Alkaline phosphatase polypeptide, and method for using the same

Abstract

To provide an alkaline phosphatase polypeptide, and a method for using the polypeptide.SOLUTION: A pharmaceutical composition contains soluble alkaline phosphatase for treating bone mineralization disorder such as hypophosphatasia (HPP) and symptoms thereof. A polypeptide contains soluble alkaline phosphatase (sALP) derived from naturally-occurring alkaline phosphatase (ALP) or fragments thereof.SELECTED DRAWING: Figure 1

Description

[Technical field]

[0001] Sequence Listing This application contains a Sequence Listing that has been submitted electronically in ASCII format and is hereby incorporated by reference in its entirety. The name of said ASCII copy, created on February 4, 2022, is 50694-094WO2_Sequence_Listing_2_4_22_ST25 and is 27,753 bytes in size. [Background technology]

[0002] Hypophosphatasia (HPP) is a rare genetic skeletal disorder with an incidence of 1 per 100,000 live births in the most severe form of the disease. The disorder is usually caused by loss-of-function mutations in the gene encoding tissue-nonspecific alkaline phosphatase (TNSALP). HPP presents with a wide range of symptoms and severity, ranging from early tooth loss to almost complete lack of bone mineralization in utero. Symptoms of HPP vary significantly from patient to patient and with the patient's age. Many HPP patients present with skeletal changes, short stature, chronic pain, pain in the legs, gait disturbances, and early non-traumatic tooth loss. Asfotase alfa (STRENSIQ®, Alexion Pharmaceuticals, Inc.), a recombinantly produced enzyme replacement therapy (ERT) containing a soluble fragment of TNSALP, is the first ERT available to HPP patients. Asfotase alfa has shown transformative effects thus far against the most severe forms of HPP, as evidenced 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 literature]

[0003] [Non-Patent Document 1] Whyte et al.,New Engl.J.Med.366:904-913,2012 Summary of the Invention

[0004] A first aspect features a pharmaceutical composition including 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 pharma- ceutically acceptable carrier. The polypeptide can include at least one mutation selected from E108M, N213Q, and N286Q (e.g., the polypeptide can include two or all three of these mutations) compared to the amino acid sequence of SEQ ID NO:1. 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 includes at least one, two, or all three of the mutations selected from E108M, N213Q, and N286Q. The pharma- ceutically acceptable carrier can include one or more of phosphate, proline, and sucrose. For example, the polypeptide can comprise or consist of the amino acid sequence of SEQ ID NO:5.

[0005] In some embodiments, the alkaline phosphatase portion 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. The polypeptide can be further linked to an Fc region (e.g., an IgG1, IgG2, IgG3, or IgG4 Fc region) and / or a polyaspartic acid region. In some embodiments, the polypeptide comprises an IgG2 / 4 Fc region, e.g., has at least 80% (e.g., at least 85%, 90%, 95%, 97%, 99%, or 100%) identity to SEQ ID NO:4. The polyaspartic acid can comprise, e.g., 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 comprises 10 aspartic acid residues (D10).

[0006] The composition may be prepared to contain a dosage of alkaline phosphatase polypeptide of 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 a volume of 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 100 mg / mL of an alkaline phosphatase polypeptide having at least 80% (e.g., at least 85%, 90%, 95%, 97%, or 99%) sequence identity to, or a sequence of, SEQ ID NO:5 (e.g., the polypeptide includes at least one, two, or all three of mutations selected from E108M, N213Q, and N286Q of SEQ ID NO:5) and a pharma- ceutically acceptable carrier.

[0007] The composition may include a phosphate (e.g., sodium phosphate) at a concentration 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 include proline and / or sucrose. The composition may further include proline. The composition may further include sucrose. For example, the composition may contain about 1 mM to about 500 mM proline, for example, about 70 mM to about 280 mM, for example, about 50 mM to about 200 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, 100 mM, 110 mM, 120 mM, 130 mM, 140 mM, 150 mM, 160 mM, 170 mM, 180 mM, 190 mM, 200 mM, 300 mM, 400 mM, or 500 mM, e.g., about 140 mM proline, and / or about 1 mM to about 500 mM sucrose, e.g., about 70 mM to about 280 mM, e.g. For example, about 50 mM to about 200 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, 100 mM, 110 mM, 120 mM, 130 mM, 140 mM, 150 mM, 160 mM, 170 mM, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 210, 220, 230, 240, 250, 260, 270, 280, 290, 300, 350, 400, 450, or 500 mM, e.g., about 140 mM sucrose, or about 210 mM sucrose. In some embodiments, the composition comprises a molar ratio of proline:sucrose of about 1:0 to about 1:3, e.g., about 1:1.In some embodiments, the formulation comprises 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 comprises about 140 mM proline. In some embodiments, the formulation comprises about 140 mM sucrose. In some embodiments, the formulation comprises about 210 mM sucrose. In some embodiments, the formulation comprises about 210 mM sucrose but does not comprise proline. In some embodiments, the formulation comprises about 140 mM proline and about 140 mM sucrose. In some embodiments, the formulation comprises about 140 mM proline, and 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 comprise about 0.01% to about 0.5% polyoxyethylene (20) sorbitan monooleate, such as about 0.01% to about 0.1% polyoxyethylene (20) sorbitan monooleate, such as about 0.05% polyoxyethylene (20) sorbitan monooleate. The polyoxyethylene (20) sorbitan monooleate may be, for example, polysorbate 80 (PS80). In some embodiments, the composition may be prepared at a pH of about pH 7.0 to about pH 7.6 (e.g., about pH 7.1, 7.2, 7.3, 7.4, 7.5, or 7.6, e.g., about pH 7.3). In some embodiments, the composition comprises about 10 mM phosphate, about 140 mM proline, about 140 mM sucrose, and about 0.05% polyoxyethylene (20) sorbitan monooleate (e.g., PS80) at a pH of about 7.3.

[0008] The composition may be a pharmaceutical composition prepared as a solution containing a polypeptide (e.g., a polypeptide of SEQ ID NO: 5, and variants thereof having at least about 85% sequence identity thereto (e.g., the polypeptide includes 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, for example, at a dosage of the polypeptide of about 0.1 mg / mL to about 10 mg / mL. The composition may be prepared in a solution having a volume of 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. The solution may contain about 10 mM phosphate, about 140 mM proline, about 140 mM sucrose, and about 0.05% polyoxyethylene (20) sorbitan monooleate (e.g., PS80) at a pH of about 7.3.

[0009] Also featured is a vial containing the pharmaceutical composition described herein. The vial may contain a solution (e.g., about 10 mM sodium phosphate, about 140 mM proline, about 140 mM sucrose, and about 0.05% polyoxyethylene (20) sorbitan monooleate (e.g., PS80) at a pH of about 7.3) containing a polypeptide (e.g., in an amount of about 0.1 mg to about 1.0 g (e.g., about 10 mg to about 200 mg)) in a volume of, for example, about 0.1 mL to about 10 mL (e.g., about 1 mL). The vial may contain the polypeptide in an amount of, for example, about 0.1 mg / mL to about 500 mg / mL, about 1 mg / mL to about 200 mg / mL, about 50 mg / mL to about 150 mg / mL, or about 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 (e.g., the polypeptide includes one, two, or all three of the mutations selected from E108M, N213Q, and N286Q of SEQ ID NO:5).

