the biomarker (e.g., VCAM-1, ICAM-1, P-NF-kB, NF-kB, ET-1, TXAS, and HO-1) is measured in the lung.

[0230] At least in the above modalities, with respect to the treatment of renal injury or vascular inflammation associated with DF, CVO, LPA and / or ARDS, the administration of ADAMTS13 may be effective in preventing the activation and / or increased expression or levels of VCAM-1, reducing the P-NFkB / NF-kB ratio and / or reducing the expression or level of at least one of ET-1 or TXAS. See Figures 3A and 3B. A Petition 870200095920, dated 07 / 31 / 2020, pp. 119 / 151110 / 132 Administration of ADAMTS13 may be effective in reducing the expression, or level of TXAS, as measured by densitometry, for example, 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 with a control (e.g., untreated individual). Administration of ADAMTS13 may be effective in reducing the P-NF-kB / NF-kB ratio, as measured by densitometry, for example, 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., untreated individual).Administration of ADAMTS13 may be effective in reducing the expression, level, or activity of VCAM-1 in an individual with DF or experiencing CVO, as measured by densitometry, for example, 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., untreated individual). In certain modalities, the biomarker (e.g., VCAM-1, P-NF-kB, NF-kB, ET-1, TXAS, and HO-1) is measured in the kidney.

[0231] At least in the above modalities, a blood sample from the individual may be obtained, for example, to monitor the treatment of DF, CVO, LPA and / or ARDS, with one or more of the following hematocrit values ​​measured: % hematocrit (Hct) and mean corpuscular volume (MCV), as indicators of erythrocyte viability; hemoglobin (Hb), mean corpuscular hemoglobin (MCH), and mean corpuscular hemoglobin concentration (MCHC), as indicators of oxygen-binding capacity; heterogeneity of Petition 870200095920, dated 07 / 31 / 2020, pages 120 / 151 111 / 132 red blood cell distribution width (HDW), as an indicator of the presence of dense red blood cells; reticulocyte count (Reties), as an indicator of anemia status; neutrophil count, as an indicator of systemic inflammatory status; and / or lactate dehydrogenase (LDH) as a general marker of cellular damage. Administration of ADAMTS13 may be effective in reducing MCHC 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., untreated individual). Administration of ADAMTS13 may be effective in increasing reties by at least 5%, at least 7%, at least 9%, at least 11%, or at least 13%, compared to a control (e.g., untreated individual).Administration of ADAMTS13 may be effective in reducing 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., untreated individual). Administration of ADAMTS13 may be effective in reducing LDH (cells / microliter) by at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, or at least 30%, compared to a control (e.g., untreated individual).

[0232] At least in the above modalities, the administration of ADAMTS13 in individuals with DF or who experience CVO, the administration of ADAMTS13 may be effective in changing the % levels of Hct, Hb, MCV, MCH and / or HDW. The administration of ADAMTS13 may be effective in increasing the % Hct in individuals with DF or who experience CVO by at least 60%, at least 65%, at least 70%, at least 75%, by Petition 870200095920, dated 07 / 31 / 2020, pages 121 / 151 112 / 132 minus 80%, or at least 85%, compared to a control (e.g., untreated individual). Administration of ADAMTS13 may be effective in increasing Hb in individuals with DF or experiencing CVO by at least 18%, at least 20%, at least 22%, at least 24%, or at least 26%, compared to a control (e.g., untreated individual). Administration of ADAMTS13 may be effective in increasing MCV in individuals with DF or experiencing CVO by at least 18%, at least 20%, at least 22%, at least 24%, or at least 26%, compared to a control (e.g., untreated individual). Administration of ADAMTS13 may be effective in increasing MCH in individuals with DF or who experience CVO 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., untreated individual).Administration of ADAMTS13 may be effective in reducing HDW in individuals with DF or who experience CVO by at least 12%, at least 14%, at least 16%, at least 18%, or at least 20%, compared to a control (e.g., untreated individual).

[0233] At least in the above modalities, administration of ADAMTS13 in individuals with DF who experience CVO may reduce or prevent DF-related tissue injury. In certain modalities, the tissue injury is caused by hypoxia. In certain modalities, the tissue injury is caused by reoxygenation. In certain modalities, the tissue is lung tissue. In certain modalities, the tissue is kidney tissue. In certain modalities, administration of ADAMTS13 reduced cellular infiltrates. Petition 870200095920, dated 07 / 31 / 2020, pages 122 / 151 113 / 132 inflammatory and / or thrombus formation in the tissue compared to the control. In certain modalities, ADAMTS13 administration reduced inflammatory cell infiltrates in lung tissue compared to the control. In certain modalities, ADAMTS13 administration reduces pulmonary inflammatory cell infiltrates by at least 20%, at least 30%, at least 40%, at least 50%, or at least 60%. In certain modalities, ADAMTS13 administration reduced thrombus formation in lung tissue compared to the control. In certain modalities, ADAMTS13 administration reduces pulmonary thrombus formation 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 certain modalities, ADAMTS13 administration reduced inflammatory cellular infiltrates in renal tissue compared to the control.In certain modalities, ADAMTS13 administration reduces renal inflammatory cell infiltrates 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 modalities, ADAMTS13 administration reduced thrombus formation in lung tissue compared to control. In certain modalities, ADAMTS13 administration reduces pulmonary thrombus formation by at least 20%, at least 30%, at least 40%, at least 50%, or at least 60%. Petition 870200095920, dated 07 / 31 / 2020, pages 123 / 151 114 / 132

