EA045023B1 - SUBCUTANEOUS ADMINISTRATION ADAMTS13 - Google Patents

Description

Cross references to related applications

This application claims benefit from U.S. Provisional Patent Application Serial No. 61/794659, filed March 15, 2013, which is incorporated herein by reference in its entirety for all purposes.

State of the art

ADAMTS (disintegrin and metalloproteinase with thrombospondin type I motifs) proteins belong to a family of metalloproteinases containing a number of conserved domains, including a zinc-dependent catalytic domain, a cysteine-rich domain, a disintegrin-like domain, and at least one (and in most cases several) thrombospondin type I repeats (see review by Nicholson et al., BMC Evol Biol. 2005 Feb. 4; 5(1):11). These proteins, evolutionarily related to the ADAM and MMP families of metalloproteinases (Jones G C, Curr Pharm Biotechnol. 2006 February; 7(1):25-31), are secreted enzymes associated with a number of diseases and conditions, including thrombotic thrombocytopenic purpura (TTP) (Moake J L, Semin Hematol. 2004 January; 41(1):4-14), connective tissue diseases, cancer, inflammation (Nicholson et al.) and severe malaria caused by Plasmodium falciparum (Larkin et al., PLoS Pathog. 2009 March;5(3):e1000349). Because of these connections, ADAMTS enzymes are considered potential therapeutic targets for a number of pathological conditions (Jones G C, Curr Pharm Biotechnol. 2006 February; 7(1):25-31).

One member of the ADAMTS family, ADAMTS13, cleaves von Willebrand factor (VWF) between residues Tyr 1605 and Met 1606. Loss of ADAMTS13 activity is associated with a number of conditions, such as TTP (Moake J L, Semin Hematol. 2004 January; 41(1):4- 14), acute and chronic inflammation (Chauhan et al., J Exp Med. 2008 Sep. 1; 205(9):2065-74), and, more recently, severe Plasmodium falciparum malaria (Larkin et al. ,PLoS Pathog.2009 Mar;5(3):e1000349).

Thrombotic thrombocytopenic purpura (TTP) is a disorder characterized by thrombotic microangiopathy, thrombocytopenia, and microvascular thrombosis, which can cause tissue ischemia and varying degrees of myocardial infarction. Clinically, TTP patients are diagnosed with symptoms such as thrombocytopenia, schizocytes (fragments of red blood cells), and elevated lactate dehydrogenase levels (Moake J L. Thrombotic microangiopathies. N Engl J Med. 2002; 347:589-600; Moake J L. von Willebrand factor, ADAMTS -13, and thrombotic thrombocytopenic purpura. Semin Hematol. 2004; 41:4-14; Sadler J E, Moake J L, Miyata T, George J N. Recent advances in thrombotic thrombocytopenic purpura. Hematology (Am Soc Hematol Educ Program). 2004: 407-423; Sadler J E. New concepts in von Willebrand disease. Annu Rev Med. 2005; 56:173-191).

There are two main types of TTP: acquired (non-hereditary/idiopathic) and familial (hereditary) (Tsai H M, Lian E C. Antibodies to von Willebrand factor-cleaving protease in acute thrombotic thrombocytopenic purpura. N Engl J. Med. 1998; 339:1585 -1594; Furlan M, Lammle B. Deficiency of von Willebrand factor-cleaving protease in familial and acquired thrombotic thrombocytopenic purpura. Baillieres Clin Haematol. 1998; 11:509-514). Genetic mutations in the ADAMTS13 gene cause familial TTP, whereas people with acquired TTP do not have the mutation. Preferably, acquired TTP is characterized by the production of specific antibodies.

In 1982, Moake et al. discovered unusually large von Willebrand factor multimers (ULVF) in the plasma of patients with chronic relapsing TTP (Moake J L, Rudy S K, Troll J H, Weinstein M J, Colannino N M, Azocar J, Seder R H, Hong S L, Deykin D. Unusually large plasma factor VIII :von Willebrand factor multimers in chronic relapsing thrombotic thrombocytopenic purpura. N Engl J Med. 1982; 307:1432-1435). The association between UL-EF and TTP was confirmed by independent studies by Furlan et al. and Tsai and Lian that the majority of patients suffering from TTP are deficient in the plasma metalloprotease now known as ADAMTS13, which degrades von Willebrand factor (Furlan M, Robles R, Solenthaler M, Wassmer M, Sandoz P, Laemmle B Deficient activity of von Willebrand factor-cleaving protease in chronic relapsing thrombotic thrombocytopenic purpura Blood 1997;89:3097-3103 Tsai H M, Sussman I I, Ginsburg D, Lankhof H, Sixma J J, Nagel R L. Proteolytic cleavage of recombinant type 2A von Willebrand factor mutants R834W and R834Q: inhibition by doxycycline and by monoclonal antibody VP-1. Blood. 1997; 89:1954-1962; Tsai H M, Lian E C Antibodies to von Willebrand factor-cleaving protease in acute thrombotic thrombocytopenic purpura N Engl J Med 1998;339:1585-1594).

ADAMTS13 protease is a 190 kDa glycosylated protein predominantly produced in the liver (Levy G G, Nichols W C, Lian E C, Foroud T, McClintick J N, McGee B M, Yang A Y, Siemieniak D R, Stark K R, Gruppo R, Sarode R, Shurin S B, Chandrasekaran V, Stabler S P, Sabio H, Bouhassira E E, Upshaw J D, Jr., Ginsburg D, Tsai H M. Mutations in a member of the ADAMTS gene family cause thrombotic thrombocytopenic purpura. Nature. 2001; 413: 488-494; Fujikawa K, Suzuki H, McMullen B, Chung D. Purification of human von Willebrand factor-cleaving protease and its identification as a new member of the metalloproteinase family. Blood. 2001; 98:1662-1666; Zheng X, Chung D, Takayama T K, Majerus E M, Sadler J E, Fujikawa K. Structure of von Willebrand factor-cleaving protease (ADAMTS13), a metalloprotease involved in thrombotic thrombocytopenic purpura. J Biol Chem. 2001;276:41059-41063; Soejima K, Mimura N, Hirashima M, Maeda H, Hamamoto T, Nakagaki T, Nozaki C. A novel human metalloprotease syn- 1 045023 thesized in the liver and secreted into the blood: possibly, the von Willebrand factor-cleaving protease; J Biochem (Tokyo). 2001; 130:475-480; Gerritsen H E, Robles R, Lammle B, Furlan M. Partial amino acid sequence of purified von Willebrand factor-cleaving protease. Blood. 2001; 98:1654-1661).

Mutations in the ADAMTS13 gene are known to cause TTP (Levy G G, Nichols W C, Lian E C, Foroud T, McClintick J N, McGee B M, Yang A Y, Siemieniak D R, Stark K R, Gruppo R, Sarode R, Shurin S B, Chandrasekaran V, Stabler S P, Sabio H, Bouhassira E E, Upshaw J D, Jr., Ginsburg D, Tsai H M. Mutations in a member of the ADAMTS gene family cause thrombotic thrombocytopenic purpura. Nature. 2001; 413:488494). Idiopathic TTP, often caused by autoantibodies that inhibit ADAMTS13 activity, is a more common disease that occurs in adults and older children and can recur at regular intervals in 11-36% of patients (Tsai H M, Lian E C. Antibodies to von Willebrand factor -cleaving protease in acute thrombotic thrombocytopenic purpura. N Engl J Med. 1998; 339:1585-1594; Furlan M, Lammle B. Deficiency of von Willebrand factor-cleaving protease in familial and acquired thrombotic thrombocytopenic purpura. Baillieres Clin Haematol. 1998; 11:509-514).

Nonneutralizing autoantibodies can also inhibit ADAMTS activity by inducing its clearance from the bloodstream (Scheiflinger F, Knobl P, Trattner B, Plaimauer B, Mohr G, Dockal M, Dorner F, Rieger M. Nonneutralizing IgM and IgG antibodies to von Willebrand factor-cleaving protease (ADAMTS-13) in a patient with thrombotic thrombocytopenic purpura. Blood. 2003;102:3241-3243). ADAMTS13 activity in plasma in healthy adults ranges from 50% to 178% (Moake J L. Thrombotic thrombocytopenic purpura and the hemolytic uremic syndrome. Arch Pathol Lab Med. 2002; 126:1430-1433). In most patients with familial or acquired TTP, plasma ADAMTS13 activity is absent or less than 5% of normal. Without treatment, mortality exceeds 90%, but plasma therapy reduces mortality to approximately 20% (Moake J L. Thrombotic thrombocytopenic purpura and the hemolytic uremic syndrome. Arch Pathol Lab Med. 2002; 126:1430-1433).

VWF, synthesized in megakaryocytes and endothelial cells, is stored in platelet granules and Weibel-Palad bodies, respectively, as ultra-large VWF (UL-VWF) (Moake J L, Rudy S K, Troll J H, Weinstein M J, Colannino N M, Azocar J, Seder R H, Hong S L, Deykin D. Unusually large plasma factor VIII: von Willebrand factor multimers in chronic relapsing thrombotic thrombocytopenic purpura. N Engl J Med. 1982; 307:1432-1435; Wagner D D, Olmsted J B, Marder V J. Immunolocalization of von Willebrand protein in Weibel-Palade bodies of human endothelial cells. J Cell Biol. 1982; 95:355-360; Wagner D D, Bonfanti R. von Willebrand factor and the endothelium. Mayo Clin Proc. 1991; 66:621-627 ; Sporn L A, Marder V J, Wagner D D. von Willebrand factor released from. Weibel-Palade bodies binds more avidly to extracellular matrix than that secreted constitutively. Blood. 1987;69:1531-1534; Tsai H M, Nagel R L, Hatcher V B , Sussman, II. Endothelial cell-derived high molecular weight von Willebrand factor is converted into the plasma multimer pattern by granulocyte proteases. Biochem Biophys Res Commun. 1989; 158:980-985; Tsai H M, Nagel R L, Hatcher V B, Sussman, II. Multimeric composition of endothelial cell-derived von Willebrand factor. Blood. 1989; 73:2074-2076). After secretion from endothelial cells, these UL-VWF multimers are cleaved by ADAMTS13 in the bloodstream into a number of smaller multimers at specific cleavage sites within the VWF molecule (Tsai H M, Nagel R L, Hatcher V B, Sussman, II. Endothelial cell-derived high molecular weight von Willebrand factor is converted into the plasma multimer pattern by granulocyte proteases. Biochem Biophys Res Commun. 1989; 158:980-985; Dent J A, Galbusera M, Ruggeri Z M. Heterogeneity of plasma von Willebrand factor multimers resulting from proteolysis of the constituent subunit J Clin Invest 1991;88:774-782 Furlan M, Robles R, Affolter D, Meyer D, Baillod P, Lammle B. Triplet structure of von Willebrand factor reflects proteolytic degradation of high molecular weight multimers. Proc Natl Acad Sci USA. 1993;90:7503-7507).

ADAMTS13 cleaves the Tyr842-Met843 bond in the central A2 domain of the mature VWF subunit; Moreover, ADAMTS13 activity requires zinc or calcium (Dent J A, Berkowitz S D, Ware J, Kasper S K, Ruggeri Z M. Identification of a cleavage site directing the immunochemical detection of molecular abnormalities in type IIA von Willebrand factor. Proc Natl Acad Sci USA. 1990;87:6306-6310). VWF exists in a filamentous ball and filamentous form, as seen by electron microscopy (Slayter H, Loscalzo J, Bockenstedt P, Handin R I. Native conformation of human von Willebrand protein. Analysis by electron microscopy and quasi-elastic light scattering J Biol Chem 1985;260:8559-8563). In addition, atomic force microscopy confirms that under static conditions the FI exists in a globular conformation and in an unfolded filamentous state after exposure to shear stress (Siedlecki S A, Lestini B J, Kottke-Marchant K K, Eppell S J, Wilson D L, Marchant R E Shear-dependent changes in the three-dimensional structure of human von Willebrand factor. Blood. 1996;88:2939-2950). This can also occur in vivo if one end of the VWF filament attaches to a surface.

Thrombi in patients with TTP consist of little fibrin and mainly VWF and platelets, suggesting VWF-mediated platelet aggregation as a cause of thrombosis (Asada Y, Sumiyoshi A, Hayashi T, Suzumiya J, Kaketani K. Immunohistochemistry of vascular lesion in thrombotic thrombocytopenic purpura, with special reference to factor VIII related antigen. Thromb Res. 1985;38:469-479). Ultra-large multimers are present in the plasma of patients with relapsed TTP. UL-vWF multimers accumulate over time because the constant presence of an inhibitor (anti-ADAMTS13 antibody) reduces ADAMTS13 activity. UL-vWF multimers are hyperactive and unfold as a result of shear stress, leading to platelet aggregation and intravascular thrombosis (Tsai H M. Von Willebrand factor, ADAMTS13, and thrombotic thrombocytopenic purpura. J

Mol Med. 2002; 80:639-647; Tsai H M. Deficiency of ADAMTS-13 in thrombotic and thrombocytopenic purpura. J Thromb Haemost. 2003; 1:2038-2040; discussion 2040-2035).

It is believed that the presence of hyperreactive UL-VWF multimers in plasma due to ADAMTS13 deficiency may be associated with an increased risk of arterial thrombosis associated with coronary artery disease. Additionally, ADAMTS13 is associated with cerebral infarction, myocardial infarction, ischemia/reperfusion injury, deep vein thrombosis, and disseminated intravascular coagulation. Accordingly, there is a need for ADAMTS13 protein-based pharmaceutical formulations useful for the treatment of various diseases and conditions associated with ADAMTS13 dysfunction and VWF.

However, pharmaceutical formulations containing very large and unstable molecules, such as ADAMTS13, can usually only be administered intravenously. This is because such pharmaceutical formulations generally have very low bioavailability due to poor absorption and severe degradation upon subcutaneous, intramuscular and intradermal administration. Accordingly, due to their low bioavailability, large and unstable proteins are typically administered intravenously to provide direct access to the bloodstream.

Although ADAMTS13 can be administered intravenously to treat various diseases and conditions associated with ADAMTS13 dysfunction and EF, it is inconvenient and challenging for patients. In particular, ADAMTS13 formulations often require regular administration throughout the life of the patient. For example, patients with familial (inherited) TTP begin treatment with intravenous ADAMTS13 in the first year of life. Accordingly, it is desirable to administer the ADAMTS13 pharmaceutical composition subcutaneously. However, the low bioavailability of large and unstable protein formulations when administered subcutaneously has hampered the development of such formulations for subcutaneous administration.

Previous studies have reported that several coagulation factors VII, VIII and IX are suitable for subcutaneous administration. For example, PCT/SE95/00348 reported a highly purified formulation of factor VIII that contains additives such as hydrolyzed gelatin, hyaluronic acid and soybean oil emulsion. Purification and additives allowed the development of a high concentration factor VIII formulation. This high concentration formulation had at least about 15% and preferably at least about 30% bioavailability after subcutaneous, intramuscular or intradermal administration compared to bioavailability after intravenous administration. However, 15-30% bioavailability for subcutaneous administration compared to intravenous administration is still very low and would be ineffective in the treatment of ADAMTS13 disorders.

In addition, previous studies have not provided a general principle for subcutaneous administration of large and unstable proteins. Rather, previous studies provide evidence that pharmaceutical compositions of large and unstable proteins for subcutaneous administration are difficult to manufacture because these compositions do not have the bioavailability required for subcutaneous administration.

Described herein is a method of subcutaneously administering an ADAMTS13 formulation for the treatment of a disease or condition associated with ADAMTS13 dysfunction and VWF. In particular, this document provides evidence of the unexpectedly high bioavailability of subcutaneous formulations of ADAMTS13, up to approximately 70%.

Brief description of the invention

In one aspect, the present disclosure provides a method of treating a blood clotting disorder in a mammal, comprising subcutaneously administering a therapeutically effective amount of a composition comprising isolated ADAMTS13 to a mammal in need thereof, wherein the therapeutically effective amount of ADAMTS13 contains 20-4000 units of activity per kilogram.

In one embodiment of the methods provided herein, the coagulation disorder is selected from the group consisting of hereditary TTP, acquired TTP, cerebral infarction, myocardial infarction, ischemia/reperfusion injury, deep vein thrombosis, and disseminated intravascular coagulation associated with sepsis.

In one embodiment of the methods provided herein, the bioavailability of ADAMTS13 after subcutaneous administration is 50-80% compared to intravenous administration standardized at a similar dose.

In one embodiment of the methods provided herein, the bioavailability of ADAMTS13 after subcutaneous administration is at least 50% compared to intravenous administration standardized at a similar dose.

In one embodiment of the methods provided herein, the bioavailability of ADAMTS13 after subcutaneous administration is at least 55% compared to intravenous administration standardized at a similar dose.

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In one embodiment of the methods provided herein, the bioavailability

ADAMTS13 after subcutaneous administration is at least 60% compared to intravenous administration normalized to the same dose.

In one embodiment of the methods provided herein, the bioavailability of ADAMTS13 after subcutaneous administration is at least 65% compared to intravenous administration standardized at a similar dose.

In one embodiment of the methods provided herein, the bioavailability of ADAMTS13 after subcutaneous administration is at least 70% compared to intravenous administration standardized at a similar dose.

In one embodiment of the methods provided herein, the bioavailability of ADAMTS13 after subcutaneous administration is at least 75% compared to intravenous administration standardized at a similar dose.

In one embodiment of the methods provided herein, the bioavailability of ADAMTS13 after subcutaneous administration is at least 80% compared to intravenous administration standardized at a similar dose.

In one embodiment of the methods provided herein, the bleeding episode is an inherited TTP.

In one embodiment of the methods provided herein, the therapeutically effective amount contains at least 20-160 units of activity per kilogram.

In one embodiment of the methods provided herein, the bleeding episode is acquired TTP.

In one embodiment of the methods provided herein, the therapeutically effective amount contains at least 40-2000 units of activity per kilogram of body weight.

In one embodiment of the methods provided herein, the bleeding episode is a cerebral infarction and/or ischemic reperfusion injury.

In one embodiment of the methods provided herein, the therapeutically effective amount contains at least 40-4000 units of activity per kilogram of body weight.

In one embodiment of the methods provided herein, the bleeding episode is a myocardial infarction and/or ischemic reperfusion injury.

In one embodiment of the methods provided herein, the therapeutically effective amount contains at least 40-2000 units of activity per kilogram.

In one embodiment of the methods provided herein, ADAMTS13 is administered as a single bolus injection, monthly, every other week, once a week, twice a week, every day, every 12 hours, every 8 hours, every six hours, every four hours or every two hours.

In one embodiment of the methods provided herein, ADAMTS13 is recombinant.

In one embodiment of the methods provided herein, ADAMTS13 is obtained from plasma.

In one embodiment of the methods provided herein, said ADAMTS13 is a human ADAMTS13 and the mammal is a human.

In one embodiment of the methods provided herein, the composition is a stable aqueous solution ready for administration.

In one embodiment of the methods provided herein, the composition is lyophilized.

In one embodiment of the methods provided herein, the composition is reconstituted with a pharmaceutically acceptable carrier suitable for injection.

In one aspect, the present disclosure provides a method of treating a bleeding episode in a mammal, comprising subcutaneously administering a therapeutically effective amount of a composition comprising isolated ADAMTS13 to a mammal in need of said administration, wherein the therapeutically effective amount of ADAMTS13 comprises at least 120-300% of the amount of a standard intravenous dose according to specific indication for use, measured in units of activity per kilogram of body weight.

In one embodiment of the methods provided herein, the specific indication for use is hereditary TTP, and the standard intravenous dose is 10-80 units of activity per kilogram of body weight.

In one embodiment of the methods provided herein, the specific indication for use is acquired TTP, and the standard intravenous dose is 20-1000 activity units per kilogram of body weight.

In one embodiment of the methods provided herein, the specific indication for use is myocardial infarction and/or ischemic reperfusion injury, and the standard intravenous dose is 20-2000 activity units per kilogram of body weight.

In one embodiment of the methods provided herein, the specific indication for use is cerebral infarction and/or ischemic reperfusion injury, and the standard intravenous dose is 20-2000 units of activity per kilogram of body weight.

In one embodiment of the methods provided herein, the bioavailability of ADAMTS13 after subcutaneous administration is 50-80% compared to intravenous administration standardized at a similar dose.

