Alkaline phosphatase polypeptide and method of use thereof

Abstract

The present invention relates to a pharmaceutical composition for treating bone mineralization disorders, such as hypophosphatasia (HPP), and symptoms thereof, comprising a soluble alkaline phosphatase (sALP) or a fragment thereof, the soluble alkaline phosphatase being derived from a naturally occurring alkaline phosphatase (ALP).

Description

Technical Field

[0001] Sequence Listing This application includes a sequence listing submitted electronically in ASCII format, which is hereby incorporated by reference in its entirety. The name of the above ASCII copy created on February 4, 2022 is 50694 - 094WO2_Sequence_Listing_2_4_22_ST25, and the size is 27,753 bytes.

Background Art

[0002] Hypophosphatasia (HPP) is a rare genetic skeletal disorder with an incidence of 1 case per 100,000 live births in its most severe form. This disorder is usually caused by loss - of - function mutations in the gene encoding tissue - nonspecific alkaline phosphatase (TNSALP). HPP presents a very wide range of symptoms and severities, from early tooth loss to almost complete absence of bone mineralization in utero. The symptoms of HPP vary significantly among patients and also vary significantly depending on the patient's age. Many HPP patients exhibit skeletal changes, short stature, chronic pain, lower limb pain, gait disturbances, and early non - traumatic tooth loss. Asfotase alfa (STRENSIQ®, Alexion Pharmaceuticals, Inc.), a recombinantly produced enzyme replacement therapy (ERT) containing soluble fragments of TNSALP, is the first ERT available to HPP patients. Asfotase alfa has shown innovative effects to date against the most severe forms of HPP, as demonstrated by improvements in bone mineralization and density, as well as respiratory and motor function, cognitive development, and muscle strength (Whyte et al., New Engl. J. Med. 366:904 - 913, 2012).

Prior Art Documents

Non - Patent Documents

[0003]

Non - Patent Document 1

[0004] The first embodiment is characterized by a pharmaceutical composition comprising an alkaline phosphatase polypeptide having at least 80% (e.g., at least 85%, 90%, 95%, 97%, 99%, or 100%) sequence identity to SEQ ID NO: 5, and a pharmaceutically acceptable carrier. The polypeptide may contain at least one mutation selected from E108M, N213Q, and N286Q compared to the amino acid sequence of SEQ ID NO: 1 (e.g., the polypeptide may contain two or all three of these mutations). For example, the polypeptide has at least 80% (e.g., at least 85%, 90%, 95%, 97%, 99%, or 100%) sequence identity to the amino acid sequence of SEQ ID NO: 5 and contains at least one, two, or all three of the mutations selected from E108M, N213Q, and N286Q. The pharmaceutically acceptable carrier may contain one or more of phosphates, proline, and sucrose. For example, a polypeptide may contain or consist of the amino acid sequence of SEQ ID NO: 5.

[0005] In some embodiments, the alkaline phosphatase moiety of the polypeptide has at least 80% (e.g., at least 85%, 90%, 95%, 97%, 99%, or 100%) sequence identity to SEQ ID NO: 3. This polypeptide may be further linked to an Fc region (e.g., an IgG1, IgG2, IgG3, or IgG4Fc region) and / or a polyaspartic acid region. In some embodiments, the polypeptide includes an IgG2 / 4Fc region and has at least 80% (e.g., at least 85%, 90%, 95%, 97%, 99%, or 100%) identity to, for example, SEQ ID NO: 4. The polyaspartic acid may include, for example, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 aspartic acid residues. In some embodiments, the polyaspartic acid includes 10 aspartic acid residues (D10).

[0006] The composition may be prepared to contain an alkaline phosphatase polypeptide in doses ranging from about 0.1 mg / mL to about 200 mg / mL (e.g., about 1, 10, 20, 25, 50, 75, 100, 125, 150, 175, or 200 mg / mL). The composition may be prepared in volumes ranging from about 0.1 mL to about 50 mL (e.g., about 0.1 to about 10 mL, e.g., about 0.1 mL, 0.2 mL, 0.3 mL, 0.4 mL, 0.5 mL, 0.6 mL, 0.7 mL, 0.8 mL, 0.9 mL, or 1.0 mL, e.g., about 1 mL to about 10 mL, e.g., about 2 mL, 3 mL, 4 mL, 5 mL, 6 mL, 7 mL, 8 mL, 9 mL, or 10 mL). In some embodiments, the composition is prepared in about 1 mL. For example, the composition may contain at least 80% (e.g., at least 85%, 90%, 95%, 97%, or 99%) sequence identity with respect to SEQ ID NO: 5, or 100 mg / mL of alkaline phosphatase polypeptide having that sequence (e.g., the polypeptide contains at least one, two, or all three mutations selected from E108M, N213Q, and N286Q of SEQ ID NO: 5), and a pharmaceutically acceptable carrier.

[0007] The composition may contain phosphate (e.g., sodium phosphate) at concentrations of, for example, about 1 mM to about 100 mM, or about 5 mM to about 20 mM, for example, about 2 mM, 3 mM, 4 mM, 5 mM, 6 mM, 7 mM, 8 mM, 9 mM, 10 mM, 20 mM, 30 mM, 40 mM, 50 mM, 60 mM, 70 mM, 80 mM, 90 mM, or 100 mM, for example, about 10 mM. The composition may further contain proline and / or sucrose. The composition may further contain proline. The composition may further contain sucrose. For example, the composition contains proline at approximately 1 mM to 500 mM, for example, approximately 70 mM to 280 mM, for example, approximately 50 mM to 200 mM, for example, approximately 2 mM, 3 mM, 4 mM, 5 mM, 6 mM, 7 mM, 8 mM, 9 mM, 10 mM, 20 mM, 30 mM, 40 mM, 50 mM, 60 mM, 70 mM, 80 mM, 90 mM, 100mM, 110mM, 120mM, 130mM, 140mM, 150mM, 160mM, 170mM, 180mM, 190mM, 200mM, 300mM, 400mM, or 500mM, for example, about 140mM of proline and / or about 1mM to about 500mM of sucrose, for example, about 70mM to about 280mM, e.g. For example, approximately 50mM to approximately 200mM, such as approximately 2mM, 3mM, 4mM, 5mM, 6mM, 7mM, 8mM, 9mM, 10mM, 20mM, 30mM, 40mM, 50mM, 60mM, 70mM, 80mM, 90mM, 100mM, 110mM, 120mM, 130mM, 140mM, 150mM, 160mM, 170mM The composition may contain sucrose in concentrations of approximately 140 mM, 210 mM, 220 mM, 230 mM, 240 mM, 250 mM, 260 mM, 270 mM, 280 mM, 290 mM, 300 mM, 350 mM, 400 mM, 450 mM, or 500 mM, for example, about 140 mM sucrose or about 210 mM sucrose. In some embodiments, the composition contains a proline:sucrose molar ratio of about 1:0 to about 1:3, for example, about 1:1.In some embodiments, the formulation contains about 1 mM to about 500 mM proline (e.g., about 50 mM to about 200 mM proline, e.g., about 140 mM proline) and about 1 mM to about 500 mM sucrose (e.g., about 40 mM to about 280 mM, e.g., about 50 mM to about 200 mM sucrose, e.g., about 140 mM or about 210 mM sucrose). In some embodiments, the formulation contains about 140 mM proline. In some embodiments, the formulation contains about 140 mM sucrose. In some embodiments, the formulation contains about 210 mM sucrose but does not contain proline. In some embodiments, the formulation contains about 140 mM proline and about 140 mM sucrose. In some embodiments, the formulation comprises about 140 mM proline, about 140 mM sucrose, and about 10 mM phosphate (e.g., sodium phosphate). In some embodiments, the formulation comprises about 210 mM sucrose and about 10 mM phosphate (e.g., sodium phosphate). The formulation may further contain about 0.01% to about 0.5% polyoxyethylene(20) monooleate sorbitan, for example, about 0.01% to about 0.1% polyoxyethylene(20) monooleate sorbitan, for example, about 0.05% polyoxyethylene(20) monooleate sorbitan. The polyoxyethylene(20) monooleate sorbitan may be, for example, polysorbate 80 (PS80). In some embodiments, the composition may be prepared at a pH of about 7.0 to about 7.6 (e.g., about 7.1, 7.2, 7.3, 7.4, 7.5, or 7.6, e.g., about 7.3). In some embodiments, the composition is at a pH of about 7.3 and contains about 10 mM phosphate, about 140 mM proline, about 140 mM sucrose, and about 0.05% sorbitan polyoxyethylene (20) monooleate (e.g., PS80).

[0008] The composition may be a pharmaceutical composition prepared as a solution containing a polypeptide (for example, the polypeptide of SEQ ID NO: 5, and its variants having at least about 85% sequence identity thereto (for example, the polypeptide contains one, two, or all three of the mutations selected from E108M, N213Q, and N286Q of SEQ ID NO: 5)). The pharmaceutical composition may contain the polypeptide in an amount of, for example, about 0.1 mg / mL to about 200 mg / mL, for example, about 100 mg / mL. The pharmaceutical composition may be prepared for subcutaneous administration in a polypeptide dose of, for example, about 0.1 mg / mL to about 10 mg / mL. The composition may be prepared in a solution of about 0.1 mL to about 50 mL (for example, about 0.1 to about 10 mL, for example, about 0.1 mL, 0.2 mL, 0.3 mL, 0.4 mL, 0.5 mL, 0.6 mL, 0.7 mL, 0.8 mL, 0.9 mL, or 1.0 mL, for example, about 1 mL to about 10 mL, for example, about 2 mL, 3 mL, 4 mL, 5 mL, 6 mL, 7 mL, 8 mL, 9 mL, or 10 mL). In some embodiments, the composition is prepared in about 1 mL. The solution may have a pH of about 7.3 and contain about 10 mM phosphate, about 140 mM proline, about 140 mM sucrose, and about 0.05% sorbitan polyoxyethylene (20) monooleate (e.g., PS80).

[0009] The specification also features vials containing the pharmaceutical compositions described herein. The vials may contain a solution (for example, at pH approximately 7.3, containing approximately 10 mM sodium phosphate, approximately 140 mM proline, approximately 140 mM sucrose, and approximately 0.05% sorbitan polyoxyethylene (20) monooleate (for example, PS80)) containing polypeptides (for example, in amounts of approximately 0.1 mg to approximately 1.0 g (for example, approximately 10 mg to approximately 200 mg)) in a volume of approximately 0.1 mL to approximately 10 mL (for example, approximately 1 mL). The vials may contain polypeptides in amounts of approximately 0.1 mg / mL to approximately 500 mg / mL, approximately 1 mg / mL to approximately 200 mg / mL, approximately 50 mg / mL to approximately 150 mg / mL, or approximately 100 mg / mL. The polypeptide may have the amino acid sequence of SEQ ID NO: 5, or a variant thereof having at least 85% sequence identity thereto (for example, the polypeptide may contain one, two, or all three of the mutations selected from E108M, N213Q, and N286Q of SEQ ID NO: 5).

[0010] The second embodiment is characterized by a method of treating symptoms of bone mineralization disorders or bone diseases in a patient (e.g., a human patient) (e.g., diseases selected from the group consisting of hypophosphatasia (HPP), fractures, osteoporosis, ossification of bone induration, chondrocalcinosis, hypotonia, Duchenne muscular dystrophy, tracheobronchomalacia, seizures, neurofibromatosis (e.g., NF-1), and craniosynostosis, or one or more of these symptoms) by administering the pharmaceutical composition of the first embodiment to a patient in need. The composition may be administered in an amount and for a duration sufficient to treat the disease or alleviate one or more of its symptoms. This treatment may promote bone formation in the patient. The polypeptide can be used to treat muscle weakness.

[0011] Polypeptides or pharmaceutical compositions containing them may be administered in doses ranging from about 0.01 mg / kg to about 60 mg / kg (for example, about 0.1 mg / kg to about 50 mg / kg, for example, about 0.1 mg / kg to about 20 mg / kg, or for example, about 0.1 mg / kg to about 10 mg / kg). Polypeptides may be administered once a day, once a week, once a month, or once a year (for example, once a week). In some embodiments, polypeptides are administered once or more every 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, or 31 days. Polypeptides may be administered once a week, once every two weeks, once every three weeks, or once every four weeks or more. Polypeptides may be administered in doses ranging from approximately 0.01 mg / kg / week to approximately 50 mg / kg / week (for example, approximately 0.01 mg / kg / week to approximately 40 mg / kg / week, for example, approximately 0.1 mg / kg / week to approximately 20 mg / kg / week, or for example, approximately 0.1 mg / kg / week to approximately 10 mg / kg / week). Polypeptides may be administered for at least one day, one week, one month, one year, or longer (for example, over the patient's lifetime).

[0012] Polypeptides or pharmaceutical compositions containing them may be administered subcutaneously, intravenously, intramuscularly, sublingually, subarachnoidally, or intradermally. In particular, polypeptides or compositions containing them may be administered subcutaneously or intravenously.

[0013] In some embodiments, the pharmaceutical composition is administered subcutaneously (e.g., to the abdomen or thigh). For example, about 10 mg to about 100 mg (e.g., about 10 mg, 15 mg, 20 mg, 25 mg, 30 mg, 35 mg, 40 mg, 45 mg, 50 mg, 55 mg, 60 mg, 65 mg, 70 mg, 75 mg, 80 mg, 85 mg, 90 mg, 95 mg, or 100 mg, for example, about 15 mg, 45 mg, or 90 mg) may be administered subcutaneously to a patient once or twice a week, for example, over at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 weeks or longer, or over a longer period (e.g., throughout the patient's lifetime). The composition may be administered in a volume of approximately 5 mL or less (for example, 4.0 mL, 3.0 mL, 2.0 mL, 1.0 mL, 0.9 mL, 0.8 mL, 0.7 mL, 0.6 mL, 0.5 mL, 0.4 mL, 0.3 mL, 0.2 mL, or 0.1 mL, or a volume in the range of approximately 5 mL to approximately 0.1 mL).

[0014] In some embodiments, the pharmaceutical composition is administered intravenously (IV). For example, about 10 mg to about 100 mg (e.g., about 10 mg, 15 mg, 20 mg, 25 mg, 30 mg, 35 mg, 40 mg, 45 mg, 50 mg, 55 mg, 60 mg, 65 mg, 70 mg, 75 mg, 80 mg, 85 mg, 90 mg, 95 mg, or 100 mg, e.g., about 15 mg, 45 mg, or 90 mg) may be administered intravenously to a patient once or twice a week, for example, over at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 weeks, or longer (e.g., over 3 weeks), or longer (e.g., over the patient's lifetime). For example, the pharmaceutical composition is administered intravenously in a volume of approximately 5 mL or less (e.g., 4.0 mL, 3.0 mL, 2.0 mL, 1.0 mL, 0.9 mL, 0.8 mL, 0.7 mL, 0.6 mL, 0.5 mL, 0.4 mL, 0.3 mL, 0.2 mL, or 0.1 mL, or a volume in the range of approximately 5 mL to approximately 0.1 mL).

[0015] In some embodiments, the pharmaceutical composition is administered intravenously and subcutaneously (e.g., to the abdomen or thigh). For example, the pharmaceutical composition is administered in a therapeutic regimen that combines intravenous and subcutaneous (e.g., to the abdomen or thigh) administration. For example, the composition may first be administered intravenously to the patient as a single dose in an amount of about 10 mg to about 100 mg (e.g., about 10 mg, 15 mg, 20 mg, 25 mg, 30 mg, 35 mg, 40 mg, 45 mg, 50 mg, 55 mg, 60 mg, 65 mg, 70 mg, 75 mg, 80 mg, 85 mg, 90 mg, 95 mg, or 100 mg, e.g., about 15 mg, 45 mg, or 90 mg), and then subcutaneously to the patient (e.g., to the abdomen or thigh) in one or more doses over time. For example, the subcutaneous dose may be approximately 10 mg to approximately 100 mg (for example, approximately 10 mg, 15 mg, 20 mg, 25 mg, 30 mg, 35 mg, 40 mg, 45 mg, 50 mg, 55 mg, 60 mg, 65 mg, 70 mg, 75 mg, 80 mg, 85 mg, 90 mg, 95 mg, or 100 mg, for example, approximately 15 mg, 45 mg, or 90 mg). The subcutaneous dose may be administered, for example, once or twice a week, once every two weeks, once every three weeks, or once every four weeks, for example, over a period of at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, or 50 weeks, or over a longer period (for example, over 1 to 10 years, or over the patient's lifetime). Intravenous and subcutaneous doses may be administered in volumes of approximately 5 mL or less (for example, 4.0 mL, 3.0 mL, 2.0 mL, 1.0 mL, 0.9 mL, 0.8 mL, 0.7 mL, 0.6 mL, 0.5 mL, 0.4 mL, 0.3 mL, 0.2 mL, or 0.1 mL, or volumes in the range of approximately 5 mL to approximately 0.1 mL).

[0016] Patients may be human subjects, including newborns, infants, children, adolescents, or adults.

[0017] In some embodiments, the TSAC of recombinant alkaline phosphatase polypeptide is about 1.0 mol / mol to about 6.0 mol / mol. In some embodiments, the TSAC is about 1.2 mol / mol to about 6.0 mol / mol relative to recombinant alkaline phosphatase. In some embodiments, the TSAC is about 1.5 mol / mol to about 6.0 mol / mol relative to recombinant alkaline phosphatase. In some embodiments, the TSAC is about 3.0 mol / mol to about 6.0 mol / mol relative to recombinant alkaline phosphatase. In some embodiments, the TSAC is about 3.2 mol / mol to about 5.9 mol / mol relative to recombinant alkaline phosphatase. In some embodiments, TSAC is expressed in concentrations of approximately 0.9 mol / mol, 1.0 mol / mol, 1.1 mol / mol, 1.2 mol / mol, 1.3 mol / mol, 1.4 mol / mol, 1.5 mol / mol, 1.6 mol / mol, 1.7 mol / mol, 1.8 mol / mol, 1.9 mol / mol, 2.0 mol / mol, 2.1 mol / mol, 2.2 mol / mol, 2.3 mol / mol, 2.4 mol / mol, 2.5 mol / mol, 2.6 mol / mol, 2.7 mol / mol, 2.8 mol / mol, 2.9 mol / mol, 3.0 mol / mol, 3.1 mol / mol, 3.2 mol / mol, and 3.3 mol / mol relative to recombinant alkaline phosphatase. ol, about 3.4mol / mol, about 3.5mol / mol, about 3.6mol / mol, about 3.7mol / mol, about 3.8mol / mol, about 3.9mol / mol, about 4.0mol / mol, about 4.1mol / mol, about 4.2mol / mol, about 4.3mol / mol, about 4.4mol / mol, about 4.5mol / mol, about 4.6mol / mol, about 4.7mol / mol, about 4.8 mol / mol, about 4.9 mol / mol, about 5.0 mol / mol, about 5.1 mol / mol, about 5.2 mol / mol, about 5.3 mol / mol, about 5.4 mol / mol, about 5.5 mol / mol, about 5.6 mol / mol, about 5.7 mol / mol, about 5.8 mol / mol, about 5.9 mol / mol, or about 6.0 mol / mol.In some embodiments, the TSAC is about 3.2 mol / mol relative to recombinant alkaline phosphatase. In some embodiments, the TSAC is about 5.0 mol / mol relative to recombinant alkaline phosphatase. In some embodiments, the TSAC is about 5.9 mol / mol relative to recombinant alkaline phosphatase.