[0234] At least in the above modalities, administration of ADAMTS13 in individuals with ALI and / or ARDS may reduce or prevent tissue injury related to ALR and / or ARDS. In certain modalities, the tissue injury is caused by hypoxia. In certain modalities, the tissue injury is caused by reoxygenation. In certain modalities, the tissue is lung tissue. In certain modalities, the tissue is kidney tissue. In certain modalities, administration of ADAMTS13 reduced inflammatory cellular infiltrates and / or thrombus formation in the tissue compared to control. In certain modalities, administration of ADAMTS13 reduced inflammatory cellular infiltrates in lung tissue compared to control. In certain modalities, administration of ADAMTS13 reduces pulmonary inflammatory cellular infiltrates by at least 20%, at least 30%, at least 40%, at least 50%, or at least 60%.In certain modalities, ADAMTS13 administration reduced thrombus formation in lung tissue compared to control. In certain modalities, ADAMTS13 administration reduces pulmonary thrombus formation by at least 20%, at least 30%, at least 40%, or at least 50%. In certain modalities, ADAMTS13 administration reduced inflammatory cell infiltrates in renal tissue compared to control. In certain modalities, ADAMTS13 administration reduces renal inflammatory cell infiltrates by at least 20%, at least 30%, at least 40%, at least 50%, or at least 60%. In certain modalities, ADAMTS13 administration reduced thrombus formation in lung tissue by... Petition 870200095920, dated 07 / 31 / 2020, pages 124 / 151 115 / 132 comparison with control. In certain modalities, ADAMTS13 administration reduces pulmonary thrombus formation by at least 20%, at least 30%, or at least 40%.

[0235] It is understood that the examples and embodiments described in this document are for illustrative purposes only and that various modifications or alterations in light thereof will be suggested for persons skilled in the art and should be included within the spirit and scope of this application and the scope of the appended claims. EXAMPLES

[0236] Additional details and aspects of the disclosure will become apparent from the following examples, which are intended to be illustrative, not limiting. EXAMPLE 1: ADAMTS13 PREVENTED THE DEATH OF MICE WITH DF EXPOSED TO INDUCED CVO DUE TO LETHAL HYPOXIA

[0237] Since acute sickle cell events are triggered by low oxygen (hypoxia), this example was conducted to evaluate the impact of ADAMTS13 on survival in a model of a sickle cell individual under hypoxia. The aim of this example was to determine whether recombinant ADAMTS13 (rADAMTS13 (BAX930 / SHP655)) could protect humanized sickle cell mice exposed to lethal hypoxia-induced CVO. It has been previously shown that exposing sickle cell mice to potentially fatal severe hypoxia / reoxygenation stress can be useful in evaluating the efficacy of innovative therapeutic treatments on the survival of sickle cell mice (Sabaa et al.). Petition 870200095920, dated 07 / 31 / 2020, pages 125 / 151 116 / 132 al., JCI 118:1924, 2008).

[0238] The experiments were performed on healthy control mice (i.e., AA) aged 4-6 weeks (Hbatm^HBvTow Hbbtm3(HBGi,HBB)Tow) and mice with DF (Hbatmi(HBA)Tow Hbbtm2(hbgi,hbb*)tow) (humanized mouse model for sickle cell disease (i.e., mice with DF or SS)). Healthy (AA) and sickle cell disease (SCD or SS) mice were treated with vehicle or rADAMTS13 at a dosage of 2940 FRETS-U / kg (~3200 IU / kg) intravenously (iv), 1 hour before severe hypoxia / reoxygenation stress (at approximately 7% oxygen for 10 hours), followed by 3 hours of reoxygenation at approximately 21% oxygen), which has previously been shown to biologically recapitulate the organ damage observed in acute CVO in human SCD patients. See similar protocol as reported by Kalish et al. (supra).More specifically, four groups (n=6) of mice with DF and AA were treated with vehicle or ADAMTS13 (BAX930 / SHP655) (2940 FRETS-U / kg (~3200 IU / kg)) and were exposed to hypoxic stress conditions.