In one embodiment of the methods provided herein, the bioavailability of ADAMTS13 after subcutaneous administration is at least 50% compared to intravenous administration standardized at a similar dose.

In one embodiment of the methods provided herein, the bioavailability of ADAMTS13 after subcutaneous administration is at least 55% compared to intravenous administration standardized at a similar dose.

In one embodiment of the methods provided herein, the bioavailability of ADAMTS13 after subcutaneous administration is at least 60% compared to intravenous administration standardized at a similar dose.

In one embodiment of the methods provided herein, the bioavailability of ADAMTS13 after subcutaneous administration is at least 65% compared to intravenous administration standardized at a similar dose.

In one embodiment of the methods provided herein, the bioavailability of ADAMTS13 after subcutaneous administration is at least 70% compared to intravenous administration standardized at a similar dose.

In one embodiment of the methods provided herein, the bioavailability of ADAMTS13 after subcutaneous administration is at least 75% compared to intravenous administration standardized at a similar dose.

In one embodiment of the methods provided herein, the bioavailability of ADAMTS13 after subcutaneous administration is at least 80% compared to intravenous administration standardized at a similar dose.

In one embodiment of the methods provided herein, ADAMTS13 is administered as a single bolus injection, monthly, every other week, once a week, twice a week, every day, every 12 hours, every 8 hours, every six hours, every four hours or every two hours.

In one embodiment of the methods provided herein, ADAMTS13 is recombinant.

In one embodiment of the methods provided herein, ADAMTS13 is obtained from plasma.

In one embodiment of the methods provided herein, said ADAMTS13 is a human ADAMTS13 and the mammal is a human.

In one embodiment of the methods provided herein, the composition is a stable aqueous solution ready for administration.

In one embodiment of the methods provided herein, the composition is lyophilized.

In one embodiment of the methods provided herein, the composition is reconstituted with a pharmaceutically acceptable carrier suitable for injection.

Brief description of the figures

Fig. 1. In FIG. Figure 1 shows the mean plasma concentrations of ADAMTS13 activity. The median _Tmax for ADAMTS13 activity after subcutaneous administration was 24 hours.

Fig. 2. In FIG. Figure 2 shows the mean plasma concentrations of ADAMTS13 activity. The median _Tmax for the ADAMTS13 antigen after subcutaneous administration was 28 hours.

Fig. 3. In FIG. Figure 3 shows observed (circles) and predicted concentrations (solid lines) for individual animals. Concentrations after IV administration were calculated using a two-component model, while concentrations after SC administration were calculated using a one-compartment model with first-order uptake and first-order elimination. Both models were modified by including an additional independent variable to model the assumed constant endogenous activity of ADAMTS13.

Fig. 4. In FIG. Figure 4 shows the observed cutoff-corrected concentrations and the corresponding predicted concentrations for individual animals. Concentrations after IV administration were predicted using a two-component model, while concentrations after SC administration were predicted using a one-compartment model with first-order uptake and first-order elimination.

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Detailed Description of the Invention

I. Introduction

ADAMTS13 (A13) is a plasma metalloprotease that cleaves von Willebrand factor (VWF) multimers and inhibits their activity towards platelet aggregation. ADAMTS13 is 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 DIC associated with sepsis.

Current treatment for these clotting disorders involves intravenous administration of ADMTS13 compounds. Treatment is currently limited to intravenous administration because ADAMTS13 is a large protein, and large proteins are typically unstable in highly bioavailable formulations suitable for subcutaneous administration. Mature ADAMTS13 has an estimated molecular mass of about 145 kDa, whereas purified plasma ADAMTS13 has an apparent molecular mass of about 180 kDa, likely due to post-translational modifications due to the presence of consensus sequences of 10 potential N-glycosylation sites, several O-glycosylation sites, and one C site -mannosylation in the TSPI-ποβτοροβ region.

The use of large and unstable proteins and molecules, such as ADAMTS13, is generally limited to intravenous administration due to the low bioavailability of the formulations when administered subcutaneously. For example, previous studies have reported that factor VIII, a 170-300 kDa protein, is typically administered intravenously because factor VIII formulations typically have very low bioavailability due to poor absorption and severe degradation when administered subcutaneously, intramuscularly, or intradermally. See PCT/SE95/00348.

For example, factor VIII concentrate, when administered intramuscularly, has been reported to achieve peak circulating levels of only 1.4% of normal plasma levels, with or without the addition of sodium citrate to prevent degradation and enhance absorption (Pool et al., New England J. Medicine, vol. 275, no. 10, pp. 547-548, 1966). Further studies showed that there were no significant differences in the activity of the enzyme in the bloodstream regardless of the addition of citrate to the drug. In a later study, highly purified factor VIII was administered intramuscularly to dogs and human volunteers with hemophilia (Johnson et al., Br. J. Hematology, vol. 21, p. 21-41, 1971). Although the doses were much higher than previously reported by Pool et al., there was no significant increase in plasma factor VIII levels in either dogs or volunteers. In fact, the concentration of factor VIII in the plasma of human volunteers with hemophilia remained below 1% of the normal plasma level, i.e. severe hemophilia A occurred even after administration in the absence of a bioavailability supplement.

There have been some advances in subcutaneous delivery of small proteins, such as factor IX, that do not degrade or aggregate like large, unstable proteins such as factor VIII and ADAMTS13. For example, subcutaneous administration of factor IX without additives is known from Berettini et al., Subcutaneous factor IX administration to patients with hemophilia B, Am. J. Hematology, 47(1):61-62, 1994. However, even factor IX, which is only 56 kDa, was characterized by weak and very slow transport into the bloodstream.

Due to the low bioavailability of proteins such as the above-described factors VIII and IX, subcutaneous delivery of large and unstable proteins is usually not carried out at all. Instead, these proteins are typically administered intravenously to allow the formulation to enter the bloodstream directly. However, it is desirable to be able to administer the drug subcutaneously, since subcutaneous administration is a minimally invasive method. Subcutaneous administration is also the most versatile route of administration, which can be used for short- and long-term therapy. Subcutaneous administration can be accomplished by injection or implantation of a sustained or scheduled release device under the surface of the skin. The injection area or device can be changed if multiple injections or devices are needed.

Accordingly, subcutaneous formulations are significantly easier for the patient to handle, especially since the formulation may need to be taken regularly throughout life (eg, starting as early as the child's first year of life). In addition, simple and rapid subcutaneous delivery improves patient compliance and provides faster access to medication when needed. Thus, there is an advantage and need for ADAMTS13 formulations for subcutaneous administration.

The present invention is based on the unexpected discovery of a successful method of subcutaneous administration of liquid and lyophilized formulations of purified ADAMTS proteins.

II. Definitions

As used herein, the terms ADAMTS13 or A13 refer to a metalloprotease of the ADAMTS (disintegrin and metalloproteinase with thrombospondin type I motifs) family of metalloproteases that cleaves von Willebrand factor (VWF) between residues Tyr 1605 and Met 1606. In the context of the present invention, ADAMTS13 protein covers any ADAMTS13 proteins, e.g. ADAMTS13 mammal, e.g. primate, human (NP620594), monkey, rabbit, pig, cattle (XP610784),

- 6 045023 rodent, mouse (NP001001322), rat (ХР342396), hamster, gerbil, dog, cat, frog (NP001083331), chicken (ХР415435) and their biologically active derivatives. As used herein, ADAMTS13 proteins refer to recombinant and plasma ADAMTS13 proteins. The term also includes mutant and variant ADAMTS13 proteins having activity, such as functional ADAMTS13 fragments and fusion proteins. In addition, the ADAMTS13 proteins of the invention may also contain markers to facilitate purification and/or detection. The ADAMTS13 proteins described herein can be modified with a therapeutic group or a group suitable for in vitro or in vivo imaging.

Human ADAMTS13 proteins include, but are not limited to, polypeptides containing the amino acid sequence of GenBank accession number NP 620594, or such polypeptides after processing, for example, a polypeptide with a signal peptide (amino acids 1-29) and/or a propeptide (amino acids 30-74) removed. Various natural variants of human ADAMTS13 are known in the art and are included in the compositions of the present invention; some of them contain mutations selected from R7W, V88M, H96D, R102C, R193W, T196I, H234Q, A250v, R268P, W390C, R398H, Q448E, Q456H, P457L, P475S, C508Y, R528G, P618A, R625H, I67 3F, R692C, A732V, E74OK, A900V, S903L, C908Y, C951G, G982R, C1024G, A1033T, R1095W, R1095W, R1123C, C1213Y, T12261, G1239V and R1336W. In addition, ADAMTS13 proteins include natural and recombinant mutant proteins, for example, those containing one or more conserved mutation at a nonessential amino acid position. Preferably, the aromatic amino acids important for the enzymatic activity of ADAMTS13 are not mutated. These include, for example, residues known or suspected to be important for binding to the metal atom, for example, residues 83, 173, 224, 228, 234, 281 and 284, and residues located in the active site of the enzyme, for example, residue 225. Similarly, Thus, in the context of the present invention, ADAMTS13 proteins include alternative isoforms, for example, isoforms without amino acids 275-305 and/or 1135-1190 of the full-length human protein.

Likewise, ADAMTS13 proteins can be subject to further modification, such as post-translational modifications (e.g., glycosylation at one or more amino acids selected from residues 142, 146, 552, 579, 614, 667, 707, 828, 1235, 1354 of the human protein, or any other natural or recombinant modification site) or chemical or enzymatic modifications ex vivo, including, without limitation, glycosylation, modification with water-soluble polymers (e.g., PEGylation, sialylation, HES modification, etc.), attachment of markers, etc.

As used herein, a coagulation disorder is defined as a disorder involving the recruitment of defective platelets as well as the recruitment of defective neutrophils. Non-limiting examples of coagulation disorders include hereditary thrombotic thrombocytopenic purpura (TTP), acquired TTP, cerebral infarction, myocardial infarction, ischemia/reperfusion injury, deep vein thrombosis, and disseminated intravascular coagulation (DIC), such as DIC associated with sepsis.

As used herein, the term one unit of ADAMTS13 activity is defined as the amount of activity in 1 ml of a pool of normal human plasma, regardless of the assay used. For example, one unit of ADAMTS13 FRETS-VWF73 activity represents the amount of activity required to cleave the same amount of FRETS-VWF73 substrate (Kokame et al., Br J Haematol. 2005 April; 129(1):93-100) that cleaves one ml pool of normal human plasma. Additional activity assays can be used to determine the activity of a single ADAMTS13 unit. For example, one can perform a direct assay for ADAMTS13 activity to detect cleavage of the full-length VWF molecule or fragments using SDS-agarose gel electrophoresis and an indirect assay for ADAMTS13 activity using a collagen binding assay.

As used herein, the terms ADAMTS13 and biologically active derivative, respectively, also include polypeptides produced by recombinant DNA technology. Alternatively, ADAMTS13 may also refer to ADAMTS13 derived from plasma purified from a human blood pool. Recombinant ADAMTS13 (rADAMTS13), for example, recombinant human ADAMTS13 (r-hu-ADAMTS13), can be produced by any method known in the art. One specific example, incorporated herein by reference with respect to a method for producing recombinant ADAMTS13, is described in WO 02/42441. These methods include any method known in the art for (i) producing recombinant DNA by genetic engineering, for example, by reverse transcription of RNA and/or DNA amplification, (ii) introducing recombinant DNA into prokaryotic or eukaryotic cells by transfection, i.e. .e. by electroporation or microinjection, (iii) culturing said transformed cells, e.g., in a continuous or batch manner, (iv) expressing ADAMTS13, e.g., constitutively or by induction, and (v) isolating said ADAMTS13, e.g., from a culture medium or by harvesting transformed cells, with the aim of (vi) obtaining substantially purified ADAMTS13, for example, by anion exchange chromatography

- 7 045023 or affinity chromatography. The term biologically active derivative also includes chimeric molecules, for example, ADAMTS13 (or a biologically active derivative thereof) in combination with an Ig to improve the biological/pharmacological properties, for example, the half-life of ADAMTS13 in the bloodstream of a mammal, particularly a human. The Ig may also contain a binding site for an optionally mutated Fc receptor.

As used herein, the term thrombus refers to a blood clot, especially a platelet-containing blood clot, microthrombus and/or embolus. Said thrombus may or may not be attached to an arterial or venous blood vessel, and may partially or completely block blood flow in the arterial or venous blood vessel.

As used herein, the term therapeutically effective amount or dose or sufficient amount or dose refers to a dose that produces the effect for which it is administered. The exact dosage depends on the purpose of treatment and is determined by one skilled in the art using known techniques (see, for example, Lieberman, Pharmaceutical Dosage Forms (vols. 1-3, 1992); Lloyd, The Art, Science and Technology of Pharmaceutical Compounding ( 1999); Pickar, Dosage Calculations (1999); and Remington: The Science and Practice of Pharmacy, ^20th Edition, 2003, Gennaro, Ed., Lippincott, Williams & Wilkins).

As used herein, physiological salt concentration refers to a pharmaceutically acceptable salt concentration in the range of about 100 mM to about 200 mM. Non-limiting examples of pharmaceutically acceptable salt include, but are not limited to, sodium and potassium chlorides, sodium and potassium acetates, sodium and potassium citrates, sodium and potassium phosphates.

As used herein, subphysiological salt concentration refers to a pharmaceutically acceptable salt concentration of less than about 100 mM. In preferred embodiments, a subphysiological salt concentration refers to a pharmaceutical salt concentration of less than about 80 mM. In another preferred embodiment, the subphysiological concentration of the pharmaceutical salt is less than about 60 mM.

As used herein, the term approximately means an approximate range of plus or minus 10% of the stated value. For example, the expression approximately 20% covers the range of 18-22%. As used herein, approximately also includes the exact amount. So approximately 20% means approximately 20% as well as 20%.

III. Compositions and compositions ADAMTS13

In one aspect, the present invention provides stabilized formulations of plasma ADAMTS13 protein and recombinant ADAMTS13 protein (rADAMTS13), as described in US Patent Application Publication No. 2011/0229455. In other embodiments, the compositions provided herein retain significant ADAMTS13 activity when stored for long periods of time. In other embodiments, the compositions of the present invention reduce or slow down the dimerization, oligomerization, or aggregation of the ADAMTS13 protein.

In one embodiment, the present invention provides ADAMTS13 formulations comprising a therapeutically effective amount or dose of ADAMTS13 protein, a subphysiological or physiological concentration of a pharmaceutically acceptable salt, a stabilizing concentration of one or more sugars or sugar alcohols, a nonionic surfactant, a buffering agent to ensure that the formulation is pH neutral, and, when if necessary, calcium and/or zinc salt. In general, the stabilized ADAMTS13 formulations provided herein are suitable for pharmaceutical administration. In a preferred embodiment, the ADAMTS13 protein is human ADAMTS13 or a biologically active derivative or fragment thereof, as described in US Patent Application Publication No. 2011/0229455.

In some embodiments, the ADAMTS13 formulations are liquid formulations. In other embodiments, ADAMTS13 formulations are lyophilized from a liquid state, as described in US Patent Application Publication No. 2011/0229455. In some embodiments of the compositions provided herein, the ADAMTS13 protein is human ADAMTS13 or recombinant human ADAMTS13 or a biologically active derivative or fragment thereof, as described in US Patent Application Publication No. 2011/0229455.

In some embodiments, ADAMTS13 is provided at a therapeutically effective dose ranging from about 0.05 mg/ml to about 10 mg/ml. In other embodiments, ADAMTS13 is present in a concentration range from about 0.1 mg/ml to about 10 mg/ml. In other embodiments, ADAMTS13 is present in a concentration range from about 0.1 mg/ml to about 5 mg/ml. In yet another embodiment, ADAMTS13 is present in a concentration range from about 0.1 mg/ml to about 2 mg/ml. In other embodiments, ADAMTS13 may be present at a concentration of about 0.01 mg/ml, or about 0.02 mg/ml, 0.03 mg/ml, 0.04 mg/ml, 0.05 mg/ml, 0.06 mg /ml, 0.07 mg/ml, 0.08 mg/ml, 0.09 mg/ml, 0.1 mg/ml, 0.2 mg/ml, 0.3 mg/ml, 0.4 mg/ ml, 0.5 mg/ml, 0.6 mg/ml, 0.7 mg/ml, 0.8 mg/ml, 0.9 mg/ml, 1.0 mg/ml, 1.1 mg/ml , 1.2 mg/ml, 1.3 mg/ml, 1.4 mg/ml, 1.5 mg/ml, 1.6 mg/ml, 1.7 mg/ml, 1.8 mg/ml, 1.9 mg/ml, 2.0 mg/ml, 2.5 mg/ml, 3.0 mg/ml, 3.5 mg/ml, 4.0 mg/ml, 4.5 mg/ml, 5 .0 mg/ml, 5.5 mg/ml, 6.0 mg/ml, 6.5 mg/ml, 7.0

- 8 045023 mg/ml, 7.5 mg/ml, 8.0 mg/ml, 8.5 mg/ml, 9.0 mg/ml, 9.5 mg/ml, 10.0 mg/ml or in higher concentration. In one embodiment, the concentration of relatively pure ADAMTS13 in the formulation can be determined by spectroscopy (i.e., measuring total protein at A280) or other determination methods (e.g., Bradford assay, silver staining, weighing of lyophilized powder, etc.). In other embodiments, the concentration of ADAMTS13 can be determined using an ADAMTS13 ELISA (eg, in mg/mL of antigen).

In other embodiments, the concentration of ADAMTS13 in a composition of the present invention may be expressed as a level of enzymatic activity. For example, in one embodiment, the ADAMTS13 formulation may contain from about 10 FRETSVWF73 activity units to about 10,000 FRETS-VWF73 activity units or other suitable number of ADAMTS13 enzyme units (MEs). In other embodiments, the formulation may contain from about 20 FRETS-VWF73 activity units ( _FV73 units) to about 8000 FRETS-VWF73 activity units, or from about 30 _{FV73 units} to about 6000 _{FV73 units} , or from about 40 _{FV73 units} to about 4000 _FV73 units , or from about 50 _{FV73 Units} to about 3000 _{FV73 Units} , or from about 75 _{FV73 Units} to about 2500 _{FV73 Units} , or from about 100 _{FV73 Units} to about 2000 _{FV73 Units} , or from about 200 _{FV73 Units} to about 1500 _{FV73 Units} , or amount of enzyme from a different range. In a preferred embodiment, the ADAMTS13 formulation provided herein contains from about 20 to about 10,000 U _{of FV73} . In some embodiments, the formulation contains approximately 10 units of FRETS-VWF73 activity, 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, 27 00, 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, 9000, 9100, 9200, 9300, 9400, 9500,

9600, 9700, 9800, 9900, 10000 or more FRETS-VWF73 activity units.

Likewise, in some embodiments, the ADAMTS13 concentration may be expressed as enzyme activity per unit volume, such as ADAMTS13 enzyme units per ml (IU/ml). For example, in one embodiment, the ADAMTS13 formulation may contain from about 10 IU/ml to about 10,000 IU/ml. In other embodiments, the formulation may contain from about 20 IU/ml to about 10,000 IU/ml, or from about 20 IU/ml to about 8,000 IU/ml, or from about 30 IU/ml to about 6,000 IU/ml, or from about 40 IU/ml to about 4000 IU/ml, or about 50 IU/ml to about 3000 IU/ml, or about 75 IU/ml to about 2500 IU/ml, or about 100 IU/ml to about 2000 IU/ml, or from about 200 IU/ml to about 1500 IU/ml, or an amount of enzyme from another range. In a preferred embodiment, the ADAMTS13 formulation provided herein contains from about 150 IU/ml to about 600 IU/ml. In yet another preferred embodiment, the ADAMTS13 formulation provided herein contains from about 100 IU/ml to about 1000 IU/ml. In some embodiments, the formulation contains about 10 IU/ml or about 20, 30, 40, 50, 60, 70, 80, 90, 100, 150, 200, 250, 300, 350, 400, 450, 500, 600, 700 , 800, 900, 1000, 1100, 1200, 1300, 1400, 1500, 1600, 1700, 1800, 1900, 2000, 2100, 2200, 2300, 2400, 2500, 2600, 2700, 2 800, 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, 9000, 9100, 9200, 9300, 9400, 9500,

9600, 9700, 9800, 9900, 10000 or more IU/ml.