[0018] In some embodiments, the composition has a pH of about 7.3 and contains about 10 mM phosphate, about 140 mM proline, about 140 mM sucrose, about 0.05% polyoxyethylene (20) sorbitan monooleate (e.g., PS80), and has a TSAC value of about 3.0 mol / mol to about 6.0 mol / mol.

[0019] In some embodiments, the method results in an AUC of about 50 μg×hour / mL to about 4000 μg×hour / mL in the patient's blood

[0021] , -9 , -7 , -8 , -5 , -6 , max , , max , ,

[0020] , , , For example, the method can result in an AUC of about 1000 μg×hour / mL to about 3000 μg×hour / mL in the patient's blood 0-168h In some embodiments, the method results in a C of about 0.5 μg / mL to about 25 μg / mL in the patient's blood max For example, the method can result in a C of about 0.6 μg / mL to about 20 μg / mL in the patient's blood max can occur.

[0020] Definition The term "about" means ±10% of the recited value. All measured values reported herein should be understood to be modified by the term "about" whether explicitly used or not, unless otherwise explicitly stated.

[0021] The term "bone targeting moiety" means that the bone targeting moiety alone has at least about 1×10 -5 M or more, for example, about 10 -6 M, about 10 -7 M, about 10 -8 M, about 10 -9This refers to an amino acid sequence of at least 3 amino acid residues that has sufficient affinity to the bone matrix to have an in vivo binding affinity of M or greater.

[0022] As used herein, the term "catalytically capable" refers to sALPs that hydrolyze inorganic pyrophosphate (PPi), a bone mineralization inhibitor, to produce inorganic phosphate (Pi), thereby reducing the extracellular concentration of PPi. Therefore, catalytically capable sALPs improve skeletal mineralization by regulating the concentration of PPi.

[0023] The term "Fc" refers to the fragment crystallized region of an immunoglobulin containing the CH2 and CH3 domains of the immunoglobulin heavy chain, e.g., IgG1, IgG2, IgG3, or IgG4. Fc may also include any portion of the hinge region connecting the Fab region and the Fc region. Fc may be derived from mammals, including humans, and may be post-translationally modified (e.g., by glycosylation or sialylation). In non-limiting embodiments, Fc may be the fragment crystallized region of human IgG2 / 4 of SEQ ID NO: 4.

[0024] "Fragment" preferably means a portion of a polypeptide or nucleic acid molecule containing at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or more of the total length of the reference nucleic acid molecule or polypeptide. Fragments are, for example, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250 , 260, 270, 280, 290, 300, 310, 320, 330, 340, 350, 360, 370, 380, 390, 400, 500, 600, 700, 800, 900, 1,000, 1100, 1200, 1300, 1400, 1500, 1600, 1700, 180 A molecule may contain 0, 1900, 2000, 2100 or more nucleotides, up to the full length of the nucleic acid molecule, or 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 260, 270, 280, 290, 300, 400, 500, 600, 700 or more amino acid residues, up to the full length of the polypeptide.

[0025] The terms “hypophosphatasia” and “HPP,” as used herein, refer to a rare hereditary skeletal disorder caused by one or more loss-of-function mutations in the ALPL (alkaline phosphatase, liver / bone / kidney) gene, which encodes tissue-nonspecific alkaline phosphatase (TNSALP). HPP may further be characterized by infantile HPP, childhood HPP, birth HPP (e.g., benign birth HPP or lethal birth HPP), dental HPP, adolescent HPP, or adult HPP. For example, “childhood HPP” refers to HPP patients aged approximately 5 to 12 years, “adolescent HPP” refers to HPP patients aged approximately 13 to 17 years, and “adult HPP” refers to HPP patients aged approximately 18 years or older. The term “adult HPP” as used herein refers to a condition or phenotype characterized by the presence of one or more of the following symptoms: elevated blood and / or urinary levels of inorganic pyrophosphate (PPi), hypocalcification, hypercalciuria, one or more skeletal deformities, hypotonia, muscle weakness, rheumatic complications, duck gait, dysgait, bone pain, pain, fracture, calcium pyrophosphate dihydrate crystal deposition, pseudogout, arthritis, pyrophosphate arthropathy, chondrocalcinosis, calcific periarthritis, and pseudofracture. The term “adolescent HPP” as used herein means a condition or phenotype characterized by elevated blood or urinary levels of PPi, PEA, or PLP; the presence of one or more of the following symptoms: osteomalacia, one or more skeletal deformities, hypotonia, weakness, rheumatoid complications, arthritis, pseudogout, duck gait, dysgait, bone pain, pain, premature tooth loss, hypocalcification, pulmonary hypoplasia, respiratory failure, seizures, hypercalciuria, short stature, and growth retardation. The term “childhood HPP” as used herein means a condition or phenotype characterized by elevated blood or urinary levels of PPi, PEA, or PLP; and the presence of one or more of the following symptoms: rickets, rickets ribs, one or more skeletal deformities, hypotonia, weakness, rheumatic complications, arthritis, pseudogout, duck gait, dysgait, bone pain, pain, premature tooth loss, hypocalcification, motor developmental delay, seizures, hypercalciuria, short stature, fractures, pseudofractures, and growth retardation.

[0026] The term “nucleic acid” or “nucleic acid molecule” means a macromolecule having a sequence of two or more covalently bonded, naturally occurring or modified nucleotides, such as RNA or DNA. Nucleic acid molecules may be, for example, single-stranded or double-stranded, and may contain modified or unmodified nucleotides, or mixtures or combinations thereof. Various salts, mixed salts, and free acids of nucleic acid molecules are also included.

[0027] "To treat," "to treat," and "treatment" mean, for example, the medical management of a patient for the purpose of treating, alleviating, stabilizing, reducing the likelihood of, or preventing a medical condition such as HPP (e.g., pediatric, adolescent, or adult HPP) or one or more of its symptoms, and / or the management of a patient who exhibits or is likely to exhibit a medical condition such as HPP, by administering a pharmaceutical composition (e.g., sALP as described herein). To treat (and other forms as used herein) includes active treatment, i.e., treatment specifically aimed at improving or relating to the cure of a disease, pathological condition, disorder, or event, and also includes causal treatment, i.e., treatment aimed at eliminating the cause of the associated disease, pathological condition, disorder, or event. In addition, this term includes palliative care, i.e., treatment designed to alleviate or improve at least one symptom rather than cure the disease, pathological condition, disorder, or event; symptomatic care, i.e., treatment targeting the systemic symptoms of the associated disease, pathological condition, disorder, or event; prophylactic care, i.e., treatment aimed at minimizing, partially or completely inhibiting, the onset of the associated disease, pathological condition, disorder, or event in, for example, a patient who is not yet diseased but is susceptible to or at risk of developing a particular disease, pathological condition, disorder, or event; and supportive care, i.e., treatment used to complement other specific treatments aimed at improving the associated disease, pathological condition, disorder, or event.

[0028] The terms “peptide,” “polypeptide,” and “protein” are used interchangeably and refer to any chain of two or more natural or non-natural amino acid residues that constitute all or part of a naturally occurring or non-natural polypeptide or peptide, regardless of post-translational modifications (e.g., glycosylation, sialylation, or phosphorylation), as described herein.

[0029] The terms “sALP,” “soluble alkaline phosphatase,” and “extracellular domain of alkaline phosphatase” are used synonymously (unless otherwise indicated in context) and refer to soluble, non-membrane-bound alkaline phosphatase, or its biologically active fragments or variants. sALP includes, for example, alkaline phosphatases lacking a C-terminal GPI signal sequence that retain alkaline phosphatase activity, such as the ability to hydrolyze PPi or other native or artificial substrates, as well as additional variants and analogues. This includes, unless otherwise specified, soluble fragments corresponding to the extracellular domains of TNSALP, PALP, GLALP, and IALP, as well as their biologically active fragments or variants. Mature sALP lacks the GPI membrane anchor and signal peptide and is cleaved during processing.

[0030] The terms "ALP" and "alkaline phosphatase" refer to naturally occurring alkaline phosphatases such as TNSALP, PALP, GLALP, and IALP that can hydrolyze PPi or other natural or artificial substrates.

[0031] The terms “pharmaceutically acceptable carrier” or “pharmaceutically acceptable excipient” mean a carrier or excipient that is physiologically acceptable to the patient being treated while maintaining the therapeutic properties of the compound administered together. One exemplary pharmaceutically acceptable carrier is physiological saline. Other physiologically acceptable carriers and their formulations are well known to those skilled in the art and are described, for example, in Remington's Pharmaceutical Sciences (Remington: The Science and Practice of Pharmacy, 22nd Ed., Allen, Ed. 2012).

[0032] The term “pharmaceutical composition” means a composition containing a polypeptide or nucleic acid molecule as described herein, prepared with pharmaceutically acceptable excipients, and which is manufactured or marketed with the approval of a government regulatory body as part of a therapeutic regimen for treating or preventing a disease or event in a patient. Pharmaceutical compositions may be prepared, for example, for subcutaneous administration (e.g., into the abdomen or thigh), for intravenous administration (e.g., as a sterile solution free of particulate embolisms and in a solvent system suitable for intravenous use), for oral administration (e.g., tablets, capsules, caplets, gel caps, or syrups), or in any other formulation form as described herein, e.g., in unit dosage forms.

[0033] The term "patient" means a mammal including, but not limited to, human mammals, or non-human mammals such as cattle, horses, dogs, sheep, or cats.

[0034] The term “therapeutic dose” means an amount of polypeptide or nucleic acid molecule described herein that is sufficient to substantially treat, prevent, delay, suppress, or inhibit any symptom of any disease or condition described herein, in particular HPP. The therapeutic dose of a composition described herein may depend on the severity of the disorder being treated, as well as the patient’s condition, weight, and overall health, and may be determined by a person skilled in the art taking such factors into consideration. The therapeutic dose of a composition described herein may be administered to a patient as a single dose or as repeated doses over a period of time.

[0035] The term “Total Sialic Acid Content” or “TSAC,” as used herein, refers to the amount of sialic acid (carbohydrate) on a particular protein molecule. This is expressed as the number of moles of sialic acid incorporated per mole of protein, i.e., “mol / mol.” TSAC concentration is measured during the purification process. For example, one method of TSAC quantification involves releasing TSAC from alkaline phosphatase using acid hydrolysis, and then detecting the released TSAC via electrochemical detection using high-speed anion exchange chromatography with pulsed current measurement detection technique (“HPAE-PAD”).

[0036] The term "sialic acid" generally refers to N-substituted or O-substituted derivatives of neuraminic acid, which is a monosaccharide having a 9-carbon backbone. Sialic acid also refers specifically to the compound N-acetylneuraminic acid, which may be abbreviated as Neu5Ac or NANA. The presence of sialic acid may affect absorption, serum half-life, and clearance of glycoproteins from serum, as well as the physical, chemical, and immunogenic properties of glycoproteins. In some embodiments of this disclosure, sialic acid associated with alkaline phosphatases, e.g., ALP201, affects the in vivo exposure and half-life of the molecule under physiological conditions. In some embodiments, precise and predictable control of the total sialic acid content (TSAC) of alkaline phosphatases serves as a quality control attribute.

[0037] As used herein, when a polypeptide or nucleic acid sequence is said to have "at least X% sequence identity" with a reference sequence, it means that, when the sequence is optimally aligned, at least X percent of amino acid residues or nucleotides in the polypeptide or nucleic acid are identical to those in the reference sequence. Optimal alignment of a sequence can be determined by various methods within the scope of the Art, for example, the Smith-Waterman alignment algorithm (Smith et al., J.Mol.Biol.147:195-7, 1981) and BLAST (Basic Local Alignment Search Tool; Altschul et al., J.Mol.Biol.215:403-10, 1990). These and other alignment algorithms are accessible using commonly available computer software, such as “Best Fit” (Smith and Waterman, Advances in Applied Mathematics, 482-489, 1981), incorporated in GENEMATCHER PLUS® (Schwarz and Dayhof, Atlas of Protein Sequence and Structure, Dayhoff, MO, Ed., pp 353-358, 1979), BLAST, BLAST-2, BLAST-P, BLAST-N, BLAST-X, WU-BLAST-2, ALIGN, ALIGN-2, CLUSTAL, or Megalign (DNASTAR). In addition, those skilled in the art can determine appropriate parameters for measuring alignment, including any algorithms required to obtain optimal alignment over the lengths of the sequences being compared.

[0038] The words “preferred” and “preferred” refer to embodiments of the disclosed compounds, compositions, and methods that may provide particular benefits under specific circumstances. However, other embodiments may also be preferred under the same or other circumstances. Furthermore, the description of one or more preferred embodiments does not mean that other embodiments are not useful, and the use of this term is not intended to exclude other embodiments from the scope of this disclosure.

[0039] In any method disclosed herein, including individual steps, the steps may be performed in any executable order, and any combination of two or more steps may be performed simultaneously as needed.

[0040] The above summary is not intended to describe all embodiments or implementations of the disclosed compounds, compositions, and methods. The following description provides more specific examples of exemplary embodiments. Throughout this application, guidance may be provided through lists of examples, which may be used in various combinations. In each case, the enumerated list serves only as a representative group and should not be interpreted as an exclusive list.

[0041] All headings are intended for the reader's convenience and, unless otherwise specified, are not used to limit the meaning of the text that follows them. [Brief explanation of the drawing]

[0042] [Figure 1] This is a pair of saturation curves showing the relationship between the PPi level of ALP201 (SEQ ID NO: 5; left) and asfotase alpha (SEQ ID NO: 6; right), which increases with the rate of PPi hydrolysis. [Figure 2] This is a pair of saturation curves showing the relationship between the rate of PLP hydrolysis and the increase in PLP levels for ALP201 (left) and asfotase alpha (right). [Figure 3]This graph shows the concentration of active alkaline phosphatase enzyme in Akp2GW(- / -) mouse plasma at the end of the study for each treatment group. Abbreviations: q1w = once a week; q2d = once every two days; qd = once a day. [Figure 4] This graph shows the alkaline phosphatase activity levels in mouse femoral tissue at the end of the experiment on day 36 / 37. Abbreviations: q2d = once every two days; qd = once a day. [Figure 5] This is a representative survival curve for a pair of Akp2GW(- / -) mice treated with ALP201 and asfotase alfa in a 36-day efficacy study. Abbreviations: MED = minimum effective dose; PBS = phosphate-buffered saline; q1w = once a week; q2d = once every two days; qd = once a day. [Figure 6] This graph shows a comparison of dose-response modeling results between ALP201 and asfotase alfa. Abbreviations: ALP = alkaline phosphatase; DR = dose-response; MED = minimum effective dose; POC = proof of concept. [Figure 7] This graph shows the simulated human ALP201 concentration-time profiles for each first-in-human (FIH) cohort. The gray area represents the 90% prediction period, i.e., the 5%–95% range, and the solid black line is the median of the simulated concentration-time profile; LLOQ is the horizontal dashed line of the PK assay, abbreviated as LLOQ = lower limit of quantification (0.15 μg / mL). [Figure 8] Graphs A and B show the mean (SD) ALP201 active plasma concentration-time profiles after intravenous administration (A) and subcutaneous administration (B) to male C57BL / 6 mice. [Figure 9] A and B are graphs showing the mean (SD) ALP201-active plasma concentration-time profiles of ALP201 (lot TSAC=5.9) after intravenous administration (A) and subcutaneous administration (B) to male C57Bl / 6 mice. [Figure 10]A and B are graphs showing the mean (SD) ALP201-active plasma concentration-time profiles of ALP201 (TSAC=5.0 lot) after intravenous administration (A) and subcutaneous administration (B) to male C57Bl / 6 mice. [Figure 11] A and B are graphs showing the mean (SD) ALP201-active plasma concentration-time profiles of ALP201 (TSAC=3.2 lot) after intravenous administration (A) and subcutaneous administration (B) to male C57Bl / 6 mice. [Figure 12] This graph shows the mean (SD) ALP201 concentration-time profile of ALP201 TSAC values ​​(3.2, 5.0, and 5.9) in active plasma after intravenous administration to male C57BL / 6 mice. [Figure 13] This graph shows the mean (SD) ALP201 TSAC values ​​in male C57BL / 6 mice, and the concentration-time profile of ALP201-active plasma. [Figure 14] This graph shows a comparison of the mean (±SD) plasma concentration-time profiles of ALP201 and asfotase alfa after a single IV and subcutaneous administration in rats. Abbreviations: IV = intravenous; SC = subcutaneous; SD = standard deviation. Source data for asfotase alfa: pooled sex, IV at 3 mg / kg, and subcutaneous. [Figure 15] This graph shows a comparison of the mean (±SD) plasma concentration-time profiles of ALP201 and asfotase alfa after a single IV and SC administration in monkeys. [Figure 16] This graph shows a comparison of dose-response modeling between ALP201 and asfotase alfa (STRENSIQ®). Abbreviations: ALP = alkaline phosphatase; DR = dose-response; MED = minimum effective dose; POC = proof of concept. [Modes for carrying out the invention]

[0043] The present invention features soluble alkaline phosphatase polypeptides (e.g., those having the sequence of SEQ ID NO: 5, and its variants having up to 80% or more sequence identity, wherein the polypeptide contains one, two, or three mutations selected from E108M, N213Q, and N286Q of SEQ ID NO: 5), fragments thereof, and fusion proteins thereof, nucleic acid molecules encoding them, and methods of using the polypeptides and nucleic acid molecules to treat disorders of bone mineralization, such as hypophosphatasia (HPP), or one or more symptoms thereof. The polypeptides include soluble alkaline phosphatase (sALP) or fragments thereof derived from naturally occurring alkaline phosphatase (ALP). Alkaline phosphatases include various isozymes that are expressed differently in different tissues. The four main ALP isozymes are tissue-nonspecific alkaline phosphatase (TNSALP), placental alkaline phosphatase (PALP), germline alkaline phosphatase (GLALP), and enteric alkaline phosphatase (IALP). Therefore, proteins derived from these ALP isozymes are also characteristic.

[0044] The polypeptides described herein are prepared, for example, in a pharmaceutical composition containing one or more of phosphates, proline, and sucrose. These components impart beneficial characteristics to the polypeptide, such as improved stability in the formulation, reduced aggregation, reduced cleavage-type protein products, and increased purity of the desired protein product.

[0045] HPP is a rare hereditary skeletal disorder with an incidence of 1 in 100,000 live births in the most severe forms of the disease. The disorder is usually caused by loss-of-function mutations in the gene encoding TNSALP. HPP presents with a very wide range of symptoms and severity, from early tooth loss to near-complete absence of bone mineralization in utero. The symptoms of HPP vary significantly from patient to patient and also significantly with age. Many HPP patients exhibit skeletal changes, short stature, chronic pain, leg pain, gait disturbances, and early non-traumatic tooth loss. Due to endogenous loss-of-function mutations in TNSALP, HPP patients require functional ALP activity from the polypeptides described herein to restore innate ALP activity and result in normal bone matrix mineralization.