[0239] Treatment with recombinant ADAMTS13 completely protected DF mice from death compared with vehicle-treated DF mice (0% mortality in DF mice treated with rADAMTS13 vs. 83.3% mortality in vehicle-treated DF mice after 10 hours of hypoxia; 0% mortality in DF mice treated with rADAMTS13 vs. 100% mortality in vehicle-treated DF mice after 10 hours of hypoxia followed by 3 hours of reoxygenation; p<0.001) (Figure 1). No difference in mouse survival was observed in Petition 870200095920, dated 07 / 31 / 2020, pages 126 / 151 117 / 132 healthy mice treated with vehicle or rADAMTS 13.

[0240] The data demonstrated that ADAMTS13 had a protective effect, including increased survival, in a model of DF after exposure to hypoxic stress. EXAMPLE 2: ADAMTS13 REDUCES STRESS-INDUCED HYPOXIA / REOXYGENATION ABOLACIES IN THE LUNG

[0241] The aim of this example was to evaluate the impact of ADAMTS13 on lung injury and vascular inflammation induced by hypoxia / reoxygenation (H / R) stress.

[0242] Healthy control mice (Hbatm1(HBA)Tow Hbbtm3(HBGI,HBB)Tow) and with DF (Hbatml (HBA)Tow Hbbtm2(HBGI,HBB*)Tow) were exposed to hypoxia / reoxygenation (H / R) stress, which has previously been shown to biologically recapitulate acute CVO and organ damage observed in acute CVO in human patients with DF. In particular, six experimental groups were used - (1) AA with untreated normoxia; (2) SS with untreated normoxia; (3) AA with vehicle plus H / R; (4) AA with ADAMTS13 (BAX930 / SHP655) plus H / R; (5) SS with vehicle plus H / R; and (6) SS with ADAMTS13 (BAX930 / SHP655) plus H / R. In this experiment, the H / R conditions were 8% oxygen for 10 h, followed by 3 h of recovery at approximately 21% oxygen, an experimental scheme that is usually not fatal for mice with DF (Kalish et al., Haematologica 100: 870-80, 2015).

[0243] Under H / R and normoxic conditions, pulmonary vascular leakage was assessed in mice by measuring protein content and leukocyte count (total leukocytes measured in cells / microliter) in bronchoalveolar lavage fluid (BAL). Petition 870200095920, dated 07 / 31 / 2020, pp. 127 / 151 118 / 132

[0244] Pulmonary vascular leakage was examined by measuring protein content and leukocyte count (i.e., number of cells) in bronchoalveolar lavage fluid (BAL). As shown in Table 1, under normoxic conditions, increased BAL protein levels and leukocyte counts were detected in mice with DF compared to healthy mice, indicating the accumulation of proteins and inflammatory cells in the alveolar space. Interestingly, in response to H / R, BAL proteins and leukocyte counts were significantly increased in mice with DF and AA. TABLE 1: Results of lung leak experiments performed in mice with DF and AA under hypoxic and normoxic conditions. Normoxic conditions Hypoxic condition (8% oxygen) Untreated AA mice (n=6) Untreated DF mice (n=6) AA mice with Vehicle (n=6) AA mice with BAX930 (0 = 6) DF mice with Vehicle (n=6) DF mice with BAX930 (0 = 6) Protein content of BAL (mg / ml) 0.9 ± 0.03 2.5 ± 0.05° 3.1 ± 0.04 1.8 ± 0.05* 5.33 ± 0.4° 3.4 ± 0.07* Leukocytes in BAL (cells / pl) 200 ± 40 832 ± 36° 567 ± 20 328 ± 12* 1977±54° ​​1198±22* AA: Healthy mice or control mice homozygous for Hb A; DF: Mice with sickle cells or mice homozygous for HbS; and BAL: Bronchoalveolar lavage; *P<0.05 compared to vehicle-treated mice; °P<0.05 compared to AA mice.

[0245] The data showed that mice with DF had a significant increase in peripheral neutrophils (cells / microliter) compared to AA mice; in Petition 870200095920, dated 07 / 31 / 2020, pp. 128 / 151 119 / 132 However, treatment with ADAMTS13 significantly reduced neutrophil counts. The data also showed that mice with DF had a higher number of leukocytes (total leukocytes in bronchoalveolar lavage (BAL) (cells / microliter)) and higher leukocyte protein content (BAL proteins (mg / ml) in bronchoalveolar lavage) compared to controls, indicating that mice with DF suffered vascular leakage. Treatment with ADAMTS13 considerably reduced this effect (Figure 2A and Table 1), indicating that ADAMTS13 reduced systemic inflammation and reduced abnormalities in pulmonary vascular dysfunction.