[0010] A second aspect features a method of treating a bone mineralization disorder or a symptom of a bone disease in a patient (e.g., a human patient) (e.g., a disease selected from the group consisting of hypophosphatasia (HPP), bone fracture, osteoporosis, sclerosteosis, chondrocalcinosis, hypotonia, Duchenne muscular dystrophy, tracheobronchomalacia, stroke, neurofibromatosis (e.g., NF-1), and craniosynostosis, or one or more symptoms thereof) by administering to a patient in need thereof a pharmaceutical composition of the first aspect. The composition may be administered in an amount and for a duration sufficient to treat the disease or alleviate one or more symptoms thereof. The treatment may promote bone formation in the patient. The polypeptides can be used to treat muscle weakness.

[0011] The polypeptide or a pharmaceutical composition containing the same may be administered at a dosage 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, or, e.g., about 0.1 mg / kg to about 10 mg / kg). The polypeptide may be administered once a day, week, month, or 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 once a week, once every two weeks, once every three weeks, once every four or more weeks. The polypeptide may be administered at a dosage of about 0.01 mg / kg / week to about 50 mg / kg / week (e.g., about 0.01 mg / kg / week to about 40 mg / kg / week, e.g., about 0.1 mg / kg / week to about 20 mg / kg / week, or, e.g., about 0.1 mg / kg / week to about 10 mg / kg / week). The polypeptide may be administered for at least one day, one week, one month, one year, or longer (e.g., for the life of the patient).

[0012] The polypeptide or a pharmaceutical composition containing it may be administered subcutaneously, intravenously, intramuscularly, sublingually, intrathecally, or intradermally. In particular, the polypeptide or a composition containing said polypeptide may be administered by subcutaneous or intravenous administration.

[0013] In some embodiments, the pharmaceutical composition is administered subcutaneously (e.g., into 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, e.g., about 15 mg, 45 mg, or 90 mg, e.g., about 15 mg, 45 mg, or 90 mg) may be administered subcutaneously to a patient once or twice a week, e.g., for at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 weeks or more, or for a longer period (e.g., for the patient's lifetime). The composition may be administered, for example, in a volume of about 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 ranging from about 5 mL to about 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, for at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 weeks, or more (e.g., for 3 weeks), or for a longer period (e.g., for the patient's life). For example, the pharmaceutical composition is administered by IV in a volume of about 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 ranging from about 5 mL to about 0.1 mL).

[0015] In some embodiments, the pharmaceutical composition is administered intravenously and subcutaneously (e.g., in the abdomen or thigh). For example, the pharmaceutical composition is administered in a treatment regimen that combines intravenous and subcutaneous (e.g., in the abdomen or thigh) administration. For example, the composition may be first administered intravenously to a patient in 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 (e.g., in the abdomen or thigh) to a patient in one or more doses over time. For example, a subcutaneous dosage may be 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, e.g., about 15 mg, 45 mg, or 90 mg). A subcutaneous dosage may be administered, for example, once or twice weekly, once every two weeks, once every three weeks, or once every four weeks, e.g., for at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, or 50 weeks or more, or for a longer period of time (e.g., over 1 to 10 years, or over the life of the patient). Intravenous and subcutaneous doses may be administered, for example, in a volume of about 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 in a volume ranging from about 5 mL to about 0.1 mL).

[0016] The patient may be a human subject, such as a neonate, infant, child, adolescent, or adult.

[0017] In some embodiments, the TSAC of the 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 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 is about 3.0 mol / mol to about 6.0 mol / mol relative to the recombinant alkaline phosphatase. In some embodiments, the TSAC is about 3.2 mol / mol to about 5.9 mol / mol relative to the recombinant alkaline phosphatase. In some embodiments, TSAC is about 0.9 mol / mol, about 1.0 mol / mol, about 1.1 mol / mol, about 1.2 mol / mol, about 1.3 mol / mol, about 1.4 mol / mol, about 1.5 mol / mol, about 1.6 mol / mol, about 1.7 mol / mol, about 1.8 mol / mol, about 1.9 mol / mol, about 2.0 mol / mol, about 2.1 mol / mol, about 2.2 mol / mol, about 2.3 mol / mol, about 2.4 mol / mol, about 2.5 mol / mol, about 2.6 mol / mol, about 2.7 mol / mol, about 2.8 mol / mol, about 2.9 mol / mol, about 3.0 mol / mol, about 3.1 mol / mol, about 3.2 mol / mol, about 3.3 mol / mol relative to the 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 the recombinant alkaline phosphatase. In some embodiments, the TSAC is about 5.0 mol / mol relative to the recombinant alkaline phosphatase. In some embodiments, the TSAC is about 5.9 mol / mol relative to the recombinant alkaline phosphatase.

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

[0019] In some embodiments, the method provides a method for determining whether a patient has an AUC of about 50 μg×hr / mL to about 4000 μg×hr / mL in blood. 0-168h For example, the method can produce an AUC of about 1000 μg × hr / mL to about 3000 μg × hr / mL in the patient's blood. 0-168h In some embodiments, the method can result 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 produce a C of about 0.6 μg / mL to about 20 μg / mL in the patient's blood. max may occur.

[0020] definition The term "about" refers to ±10% of the recited value. All measurements reported herein should be understood to be modified by the term "about," unless expressly stated otherwise, whether expressly used or not.

[0021] The term "bone-targeting moiety" refers to a bone-targeting moiety that alone binds to bone matrix 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 -9By "bone matrix" is meant an amino acid sequence at least three amino acid residues in length with sufficient affinity for bone matrix so as to have an in vivo binding affinity of M or greater.

[0022] The term "catalytically competent" as used herein refers to sALP that hydrolyzes inorganic pyrophosphate (PPi), a bone mineralization inhibitor, to generate inorganic phosphate (Pi), thereby reducing the extracellular concentration of PPi. Thus, catalytically competent sALP improves skeletal mineralization by regulating the concentration of PPi.