[0046] Soluble alkaline phosphatase polypeptide The polypeptides described herein include soluble alkaline phosphatases (sALP), such as mutant tissue-nonspecific alkaline phosphatases (TNSALP) or fragments thereof. sALP may be fused to a polyaspartic acid of an Fc region and sequence n ("Dn", where n is, for example, equal to 3-20) (sALP-Fc-Dn). The polypeptide may contain human TNSALP, such as a soluble fragment of human TNSALP (e.g., residues 1-491 or 1-485 of SEQ ID NO: 1). The polypeptide may contain one, two, or three of the mutants E108M, N213Q, and / or N286Q compared to SEQ ID NO: 1. For example, sALP may have at least 80% (e.g., at least 85%, 90%, 95%, 97%, 99%, or 100%) identity with SEQ ID NO: 2 or 3. The Fc region may be an IgG2 / 4Fc region. For example, the Fc region may have at least 80% identity (e.g., at least 85%, 90%, 95%, 97%, 99%, or 100%) with respect to SEQ ID NO: 4. The polyaspartic acid may contain, for example, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 aspartic acid residues. In some embodiments, the polyaspartic acid contains 10 aspartic acid residues (D10). In some embodiments, the polypeptide has at least 80% identity (e.g., at least 85%, 90%, 95%, 97%, 99%, or 100%) with respect to SEQ ID NO: 5. For example, the polypeptide may contain or consist of the polypeptide of SEQ ID NO: 5. The polypeptide may consist of SEQ ID NO: 5.

[0047] In some embodiments, the alkaline phosphatase moiety of the polypeptide has at least 80% (e.g., at least 85%, 90%, 95%, 97%, 99%, or 100%) sequence identity to SEQ ID NO: 3. This polypeptide may be further linked to an Fc region (e.g., an IgG1, IgG2, IgG3, or IgG4Fc region) and / or a polyaspartic acid region. In some embodiments, the polypeptide includes an IgG2 / 4Fc region and has at least 80% (e.g., at least 85%, 90%, 95%, 97%, 99%, or 100%) identity to, for example, SEQ ID NO: 4. The polyaspartic acid may include, for example, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 aspartic acid residues. In some embodiments, the polyaspartic acid includes 10 aspartic acid residues (D10).

[0048] Sequence ID 1 - Human TNSALP lacking signal peptide (UniProt P05186.4) LVPEKEKDPKYWRDQAQETLKYALELQKLNTNVAKNVIMFLGDGMGVSTVTAARILKGQLHHNPGEETRLEMDKFPFVALSKTYNTNAQVPDSAGTATAYLCGVKANEGTVGVSAATERSRCNTTQ GNEVTSILRWAKDAGKSVGIVTTTRVNHATPSAAYAHSADRDWYSDNEMPPEALSQGCKDIAYQLMHNIRDIDVIMGGGRKYMYPKNKTDVEYESDEKARGTRLDGLDLVDTWKSFKPRYKHSHFIW NRTELLTLDPHNVDYLLGLFEPGDMQYELNRNNVTDPSLSEMVVVAIQILRKNPKGFFLLVEGGRIDHGHHEGKAKQALHEAVEMDRAIGQAGSLTSSEDTLTVVTADHSHVFTFGGYTPRGNSIFG LAPMLSDTDKKPFTAILYGNGPGYKVVGGERENVSMVDYAHNNYQAQSAVPLRHETHGGEDVAVFSKGPMAHLLHGVHEQNYVPHVMAYAACIGANLGHCAPASSAGSLAAGPLLLALALYPLSVLF Sequence ID 2 - Human TNSALP (1-485; E108M, N213Q, N286Q) LVPEKEKDPKYWRDQAQETLKYALELQKLNTNVAKNVIMFLGDGMGVSTVTAARILKGQLHHNPGEETRLEMDKFPFVALSKTYNTNAQVPDSAGTATAYLCGVKANMGTVGVSAATERSR CNTTQGNEVTSILRWAKDAGKSVGIVTTTRVNHATPSAAYAHSADRDWYSDNEMPPEALSQGCKDIAYQLMHNIRDIDVIMGGGRKYMYPKQKTDVEYESDEKARGTRLDGLDLVDTWKSF KPRYKHSHFIWNRTELLTLDPHNVDYLLGLFEPGDMQYELNRNQVTDPSLSEMVVVAIQILRKNPKGFFLLVEGGRIDHGHHEGKAKQALHEAVEMDRAIGQAGSLTSSEDTLTVVTADHS HVFTFGYTPRGNSIFGLAPMLSDTDKKPFTAILYGNGPGYKVVGGERENVSMVDYAHNNYQAQSAVPLRHETHGGEDVAVFSKGPMAHLLHGVHEQNYVPHVMAYAACIGANLGHCAPASS Sequence ID 3 - Human TNSALP (1-491; E108M, N213Q, N286Q) LVPEKEKDPKYWRDQAQETLKYALELQKLNTNVAKNVIMFLGDGMGVSTVTAARILKGQLHHNPGEETRLEMDKFPFVALSKTYNTNAQVPDSAGTATAYLCGVKANMGTVGVSAATERSRC NTTQGNEVTSILRWAKDAGKSVGIVTTTRVNHATPSAAYAHSADRDWYSDNEMPPEALSQGCKDIAYQLMHNIRDIDVIMGGGRKYMYPKQKTDVEYESDEKARGTRLDGLDLVDTWKSFKPR YKHSHFIWNRTELLTLDPHNVDYLLGLFEPGDMQYELNRNQVTDPSLSEMVVVAIQILRKNPKGFFLLVEGGRIDHGHHEGKAKQALHEAVEMDRAIGQAGSLTSSEDTLTVVTADHSHVFTF GGYTPRGNSIFGLAPMLSDTDKKPFTAILYGNGPGYKVVGGERENVSMVDYAHNNYQAQSAVPLRHETHGGEDVAVFSKGPMAHLLHGVHEQNYVPHVMAYAACIGANLGHCAPASSAGSLAA Sequence ID 4-IgG2 / 4Fc VECPPCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKT ISKAKGQPREPQVYTLPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK Sequence ID 5-ALP201 LVPEKEKDPKYWRDQAQETLKYALELQKLNTNVAKNVIMFLGDGMGVSTVTAARILKGQLHHNPGEETRLEMDKFPFVALSKTYNTNAQVPDSAGTATAYLCGVKANMGTVGVSAATERSRCNTTQGNEVTSILRWAKDAGKSVGIVTTTRVNHATPSAAYAHSADRDWYSDNEMPPEALS QGCKDIAYQLMHNIRDIDVIMGGGRKYMYPKQKTDVEYESDEKARGTRLDGLDLVDTWKSFKPRYKHSHFIWNRTELLTLDPHNVDYLLGLFEPGDMQYELNRNQVTDPSLSEMVVVAIQILRKNPKGFFLLVEGGRIDHGHHEGKAKQALHEAVEMDRAIGQAGSLTSSEDTLTVVTADH SHVFTFGYTPRGNSIFGLAPMLSDTDKKPFTAILYGNGPGYKVVGGERENVSMVDYAHNNYQAQSAVPLRHETHGGEDVAVFSKGPMAHLLHGVHEQNYVPHVMAYAACIGANLGHCAPASSAGSLAAVECPCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFN WYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGKDDDDDDDDDD SEQ ID NO: 6 - Asfotase alpha LVPEKEKDPKYWRDQAQETLKYALELQKLNTNVAKNVIMFLGDGMGVSTVTAARILKGQLHHNPGEETRLEMDKFPFVALSKTYNTNAQVPDSAGTATAYLCGVKANEGTVGVSAATERS RCNTTQGNEVTSILRWAKDAGKSVGIVTTTRVNHATPSAAYAHSADRDWYSDNEMPPEALSQGCKDIAYQLMHNIRDIDVIMGGGRKYMYPKNKTDVEYESDEKARGTRLDGLDLVDTWK SFKPRYKHSHFIWNRTELLTLDPHNVDYLLGLFEPGDMQYELNRNNVTDPSLSEMVVVAIQILRKNPKGFFLLVEGGRIDHGHHEGKAKQALHEAVEMDRAIGQAGSLTSSEDTLTVVTA DHSHVFTFGYTPRGNSIFGLAPMLSDTDKKPFTAILYGNGPGYKVVGGERENVSMVDYAHNNYQAQSAVPLRHETHGGEDVAVFSKGPMAHLLHGVHEQNYVPHVMAYAACIGANLGHC APASSLKDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTI SKAKGQPREPQVYTLPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKDIDDDDDDDDDD

[0049] Total sialic acid content As described herein, TSAC can affect the half-life of recombinant alkaline phosphatase under physiological conditions. Therefore, TSAC levels may function as a quality attribute for recombinant alkaline phosphatases, such as ALP201. Controlling the TSAC range during polypeptide production can improve batch-to-batch reproducibility and reduce heterogeneity in the produced polypeptides. In some embodiments, the TSAC is about 0.8 mol / mol to about 8.0 mol / mol relative to the recombinant alkaline phosphatase. In some embodiments, the TSAC is about 0.9 mol / mol to about 7.0 mol / mol relative to the recombinant alkaline phosphatase. In some embodiments, the TSAC is about 1.0 mol / mol to about 6.0 mol / mol relative to the recombinant alkaline phosphatase. In some embodiments, the TSAC is about 1.2 mol / mol to about 6.0 mol / mol relative to the recombinant alkaline phosphatase. In some embodiments, the TSAC is about 1.5 mol / mol to about 6.0 mol / mol relative to the recombinant alkaline phosphatase. In some embodiments, the TSAC concentration is approximately 2.0 mol / mol to approximately 6.0 mol / mol relative to recombinant alkaline phosphatase. In some embodiments, the TSAC concentration is approximately 3.2 mol / mol to approximately 5.9 mol / mol relative to recombinant alkaline phosphatase.In some embodiments, TSAC is expressed in concentrations of approximately 0.9 mol / mol, 1.0 mol / mol, 1.1 mol / mol, 1.2 mol / mol, 1.3 mol / mol, 1.4 mol / mol, 1.5 mol / mol, 1.6 mol / mol, 1.7 mol / mol, 1.8 mol / mol, 1.9 mol / mol, 2.0 mol / mol, 2.1 mol / mol, 2.2 mol / mol, 2.3 mol / mol, 2.4 mol / mol, 2.5 mol / mol, 2.6 mol / mol, 2.7 mol / mol, 2.8 mol / mol, 2.9 mol / mol, 3.0 mol / mol, 3.1 mol / mol, 3.2 mol / mol, and 3.3 mol / mol relative to recombinant alkaline phosphatase. The concentrations are approximately 3.4 mol / mol, 3.5 mol / mol, 3.6 mol / mol, 3.7 mol / mol, 3.8 mol / mol, 3.9 mol / mol, 4.0 mol / mol, 4.1 mol / mol, 4.2 mol / mol, 4.3 mol / mol, 4.4 mol / mol, 4.5 mol / mol, 4.6 mol / mol, 4.7 mol / mol, 4.8 mol / mol, 4.9 mol / mol, 5.0 mol / mol, 5.1 mol / mol, 5.2 mol / mol, 5.3 mol / mol, 5.4 mol / mol, 5.5 mol / mol, 5.6 mol / mol, 5.7 mol / mol, 5.8 mol / mol, 5.9 mol / mol, or 6.0 mol / mol. In some embodiments, TSAC is approximately 3.2 mol / mol relative to recombinant alkaline phosphatase. In some embodiments, the TSAC concentration is approximately 5.0 mol / mol relative to recombinant alkaline phosphatase. In some embodiments, the TSAC concentration is approximately 5.9 mol / mol relative to recombinant alkaline phosphatase.

[0050] Pharmaceutical composition Polypeptides described herein (for example, polypeptides having the sequence of SEQ ID NO: 5, or variants thereof having at least 85% sequence identity thereto (for example, polypeptides containing one or more or all of the mutations E108M, N213Q, and N286Q compared to SEQ ID NO: 1 or SEQ ID NO: 5)) can be prepared as pharmaceutical compositions by various methods known in the art.

[0051] The composition may contain one or more or all of phosphates, proline, and sucrose. For example, the composition may contain phosphates and sucrose. For example, the composition may contain phosphates (e.g., sodium phosphate) at concentrations of, for example, about 1 mM to about 100 mM, or about 5 mM to about 20 mM phosphate, for example, about 2 mM, 3 mM, 4 mM, 5 mM, 6 mM, 7 mM, 8 mM, 9 mM, 10 mM, 20 mM, 30 mM, 40 mM, 50 mM, 60 mM, 70 mM, 80 mM, 90 mM, or 100 mM, for example, about 10 mM. The composition may further contain proline and / or sucrose. The composition may further contain proline. The composition may further contain sucrose. For example, the composition contains approximately 1 mM to 500 mM proline, for example, approximately 70 mM to 280 mM, for example, approximately 50 mM to 200 mM, for example, approximately 2 mM, 3 mM, 4 mM, 5 mM, 6 mM, 7 mM, 8 mM, 9 mM, 10 mM, 20 mM, 30 mM, 40 mM, 50 mM, 60 mM, 70 mM, 80 mM, 90 mM, 100mM, 110mM, 120mM, 130mM, 140mM, 150mM, 160mM, 170mM, 180mM, 190mM, 200mM, 300mM, 400mM, or 500mM, for example, about 140mM of proline and / or about 1mM to about 500mM of sucrose, for example, about 70mM to about 280mM, for example. For example, approximately 50mM to approximately 200mM, such as approximately 2mM, 3mM, 4mM, 5mM, 6mM, 7mM, 8mM, 9mM, 10mM, 20mM, 30mM, 40mM, 50mM, 60mM, 70mM, 80mM, 90mM, 100mM, 110mM, 120mM, 130mM, 140mM, 150mM, 160mM, 170mM The composition may contain sucrose in concentrations of approximately 140 mM, 190 mM, 200 mM, 210 mM, 220 mM, 230 mM, 240 mM, 250 mM, 260 mM, 270 mM, 280 mM, 290 mM, 300 mM, 350 mM, 400 mM, 450 mM, or 500 mM, for example, about 140 mM sucrose or about 210 mM sucrose. In some embodiments, the composition contains a molar ratio of proline:sucrose of approximately 1:0 to about 1:3, for example, about 1:1. In some embodiments, the composition contains about 140 mM proline. In some embodiments, the formulation contains about 140 mM sucrose.In some embodiments, the formulation contains about 210 mM sucrose. In some embodiments, the formulation contains about 210 mM sucrose but does not contain proline. In some embodiments, the composition contains about 210 mM sucrose and about 10 mM phosphate (e.g., sodium phosphate). The formulation may further contain about 0.01% to about 0.5%, for example, about 0.01% to about 0.1%, for example, about 0.05% of sorbitan polyoxyethylene (20) monooleate (e.g., polysorbate 80 (PS80)). In some embodiments, the composition is prepared at a pH of about 7.0 to about 7.6 (e.g., about 7.1, 7.2, 7.3, 7.4, 7.5, or 7.6, for example, about 7.3). In some embodiments, the composition has a pH of about 7.3 and comprises about 10 mM phosphate, about 140 mM proline, about 140 mM sucrose, and about 0.05% sorbitan polyoxyethylene (20) monooleate (e.g., PS80).

[0052] The composition may be prepared as a solution containing polypeptide in an amount of, for example, about 0.1 mg / mL to about 200 mg / mL, for example, about 100 mg / mL. The composition may be prepared for intravenous or subcutaneous administration with polypeptide dosages of, for example, about 0.1 mg / mL to about 10 mg / mL. The composition may be prepared as a solution in a volume of about 0.1 mL to about 50 mL (for example, about 0.1 to about 10 mL, for example, about 0.1 mL, 0.2 mL, 0.3 mL, 0.4 mL, 0.5 mL, 0.6 mL, 0.7 mL, 0.8 mL, 0.9 mL, or 1.0 mL, for example, about 1 mL to about 10 mL, for example, about 2 mL, 3 mL, 4 mL, 5 mL, 6 mL, 7 mL, 8 mL, 9 mL, or 10 mL). In some embodiments, the composition is prepared in about 1 mL.

[0053] This disclosure also features vials containing pharmaceutical compositions as described herein. The vials may contain the solution in a volume of, for example, about 0.1 mL to about 10 mL (e.g., about 1 mL). The vials may contain the polypeptide in an amount of, for example, about 0.1 mg / mL to about 500 mg / mL, for example, about 1 mg / mL to about 200 mg / mL, for example, about 50 mg / mL to about 150 mg / mL, for example, about 100 mg / mL of the polypeptide (e.g., the polypeptide of SEQ ID NO: 5, or a variant having at least 85% sequence identity thereto).

[0054] For example, the vial may contain a solution of about 0.25 mL, about 0.5 mL, about 0.75 mL, or about 1.0 mL in volume, containing the polypeptide of Sequence ID No. 5 at a concentration of about 50 to about 100 mg / mL, wherein the solution has a pH of about 7.3 and contains about 10 mM phosphate, about 140 mM proline, about 140 mM sucrose, and about 0.05% sorbitan polyoxyethylene (20) monooleate (e.g., PS80).

[0055] formulation Compositions comprising sALP and sALP fusion polypeptides (e.g., a polypeptide having the sequence of SEQ ID NO: 5, or a variant thereof having at least 85% sequence identity thereto (e.g., a polypeptide containing one or more or all of the mutations E108M, N213Q, and N286Q compared to SEQ ID NO: 1 or SEQ ID NO: 5)) can be prepared according to standard methods. For example, an sALP composition may be prepared, for example, as a buffer solution suitable for storage at 2–8°C (e.g., 4°C) at a suitable concentration. An sALP composition may also be prepared for storage at temperatures below 0°C (e.g., -20°C or -80°C). An sALP composition may further be prepared for storage at 2–8°C (e.g., 4°C) for up to two years (e.g., one month, two months, three months, four months, five months, six months, seven months, eight months, nine months, ten months, eleven months, one year, one and a half years, or two years). Therefore, the compositions described herein can be prepared to be stable when stored at 2–8°C (e.g., 4°C) for at least one year. The compositions can be prepared in suitable volumes, for example, about 0.1 mL to about 10 mL.

[0056] A composition containing sALP and sALP fusion polypeptides (for example, a polypeptide having the sequence of SEQ ID NO: 5, or a variant thereof having at least 85% sequence identity thereto (for example, a polypeptide containing one or more or all of the mutations E108M, N213Q, and N286Q compared to SEQ ID NO: 1 or SEQ ID NO: 5)) can be in liquid form.

[0057] For example, compositions intended for systemic or local delivery may be in the form of injectable solutions or infusible solutions. Therefore, sALP compositions (e.g., compositions containing a polypeptide having the sequence of SEQ ID NO: 5, or a variant thereof having at least 85% sequence identity thereto (e.g., a polypeptide containing one or more or all of the mutations E108M, N213Q, and N286Q compared to SEQ ID NO: 1 or SEQ ID NO: 5)) may be prepared for parenteral administration (e.g., subcutaneous, intravenous, intraperitoneal, or intramuscular injection). When used herein, “parenteral administration,” “administer parenterally,” and other grammatically equivalent terms refer to modes of administration other than enteral and topical administration, which are typically by injection, but are not limited to subcutaneous, intradermal, intravenous, intranasal, intraocular, pulmonary, intramuscular, intraarterial, subarachnoid, intra-articular, intraorbital, intracardiac, intradermal, intrapulmonary, intraperitoneal, transtracheal, subepidermal, intra-articular, subcapsular, subarachnoid, intrathecal, epidural, intracerebral, intracranial, carotid, and intrasternal injections and infusions. Specific routes of administration include intravenous and subcutaneous administration.

[0058] The composition can be prepared as a lyophilized composition. The composition can be rehydrated with a solution (e.g., as described herein) before administration.