[0246] These data indicate that ADAMTS13 prevented hypoxia-induced inflammatory vasculopathy and pulmonary vascular leakage anomalies in the lungs of mice with DF during acute vaso-occlusive crisis. Furthermore, ADAMTS13 significantly reduced BAL protein content and leukocyte count in mice with DF and AA compared to vehicle-treated controls, indicating that ADAMTS13 had a protective effect on the lung under hypoxic conditions. EXAMPLE 3: ADAMTS13 reduced stress-induced pulmonary vascular activation by Hypoxia / Reoxygenation

[0247] In order to study the effects of ADAMTS13 on vascular lung injury and inflammation, additional experiments were conducted with the same six experimental groups as described in Example 2 (i.e., (1) AA with untreated normoxia; (2) SS with untreated normoxia; (3) AA with Petition 870200095920, dated 07 / 31 / 2020, pp. 129 / 151 120 / 132 vehicle plus H / R; (4) AA with ADAMTS13 (BAX930 / SHP655) plus H / R; (5) SS with vehicle plus H / R; and (6) SS with ADAMTS13 (BAX930 / SHP655) plus H / R). In this example, as those reported in Example 2, vehicle or ADAMTS13 were administered to the animals and then exposed to 8% oxygen for 10 h followed by 3 h of recovery at 21% oxygen. Additional controls (AA and DF) were also subjected to normoxic conditions without vehicle or ADAMTS13.

[0248] Immunoblot analyses with specific antibodies against several markers of inflammation, vasoconstriction, and platelet aggregation (i.e., nuclear factor kappa B (NF-kB), endothelin-1 (ET-1), heme oxygenase 1 (HO-1), intracellular adhesion molecule 1 (ICAM-1), thromboxane synthase (TXAS), and vascular cell adhesion molecule 1 (VCAM-1)) were performed to measure the expression of these proteins in the lungs of healthy control mice (AA) and mice with DF treated with vehicle or rADAMTS13 after exposure to normoxic or hypoxic conditions (e.g., H / R).

[0249] The data from this example showed that ADAMTS13 prevented hypoxia-induced NF-kB activation in lung tissues of mice with DF and AA, indicating that ADAMTS13 reduces the process of pulmonary inflammation triggered by hypoxia (Figure 2B). In lungs of mice with DF under hypoxia, ADAMTS13 prevented the activation of VCAM1 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).

[0250] Table 2 reports the densitometric values Petition 870200095920, dated 07 / 31 / 2020, pp. 130 / 151 121 / 132 obtained through immunoblot analyses with specific antibodies against nuclear factor kappa B (NF-kB) and its activated form (P-NF-kB), endothelin 1 (ET-1), heme oxygenase 1 (HO-1), intracellular adhesion molecule 1 (ICAM-1), thromboxane synthase (TXAS), and vascular cell adhesion molecule 1 (VCAM-1) in the lungs of healthy control mice (AA) and sickle cell mice (SC) treated with vehicle or rADAMTS13 and exposed to hypoxia / reoxygenation or normoxic stress.

[0251] As shown in Table 2, under normoxic conditions, all measured protein markers (except ICAM-1) showed increased protein expression in SC mice compared to AA mice. Under hypoxic conditions, the expression of all measured markers was further increased in both healthy control and SC mice.However, treatment with ADAMTS13 (i.e., BAX930 / SHP655) had a protective effect in mice with DF and AA, as demonstrated by lower levels of all markers of inflammation, vasoconstriction, and platelet aggregation tested. TABLE 2. Normoxic conditions Hypoxic condition (8% oxygen) Untreated AA mice (n=6) Untreated DF mice (n=6) s AA mice with Vehicle (n=6) AA mice with BAX930 (n=6) DF mice with Vehicle (n=6) s DF mice with BAX930 (n=6) TXAS (DU) 0.9 ± 0.09 2±0.025° 2.4±0.05 0.7±0.04* 3.7±0.05° 0.8±0.034* ET-1 (DU) 0.55±0.06 1.65±0.07° 1.7±0.025 0.4±0.042* 2±0.06° 0.6±0.03* VCAM-1 (DU) 0.5±0.03 1.4±0.07° 2.1 ± 0.04 2.0±0.02 3.2±0.09° 1.8±0.03* ICAM-1 (DU) 0.8±0.03 0.9±0.07 1.7±0.045 0.75±0.04* 2.3±0.081 ° 0.85±0.07* HO-1 (DU) 0.4±0.06 1.1±0.03° 2.8±0.02 1.5±0.07* 3.0±0.04 1.44±0.09* P-NFkB / NF-kB ratio (DU) 0.8±0.03 1.6±0.08° 2±0.6 0.7±0.05* 3.8±0.34° 0.6±0.03* AA: Healthy mice or control mice Petition 870200095920, dated 07 / 31 / 2020, pages 131 / 151 122 / 132 homozygous for Hb A; DF: Sickle cell mice or HbS homozygous 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-kB: Phosphonuclear Factor kappa B; and NF-kB: Nuclear Factor kappa B. *P<0.05 compared to vehicle-treated mice; ° P<0.05 compared to AA mice.