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

[0024] By "fragment" is meant a portion of a polypeptide or nucleic acid molecule, preferably comprising at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or more of the full length of a reference nucleic acid molecule or polypeptide. Fragments can be, 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, 310, 320, 330, 340, 350, 360, 370, 380, 390, 400, 410, 420, 430, 440, 450, 460, 470, 480, 490, 500, 510, 520, 530, 540, 550, 560, 570, 580, 590, 600, 610, 620, 630, 640, 650, 660, 670, 680, 690, 700, 710, 720, 730, 740, 750, 760 , 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 In some embodiments, the nucleic acid 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 genetic skeletal disorder caused by one or more loss-of-function mutations in the ALPL (alkaline phosphatase, liver / bone / kidney) gene, which encodes, for example, tissue-nonspecific alkaline phosphatase (TNSALP). HPP may be further characterized as infantile HPP, pediatric HPP, perinatal HPP (e.g., benign perinatal HPP or lethal perinatal HPP), odontogenic HPP, adolescent HPP, or adult HPP. For example, "pediatric HPP" refers to HPP patients between about 5 and about 12 years of age, "adolescent HPP" refers to HPP patients between about 13 and about 17 years of age, and "adult HPP" refers to HPP patients aged about 18 years or older. The term "adult HPP", as used herein, refers to a disease state or phenotype characterized by the presence of one or more of the following symptoms: elevated blood and / or urinary levels of inorganic pyrophosphate (PPi), hypomineralization, hypercalciuria, one or more skeletal deformities, hypotonia, muscle weakness, rheumatoid complications, duck gait, lameness, bone pain, pain, fractures, calcium pyrophosphate dihydrate crystal deposition, pseudogout, arthritis, pyrophosphate arthropathy, chondrocalcinosis, calcific periarthritis, and pseudofractures. The term "adolescent HPP", as used herein, refers to a condition or phenotype characterized by the presence of one or more of the following symptoms: elevated blood or urinary levels of PPi, PEA, or PLP; osteomalacia, one or more skeletal deformities, hypotonia, muscle weakness, rheumatoid complications, arthritis, pseudogout, duck gait, lameness, bone pain, pain, premature tooth loss, hypomineralization, pulmonary hypoplasia, respiratory insufficiency, seizures, hypercalciuria, short stature, and growth retardation. The term "pediatric HPP", as used herein, refers to a disease state or phenotype characterized by the presence of one or more of the following symptoms: elevated blood or urinary levels of PPi, PEA, or PLP; rickets, rachitic ribs, one or more skeletal deformities, hypotonia, muscle weakness, rheumatoid complications, arthritis, pseudogout, duck gait, lameness, bone pain, pain, early tooth loss, hypomineralization, delayed motor development, seizures, hypercalciuria, short stature, fractures, pseudofractures, and growth retardation.

[0026] The term "nucleic acid" or "nucleic acid molecule" refers to a polymer having a sequence of two or more covalently linked naturally occurring or modified nucleotides, such as RNA or DNA. Nucleic acid molecules can be, for example, single-stranded or double-stranded, and can contain modified or unmodified nucleotides, or mixtures or combinations thereof. Various salts, mixed salts, and free acid forms of nucleic acid molecules are also included.

[0027] "Treating", "treat", and "treatment" refer to the medical management of a patient with the intent to cure, alleviate, stabilize, reduce the likelihood of, or prevent a condition, such as HPP (e.g., pediatric, adolescent, or adult HPP), or one or more symptoms thereof, by, for example, administering a pharmaceutical composition (e.g., sALP as described herein), and / or the management of a patient exhibiting or likely to have a condition, such as HPP. Treating (and other forms as used herein) includes active treatment, i.e., treatment that is specifically aimed at ameliorating or associated with curing a disease, pathological condition, disorder, or event, and also includes causal treatment, i.e., treatment that is aimed at eliminating the cause of the associated disease, pathological condition, disorder, or event. In addition, the term also includes palliative treatment, i.e., treatment designed to relieve or ameliorate at least one symptom instead of curing the disease, pathological condition, disorder, or event; symptomatic treatment, i.e., treatment directed to the systemic symptoms of the relevant disease, pathological condition, disorder, or event; prophylactic treatment, i.e., treatment aimed at minimizing or partially or completely inhibiting the onset of the relevant disease, pathological condition, disorder, or event, for example, in patients who are not yet ill but who are susceptible to or at risk for the particular disease, pathological condition, disorder, or event; and supportive treatment, i.e., treatment utilized to supplement another specific therapy aimed at ameliorating the relevant 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 naturally occurring or non-naturally occurring amino acid residues that constitutes all or part of a naturally occurring or non-naturally occurring polypeptide or peptide, regardless of post-translational modification (e.g., glycosylation, sialylation, or phosphorylation), as described herein.

[0029] The terms "sALP", "soluble alkaline phosphatase" and "extracellular domain of alkaline phosphatase" are used interchangeably (unless the context indicates otherwise) and refer to soluble, non-membrane-bound alkaline phosphatase, or biologically active fragments or variants thereof. sALP includes, for example, alkaline phosphatases lacking the C-terminal GPI signal sequence, as well as additional variants and analogs, that retain alkaline phosphatase activity, e.g., the ability to hydrolyze PPi or other natural or artificial substrate(s). This includes, unless otherwise indicated, soluble fragments corresponding to the extracellular domains of TNSALP, PALP, GLALP, and IALP, as well as biologically active fragments or variants thereof. Mature sALP lacks the GPI membrane anchor and signal peptide, which are cleaved during processing.

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

[0031] The term "pharmaceutically acceptable carrier" or "pharmaceutically acceptable excipient" refers to a carrier or excipient that is physiologically acceptable to the patient being treated while retaining the therapeutic properties of the compound with which it is administered. One exemplary pharmaceutically acceptable carrier material 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" refers to a composition containing a polypeptide or nucleic acid molecule described herein prepared with a pharma- ceutical acceptable excipient, and includes those manufactured or sold with the approval of a government regulatory agency as part of a treatment regimen for treating or preventing a disease or event in a patient. Pharmaceutical compositions can 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 emboli and in a solvent system suitable for intravenous use), for oral administration (e.g., tablets, capsules, caplets, gelcaps, or syrups), or in any other formulation form described herein, e.g., unit dosage form.

[0033] The term "patient" means a mammal, including, but not limited to, a human mammal, or a non-human mammal, such as a cow, horse, dog, sheep, or cat.

[0034] The term "therapeutically effective amount" refers to an amount of a polypeptide or nucleic acid molecule described herein sufficient to substantially treat, prevent, delay, inhibit, or abrogate any symptoms of a disease or condition described herein, particularly HPP. The therapeutically effective amount of a composition described herein may depend on the severity of the disorder to be treated, as well as the pathology, weight, and general condition of the patient, and can be determined by one of skill in the art taking such factors into consideration. The therapeutically effective amount of a composition described herein may be administered to a patient in a single dose or in multiple doses administered 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. It 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 uses acid hydrolysis to release TSAC from alkaline phosphatase, and then detects the released TSAC via electrochemical detection using high performance anion exchange chromatography with pulsed amperometric detection technology ("HPAE-PAD").