In some embodiments, the ADAMTS13 compositions provided herein may further contain one or more pharmaceutically acceptable excipients, carriers, and/or diluents, as described in US Patent Application No. 20110229455. Additionally, in one embodiment, the ADAMTS13 compositions provided herein herein have a tonicity within the range described in US Patent Application Publication No. 2011/0229455.

Additional embodiments of the present invention provide ADAMTS13 formulations including the exemplary formulations described in Section III (ADAMTS13 Compositions and Formulations) of US Patent Application Publication No. 2011/0229455.

In some embodiments, the resulting ADAMTS13 formulations contain additives. The methods for producing ADAMTS13 and its compositions described in sections IV and V of US Patent Application Publication No. 2011/0229455 are incorporated herein by reference.

IV. Treatment methods

The compositions described herein are suitable for subcutaneous administration for therapeutic or prophylactic purposes. Typically, for therapeutic use, the formulations are administered to a subject with a disease or condition associated with dysfunction of ADAMTS13 or VWF, or otherwise in need thereof, at a therapeutically effective dose. The compositions and amounts effective for these purposes depend on the severity of the disease or condition and the general health of the patient. The formulations may be administered single or multiple times depending on the dosage and frequency required and tolerated by the patient.

The patient or subject for the purposes of the present invention includes humans and other animals, in particular mammals. Thus, the compositions, formulations and methods can be used for both medical and veterinary purposes. In a particular embodiment, the patient is a mammal, and in one embodiment, a human. In combination with the compositions and methods proposed in the present invention, other known methods and means of treating conditions associated with ADAMTS13 dysfunction or VWF can be used.

In some embodiments, the ADAMTS13 subcutaneous formulation is administered by subcutaneous injection. In specific embodiments, the ADAMTS13 subcutaneous formulation is administered by subcutaneous injection into the same area of the patient's body (e.g., shoulder, anterior thigh, lower abdomen, or upper back) in repeated or continuous injections. In other embodiments, the ADAMTS13 subcutaneous formulation is administered by subcutaneous injection into different areas of the patient's body or alternately from different sides. In some embodiments, the subcutaneous formulation of ADAMTS13 is administered via a device implanted under the skin. In some embodiments, the implanted device releases the ADAMTS13 formulation at a specific time. In some embodiments, the implanted device provides continuous release of the ADAMTS13 formulation.

In some embodiments, the ADAMTS13 composition described herein is used to treat and prevent ADAMTS13 dysfunction and VWF. In some embodiments, the ADAMTS13 composition described herein is used for the treatment and prevention of thrombotic diseases and conditions. In some embodiments, the ADAMTS13 composition described herein is used for the treatment and prevention of infarction.

In one embodiment, ADAMTS13 is administered at a dose of 2 _FV73 Units/kg body weight to 4000 _FV73 Units/kg body weight. In one embodiment, ADAMTS13 is administered at a dose of 20 _FV73 Units/kg body weight to 2000 _FV73 Units/kg body weight. In one embodiment, ADAMTS13 is administered at a dose of 20 _FV73 Units/kg body weight to 1000 _FV73 Units/kg body weight. In one embodiment, ADAMTS13 is administered at a dose of 20 _FV73 Units/kg body weight to 500 _FV73 Units/kg body weight. In one embodiment, ADAMTS13 is administered at a dose of 20 _FV73 Units/kg body weight to 200 _FV73 Units/kg body weight. In one embodiment, ADAMTS13 is administered at a dose of 20 _FV73 Units/kg body weight to 100 _FV73 Units/kg body weight. In one embodiment, ADAMTS13 is administered at a dose of 40 _FV73 Units/kg body weight to 200 _FV73 Units/kg body weight. In one embodiment, ADAMTS13 is administered at a dose of 40 _FV73 Units/kg body weight to 100 _FV73 Units/kg body weight. In other embodiments, ADAMTS13 is administered in an amount of approximately 20 _FV73 Units/kg body weight, 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, 27 00, 2800, 2900, 3000, 3100 , 3200, 3300, 3400, 3500, 3600, 3700, 3800, 3900, 4000 _FV73 units/kg body weight, or at an intermediate concentration or range of concentrations.

In general, the dose of ADAMTS13 administered to a mammal depends on, among other factors, the disease or condition being treated, the species of the mammal, the age of the mammal, and the general health of the mammal. One skilled in the art will readily be able to convert dosages for different mammals, for example, a mouse dose to a human dose. One means for extrapolating a human dose from an animal dose involves using body surface area, which is known to correlate well with several metabolic parameters, such as blood volume, circulating plasma, and renal function in various mammals. Thus, a conversion factor (e.g., Km) that correlates the average mass of a mammal with the average body surface area can be used to correlate a drug dosage (e.g., ADAMTS13 dosage) expressed in units of protein (e.g., mass or activity) per unit body weight mammal (eg, kg) used for the first mammal species (eg, mouse), and the corresponding dose for the second mammal species (eg, human). See review by Reagan-Shaw et al., FASEB, 22:659-62 (2007). For example, this can be done by first multiplying the dose of drug administered to a first mammal species by the conversion factor established for that mammal, and then dividing the result by the conversion factor established for the second mammal species. Examples of such conversion factors are given in table below. 1, taken from the US Department of Health and Human Services guidance for estimating the maximum safe starting dose in early-stage clinical trials of therapeutic agents in adult healthy volunteers.

- 10 045023

Table 1 Conversion of animal doses to equivalent human doses based on body surface area

Species To Convert Animal Dose in mgkg to Dose in mg in ² . Multiply by km To Convert Animal Dose in mg. kg to HED ^a in m&ki, Either: Divide Animal Dose By Multiply Animal Dose By Human 37 — — Child (20 kg) ^b 25 — — Mouse 3 12.3 0.08 Hamster 5 7.4 0.13 Rat 6 6.2 0.16 Ferret 7 5.3 0.19 Guinea pig 8 4.6 0.22 Rabbit 12 3.1 0.32 Dog 20 1.8 0.54 Primates: Monkeys ^c 12 3.1 0.32 Mannoset 6 6.2 0.16 Squirrel monkey 7 5.3 0.19 Baboon 20 1.8 0.54 Micro-pig 27 1.4 0.73 Mini-pig 35 1.1 0.95

^a Represents 60 kg of human weight. For unlisted species or for weights outside standard ranges, the HED can be calculated using the formula: HED=animal dose in mg/kg(animal weight in kg/human weight in kg) ^0.33 .

^b The km value is for reference only because healthy children (pups) rarely volunteer in phase 1 studies.

^c For example, cynomolgus, rhesus and kulturval.

In one embodiment, the bioavailability of ADAMTS13 after subcutaneous administration is at least 50% to at least 80% compared to intravenous administration standardized to a similar dose. In one embodiment, the bioavailability of ADAMTS13 after subcutaneous administration is at least 60% to at least 80% compared to intravenous administration standardized to a similar dose. In one embodiment, the bioavailability of ADAMTS13 after subcutaneous administration is at least 50% to 70% compared to intravenous administration standardized to a similar dose. In one embodiment, the bioavailability of ADAMTS13 after subcutaneous administration is at least 55% to 65% compared to intravenous administration standardized to a similar dose. In one embodiment, the bioavailability of ADAMTS13 after subcutaneous administration is at least 55% to 70% compared to intravenous administration standardized to a similar dose.

In some embodiments, the bioavailability of ADAMTS13 after subcutaneous administration is at least 40%, or at least 45%, or at least 50%, or at least 51%, or at least 52%, or at least 53% , or at least 54%, or at least 55%, or at least 56%, or at least 57%, or at least 58%, or at least 59%, or at least 60%, or at least 61%, or at least 62%, or at least 63%, or at least 64%, or at least 65%, or at least 66%, or at least 67%, or at least 68%, or at least 69%, or at least 70%, or at least 71%, or at least 72%, or at least 73%, or at least 74%, or according at least 75%, or at least 7 6%, or at least 77%, or at least 78%, or at least 79%, or at least 80%, or at least 81%, or according at least 82%, or at least 83%, or at least 84%, or at least 85% compared to intravenous administration normalized to a similar dose.

In some embodiments, the ADAMTS13 formulation is administered by subcutaneous administration as a single bolus injection. In some embodiments, the ADAMTS13 formulation is administered by subcutaneous administration once a month. In some embodiments, the ADAMTS13 formulation is administered by subcutaneous administration once every two weeks. In some embodiments, the ADAMTS13 formulation is administered by subcutaneous administration once a week. In some embodiments, the ADAMTS13 formulation is administered by subcutaneous administration twice per week. In some embodiments, the ADAMTS13 composition is administered

- 11 045023 by subcutaneous injection once a day. In some embodiments, AD AMTS 13 is administered by subcutaneous injection every 12 hours. In some embodiments, ADAMTS13 is administered by subcutaneous injection every 8 hours. In some embodiments, AD AMTS 13 is administered by subcutaneous injection every six hours. In some embodiments, the AD AMTS 13 formulation is administered by subcutaneous administration once every four hours. In some embodiments, the AD AMTS 13 formulation is administered by subcutaneous administration once every two hours.

In one embodiment, the AD AMTS 13 formulation is administered by subcutaneous administration at a dose and frequency combination selected from options 1-1133 in Table. 2. In general, the dose and frequency of AD AMTS 13 administered to a mammal depends on, among other factors, the disease or condition being treated, the species of the mammal, the age of the mammal, and the general health of the mammal.

table 2

Beneficial Dosage and Frequency Combinations of ADAMTS13 for Subcutaneous Administration

Dose (URU73/kg body weight) Once every month Twice a month Three times a month Once every a week Two times a week In one day Once a day Twice a day Three times a day Four times a day Six times a day Var. 1 Var. 104 Var. 207 Var. 310 Var. 413 Var. 516 Var. 619 Var. 722 Var. 825 Var. 928 Var. 1031 Var. 2 Var. 105 Var. 208 Var. 311 Var. 414 Var. 517 Var. 620 Var. 723 Var. 826 Var. 929 Var. 1032 Var. 3 Var. 106 Var. 209 Var. 312 Var. 415 Var. 518 Var. 621 Var. 724 Var. 827 Var. 930 Var. 1033 Var. 4 Var. 107 Var. 210 Var. 313 Var. 416 Var. 519 Var. 622 Var. 725 Var. 828 Var. 931 Var. 1034 Var. 5 Var. 108 Var. 211 Var. 314 Var. 417 Var. 520 Var. 623 Var. 726 Var. 829 Var. 932 Var. 1035 Var. 6 Var. 109 Var. 212 Var. 315 Var. 418 Var. 521 Var. 624 Var. 727 Var. 830 Var. 933 Var. 1036

- 12045023

Bap. 7 Bap. 110 Bap. 213 Bap. 316 Bap. 419 Bap. 522 Bap. 625 Bap. 728 Bap. 831 Bap. 934 Bap. 1037 Bap. 8 Bap. Ill Bap. 214 Bap. 317 Bap. 420 Bap. 523 Bap. 626 Bap. 729 Bap. 832 Bap. 935 Bap. 1038 Bap. 9 Bap. 112 Bap. 215 Bap. 318 Bap. 421 Bap. 524 Bap. 627 Bap. 730 Bap. 833 Bap. 936 Bap. 1039 Bap. 10 Bap. 113 Bap. 216 Bap. 319 Bap. 422 Bap. 525 Bap. 628 Bap. 731 Bap. 834 Bap. 937 Bap. 1040 Bap. eleven Bap. 114 Bap. 217 Bap. 320 Bap. 423 Bap. 526 Bap. 629 Bap. 732 Bap. 835 Bap. 938 Bap. 1041 Bap. 12 Bap. 115 Bap. 218 Bap. 321 Bap. 424 Bap. 527 Bap. 630 Bap. 733 Bap. 836 Bap. 939 Bap. 1042 Bap. 13 Bap. 116 Bap. 219 Bap. 322 Bap. 425 Bap. 528 Bap. 631 Bap. 734 Bap. 837 Bap. 940 Bap. 1043 Bap. 14 Bap. 117 Bap. 220 Bap. 323 Bap. 426 Bap. 529 Bap. 632 Bap. 735 Bap. 838 Bap. 941 Bap. 1044 Bap. 15 Bap. 118 Bap. 221 Bap. 324 Bap. 427 Bap. 530 Bap. 633 Bap. 736 Bap. 839 Bap. 942 Bap. 1045 Bap. 16 Bap. 119 Bap. 222 Bap. 325 Bap. 428 Bap. 531 Bap. 634 Bap. 737 Bap. 840 Bap. 943 Bap. 1046 Bap. 17 Bap. 120 Bap. 223 Bap. 326 Bap. 429 Bap. 532 Bap. 635 Bap. 738 Bap. 841 Bap. 944 Bap. 1047 Bap. 18 Bap. 121 Bap. 224 Bap. 327 Bap. 430 Bap. 533 Bap. 636 Bap. 739 Bap. 842 Bap. 945 Bap. 1048 Bap. 19 Bap. 122 Bap. 225 Bap. 328 Bap. 431 Bap. 534 Bap. 637 Bap. 740 Bap. 843 Bap. 946 Bap. 1049 Bap. 20 Bap. 123 Bap. 226 Bap. 329 Bap. 432 Bap. 535 Bap. 638 Bap. 741 Bap. 844 Bap. 947 Bap. 1050 Bap. 21 Bap. 124 Bap. 227 Bap. 330 Bap. 433 Bap. 536 Bap. 639 Bap. 742 Bap. 845 Bap. 948 Bap. 1051 Bap. 22 Bap. 125 Bap. 228 Bap. 331 Bap. 434 Bap. 537 Bap. 640 Bap. 743 Bap. 846 Bap. 949 Bap. 1052 Bap. 23 Bap. 126 Bap. 229 Bap. 332 Bap. 435 Bap. 538 Bap. 641 Bap. 744 Bap. 847 Bap. 950 Bap. 1053 Bap. 24 Bap. 127 Bap. 230 Bap. 333 Bap. 436 Bap. 539 Bap. 642 Bap. 745 Bap. 848 Bap. 951 Bap. 1054

- 13 045023

Bap. 25 Bap. 128 Bap. 231 Bap. 334 Bap. 437 Bap. 540 Bap. 643 Bap. 746 Bap. 849 Bap. 952 Bap. 1055 Bap. 26 Bap. 129 Bap. 232 Bap. 335 Bap. 438 Bap. 541 Bap. 644 Bap. 747 Bap. 850 Bap. 953 Bap. 1056 Bap. 27 Bap. 130 Bap. 233 Bap. 336 Bap. 439 Bap. 542 Bap. 645 Bap. 748 Bap. 851 Bap. 954 Bap. 1057 Bap. 28 Bap. 131 Bap. 234 Bap. 337 Bap. 440 Bap. 543 Bap. 646 Bap. 749 Bap. 852 Bap. 955 Bap. 1058 Bap. 29 Bap. 132 Bap. 235 Bap. 338 Bap. 441 Bap.54 4 Bap. 647 Bap. 750 Bap. 853 Bap. 956 Bap. 1059 Bap. thirty Bap. 133 Bap. 236 Bap. 339 Bap. 442 Bap. 545 Bap. 648 Bap. 751 Bap. 854 Bap. 957 Bap. 1060 Bap. 31 Bap. 134 Bap. 237 Bap. 340 Bap. 443 Bap. 546 Bap. 649 Bap. 752 Bap. 855 Bap. 958 Bap. 1061 Bap. 32 Bap. 135 Bap. 238 Bap. 341 Bap. 444 Bap. 547 Bap. 650 Bap. 753 Bap. 856 Bap. 959 Bap. 1062 Bap. 33 Bap. 136 Bap. 239 Bap. 342 Bap. 445 Bap. 548 Bap. 651 Bap. 754 Bap. 857 Bap. 960 Bap. 1063 Bap. 34 Bap. 137 Bap. 240 Bap. 343 Bap. 446 Bap. 549 Bap. 652 Bap. 755 Bap. 858 Bap. 961 Bap. 1064 Bap. 35 Bap. 138 Bap. 241 Bap. 344 Bap. 447 Bap. 550 Bap. 653 Bap. 756 Bap. 859 Bap. 962 Bap. 1065 Bap. 36 Bap. 139 Bap. 242 Bap. 345 Bap. 448 Bap. 551 Bap. 654 Bap. 757 Bap. 860 Bap. 963 Bap. 1066 Bap. 37. Bap. 140 Bap. 243 Bap. 346 Bap. 449 Bap. 552 Bap. 655 Bap. 758 Bap. 861 Bap. 964 Bap. 1067 Bap. 38 Bap. 141 Bap. 244 Bap. 347 Bap. 450 Bap. 553 Bap. 656 Bap. 759 Bap. 862 Bap. 965 Bap. 1068 Bap. 39 Bap. 142 Bap. 245 Bap. 348 Bap. 451 Bap. 554 Bap. 657 Bap. 760 Bap. 863 Bap. 966 Bap. 1069 Bap. 40 Bap. 143 Bap. 246 Bap. 349 Bap. 452 Bap. 555 Bap. 658 Bap. 761 Bap. 864 Bap. 967 Bap. 1070 Bap. 41 Bap. 144 Bap. 247 Bap. 350 Bap. 453 Bap. 556 Bap. 659 Bap. 762 Bap. 865 Bap. 968 Bap. 1071 Bap. 42 Bap. 145 Bap. 248 Bap. 351 Bap. 454 Bap. 557 Bap. 660 Bap. 763 Bap. 866 Bap. 969 Bap. 1072

- 14 045023

Bap. 43 Bap. 146 Bap. 249 Bap. 352 Bap. 455 Bap. 558 Bap. 661 Bap. 764 Bap. 867 Bap. 970 Bap. 1073 Bap. 44 Bap. 147 Bap. 250 Bap. 353 Bap. 456 Bap. 559 Bap. 662 Bap. 765 Bap. 868 Bap. 971 Bap. 1074 Bap. 45 Bap. 148 Bap. 251 Bap. 354 Bap. 457 Bap. 560 Bap. 663 Bap. 766 Bap. 869 Bap. 972 Bap. 1075 Bap. 46 Bap. 149 Bap. 252 Bap. 355 Bap. 458 Bap. 561 Bap. 664 Bap. 767 Bap. 870 Bap. 973 Bap. 1076 Bap. 47 Bap. 150 Bap. 253 Bap. 356 Bap. 459 Bap. 562 Bap. 665 Bap. 768 Bap. 871 Bap. 974 Bap. 1077 Bap. 48 Bap. 151 Bap. 254 Bap. 357 Bap. 460 Bap. 563 Bap. 666 Bap. 769 Bap. 872 Bap. 975 Bap. 1078 Bap. 49 Bap. 152 Bap. 255 Bap. 358 Bap. 461 Bap. 564 Bap. 667 Bap. 770 Bap. 873 Bap. 976 Bap. 1079 Bap. 50 Bap. 153 Bap. 256 Bap. 359 Bap. 462 Bap. 565 Bap. 668 Bap. 771 Bap. 874 Bap. 977 Bap. 1080 Bap. 51 Bap. 154 Bap. 257 Bap. 360 Bap. 463 Bap. 566 Bap. 669 Bap. 772 Bap. 875 Bap. 978 Bap. 1081 Bap. 52 Bap. 155 Bap. 258 Bap. 361 Bap. 464 Bap. 567 Bap. 670 Bap. 773 Bap. 876 Bap. 979 Bap. 1082 Bap. 53 Bap. 156 Bap. 259 Bap. 362 Bap. 465 Bap. 568 Bap. 671 Bap. 774 Bap. 877 Bap. 980 Bap. 1083 Bap. 54 Bap. 157 Bap. 260 Bap. 363 Bap. 466 Bap. 569 Bap. 672 Bap. 775 Bap. 878 Bap. 981 Bap. 1084 Bap. 55 Bap. 158 Bap. 261 Bap. 364 Bap. 467 Bap. 570 Bap. 673 Bap. 776 Bap. 879 Bap. 982 Bap. 1085 Bap. 56 Bap. 159 Bap. 262 Bap. 365 Bap. 468 Bap. 571 Bap. 674 Bap. 777 Bap. 880 Bap. 983 Bap. 1086 Bap. 57 Bap. 160 Bap. 263 Bap. 366 Bap. 469 Bap. 572 Bap. 675 Bap. 778 Bap. 881 Bap. 984 Bap. 1087 Bap. 58 Bap. 161 Bap. 264 Bap. 367 Bap. 470 Bap. 573 Bap. 676 Bap. 779 Bap. 882 Bap. 985 Bap. 1088 Bap. 59 Bap. 162 Bap. 265 Bap. 368 Bap. 471 Bap. 574 Bap. 677 Bap. 780 Bap. 883 Bap. 986 Bap. 1089 Bap. 60 Bap. 163 Bap. 266 Bap. 369 Bap. 472 Bap. 575 Bap. 678 Bap. 781 Bap. 884 Bap. 987 Bap. 1090