[0252] Recombinant ADAMTS13 significantly reduced the expression levels of each of the protein markers tested in mice with DF (i.e., compared to vehicle-treated DF mice) (Table 2). Furthermore, recombinant ADAMTS13 reduced the pulmonary expression of all protein markers tested, with the exception of VCAM-1, in healthy control mice (AA) (i.e., compared to vehicle-treated control mice).

[0253] These data indicate that ADAMTS13 prevented hypoxia-induced inflammatory vasculopathy and pulmonary vascular leakage abnormalities in the lungs of mice with SCD during acute vaso-occlusive crisis. Furthermore, ADAMTS13 prevented the hypoxia-induced increased expression of potent vascular tone modulators, such as ET1 and TXAS, indicated as contributing factors to the vascular dysfunction described in SCD during acute events. Moreover, the data also showed that ADAMTS13 had a protective effect on lung tissue in healthy animals subjected to hypoxic conditions. Thus, the data indicate that ADAMTS13 reduces vascular activation and inflammatory responses related to hypoxic stress in the lungs. Petition 870200095920, dated 07 / 31 / 2020, pages 132 / 151 123 / 132 of healthy mice and mice with DF. EXAMPLE 4: ADAMTS13 REDUCES HYPOXIA / REOXYGENATION STRESS-INDUCED RENAL VASCULAR ACTIVATION

[0254] In order to study the effects of ADAMTS13 on vascular injury and inflammation in the kidney, additional experiments were conducted with the same six experimental groups as described in Example 2 (i.e., (1) AA with untreated normoxia; (2) SS with untreated normoxia; (3) AA with vehicle plus H / R; (4) AA with ADAMTS13 (BAX930 / SHP655) plus H / R; (5) SS with vehicle plus H / R; and (6) SS with ADAMTS13 (BAX930 / SHP655) plus H / R). In this example, as those reported in Examples 2 and 3, the animals were administered the vehicle or ADAMTS13 and then exposed to 8% oxygen for 10 h followed by 3 h of recovery at approximately 21% oxygen, which has previously been shown to biologically recapitulate acute CVO and organ damage observed in acute CVO in human patients with DF. Additional controls (AA and DF) were also subjected to normoxia conditions without vehicle or ADAMTS13.

[0255] Immunoblot analyses with antibodies specific against NF-kB and its activated form, P-NF-kB, as well as ET-1, TXAS, and VCAM-1 were performed to measure the expression of these proteins in the kidneys of mice with DF and AA treated with vehicle or rADAMTS13.

[0256] Table 3 reports the densitometric values ​​obtained through immunoblot analyses with specific antibodies against nuclear factor kappa B (NF-kB) and its activated form (P-NF-kB), endothelin 1 (ET-1), thromboxane synthase (TXAS), and vascular cell adhesion molecule 1. Petition 870200095920, dated 07 / 31 / 2020, pages 133 / 151 124 / 132 (VCAM-1) in the kidneys of healthy control (AA) mice and sickle cell (SC) mice treated with vehicle or rADAMTS13 and exposed to hypoxic or normoxic conditions (hypoxia / reoxygenation stress). As can be seen in Table 3, under normoxic conditions, all protein marker levels were higher in SC mice than in AA mice. Under hypoxic conditions, the expression levels of all protein markers, except VCAM-1, were further increased in both SC and AA mice. TABLE 3 Normoxic conditions Hypoxic condition (8% oxygen) Untreated AA mice (n=6) Untreated DF mice (n=6) AA mice with Vehicle (n=6) AA mice with BAX930 (n=6) DF mice with Vehicle (n=6) DF mice with BAX930 (n=6) TXAS (DU) 0.6±0.05 0.97±0.03° 1.8±0.07 0.4±0.022* 2.5±0.041° 0.6±0.02* ET-1 (DU) 0.5±0.051 0.98±0.05° 1.1±0.012 0.99±0.02 1.5±0.03° 1.4±0.02 VCAM-1 (DU) 1.1 ± 0.02 1.9±0.06° 1.05±0.08 0.9±0.05 2.1±0.08° 0.8±0.03* P-NFkB / NF-kB ratio (DU) 0.4±0.02 1.5±0.045° 2.4±0.08 0.6±0.055* 2.3±0.023 0.7±0.08* AA: Healthy mice or control mice homozygous for Hb A; DF: Sickle cell mice or mice homozygous for HbS; TXAS: Thromboxane synthase; ET-1: Endothelin 1; VCAM-1: Vascular Cell Adhesion Molecule 1; P-NF-kB: Phosphonuclear Factor kappa B; and NF-kB: Nuclear Factor kappa B. *P<0.05 compared to vehicle-treated mice; °P<0.05 compared to AA mice.