[0036] The term "sialic acid" generally refers to N- or O-substituted derivatives of neuraminic acid, a monosaccharide with a nine-carbon backbone. Sialic acid also specifically refers to the compound N-acetylneuraminic acid, and may be abbreviated as Neu5Ac or NANA. The presence of sialic acid can affect the 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 the present disclosure, sialic acid associated with alkaline phosphatase, e.g., ALP201, affects the in vivo exposure and half-life of the molecule in physiological conditions. In some embodiments, precise and predictable control of the total sialic acid content (TSAC) of alkaline phosphatase 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 at least X percent of the amino acid residues or nucleotides in the polypeptide or nucleic acid are identical to those in the reference sequence when the sequences are optimally aligned. Optimal alignment of sequences can be determined by a variety of methods within the art, such as 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, such as GENEMATCHER PLUS™ (Schwarz Alignment can be performed using publicly available computer software such as "Best Fit" (Smith and Waterman, Advances in Applied Mathematics, 482-489, 1981), BLAST, BLAST-2, BLAST-P, BLAST-N, BLAST-X, WU-BLAST-2, ALIGN, ALIGN-2, CLUSTAL, or Megalign (DNASTAR), as incorporated in "Best Fit" (Smith and Waterman, Advances in Applied Mathematics, 482-489, 1981), as incorporated in "Best Fit" (Smith and Dayhof, Atlas of Protein Sequence and Structure, Dayhoff, MO, Ed., pp 353-358, 1979). In addition, those skilled in the art can determine appropriate parameters for measuring alignment, including any algorithms needed to obtain optimal alignment over the length of the sequences being compared.

[0038] The words "preferred" and "preferably" refer to embodiments of the disclosed compounds, compositions, and methods that may provide certain benefits, under certain circumstances. However, other embodiments may also be preferred, under the same or other circumstances. Furthermore, the recitation of one or more preferred embodiments does not imply that other embodiments are not useful, and use of the term is not intended to exclude other embodiments from the scope of the present disclosure.

[0039] In any method disclosed herein that includes individual steps, the steps may be performed in any order practicable, and any combination of two or more steps may be performed simultaneously, as appropriate.

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

[0041] All headings are for the convenience of the reader and should not be construed as limiting the meaning of the text that follows the heading, unless specifically stated. [Brief description of the drawings]

[0042] [Figure 1] A set of saturation curves showing the relationship of increasing PPi levels with the rate of PPi hydrolysis for ALP201 (SEQ ID NO:5; left) and asfotase alpha (SEQ ID NO:6; right). [Diagram 2] A set of saturation curves showing the relationship of increasing PLP levels with the rate of PLP hydrolysis for ALP201 (left) and asfotase alfa (right). [Diagram 3]1 is a graph showing the concentration of active alkaline phosphatase enzyme in the plasma of Akp2GW(- / -) mice at the end of the study by treatment group. Abbreviations: q1w = once a week; q2d = once every 2 days; qd = once a day. [Figure 4] 1 is a graph showing alkaline phosphatase activity levels in mouse femur tissue at the end of the study on days 36 / 37. Abbreviations: q2d = once every 2 days; qd = once a day. [Diagram 5] Representative survival curves for a set of Akp2GW(- / -) mice treated with ALP201 and asfotase alfa in a 36-day efficacy study. Abbreviations: MED = minimal effective dose; PBS = phosphate buffered saline; q1w = once a week; q2d = once every 2 days; qd = once a day. [Figure 6] Graph showing a comparison of dose-response modeling results between ALP201 and asfotase alfa. Abbreviations: ALP = alkaline phosphatase; DR = dose-response; MED = minimal effective dose; POC = proof of concept. [Figure 7] Figure 1 shows the simulated human ALP201 concentration-time profiles per first-in-human (FIH) cohort. The grey area represents the 90% predicted period, i.e., the 5%-95% range, the solid black line is the median of the simulated concentration-time profiles; LLOQ is the horizontal dashed line of the PK assay, abbreviation: LLOQ = lower limit of quantification (0.15 µg / mL). [Figure 8] A and B are graphs showing mean (SD) ALP201 activity plasma concentration versus time profiles following intravenous (A) and subcutaneous (B) administration to male C57BL / 6 mice. [Figure 9] A and B are graphs showing the mean (SD) ALP201 activity plasma concentration versus time profiles of ALP201 (lot with TSAC=5.9) following intravenous (A) and subcutaneous (B) administration to male C57Bl / 6 mice. [Figure 10]A and B are graphs showing the mean (SD) ALP201 activity plasma concentration versus time profiles of ALP201 (lots with TSAC=5.0) following intravenous (A) and subcutaneous (B) administration to male C57Bl / 6 mice. [Figure 11] A and B are graphs showing the mean (SD) ALP201 activity plasma concentration versus time profiles of ALP201 (lot TSAC=3.2) following intravenous (A) and subcutaneous (B) administration to male C57Bl / 6 mice. [Figure 12] FIG. 13 is a graph showing the mean (SD) ALP201 activity plasma concentration versus time profiles of ALP201 TSAC values ​​(3.2, 5.0, and 5.9) following intravenous administration to male C57BL / 6 mice. [Figure 13] Graph showing mean (SD) ALP201 activity plasma concentration vs. time profile of ALP201 TSAC values ​​following subcutaneous administration to male C57BL / 6 mice. [Figure 14] Graph showing comparison of mean (±SD) plasma concentration vs. time profiles of ALP201 and asfotase alfa after single IV and SC administration in rats. Abbreviations: IV intravenous; SC = subcutaneous, SD = standard deviation. Asfotase alfa source data: pooled gender, IV at 3 mg / kg, and SC. [Figure 15] FIG. 1 is a graph showing a comparison of mean (±SD) plasma concentration versus time profiles of ALP201 and asfotase alfa following single IV and SC administration in monkeys. [Figure 16] Graph showing dose-response modeling comparison of ALP201 with asfotase alfa (STRENSIQ®). Abbreviations: ALP = alkaline phosphatase; DR = dose-response; MED = minimal effective dose; POC = proof of concept. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0043] The present invention features soluble alkaline phosphatase polypeptides (e.g., those having the sequence of SEQ ID NO:5 and variants thereof having up to 80% or more sequence identity thereto, wherein the polypeptide comprises 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 same, and methods of using the polypeptides and nucleic acid molecules to treat diseases such as bone mineralization disorders, e.g., hypophosphatasia (HPP), or one or more symptoms thereof. The polypeptides include soluble alkaline phosphatase (sALP) or fragments thereof, which are derived from naturally occurring alkaline phosphatase (ALP). Alkaline phosphatase includes various isozymes that are differentially expressed in different tissues. The four major ALP isozymes include tissue non-specific alkaline phosphatase (TNSALP), placental alkaline phosphatase (PALP), germline alkaline phosphatase (GLALP), and intestinal alkaline phosphatase (IALP). Thus, proteins derived from these ALP isozymes are also characterized.

[0044] The polypeptides described herein are formulated in pharmaceutical compositions containing, for example, one or more of phosphate, proline, and sucrose, which confer beneficial characteristics to the polypeptide, such as increased stability in the formulation, reduced aggregation, reduced truncated protein products, and increased purity of the desired protein product.