[0059] Dosage The sALP polypeptide described herein (for example, a polypeptide having the sequence of SEQ ID NO: 5, or a variant thereof having at least 85% sequence identity thereto (for example, a polypeptide containing one or more or all of the mutations E108M, N213Q, and N286Q compared to SEQ ID NO: 1)) is administered to patients suffering from or prone to bone mineralization disorders such as HPP, for example, at doses of 0.01 mg / kg to 500 mg / kg (for example, 0.05 mg / kg to 500 mg / kg, 0.1 mg / kg to 60 mg / kg, 0.1 mg / kg to 50 mg / kg, 0.1 mg / kg to 20 mg / kg, 5 mg / kg to 500 mg / kg, 0.1 mg / kg to 100 mg / kg, 10 mg / kg to 100 mg / kg, 0.1 mg / kg to 50 mg / kg, 0 (0.5 mg / kg to 25 mg / kg, 1.0 mg / kg to 10 mg / kg, 1.5 mg / kg to 5 mg / kg, or 2.0 mg / kg to 3.0 mg / kg), or 1 μg / kg to 1,000 μg / kg (for example, 5 μg / kg to 1,000 μg / kg, 1 μg / kg to 750 μg / kg, 5 μg / kg to 750 μg / kg, 10 μg / kg to 750 μg / kg, 1 μg It can be administered in individual doses within the range of 1 / kg~500μg / kg, 5μg / kg~500μg / kg, 10μg / kg~500μg / kg, 1μg / kg~100μg / kg, 5μg / kg~100μg / kg, 10μg / kg~100μg / kg, 1μg / kg~50μg / kg, 5μg / kg~50μg / kg, or 10μg / kg~50μg / kg.

[0060] Exemplary doses of sALP include, for example, 0.01, 0.05, 0.1, 0.5, 1, 2, 2.5, 5, 10, 20, 25, 50, 100, 125, 150, 200, 250, or 500 mg / kg; or 1, 2, 2.5, 5, 10, 20, 25, 50, 100, 125, 150, 200, 250, 500, 750, 900, or 1,000 μg / kg. For all doses or ranges listed herein, the term “about” may be used to modify these doses to be within ±10% of the endpoints of the listed values ​​or ranges. In particular, the compositions according to this disclosure (for example, a polypeptide having the sequence of SEQ ID NO: 5, or a variant thereof having at least 85% sequence identity thereto (for example, a polypeptide containing one or more or all of the mutations E108M, N213Q, and N286Q compared to SEQ ID NO: 1 or SEQ ID NO: 5)) can be administered to a patient in doses ranging from about 0.001 mg / kg / day to about 500 mg / kg / day, about 0.01 mg / kg / day to about 100 mg / kg / day, or about 0.01 mg / kg / day to about 20 mg / kg / day. For example, a sALP composition (e.g., a polypeptide having the sequence of SEQ ID NO: 5, or a variant thereof having at least 85% sequence identity thereto (e.g., a polypeptide containing one or more or all of the mutations E108M, N213Q, and N286Q compared to SEQ ID NO: 1 or SEQ ID NO: 5)) can be administered to a patient in weekly doses ranging from approximately 0.5 mg / kg / week to approximately 140 mg / kg / week, for example, approximately 0.8 mg / kg / week to approximately 50 mg / kg / week, or approximately 1 mg / kg / week to approximately 10 mg / kg / week (e.g., approximately 6 to approximately 9 mg / kg / week). In particular, sALP can be administered at least once a week (for example, 1, 2, 3, 4, 5, 6, 7 times or more per week), at least once every two weeks, or at least once a month (for example, once every 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 days). In some embodiments, the formulation is administered once a week. In some embodiments, the formulation is administered once every two weeks.

[0061] In particular, sALP (e.g., a polypeptide having the sequence of SEQ ID NO: 5, or a variant thereof having at least 85% sequence identity thereto (e.g., a polypeptide containing one or more or all of the mutations E108M, N213Q, and N286Q compared to SEQ ID NO: 1 or SEQ ID NO: 5)) can be administered at doses of 2 mg / kg three times per week (total dose 6 mg / kg / week), 1 mg / kg six times per week (total dose 6 mg / kg / week), 3 mg / kg three times per week (total dose 9 mg / kg / week), 0.5 mg / kg three times per week (total dose 1.5 mg / kg / week), or 9.3 mg / kg three times per week (total dose 28 mg / kg / week). The dose may be adjusted by a clinician based on conventional factors such as the severity of the disease and different parameters in patients with or prone to bone mineralization disorders such as HPP. Alternatively, approximately 0.1 mg / kg to approximately 20 mg / kg (e.g., approximately 0.1 mg / kg to approximately 9 mg / kg) may be administered once a week or once every two weeks. The sALP composition can also be administered 1 to 10 times per week or every two weeks (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10 times) in doses of 1 to 90 mg per dose (e.g., 0.1 mL to 100 mL). In some embodiments, the sALP composition is administered once a week. In some embodiments, the sALP composition is administered once every two weeks.

[0062] Compositions containing sALP or sALP-fusion polypeptides (e.g., polypeptides having the sequence of SEQ ID NO: 5, or variants thereof having at least 85% sequence identity thereto (e.g., polypeptides containing one or more or all of the mutations E108M, N213Q, and N286Q compared to SEQ ID NO: 1 or SEQ ID NO: 5)) can be administered to patients in single-dose or repeated-dose regimens. Each dose may be administered, for example, once every hour, every other hour, once daily, every other day, twice a week, three times a week, four times a week, five times a week, six times a week, once a week, every other week, once a month, every other month, or once a year. Alternatively, each dose may be administered, for example, two, three, four, five, six, seven, eight, nine, ten, eleven, or twelve times a day, week, or month. In particular, the administration regimen is once, twice, or three times per week. The duration of the administration regimen can be, for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 days (multiple), weeks (multiple), or months (multiple), or for the duration of the life of the patient who has or is prone to having osteocalcinosis, such as HPP. The dose, frequency, and duration of administration can be adjusted by the clinician based on conventional factors such as the severity of the disease and different parameters in the patient who has or is prone to having osteocalcinosis, such as HPP.

[0063] For example, the dosage of sALP or sALP fusion polypeptide (e.g., a polypeptide having the sequence of SEQ ID NO: 5, or a variant thereof having at least 85% sequence identity thereto (e.g., a polypeptide containing one or more or all of the mutations E108M, N213Q, and N286Q compared to SEQ ID NO: 1 or SEQ ID NO: 5)) may be approximately 0.1 mg / kg to approximately 10 mg / kg per body weight, administered subcutaneously or intravenously at least once a week (e.g., 2, 3, 4, 5, 6, or 7 times).

[0064] In some specific embodiments, a polypeptide (e.g., a polypeptide having the sequence of SEQ ID NO: 5, or a variant thereof having at least 85% sequence identity thereto (e.g., a polypeptide containing one or more or all of the mutations E108M, N213Q, and N286Q compared to SEQ ID NO: 1 or SEQ ID NO: 5)) or a pharmaceutical composition containing the same may be administered in doses of about 0.01 mg / kg to about 60 mg / kg (e.g., about 0.1 mg / kg to about 50 mg / kg, e.g., about 0.1 mg / kg to about 20 mg / kg, e.g., about 0.1 mg / kg to about 10 mg / kg). The polypeptide may be administered once a day, once a week, once a month, or once a year (e.g., once a week). In some embodiments, the polypeptide is administered once or more every 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, or 31 days. The polypeptide may be administered in doses ranging from approximately 0.01 mg / kg / week to approximately 50 mg / kg / week (e.g., approximately 0.01 mg / kg / week to approximately 40 mg / kg / week, e.g., approximately 0.1 mg / kg / week to approximately 20 mg / kg / week, e.g., approximately 0.1 mg / kg / week to approximately 10 mg / kg / week). The polypeptide may be administered over a period of at least 1 day, 1 week, 1 month, 1 year, or longer (e.g., over 1 to 5 years, or over the patient's lifetime).

[0065] In some embodiments, the pharmaceutical composition is administered subcutaneously. For example, about 10 mg to about 100 mg (e.g., about 10 mg, 15 mg, 20 mg, 25 mg, 30 mg, 35 mg, 40 mg, 45 mg, 50 mg, 55 mg, 60 mg, 65 mg, 70 mg, 75 mg, 80 mg, 85 mg, 90 mg, 95 mg, or 100 mg, e.g., about 15 mg, 45 mg, or 90 mg) may be administered subcutaneously to a patient, for example, once or twice a week, for example, for at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 weeks, or longer.

[0066] In some embodiments, the pharmaceutical composition is administered intravenously. For example, about 10 mg to about 100 mg (e.g., about 10 mg, 15 mg, 20 mg, 25 mg, 30 mg, 35 mg, 40 mg, 45 mg, 50 mg, 55 mg, 60 mg, 65 mg, 70 mg, 75 mg, 80 mg, 85 mg, 90 mg, 95 mg, or 100 mg, e.g., about 15 mg, 45 mg, or 90 mg) may be administered intravenously to a patient, for example, once or twice a week, for example, for at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 weeks, or longer.

[0067] In some embodiments, the pharmaceutical composition is administered intravenously and subcutaneously. In some embodiments, the pharmaceutical composition is administered intravenously and subcutaneously simultaneously. In some embodiments, the pharmaceutical composition is administered intravenously and then subcutaneously. In some embodiments, the pharmaceutical composition is administered subcutaneously and then intravenously. For example, the pharmaceutical composition may be administered intravenously to the patient in one or more doses (e.g., a single dose) as a loading dose, and then subcutaneously to the patient one or more times in maintenance doses (e.g., each dose administered approximately once a week over the course of treatment).

[0068] Treatment method This specification provides methods for treating or alleviating at least one symptom of bone mineralization disorders such as HPP. Other diseases or disorders such as fractures, osteoporosis, indurated ossification, chondrocalcinosis, hypotonia, Duchenne muscular dystrophy, tracheobronchomalacia, seizures, neurofibromatosis 1 (NF-1), and craniosynostosis can also be treated by the compositions and methods described herein. Patients may have muscle weakness. Patients may have muscle weakness disorders such as calcium pyrophosphate deposition (CPPD) or familial hypophosphatemia. Such treatment may involve administering alkaline phosphatase (e.g., a pharmaceutical composition containing alkaline phosphatase) or a polypeptide with alkaline phosphatase activity to reduce elevated PPi concentrations in such patients. For example, soluble alkaline phosphatase (sALP, e.g., a polypeptide having the sequence of SEQ ID NO: 5, or a variant thereof having at least 85% sequence identity thereto (e.g., a polypeptide containing one or more or all of the mutations E108M, N213Q, and N286Q compared to SEQ ID NO: 1 or SEQ ID NO: 5)) may be administered to neonates, infants, children, adolescents, or adults.

[0069] Patients may have been diagnosed with osteocalcinosis disorders (e.g., HPP) prior to administration of alkaline phosphatase or alkaline phosphatase-active polypeptides (e.g., sALP, e.g., a polypeptide having the sequence of SEQ ID NO: 5, or a variant thereof having at least 85% sequence identity (e.g., a polypeptide containing one or more or all of the mutations E108M, N213Q, and N286Q compared to SEQ ID NO: 1 or SEQ ID NO: 5)). In addition, patients with osteocalcinosis disorders such as HPP, or those prone to them, may be untreated patients who have not previously received treatment with sALP (e.g., a polypeptide having the sequence of SEQ ID NO: 5, or a variant thereof having at least 85% sequence identity (e.g., a polypeptide containing one or more or all of the mutations E108M, N213Q, and N286Q compared to SEQ ID NO: 1 or SEQ ID NO: 5)).

[0070] One or more symptoms of the disease may first appear in the patient during neonatal, infant, or childhood. In other embodiments, one or more symptoms of the disease may first appear in the patient during adulthood. In some embodiments, no detectable symptoms of the disease appear in the patient before adulthood. In some embodiments, detectable symptoms of the disease appear in the patient before adulthood, but the disease condition remains undiagnosed until adulthood.

[0071] The method involves administering alkaline phosphatase or an alkaline phosphatase-active polypeptide (e.g., sALP, e.g., a polypeptide having the sequence of SEQ ID NO: 5, or a variant thereof having at least 85% sequence identity thereto (e.g., a polypeptide containing one or more or all of the mutations E108M, N213Q, and N286Q compared to SEQ ID NO: 1 or SEQ ID NO: 5)) to patients suffering from or prone to bone mineralization disorders such as HPP, either as a single dose or as repeated doses over a certain period of time. In particular, sALPs such as polypeptides having the sequence of Sequence ID No. 5 can be administered to patients who have been determined to have elevated inorganic pyrophosphate (PPi) levels or at least one predetermined biomarker / score for HPP symptoms (e.g., muscle weakness), e.g., an average BOT-2 muscle strength score of less than 10, an average BOT-2 running speed score and agility score of less than 5, an average CHAQ index score greater than approximately 0.8, and / or an average PODCI score of less than approximately 40, an average 6MWT of less than approximately 80% of the predicted 6MWT value, a muscle strength grade of less than 5, and / or an average HHD value of less than approximately 80% of the predicted HHD value (e.g., average HHD muscle strength value or grip strength value). For example, sALP can be administered to patients whose PPi concentration in a sample (e.g., plasma sample) has been determined to be greater than approximately 5.71 μM in infants or children (e.g., patients under approximately 12 years of age); greater than approximately 4.78 μM in adolescents (e.g., patients between approximately 13 and 18 years of age); or greater than approximately 5.82 μM in adults (e.g., patients over approximately 18 years of age). In other embodiments, the bone mineralization disorder such as HPP described herein is caused by an elevated concentration of at least one alkaline phosphatase substrate (e.g., PPi, PLP, PEA, etc.).Alternatively, alkaline phosphatase or an alkaline phosphatase-active polypeptide (e.g., sALP, e.g., a polypeptide having the sequence of SEQ ID NO: 5, or a variant thereof having at least 85% sequence identity thereto (e.g., a polypeptide containing one or more or all of the mutations E108M, N213Q, and N286Q compared to SEQ ID NO: 1 or SEQ ID NO: 5)) may be administered to patients suffering from or prone to bone mineralization disorders such as HPP, prior to the assessment of muscle weakness scores (e.g., using BOT-2 muscle strength score, BOT-2 running speed score and agility score, CHAQ index score, BSID-III scale score, PDMS-2 standard score, muscle strength score, 6MWT value, and / or HHD value). Treatment with ALP according to the method described herein promotes, for example, increased ADL activity, reduced pain, and / or improved motor development.

[0072] In addition, the effectiveness of treatment with sALP (e.g., a polypeptide having the sequence of SEQ ID NO: 5, or a variant thereof having at least 85% sequence identity thereto (e.g., a polypeptide containing one or more or all of the mutations E108M, N213Q, and N286Q compared to SEQ ID NO: 5)) can be evaluated by using each of the listed scores (e.g., BOT-2 muscle strength score, BOT-2 running speed score and agility score, CHAQ index score, BSID-III scale score, PDMS-2 standard score, 6MWT, 12-POMA-G, modified mobility-oriented assessment (mPOMA-G, as exemplified in Phillips et al. 2015 Bone Abstracts 4: P136), or HHD value) alone or in any combination thereto, in patients suffering from or prone to bone mineralization disorders such as HPP as described herein, and the effectiveness of sALP for the treatment of bone mineralization disorders such as HPP can be demonstrated by the improvement compared to a specific test score.

[0073] For example, if alkaline phosphatase or an alkaline phosphatase-active polypeptide (e.g., sALP, e.g., a polypeptide having the sequence of SEQ ID NO: 5, or a variant thereof having at least 85% sequence identity thereto (e.g., a polypeptide containing one or more or all of the mutations E108M, N213Q, and N286Q compared to SEQ ID NO: 1 or SEQ ID NO: 5)) is administered to a patient suffering from or prone to osteocalcinosis disorders such as HPP, and as a result the BOT-2 muscle strength score increases by an average of approximately 10 or more, while the patient's average BOT-2 muscle strength score was previously less than approximately 10, then treatment with alkaline phosphatase or an alkaline phosphatase-active polypeptide is effective, for example, in the treatment of physical functional impairments associated with osteocalcinosis disorders such as HPP. Alternatively, if sALP administration does not result in an average increase of approximately 10 or more in the BOT-2 muscle strength score, the effective dose of alkaline phosphatase or alkaline phosphatase-active polypeptide for the patient can be determined by changing the dose and / or frequency of administration (e.g., increasing it indefinitely or over a short period (e.g., 1 to 6 months, or up to 1 year or more)). For example, the dosage of sALP (e.g., a polypeptide having the sequence of SEQ ID NO: 5, or a variant thereof having at least 85% sequence identity thereto (e.g., a polypeptide containing one or more or all of the mutations E108M, N213Q, and N286Q compared to SEQ ID NO: 1 or SEQ ID NO: 5)) can be increased, for example, from about 0.1-1 mg / kg / week to about 1-2 mg / kg / week, from about 0.5-3 mg / kg / week to about 3-6 mg / kg / week, or from about 3-6 mg / kg / week to about 6-9 mg / kg / week. Similarly, the dosage can be increased, for example, from once every 3 weeks to once every 2 weeks, or from once every 2 weeks to once every week.Alternatively, if an improvement is achieved in one of the metrics described herein, the dosage and / or frequency of administration may be continued as is, or reduced, for example, from approximately 6-9 mg / kg / week to approximately 3-6 mg / kg / week, from approximately 3-6 mg / kg / week to approximately 0.5-3 mg / kg / week, or from approximately 0.5-3 mg / kg / week to approximately 0.1-1 mg / kg / week. Similarly, the frequency of administration may also be reduced from approximately once a week to approximately once every two weeks, or from approximately once every two weeks to approximately once every three weeks.

[0074] In addition, if alkaline phosphatase or a polypeptide with alkaline phosphatase activity (e.g., sALP, e.g., a polypeptide having the sequence of SEQ ID NO: 5, or a variant thereof having at least 85% sequence identity (e.g., a polypeptide containing one or more or all of the mutations E108M, N213Q, and N286Q compared to SEQ ID NO: 1 or SEQ ID NO: 5)) is administered to patients suffering from or prone to osteocalcinosis disorders such as HPP, resulting in an improvement in the muscle strength grade classification of one or more patients (e.g., an improvement of 1, 2, 3, 4, or 5 muscle strength grades from a previously low muscle strength grade), and the patient's average muscle strength grade was previously less than approximately 5, then treatment with alkaline phosphatase or a polypeptide with alkaline phosphatase activity is effective, for example, in the treatment of physical functional impairments associated with osteocalcinosis disorders such as HPP. Alternatively, if sALP administration does not result in an improvement in the muscle strength grade classification of one or more patients from a previously low muscle strength grade, the effective dose of alkaline phosphatase or alkaline phosphatase-active polypeptide for the patient can be determined by changing (e.g., increasing) the dose and / or frequency of administration of alkaline phosphatase or alkaline phosphatase-active polypeptide. For example, the dose of sALP (e.g., a polypeptide having the sequence of SEQ ID NO: 5, or a variant thereof having at least 85% sequence identity thereto (e.g., a polypeptide containing one or more or all of the mutations E108M, N213Q, and N286Q compared to SEQ ID NO: 1 or SEQ ID NO: 5)) can be increased, for example, from about 0.1-1 mg / kg / week to about 1-2 mg / kg / week, from about 0.5-3 mg / kg / week to about 3-6 mg / kg / week, or from about 3-6 mg / kg / week to about 6-9 mg / kg / week. Similarly, the frequency of administration can be increased, for example, from once every three weeks to once every two weeks, or from once every two weeks to once every week.Alternatively, if an improvement is achieved in one of the metrics described herein, the dosage and / or frequency of administration may be continued as is, or reduced, for example, from approximately 6-9 mg / kg / week to approximately 3-6 mg / kg / week, from approximately 3-6 mg / kg / week to approximately 0.5-3 mg / kg / week, or from approximately 0.5-3 mg / kg / week to approximately 0.1-1 mg / kg / week. Similarly, the frequency of administration may also be reduced from approximately once a week to approximately once every two weeks, or from approximately once every two weeks to approximately once every three weeks.