[0257] The data from this example showed that ADAMTS13 prevented hypoxia-induced NF-kB activation in the kidneys of mice with DF and AA, as well as mice with DF under normoxic conditions (Table 3 and Figure 3A). In Petition 870200095920, dated 07 / 31 / 2020, pp. 134 / 151 In 125 / 132 mice with DF exposed to hypoxia, there was an increase in the expression of VCAM-1, ET-1, and TXAS. ADAMTS13 prevented the hypoxia-induced increased expression of VCAM-1 and TXAS in mouse kidney strains and ET-1 levels in AA mouse kidneys (Table 3 and Figure 3B).

[0258] These data indicate that ADAMTS13 prevented hypoxia-induced increased expression of potent modulators of vascular tone, and / or factors that contribute to the vascular dysfunction described in SCD during acute events. The data indicate that ADAMTS13 reduces vascular activation and inflammatory responses related to hypoxic stress in the kidneys of healthy mice and mice with SCD. The example showed that rADAMTS13 can reduce acute events related to sickle cells, such as vasoconstriction and inflammatory vasculopathy in the kidney. EXAMPLE 5: ADAMTS13 IMPROVES HYPOXIA / REOXYGENATION STRESS-INDUCED ABNORMALITIES IN VARIOUS HEMATOLOGICAL PARAMETERS

[0259] In order to study the effects of ADAMTS13 on various hematological parameters, additional experiments were conducted with the same six experimental groups as described in Example 2 (i.e., (1) AA with untreated normoxia; (2) SS with untreated normoxia; (3) AA with vehicle plus H / R; (4) AA with ADAMTS13 (BAX930 / SHP655) plus H / R; (5) SS with vehicle plus H / R; and (6) SS with ADAMTS13 (BAX930 / SHP655) plus H / R). In this example, as those reported in Examples 2-4, animals received vehicle or ADAMTS13 administration and were then exposed to normoxic or H / R conditions (8% oxygen for 10 h followed by 3 h of recovery at approximately 21% Petition 870200095920, dated 07 / 31 / 2020, pp. 135 / 151 126 / 132 of oxygen).

[0260] The following hematological parameters were determined: % hematocrit (Hct) and mean corpuscular volume (MCV), as indicators of erythrocyte viability; hemoglobin (Hb), mean corpuscular hemoglobin (MCH), and mean corpuscular hemoglobin concentration (MCHC), as indicators of oxygen-binding capacity; red cell distribution heterogeneity (HDW), as an indicator of the presence of dense red cells; reticulocyte count, as an indicator of anemia status; neutrophil count, as an indicator of systemic inflammatory status; and lactate dehydrogenase (LDH) as a general marker of cellular damage.

[0261] Hematocrit is the ratio of red blood cell volume to total blood volume. MCV is the mean corpuscular volume (RCB). Hemoglobin is the protein responsible for transporting oxygen in the blood, and MCHC is the mean amount of hemoglobin per RBC in a blood sample; MCHC reflects the hemoglobin content within intact RBCs. Hemoglobin distribution width (HDW) is a measurement of the heterogeneity of hemoglobin concentration in red blood cells. Reticulocytes are relatively immature, newly produced red blood cells; reticulocyte count indicates whether sufficient red blood cells are being produced in the bone marrow. Neutrophils are recruited to the site of injury within minutes of trauma; thus, neutrophils are evidence of acute inflammation, and neutrophil count indicates an inflammatory state.