- 15 045023

Bap. 61 Bap. 164 Bap. 267 Bap. 370 Bap. 473 Bap. 576 Bap. 679 Bap. 782 Bap. 885 Bap. 988 Bap. 1091 Bap. 62 Bap. 165 Bap. 268 Bap. 371 Bap. 474 Bap. 577 Bap. 680 Bap. 783 Bap. 886 Bap. 989 Bap. 1092 Bap. 63 Bap. 166 Bap. 269 Bap. 372 Bap. 475 Bap. 578 Bap. 681 Bap. 784 Bap. 887 Bap. 990 Bap. 1093 Bap. 64 Bap. 167 Bap. 270 Bap. 373 Bap. 476 Bap. 579 Bap. 682 Bap. 785 Bap. 888 Bap. 991 Bap. 1094 Bap. 65 Bap. 168 Bap. 271 Bap. 374 Bap. 477 Bap. 580 Bap. 683 Bap. 786 Bap. 889 Bap. 992 Bap. 1095 Bap. 66 Bap. 169 Bap. 272 Bap. 375 Bap. 478 Bap. 581 Bap. 684 Bap. 787 Bap. 890 Bap. 993 Bap. 1096 Bap. 67 Bap. 170 Bap. 273 Bap. 376 Bap. 479 Bap. 582 Bap. 685 Bap. 788 Bap. 891 Bap. 994 Bap. 1097 Bap. 68 Bap. 171 Bap. 274 Bap. 377 Bap. 480 Bap. 583 Bap. 686 Bap. 789 Bap. 892 Bap. 995 Bap. 1098 Bap. 69 Bap. 172 Bap. 275 Bap. 378 Bap. 481 Bap. 584 Bap. 687 Bap. 790 Bap. 893 Bap. 996 Bap. 1099 Bap. 70 Bap. 173 Bap. 276 Bap. 379 Bap. 482 Bap. 585 Bap. 688 Bap. 791 Bap. 894 Bap. 997 Bap. 1100 Bap. 71 Bap. 174 Bap. 277 Bap. 380 Bap. 483 Bap. 586 Bap. 689 Bap. 792 Bap. 895 Bap. 998 Bap. 1101 Bap. 72 Bap. 175 Bap. 278 Bap. 381 Bap. 484 Bap. 587 Bap. 690 Bap. 793 Bap. 896 Bap. 999 Bap. 1102 Bap. 73 Bap. 176 Bap. 279 Bap. 382 Bap. 485 Bap. 588 Bap. 691 Bap. 794 Bap. 897 Bap. 1000 Bap. 1103 Bap. 74 Bap. 177 Bap. 280 Bap. 383 Bap. 486 Bap. 589 Bap. 692 Bap. 795 Bap. 898 Bap. 1001 Bap. 1104 Bap. 75 Bap. 178 Bap. 281 Bap. 384 Bap. 487 Bap. 590 Bap. 693 Bap. 796 Bap. 899 Bap. 1002 Bap. 1105 Bap. 76 Bap. 179 Bap. 282 Bap. 385 Bap. 488 Bap. 591 Bap. 694 Bap. 797 Bap. 900 Bap. 1003 Bap. 1106 Bap. 77 Bap. 180 Bap. 283 Bap. 386 Bap. 489 Bap. 592 Bap. 695 Bap. 798 Bap. 901 Bap. 1004 Bap. 1107 Bap. 78 Bap. 181 Bap. 284 Bap. 387 Bap. 490 Bap. 593 Bap. 696 Bap. 799 Bap. 902 Bap. 1005 Bap. 1108

- 16 045023

Bap. 79 Bap. 182 Bap. 285 Bap. 388 Bap. 491 Bap. 594 Bap. 697 Bap. 800 Bap. 903 Bap. 1006 Bap. 1109 Bap. 80 Bap. 183 Bap. 286 Bap. 389 Bap. 492 Bap. 595 Bap. 698 Bap. 801 Bap. 904 Bap. 1007 Bap. 1110 Bap. 81 Bap. 184 Bap. 287 Bap. 390 Bap. 493 Bap. 596 Bap. 699 Bap. 802 Bap. 905 Bap. 1008 Bap. 1111 Bap. 82 Bap. 185 Bap. 288 Bap. 391 Bap. 494 Bap. 597 Bap. 700 Bap. 803 Bap. 906 Bap. 1009 Bap. 1112 Bap. 83 Bap. 186 Bap. 289 Bap. 392 Bap. 495 Bap. 598 Bap. 701 Bap. 804 Bap. 907 Bap. 1010 Bap. 1113 Bap. 84 Bap. 187 Bap. 290 Bap. 393 Bap. 496 Bap. 599 Bap. 702 Bap. 805 Bap. 908 Bap. 1011 Bap. 1114 Bap. 85 Bap. 188 Bap. 291 Bap. 394 Bap. 497 Bap. 600 Bap. 703 Bap. 806 Bap. 909 Bap. 1012 Bap. 1115 Bap. 86 Bap. 189 Bap. 292 Bap. 395 Bap. 498 Bap. 601 Bap. 704 Bap. 807 Bap. 910 Bap. 1013 Bap. 1116 Bap. 87 Bap. 190 Bap. 293 Bap. 396 Bap. 499 Bap. 602 Bap. 705 Bap. 808 Bap. 911 Bap. 1014 Bap. 1117 Bap. 88 Bap. 191 Bap. 294 Bap. 397 Bap. 500 Bap. 603 Bap. 706 Bap. 809 Bap. 912 Bap. 1015 Bap. 1118 Bap. 89 Bap. 192 Bap. 295 Bap. 398 Bap. 501 Bap. 604 Bap. 707 Bap. 810 Bap. 913 Bap. 1016 Bap. 1119 Bap. 90 Bap. 193 Bap. 296 Bap. 399 Bap. 502 Bap. 605 Bap. 708 Bap. 811 Bap. 914 Bap. 1017 Bap. 1120 Bap. 91 Bap. 194 Bap. 297 Bap. 400 Bap. 503 Bap. 606 Bap. 709 Bap. 812 Bap. 915 Bap. 1018 Bap. 1121 Bap. 92 Bap. 195 Bap. 298 Bap. 401 Bap. 504 Bap. 607 Bap. 710 Bap. 813 Bap. 916 Bap. 1019 Bap. 1122 Bap. 93 Bap. 196 Bap. 299 Bap. 402 Bap. 505 Bap. 608 Bap. 711 Bap. 814 Bap. 917 Bap. 1020 Bap. 1123 Bap. 94 Bap. 197 Bap. 300 Bap. 403 Bap. 506 Bap. 609 Bap. 712 Bap. 815 Bap. 918 Bap. 1021 Bap. 1124 Bap. 95 Bap. 198 Bap. 301 Bap. 404 Bap. 507 Bap. 610 Bap. 713 Bap. 816 Bap. 919 Bap. 1022 Bap. 1125 Bap. 96 Bap. 199 Bap. 302 Bap. 405 Bap. 508 Bap. 611 Bap. 714 Bap. 817 Bap. 920 Bap. 1023 Bap. 1126

- 17045023

Var. 97 Var. 200 Var. 303 Var. 406 Var. 509 Var. 612 Var. 715 Var. 818 Var. 921 Var. 1024 Var. 1127 Var. 98 Var. 201 Var. 304 Var. 407 Var. 510 Var. 613 Var. 716 Var. 819 Var. 922 Var. 1025 Var. 1128 Var. 99 Var. 202 Var. 305 Var. 408 Var. 511 Var. 614 Var. 717 Var. 820 Var. 923 Var. 1026 Var. 1129 Var. 100 Var. 203 Var. 306 Var. 409 Var. 512 Var. 615 Var. 718 Var. 821 Var. 924 Var. 1027 Var. ISO Var. 101 Var. 204 Var. 307 Var. 410 Var. 513 Var. 616 Var. 719 Var. 822 Var. 925 Var. 1028 Var. 1131 Var. 102 Var. 205 Var. 308 Var. 411 Var. 514 Var. 617 Var. 720 Var. 823 Var. 926 Var. 1029 Var. 1132 Var. 103 Var. 206 Var. 309 Var. 412 Var. 515 Var. 618 Var. 721 Var. 824 Var. 927 Var. 1030 Var. 1133

In some embodiments, approximately 120-300% of the amount of a standard intravenous dose of the ADAMTS13 formulation for a particular indication, measured in units of activity per kilogram, is administered subcutaneously. In a specific embodiment, approximately 120% of the amount of a standard intravenous dose of the ADAMTS13 formulation for a particular indication, measured in units of activity per kilogram, is administered subcutaneously. In a specific embodiment, approximately 130% of the amount of a standard intravenous dose of the ADAMTS13 formulation for a particular indication, measured in units of activity per kilogram, is administered subcutaneously. In a specific embodiment, approximately 140% of the amount of a standard intravenous dose of the ADAMTS13 formulation for a particular indication, measured in units of activity per kilogram, is administered subcutaneously. In a specific embodiment, approximately 150% of the amount of a standard intravenous dose of the ADAMTS13 formulation for a particular indication, measured in units of activity per kilogram, is administered subcutaneously. In a specific embodiment, approximately 160% of the amount of a standard intravenous dose of the ADAMTS13 formulation for a particular indication, measured in units of activity per kilogram, is administered subcutaneously. In a specific embodiment, approximately 170% of the amount of a standard intravenous dose of the ADAMTS13 formulation for a particular indication, measured in units of activity per kilogram, is administered subcutaneously. In a specific embodiment, approximately 180% of the amount of a standard intravenous dose of the ADAMTS13 formulation for a particular indication, measured in units of activity per kilogram, is administered subcutaneously. In a specific embodiment, approximately 190% of the amount of a standard intravenous dose of the ADAMTS13 formulation for a particular indication, measured in units of activity per kilogram, is administered subcutaneously.

In a specific embodiment, approximately 200% of the amount of a standard intravenous dose of the ADAMTS13 formulation for a particular indication, measured in units of activity per kilogram, is administered subcutaneously. In a specific embodiment, approximately 210% of the amount of a standard intravenous dose of the ADAMTS13 formulation for a particular indication, measured in units of activity per kilogram, is administered subcutaneously. In a specific embodiment, approximately 220% of the amount of a standard intravenous dose of the ADAMTS13 formulation for a particular indication, measured in units of activity per kilogram, is administered subcutaneously. In a specific embodiment, approximately 230% of the amount of a standard intravenous dose of the ADAMTS13 formulation for a particular indication, measured in units of activity per kilogram, is administered subcutaneously. In a specific embodiment, approximately 240% of the amount of a standard intravenous dose of the ADAMTS13 formulation for a particular indication, measured in units of activity per kilogram, is administered subcutaneously. In a specific embodiment, approximately 250% of the amount of a standard intravenous dose of the ADAMTS13 formulation for a particular indication, measured in units of activity per kilogram, is administered subcutaneously. In a specific embodiment, approximately 260% of the amount of a standard intravenous dose of the ADAMTS13 formulation for a particular indication, measured in units of activity per kilogram, is administered subcutaneously. In a specific embodiment, approximately 270% of the amount of a standard intravenous dose of the ADAMTS13 formulation for a particular indication, measured in units of activity per kilogram, is administered subcutaneously. In a specific embodiment, approximately 280% of the amount of a standard intravenous dose of the ADAMTS13 formulation for a particular indication, measured in units of activity per kilogram, is administered subcutaneously. In a specific embodiment, approximately 290% of the amount of a standard intravenous dose of the ADAMTS13 formulation for a particular indication, measured in units of activity per kilogram, is administered subcutaneously. In a specific embodiment, approximately 300% of the amount of a standard intravenous dose of the formulation is administered subcutaneously.

ADAMTS13 for a specific reading, measured in units of activity per kilogram.

In some embodiments, approximately 120%, approximately 122%, approximately 125%, approximately 127%, approximately 130%, approximately 132%, approximately 135%, approximately 137%, approximately 140%, approximately 142%, approximately 145% are administered subcutaneously. approximately 147%, approximately 150%, approximately 152%, approximately 155%, approximately 157%, approximately 160%, approximately 162%, approximately 165%, approximately 167%, approximately 170%, approximately 172%, approximately 175%, approximately 177 %, approximately 180%, approximately 182%, approximately 185%, approximately 187%, approximately 190%, approximately 192%, approximately 195%, approximately 97%, approximately 200%, approximately 202%, approximately 205%, approximately 207%, approximately 210%, approximately 212%, approximately 215%, approximately 217%, approximately 220%, approximately 222%, approximately 225%, approximately 227%, approximately 230%, approximately 232%, approximately 235%, approximately 237%, approximately 240 %, approximately 242%, approximately 245%, approximately 247%, approximately 250%, approximately 252%, approximately 255%, approximately 257%, approximately 260%, approximately 262%, approximately 265%, approximately 267%, approximately 270%, approximately 272%, approximately 275%, approximately 277%, approximately 280%, approximately 282%, approximately 285%, approximately 287%, approximately 290%, approximately 292%, approximately 295%, approximately 297% or approximately 300% of the standard amount intravenous dose of the ADAMTS13 formulation for a specific indication, measured in units of activity per kilogram.

In one embodiment, the ADAMTS13 formulation is administered by subcutaneous administration to reduce inflammation caused by a clotting disorder (eg, infarction), thereby preventing or reducing tissue damage (eg, brain damage) and/or to reduce reperfusion injury by preventing infiltration and damaging actions of leukocytes. In one embodiment, the ADAMTS13 formulation is administered by subcutaneous administration to protect against secondary tissue damage in the infarcted area (eg, brain tissue and myocardial tissue) caused by reperfusion.

Hereditary TTP

In one embodiment, the ADAMTS13 formulation described herein is used for the treatment and prevention of hereditary TTP. Hereditary TTP is caused by genetic mutations in the ADAMTS13 gene. Hereditary TTP can result in neurological manifestations (eg, altered mental status, stroke, seizures, hemiplegia, paresthesia, visual disturbances, and aphasia), fatigue, and severe bleeding. If left untreated, acquired TTP can lead to death or long-term physiological impairment. In addition, since familial TTP is caused by a genetic mutation, lifelong treatment and patient compliance are required. Although intravenous delivery of ADAMTS13 formulations is effective in the treatment of hereditary TTP, intravenous drug administration is challenging for patients (especially children with hereditary TTP) and reduces patient adherence to treatment. Accordingly, it is desirable to develop a subcutaneous formulation of ADAMTS13 and a method for subcutaneous delivery of ADAMTS13.

In one embodiment, the present disclosure provides a method of treating hereditary TTP in a mammal in need thereof, comprising subcutaneously administering a therapeutically effective amount of a composition comprising isolated ADAMTS13 to the mammal, wherein the therapeutically effective amount of ADAMTS13 contains from 20 to 4000 units of FRETSVWF73 activity per kilogram of mammal body weight ( Units _FV73 /kg). In a particular embodiment, the mammal is a human. In one embodiment, from 20 Units _{of FV73} to 4000 Units _{of FV73} /kg ADAMTS13 is administered approximately once per month. In one embodiment, 20 to 4000 U _FV73 /kg ADAMTS13 is administered approximately twice a month. In one embodiment, 20 to 4000 U _FV73 /kg ADAMTS13 is administered approximately once per week. In one embodiment, 20 to 4000 U _FV73 /kg ADAMTS13 is administered approximately twice per week. In one embodiment, 20 to 4000 U _FV7 /kg ADAMTS13 is administered approximately once every 48 hours. In one embodiment, 20 to 4000 U _FV73 /kg ADAMTS13 is administered approximately once every 24 hours. In one embodiment, 20 to 4000 U _FV73 /kg ADAMTS13 is administered approximately every 12 hours.

In one embodiment, the present disclosure provides a method of treating hereditary TTP in a mammal in need thereof, comprising subcutaneously administering a therapeutically effective amount of a composition comprising isolated ADAMTS13 to the mammal, wherein the therapeutically effective amount of ADAMTS13 contains from 20 to 2000 units of FRETSVWF7 3 activity per kilogram of body weight of the mammal (Unit _FV73 /kg). In a particular embodiment, the mammal is a human. In one embodiment, from 20 _FV73 Units to 2000 _FV73 Units/kg

- 19 045023

ADAMTS13 is administered approximately once a month. In one embodiment, 20 to 2000 U _FV73 /kg ADAMTS13 is administered approximately twice a month. In one embodiment, 20 to 2000 U _FV73 /kg ADAMTS13 is administered approximately once per week. In one embodiment, 20 to 2000 U _FV73 /kg ADAMTS13 is administered approximately twice per week. In one embodiment, 20 to 2000 _UFV73 /kg ADAMTS13 is administered approximately once every 48 hours. In one embodiment, 20 to 2000 UF _V73 /kg ADAMTS13 is administered approximately once every 24 hours. In one embodiment, 20 to 2000 U _FV73 /kg ADAMTS13 is administered approximately once every 12 hours.

In one embodiment, the present disclosure provides a method of treating hereditary TTP in a mammal in need thereof, comprising subcutaneously administering a therapeutically effective amount of a composition comprising isolated ADAMTS13 to the mammal, wherein the therapeutically effective amount of ADAMTS13 contains from 20 to 1000 units of FRETSVWF73 activity per kilogram of mammal body weight ( Units _FV73 /kg). In a particular embodiment, the mammal is a human. In one embodiment, from 20 Units _{of FV73} to 1000 Units _{of FV73} /kg ADAMTS13 is administered approximately once per month. In one embodiment, 20 to 1000 U _FV73 /kg ADAMTS13 is administered approximately twice a month. In one embodiment, 20 to 1000 U _FV73 /kg ADAMTS13 is administered approximately once per week. In one embodiment, 20 to 1000 U _FV73 /kg ADAMTS13 is administered approximately twice per week. In one embodiment, 20 to 1000 Units of _FV73 /kg ADAMTS13 is administered approximately once every 48 hours. In one embodiment, 20 to 1000 Units of _FV73 /kg ADAMTS13 is administered approximately once every 24 hours. In one embodiment, 20 to 1000 Units of _FV73 /kg ADAMTS13 is administered approximately every 12 hours.

In one embodiment, the present disclosure provides a method of treating hereditary TTP in a mammal in need thereof, comprising subcutaneously administering a therapeutically effective amount of a composition comprising isolated ADAMTS13 to the mammal, wherein the therapeutically effective amount of ADAMTS13 contains from 20 to 500 units of FRETSVWF73 activity per kilogram of mammal body weight ( Units _FV73 /kg). In a particular embodiment, the mammal is a human. In one embodiment, from 20 Units _{of FV73} to 500 Units _{of FV73} /kg ADAMTS13 is administered approximately once per month. In one embodiment, 20 to 500 U _FV73 /kg ADAMTS13 is administered approximately twice a month. In one embodiment, 20 to 500 U _FV73 /kg ADAMTS13 is administered approximately once per week. In one embodiment, 20 to 500 U _FV73 /kg ADAMTS13 is administered approximately twice per week. In one embodiment, 20 to 500 Units of _FV73 /kg ADAMTS13 is administered approximately once every 48 hours. In one embodiment, 20 to 500 Units of _FV73 /kg ADAMTS13 is administered approximately once every 24 hours. In one embodiment, 20 to 500 Units of _FV73 /kg ADAMTS13 is administered approximately every 12 hours.

In one embodiment, the present disclosure provides a method of treating hereditary TTP in a mammal in need thereof, comprising subcutaneously administering a therapeutically effective amount of a composition comprising isolated ADAMTS13 to the mammal, wherein the therapeutically effective amount of ADAMTS13 contains from 20 to 200 units of FRETSVWF73 activity per kilogram of mammal body weight ( Units _FV73 /kg). In a particular embodiment, the mammal is a human. In one embodiment, from 20 Units _{of FV73} to 200 Units _{of FV73} /kg ADAMTS13 is administered approximately once per month. In one embodiment, 20 to 200 U _FV73 /kg ADAMTS13 is administered approximately twice a month. In one embodiment, 20 to 200 U _FV73 /kg ADAMTS13 is administered approximately once per week. In one embodiment, 20 to 200 U _FV73 /kg ADAMTS13 is administered approximately twice per week. In one embodiment, 20 to 200 Units of _FV73 /kg ADAMTS13 is administered approximately once every 48 hours. In one embodiment, 20 to 200 Units of _FV73 /kg ADAMTS13 is administered approximately once every 24 hours. In one embodiment, 20 to 200 Units of _FV73 /kg ADAMTS13 is administered approximately every 12 hours.