[0075] Pharmacokinetic (PK) parameters In some embodiments, this treatment method results in an AUC of approximately 50 μg × hour / mL to approximately 4000 μg × hour / mL in the patient's blood. 0-168h For example, this method produces an AUC of approximately 1000 μg × hour / mL to approximately 3000 μg × hour / mL in the patient's blood. 0-168h This may occur. In some embodiments, this treatment method can result in a C concentration of approximately 0.5 μg / mL to approximately 25 μg / mL in the patient's blood. max This occurs. For example, this treatment method can result in a C concentration of approximately 0.6 μg / mL to 20 μg / mL in the patient's blood. max This can occur. [Examples]

[0076] This disclosure is illustrated by the following non-limiting examples. It should be understood that specific examples, substances, quantities, and procedures are to be interpreted broadly in accordance with the scope and intent of the disclosures set forth herein.

[0077] Example 1 ALP201 is a human recombinant TNSALP-Fc-decaspartate fusion protein. It is a soluble glycoprotein composed of two polypeptide chains of 724 amino acids each, prepared from the catalytic domain of human TNSALP (SwissProt, P05186), the human immunoglobulin (Ig) G2 / 4Fc domain (SwissProt, P01859, P01861) (for purification and half-life extension), and the decaaspartate peptide (for bone targeting).

[0078] ALP201 is an ERT that addresses the underlying cause of bone mineralization disorders such as HPP by replacing a defective alkaline phosphatase enzyme. ALP201 shares some structural similarities with asfotase alpha, another TNSALP-Fc-decaspartate fusion protein ERT, which is the only approved treatment for pediatric HPP patients (marketed under the trademark STRENSIQ®).

[0079] ALP201 is a next-generation HPP therapy that has equivalent potency to asfotase alfa and exhibits improved activity compared to asfotase alfa. Furthermore, ALP201 increases exposure due to its longer half-life, reduced α-phase clearance, and increased bioavailability. These improved characteristics support lower doses and longer dosing intervals for ALP201 compared to asfotase alfa. Patient experience is expected to improve with reduced injection volume and frequency, which may also lead to a reduction in injection site reactions.

[0080] ALP201 Physical and chemical properties ALP201 is a soluble Fc fusion protein with a molecular weight of approximately 160 kDa, composed of two polypeptide chains covalently linked by two disulfide bonds. Each polypeptide chain contains 724 amino acids and is composed of three segments. The N-terminal region of the polypeptide, amino acids L1-A491, is the soluble portion of a human tissue-nonspecific alkaline phosphatase enzyme and contains the catalytic enzyme TNSALP. A single point mutation (E108M) was introduced within the enzyme region of each polypeptide chain to improve enzyme activity, and two N-linked glycosylation sites (N213Q and N286Q) were removed for process improvement. The second part of the polypeptide, amino acids V492-K714, contains the Fc portion of human immunoglobulin gamma 2 / 4 (IgG2 / 4), which includes the hinge, CH2, and CH3 domains. The C-terminal region of the polypeptide, amino acids D715-D724, contains 10 aspartic acid molecules. This peptide sequence facilitates the binding of ALP201 to the mineral phase of bone.

[0081] Each polypeptide chain of ALP201 contains four glycosylation sites (N123, N254, N413, and N564) and eleven cysteine ​​(Cys) residues. Cys102 exists as free cysteine. Each polypeptide chain contains four intrachain disulfide bonds between Cys122 and Cys184, between Cys472 and Cys480, between Cys528 and Cys588, and between Cys634 and Cys692. Two polypeptide chains are linked by two interchain disulfide bonds between Cys494 and Cys494, and between Cys497 and Cys497. In addition to these covalent structural features, mammalian alkaline phosphatases generally have four metal-binding sites on each polypeptide chain (two for zinc, one for magnesium, and one for calcium).

[0082] General characteristics of ALP201 Table 1 lists the general properties of ALP201. The theoretical chemical formula and theoretical average molecular weight were calculated assuming that all but two cysteine ​​residues are disulfide bonds. [Table 1]

[0083] Pharmaceuticals ALP201 100mg / mL vial The ALP201 drug (100 mg / mL) is a sterile, preservative-free formulation liquid solution containing ALP201 and excipients, packaged in a single-dose 2 mL vial. The drug does not contain any novel excipients of animal or human origin. Each 2 mL vial typically contains 1.2 mL (overfilled) of the drug to deliver 100 mg of ALP201 per vial. The drug is filled into a 2 mL Type I clear glass vial with a 13 mm chlorobutyl stopper and aluminum seal. The quantitative composition of the ALP201 drug is shown in Table 2. [Table 2]

[0084] In vivo testing ALP201 was evaluated using in vivo studies to clarify its pharmacological properties, PK / toxicokinetic (TK) characteristics, local tolerability, and potential systemic toxicity. Pharmacological studies included repeated-dose studies in HPP mice (Akp2GW(- / -) mice) to determine the minimum effective dose of ALP201. Single-dose PK studies were conducted in mice, rats, and monkeys to understand the PK properties of ALP201. Toxicity studies evaluated the systemic toxicity of SC ALP201 administration to rats and monkeys. These studies included safety pharmacological endpoints (cardiovascular, respiratory, and nervous system function in monkey studies, and nervous system function in rat studies), TK assessment, and recovery periods to assess the reversibility of treatment-related effects.

[0085] Table 3 shows an overview of the in vivo trials conducted using ALP201, which support its use in humans. [Table 3-1] [Table 3-2] [Table 3-3] [Table 3-4]

[0086] In vitro pharmacological properties Pyrophosphate (PPi) is an important natural substrate of TNSALP, and in HPP patients, low TNSALP levels lead to elevated circulating PPi levels in the blood. Elevated pyrophosphate levels interfere with proper bone mineralization, causing the abnormal bone symptoms observed in HPP patients. Therefore, PPi was considered a natural substrate to be targeted at higher enzymatic activity levels by a modified second-generation asfotase alpha molecule.

[0087] Figure 1 shows the substrate saturation curves for pyrophosphate hydrolysis by ALP201 and asfotase alpha. ALP201 maintains a Km value (47 mM) for pyrophosphate similar to that of asfotase alpha (53 mM), but ALP201 functions at an extremely high turnover rate (ALP201 kcat = 11,619 min). -1 In contrast, asfotase alfa showed a reaction of 6,714 min -1 The similar Km values ​​of the wild-type TNSALP catalytic domains of ALP201 and asfotase alfa suggest that, when present at comparable serum alkaline phosphatase activity levels, ALP201 is less likely to cause dangerously low circulating PPi levels than asfotase alfa.

[0088] Pyridoxil-5'-phosphate is the second natural substrate of TNSALP, which is clinically relevant to HPP. TNSALP cleaves PLP to form pyridoxal, the B6 vitamer most readily taken up by tissues. When serum alkaline phosphatase levels are very low, systemic vitamin B6 metabolism may be impaired. In HPP, PLP deficiency in the brain manifests as seizures, and systemic deficiency of PLP hydrolysis by TNSALP may contribute to the pain, muscle weakness, and hypotonia experienced by some HPP patients. Therefore, using ERT for the treatment of HPP can provide sufficient PLP hydrolysis activity.

[0089] The substrate saturation curves for PLP hydrolysis by ALP201 and asfotase alfa are shown in Figure 2. Although ALP201 has a slightly weaker Km value for PLP (2.76 mM) compared to asfotase alfa (1.71 mM), ALP201 functions with an extremely high turnover rate (ALP201 kcat = 3,324 min). -1 In contrast, asfotase alpha showed a reaction of 2,623 min -1 As a result, the PLP activity saturation curves of the two molecules are very similar. The substrate reaction rate parameters are shown in Table 4. [Table 4]

[0090] In vivo pharmacological properties A preclinical efficacy study for ALP201 was conducted using Akp2GW(- / -) mice, an animal model of human HPP. The Akp2GW(- / -) mice possess the same HPP-induced TNSALP mutation as those used in the Akp2(- / -) mice previously used in the preclinical evaluation of asfotase alfa. Spontaneous follow-up studies conducted at Alexion during the development of Akp2GW(- / -) mice revealed that homozygous TNSALP-active knockout Akp2GW(- / -) mice exhibit nearly identical bone mineralization and survival phenotypes to Akp2(- / -) mice (Akp2GW-NH-01-02).

[0091] In efficacy studies used to evaluate the efficacy of ALP201 in the Akp2GW(- / -) mouse model, the test substance was administered subcutaneously from day 1 to day 35 of life. Results reported in all studies included overall survival, weight gain rate, hindlimb bone mineralization at day 36 (or at death if before end of study [EOS]), and EOS trough plasma enzyme activity levels (recorded at day 36 for once-daily [qd] and once-weekly [q1w] administration groups, and at day 37 for every-two [q2d] administration groups). In some studies, femoral and tibia lengths, as well as alkaline phosphatase activity in the mouse femur, were determined at EOS.

[0092] In previous studies using Akp2(- / -) mice, asfotase alfa demonstrated efficacy in both bone mineralization and overall survival endpoints at doses of 7–10 mg / kg / day of the active preparation of asfotase alfa, depending on the specific activity of the test substance. The estimated minimum effective dose (MED) of asfotase alfa was defined as the dose at which 85% of measurable mice in the group achieved a normal bone mineralization score by the end of the study, or at the time of death if death occurred before the end of the study. Analysis of efficacy data showed that this value was approximately 2.0–2.5 mg / kg / day, depending on the specific activity of the test substance.

[0093] The efficacy of ALP201 in a mouse model of HPP was tested in Akp2GW(- / -) mice in two repeated-dose studies at various SC doses and dosing intervals. In these studies, the efficacy of ALP201 was compared to the efficacy observed in a positive control group that received daily SC administration of asfotase alfa.

[0094] In previous studies, asfotase alfa was administered via subcutaneous injection (SC) at the maximum effective dose of 9.8 mg / kg / day on a qd dosing schedule. An equivalent 4-MUP active dose of ALP201 was administered via SC in PBS at qd, q2d, and q1w dosing intervals. In one dosing group, the dose of ALP201 was logarithmically reduced to half after weaning of mice on day 25. Subcutaneous administration of PBS on a qd schedule was used as a negative control. A list of these dosing groups can be found in Table 5.

[0095] In the HPP-MED-01 study, asfotase alfa was administered via SC at the minimum effective dose of 2.5 mg / kg / day on a qd dosing schedule. ALP201 was administered subcutaneously in PBS at doses of 2.0, 0.8, 0.3, and 0.15 mg / kg on a q2d dosing schedule. Subcutaneous administration of PBS on a q2d schedule was used as a negative control. A list of these dosing groups can be found in Table 5. [Table 5]

[0096] Results of bone mineralization Radiographic analysis of the hind limbs of treated Akp2GW(- / -) mice determined the outcome of bone mineralization at days 36 / 37. Radiographic visualizations of hind limb bone mineralization were compared to reference images at day 36, illustrating four classification categories: no effect, mild impairment, moderate impairment, and severe impairment. A detailed explanation of these classifications can be found in Table 6. Blinded individuals assigned scores to each mouse image, and scores were aggregated within individual treatment groups after completion. [Table 6] The distribution of observed EOS hindlimb bone mineralization index scores for each treatment group can be seen in Table 7. [Table 7]

[0097] Treatment of Akp2GW(- / -) mice with ALP201 showed a dose-response, with an increasing proportion of mice having unaffected hindlimb bone mineralization as the ALP201 dose increased over a Q2D dosing interval. All ALP201 groups showed a statistically significant improvement compared to PBS-treated Akp2GW(- / -) controls (p<0.001 in one-way ANOVA). These data were used to inform model-based analyses and predict human dose prospects.

[0098] Results of trough plasma reactive oxygen species concentration at the end of the test The trough plasma active TNSALP enzyme concentration was determined by measuring the TNSALP activity level in plasma samples on days 36 and 37. The measured TNSALP activity was fitted to a standard curve of known enzyme activity and concentration to quantify the active enzyme level per unit volume of plasma. Figure 3 shows the distribution of EOS trough plasma active enzyme concentrations. Table 8 shows a summary of the average EOS trough plasma active enzyme concentrations for each treatment group. [Table 8-1] [Table 8-2]

[0099] Analysis of EOS trough plasma reactive enzymes and corresponding enzyme activity levels after repeated administration in treated Akp2GW(- / -) mice clearly shows greater absolute and dose-normalized accumulation of ALP201 compared to asfotase alfa, which is likely due to the superior PK profile of ALP201 after SC administration.

[0100] Bone enzyme activity levels at the end of the study in Akp2GW(- / -) mice treated on day 36 / 37 Alkaline phosphatase activity levels in treated Akp2GW(- / -) mouse femoral tissue were determined by ex vivo assay using 4-MUP as a substrate at the end of the HPP-MED-01 study. To avoid potential contamination from blood or tissues with high blood flow that may still contain high levels of ALP201 or asfotase alpha, only the calcified portion of the femur was assayed for activity. Figure 4 shows the distribution of enzyme activity levels associated with treated Akp2GW(- / -) mouse femurs. Here, the data represent 4-MUP hydrolytic activity per 1 mg of calcified femoral tissue in microunits. [Table 9]

[0101] The bone activity data showed a dose-response pattern, with EOS bone tissue activity levels increasing with increasing ALP201 dose. At the end of the study, mice in the 0.15 mg / kg q2d group of ALP201 recovered 22.6% of wild-type alkaline phosphatase activity, compared to 26.9% in the 2.5 mg / kg q2d group of asfotase alfa. Administration of ALP201 at 0.3, 0.8, and 2.0 mg / kg q2d restored wild-type activity by 34.4%, 37.6%, and 44.9%, respectively. The difference in bone activity levels at the end of the study between the 0.8 and 2.0 mg / kg q2d ALP201 doses was statistically significant compared to the 2.5 mg / kg q2d group of asfotase alfa, with adjusted p-values ​​of 0.0048 and <0.0001, respectively, when analyzed by one-way ANOVA.

[0102] Survival results of treated Akp2GW(- / -) mice All Akp2GW(- / -) mice treated with PBS died on or before day 26 of the study, with a median survival time of 20 days. Treatment of Akp2GW(- / -) mice with ALP201 significantly improved 36-day survival in all dose groups compared to the PBS solvent control (Figure 5 and Table 10). All ALP201 dose groups achieved an EOS survival rate of at least 69%, and overall survival was ≥88% in all qd and q2d interval dosing groups with doses exceeding 0.15 mg / kg / day. The survival curves for the 4.8 mg / kg ALP201 q1w group and the 9.8 mg / kg asfotase alfa qd group showed very similar results. [Table 10]

[0103] Weight results The mean body weight of Akp2GW(- / -) mice treated with ALP201 and asfotase alfa was consistently lower than the mean body weight of their wild-type littermates treated with PBS in all groups. There was no statistically significant difference in body weight at the end of the study between the ALP201-treated groups and the asfotase alfa-treated groups.

[0104] Safety pharmacological properties Independent safety pharmacological characterization studies were not conducted for ALP201. However, cardiovascular, respiratory, and blood pressure endpoints were measured as part of a central GLP-compliant monkey toxicity study, and neurological function endpoints were evaluated as part of a rat GLP toxicity study. ALP201 treatment resulted in an increase in heart rate only in the low-dose group (28% compared to the control group), accompanied by a decrease in RR, PR, and QT intervals, but no significant change in QRS duration. No biologically significant changes were observed in the medium and high-dose groups. Therefore, there was no dose-response relationship between the slight increase in heart rate and the ALP201 dose. Because the degree of increase in heart rate was slight, these findings are considered non-harmful. Furthermore, after SC administration every 3 days for 28 days up to the highest concentration evaluated in this study of 20 mg / kg, no ALP201-related findings were observed in ECG, blood pressure, or respiratory parameters evaluated in monkeys. ALP201 did not affect behavioral indicators or motor activity in rats after administration of SC once every three days for 28 days, up to the highest concentration evaluated in this study of 30 mg / kg.

[0105] Pharmacokinetics and drug metabolism in animals Single-dose PK studies were conducted in mice, rats, and monkeys. Data from these studies were used to evaluate the kinetics (absorption, distribution, and elimination) of ALP201 in preclinical species.

[0106] absorption The pharmacokinetics (PK) of ALP201 were evaluated in Sprague-Dawley rats and cynomolgus monkeys. A summary of non-GLP and GLP studies following single-dose and repeated-dose administration of ALP201 is presented herein.

[0107] The average bioavailability was 96%, 58%, and 78% in mice, rats, and monkeys, respectively. The time to reach peak concentration (t) max The period of absorption was in the range of 17 to 48 hours after administration, which suggests that the absorption of ALP201 from the SC injection site is slow.

[0108] distribution After a single intravenous administration to mice, rats, and monkeys, the apparent volume of distribution of ALP201 ranged from 0.08 to 0.15 L / kg. This value was significantly larger (>2 times) than the plasma volume in these species, suggesting that ALP201 is distributed beyond the intravascular compartment.

[0109] Pharmacokinetics in single-dose studies PK in male C57BL / 6 mice after single dose (Experiment HPP-PK-01) The pharmacokinetics (PK) of ALP201 were evaluated after a single IV or SC administration to male C57BL / 6 mice. Sixteen animals received a single IV or SC administration of ALP201 at a dose of 4 mg / kg. For each administration route, the mice were randomly subdivided into four sample collection cohorts of four mice each. Blood samples were collected up to 20 days post-administration using a semi-continuous sample collection design. A summary of the PK parameters of ALP201 is shown in Table 11. [Table 11]

[0110] Following IV administration, the concentration-time profile decreased exponentially. CL was 0.0019 L / h / kg, and the apparent terminal phase t1 / 2 was 48 hours (Table 11). d The value is 0.13 L / kg, which is greater than the plasma volume of mice showing ALP201 distribution beyond intravascular space. Time to reach peak concentration after SC administration (t max The absorption rate was 48 hours after administration. This suggests that absorption from the SC injection site is relatively slow. The absolute bioavailability after SC administration was 96%.

[0111] Single-dose PK study in rats The pharmacokinetics (PK) of ALP201 were evaluated after a single IV or SC administration to rats. ALP201 was administered as a single IV or SC escalation (IV bolus at doses of 1, 3, 9, or 27 mg / kg, or SC at doses of 2 or 27 mg / kg) to four animals (2 animals per sex / dose). Plasma concentrations of ALP201 were quantifiable up to 28 days post-administration. No significant sex-specific differences were observed in the PK parameters of ALP201; therefore, pooled sex values ​​are presented. Descriptive statistics for the PK parameters of ALP201 are summarized in Table 12 for each pooled sex group. The mean plasma concentration-time profiles of ALP201 are shown in Figure 14. Note that background data for asfotase alfa has been added for comparison. [Table 12]

[0112] In the case of IV administration, the PK exposure amount of ALP201 (C max and AUC ∞ ) increased slightly below the dose ratio within the test dose range of 1 mg / kg to 27 mg / kg. The mean CL and V of ALP201 d This remained consistent throughout the entire dose. The mean CL value ranged from 0.0012 to 0.0019 L / h / kg, and the mean V d The values ​​ranged from 0.12 to 0.15 L / kg. Similarly, the t1 / 2 of ALP201, which remained relatively similar across the entire IV dose, was approximately 2 days on average. In the case of SC administration, CL / F, V d / F and t 1 / 2 The values ​​were similar to those estimated from IV administration, except for the SC administration group of 27 mg / kg, which was considered to have abnormal values. max The absorption period ranged from 30 to 48 hours after administration. This suggests that absorption from the SC injection site is relatively slow. The absolute bioavailability after SC administration was 61% and 54% at doses of 2 and 27 mg / kg, respectively.