[0262] Table 4 shows the hematological parameters in Petition 870200095920, dated 07 / 31 / 2020, pp. 136 / 151 127 / 132 healthy control (AA) and sickle cell (DC) mice were subjected to 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, while MCV and HDW levels were higher, as were reticulocyte and neutrophil counts, in DC mice compared to control (AA) mice. In healthy control mice, hypoxic conditions increased reticulocyte and neutrophil counts. Administration of ADAMTS13 to control mice enhanced the large increase in neutrophil count, indicating a reduction in inflammation. In mice with sickle cell disease, hypoxic conditions reduced hematocrit, hemoglobin, MCV, and MCH, and increased MCHC, HDW, and neutrophil count.Administration of ADAMTS13 to mice with DF improved the reduction in Hct, Hb, MCV, and MCH, and improved the increase in MCHC, HDW, and neutrophil count. TABLE 4 Normoxic conditions Hypoxic condition (8% oxygen) Untreated AA mice (n=6) Untreated DF mice (n=6) AA mice with Vehicle (n=6) AA mice with BAX930 (n=5) DF mice with Vehicle (n=6) DF mice with BAX930 (n=5) Hct (%) 46.3±0.95 35.6±1.6° 45.3±0.8 44.3±0.4 15.8±2.3° 27.9±0.8* Hb (g / dl) 13.8±1.3 8.7±0.51° 13.4±0.1 13.1±0.5 5.99±0.5° 7.4±0.5* MCV (fl) 37.9±0.2 50.9±1.8° 38.3±0.3 38.5±0.3 41.3±1.7° 51.2±1.8* MCH (g / dl) 12.0±0.5 11.8±1.2 12.0±0.4 11.2±0.2 9.1±0.2° 9.7±0.4 CHCM (g / dl) 25.2±0.1 25.1±0.4 25.1±0.6 23.3±0.9 26.8±0.3° 24.8±0.2* HDW (g / dl) 2.88+0.03 4.72±0.08° 2.86±0.03 2.9±0.04 5.63±0.06° 4.78±0.04* Rhetics (%) 7.22±0.5 43.1±11 ° 8.59±0.7 9, 6±0.21 42.1±12° 45±2.9 Neutrophils (cells / pl) 841±135 3251±488° 3600±120 1768±299* 6800±250° 4399±133* Petition 870200095920, dated 07 / 31 / 2020, pp. 137 / 151 128 / 132 Normoxic conditions Hypoxic condition (8% oxygen) Untreated AA mice 0=6) Untreated DF mice 0=6) AA mice with Vehicle 0=6) AA mice with BAX930 0=5) DF mice with Vehicle 0=6) DF mice with BAX930 0=5) LDH (U / l) 288±12 573±30° 293±15 277±26 1234±81° 852 ±19* AA: Healthy mice or control mice homozygous for Hb A; DF: Sickle cell mice or mice homozygous for HbS; Hct: hematocrit; Hb: hemoglobin; MCV: mean corpuscular volume; MCH: mean corpuscular hemoglobin; MCHC: cellular hemoglobin concentration; HDW: red blood cell distribution heterogeneity; Retics: reticulocytes; and LDH: lactate dehydrogenase. *P<0.002 compared to vehicle-treated mice; °P<0.005 compared to AA mice. EXAMPLE 6: ADAMTS13 IMPROVES HYPOXIA / REOXYGENATION STRESS-INDUCED ANOMALIES IN VARIOUS HISTOPATHOLOGICAL PARAMETERS

[0263] In order to study the effects of ADAMTS13 on various histopathological parameters, additional experiments were conducted with four experimental groups - (1) AA with vehicle plus H / R; (2) AA with ADAMTS13 (BAX930 / SHP655) plus H / R; (3) SS with vehicle plus H / R; and (4) SS with ADAMTS13 (BAX930 / SHP655) plus H / R). In this example, animals received either vehicle or ADAMTS13 and were then exposed to H / R conditions (8% oxygen for 10 h followed by 3 h of recovery at approximately 21% oxygen).

[0264] The lungs and kidneys were collected after 3 h of reoxygenation. Lung and kidney pathology were analyzed, as well as inflammatory cellular infiltrate and the presence of thrombi. Petition 870200095920, dated 07 / 31 / 2020, pages 138 / 151 129 / 132 were determined.

[0265] Histological analysis revealed that H / R stress induced severe DF-related tissue damage in the lung and kidney of mice with DF. In the lung, H / R induced inflammatory cell infiltration and thrombus formation in all mice with DF (Table 5). In AA mice, H / R induced modest inflammatory cell infiltration and some thrombus formation in a few mice. In mice with DF, ADAMTS13 (BAX930 / SHP655) reduced inflammatory cell infiltration and thrombus formation compared to vehicle-treated mice with DF. In AA mice, ADAMTS13 (BAX930 / SHP655) reduced inflammatory cell infiltration in the lung.

[0266] In the kidney, H / R induced inflammatory cell infiltration and thrombus formation in all mice with DF. In AA mice, H / R induced limited inflammatory cell infiltration with little thrombus formation in a small number of mice. ADAMTS13 (BAX930 / SHP655) reduced inflammatory cell infiltrates in the kidney of mice with DF exposed to H / R, also impacting thrombus formation. In AA mice, ADAMTS13 (BAX930 / SHP655) reduced inflammatory cell infiltrates without any effect on thrombus formation (Table 5). TABLE 5 AA mice Mice with DF H / R and vehicle H / R and BAX930 H / R and vehicle H / R and BAX930 Lung (n=5) (n=5) (n=5) (n=4) Petition 870200095920, dated 07 / 31 / 2020, pages 139 / 151 130 / 132 AA Mice Mice with DF H / R and vehicle H / R and BAX930 H / R and vehicle H / R and BAX930 Inflammatory cellular infiltrates + (4 / 5) + (2 / 5) + (2 / 5) ++ (3 / 5) + (1 / 4) Thrombi + (2 / 5) 2.5 per observation field + (5 / 5) 2.2 per observation field + (5 / 5) 3 per observation field + (1 / 4) 3 per observation field Kidney (n=5) (n=5) (n=5) (n=4) Inflammatory cellular infiltrates + (2 / 5) + (1 / 5) + (2 / 5) ++ (1 / 5) 0 Thrombi + (4 / 5) 3.8 per observation field + (5 / 5) 3.2 per observation field + (5 / 5) 5 per observation field + (4 / 4) 2.5 per observation field observation H / R: hypoxia / reoxygenation stress; presence of thrombi per 250x magnification field given in numbers; presence of inflammatory cellular infiltrates per magnification field (250x for lung tissue, 400x for kidneys): + 1-10 cells per magnification field; ++ 10-50 cells per magnification field; number of animals with findings declared in parentheses. EXAMPLE 7: ADAMTS13 REDUCES ORGAN DAMAGE IN INDIVIDUALS SUFFERING FROM HYPOXEMIA AND IN INDIVIDUALS AT RISK OF DEVELOPING ARDS