[0045] HPP is a rare genetic skeletal disease with an incidence of 1 case per 100,000 live births in the most severe form 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, ranging from early tooth loss to almost complete lack of bone mineralization in utero. Symptoms of HPP vary significantly from patient to patient and also by patient age. Many HPP patients exhibit skeletal changes, short stature, chronic pain, leg pain, gait disturbance, and early non-traumatic tooth loss. Due to endogenous TNSALP loss-of-function mutations, HPP patients require functional ALP activity of the polypeptides described herein to restore native ALP activity and result in normal bone matrix mineralization.

[0046] Soluble alkaline phosphatase polypeptides The polypeptides described herein include soluble alkaline phosphatases (sALPs), such as mutant tissue non-specific alkaline phosphatases (TNSALPs) or fragments thereof. The sALPs can be fused to an Fc region and polyaspartic acid of sequence n ("Dn", where n is, for example, equal to 3-20) (sALP-Fc-Dn). The polypeptides can include human TNSALP, such as a soluble fragment of human TNSALP (e.g., residues 1-491 or 1-485 of SEQ ID NO: 1). The polypeptides can include one, two, or three of the mutations E108M, N213Q, and / or N286Q, relative to SEQ ID NO: 1. For example, the sALP can have at least 80% (e.g., at least 85%, 90%, 95%, 97%, 99%, or 100%) identity to SEQ ID NO: 2 or 3. The Fc region can be an IgG2 / 4 Fc region. For example, the Fc region may have at least 80% (e.g., at least 85%, 90%, 95%, 97%, 99%, or 100%) identity to 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). In some embodiments, the polypeptide has at least 80% (e.g., at least 85%, 90%, 95%, 97%, 99%, or 100%) identity to SEQ ID NO:5. For example, the polypeptide may include 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 portion 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. The polypeptide can be further linked to an Fc region (e.g., an IgG1, IgG2, IgG3, or IgG4 Fc region) and / or a polyaspartic acid region. In some embodiments, the polypeptide comprises an IgG2 / 4 Fc region, e.g., has at least 80% (e.g., at least 85%, 90%, 95%, 97%, 99%, or 100%) identity to SEQ ID NO:4. The polyaspartic acid can comprise, e.g., 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 comprises 10 aspartic acid residues (D10).

[0048] SEQ ID NO:1 - Human TNSALP lacking the signal peptide (UniProt P05186.4) LVPEKEKDPKYWRDQAQETLKYALELQKLNTNVAKNVIMFLGDGMGVSTVTAARILKGQLHHNPGEETRLEMDKFPFVALSKTYNTNAQVPDSAGTATAYLCGVKANEGTVGVSAATERSRCNTTQ GNEVTSILRWAKDAGKSVGIVTTTRVNHATPSAAYAHSADRDWYSDNEMPPEALSQGCKDIAYQLMHNIRDIDVIMGGGRKYMYPKNKTDVEYESDEKARGTRLDGLDLVDTWKSFKPRYKHSHFIW NRTELLTLDPHNVDYLLGLFEPGDMQYELNRNNVTDPSLSEMVVVAIQILRKNPKGFFLLVEGGRIDHGHHEGKAKQALHEAVEMDRAIGQAGSLTSSEDTLTVVTADHSHVFTFGGYTPRGNSIFG LAPMLSDTDKKPFTAILYGNGPGYKVVGGERENVSMVDYAHNNYQAQSAVPLRHETHGGEDVAVFSKGPMAHLLHGVHEQNYVPHVMAYAACIGANLGHCAPASSAGSLAAGPLLLALALYPLSVLF SEQ ID NO:2 - Human TNSALP (1-485; E108M, N213Q, N286Q) LVPEKEKDPKYWRDQAQETLKYALELQKLNTNVAKNVIMFLGDGMGVSTVTAARILKGQLHHNPGEETRLEMDKFPFVALSKTYNTNAQVPDSAGTATAYLCGVKANMGTVGVSAATERSR CNTTQGNEVTSILRWAKDAGKSVGIVTTTRVNHATPSAAYAHSADRDWYSDNEMPPEALSQGCKDIAYQLMHNIRDIDVIMGGGRKYMYPKQKTDVEYESDEKARGTRLDGLDLVDTWKSF KPRYKHSHFIWNRTELLTLDPHNVDYLLGLFEPGDMQYELNRNQVTDPSLSEMVVVAIQILRKNPKGFFLLVEGGRIDHGHHEGKAKQALHEAVEMDRAIGQAGSLTSSEDTLTVVTADHS HVFTFGYTPRGNSIFGLAPMLSDTDKKPFTAILYGNGPGYKVVGGERENVSMVDYAHNNYQAQSAVPLRHETHGGEDVAVFSKGPMAHLLHGVHEQNYVPHVMAYAACIGANLGHCAPASS SEQ ID NO:3 - Human TNSALP (1-491; E108M, N213Q, N286Q) LVPEKEKDPKYWRDQAQETLKYALELQKLNTNVAKNVIMFLGDGMGVSTVTAARILKGQLHHNPGEETRLEMDKFPFVALSKTYNTNAQVPDSAGTATAYLCGVKANMGTVGVSAATERSRC NTTQGNEVTSILRWAKDAGKSVGIVTTTRVNHATPSAAYAHSADRDWYSDNEMPPEALSQGCKDIAYQLMHNIRDIDVIMGGGRKYMYPKQKTDVEYESDEKARGTRLDGLDLVDTWKSFKPR YKHSHFIWNRTELLTLDPHNVDYLLGLFEPGDMQYELNRNQVTDPSLSEMVVVAIQILRKNPKGFFLLVEGGRIDHGHHEGKAKQALHEAVEMDRAIGQAGSLTSSEDTLTVVTADHSHVFTF GGYTPRGNSIFGLAPMLSDTDKKPFTAILYGNGPGYKVVGGERENVSMVDYAHNNYQAQSAVPLRHETHGGEDVAVFSKGPMAHLLHGVHEQNYVPHVMAYAACIGANLGHCAPASSAGSLAA SEQ ID NO:4 - IgG2 / 4Fc VECPPCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKT ISKAKGQPREPQVYTLPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK SEQ ID NO:5-ALP201 LVPEKEKDPKYWRDQAQETLKYALELQKLNTNVAKNVIMFLGDGMGVSTVTAARILKGQLHHNPGEETRLEMDKFPFVALSKTYNTNAQVPDSAGTATAYLCGVKANMGTVGVSAATERSRCNTTQGNEVTSILRWAKDAGKSVGIVTTTRVNHATPSAAYAHSADRDWYSDNEMPPEALS QGCKDIAYQLMHNIRDIDVIMGGGRKYMYPKQKTDVEYESDEKARGTRLDGLDLVDTWKSFKPRYKHSHFIWNRTELLTLDPHNVDYLLGLFEPGDMQYELNRNQVTDPSLSEMVVVAIQILRKNPKGFFLLVEGGRIDHGHHEGKAKQALHEAVEMDRAIGQAGSLTSSEDTLTVVTADH SHVFTFGYTPRGNSIFGLAPMLSDTDKKPFTAILYGNGPGYKVVGGERENVSMVDYAHNNYQAQSAVPLRHETHGGEDVAVFSKGPMAHLLHGVHEQNYVPHVMAYAACIGANLGHCAPASSAGSLAAVECPCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFN WYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGKDDDDDDDDDD SEQ ID NO:6 - Asfotase alfa LVPEKEKDPKYWRDQAQETLKYALELQKLNTNVAKNVIMFLGDGMGVSTVTAARILKGQLHHNPGEETRLEMDKFPFVALSKTYNTNAQVPDSAGTATAYLCGVKANEGTVGVSAATERSRCNTTQGNEVTSILRWAKDAGKSVGIVTTTRVNHATPSAAYAHSADRDWYSDNEMPPEALS QGCKDIAYQLMHNIRDIDVIMGGGRKYMYPKNKTDVEYESDEKARGTRLDGLDLVDTWKSFKPRYKHSHFIWNRTELLTLDPHNVDYLLGLFEPGDMQYELNRNVTDPSLSEMVVVAIQILRKNPKGFFLLVEGGRIDHGHHEGKAKQALHEAVEMDRAIGQAGSLTSSEDTLTVVTADHS HVFTFGGYTPRGNSIFGLAPMLSDTDKKPFTAILYGNGPGYKVVGGERENVSMVDYAHNNYQAQSAVPLRHETHGGEDVAVFSKGPMAHLLHGVHEQNYVPHVMAYAACIGANLGHCAPASSLKDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNW YVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKDIDDDDDDDDDD

[0049] Total Sialic Acid Content As described herein, TSAC may affect the half-life of recombinant alkaline phosphatase under physiological conditions. Thus, TSAC levels may serve as a quality attribute for recombinantly produced alkaline phosphatases, such as ALP201. Control of the TSAC range during polypeptide production can improve batch-to-batch reproducibility and reduce heterogeneity in the produced polypeptide. In some embodiments, TSAC is about 0.8 mol / mol to about 8.0 mol / mol for recombinant alkaline phosphatase. In some embodiments, TSAC is about 0.9 mol / mol to about 7.0 mol / mol for recombinant alkaline phosphatase. In some embodiments, TSAC is about 1.0 mol / mol to about 6.0 mol / mol for recombinant alkaline phosphatase. In some embodiments, TSAC is about 1.2 mol / mol to about 6.0 mol / mol for recombinant alkaline phosphatase. In some embodiments, TSAC is about 1.5 mol / mol to about 6.0 mol / mol for recombinant alkaline phosphatase. In some embodiments, TSAC is about 2.0 mol / mol to about 6.0 mol / mol relative to the recombinant alkaline phosphatase, hi some embodiments, TSAC is about 3.2 mol / mol to about 5.9 mol / mol relative to the recombinant alkaline phosphatase.In some embodiments, TSAC is about 0.9 mol / mol, about 1.0 mol / mol, about 1.1 mol / mol, about 1.2 mol / mol, about 1.3 mol / mol, about 1.4 mol / mol, about 1.5 mol / mol, about 1.6 mol / mol, about 1.7 mol / mol, about 1.8 mol / mol, about 1.9 mol / mol, about 2.0 mol / mol, about 2.1 mol / mol, about 2.2 mol / mol, about 2.3 mol / mol, about 2.4 mol / mol, about 2.5 mol / mol, about 2.6 mol / mol, about 2.7 mol / mol, about 2.8 mol / mol, about 2.9 mol / mol, about 3.0 mol / mol, about 3.1 mol / mol, about 3.2 mol / mol, about 3.3 mol / mol relative to the recombinant alkaline phosphatase. ol, about 3.4 mol / mol, about 3.5 mol / mol, about 3.6 mol / mol, about 3.7 mol / mol, about 3.8 mol / mol, about 3.9 mol / mol, about 4.0 mol / mol, about 4.1 mol / mol, about 4.2 mol / mol, about 4.3 mol / mol, about 4.4 mol / mol, about 4.5 mol / mol, about 4.6 mol / mol, about 4.7 mol / 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 the recombinant alkaline phosphatase. In some embodiments, the TSAC is about 5.0 mol / mol relative to the recombinant alkaline phosphatase. In some embodiments, the TSAC is about 5.9 mol / mol relative to the recombinant alkaline phosphatase.

[0050] Pharmaceutical Compositions The polypeptides described herein (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 comprising 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 include one or more or all of phosphate, proline, and sucrose. The composition may include, for example, phosphate and sucrose. For example, the composition may include phosphate (e.g., sodium phosphate) at a concentration 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 include proline and / or sucrose. The composition may further include proline. The composition may further include sucrose. For example, the composition may contain about 1 mM to about 500 mM proline, for example, about 70 mM to about 280 mM, for example, about 50 mM to about 200 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, 100 mM, 110 mM, 120 mM, 130 mM, 140 mM, 150 mM, 160 mM, 170 mM, 180 mM, 190 mM, 200 mM, 300 mM, 400 mM, or 500 mM, for example, about 140 mM proline, and / or about 1 mM to about 500 mM sucrose, for example, about 70 mM to about 280 mM, e.g. For example, about 50 mM to about 200 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, 100 mM, 110 mM, 120 mM, 130 mM, 140 mM, 150 mM, 160 mM, 170 mM, 1 mM, 180 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, e.g., about 140 mM sucrose, or about 210 mM sucrose. In some embodiments, the composition comprises a molar ratio of proline:sucrose of about 1:0 to about 1:3, e.g., about 1:1. In some embodiments, the composition comprises about 140 mM proline. In some embodiments, the formulation comprises about 140 mM sucrose.In some embodiments, the formulation comprises about 210 mM sucrose. In some embodiments, the formulation comprises about 210 mM sucrose, but does not comprise proline. In some embodiments, the composition comprises about 210 mM sucrose, and about 10 mM phosphate (e.g., sodium phosphate). The formulation may further comprise about 0.01% to about 0.5%, e.g., about 0.01% to about 0.1%, e.g., about 0.05% polyoxyethylene (20) sorbitan monooleate (e.g., polysorbate 80 (PS80)), etc. In some embodiments, the composition is prepared at a pH of about pH 7.0 to about pH 7.6 (e.g., about pH 7.1, 7.2, 7.3, 7.4, 7.5, or 7.6, e.g., about 7.3). In some embodiments, the composition comprises about 10 mM phosphate, about 140 mM proline, about 140 mM sucrose, and about 0.05% polyoxyethylene (20) sorbitan monooleate (e.g., PS80) at a pH of about 7.3.

[0052] The composition may be prepared as a solution containing the polypeptide, for example, in an amount of 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, for example, in a dosage of about 0.1 mg / mL to about 10 mg / mL of the polypeptide. The composition may be prepared in a solution with 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] The present disclosure also features a vial containing a pharmaceutical composition as described herein. The vial may contain a solution, for example, in a volume of about 0.1 mL to about 10 mL (e.g., about 1 mL). The vial may contain a polypeptide, for example, in an amount of 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., a polypeptide of SEQ ID NO: 5, or a variant having at least 85% sequence identity thereto).