One embodiment of the present disclosure provides a method of treating hereditary TTP in a mammal in need thereof, comprising subcutaneously administering a therapeutically effective amount of a composition comprising isolated ADAMTS13 to the mammal, wherein the therapeutically effective amount of ADAMTS13 contains from 20 to 100 units of FRETSVWF73 activity per kilogram of mammal body weight ( Units _FV73 /kg). In a particular embodiment, the mammal is a human. In one embodiment, from 20 Units _{of FV73} to 100 Units _{of FV73} /kg ADAMTS13 is administered approximately once per month. In one embodiment, 20 to 100 U _FV73 /kg ADAMTS13 is administered approximately twice a month. In one embodiment, 20 to 100 U _FV73 /kg ADAMTS13 is administered approximately once per week. In one embodiment, 20 to 100 U _FV73 /kg ADAMTS13 is administered approximately twice per week. In one embodiment, 20 to 100 Units of _FV73 /kg ADAMTS13 is administered approximately once every 48 hours. In one embodiment, 20 to 100 Units of _FV73 /kg ADAMTS13 is administered approximately once every 24 hours. In one embodiment, 20 to 100 Units of _FV73 /kg ADAMTS13 is administered approximately every 12 hours.

In one embodiment, the present disclosure provides a method of treating hereditary TTP in a mammal in need thereof, comprising subcutaneously administering a therapeutically effective amount of a composition comprising isolated ADAMTS13 to the mammal, wherein

- 20 045023 a therapeutically effective amount of ADAMTS13 contains from 40 to 200 units of FRETSVWF73 activity per kilogram of mammalian body weight ( _FV73 Units/kg). In a particular embodiment, the mammal is a human. In one embodiment, from 40 U _FV73 to 200 U _FV 7 ₃ /kg ADAMTS13 is administered approximately once a month. In one embodiment, 40 to 200 U _FV 7 ₃ /kg ADAMTS13 is administered approximately twice a month. In one embodiment, 40 to 200 U^ ₇₃ /kg ADAMTS13 is administered approximately once per week. In one embodiment, 40 to 200 U _FV73 /kg ADAMTS13 is administered approximately twice per week. In one embodiment, 40 to 200 Units of _FV73 /kg ADAMTS13 is administered approximately once every 48 hours. In one embodiment, 40 to 200 Units of _FV73 /kg ADAMTS13 is administered approximately once every 24 hours. In one embodiment, 40 to 200 Units of _FV73 /kg ADAMTS13 is administered approximately every 12 hours.

In one embodiment, the present disclosure provides a method of treating hereditary TTP in a mammal in need thereof, comprising subcutaneously administering a therapeutically effective amount of a composition comprising isolated ADAMTS13 to the mammal, wherein the therapeutically effective amount of ADAMTS13 and the dosing frequency are selected from options 1-1133 in Table. 2. In a particular embodiment, the mammal is a human.

Acquired TTP

In specific embodiments, the ADAMTS13 composition described herein is used for the treatment and prevention of acquired TTP. In acquired TTP, patients are characterized by low ADAMTS13 activity due to the formation of autoimmune antibodies against ADAMTS13. ADAMTS13 as part of immunocomplexes is inactivated, neutralized and/or removed from the patient’s bloodstream and plasma. Reduced ADAMTS13 activity leads to the accumulation of large uncleaved VWF multimers, which can spontaneously attach to platelets and lead to the formation of thrombi with a high content of platelets and VWF in the microvasculature. Like hereditary TTP, acquired TTP can also lead to neurological manifestations (eg, altered mental status, stroke, seizures, hemiplegia, paresthesia, visual disturbances, and aphasia), fatigue, and severe bleeding. If left untreated, acquired TTP can lead to death or long-term physiological impairment. Accordingly, patient compliance with ADAMTS13 is essential to prevent irreversible damage and possible fatal outcome. Thus, it is desirable to develop a subcutaneous formulation of ADAMTS13 and a method for subcutaneous delivery of ADAMTS13 to reduce complications and increase patient compliance with treatment as described above.

In one embodiment, the present disclosure provides a method of treating acquired TTP in a mammal in need thereof, comprising subcutaneously administering a therapeutically effective amount of a composition comprising isolated ADAMTS13 to the mammal, wherein the therapeutically effective amount of ADAMTS13 contains from 20 to 4000 units of FRETS-VWF73 activity per kilogram of body weight mammal (U _FV73 /kg). In a particular embodiment, the mammal is a human. In one embodiment, from 20 Units _{of FV73} to 4000 Units _{of FV73} /kg ADAMTS13 is administered approximately once per month. In one embodiment, 20 to 4000 U _FV73 /kg ADAMTS13 is administered approximately twice a month. In one embodiment, 20 to 4000 U _FV73 /kg ADAMTS13 is administered approximately once per week. In one embodiment, 20 to 4000 U _FV73 /kg ADAMTS13 is administered approximately twice per week. In one embodiment, 20 to 4000 Units of _FV73 /kg ADAMTS13 is administered approximately once every 48 hours. In one embodiment, 20 to 4000 Units of _FV73 /kg ADAMTS13 is administered approximately once every 24 hours. In one embodiment, 20 to 4000 Units of _FV73 /kg ADAMTS13 is administered approximately every 12 hours.

In one embodiment, the present disclosure provides a method of treating acquired TTP in a mammal in need thereof, comprising subcutaneously administering a therapeutically effective amount of a composition comprising isolated ADAMTS13 to the mammal, wherein the therapeutically effective amount of ADAMTS13 contains from 20 to 2000 units of FRETS-VWF73 activity per kilogram of body weight mammal (U _FV73 /kg). In a particular embodiment, the mammal is a human. In one embodiment, from 20 Units _{of FV73} to 2000 Units _{of FV73} /kg ADAMTS13 is administered approximately once per month. In one embodiment, 20 to 2000 U _FV73 /kg ADAMTS13 is administered approximately twice a month. In one embodiment, 20 to 2000 _FV73 Units/kg ADAMTS13 is administered approximately once per week. In one embodiment, 20 to 2000 U _FV73 /kg ADAMTS13 is administered approximately twice per week. In one embodiment, 20 to 2000 Units of _FV73 /kg ADAMTS13 is administered approximately once every 48 hours. In one embodiment, 20 to 2000 Units of _FV73 /kg ADAMTS13 is administered approximately once every 24 hours. In one embodiment, 20 to 2000 Units of _FV73 /kg ADAMTS13 is administered approximately every 12 hours.

In one embodiment, the present disclosure provides a method of treating acquired TTP in a mammal in need thereof, comprising subcutaneously administering a therapeutically effective amount of a composition comprising isolated ADAMTS13 to the mammal, wherein the therapeutically effective amount of ADAMTS13 contains from 20 to 1000 units of FRETS-VWF73 activity per kilogram of body weight mammal (U _FV73 /kg). In a particular embodiment, a mammal

- 21 045023 is human. In one embodiment, 20 U^ ₇₃ to 1000 U^ ₇₃ /kg ADAMTS13 is administered approximately once per month. In one embodiment, 20 to 1000 U _FV73 /kg ADAMTS13 is administered approximately twice a month. In one embodiment, 20 to 1000 U _FV73 /kg ADAMTS13 is administered approximately once per week. In one embodiment, 20 to 1000 U _FV73 /kg ADAMTS13 is administered approximately twice per week. In one embodiment, 20 to 1000 Units of _FV73 /kg ADAMTS13 is administered approximately once every 48 hours. In one embodiment, 20 to 1000 Units of _FV73 /kg ADAMTS13 is administered approximately once every 24 hours. In one embodiment, 20 to 1000 Units of _FV73 /kg ADAMTS13 is administered approximately every 12 hours.

In one embodiment, the present disclosure provides a method of treating acquired TTP in a mammal in need thereof, comprising subcutaneously administering a therapeutically effective amount of a composition comprising isolated ADAMTS13 to the mammal, wherein the therapeutically effective amount of ADAMTS13 contains from 20 to 500 units of FRETS-VWF73 activity per kilogram of body weight mammal (U _FV73 /kg). In a particular embodiment, the mammal is a human. In one embodiment, from 20 Units _{of FV73} to 500 Units _{of FV73} /kg ADAMTS13 is administered approximately once per month. In one embodiment, 20 to 500 U _FV73 /kg ADAMTS13 is administered approximately twice a month. In one embodiment, 20 to 500 U _FV73 /kg ADAMTS13 is administered approximately once per week. In one embodiment, 20 to 500 U _FV73 /kg ADAMTS13 is administered approximately twice per week. In one embodiment, 20 to 500 Eg _FV73 /kg ADAMTS13 is administered approximately once every 48 hours. In one embodiment, 20 to 500 Eg _FV7 w/kg ADAMTS13 is administered approximately once every 24 hours. In one embodiment, 20 to 500 Eg FV73/kg ADAMTS13 is administered approximately once every 24 hours. _FV73 /kg ADAMTS13 is administered approximately every 12 hours.

In one embodiment, the present disclosure provides a method of treating acquired TTP in a mammal in need thereof, comprising subcutaneously administering a therapeutically effective amount of a composition comprising isolated ADAMTS13 to the mammal, wherein the therapeutically effective amount of ADAMTS13 contains from 20 to 200 units of FRETS-VWF73 activity per kilogram of body weight mammal (Eg _FV7 h/kg). In a particular embodiment, the mammal is a human. In one embodiment, from 20 Units _{of FV73} to 200 Units _{of FV73} /kg ADAMTS13 is administered approximately once per month. In one embodiment, 20 to 200 U _FV73 /kg ADAMTS13 is administered approximately twice a month. In one embodiment, 20 to 200 U _FV73 /kg ADAMTS13 is administered approximately once per week. In one embodiment, 20 to 200 U _FV73 /kg ADAMTS13 is administered approximately twice per week. In one embodiment, 20 to 200 Units of _FV73 /kg ADAMTS13 is administered approximately once every 48 hours. In one embodiment, 20 to 200 Units of _FV73 /kg ADAMTS13 is administered approximately once every 24 hours. In one embodiment, 20 to 200 Units of _FV73 /kg ADAMTS13 is administered approximately every 12 hours.

In one embodiment, the present disclosure provides a method of treating acquired TTP in a mammal in need thereof, comprising subcutaneously administering a therapeutically effective amount of a composition comprising isolated ADAMTS13 to the mammal, wherein the therapeutically effective amount of ADAMTS13 contains from 20 to 100 units of FRETS-VWF73 activity per kilogram of body weight mammal (Eg _FV7 h/kg). In a particular embodiment, the mammal is a human. In one embodiment, from 20 Units _{of FV73} to 100 Units _{of FV73} /kg ADAMTS13 is administered approximately once per month. In one embodiment, 20 to 100 U _FV73 /kg ADAMTS13 is administered approximately twice a month. In one embodiment, 20 to 100 U _FV73 /kg ADAMTS13 is administered approximately once per week. In one embodiment, 20 to 100 U _FV73 /kg ADAMTS13 is administered approximately twice per week. In one embodiment, 20 to 100 Units of _FV73 /kg ADAMTS13 is administered approximately once every 48 hours. In one embodiment, 20 to 100 Units of _FV73 /kg ADAMTS13 is administered approximately once every 24 hours. In one embodiment, 20 to 100 Units of _FV73 /kg ADAMTS13 is administered approximately every 12 hours.

In one embodiment, the present disclosure provides a method of treating acquired TTP in a mammal in need thereof, comprising subcutaneously administering a therapeutically effective amount of a composition comprising isolated ADAMTS13 to the mammal, wherein the therapeutically effective amount of ADAMTS13 contains from 40 to 200 units of FRETS-VWF73 activity per kilogram of body weight mammal (Eg _FV7 h/kg). In a particular embodiment, the mammal is a human. In one embodiment, from 40 Units of _FV73 to 200 Units _{of FV73} /kg ADAMTS13 is administered approximately once per month. In one embodiment, 40 to 200 U _FV73 /kg ADAMTS13 is administered approximately twice a month. In one embodiment, 40 to 200 U _FV73 /kg ADAMTS13 is administered approximately once per week. In one embodiment, 40 to 200 U _FV73 /kg ADAMTS13 is administered approximately twice per week. In one embodiment, 40 to 200 Units of _FV73 /kg ADAMTS13 is administered approximately once every 48 hours. In one embodiment, 40 to 200 Units of _FV73 /kg ADAMTS13 is administered approximately once every 24 hours. In one embodiment, 40 to 200 Units of _FV73 /kg ADAMTS13 is administered approximately every 12 hours.

In one embodiment, the present disclosure provides a method of treating acquired TTP in a mammal in need thereof, comprising subcutaneously administering a therapeutically effective amount of a composition comprising isolated ADAMTS13 to the mammal, wherein the therapeutically effective amount of ADAMTS13 and the dosing frequency are selected from options 1-1133 in table 2. In a particular embodiment, the mammal is a human.

Myocardial infarction and reperfusion injury

In specific embodiments, the ADAMTS13 composition described herein is used for the treatment and prevention of myocardial infarction. In specific embodiments, the ADAMTS13 composition described herein is used for the treatment and prevention of ischemic reperfusion injury. Reperfusion is the restoration of blood supply to tissues that have undergone ischemia due to a decrease in blood supply. Reperfusion is a procedure for the treatment of infarction (eg, myocardial infarction and cerebral infarction) or other ischemia, allowing the restoration of viable ischemic tissue, thereby limiting further necrosis. However, reperfusion itself can lead to further damage to ischemic tissue, causing reperfusion injury. For example, acute myocardial infarction (AMI) is caused by thrombotic occlusion of a coronary artery. In addition to the direct damage that occurs during loss of blood flow, ischemia/reperfusion injury includes tissue damage that occurs after blood flow is restored due to reperfusion.

Additionally, ADAMTS13 has been reported to have anti-inflammatory effects that prevent or reduce secondary injury during ischemic reperfusion. De Meyer et al. (Protective anti-inflammatory effect of ADAMTS13 on myocardial ischemia/reperfusion injury in mice, Blood, 2012, 120(26):5217-5223). As described by De Meyer et al., VWF and ADAMTS13 are involved in platelet adhesion and thrombus formation, as ADAMTS13 cleaves the most thrombogenic VWF multimers into smaller and less hemostatically active VWF fragments. De Meyer et al. also describe the role of ADAMTS in suppressing inflammatory responses. Additionally, ADAMTS13 has been shown to reduce thrombosis and inflammation (eg, atherosclerosis). Chauhan et al. (ADAMTS13: a new link between thrombosis and inflammation, J Exp Med., 2008, 205:2065-2074); Chauhan et al. (Systemic antithrombotic effects of ADAMTS13, J Exp Med., 2006, 203:767-776; Gandhi et al. (ADAMTS13 reduces vascular inflammation and the development of early atherosclerosis in mice, Blood, 2012, 119 (10):2385-2391 De Meyer et al suggest that ADAMTS13 prevents excess VWF-mediated recruitment of platelets and leukocytes in ischemic myocardium by cleaving VWF.Based on this hypothesis, De Meyer et al showed that myocardial infiltration of neutrophils in animals with induced myocardial infarction was ninefold times lower when animals were treated with ADAMTS13. Accordingly, De Meyer et al. showed that ADAMTS13 reduces inflammatory responses in ischemic myocardium. This reduction in inflammation also reduces reperfusion injury by preventing leukocyte infiltration and damage caused by them. Thus, it is desirable to administer ADAMTS13 formulations subcutaneously patients to avoid inflammation leading to tissue damage during infarction (eg, myocardial infarction and cerebral infarction) and reperfusion, since subcutaneous administration is easier and faster than the administration of intravenous compositions, which are typically administered by a healthcare professional.

In some embodiments, the pharmaceutical composition for treating infarction and/or reperfusion injury is administered immediately upon detection of myocardial infarction, e.g., within 5 minutes, 10 minutes, 15 minutes, 20 minutes, 25 minutes, 30 minutes, 35 minutes, 40 minutes, 45 min, 50 min, 55 min, 60 min, 90 min, 110 min, 120 min, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 9 hours, 10 hours, 11 hours, 12 hours, 13 hours, 14 hours, 15 hours, 16 hours, 17 hours, 18 hours, 19 hours, 20 hours, 21 hours, 22 hours, 23 hours, 24 hours, 25 hours or more, or any combination thereof. Accordingly, it is important to have a pharmaceutical composition that can be administered quickly and easily.

In one embodiment, the present disclosure provides a method of treating myocardial infarction in a mammal in need thereof, comprising subcutaneously administering a therapeutically effective amount of a composition comprising isolated ADAMTS13 to the mammal, wherein the therapeutically effective amount of ADAMTS13 contains from 20 to 4000 units of FRETS-VWF73 activity per kilogram of body weight mammal (U _FV7 3/kg). In a particular embodiment, the mammal is a human. In one embodiment, from 20 U _{of FV73} to 4000 U of _FV73 /kg ADAMTS13 is administered within 10 minutes of detection of myocardial infarction in the mammal. In one embodiment, 20 to 4000 U _FV73 /kg ADAMTS13 is administered within 30 minutes of detection of myocardial infarction in the mammal. In one embodiment, 20 to 4000 U _FV73 /kg ADAMTS13 is administered within 60 minutes of detection of myocardial infarction in the mammal. In one embodiment, 20 to 4000 U _FV73 /kg ADAMTS13 is administered within 4 hours of detection of myocardial infarction in the mammal. In one embodiment, 20 to 4000 U _FV73 /kg ADAMTS13 is administered within 12 hours of detection of myocardial infarction in the mammal. In one embodiment, 20 to 4000 U _FV73 /kg ADAMTS13 is administered within 24 hours of detection of myocardial infarction in the mammal.

In one embodiment, the present disclosure provides a method of treating myocardial infarction in a mammal in need thereof, comprising subcutaneously administering a therapeutically effective amount of a composition comprising isolated ADAMTS13 to the mammal, wherein the therapeutically effective amount of ADAMTS13 contains from 20 to 2000 units of FRETS-VWF73 activity. per kilogram of mammal body weight (U _FV73 /kg). In a particular embodiment, the mammal is a human. In one embodiment, from 20 _FV73 Units to 2000 _FV73 Units/kg ADAMTS13 is administered within 10 minutes of detection of myocardial infarction in the mammal. In one embodiment, 20 to 2000 U _FV 7 ₃ /kg ADAMTS13 is administered within 30 minutes of detection of myocardial infarction in the mammal. In one embodiment, 20 to 2000 U^ ₇₃ /kg ADAMTS13 is administered within 60 minutes of detection of myocardial infarction in the mammal. In one embodiment, 20 to 2000 U _FV73 /kg ADAMTS13 is administered within 4 hours of detection of myocardial infarction in the mammal. In one embodiment, 20 to 2000 U _FV73 /kg ADAMTS13 is administered within 12 hours of detection of myocardial infarction in the mammal. In one embodiment, 20 to 2000 U _FV73 /kg ADAMTS13 is administered within 24 hours of detection of myocardial infarction in the mammal.

In one embodiment, the present disclosure provides a method of treating myocardial infarction in a mammal in need thereof, comprising subcutaneously administering a therapeutically effective amount of a composition comprising isolated ADAMTS13 to the mammal, wherein the therapeutically effective amount of ADAMTS13 contains from 20 to 1000 units of FRETS-VWF73 activity per kilogram of body weight mammal (U _FV73 /kg). In a particular embodiment, the mammal is a human. In one embodiment, from 20 Units _{of FV73} to 1000 Units _{of FV73} /kg ADAMTS13 is administered within 10 minutes of detection of myocardial infarction in the mammal. In one embodiment, 20 to 1000 U _FV73 /kg ADAMTS13 is administered within 30 minutes of detection of myocardial infarction in the mammal. In one embodiment, 20 to 1000 U _FV73 /kg ADAMTS13 is administered within 60 minutes of detection of myocardial infarction in the mammal. In one embodiment, 20 to 1000 U _FV73 /kg ADAMTS13 is administered within 4 hours of detection of myocardial infarction in the mammal. In one embodiment, 20 to 1000 U _FV73 /kg ADAMTS13 is administered within 12 hours of detection of myocardial infarction in the mammal. In one embodiment, 20 to 1000 U _FV73 /kg ADAMTS13 is administered within 24 hours of detection of myocardial infarction in the mammal.