[0267] ADAMTS13 has been shown to reduce end-organ injury in a mouse model of severe hypoxemia. Petition 870200095920, dated 07 / 31 / 2020, pages 140 / 151 131 / 132 (8% oxygen for 10 h followed by 3 h of recovery at approximately 21% oxygen). Given that severe hypoxemia injury is a contributing factor to the pathophysiology observed in patients suffering from acute lung injury (ALI) and acute respiratory distress syndrome (ARDS) (ALI / ARDS), it is hypothesized that administration of ADAMTS13 to patients at risk of or who have developed ALI and / or ARDS (ALI / ARDS) may prevent, treat, or improve the disease process and have enhanced outcomes such as survival, long-term lung function, and avoidance of further end-organ damage.

[0268] Mice receive LPS administration, either directly into the lungs via intratracheal injection or inhalation, or intraperitoneally or intravenously to elicit a systemic inflammatory response. Mice treated with intratracheal LPS have a robust and acute inflammatory cellular influx into the lung with resolution within 48 hours. LPS activates systemic inflammation and is associated with mild lung injury. This injury can be increased with repeated LPS injections or implantation of an LPS pump in the peritoneal cavity to continuously release LPS for hours, or even days.

[0269] ADAMTS13 is administered in doses of approximately 50, 100, 200, 500, 1000, 2000, and 3000 international units per kilogram of body weight before LPS treatment and within 12, 24, 48, 72, and 96 hours after LPS treatment. Doses of ADAMTS13 are administered daily or every 12 hours subcutaneously or intravenously until subjects are sacrificed to examine the inflammatory response in Petition 870200095920, dated 07 / 31 / 2020, pages 141 / 151 132 / 132 lung and organ damage.

[0270] Treatment with ADAMTS13 reduces the inflammatory response, including inflammatory cell influx to the lungs, as measured by the reduced number of neutrophils, macrophages, monocytes, mast cells, eosinophils and / or basophils present in the lungs of mice treated with ADAMTS13. Treatment with ADAMTS13 also reduces organ damage, as measured by blood urea nitrogen (BUN), creatinine, BUN / creatinine ratio, troponin, neuron-specific enolase (NSE).

[0271] The invention has been described in terms of particular embodiments found or proposed as comprising specific modes for carrying out the invention. Various modifications and variations of the invention described will be apparent to those skilled in the art without departing from the scope and spirit of the invention. Although the invention has been described in conjunction with specific embodiments, it should be understood that the invention as claimed should not be inappropriately limited to those specific embodiments. Indeed, various modifications of the described modes for carrying out the invention that are obvious to those skilled in the relevant fields are intended to be within the scope of the following claims. Petition 870200095920, dated 07 / 31 / 2020, pp. 142 / 151

Claims

1 / 2 CLAIMS 1. Use of a composition comprising a Disintegrin and Metalloproteinase with Thrombospondin domains type 1, member 13 (ADAMTS13), characterized in being in the preparation of a medicament to treat or prevent a vaso-occlusive crisis associated with sickle cell disease in an individual, wherein the vaso-occlusive crisis is an acute event of microvascular occlusion with tissue ischemia and acute pain.

2. Use according to claim 1, characterized in that the amount of ADAMTS13 in the composition is between 10 IU / mL and 10,000 IU / mL.

3. Use according to claim 1, characterized in that the amount of ADAMTS13 in the composition is between 50 IU / mL and 3,000 IU / mL.

4. Use according to claim 1, characterized in that the amount of ADAMTS13 in the composition is between 100 IU / mL and 2,000 IU / mL.

5. Use according to claim 1, characterized in that the amount of ADAMTS13 in the composition is between 100 IU / mL and 1,000 IU / mL.

6. Use, according to any one of claims 1 to 5, characterized in that ADAMTS13 is recombinant ADAMTS13.

7. Use, according to any one of claims 1 to 6, characterized in that ADAMTS13 is derived from plasma.

8. Use, according to any one of claims 1 to 7, characterized in that the individual is a mammal. Petition 870260063825, dated 06 / 29 / 2026, page 22 / 38 2 / 2 9. Use, according to any one of claims 1 to 8, characterized in that the individual is a human being.

10. Use, according to any one of claims 1 to 9, characterized in that the composition is in a stable aqueous solution ready for use. Petition 870260063825, dated 06 / 29 / 2026, p. 23 / 38

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