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

[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 comprising 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, sALP compositions can be prepared, for example, in a buffer solution suitable for storage at 2-8°C (e.g., 4°C) at a suitable concentration. sALP compositions can also be prepared for storage at temperatures below 0°C (e.g., -20°C or -80°C). sALP compositions can further be prepared for storage at 2-8°C (e.g., 4°C) for up to 2 years (e.g., 1 month, 2 months, 3 months, 4 months, 5 months, 6 months, 7 months, 8 months, 9 months, 10 months, 11 months, 1 year, 1.5 years, or 2 years). Thus, the compositions described herein can be prepared to be stable for at least one year upon storage at 2-8° C. (e.g., 4° C.) The compositions can be prepared in a suitable volume, for example, from about 0.1 mL to about 10 mL.

[0056] 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 comprising 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 can be in the form of an injectable or infusible solution. Thus, 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)) can be prepared for administration by parenteral modes (e.g., subcutaneous, intravenous, intraperitoneal, or intramuscular injection). "Parenteral administration", "administer parenterally", and other grammatically equivalent phrases, as used herein, refer to modes of administration other than enteral and topical administration, usually by injection, including, but not limited to, subcutaneous, intradermal, intravenous, intranasal, intraocular, pulmonary, intramuscular, intraarterial, intrathecal, intraarticular, intraorbital, intracardiac, intradermal, intrapulmonary, intraperitoneal, transtracheal, subcuticular, intraarticular, subcapsular, subarachnoid, intrathecal, epidural, intracerebral, intracranial, intracarotid, and intrasternal injection and infusion. Specific routes of administration include intravenous and subcutaneous administration.

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

[0059] Dosage An sALP polypeptide described herein (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 including one or more or all of the mutations E108M, N213Q, and N286Q relative to SEQ ID NO:1)) may be administered to a patient suffering from or prone to a bone mineralization disorder, such as HPP, at a dose of, for example, 0.01 mg / kg to 500 mg / kg (e.g., 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 ... 0.5mg / kg to 25mg / kg, 1.0mg / kg to 10mg / kg, 1.5mg / kg to 5mg / kg, or 2.0mg / kg to 3.0mg / kg), or 1μg / kg to 1,000μg / kg (e.g., 5μg / kg to 1,000μg / kg, 1μg / kg to 750μg / kg, 5μg / kg to 750μg / kg, 10μg / kg to 750μg / kg, 1μg In some embodiments, the therapeutic agent may be administered in individual doses ranging from 1 μg / kg to 500 μg / kg, 5 μg / kg to 500 μg / kg, 10 μg / kg to 500 μg / kg, 1 μg / kg to 100 μg / kg, 5 μg / kg to 100 μg / kg, 10 μg / kg to 100 μg / kg, 1 μg / kg to 50 μg / kg, 5 μg / kg to 50 μg / kg, or 10 μg / kg to 50 μg / 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 recited herein, the term "about" may be used to modify these doses by ±10% of the endpoints of the recited value or range. In particular, compositions according to the present disclosure (e.g., a polypeptide having a sequence of SEQ ID NO:5, or a variant thereof having at least 85% sequence identity thereto (e.g., a polypeptide comprising 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 at a dose ranging from about 0.001 mg / kg / day to about 500 mg / kg / day, from about 0.01 mg / kg / day to about 100 mg / kg / day, or from about 0.01 mg / kg / day to about 20 mg / kg / day. For example, an 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 including 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 at a weekly dose ranging, for example, from about 0.5 mg / kg / week to about 140 mg / kg / week, e.g., from about 0.8 mg / kg / week to about 50 mg / kg / week, or from about 1 mg / kg / week to about 10 mg / kg / week (e.g., from about 6 to about 9 mg / kg / week). In particular, sALP can be administered once or more per week (e.g., 1, 2, 3, 4, 5, 6, 7 or more times per week), once or more per every other week, or once or more per month (e.g., 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 per 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 comprising one or more or all of the mutations E108M, N213Q, and N286Q, as compared to SEQ ID NO:1 or SEQ ID NO:5)) can be administered at a dosage of 2 mg / kg three times a week (total dose of 6 mg / kg / week), 1 mg / kg six times a week (total dose of 6 mg / kg / week), 3 mg / kg three times a week (total dose of 9 mg / kg / week), 0.5 mg / kg three times a week (total dose of 1.5 mg / kg / week), or 9.3 mg / kg three times a week (total dose of 28 mg / kg / week). The dosage may be adjusted by the clinician based on conventional factors such as the extent of the disease and different parameters of the patient suffering from or prone to bone mineralization disorders such as HPP. Alternatively, about 0.1 mg / kg to about 20 mg / kg (e.g., about 0.1 mg / kg to about 9 mg / kg) may be administered once a week or once every two weeks. The sALP composition may also be administered 1 to 10 times (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10 times) every week or every two weeks at a dose of 1 to 90 mg per administration (e.g., in a volume of 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] A composition containing sALP or sALP fusion polypeptide (e.g., a polypeptide having a sequence of SEQ ID NO:5, or a variant thereof having at least 85% sequence identity thereto (e.g., a polypeptide including 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 a single dose regimen or in a repeated dose regimen. Each dose can be administered, for example, hourly, every other hour, 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, once every other week, once a month, once every other month, or once a year. Alternatively, each dose can 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(s), weeks(s), or months(s), or for the lifespan of a patient suffering from or prone to bone mineralization disorders, such as HPP. The amount, frequency, and duration of administration can be adjusted by a clinician based on conventional factors, such as the extent of disease and different parameters of a patient suffering from or prone to bone mineralization disorders, such as HPP.

[0063] For example, dosages of sALP or 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 including one or more or all of the mutations E108M, N213Q, and N286Q compared to SEQ ID NO:1 or SEQ ID NO:5)) may range from about 0.1 mg / kg body weight to about 10 mg / kg body weight, administered subcutaneously or intravenously one or more times per week (e.g., 2, 3, 4, 5, 6, or 7 times).

[0064] In some particular 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 comprising 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 same may be administered at a dosage 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 daily, weekly, monthly, or yearly (e.g., once a week). In some embodiments, the polypeptide is administered one or more times 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 at a dosage of about 0.01 mg / kg / week to about 50 mg / kg / week (e.g., about 0.01 mg / kg / week to about 40 mg / kg / week, e.g., about 0.1 mg / kg / week to about 20 mg / kg / week, e.g., about 0.1 mg / kg / week to about 10 mg / kg / week). The polypeptide may be administered for at least one day, one week, one month, one year, or more (e.g., one to five years, or for the life of the patient).

[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 more.

[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 more.

[0067] In some embodiments, the pharmaceutical composition is administered intravenously and subcutaneously. In some embodiments, the pharmaceutical composition is administered intravenously and subcutaneously at the same time. 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., in a single dose), e.g., as a loading dose, followed by one or more subcutaneous doses (e.g., each dose administered about once a week over the treatment period).