In one embodiment, the present disclosure provides a method of treating myocardial infarction in a mammal in need thereof, comprising subcutaneously administering a therapeutically effective amount of a composition comprising isolated ADAMTS13 to the mammal, wherein the therapeutically effective amount of ADAMTS13 contains from 20 to 500 units of FRETS-VWF73 activity per kilogram of body weight mammal (U _FV73 /kg). In a particular embodiment, the mammal is a human. In one embodiment, from 20 U _{of FV73} to 500 U of _FV73 /kg ADAMTS13 is administered within 10 minutes of detection of myocardial infarction in the mammal. In one embodiment, 20 to 500 U _{of FV73} /kg ADAMTS13 is administered within 30 minutes of detection of myocardial infarction in the mammal. In one embodiment, 20 to 500 U _FV73 /kg ADAMTS13 is administered within 60 minutes of detection of myocardial infarction in the mammal. In one embodiment, 20 to 500 U _FV73 /kg ADAMTS13 is administered within 4 hours of detection of myocardial infarction in the mammal. In one embodiment, 20 to 500 U _FV73 /kg ADAMTS13 is administered within 12 hours of detection of myocardial infarction in the mammal. In one embodiment, 20 to 500 U _FV73 /kg ADAMTS13 is administered within 24 hours of detection of myocardial infarction in the mammal.

In one embodiment, the present disclosure provides a method of treating myocardial infarction in a mammal in need thereof, comprising subcutaneously administering a therapeutically effective amount of a composition comprising isolated ADAMTS13 to the mammal, wherein the therapeutically effective amount of ADAMTS13 contains from 20 to 200 units of FRETS-VWF73 activity per kilogram of body weight mammal (U _FV73 /kg). In a particular embodiment, the mammal is a human. In one embodiment, from 20 Units _{of FV73} to 200 Units _{of FV73} /kg ADAMTS13 is administered within 10 minutes of detection of myocardial infarction in the mammal. In one embodiment, 20 to 200 U _FV73 /kg ADAMTS13 is administered within 30 minutes of detection of myocardial infarction in the mammal. In one embodiment, 20 to 200 U _FV73 /kg ADAMTS13 is administered within 60 minutes of detection of myocardial infarction in the mammal. In one embodiment, 20 to 200 U _FV73 /kg ADAMTS13 is administered within 4 hours of detection of myocardial infarction in the mammal. In one embodiment, 20 to 200 U _FV73 /kg ADAMTS13 is administered within 12 hours of detection of myocardial infarction in the mammal. In one embodiment, 20 to 200 U _FV73 /kg ADAMTS13 is administered within 24 hours of detection of myocardial infarction in the mammal.

In one embodiment, the present disclosure provides a method of treating myocardial infarction in a mammal in need thereof, comprising subcutaneously administering a therapeutically effective amount of a composition comprising isolated ADAMTS13 to the mammal, wherein the therapeutically effective amount of ADAMTS13 contains from 20 to 100 units of FRETS-VWF73 activity per

- 24,045,023 kilograms of mammal body weight ( _FV73 units/kg). In a particular embodiment, the mammal is a human. In one embodiment, 20 U _FV73 to 100 U F _V7 3/kg ADAMTS13 is administered within 10 minutes of detection of myocardial infarction in the mammal. In one embodiment, 20 to 100 U _FV7 g/kg ADAMTS13 is administered within 30 minutes of detection of myocardial infarction in the mammal. In one embodiment, 20 to 100 U _FV73 /kg ADAMTS13 is administered within 60 minutes of detection of myocardial infarction in the mammal. In one embodiment, 20 to 100 U _FV73 /kg ADAMTS13 is administered within 4 hours of detection of myocardial infarction in the mammal. In one embodiment, 20 to 100 U _FV73 /kg ADAMTS13 is administered within 12 hours of detection of myocardial infarction in the mammal. In one embodiment, 20 to 100 U _FV73 /kg ADAMTS13 is administered within 24 hours of detection of myocardial infarction in the mammal.

In one embodiment, the present disclosure provides a method of treating myocardial infarction in a mammal in need thereof, comprising subcutaneously administering a therapeutically effective amount of a composition comprising isolated ADAMTS13 to the mammal, wherein the therapeutically effective amount of ADAMTS13 contains from 40 to 200 units of FRETS-VWF73 activity per kilogram of body weight mammal (U _FV73 /kg). In a particular embodiment, the mammal is a human. In one embodiment, 40 U of _FV73 to 200 U of _FV73 /kg ADAMTS13 is administered within 10 minutes of detection of myocardial infarction in the mammal. In one embodiment, 40 to 200 U _FV73 /kg ADAMTS13 is administered within 30 minutes of detection of myocardial infarction in the mammal. In one embodiment, 40 to 200 U _FV73 /kg ADAMTS13 is administered within 60 minutes of detection of myocardial infarction in the mammal. In one embodiment, 40 to 200 U _FV73 /kg ADAMTS13 is administered within 4 hours of detection of myocardial infarction in the mammal. In one embodiment, 40 to 200 U _FV73 /kg ADAMTS13 is administered within 12 hours of detection of myocardial infarction in the mammal. In one embodiment, 40 to 200 U _FV73 /kg ADAMTS13 is administered within 24 hours of detection of myocardial infarction in the mammal.

Cerebral infarction

In one embodiment, the ADAMTS13 formulation described herein is used for the treatment and/or prevention of cerebral infarction. A cerebral infarction, commonly called a stroke, occurs when blood flow to part of the brain is blocked. A cerebral infarction can occur, for example, when a blood vessel that supplies blood to the brain is blocked by a blood clot. Cerebral infarction can also occur due to blunt force trauma and mechanical trauma. It can be caused by a blood clot in an artery in the brain (thrombotic stroke) or a blood clot from another part of the body that travels to the brain (embolic stroke). Accordingly, in some embodiments of the present invention there is provided a method of improving recovery (or reducing damage) of sensory and/or motor function in a patient after a cerebral infarction, comprising the step of administering to said individual a pharmaceutical composition containing a therapeutically effective amount of ADAMTS13 protein or a biologically active derivative thereof, thereby thereby improving recovery (or reducing damage) to sensory and/or motor function in an individual following cerebral infarction.

In some embodiments, the pharmaceutical composition is administered immediately after detection of cerebral infarction, for example, within 5 minutes, 10 minutes, 15 minutes, 20 minutes, 25 minutes, 30 minutes, 35 minutes, 40 minutes, 45 minutes, 50 minutes, 55 minutes, 60 min, 90 min, 110 min, 120 min, 3 hrs, 4 hrs, 5 hrs, 6 hrs, 7 hrs, 8 hrs, 9 hrs, 10 hrs, 11 hrs, 12 hrs, 13 hrs, 14 hrs, 15 hrs , 16 hours, 17 hours, 18 hours, 19 hours, 20 hours, 21 hours, 22 hours, 23 hours, 24 hours, 25 hours or more, or any combination thereof. Accordingly, it is important to have a pharmaceutical composition that can be administered quickly and easily.

One embodiment of the present disclosure provides a method of treating cerebral infarction in a mammal in need thereof, comprising subcutaneously administering a therapeutically effective amount of a composition comprising isolated ADAMTS13 to the mammal, wherein the therapeutically effective amount of ADAMTS13 contains from 20 to 4000 units of FRETSVWF73 activity per kilogram of body weight of the mammal ( Units _FV73 /kg). In a particular embodiment, the mammal is a human. In one embodiment, from 20 U _{of FV73} to 4000 U of _FV73 /kg ADAMTS13 is administered within 10 minutes of detection of cerebral infarction in the mammal. In one embodiment, 20 to 4000 U _FV73 /kg ADAMTS13 is administered within 30 minutes of detection of cerebral infarction in the mammal. In one embodiment, 20 to 4000 U _FV73 /kg ADAMTS13 is administered within 60 minutes of detection of cerebral infarction in the mammal. In one embodiment, 20 to 4000 U _FV73 /kg ADAMTS13 is administered within 4 hours of detection of cerebral infarction in the mammal. In one embodiment, 20 to 4000 U _FV73 /kg ADAMTS13 is administered within 12 hours of detection of cerebral infarction in the mammal. In one embodiment, 20 to 4000 U _FV73 /kg ADAMTS13 is administered within 24 hours of detection of cerebral infarction in the mammal.

In one embodiment of the present disclosure, a method for treating cerebral infarction is provided.

- 25 045023 farct in a mammal in need thereof, comprising subcutaneous administration of a therapeutically effective amount of a composition containing isolated ADAMTS13 to the mammal, wherein the therapeutically effective amount of ADAMTS13 contains from 20 to 2000 units of FRETSVWF73 activity per kilogram of mammalian body weight ( _FV73 Units/kg). In a particular embodiment, the mammal is a human. In one embodiment, from 20 U _FV73 to 2000 U _FV 7 ₃ /kg ADAMTS13 is administered within 10 minutes of detection of cerebral infarction in the mammal. In one embodiment, 20 to 2000 U _FV 7 ₃ /kg ADAMTS13 is administered within 30 minutes of detection of cerebral infarction in the mammal. In one embodiment, 20 to 2000 U _FV 7 ₃ /kg ADAMTS13 is administered within 60 minutes of detection of cerebral infarction in the mammal. In one embodiment, 20 to 2000 U _FV 7 ₃ /kg ADAMTS13 is administered within 4 hours of detection of cerebral infarction in the mammal. In one embodiment, 20 to 2000 U _FV 7 ₃ /kg ADAMTS13 is administered within 12 hours of detection of cerebral infarction in the mammal. In one embodiment, 20 to 2000 U _FV 7 ₃ /kg ADAMTS13 is administered within 24 hours of detection of cerebral infarction in the mammal.

In one embodiment, the present disclosure provides a method of treating cerebral infarction in a mammal in need thereof, comprising subcutaneously administering a therapeutically effective amount of a composition comprising isolated ADAMTS13 to the mammal, wherein the therapeutically effective amount of ADAMTS13 contains from 20 to 1000 units of FRETSVWF73 activity per kilogram of body weight of the mammal ( U^ ₇₃ /kg). In a particular embodiment, the mammal is a human. In one embodiment, from 20 Units _{of FV73} to 1000 Units _{of FV73} /kg ADAMTS13 is administered within 10 minutes of detection of cerebral infarction in the mammal. In one embodiment, 20 to 1000 U _FV73 /kg ADAMTS13 is administered within 30 minutes of detection of cerebral infarction in the mammal. In one embodiment, 20 to 1000 U _FV73 /kg ADAMTS13 is administered within 60 minutes of detection of cerebral infarction in the mammal. In one embodiment, 20 to 1000 U _FV73 /kg ADAMTS13 is administered within 4 hours of detection of cerebral infarction in the mammal. In one embodiment, 20 to 1000 U _FV73 /kg ADAMTS13 is administered within 12 hours of detection of cerebral infarction in the mammal. In one embodiment, 20 to 1000 Eg _FV7 g/kg ADAMTS13 is administered within 24 hours of detection of cerebral infarction in the mammal.

In one embodiment, the present disclosure provides a method of treating cerebral infarction in a mammal in need thereof, comprising subcutaneously administering a therapeutically effective amount of a composition comprising isolated ADAMTS13 to the mammal, wherein the therapeutically effective amount of ADAMTS13 contains from 20 to 500 units of FRETSVWF73 activity per kilogram of body weight of the mammal ( Units _FV73 /kg). In a particular embodiment, the mammal is a human. In one embodiment, from 20 U _{of FV73} to 500 U of _FV73 /kg ADAMTS13 is administered within 10 minutes of detection of cerebral infarction in the mammal. In one embodiment, 20 to 500 U _FV73 /kg ADAMTS13 is administered within 30 minutes of detection of cerebral infarction in the mammal. In one embodiment, 20 to 500 U _FV73 /kg ADAMTS13 is administered within 60 minutes of detection of cerebral infarction in the mammal. In one embodiment, 20 to 500 U _FV73 /kg ADAMTS13 is administered within 4 hours of detection of cerebral infarction in the mammal. In one embodiment, 20 to 500 U _FV73 /kg ADAMTS13 is administered within 12 hours of detection of cerebral infarction in the mammal. In one embodiment, 20 to 500 U _FV73 /kg ADAMTS13 is administered within 24 hours of detection of cerebral infarction in the mammal.

In one embodiment, the present disclosure provides a method of treating cerebral infarction in a mammal in need thereof, comprising subcutaneously administering a therapeutically effective amount of a composition comprising isolated ADAMTS13 to the mammal, wherein the therapeutically effective amount of ADAMTS13 contains from 20 to 200 units of FRETSVWF73 activity per kilogram of body weight of the mammal ( Units _FV73 /kg). In a particular embodiment, the mammal is a human. In one embodiment, from 20 Units _{of FV73} to 200 Units _{of FV73} /kg ADAMTS13 is administered within 10 minutes of detection of cerebral infarction in the mammal. In one embodiment, 20 to 200 Eg _FV7 g/kg ADAMTS13 is administered within 30 minutes of detection of cerebral infarction in the mammal. In one embodiment, 20 to 200 U _FV73 /kg ADAMTS13 is administered within 60 minutes of detection of cerebral infarction in the mammal. In one embodiment, 20 to 200 U _FV73 /kg ADAMTS13 is administered within 4 hours of detection of cerebral infarction in the mammal. In one embodiment, 20 to 200 U _FV73 /kg ADAMTS13 is administered within 12 hours of detection of cerebral infarction in the mammal. In one embodiment, 20 to 200 U _FV73 /kg ADAMTS13 is administered within 24 hours of detection of cerebral infarction in the mammal.

In one embodiment, the present disclosure provides a method of treating cerebral infarction in a mammal in need thereof, comprising subcutaneously administering a therapeutically effective amount of a composition comprising isolated ADAMTS13 to the mammal, wherein

- 26 045023 a raphetically effective amount of ADAMTS13 contains from 20 to 100 units of FRETSVWF73 activity per kilogram of mammalian body weight ( _FV73 Units/kg). In a particular embodiment, the mammal is a human. In one embodiment, from 20 U _FV 7 ₃ to 100 U _FV 7 ₃ /kg ADAMTS13 is administered within 10 minutes of detection of cerebral infarction in the mammal. In one embodiment, 20 to 100 U _FV73 /kg ADAMTS13 is administered within 30 minutes of detection of cerebral infarction in the mammal. In one embodiment, 20 to 100 U _FV73 /kg ADAMTS13 is administered within 60 minutes of detection of cerebral infarction in the mammal. In one embodiment, 20 to 100 U _FV73 /kg ADAMTS13 is administered within 4 hours of detection of cerebral infarction in the mammal. In one embodiment, 20 to 100 U _FV73 /kg ADAMTS13 is administered within 12 hours of detection of cerebral infarction in the mammal. In one embodiment, 20 to 100 U _FV73 /kg ADAMTS13 is administered within 24 hours of detection of cerebral infarction in the mammal.

In one embodiment, the present disclosure provides a method of treating cerebral infarction in a mammal in need thereof, comprising subcutaneously administering a therapeutically effective amount of a composition comprising isolated ADAMTS13 to the mammal, wherein the therapeutically effective amount of ADAMTS13 contains from 40 to 200 units of FRETSVWF73 activity per kilogram of body weight of the mammal ( Units _FV73 /kg). In a particular embodiment, the mammal is a human. In one embodiment, from 40 U _FV73 to 200 U _FV73 /kg ADAMTS13 is administered within 10 minutes of detection of cerebral infarction in the mammal. In one embodiment, 40 to 200 U _FV73 /kg ADAMTS13 is administered within 30 minutes of detection of cerebral infarction in the mammal. In one embodiment, 40 to 200 U _FV73 /kg ADAMTS13 is administered within 60 minutes of detection of cerebral infarction in the mammal. In one embodiment, 40 to 200 U _FV73 /kg ADAMTS13 is administered within 4 hours of detection of cerebral infarction in the mammal. In one embodiment, 40 to 200 U _FV73 /kg ADAMTS13 is administered within 12 hours of detection of cerebral infarction in the mammal. In one embodiment, 40 to 200 U _FV73 /kg ADAMTS13 is administered within 24 hours of detection of cerebral infarction in the mammal.

Deep vein thrombosis

In one embodiment, the ADAMTS13 formulation described herein is used for the treatment and/or prevention of deep vein thrombosis (DVT). DVT is a blood clot that forms in a vein inside the body. Although most deep vein blood clots occur in the calf or thigh, they can form throughout the body. DVT is particularly dangerous because the clot can break off and travel through the bloodstream (embolus), for example to the heart, lungs or brain. This embolism can cause organ damage and death. Accordingly, as described above, ADAMTS13 formulations can be used to treat DVT and resulting embolism. In addition, since DVT can develop rapidly and cause damage, it is important to have a pharmaceutical composition that can be administered quickly and easily. Therefore, it is desirable to develop a subcutaneous formulation of ADAMTS13 and a method for subcutaneous delivery of ADAMTS13.

In some embodiments, the ADAMTS13 pharmaceutical composition is administered immediately upon detection of deep vein thrombosis, for example, within 5 minutes, 10 minutes, 15 minutes, 20 minutes, 25 minutes, 30 minutes, 35 minutes, 40 minutes, 45 minutes, 50 minutes, 55 min, 60 min, 90 min, 110 min, 120 min, 3 hrs, 4 hrs, 5 hrs, 6 hrs, 7 hrs, 8 hrs, 9 hrs, 10 hrs, 11 hrs, 12 hrs, 13 hrs, 14 hrs, 15 hours, 16 hours, 17 hours, 18 hours, 19 hours, 20 hours, 21 hours, 22 hours, 23 hours, 24 hours, 25 hours or more, or any combination thereof. Accordingly, it is important to have a pharmaceutical composition that can be administered quickly and easily.

In one embodiment, the present disclosure provides a method of treating deep vein thrombosis in a mammal in need thereof, comprising subcutaneously administering a therapeutically effective amount of a composition comprising isolated ADAMTS13 to the mammal, wherein the therapeutically effective amount of ADAMTS13 contains from 20 to 4000 units of FRETS-VWF73 activity per kilogram of body weight body of a mammal (U _FV73 /kg). In a particular embodiment, the mammal is a human. In one embodiment, from 20 Units _{of FV73} to 4000 Units _{of FV73} /kg ADAMTS13 is administered within 10 minutes of detection of deep vein thrombosis in a mammal. In one embodiment, 20 to 4000 U of _FV73 /kg ADAMTS13 is administered within 30 minutes of detection of deep vein thrombosis in a mammal. In one embodiment, 20 to 4000 U of _FV73 /kg ADAMTS13 is administered within 60 minutes of detection of deep vein thrombosis in a mammal. In one embodiment, 20 to 4000 U _FV73 /kg ADAMTS13 is administered within 4 hours of detection of deep vein thrombosis in a mammal. In one embodiment, 20 to 4000 U _FV73 /kg ADAMTS13 is administered within 12 hours of detection of deep vein thrombosis in a mammal. In one embodiment, 20 to 4000 U _FV73 /kg ADAMTS13 is administered within 24 hours of detection of deep vein thrombosis in a mammal.

In one embodiment, the present disclosure provides a method of treating deep vein thrombosis in a mammal in need thereof, comprising subcutaneously administering a therapeutically effective amount of a composition comprising isolated ADAMTS13 to the mammal, wherein the treatment

- 27 045023 a highly effective amount of ADAMTS13 contains from 20 to 2000 units of FRETS-VWF73 activity per kilogram of mammalian body weight ( _FV73 units/kg). In a particular embodiment, the mammal is a human. In one embodiment, from 20 U _{of FV73} to 2000 U of _FV73 /kg ADAMTS13 is administered within 10 minutes of detection of deep vein thrombosis in a mammal. In one embodiment, 20 to 2000 U _FV73 /kg ADAMTS13 is administered within 30 minutes of detection of deep vein thrombosis in a mammal. In one embodiment, 20 to 2000 U _FV73 /kg ADAMTS13 is administered within 60 minutes of detection of deep vein thrombosis in a mammal. In one embodiment, 20 to 2000 U _FV73 /kg ADAMTS13 is administered within 4 hours of detection of deep vein thrombosis in the mammal. In one embodiment, 20 to 2000 U of _FV73 /kg ADAMTS13 is administered within 12 hours of detection of deep vein thrombosis in the mammal. In one embodiment, 20 to 2000 U _FV73 /kg ADAMTS13 is administered within 24 hours of detection of deep vein thrombosis in a mammal.