[0113] Single-dose PK study in monkeys The pharmacokinetics (PK) of ALP201 were evaluated after a single IV or SC administration to monkeys. Three male monkeys received a single IV or SC escalation of ALP201 (IV bolus at doses of 2, 6, or 20 mg / kg, or SC at doses of 2 or 20 mg / kg). Plasma concentrations of ALP201 were quantifiable up to 28 days post-administration. Descriptive statistics of the PK parameters of ALP201 are summarized in Table 13. The mean plasma concentration-time profile of ALP201 is shown in Figure 15. Note that background data for asfotase alfa has been added for comparison. [Table 13]

[0114] In the case of IV administration, C max and AUC ∞ The increase in values ​​was close to the dose ratio within the test dose range of 2 mg / kg to 20 mg / kg. CL and V of ALP201 d This remained consistent throughout the entire dose, with the mean CL value ranging from 0.0008 to 0.0011 L / h / kg, and the mean V d The values ​​ranged from 0.08 to 0.10 L / kg. Similarly, the values ​​of ALP201 remained relatively similar across the entire dose. 1 / 2 The average duration was approximately 3 days. In the case of SC administration, the average CL / F and V were... d / F and t 1 / 2 The values ​​were consistent with those estimated after IV administration. Mean t max The absorption period was in the range of 17–19 hours after administration. This suggests that absorption from the SC injection site is relatively slow. The absolute bioavailability was 69.6% and 86.9% at doses of 2 mg / kg and 20 mg / kg, respectively.

[0115] Repeated-dose toxicology study using ALP201 4-week SC administration to rats (1727-227): ALP201 was administered to rats via SC at doses of 2, 10, or 30 mg / kg / dose, every 3 days for 4 weeks. In addition, to compare bioavailability via IV and SC pathways, ALP201 was administered intravenously to rats as a single dose of 10 mg / kg. Pharmacokinetic parameters calculated based on combined plasma ALP201 concentrations after administration on day 0 and day 24 using non-compartmental analysis are shown in Table 14.

[0116] Systemic exposure to ALP201 appeared to be independent of sex after SC administration of ALP201 on day 0 and day 24, and after a single IV bolus injection of ALP201 on day 0. After SC administration of ALP201 every three days, C of ALP201 on day 0 and day 24 max and AUC 0-72h The value increased with increasing dose, slightly below the dose ratio. ALP201 total body exposure (AUC) 0-72h Subcutaneous bioavailability (AUC at 10 mg / kg) of ALP201 appeared to decrease after repeated SC administration. The reduction in exposure appeared to be dose-dependent, with the maximum reduction after repeated SC administration of 30 mg / kg of ALP201 being up to 2 times. 0-72h The percentage (based on the value) was approximately 44.2%. [Table 14-1] [Table 14-2]

[0117] SC administration to monkeys for 4 weeks: ALP201 was administered to monkeys as SC at doses of 1, 5, or 20 mg / kg / dose, every 3 days for 4 weeks. PK parameters calculated using non-compartmental analysis based on ALP201 plasma concentrations after administration on day 1 and day 24 are shown in Table 15. [Table 15]

[0118] Systemic exposure to ALP201 appeared to be independent of sex after SC administration of ALP201 on day 0 and day 24. After subcutaneous administration of ALP201 every three days, C exposure to ALP201 on day 0 and day 24 was observed. max and AUC 0-72h The values ​​increased with increasing dose, almost proportionally to the dose. ALP201 whole-body exposure (AUC) 0-72h The value appeared to increase after repeated subcutaneous administration of ALP201 on day 24.

[0119] Pharmacokinetics, pharmacodynamics, and immunogenicity in repeated-dose studies 28-day toxicity study in rats with a 28-day recovery period In male and female rats, the TK / ADA of ALP201 was evaluated after 2, 10, or 30 mg / kg SC administration (10 / sex / administration group, 5 / sex / time point) every 3 days for 4 weeks (total of 10 administrations), and after a single IV administration of 10 mg / kg (total of 1 administration). Blood samples for TK analysis were collected from all animals over a 72-hour period starting on day 0 and day 24. A summary of the PK parameters of ALP201 is shown in Table 16. [Table 16]

[0120] Systemic exposure to ALP201 appeared to be independent of sex after repeated SC administration of ALP201 and after a single IV bolus injection of ALP201. Therefore, Table 16 includes pooled sex results. ALP201 exposure (C) on day 0 and day 24 after SC administration of ALP201 every 3 days (q3d). max and AUC 72h ) increased with increasing dose, but slightly less than the dose ratio. Total exposure (C) on day 24 max and AUC 72hThe exposure appeared to decrease after repeated SC administration. The reduction in exposure appeared to be dose-dependent, with a maximum reduction of up to 60% after repeated SC administration of 30 mg / kg ALP201. The subcutaneous bioavailability of ALP201 was approximately 44.2%.

[0121] The ADA response was negative in all pre-administration samples. The incidence of ADA was 58% and 90% for post-administration ADA samples on D28 and D56, respectively. The reduction in exposure (60%) on day 24 with a 30 mg / kg dose was thought to be due to the immunogenic response.

[0122] 28-day toxicity study in monkeys with a 28-day recovery period. In male and female monkeys, TK / ADA of ALP201 was evaluated after SC administration at 1, 5, or 20 mg / kg every 3 days for 4 weeks (total of 10 doses) (5 / sex / administration group). Blood samples for TK analysis were collected on day 0 and day 24. Descriptive statistics of ALP201 PK parameters are summarized in Table 17. [Table 17]

[0123] There was no sex difference in systemic exposure to ALP201 on day 0 and day 24. Therefore, Table 17 includes pooled sex results. After subcutaneous administration of ALP201 every three days, ALP201 (C) levels on day 0 and day 24 were measured. max and AUC 72h The AUC (Apex Circulation) increased almost proportionally with the dose. 72h The value) appeared to increase after repeated subcutaneous administration of ALP201 on day 24, and the mean accumulated AUC 72h The rates were 2.12, 1.83, and 2.15 for 1, 5, and 20 mg / kg, respectively.

[0124] The ADA response was negative in pre-administration samples. The incidence of ADA was 23% and 92% for post-administration ADA samples on D28 and D56, respectively. Since ADA samples were not collected on the TK collection day after repeated administration, there was no direct correlation between a positive ADA response and the concentration-time profile of systemic exposure. However, abnormal concentration-time profiles in some animals at ≤5 mg / kg appeared to be influenced by anti-ALP201 antibodies.

[0125] Model-based analysis and predicted human dose regimens ALP201 Modeling Population pharmacokinetic (Pop-PK) model To predict human ALP201 exposure from mouse dose-response results, we developed a Pop-PK model that predicts human PK parameter estimates by using body weight-based relative growth scaling and pooling mouse, rat, and monkey PK data. Mouse PK data included single C2C mice from a single-dose wild-type mouse study (HPP-PK-01) and two-dose repeated-dose Akp2GW(- / -) mouse efficacy studies (HPP-PoC-01 and HPP-MED-01). トラフ Measurements (D36 / D37) were included. For rats and monkeys, single-dose studies, dose-ranging studies in rats and cynomolgus monkeys, and 4-week GLP toxicity studies with repeated doses in rats and cynomolgus monkeys were also included.

[0126] The current Pop-PK model was developed using the NONMEM software program, version 7.2 (ICON solutions), which simultaneously analyzes IV and SC PK data from three animal species, taking into account body weight differences using the relative growth principle. Here, animal body weight was centered at 70 kg, and the relative growth index was fixed at 0.75 for the clearance parameter and 1.0 for the volume of distribution parameter. The effect of ADA on PK was evaluated using CL based on 4 weeks of TK, and it appeared that ALP201 concentrations decreased in ADA+ animals in rats and, to a lesser extent, in monkeys. The best current Pop-PK model is a two-compartment model with loss of linearity. Testing the effect of ADA+ on ALP201 concentration was inconclusive. The estimated bioavailability in humans was approximately 75%, and the calculated effective half-life for humans was 7-9 days. Human PK simulations used variability estimated from the human asfotase alfa Pop-PK model, as well as the mean (and standard deviation) adult baseline body weight of HPP patients participating in clinical trials evaluating asfotase alfa as a treatment for HPP.

[0127] Dose-response model E max We selected a development dose-response model by testing family models. Most dose-response relationships are E max This can be explained by one of the model parameterizations. The current best dose-response characterization is E maxThe +E0 (baseline) model was used. The efficacy endpoint, bone mineralization, was evaluated by radiography, which was chosen because it is the same as the clinical definition of efficacy of asfotase alfa used in nonclinical dose-response evaluations. Bone mineralization after treatment with asfotase alfa or ALP201 based on two efficacy trials (HPP-PoC-01 and HPP-MED-01) was plotted against dose (Figure 6). Asfotase alfa data from previous efficacy trials were included for comparison. The y-axis represents the normal value %, defined as the percentage of mice in the treatment group with a bone mineralization score of 4. The x-axis represents the dose normalized to mg / kg / day. The dose that resulted in normal mineralization in 85% of the treated population (ED85) was selected as the target effective dose.

[0128] Dose conversion from mouse to human and proposed starting dose for humans. Relative growth scaling was applied to the target effective dose (ED85) for mice to determine the human dose. The formula used to predict the human equivalent dose (HED, mg / kg / day) from mouse ED85 was ED85 × (0.025 kg mouse / 70 kg human). 0.25 This was the result. Converted to HED, it was a constant weekly dose of 45 mg / week. This will be used as the dose for FIH trial cohort 2 (both IV and SC). For cohort 3 (IV and SC), 90 mg / week will be used. For cohort 1, a NOAEL-based starting dose of 15 mg / week will be used (for both IV and SC).

[0129] Predicted human exposure and PK safety margin for ALP201 Using the Pop-PK model, a detailed PK profile was simulated for humans based on doses adjusted for relative growth (Figure 7). The simulated PK data was used to define the exposure metric C. max And AUC was calculated.

[0130] Predicted human exposure (C) for the proposed FIH ALP201 dose max and AUC 168hThe predicted PK safety margins are shown in Table 18. The PK safety margin for the proposed FIH ALP201 dose was calculated using the predicted human exposure and the observed NOAEL exposure from a 4-week GLP monkey toxicity study (NOAEL dose for SC administration of 20 mg / kg / Q3D). [Table 18]

[0131] Toxicity testing Non-clinical safety studies were conducted in rats and monkeys to evaluate local tolerability, systemic toxicity, and safety pharmacological parameters after SC administration of ALP201 for up to 28 days. Furthermore, to clarify the bioavailability characteristics of SC ALP201, the non-clinical safety of a single IV dose of ALP201 was also evaluated in a 28-day toxicity study in rats.

[0132] Single-dose toxicity study Independent single-dose tolerability studies for ALP201 were not conducted. However, ALP201 tolerability in rats and monkeys was evaluated as part of single-dose pharmacokinetic studies. ALP201 tolerability was assessed by clinical observation, including injection site reactions and clinicopathological data. No notable ALP201-related injection site reactions, clinical observations, or clinicopathological observations were observed in the rat or monkey single-dose pharmacokinetic studies. In summary, single doses of ALP201 were well-tolerated when administered intravenously or via seroconduct at a maximum of 27 mg / kg in rats and 20 mg / kg in monkeys. The dose for the repeated-dose determination (GLP) toxicity study of ALP201 was selected based on the obtained tolerability and clinicopathological data.

[0133] Repeated-dose toxicity study Definitive or GLP-compliant 28-day toxicity studies were conducted in Sprague-Dawley rats and cynomolgus monkeys. In the 28-day toxicity study in rats, administration of ALP201 via subcutaneous injection every three days for 28 days (days 1, 4, 7, 10, 13, 16, 19, 22, 25, and 28) to male and female CD® rats was well tolerated up to the highest SC dose evaluated in this study, 30 mg / kg / dose. In addition, intravenous administration of 10 mg / kg of ALP201 was also well tolerated after a single injection. No changes related to ALP201 were observed in clinical observations, body weight, body weight gain, quantitative food intake, ophthalmological examinations, overall functional observations, hematological examinations, urinalysis, gross findings, or organ weights. At the end of the administration phase, dose-related increases in alkaline phosphatase (ALP) activity (expected pharmacological effect) were observed in SCs with ALP201 doses of ≥2 mg / kg / dose, but complete recovery was achieved after a 28-day recovery period. Non-adverse microscopic changes were associated with injection therapy, accompanied by reactive changes in afferent lymph nodes. No recognizable ALP201-related changes were observed at the injection site or afferent lymph nodes in terms of incidence, severity, or microscopic features of the changes. Microscopic changes at the injection site and afferent lymph nodes in the recovered animals were similar to those shown in necropsy animals at the end of the phase, although less pronounced. In conclusion, a dose level of 30 mg / kg / dose is considered to be the no-observed-adverse-effect level (NOAEL) for subcutaneous administration, compared to 43.9 μg / mL C in a combined male and female population at day 24. max Value and AUC of 2120hr*μg / mL 0-72hr It corresponds to a value.

[0134] AUC after a single IV bolus injection of 10 mg / kg of ALP2-1 into rats. 0-72hr The concentration was 3690 h*μg / mL. SC bioavailability of ALP201 in rats (AUC of SC at 10 mg / kg / dose) 0-72hr The percentage (based on the value) was approximately 44.2%.

[0135] In a 28-day toxicity study in monkeys, administration of ALP201 via SC injection every three days for 28 days (10 times in total, on days 1, 4, 7, 10, 13, 16, 19, 22, 25, and 28) at doses of 1 mg / kg, 5 mg / kg, or 20 mg / kg to male and female cynomolgus monkeys was well tolerated up to the highest dose evaluated in this study, 20 mg / kg / dose. No changes associated with ALP201 were observed in injection site reactions (skin scoring), body weight, body weight gain, qualitative food intake, ophthalmological examination, manual respiration rate, indirect blood pressure, qualitative electrocardiogram, hematological examination, urinalysis, gross findings, and organ weights. At the end of the administration phase, a dose-related increase in alkaline phosphatase (ALP) activity (expected pharmacological effect) was observed, but after a 28-day recovery period, it recovered completely (at 1 mg / kg / dose) or almost completely (at ≥5 mg / kg / dose). Non-adverse ALP201-related microscopic changes were limited to the injection site, showing mild to mild degeneration / necrosis, calcification, and mixed cell inflammation / infiltration. Partial recovery of degeneration / necrosis and mixed cell inflammation / infiltration at the injection site(s) were observed in the recovery group, with mild to mild calcification observed at ≥1 mg / kg / dose in males and 20 mg / kg / dose in females. Increased heart rate was observed at 1 mg / kg / dose in all animals, and at ≥5 mg / kg / dose in some animals. These increases in heart rate are not considered harmful, given the discrepancies in whole-body exposure data and the fact that heart rate and ECG values ​​remained within the normal range of biological variation in monkeys of this age. In conclusion, a dose level of 20 mg / kg / dose is considered to be the no-observed-adverse-effect level (NOAEL), which is based on a combined dose of 254 μg / mL C for males and females at day 24, respectively. max Values ​​and mean AUC of 15400hr*μg / mL 0-72hr It corresponds to a value.

[0136] Local tolerability test Independent non-clinical studies were not conducted to evaluate local tolerability. However, injection site evaluation was performed in general toxicity studies of SC ALP201 in rats and monkeys, and in IV ALP201 in general toxicity studies in rats. Subcutaneous and IV administration of ALP201 did not result in any adverse findings at the injection site, and local tolerability was good in monkeys.

[0137] Summary of non-clinical observational results related to the ALP201 clinical trial. Safety evaluation No changes related to ALP201 were observed in clinical observations, body weight, body weight gain, quantitative food intake, ophthalmic examinations, comprehensive functional assessment (CNS) results, cardiovascular assessment items, respiratory assessment items, hematological tests, urinalysis, gross findings, and organ weight in the 28-day toxicity study. At the end of the administration phase, a significant dose-related increase in alkaline phosphatase (ALP) activity, consistent with the expected pharmacological effect, was observed in rats or monkeys. No notable systemic organ toxicity was observed in rats or monkeys at any dose evaluated in the corresponding studies.

[0138] Following SC administration to rats and monkeys, ALP201 was well-tolerated locally. Non-adverse ALP201-related microscopic changes in monkey studies were limited to injection sites, with mild to mild degeneration / necrosis observed in males and females. Degeneration / necrosis and mixed cell inflammation / infiltration at injection sites(s) were less frequent and severe in the recovery group, or exhibited characteristics of chronicity (calcification) and partial recovery. Non-adverse microscopic changes in rat studies were limited to injection therapy accompanied by reactive changes in afferent lymph nodes. No recognizable ALP201-related changes with significant incidence, severity, or microscopic characteristics were observed at the injection site or afferent lymph nodes. While less pronounced in the recovery group, microscopic changes at the injection site and afferent lymph nodes in recovering animals were similar to those observed in terminal necropsy animals.

[0139] Based on GLP SC monkey toxicity studies, the observed NOAEL exposure at a 20 mg / kg / dose was approximately 113 times (for IV) and 83 times (for SC) higher than the predicted AUC exposure for a proposed single introductory dose of 15 mg IV or 15 mg qw×3SC in humans (Table 18). Given the safety margin and lack of systemic toxicity or local tolerability findings in 28-day rat and monkey toxicity studies using ALP201, the safety risk to humans for an introductory dose of 15 mg is very low.

[0140] immunogenicity The potential immunogenicity of ALP201 was evaluated by measuring ALP201 anti-drug antibodies (ADA) in serum collected from 28-day toxicity studies in GLP rats and monkeys.

[0141] In rat toxicity studies, on day 28 of PK, the ADA response was positive in 9 out of 20 animals (6 males, 3 females) in the control group, 10 out of 19 animals (6 males, 4 females) in the 2 mg / kg / dose group, 12 out of 20 animals (7 males, 5 females) in the 10 mg / kg / dose group, and 12 out of 20 animals (6 males, 6 females) in the 30 mg / kg / dose group. Furthermore, on day 28 of PK, the ADA response was positive in 14 out of 20 animals (8 males; 6 females) after a single 10 mg / kg IV ALP201 dose. While ADA may have contributed to the significant decrease in systemic exposure at 30 mg / kg / dose on day 24, the positive ADA response did not consistently appear to affect the composite plasma concentration-time profiles of the 2 and 10 mg / kg / dose SC groups on day 24. The investigation concluded that the positive ADA response in control animals was unlikely to be due to ALP201 misdose. A dose-dependent increase in ALP, the expected pharmacological effect of ALP201, was observed in the group administered ≥2 mg / kg SC before terminal necropsy, but this recovered by convalescent necropsy. The ALP results in the control group animals were similar both before terminal necropsy and convalescent necropsy. Furthermore, there were no survival findings related to ALP201 during administration or the recovery phase. Therefore, in summary, the positive ADA response observed in control animals on PK days 28 and 56 (days 29 and 57 of the study) was most likely due to contamination during blood collection and is not considered to affect the study.