In one embodiment, the present disclosure provides a method of treating deep vein thrombosis in a mammal in need thereof, comprising subcutaneously administering a therapeutically effective amount of a composition comprising isolated ADAMTS13 to the mammal, wherein the therapeutically effective amount of ADAMTS13 contains from 20 to 1000 units of FRETS-VWF73 activity per kilogram of body weight body of a mammal (U _FV73 /kg). In a particular embodiment, the mammal is a human. In one embodiment, from 20 U _{of FV73} to 1000 U of _FV73 /kg ADAMTS13 is administered within 10 minutes of detection of deep vein thrombosis in the mammal. In one embodiment, 20 to 1000 Units of _FV73 /kg ADAMTS13 is administered within 30 minutes of detection of deep vein thrombosis in a mammal. In one embodiment, 20 to 1000 Units of _FV73 /kg ADAMTS13 is administered within 60 minutes of detection of deep vein thrombosis in a mammal. In one embodiment, 20 to 1000 U of _FV73 /kg ADAMTS13 is administered within 4 hours of detection of deep vein thrombosis in the mammal. In one embodiment, 20 to 1000 U _FV73 /kg ADAMTS13 is administered within 12 hours of detection of deep vein thrombosis in the mammal. In one embodiment, 20 to 1000 U _FV73 /kg ADAMTS13 is administered within 24 hours of detection of deep vein thrombosis in a mammal.

In one embodiment, the present disclosure provides a method of treating deep vein thrombosis in a mammal in need thereof, comprising subcutaneously administering a therapeutically effective amount of a composition comprising isolated ADAMTS13 to the mammal, wherein the therapeutically effective amount of ADAMTS13 contains from 20 to 500 units of FRETS-VWF73 activity per kilogram of body weight body of a mammal (U _FV73 /kg). In a particular embodiment, the mammal is a human. In one embodiment, from 20 U _{of FV73} to 500 U of _FV73 /kg ADAMTS13 is administered within 10 minutes of detection of deep vein thrombosis in a mammal. In one embodiment, 20 to 500 U of _FV73 /kg ADAMTS13 is administered within 30 minutes of detection of deep vein thrombosis in a mammal. In one embodiment, 20 to 500 U _{of FV73} /kg ADAMTS13 is administered within 60 minutes of detection of deep vein thrombosis in a mammal. In one embodiment, 20 to 500 U _FV73 /kg ADAMTS13 is administered within 4 hours of detection of deep vein thrombosis in a mammal. In one embodiment, 20 to 500 U _FV73 /kg ADAMTS13 is administered within 12 hours of detection of deep vein thrombosis in a mammal. In one embodiment, 20 to 500 U _{of FV73} /kg ADAMTS13 is administered within 24 hours of detection of deep vein thrombosis in a mammal.

In one embodiment, the present disclosure provides a method of treating deep vein thrombosis in a mammal in need thereof, comprising subcutaneously administering a therapeutically effective amount of a composition comprising isolated ADAMTS13 to the mammal, wherein the therapeutically effective amount of ADAMTS13 contains from 20 to 200 units of FRETS-VWF73 activity per kilogram of body weight body of a mammal (U _FV73 /kg). In a particular embodiment, the mammal is a human. In one embodiment, 20 U of _FV73 to 200 U _{of FV73} /kg ADAMTS13 is administered within 10 minutes of detection of deep vein thrombosis in the mammal. In one embodiment, 20 to 200 U _FV73 /kg ADAMTS13 is administered within 30 minutes of detection of deep vein thrombosis in a mammal. In one embodiment, 20 to 200 U of _FV73 /kg ADAMTS13 is administered within 60 minutes of detection of deep vein thrombosis in a mammal. In one embodiment, 20 to 200 U _{of FV73} /kg ADAMTS13 is administered within 4 hours of detection of deep vein thrombosis in the mammal. In one embodiment, 20 to 200 U _FV73 /kg ADAMTS13 is administered within 12 hours of detection of deep vein thrombosis in the mammal. In one embodiment, 20 to 200 U _FV73 /kg ADAMTS13 is administered within 24 hours of detection of deep vein thrombosis in a mammal.

In one embodiment, the present disclosure provides a method of treating deep vein thrombosis in a mammal in need thereof, comprising subcutaneously administering a therapeutically effective amount of a composition comprising isolated ADAMTS13 to the mammal, wherein the therapeutically effective amount of ADAMTS13 contains from 20 to 100 units of FRETS-VWF73 activity per kilogram of body weight body of a mammal (U _FV73 /kg). In a particular embodiment, a mammal

- 28 045023 This is a person. In one embodiment, 20 U^ ₇₃ to 100 Eg _FV7 g/kg ADAMTS13 is administered within 10 minutes of detection of deep vein thrombosis in the mammal. In one embodiment, 20 to 100 U of _FV73 /kg ADAMTS13 is administered within 30 minutes of detection of deep vein thrombosis in the mammal. In one embodiment, 20 to 100 U of _FV73 /kg ADAMTS13 is administered within 60 minutes of detection of deep vein thrombosis in the mammal. In one embodiment, 20 to 100 U of _FV73 /kg ADAMTS13 is administered within 4 hours of detection of deep vein thrombosis in the mammal. In one embodiment, 20 to 100 U of _FV73 /kg ADAMTS13 is administered within 12 hours of detection of deep vein thrombosis in the mammal. In one embodiment, 20 to 100 U of _FV73 /kg ADAMTS13 is administered within 24 hours of detection of deep vein thrombosis in a mammal.

In one embodiment, the present disclosure provides a method of treating deep vein thrombosis in a mammal in need thereof, comprising subcutaneously administering a therapeutically effective amount of a composition comprising isolated ADAMTS13 to the mammal, wherein the therapeutically effective amount of ADAMTS13 contains from 40 to 200 units of FRETS-VWF73 activity per kilogram of body weight body of a mammal (Eg _FV7 h/kg). In a particular embodiment, the mammal is a human. In one embodiment, from 40 U _FV73 to 200 U _FV73 /kg ADAMTS13 is administered within 10 minutes of detection of deep vein thrombosis in a mammal. In one embodiment, 40 to 200 U of _FV73 /kg ADAMTS13 is administered within 30 minutes of detection of deep vein thrombosis in a mammal. In one embodiment, 40 to 200 Eg _FV7 g/kg ADAMTS13 is administered within 60 minutes of detection of deep vein thrombosis in a mammal. In one embodiment, 40 to 200 U of _FV73 /kg ADAMTS13 is administered within 4 hours of detection of deep vein thrombosis in the mammal. In one embodiment, 40 to 200 U of _FV73 /kg ADAMTS13 is administered within 12 hours of detection of deep vein thrombosis in the mammal. In one embodiment, 40 to 200 U of _FV73 /kg ADAMTS13 is administered within 24 hours of detection of deep vein thrombosis in a mammal.

In one embodiment, the present disclosure provides a method of prophylactically treating a mammal at risk for deep vein thrombosis, comprising subcutaneously administering a therapeutically effective amount of a composition comprising isolated ADAMTS13 to a mammal in need thereof, wherein the therapeutically effective amount of ADAMTS13 contains from 20 to 4000 units of FRETS- activity. VWF73 per kilogram of mammal body weight (U _FV73 /kg). In a particular embodiment, the mammal is a human. In one embodiment, from 20 Units of _FV73 to 4000 Units _{of FV73} /kg ADAMTS13 is administered approximately once per month. In one embodiment, 20 to 4000 U _FV73 /kg ADAMTS13 is administered approximately twice a month. In one embodiment, 20 to 4000 Units of _FV73 /kg ADAMTS13 is administered approximately once per week. In one embodiment, 20 to 4000 U _FV73 /kg ADAMTS13 is administered approximately twice per week. In one embodiment, from 20 to 4000 Eg _FV73 /kg ADAMTS13 is administered approximately once every 48 hours. In one embodiment, from 20 to 4000 Eg _FV73 /kg ADAMTS13 is administered approximately once every 24 hours. In one embodiment, from 20 to 4000 Eg _FV7 3/kg ADAMTS13 is administered approximately once every 12 hours.

In one embodiment, the present disclosure provides a method of prophylactically treating a mammal at risk for deep vein thrombosis, comprising subcutaneously administering a therapeutically effective amount of a composition comprising isolated ADAMTS13 to a mammal in need thereof, wherein the therapeutically effective amount of ADAMTS13 contains from 20 to 2000 units of FRETS- activity VWF73 per kilogram of mammal body weight (U _FV73 /kg). In a particular embodiment, the mammal is a human. In one embodiment, from 20 Units _{of FV73} to 2000 Units _{of FV73} /kg ADAMTS13 is administered approximately once per month. In one embodiment, 20 to 2000 U _FV73 /kg ADAMTS13 is administered approximately twice a month. In one embodiment, 20 to 2000 Units of _FV73 /kg ADAMTS13 is administered approximately once per week. In one embodiment, 20 to 2000 U _FV73 /kg ADAMTS13 is administered approximately twice per week. In one embodiment, 20 to 2000 Eg _FV73 /kg ADAMTS13 is administered approximately once every 48 hours. In one embodiment, 20 to 2000 Eg _FV73 /kg ADAMTS13 is administered approximately once every 24 hours. In one embodiment, 20 to 2000 Eg _FV7 h/kg ADAMTS13 is administered approximately once every 12 hours.

In one embodiment, the present disclosure provides a method of prophylactically treating a mammal at risk for deep vein thrombosis, comprising subcutaneously administering a therapeutically effective amount of a composition comprising isolated ADAMTS13 to the mammal in need thereof, wherein the therapeutically effective amount of ADAMTS13 contains from 20 to 1000 units of FRETS- activity VWF73 per kilogram of mammal body weight (U _FV73 /kg). In a particular embodiment, the mammal is a human. In one embodiment, from 20 Units of _FV73 to 1000 Units _{of FV73} /kg ADAMTS13 is administered approximately once per month. In one embodiment, 20 to 1000 U _FV73 /kg ADAMTS13 is administered approximately twice a month. In one embodiment, 20 to 1000 U _FV73 /kg ADAMTS13 is administered approximately once per week. In one embodiment, 20 to 1000 U _FV73 /kg ADAMTS13 is administered approximately twice per week. In one embodiment, 20 to 1000 U _FV73 /kg ADAMTS13 is administered approximately once every 48 hours. In one embodiment, 20 to 1000 U^7 ₃ /kg ADAMTS13 is administered approximately once every 24 hours. In one embodiment from 20 to 1000 U _FV7 3/kg ADAMTS13 is administered approximately once every 12 hours.

In one embodiment, the present disclosure provides a method of prophylactically treating a mammal at risk for deep vein thrombosis, comprising subcutaneously administering a therapeutically effective amount of a composition comprising isolated ADAMTS13 to the mammal in need thereof, wherein the therapeutically effective amount of ADAMTS13 contains from 20 to 500 units of FRETS- activity VWF73 per kilogram of mammal body weight ( _FV73 units/kg). In a particular embodiment, the mammal is a human. In one embodiment, from 20 Units _{of FV73} to 500 Units _{of FV73} /kg ADAMTS13 is administered approximately once per month. In one embodiment, 20 to 500 U _FV73 /kg ADAMTS13 is administered approximately twice a month. In one embodiment, 20 to 500 U _FV73 /kg ADAMTS13 is administered approximately once per week. In one embodiment, 20 to 500 U _FV73 /kg ADAMTS13 is administered approximately twice per week. In one embodiment, 20 to 500 Units of _FV73 /kg ADAMTS13 is administered approximately once every 48 hours. In one embodiment, 20 to 500 Units of _FV73 /kg ADAMTS13 is administered approximately once every 24 hours. In one embodiment, 20 to 500 Units of _FV73 /kg ADAMTS13 is administered approximately every 12 hours.

In one embodiment, the present disclosure provides a method of prophylactically treating a mammal at risk for deep vein thrombosis, comprising subcutaneously administering a therapeutically effective amount of a composition comprising isolated ADAMTS13 to a mammal in need thereof, wherein the therapeutically effective amount of ADAMTS13 contains from 20 to 200 units of FRETS- activity. VWF73 per kilogram of mammal body weight ( _FV73 units/kg). In a particular embodiment, the mammal is a human. In one embodiment, from 20 Units _{of FV73} to 200 Units _{of FV73} /kg ADAMTS13 is administered approximately once per month. In one embodiment, 20 to 200 U _FV73 /kg ADAMTS13 is administered approximately twice a month. In one embodiment, 20 to 200 U _FV73 /kg ADAMTS13 is administered approximately once per week. In one embodiment, 20 to 200 U _FV73 /kg ADAMTS13 is administered approximately twice per week. In one embodiment, 20 to 200 Units of _FV73 /kg ADAMTS13 is administered approximately once every 48 hours. In one embodiment, 20 to 200 Units of _FV73 /kg ADAMTS13 is administered approximately once every 24 hours. In one embodiment, 20 to 200 Units of _FV73 /kg ADAMTS13 is administered approximately every 12 hours.

In one embodiment, the present disclosure provides a method of prophylactically treating a mammal at risk for deep vein thrombosis, comprising subcutaneously administering a therapeutically effective amount of a composition comprising isolated ADAMTS13 to a mammal in need thereof, wherein the therapeutically effective amount of ADAMTS13 contains from 20 to 100 units of FRETS- activity. VWF73 per kilogram of mammal body weight ( _FV73 units/kg). In a particular embodiment, the mammal is a human. In one embodiment, from 20 Units _{of FV73} to 100 Units _{of FV73} /kg ADAMTS13 is administered approximately once per month. In one embodiment, 20 to 100 U _FV73 /kg ADAMTS13 is administered approximately twice a month. In one embodiment, 20 to 100 U _FV73 /kg ADAMTS13 is administered approximately once per week. In one embodiment, 20 to 100 U _FV73 /kg ADAMTS13 is administered approximately twice per week. In one embodiment, 20 to 100 Units of _FV73 /kg ADAMTS13 is administered approximately once every 48 hours. In one embodiment, 20 to 100 Units of _FV73 /kg ADAMTS13 is administered approximately once every 24 hours. In one embodiment, 20 to 100 Units of _FV7 3/kg ADAMTS13 is administered approximately once every 12 hours.

In one embodiment, the present disclosure provides a method of prophylactically treating a mammal at risk for deep vein thrombosis, comprising subcutaneously administering a therapeutically effective amount of a composition comprising isolated ADAMTS13 to the mammal in need thereof, wherein the therapeutically effective amount of ADAMTS13 contains from 40 to 200 units of FRETS- activity. VWF73 per kilogram of mammal body weight ( _FV73 units/kg). In a particular embodiment, the mammal is a human. In one embodiment, from 40 Units _{of FV73} to 200 Units _{of FV73} /kg ADAMTS13 is administered approximately once per month. In one embodiment, 40 to 200 U _FV73 /kg ADAMTS13 is administered approximately twice a month. In one embodiment, 40 to 200 U _FV73 /kg ADAMTS13 is administered approximately once per week. In one embodiment, 40 to 200 U _FV73 /kg ADAMTS13 is administered approximately twice per week. In one embodiment, 40 to 200 Units of _FV7 /kg ADAMTS13 is administered approximately once every 48 hours. In one embodiment, 40 to 200 Units of _FV73 /kg ADAMTS13 is administered approximately once every 24 hours. In one embodiment, 40 to 200 Units _FV7 3/kg ADAMTS13 is administered approximately every 12 hours.

In one embodiment, the present disclosure provides a method of prophylactically treating a mammal at risk for deep vein thrombosis, comprising subcutaneously administering a therapeutically effective amount of a composition comprising isolated ADAMTS13 to the mammal, wherein the therapeutically effective amount of ADAMTS13 and the dosing frequency are selected from options 1-1133 in Table. 2. In a particular embodiment, the mammal is a human.

Disseminated intravascular coagulation

In specific embodiments, the ADAMTS13 composition described herein is used for the treatment and prevention of disseminated intravascular coagulation (DIC),

- 30 045023 more specifically, DIC associated with sepsis. DIC is a condition in which blood clots form in small blood vessels throughout the body. These blood clots can reduce or block blood flow throughout the body and cause tissue and organ damage. Blood clots in small blood vessels occur due to increased thrombotic activity. This increase in activity places excess stress on platelets and clotting factors, thereby increasing the likelihood of serious internal and external bleeding by depleting available sources of platelets and clotting factors. Accordingly, patients with DIC often suffer from blood clots and disorders accompanied by severe bleeding.

Certain illnesses, such as sepsis, surgery/trauma, cancer, childbirth/pregnancy complications, venomous snake bites (rattlesnakes and pit vipers), frostbite, and burns, can cause overactive clotting factors and lead to disseminated intravascular coagulation. DIC can be acute (developing rapidly over a few hours or days) or chronic (developing over several weeks or months). Although both types of DIC require treatment, acute DIC must be treated immediately to prevent excess blood clotting in small blood vessels, which quickly leads to severe bleeding. Accordingly, it is important to have a pharmaceutical composition that can be quickly and easily administered for the treatment of DIC, especially acute DIC, such as DIC associated with sepsis. Therefore, it is desirable to develop a subcutaneous formulation of ADAMTS13 and a method for subcutaneous delivery of ADAMTS13.

In some embodiments, the pharmaceutical composition is administered immediately after detection of disseminated intravascular coagulation, for example, within 5 minutes, 10 minutes, 15 minutes, 20 minutes, 25 minutes, 30 minutes, 35 minutes, 40 minutes, 45 minutes, 50 minutes, 55 minutes , 60 min, 90 min, 110 min, 120 min, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 9 hours, 10 hours, 11 hours, 12 hours, 13 hours, 14 hours, 15 hours, 16 hours, 17 hours, 18 hours, 19 hours, 20 hours, 21 hours, 22 hours, 23 hours, 24 hours, 25 hours or more, or any combination thereof. Accordingly, it is important to have a pharmaceutical composition that can be administered quickly and easily.

In one embodiment, the present disclosure provides a method of treating disseminated intravascular coagulation in a mammal in need thereof, comprising subcutaneously administering a therapeutically effective amount of a composition comprising isolated ADAMTS13 to the mammal, wherein the therapeutically effective amount of ADAMTS13 contains from 20 to 4000 units of FRETS-VWF73 activity per kilogram of body weight body of a mammal (U _FV73 /kg). In a particular embodiment, the mammal is a human. In one embodiment, 20 U _FV73 to 4000 U _FV7 g/kg ADAMTS13 is administered within 10 minutes of detection of disseminated intravascular coagulation in the mammal. In one embodiment, 20 to 4000 U _FV73 /kg ADAMTS13 is administered within 30 minutes of detection of disseminated intravascular coagulation in the mammal. In one embodiment, 20 to 4000 U _FV73 /kg ADAMTS13 is administered within 60 minutes of detection of disseminated intravascular coagulation in the mammal. In one embodiment, 20 to 4000 U of _FV73 /kg ADAMTS13 is administered within 4 hours of detection of disseminated intravascular coagulation in the mammal. In one embodiment, 20 to 4000 U _FV7 g/kg ADAMTS13 is administered within 12 hours of detection of disseminated intravascular coagulation in the mammal. In one embodiment, 20 to 4000 U _FV73 /kg ADAMTS13 is administered within 24 hours of detection of disseminated intravascular coagulation in the mammal.

In one embodiment, the present disclosure provides a method of treating disseminated intravascular coagulation in a mammal in need thereof, comprising subcutaneously administering a therapeutically effective amount of a composition comprising isolated ADAMTS13 to the mammal, wherein the therapeutically effective amount of ADAMTS13 contains from 20 to 2000 units of FRETS-VWF73 activity per kilogram of body weight body of a mammal (U _FV73 /kg). In a particular embodiment, the mammal is a human. In one embodiment, from 20 _FV73 Units to 2000 _FV73 Units/kg ADAMTS13 is administered within 10 minutes of detection of disseminated intravascular coagulation in the mammal. In one embodiment, 20 to 2000 U _FV73 /kg ADAMTS13 is administered within 30 minutes of detection of disseminated intravascular coagulation in the mammal. In one embodiment, 20 to 2000 U _FV73 /kg ADAMTS13 is administered within 60 minutes of detection of disseminated intravascular coagulation in the mammal. In one embodiment, 20 to 2000 U _FV7 g/kg ADAMTS13 is administered within 4 hours of detection of disseminated intravascular coagulation in the mammal. In one embodiment, 20 to 2000 U _FV73 /kg ADAMTS13 is administered within 12 hours of detection of disseminated intravascular coagulation in the mammal. In one embodiment, 20 to 2000 U _FV73 /kg ADAMTS13 is administered within 24 hours of detection of disseminated intravascular coagulation in the mammal.

In one embodiment, the present disclosure provides a method of treating disseminated intravascular coagulation in a mammal in need thereof, comprising subcutaneously administering a therapeutically effective amount of a composition comprising isolated ADAMTS13 to the mammal, wherein the therapeutically effective amount of ADAMTS13 comprises from 20 to 1000 units

- 31 045023 nits of FRETS-VWF73 activity per kilogram of mammal body weight (Ed^/kg). In a particular embodiment, the mammal is a human. In one embodiment, from 20 U _{of FV73} to 1000 U _{of FV73} /kg ADAMTS13 is administered within 10 minutes of detection of disseminated intravascular coagulation in the mammal. In one embodiment, 20 to 1000 U _FV73 /kg ADAMTS13 is administered within 30 minutes of detection of disseminated intravascular coagulation in the mammal. In one embodiment, 20 to 1000 U _FV73 /kg ADAMTS13 is administered within 60 minutes of detection of disseminated intravascular coagulation in the mammal. In one embodiment, 20 to 1000 U _FV73 /kg ADAMTS13 is administered within 4 hours of detection of disseminated intravascular coagulation in the mammal. In one embodiment, 20 to 1000 U _FV73 /kg ADAMTS13 is administered within 12 hours of detection of disseminated intravascular coagulation in the mammal. In one embodiment, 20 to 1000 U of _FV73 /kg ADAMTS13 is administered within 24 hours of detection of disseminated intravascular coagulation in the mammal.

In one embodiment, the present disclosure provides a method of treating disseminated intravascular coagulation in a mammal in need thereof, comprising subcutaneously administering a therapeutically effective amount of a composition comprising isolated ADAMTS13 to the mammal, wherein the therapeutically effective amount of ADAMTS13 contains from 20 to 500 units of FRETS-VWF73 activity per kilogram of body weight body of a mammal (U _FV73 /kg). In a particular embodiment, the mammal is a human. In one embodiment, from 20 U _{of FV73} to 500 U of _FV73 /kg ADAMTS13 is administered within 10 minutes of detection of disseminated intravascular coagulation in the mammal. In one embodiment, 20 to 500 U _FV73 /kg ADAMTS13 is administered within 30 minutes of detection of disseminated intravascular coagulation in the mammal. In one embodiment, 20 to 500 U _FV73 /kg ADAMTS13 is administered within 60 minutes of detection of disseminated intravascular coagulation in the mammal. In one embodiment, 20 to 500 U _FV73 /kg ADAMTS13 is administered within 4 hours of detection of disseminated intravascular coagulation in the mammal. In one embodiment, 20 to 500 U of _FV73 /kg ADAMTS13 is administered within 12 hours of detection of disseminated intravascular coagulation in the mammal. In one embodiment, 20 to 500 U of _FV73 /kg ADAMTS13 is administered within 24 hours of detection of disseminated intravascular coagulation in the mammal.

In one embodiment, the present disclosure provides a method of treating disseminated intravascular coagulation in a mammal in need thereof, comprising subcutaneously administering a therapeutically effective amount of a composition comprising isolated ADAMTS13 to the mammal, wherein the therapeutically effective amount of ADAMTS13 contains from 20 to 200 units of FRETS-VWF73 activity per kilogram of body weight body of a mammal (U _FV73 /kg). In a particular embodiment, the mammal is a human. In one embodiment, 20 U _FV73 to 200 U _FV73 /kg ADAMTS13 is administered within 10 minutes of detection of disseminated intravascular coagulation in the mammal. In one embodiment, 20 to 200 U _FV73 /kg ADAMTS13 is administered within 30 minutes of detection of disseminated intravascular coagulation in the mammal. In one embodiment, 20 to 200 U _FV73 /kg ADAMTS13 is administered within 60 minutes of detection of disseminated intravascular coagulation in the mammal. In one embodiment, 20 to 200 U _FV73 /kg ADAMTS13 is administered within 4 hours of detection of disseminated intravascular coagulation in the mammal. In one embodiment, 20 to 200 U _FV73 /kg ADAMTS13 is administered within 12 hours of detection of disseminated intravascular coagulation in the mammal. In one embodiment, 20 to 200 U _FV73 /kg ADAMTS13 is administered within 24 hours of detection of disseminated intravascular coagulation in the mammal.

In one embodiment, the present disclosure provides a method of treating disseminated intravascular coagulation in a mammal in need thereof, comprising subcutaneously administering a therapeutically effective amount of a composition comprising isolated ADAMTS13 to the mammal, wherein the therapeutically effective amount of ADAMTS13 contains from 20 to 100 units of FRETS-VWF73 activity per kilogram of body weight body of a mammal (U _FV73 /kg). In a particular embodiment, the mammal is a human. In one embodiment, 20 U of _FV73 to 100 U _{of FV73} /kg ADAMTS13 is administered within 10 minutes of detection of disseminated intravascular coagulation in the mammal. In one embodiment, 20 to 100 U _FV73 /kg ADAMTS13 is administered within 30 minutes of detection of disseminated intravascular coagulation in the mammal. In one embodiment, 20 to 100 U _FV73 /kg ADAMTS13 is administered within 60 minutes of detection of disseminated intravascular coagulation in the mammal. In one embodiment, 20 to 100 U _FV73 /kg ADAMTS13 is administered within 4 hours of detection of disseminated intravascular coagulation in the mammal. In one embodiment, 20 to 100 U _FV73 /kg ADAMTS13 is administered within 12 hours of detection of disseminated intravascular coagulation in the mammal. In one embodiment

- 32 045023 from 20 to 100 Eg _FV7 3/kg ADAMTS13 is administered within 24 hours from the moment of detection of disseminated intravascular coagulation in a mammal.

In one embodiment, the present disclosure provides a method of treating disseminated intravascular coagulation in a mammal in need thereof, comprising subcutaneously administering a therapeutically effective amount of a composition comprising isolated ADAMTS13 to the mammal, wherein the therapeutically effective amount of ADAMTS13 contains from 40 to 200 units of FRETS-VWF73 activity per kilogram of body weight body of a mammal (U _FV73 /kg). In a particular embodiment, the mammal is a human. In one embodiment, 40 U _FV73 to 200 U _FV73 /kg ADAMTS13 is administered within 10 minutes of detection of disseminated intravascular coagulation in the mammal. In one embodiment, 40 to 200 U of _FV73 /kg ADAMTS13 is administered within 30 minutes of detection of disseminated intravascular coagulation in the mammal. In one embodiment, 40 to 200 U of _FV73 /kg ADAMTS13 is administered within 60 minutes of detection of disseminated intravascular coagulation in the mammal. In one embodiment, 40 to 200 U _FV73 /kg ADAMTS13 is administered within 4 hours of detection of disseminated intravascular coagulation in the mammal. In one embodiment, 40 to 200 U of _FV73 /kg ADAMTS13 is administered within 12 hours of detection of disseminated intravascular coagulation in the mammal. In one embodiment, 40 to 200 Eg _FV73 /kg ADAMTS13 is administered within 24 hours of detection of disseminated intravascular coagulation in the mammal.

V. Kits based on ADAMTS13

In yet another aspect, kits are provided for treating a disease or condition associated with ADAMTS13 dysfunction or VWF. In one embodiment, the kit contains the composition rADAMTS13. In some embodiments, the kits provided herein may contain one or more doses of a liquid or lyophilized formulation provided herein. If the kits contain a lyophilized formulation of rADAMTS13, the kits generally must also contain a suitable liquid for reconstituting the liquid formulation, such as sterile water or a pharmaceutically acceptable buffer. In some embodiments, the kit contains the ADAMTS13 formulation packaged in syringes for subcutaneous administration by a healthcare professional or for home use.

In one embodiment, a kit is provided containing from about 10 units of FRETS-VWF73 activity to about 10,000 units of FRETS-VWF73 activity. In other embodiments, the kit may contain, for example, from about 20 FRETS-VWF73 activity units ( _FV73 Units) to about 8000 FRETS-VWF73 Units, or from about 30 _FV73 Units to about 6000 _FV73 Units, or from about 40 _FV73 Units to about 4000 Units _FV73 or from approximately 50 Units _FV73 to approximately 3000 Units _FV73 , or from approximately 75 Units _FV73 to approximately 2500 Units _FV73 , or from approximately 100 Units _FV73 to approximately 2000 Units _FV73 , or from approximately 200 Units _FV73 to approximately 1500 Units _FV73 , or an amount of enzyme from another range. In some embodiments, the set may contain approximately 10 units of FRETSVWF73 activity, 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, 9000, 9100, 9200, 9300, 9400, 9500, 9600,

9700, 9800, 9900, 10000, 11000, 12000, 13000, 14000, 15000, 16000, 17000, 18000, 19000, 20000 or more FRETS-VWF73 activity units.

In some embodiments, the kit is for a single administration or dose of ADAMTS13. In other embodiments, the kit may contain multiple doses of ADAMTS13 for subcutaneous administration. In one embodiment, the kit may contain the ADAMTS13 formulation packaged in syringes for subcutaneous administration by a healthcare professional or for home use.

VI. Examples

Example 1: Pharmacokinetic properties of rADAMTS13 when administered intravenously and subcutaneously.

This study was conducted to evaluate the pharmacokinetic properties of recombinant ADAMTS13 in Göttingen minipigs following intravenous (IV) or subcutaneous (SC) administration. In this study, 4 male Gottingen miniature pigs were administered ADMTS13 IV (intravenous group) and 4 male Gottingen minipigs were administered ADMTS13 SC (subcutaneous group).

Male Göttingen miniature pigs were conditioned to cooperate with study procedures before the start of the study. Thus, animals were not immobilized during administration of anesthetics or test samples and collection of blood samples.

Animals were anesthetized for catheter implantation using a Zoletil mixture consisting of

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1.11 mg/kg tiletamine, 1.11 mg/kg zolazepam, 0.56 mg/kg xylazine, 0.56 mg/kg ketamine and 0.11 mg/kg butorphanol (IM). If necessary, isoflurane was used to maintain anesthesia. During surgery, animals received pure oxygen through a mask or endotracheal tube. The jugular area was shaved and disinfected. The central catheter was inserted into the cranial vena cava according to Seldinger. Before recovery from anesthesia, 4 mg/kg carprofen was administered for pain relief.

The intravenous group received a nominal dose of 200 FRETS-U/kg rADAMTS13 IV (0.085 ml/kg). The subcutaneous group received a nominal dose of 1000 FRETS-U/kg rADAMTS13 SC (0.456 ml/kg). Blood samples were collected through a central venous catheter before administration of the test sample and 5 minutes, 1 hour, 3 hours, 6 hours, 9 hours, 24 hours, 32 hours, 48 hours, 56 hours and 72 hours after administration of the test sample. Blood samples were obtained as follows: the first 0.5-0.7 ml was discarded to avoid dilution with the catheter priming solution (saline). Then 0.8 ml of blood was collected and mixed with 0.2 ml of sodium citrate. The blood sample was centrifuged at 5700 rpm (1x10 min, 1x5 min). Plasma was stored at temperatures below -60°C.

ADAMTS13 activity was determined by FRETS assay and antigen detection by ELISA. In addition, the potential formation of binding antibodies against human rADAMTS13 and the effect of rADAMTS13 on the pattern of endogenous VWF multimers were assessed.

As expected, after IV administration, the median _Tmax (time to reach _Cmax ) was 5 minutes, indicating immediate bioavailability. The median _Tmax after subcutaneous administration was 24 hours for ADAMTS13 activity and 28 hours for ADAMTS13 antigen. The mean plasma concentrations of ADAMTS13 activity are shown in FIG. 1, and the average concentrations of the ADAMTS13 antigen are shown in Fig. 2.

PPK ₀ . _tnO c _Λ [h-U/ml/U/kg and h-μg/ml/μg/kg] adjusted for dose after IV administration was 0.306 for activity and 0.373 for antigen. AUC ₀ - _tnO c _Λ was calculated using the trapezoidal rule from time 0 to the last sampling time (Shchosl) (Hauschke et al., 2007). PPK ₀ . _tnO c _Λ adjusted for dose after SC administration was 0.198 for activity and 0.243 for antigen. Thus, the dose-adjusted AUC- _tn0 to _Λ ratio after subcutaneous and intravenous administration was 0.646 for activity and 0.651 for antigen. IVR [%] was 55.8 (activity) and 79.3 (antigen) after intravenous administration and 13.8 (activity) and 17.3 (antigen) after subcutaneous administration.

Because plasma rADAMTS13 levels did not decrease sufficiently during the observation period, estimates of the terminal and initial half-lives should be considered with caution. Thus, dependent parameters (MPT, Vss, Cls, AUC ₀ . _inf ) may also be unreliable. A follow-up study will be conducted to evaluate these parameters. Evaluation of binding antibodies against rADAMTS13 was negative for all samples from all animals.

The results of assessing the pattern of VWF multimers in miniature pigs indicated extremely limited cleavage of endogenous VWF by recombinant ADAMTS13 (table). The intensity of the satellite band close to the lower VWM multimer increased slightly over time in animals receiving SC administration, but not in animals receiving IV administration. The height of the VWF multimer pattern did not change in any group.

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Table 3

Multimeric analysis of recombinant ADAMTS13 in low and high resolution agarose gels

Experimental group Number of animals VWF splitting Intravenous group (200 IU/kg i.v.) 1 No 2 No 3 No 4 No Subcutaneous group (1000 IU/kg s.c.) 1 Minor increase intensity of one satellite band 2 Minor increase intensity of one satellite band 3 Minor increase intensity of one satellite band 4 Minor increase intensity of one satellite band

Example 2: Pharmacokinetic properties of rADAMTS13 when administered intravenously and subcutaneously.

This study was conducted to evaluate the pharmacokinetic properties of recombinant ADAMTS13 in Göttingen minipigs following intravenous (IV) or subcutaneous (SC) administration. In this study, 4 male Gottingen miniature pigs were administered ADMTS13 IV (intravenous group) and 4 male Gottingen minipigs were administered ADMTS13 SC (subcutaneous group).

Male Göttingen miniature pigs were acclimated to study procedures and anesthetized for catheter implantation as described in Example 1.

In this study, 3 and 5 male Göttingen miniature pigs were used per group. Both groups received a nominal dose of 200 FRETS-U/kg rADAMTS13. Three animals were administered the substance intravenously, and the other five animals were administered subcutaneously. Blood samples were collected through a central venous catheter before administration of the test sample and after 5 minutes, 1 hour, 2 hours, 6 hours, 5 hours, 23 hours, 30 hours, 47 hours, 54 hours, 71 hours, 78 hours, 95 hours and 102 hours. h after administration of the test sample.

Blood samples were obtained as follows: The first 0.5-0.7 ml was discarded to avoid dilution with the catheter priming solution (saline). Then 0.8 ml of blood was collected and mixed with 0.2 ml of sodium citrate. The blood sample was centrifuged at 5700 rpm (1x10 min, 1x5 min). Plasma was stored at temperatures below -60°C.

ADAMTS13 activity was determined by FRETS assay and antigen detection by ELISA.

As expected, after IV administration, the median _Tmax (time to reach _Cmax ) was 5 minutes, indicating immediate bioavailability. The median _Tmax after subcutaneous administration was 30 hours. The average concentrations of ADAMTS13 activity in plasma are shown in Fig. 3, and the average concentrations of the ADAMTS13 antigen are shown in FIG. 4.

PPCo.tafter [U/ml/-h and µg/ml/-h] after IV administration was 68.25 for activity and 39.81 for antigen. PPK ₀ . _SS after subcutaneous administration was 47.16 for activity and 26.23 for antigen. IVR [%] was 61.0 (activity) and 45.6 (antigen) after intravenous administration and 13.0 (activity) and 9.6 (antigen) after subcutaneous administration.

The terminal half-life [h] after IV administration was 46.96 for activity and 48.83 for antigen. The terminal half-life after SC administration was 56.76 for activity and 41.25 for antigen. MRT [h] was 64.08 (activity) and 66.26 (antigen) after intravenous administration and 104.2 (activity) and 77.52 (antigen) after subcutaneous administration.

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Claims (28)

Thus, bioavailability with subcutaneous administration compared to intravenous administration was 65.9% and 69.1% for ADAMTS13 antigen and ADAMTS13 activity, respectively. CLAIM 1. A method of treating a disease or disorder, wherein the disease or disorder is thrombotic thrombocytopenic purpura (TTP), cerebral infarction, myocardial infarction, reperfusion injury, deep vein thrombosis or disseminated intravascular coagulation associated with sepsis in a mammal, comprising subcutaneous administration of a therapeutically effective an amount of a composition comprising ADAMTS13 proteins and at least one compound selected from the group consisting of a pharmaceutically acceptable salt, a sugar, a sugar alcohol, a nonionic surfactant and a buffering agent, wherein the therapeutically effective amount of ADAMTS13 is from 20 to 4000 units of activity per kilogram of body weight. 2. The method of claim 1, wherein said disease or disorder is TTP. 3. The method according to claim 2, wherein TTP is hereditary TTP. 4. The method according to claim 2, wherein the TTP is an acquired TTP. 5. The method of claim 1, wherein the disease or disorder is cerebral infarction. 6. The method of claim 1, wherein the disease or disorder is myocardial infarction. 7. The method of claim 1, wherein the disease or disorder is ischemic reperfusion injury. 8. The method of claim 1, wherein the disease or disorder is deep vein thrombosis. 9. The method of claim 1, wherein the disease or disorder is disseminated intravascular coagulation. 10. A method of preventing or reducing tissue damage in a mammal, comprising subcutaneously administering a therapeutically effective amount of a composition comprising ADAMTS13 proteins, wherein the tissue damage is secondary tissue damage to the infarcted area. 11. The method according to claim 10, wherein the infarction is caused by a blood clotting disorder. 12. The method according to claim 10, where the heart attack is caused by trauma from a blunt object or mechanical trauma. 13. A method of preventing or reducing tissue damage caused by a blood clotting disorder in a mammal, comprising administering a therapeutically effective amount of a composition comprising ADAMTS13 proteins. 14. A method of improving recovery and/or reducing damage to sensory and/or motor function after cerebral infarction in a mammal, comprising subcutaneous administration of a therapeutically effective amount of a composition containing ADAMTS13 proteins. 15. The method according to claim 14, wherein the cerebral infarction is caused by a blood clotting disorder. 16. The method of claim 14, wherein the cerebral infarction is caused by blunt force trauma or mechanical trauma. 17. Method according to any one of paragraphs. 11, 13, or 15, wherein the coagulation disorder is selected from the group consisting of hereditary thrombotic thrombocytopenic purpura (TTP), acquired TTP, cerebral infarction, myocardial infarction, ischemia/reperfusion injury, deep vein thrombosis, and disseminated intravascular coagulation. 18. The method of claim 17, wherein the clotting disorder is hereditary TTP. 19. The method of claim 17, wherein the clotting disorder is acquired TTP. 20. The method of claim 17, wherein the coagulation disorder is a cerebral infarction. 21. The method of claim 17, wherein the coagulation disorder is ischemic reperfusion injury. 22. The method of claim 17, wherein the coagulation disorder is a myocardial infarction. 23. The method of claim 17, wherein the coagulation disorder is disseminated intravascular coagulation. 24. A method of reducing the neurological manifestation, fatigue or severe bleeding associated with thrombotic thrombocytopenic purpura (TTP) in a mammal, comprising subcutaneously administering a therapeutically effective amount of a composition containing ADAMTS13 proteins. 25. The method of claim 24, wherein the method includes reducing the neurological manifestations associated with TTP. 26. The method of claim 24 or 25, wherein the neurological manifestation is a change in mental status, stroke, seizures, hemiplegia, paresthesia, visual impairment and/or aphasia. 27. The method of claim 24, wherein the method includes reducing fatigue associated with TTP. 28. The method of claim 24, wherein the method includes reducing severe bleeding associated with TTP. -