[0142] In toxicity studies in monkeys, the anti-ALP201 antibody response was negative in all pre-administration samples on day 0. The ADA response was positive on day 28 in 4 out of 10 animals at 1 mg / kg / dose, 6 out of 10 animals at 5 mg / kg / dose, and 5 out of 10 animals at 20 mg / kg / dose. After repeated administration, ADA samples were not collected on the TK collection day, so there was no direct correlation between a positive ADA response and the concentration-time profile of systemic exposure. However, the concentration-time profiles of some animals at ≤5 mg / kg appeared to be affected by the anti-ALP201 antibody. Three animals (2 males and 1 female) at 1 mg / kg / dose and three animals (3 females) at 5 mg / kg / dose were affected by the anti-ALP201 antibody, and for these animals, the days that affected the concentration-time profile ranged from day 18 to day 24.

[0143] Example 2 The pharmacokinetic profiles of asfotase alfa in multiple species suggest that frequent administration is necessary due to a combination of reduced absolute bioavailability and half-life. The catalytic domain of human TNSALP is a highly glycosylated molecule. The presence of a non-sialylated glycan leads to clearance in the liver via the asialoglycoprotein receptor (ASGPR).

[0144] With these considerations in mind, we developed ALP201 as a next-generation alkaline phosphatase ERT. ALP201 retains the TNSALP-IgG-Fc-D10 structure used in asfotase alfa, while incorporating the removal of two non-essential N-linked glycans, a change in the human Fc isotype to IgG2 / 4, and numerous process improvements in molecular expression, resulting in higher TSAC uptake. This report presents the pharmacokinetic parameters of ALP201 as determined in single-dose studies in male C57BL / 6 mice via intravenous and subcutaneous administration. We also report the PK parameters of multiple lots of purified ALP201 expressed at various levels of TSAC uptake to help determine which PK parameters were most strongly affected by non-sialylated glycan clearance.

[0145] Materials and methods animal lineage To maintain consistency with these studies and previous pharmacokinetic studies conducted for asfotase alfa, this study was performed using male C57BL / 6 mice approximately 11–12 weeks old at the time of administration. Information on the test molecules is shown in Table 19. [Table 19]

[0146] Administration to animals and blood sample collection Animals aged 11–12 weeks were randomized according to body weight and assigned to four groups. A 4 mg / kg dose of ALP201 was administered to 16 mice / group on day 0 by intravenous delivery (IV) (group 1) or subcutaneous delivery (SC) (group 2). Equivalent volume of PBS was administered to 4 mice / group on day 0 by IV (group 3) or SC (group 4). IV administration was performed via the tail vein. SC bolus administration was performed in the scapular region above the shoulder.

[0147] A semi-sequential sampling design was used, with four cohorts per group, n=4 per cohort, and one additional mouse per group. In the Alexion study, blood was collected three times from each mouse, including a terminal blood sample. The time points (time after administration) for each cohort are shown in Table 20.

[0148] For non-terminal blood collection, 100 mL of whole blood was collected submandibularly into a pre-coated lithium heparinized tube, and at least 50 μL of plasma was collected at each time point in each animal. At terminal blood collection, as much blood as possible was collected by cardiac puncture into a lithium heparinized collection tube. The blood was maintained at 4°C, and the plasma was processed by centrifugation as soon as possible after collection. Each plasma sample was divided into two equivolute aliquots, then rapidly frozen in CO2 / ethanol and stored at -80°C. [Table 20] [Table 21] [Table 22]

[0149] In lots 2, 3, and 4, groups of 32 mice were divided into eight cohorts of four animals per time point for each administration route. In the IV administration group, blood samples were taken twice from each cohort at approximately 0.5, 1, 3, 8, 12, 21, 26, 32, 45, 49, 72, 96, 120, 192, 264, and 336 hours post-administration (first sample taken from a living individual, second at terminal). In the SC administration group, blood samples were taken twice from each cohort at approximately 1, 3, 6, 8, 12, 21, 26, 32, 45, 49, 72, 96, 120, 192, 264, and 336 hours post-administration (first sample taken from a living individual, second at terminal).

[0150] Determination of active ALP201 concentration in plasma Plasma samples were assayed for alkaline phosphatase activity using the artificial substrate 4-methylumbelliferyl phosphate (4-MUP). Hydrolysis of the phosphate ester bond of 4-MUP releases the fluorescent compound 4-methylumbelliferone (4-MU), which is readily detectable by fluoroscopy. Enzyme activity was quantified via a standardized curve.

[0151] Sample analysis of in-house PK testing In the 4-MUP assay, enzyme activity was quantified via standardized enzyme activity curves created using serial dilutions of asfotase alpha protein reference standards with known activity on the same plate. Thawed plasma samples were diluted 100- to 2,000-fold in assay buffer (50 mM HEPES, 150 mM NaCl, 1 mM MgCl2, pH 7.4, and 1 mg / mL BSA) and assayed to determine the active ALP201 enzyme concentration. The final protein standardized range for each plate was 4-80 ng / mL.

[0152] Diluted samples were quantified as follows. All diluted samples were brought to 37°C before the start of the assay by adding 4-MUP to the protein sample to obtain a final concentration of 10 mM 4-MUP. The production of 4-MU was measured at an excitation wavelength of 360 nm and an emission wavelength of 465 nm. Data was collected using a plate reader maintained at 37°C. Plates were read every 40 seconds for a total of 20 minutes. The reaction rates of plasma and standard samples were calculated using Microsoft Excel by linear regression of the reaction gradient in units of relative fluorescence units (RFU) per minute. A linear standard curve was created using the reaction rate of the standard sample in units of RFU / min / U. The plasma activity of ALP201 in U / mL was determined by comparing the reaction rates measured in plasma samples with the standard curve. The sample activity in units / mL was converted to mg / L by dividing the sample activity by the specific activity (units U / mg) of the protein sample tested. To consolidate data from each independent sample collection point, the sampling was performed twice, with two samples collected at a time.

[0153] PK test sample analysis The same 4-MUP hydrolysis assay was performed. On the day of the assay, the standard, quality control (QC), dilution control (DC), and blank were diluted 250-fold with assay buffer. Furthermore, the standard, QC, DC, and blank were diluted 2-fold with the substrate 4-methylumbelliferyl phosphate (4-MUP) to a total minimum required dilution (MRD) of 500-fold. Before performing the MRD, the DC was diluted 200-fold with mouse plasma. ALP201 was quantified based on the fluorescent product methylumbelliferone, which results from the hydrolysis of the 4-MUP substrate used in the assay. The plate was placed on a plate reader and read every 60 seconds for 25 minutes at 37°C with dynamic fluorescence settings of 360 nm (excitation), 455 nm (cutoff), and 465 nm (emission). Enzyme activity was directly proportional to the substrate reaction rate. Vmax (rate in fluorescence intensity / min) was determined from the line that best fit across the data. Blanks were not included in the curve fitting. Results were reported in mg / L. Values ​​were corrected for differences in specific activity between the test substance and the protein activity standard.

[0154] Pharmacokinetic analysis PK analysis was performed using Phoenix WinNonlin v8.0 (Certara). Pharmacokinetic parameters were calculated using non-compartmental analysis.

[0155] The following non-compartmental PK parameters were calculated: Area under the concentration-time curve (AUC) from time 0 (administration) to the final detectable concentration. t ), and the area under the dose-normalized concentration-time curve (AUC) from time 0 (administration) to the final detectable concentration. t Area under the concentration-time curve (AUC) extrapolated to time 0 (administration) or infinity ( / dose). inf ), the area under the dose-normalized concentration-time curve (AUC) extrapolated to time 0 (administration) or infinity. inf ( / dose), observed highest plasma concentration (C) max ), dose-normalized observed peak plasma concentration (C max ( / dose), time to the highest observed plasma concentration (Tmax ), terminal elimination half-life (t 1 / 2 ), total clearance (CL), apparent volume of distribution (V d ), and bioavailability (F). PK parameters were calculated using actual sample collection time. Dose-normalized PK parameters were calculated using actual doses.

[0156] Results and Discussion Pharmacokinetic parameters of ALP201 after IV and SC administration in mice The pharmacokinetic activity (PK) of ALP201 was evaluated after a single IV or SC administration to male C57BL / 6 mice. Blood samples were collected at 0.25, 1, 6, 24, 48, 72, 96, 120, 192, 264, 336, and 480 hours post-administration from cohorts of 16 mice per group, further subdivided into groups of 4 mice.

[0157] Plasma concentrations of ALP201 were measured using an enzyme activity assay that measures the alkaline phosphatase catalytic activity of ALP201. The enzyme activity of ALP201 was then converted to mass units (mg / L) for reporting.

[0158] The mean active ALP201 plasma concentration-versus-time profiles after IV and SC administration are shown in Figures 8A and 8B on a semi-logarithmic scale. An overview of the PK parameters of ALP201 is shown in Tables 7 and 11 above.

[0159] The individual plasma activity profiles of some mice in the IV group suggest that a portion of the dose may have been delivered subcutaneously, which could explain the relatively high variability observed at the early stages of the IV dose profile. If this occurred, the calculated C for the IV dose group would be... max The value of can also be artificially lowered. After IV administration, the concentration-time profile decreased exponentially (Figures 8A and 8B). CL was 0.0019 L / h / kg, and the apparent terminal phase t1 / 2 was 48 hours. The time to reach peak concentration (t) after SC administration was also measured. maxThe absorption rate was 48 hours after administration. This suggests that absorption from the SC injection site is relatively slow. The absolute bioavailability after SC administration was 96%.

[0160] Pharmacokinetic parameters of ALP201 lots with various total sialic acid content values ​​in mice Three separate lots of ALP201 purified with 5.9, 5.0, and 3.2 moles of sialic acid per mole of protein monomer were administered to 32 mice via IV and SC administration. From cohorts subdivided into groups of four mice each, blood samples were collected at 0.5, 1, 3, 8, 12, 21, 26, 32, 45, 49, 72, 96, 120, 192, 264, and 336 hours post-administration for IV administration, and at 1, 3, 6, 8, 12, 21, 26, 32, 45, 49, 72, 96, 120, 192, 264, and 336 hours post-administration for SC administration, with two blood samples taken from each mouse.

[0161] Plasma concentrations of ALP201 were measured using an assay that measures the alkaline phosphatase catalytic activity of ALP201. The enzyme activity of ALP201 was reported by Charles River in units of mass (mg / L).

[0162] The mean active ALP201 plasma concentration-time profiles after IV and SC administration for each protein lot are shown on a semi-logarithmic scale in Figures 9A and 9B, 10A and 10B, and 11A and 11B. Figure 12 shows the collection of plasma concentration-time profiles for IV administration at various TSAC values. Figure 13 shows the collection of plasma concentration-time profiles for SC administration. Tables 21 and 22 summarize the mean PK parameters and mean dose-normalized PK parameters after IV and SC administration, respectively. [Table 23] [Table 24]

[0163] ALP201 protein lots with TSAC values ​​of 5.9, 5.0, and 3.2 mol / mol correspond to 1.48, 1.25, and 0.80 mol of sialic acid per common N-linked glycan site, and the PK profiles of all lots (both IV and SC administrations) were similar in shape. max and t 1 / 2 The values ​​were also similar.

[0164] Increasing the TSAC value slightly reduces clearance, and C occurs through both the IV and SC pathways. max And AUC increased, and volume of distribution decreased. All of these changes are consistent with the idea that lower TSAC values ​​indicate higher levels of immature N-linked glycans, which can be rapidly removed by cell receptors such as ASGPR, and lead to increased clearance of the protein sample, C max The AUC decreases. The changes in these parameters are relatively small, with a maximum difference of twofold.

[0165] Comparison of PK parameters of ALP201 and asfotase alfa in mice. As a second-generation alkaline phosphatase ERT, ALP201 was designed to improve upon the PK parameters of asfotase alfa, which were previously tested in a mouse PK study using male C57BL / 6 mice. A summary of the PK parameters of asfotase alfa is shown in Table 25. [Table 25]

[0166] In all PK datasets collected for ALP201 in male C57BL / 6 mice, ALP201 exhibited excellent in vivo half-life (t 1 / 2The study showed in vivo exposure (measured by Cmax and AUC) and bioavailability. It also demonstrated that ALP201 had significantly lower clearance than asfotase alfa in mice in all tested samples.

[0167] The release specifications for asfotase alfa restrict the molecular TSAC value to 1.2–3.0 mol / mol, and since the molecule has six N-linked glycosylation sites per monomer, the amount of sialic acid incorporated by asfotase alfa is only 0.20–0.50 moles per glycan. This suggests that many glycans on asfotase alfa do not contain sialic acid sites and may be effective substrates for ASPGR clearance. This may explain the higher clearance and lower dose-normalized Cmax and AUC with asfotase alfa compared to ALP201.

[0168] All of these ALP201 samples were far more bioavailable than asfotase alfa, with the smallest bioavailable ALP201 sample being approximately twice the observed bioavailability of asfotase alfa (72.1% vs. 38.8%). Differences in human IgG Fc domain isotypes between the two constructs may be the cause of this observation. We have previously shown that the IgG2 / 4 Fc domain of ALP201 does not bind to a common panel of Fcg receptors, while the IgG1 Fc domain of asfotase alfa strongly binds to at least two of them (Source: Research Technical Report 036). If these Fcg receptors are present in the subcutaneous space, asfotase alfa may already be cleared before entering the systemic circulation, thereby reducing its bioavailability and exposure.

[0169] conclusion The findings from these trials indicate that ALP201 has higher bioavailability and dose-normalized C maxAnd, with dose-normalized exposure, shows a substantial improvement in the dose-corrected pharmacokinetic profile in mice compared to asparaginase alpha after IV or SC administration. Additional PK studies using ALP201 samples with various levels of TSAC uptake showed that as the TSAC level increased, C max and in vivo exposure increased slightly. The elimination half-life, T max , and bioavailability did not appear to be significantly affected by changes in the TSAC value.

[0170] Example 3 Summary of single-dose PK study using ALP201 As part of the asparaginase alpha non-clinical development program, two single-dose PK studies were conducted in Sprague-Dawley rats and cynomolgus monkeys, respectively (Table 26). The ALP201 single-dose study was supported by these tests.

Table 26

[0171] After single-dose IV administration, the systemic clearance (CL) of asparaginase alpha was in the range of 0.0193 - 0.0492 L / h / kg, and the apparent half-life was in the range of 27 - 34 hours. The volume of distribution at steady state (V ss ) was in the range of 0.432 - 1.39 L / kg. After single-dose SC administration to rats and monkeys, the time to reach the maximum concentration (t max ) was 10 - 32 hours after administration, suggesting slow absorption of asparaginase alpha from the SC injection site. The estimated bioavailability of the SC administration route was in the range of 26% - 35% in rats and monkeys.

[0172] Conclusions based on single-dose PK study ALP201 was designed to improve based on the characteristics of approved product asparaginase alpha, with improvements in efficacy / activity, systemic exposure, absolute bioavailability, and half-life. The Ig Fc domain isotype in ALP201 is G2 / 4 instead of the IgG1 Fc domain present in asparaginase alpha. This modification was designed to improve the pharmacokinetic properties of ALP201, including systemic PK exposure, half-life, and bioavailability.

[0173] Preclinical data from three animal species (mouse, rat, and monkey) support the evidence that these objectives have been achieved. Compared with asparaginase alpha, ALP201 showed significant improvements in PK exposure (>10-fold), absolute bioavailability (2-fold), and half-life (2-fold) in three animal species after IV / SC administration. Preclinical population PK modeling also anticipates that the improvement in the PK properties of ALP201 will apply to humans, which will facilitate patient treatment through reduced injection volume, reduced dosing frequency, and reduced annual total dose, thereby predicting a reduction in the treatment burden on patients.

[0174] Nonclinical safety studies Summary of toxicity tests to support the first-in-human study An independent single-dose toxicity / tolerance study for ALP201 was not conducted. The ALP201 doses in the repeated-dose GLP toxicity study were selected based on data obtained from the PK studies of single-dose IV and SC administration of ALP2 in rats and monkeys. The tolerance of ALP201 was evaluated by clinical observations including injection site reactions and clinical pathology data in the PK studies of single-dose IV and SC administration in rats and monkeys. In summary, ALP201 had good tolerance when administered as a single dose up to 27 mg / kg in rats and 20 mg / kg in monkeys via IV or SC.

[0175] Definitive GLP-compliant 28-day toxicity studies of ALP201 in cynomolgus monkeys and Sprague-Dawley rats are ongoing. The survival portion, including the recovery phase, of the rat and monkey studies is complete, and reports are being prepared. Audited draft reports for these studies are not available, but all data from both studies, excluding ADA data, are available, and a summary of the findings is shown in Table 27. In summary, the results from the 28-day toxicity studies of ALP201 in rats and monkeys indicate that ALP201 treatment did not result in any notable / biologically significant treatment-related adverse effects from any of the toxicity endpoints evaluated in both studies, including safety pharmacological endpoints. [Table 27-1] [Table 27-2]

[0176] Non-clinical safety strategy for ALP201 in late-stage clinical development ALP201 is an improved ERT compared to the ERT product Asfotase Alpha (STRENSIQ®) marketed by Alexion. The human TNSALP catalytic domain of ALP201 contains three rationally designed modifications to confer higher enzymatic activity. The Fc portion of ALP201 is human immunoglobulin gamma 2 / 4 (IgG2 / 4), while the Fc portion of Asfotase Alpha is human immunoglobulin IgG1. The C-terminal regions of ALP201 and Asfotase Alpha that target bone are identical.

[0177] The findings obtained from ALP201 and the selected asfotase alpha test are as follows: 1. The toxic findings of ALP201 in GLP 28-day toxicity studies in rats and monkeys are very similar (Table 31). 2. Toxicity findings from toxicity tests (4 weeks / 28 days) of ALP201 and asfotase alfa in monkeys and rats over the same period were very similar (Tables 32 and 33, respectively). 3. The complete non-clinical safety package of asfotase alfa has shown only transient local tolerability findings in rats, and there have been no other notable adverse findings associated with asfotase alfa treatment.

[0178] Long-term / 6-month general toxicity testing strategy for ALP201 The long-term toxicity testing strategy for a single species is supported by the following observations. 4. In 28-day GLP toxicity studies in rats and monkeys, no significant systemic toxicity and / or local tolerability findings were observed after 10 repeated administrations of ALP201 via SC every 3 days (Q3D); however, systemic exposure to ALP201 was significantly higher than that to asfotase alfa. A summary of systemic exposures is shown in Tables 31 and 32. 5. The toxic findings from the ALP201 rat and monkey 28-day GLP toxicity studies were very similar. A summary of the findings from these studies is shown in Table 28. 6. When toxicological findings from short-term general toxicity tests are similar, a long-term general toxicity test on a single substance is usually sufficient. 7. Rats are a preferred species for long-term toxicity studies using ALP201. In addition, rats are a preferred species when only one species is used to conduct long-term general toxicity studies. [Table 28-1] [Table 28-2] [Table 28-3] [Table 29-1]

Table 29-2

Table 29-3

[0179] Model-based analysis and predicted human dosing regimen ALP201 modeling Data used in the current model To perform dose extrapolation from mouse (disease model species) to human, sufficient PK analysis to simulate human exposure from doses adjusted based on relative growth is required. A single C トラフ measurement value (D36 / D37) was available from limited mouse PK data, a single-dose wild-type mouse study (HPP-PK-01) and two repeated-dose Akp2GW(- / -) mouse efficacy studies (HPP-PoC-01 and HPP-MED-01). To create a reliable PK characterization of ALP201, mouse data was augmented with PK data from dose-range finding studies in rats and cynomolgus monkeys. Table 30 shows the data (47 animals and 387 PK concentrations) analyzed so far using the population pharmacokinetic (Pop-PK) modeling approach. For dose-response modeling, efficacy (bone mineralization) data from two Akp2GW(- / -) mouse efficacy studies (HPP-PoC-01 and HPP-MED-01) were used. <0OO0978>

Table 30

[0180] Current Pop-PK model Considering species differences in PK dynamics based on body weight according to the relative growth principle, the current Pop-PK model was developed using the NONMEM software program, version 7.2 (ICON solutions), which simultaneously analyzes PK data from three animal species. Here, animal body weight was centered at 70 kg, and the relative growth index was fixed at 0.75 for the clearance parameter and 1.0 for the volume of distribution parameter. IV and SC data were also fitted simultaneously to estimate extravascular parameters, such as absolute bioavailability after SC injection. The current model is a two-compartment model with loss of linearity, and the estimated model parameters are shown in Table 31. [Table 31]

[0181] Standard or mean parameter estimates for PK parameters were well estimated. CL variability (BSV) was within acceptable limits and estimated well, but Ka and F variability was high (86% and 76%, respectively) and not estimated well. Contraction assessed how the data indicated the PK parameter estimates, with a value of <30% being considered good. Given the high contraction values ​​for Ka and F (40% and 59%, respectively), additional data from the 4-week GLP toxicity study should improve the estimates for the Ka and F parameters. Overall, the current Pop-PK model should provide reasonable dose simulations given the good mean parameter estimates. However, the variability range may be excessively broad.

[0182] Current dose-response model E max We selected a development dose-response model by testing family models. Most dose-response relationships are E max This can be explained by one of the model parameterizations. The current best dose-response characterization is E maxIt uses the +E0 (baseline) model. Bone mineralization, which is an efficacy evaluation item, is an evaluation by X-ray imaging and was selected because it is the same as the clinical definition of the efficacy of asfotase alfa used in non-clinical dose-response evaluations. Bone mineralization after treatment with asfotase alfa or ALP201 based on two efficacy trials (HPP-PoC-01 and HPP-MED-01) was plotted against the dose (Figure 16). Asfotase alfa data from the previous efficacy trial ALP-PT-12 was included for comparison. The y-axis represents the % normal value defined as the proportion of mice in the dosing group with a bone mineralization score of 4. The x-axis represents the dose normalized to mg / kg / day. The dose that brings normal mineralization to 85% of the treated population (ED85) was selected as the target effective dose.

[0183] Dose conversion from mouse to human and proposal of human starting dose Relative growth scaling was applied to the mouse target effective dose (ED85) to determine the human dose. The formula for predicting the human equivalent dose (HED, mg / kg / day) from mouse ED85 was ED85 × (0.025 kg mouse / 70 kg human) 0.25 which was. When converted, it is a constant weekly dose of 45 mg / week, which is used as the starting dose for the MAD arm of the FIH trial. After considering the results from the NOAEL and MABEL methods, the maximum recommended starting dose (MRSD) for the SAD arm was selected. The test dose that brings the minimum on-target pharmacological response was selected and it was determined to convert to a fixed human dose (5 mg).

[0184] Other embodiments The forms and examples for implementing the foregoing invention were presented only for the purpose of clarifying understanding. No unnecessary limitations should be understood therefrom. The present disclosure is not strictly limited to the details illustrated and described, and modifications apparent to those skilled in the art are intended to be included within the scope defined by the claims.

[0185] Unless otherwise stated, all figures representing quantities, molecular weights, etc., of components used herein and in the claims should be understood in all cases as being modified by the term “approximately.” Therefore, unless otherwise stated, the numerical parameters described herein and in the claims are approximations that may vary depending on the desired properties to be obtained. At a minimum, and not in an attempt to limit the principle of equivalents which is equivalent to the claims, each numerical parameter should be interpreted with respect to the reported number of significant figures, by applying at least common rounding techniques.

[0186] Although the numerical ranges and parameters representing the broad scope of this disclosure are approximations, the numerical values ​​shown in specific examples are reported as accurately as possible. However, all numerical values ​​inherently contain a certain range that inevitably arises from the standard deviations observed in their corresponding test measurements.

[0187] The complete disclosure of all patents, patent applications including provisional patent applications, publications including patent and non-patent publications, and electronically available materials (including, for example, nucleotide sequence submissions in GenBank and RefSeq, and, for example, amino acid sequence submissions in SwissProt, PIR, PRF, and PDB, and translations from annotated code areas in GenBank and RefSeq) described herein is incorporated by reference. The modes and examples for carrying out the invention described above are presented solely for the purpose of clarifying understanding. No unnecessary limitations should be derived therefrom. This disclosure is not strictly limited to the details illustrated and described, and variations that are obvious to those skilled in the art are included within the scope of the embodiments defined by the claims. The present invention provides, for example, the following items: (Item 1) A pharmaceutical composition comprising a polypeptide having at least 80% sequence identity with respect to SEQ ID NO: 5 and at least one mutation selected from E108M, N213Q, and N286Q compared to SEQ ID NO: 1, and a pharmaceutically acceptable carrier comprising one or more of phosphate, proline, and sucrose. (Item 2) The pharmaceutical composition according to item 1, wherein the polypeptide has at least 85%, 90%, 95%, 97%, or 99% sequence identity with respect to SEQ ID NO: 5. (Item 3) The pharmaceutical composition according to item 2, wherein the polypeptide comprises or consists of the sequence of Sequence ID No. 5. (Item 4) The pharmaceutical composition according to any one of items 1 to 3, wherein the polypeptide comprises two or three mutations E108M, N213Q, and N286Q compared to SEQ ID NO: 1. (Item 5) The aforementioned composition is a pharmaceutical composition according to any one of items 1 to 4, comprising about 1 mM to about 100 mM of phosphate. (Item 6) The aforementioned composition is the pharmaceutical composition described in item 5, comprising approximately 5 mM to approximately 20 mM of phosphate. (Item 7) The aforementioned composition is the pharmaceutical composition described in item 6, comprising approximately 10 mM of phosphate. (Item 8) The pharmaceutical composition according to any one of items 1 to 7, wherein the phosphate is sodium phosphate. (Item 9) The composition is a pharmaceutical composition according to any one of items 1 to 8, comprising approximately 1 mM to approximately 500 mM proline. (Item 10) The aforementioned composition is the pharmaceutical composition described in item 9, comprising approximately 50 mM to approximately 200 mM proline. (Item 11) The composition is the pharmaceutical composition described in item 10, comprising approximately 140 mM proline. (Item 12) The aforementioned composition is a pharmaceutical composition according to any one of items 1 to 11, comprising approximately 1 mM to approximately 500 mM sucrose. (Item 13) The aforementioned composition is the pharmaceutical composition described in item 12, comprising approximately 50 mM to approximately 200 mM sucrose. (Item 14) The aforementioned composition is the pharmaceutical composition described in item 13, comprising approximately 140 mM sucrose. (Item 15) The composition is a pharmaceutical composition according to any one of items 6 to 14, comprising about 10 mM phosphate, about 140 mM proline, and about 140 mM sucrose. (Item 16) The composition is a pharmaceutical composition according to any one of items 1 to 15, comprising about 0.01% to about 0.5% sorbitan polyoxyethylene (20) monooleate. (Item 17) The pharmaceutical composition according to item 16, wherein the composition comprises about 0.01% to about 0.1% of sorbitan polyoxyethylene (20) monooleate. (Item 18) The pharmaceutical composition described in item 17, wherein the composition contains about 0.05% sorbitan polyoxyethylene (20) monooleate. (Item 19) The pharmaceutical composition according to item 18, comprising about 140 mM proline, about 140 mM sucrose, and about 0.05% sorbitan polyoxyethylene (20) monooleate. (Item 20) The polypeptide comprises a total sialic acid content (TSAC) of approximately 1.0 mol / mol to approximately 6.0 mol / mol, as described in any one of items 1 to 19. (Item 21) The pharmaceutical composition described in item 20, wherein the TSAC is approximately 3.0 mol / mol to approximately 6.0 mol / mol. (Item 22) The pharmaceutical composition according to item 21, wherein the TSAC is approximately 3.2 mol / mol, 5.0 mol / mol, or approximately 5.9 mol / mol. (Item 23) The composition is a pharmaceutical composition according to any one of items 1 to 22, having a pH of approximately 7.3. (Item 24) The aforementioned composition is a pharmaceutical composition according to any one of items 1 to 23, prepared at a concentration of about 0.1 mg / mL to about 200 mg / mL. (Item 25) The composition is a pharmaceutical composition according to any one of items 1 to 24, prepared in a volume of about 0.1 mL to about 50 mL. (Item 26) The pharmaceutical composition described in item 24 or 25, which is prepared at a concentration of about 100 mg / mL in a volume of about 1 mL. (Item 27) A vial containing the pharmaceutical composition described in any one of items 1 to 23. (Item 28) The composition is prepared in a vial as described in item 27, in a volume of approximately 0.1 mL to approximately 50 mL. (Item 29) The composition is prepared in a vial as described in item 28, with a volume of approximately 10 mL. (Item 30) The composition is prepared in a vial according to any one of items 27 to 29, at a concentration of about 0.1 mg / mL to about 200 mg / mL. (Item 31) The composition is prepared in a vial as described in item 30, at a concentration of approximately 100 mg / mL. (Item 32) The composition is prepared in a vial according to any one of items 27 to 31, with a concentration of approximately 100 mg / mL in a volume of approximately 1 mL. (Item 33) A method for treating a bone mineralization disorder in a patient in need thereof, comprising administering to the patient a dose of a pharmaceutical composition described in any one of items 1 to 26. (Item 34) The aforementioned bone mineralization disorder is selected from hypophosphatasia (HPP), fracture, osteoporosis, indurative ossification, chondrocalcinosis, hypotonia, Duchenne muscular dystrophy, tracheobronchomalacia, seizures, neurofibromatosis, and craniosynostosis, as described in item 33. (Item 35) The method according to item 33 or 34, wherein the polypeptide is administered in an amount and for a duration sufficient to treat the bone mineralization disorder. (Item 36) The treatment described herein is the method described in any one of items 33 to 35, which promotes bone formation in the patient. (Item 37) The polypeptide is administered in a dose of approximately 0.01 mg / kg to approximately 20 mg / kg, according to the method described in any one of items 33 to 36. (Item 38) The polypeptide is administered in a dose of approximately 0.1 mg / kg to approximately 10 mg / kg, as described in item 37. (Item 39) The polypeptide is administered once daily, once weekly, once monthly, or once year, according to the method described in any one of items 33 to 38. (Item 40) The method according to any one of items 33 to 39, wherein approximately 10 to approximately 100 mg of the polypeptide is administered per week. (Item 41) The method described in item 40, in which approximately 15 to 90 mg of polypeptide is administered per week. (Item 42) The method described in item 41, in which approximately 15 mg, 30 mg, 45 mg, or 90 mg of polypeptide is administered per week. (Item 43) The polypeptide is administered for at least one day, one week, one month, one year, or longer, according to any one of items 33 to 42. (Item 44) The composition is administered subcutaneously, intravenously, intramuscularly, sublingually, subarachnoidally, or intradermally according to any one of items 33 to 43. (Item 45) The composition is administered subcutaneously or intravenously according to the method described in item 44. (Item 46) The patient is human, as described in any one of items 33-45. (Item 47) The method according to item 46, wherein the person is a newborn, child, adolescent, or adult. (Item 48) By the above method, the AUC in the patient's blood ranges from approximately 50 μg × hour / mL to approximately 4000 μg × hour / mL. 0-168h The method described in any one of items 33-47, which results in the occurrence of the method described in any one of items 33-47. (Item 49) By the above method, the AUC in the patient's blood ranges from approximately 1000 μg × hour / mL to approximately 3000 μg × hour / mL. 0-168h The method described in item 48, which results in the occurrence of the event. (Item 50) By the above method, approximately 0.5 μg / mL to approximately 25 μg / mL of C is obtained in the patient's blood. max The method described in any one of items 33-48, which results in the occurrence of the method described in any one of items 33-48. (Item 51) By the above method, approximately 0.6 μg / mL to approximately 20 μg / mL of C is obtained in the patient's blood. max The method described in item 49, which results in the occurrence of the event.

Claims

1. A pharmaceutical composition comprising a polypeptide having at least 90% sequence identity with respect to Sequence ID No. 5 and E108M, N213Q, and N286Q mutations compared to Sequence ID No. 1, and a pharmaceutically acceptable carrier comprising one or more of phosphate, proline, and sucrose, wherein the polypeptide hydrolyzes inorganic pyrophosphate (PPi) to provide inorganic phosphoric acid (Pi).

2. The pharmaceutical composition according to claim 1, wherein the polypeptide has at least 95%, 97%, or 99% sequence identity with respect to SEQ ID NO:

5.

3. The pharmaceutical composition according to claim 2, wherein the polypeptide comprises or is composed of the sequence of Sequence ID No.

5.

4. The pharmaceutical composition according to any one of claims 1 to 3, wherein the composition contains about 1 mM to about 100 mM of phosphate.

5. The pharmaceutical composition according to claim 4, wherein the composition comprises about 5 mM to about 20 mM of phosphate.

6. The pharmaceutical composition according to claim 5, wherein the composition comprises about 10 mM of phosphate.

7. The pharmaceutical composition according to any one of claims 1 to 6, wherein the phosphate is sodium phosphate.

8. The pharmaceutical composition according to any one of claims 1 to 7, wherein the composition contains about 1 mM to about 500 mM proline.

9. The pharmaceutical composition according to claim 8, wherein the composition contains about 50 mM to about 200 mM proline.

10. The pharmaceutical composition according to claim 9, wherein the composition contains about 140 mM proline.

11. The pharmaceutical composition according to any one of claims 1 to 10, wherein the composition contains about 1 mM to about 500 mM sucrose.

12. The pharmaceutical composition according to claim 11, wherein the composition contains about 50 mM to about 200 mM sucrose.

13. The pharmaceutical composition according to claim 12, wherein the composition contains about 140 mM sucrose.

14. The pharmaceutical composition according to any one of claims 5 to 13, wherein the composition comprises about 10 mM phosphate, about 140 mM proline, and about 140 mM sucrose.

15. The pharmaceutical composition according to any one of claims 1 to 14, wherein the composition comprises about 0.01% to about 0.5% of sorbitan polyoxyethylene (20) monooleate.

16. The pharmaceutical composition according to claim 15, wherein the composition comprises about 0.01% to about 0.1% of sorbitan polyoxyethylene (20) monooleate.

17. The pharmaceutical composition according to claim 16, wherein the composition comprises about 0.05% sorbitan polyoxyethylene (20) monooleate.

18. The pharmaceutical composition according to claim 17, wherein the composition comprises about 140 mM proline, about 140 mM sucrose, and about 0.05% sorbitan polyoxyethylene (20) monooleate.

19. The pharmaceutical composition according to any one of claims 1 to 18, wherein the polypeptide comprises a total sialic acid content (TSAC) of about 1.0 mol / mol to about 6.0 mol / mol.

20. The pharmaceutical composition according to claim 19, wherein the TSAC is approximately 3.0 mol / mol to approximately 6.0 mol / mol.

21. The pharmaceutical composition according to claim 20, wherein the TSAC is approximately 3.2 mol / mol, 5.0 mol / mol, or approximately 5.9 mol / mol.

22. The composition is a pharmaceutical composition according to any one of claims 1 to 21, comprising a pH of about 7.

3.

23. The pharmaceutical composition according to any one of claims 1 to 22, wherein the composition is prepared at a concentration of about 0.1 mg / mL to about 200 mg / mL.

24. The pharmaceutical composition according to any one of claims 1 to 23, wherein the composition is prepared in a volume of about 0.1 mL to about 50 mL.

25. The pharmaceutical composition according to claim 23 or 24, wherein the composition is prepared at a concentration of about 100 mg / mL in a volume of about 1 mL.

26. A vial containing the pharmaceutical composition according to any one of claims 1 to 25.

27. The vial according to claim 26, wherein the composition is prepared in a volume of about 0.1 mL to about 50 mL.

28. The vial according to claim 27, wherein the composition is prepared in a volume of about 10 mL.

29. The vial according to any one of claims 26 to 28, wherein the composition is prepared at a concentration of about 0.1 mg / mL to about 200 mg / mL.

30. The vial according to claim 29, wherein the composition is prepared at a concentration of about 100 mg / mL.

31. The vial according to any one of claims 26 to 30, wherein the composition is prepared at a concentration of about 100 mg / mL in a volume of about 1 mL.

32. A pharmaceutical composition according to any one of claims 1 to 25 for treating hypophosphatasia (HPP) in a patient in need thereof, characterized in that a dose of the pharmaceutical composition is administered to the patient.

33. The pharmaceutical composition according to claim 32, wherein the patient further has a disorder selected from fracture, osteoporosis, indurative ossification, chondrocalcinosis, hypotonia, Duchenne muscular dystrophy, tracheobronchomalacia, seizures, neurofibromatosis, and craniosynostosis.

34. The pharmaceutical composition according to claim 32 or 33, characterized in that the polypeptide is administered in an amount and for a period of time sufficient to treat the HPP.

35. The pharmaceutical composition according to any one of claims 32 to 34, wherein the treatment described herein promotes bone formation in the patient.

36. The pharmaceutical composition according to any one of claims 32 to 35, characterized in that the polypeptide is administered in a dose of about 0.01 mg / kg to about 20 mg / kg.

37. The pharmaceutical composition according to claim 36, characterized in that the polypeptide is administered in a dose of about 0.1 mg / kg to about 10 mg / kg.

38. The pharmaceutical composition according to any one of claims 32 to 37, characterized in that the polypeptide is administered once a day, once a week, once a month, or once a year.

39. The pharmaceutical composition according to any one of claims 32 to 38, characterized in that approximately 10 to approximately 100 mg of the polypeptide is administered per week.

40. The pharmaceutical composition according to claim 39, characterized in that approximately 15 to approximately 90 mg of polypeptide is administered per week.

41. The pharmaceutical composition according to claim 40, characterized in that approximately 15 mg, approximately 30 mg, approximately 45 mg, or approximately 90 mg of polypeptide is administered per week.

42. The pharmaceutical composition according to any one of claims 32 to 41, characterized in that the polypeptide is administered for at least one day, one week, one month, one year, or longer.

43. The pharmaceutical composition according to any one of claims 32 to 42, characterized in that the composition is administered subcutaneously, intravenously, intramuscularly, sublingually, subarachnoidally, or intradermally.

44. The pharmaceutical composition according to claim 43, characterized in that the composition is administered subcutaneously or intravenously.

45. The pharmaceutical composition according to any one of claims 32 to 44, wherein the patient is a human.

46. The pharmaceutical composition according to claim 45, wherein the human is a newborn, a child, an adolescent, or an adult.

47. The pharmaceutical composition produces an AUC of approximately 50 μg × hour / mL to approximately 4000 μg × hour / mL in the patient's blood. ０－１６８ｈ A pharmaceutical composition according to any one of claims 32 to 46, which produces the following.

48. The pharmaceutical composition results in an AUC of approximately 1000 μg × hour / mL to approximately 3000 μg × hour / mL in the patient's blood. ０－１６８ｈ The pharmaceutical composition according to claim 47, wherein the following occurs.

49. The pharmaceutical composition provides the patient with approximately 0.5 μg / mL to approximately 25 μg / mL of C in their blood. ｍａｘ A pharmaceutical composition according to any one of claims 32 to 47, which produces the following.

50. The pharmaceutical composition provides the patient with approximately 0.6 μg / mL to approximately 20 μg / mL of C in their blood. ｍａｘ The pharmaceutical composition according to claim 48, wherein the following occurs.

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