[0068] Treatment method Provided herein is a method for treating or alleviating at least one symptom in a patient with a bone mineralization disorder, such as HPP. Other diseases or disorders, such as bone fracture, osteoporosis, sclerosteosis, chondrocalcinosis, hypotonia, Duchenne muscular dystrophy, tracheobronchomalacia, stroke, neurofibromatosis 1 (NF-1), and craniosynostosis, can also be treated by the compositions and methods described herein. The patient may suffer from muscle weakness. The patient may suffer from a muscle weakness disease, such as calcium pyrophosphate deposition disease (CPPD) or familial hypophosphatemia. Such treatment can include administering alkaline phosphatase (e.g., a pharmaceutical composition containing alkaline phosphatase) or a polypeptide with alkaline phosphatase activity to reduce elevated PPi levels in such patients. For example, a 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 including 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 a neonate, infant, child, adolescent, or adult.

[0069] The patient may have been diagnosed with a bone mineralization disorder (e.g., HPP) prior to administration of alkaline phosphatase or a polypeptide with alkaline phosphatase activity (e.g., sALP, e.g., a polypeptide having a sequence of SEQ ID NO:5, or a variant thereof having at least 85% sequence identity thereto (e.g., a polypeptide comprising one or more or all of the mutations E108M, N213Q, and N286Q, as compared to SEQ ID NO:1 or SEQ ID NO:5)). In addition, a patient suffering from or prone to a bone mineralization disorder such as HPP may be a treatment-naïve patient who has not previously been treated with sALP (e.g., a polypeptide having a sequence of SEQ ID NO:5, or a variant thereof having at least 85% sequence identity thereto (e.g., a polypeptide comprising one or more or all of the mutations E108M, N213Q, and N286Q, as compared to SEQ ID NO:1 or SEQ ID NO:5)).

[0070] One or more symptoms of the disease may first appear during the patient's neonatal, infant, or childhood. In other embodiments, one or more symptoms of the disease may first appear during the patient's adulthood. In some embodiments, the patient does not exhibit detectable symptoms of the disease before reaching adulthood. In some embodiments, the patient exhibits detectable symptoms of the disease before reaching adulthood, but the disease condition remains undiagnosed until adulthood.

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

[0072] In addition, each of the described scores (e.g., BOT-2 strength score, BOT-2 running speed and agility score, CHAQ index score, BSID-III scale score, PDMS-2 standard score, 6MWT, 12-POMA-G, modified Locomotion Oriented Assessment (mPOMA-G, as exemplified in Phillips et al. 2015 Bone Abstracts 4:P136), or HHD value) can be used alone or in any combination to evaluate the effectiveness of treatment with sALP (e.g., a polypeptide having a sequence of SEQ ID NO:5, or a variant thereof having at least 85% sequence identity thereto (e.g., a polypeptide comprising one or more or all of the mutations E108M, N213Q, and N286Q compared to SEQ ID NO:1 or SEQ ID NO:5)) in patients suffering from or prone to a bone mineralization disorder such as HPP as described herein, where an improvement compared to the particular test score demonstrates that sALP is effective for treating a bone mineralization disorder such as HPP.

[0073] For example, if an alkaline phosphatase or a polypeptide with alkaline phosphatase activity (e.g., sALP, e.g., a polypeptide having a sequence of SEQ ID NO:5, or a variant thereof having at least 85% sequence identity thereto (e.g., a polypeptide including 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 a bone mineralization disorder, such as HPP, resulting in an average increase in BOT-2 muscle strength score to about or greater than about 10, where the patient's average BOT-2 muscle strength score was previously less than about 10, then treatment with the alkaline phosphatase or a polypeptide with alkaline phosphatase activity is effective in treating a physical impairment associated with a bone mineralization disorder, such as HPP. Alternatively, if administration of sALP does not result in an average increase in BOT-2 muscle strength score of about 10 or more than about 10, the dosage and / or frequency of administration of alkaline phosphatase or a polypeptide having alkaline phosphatase activity can be altered (e.g., increased indefinitely or over a short period of time (e.g., 1-6 months, or up to a year or more)) to determine the effective amount of alkaline phosphatase or a polypeptide having alkaline phosphatase activity for the patient. 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 including 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 about once every 3 weeks to about once every 2 weeks, or from about once every 2 weeks to about once a week.Alternatively, if improvement in one of the metrics described herein is achieved, the dosage and / or frequency can be continued or reduced, e.g., from about 6-9 mg / kg / week to about 3-6 mg / kg / week, from about 3-6 mg / kg / week to about 0.5-3 mg / kg / week, or from about 0.5-3 mg / kg / week to about 0.1-1 mg / kg / week. Similarly, the frequency of administration can be reduced from about once per week to about once every two weeks, or from about once every two weeks to about once every three weeks.

[0074] In addition, an alkaline phosphatase or a polypeptide with alkaline phosphatase activity (e.g., sALP, e.g., a polypeptide having a sequence of SEQ ID NO:5, or a variant thereof having at least 85% sequence identity thereto (e.g., a polypeptide including 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 a patient suffering from or prone to a bone mineralization disorder, such as HPP, resulting in an improvement in one or more patient strength grade classifications (e.g., an improvement in strength grades of 1, 2, 3, 4, or 5 from a previously low strength grade), where the patient's average strength grade was previously less than about 5, and where treatment with an alkaline phosphatase or a polypeptide with alkaline phosphatase activity is effective in treating, for example, a physical impairment associated with a bone mineralization disorder, such as HPP. Alternatively, if administration of sALP does not result in one or more patients' muscle grade classifications improving from a previous low muscle grade, the dosage and / or frequency of administration of alkaline phosphatase or a polypeptide with alkaline phosphatase activity can be altered (e.g., increased) to determine the effective amount of alkaline phosphatase or a polypeptide with alkaline phosphatase activity for the patient. For example, the dosage of sALP (e.g., a polypeptide having a sequence of SEQ ID NO: 5, or a variant thereof having at least 85% sequence identity thereto (e.g., a polypeptide including 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 about once every three weeks to about once every two weeks, or from about once every two weeks to about once a week.Alternatively, if improvement in one of the metrics described herein is achieved, the dosage and / or frequency can be continued or reduced, e.g., from about 6-9 mg / kg / week to about 3-6 mg / kg / week, from about 3-6 mg / kg / week to about 0.5-3 mg / kg / week, or from about 0.5-3 mg / kg / week to about 0.1-1 mg / kg / week. Similarly, the frequency of administration can be reduced from about once per week to about once every two weeks, or from about once every two weeks to about once every three weeks.

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

[0076] The present disclosure is illustrated by the following non-limiting examples. It should be understood that the specific examples, materials, amounts, and procedures are to be interpreted broadly in accordance with the scope and spirit of the disclosure described herein.

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

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

[0079] ALP201 is a next-generation HPP therapy with equivalent efficacy and improved activity compared to asfotase alfa. ALP201 also provides increased exposure due to longer half-life, reduced alpha-phase clearance, and increased bioavailability. These improved properties support lower doses and longer dosing intervals for ALP201 compared to asfotase alfa. Patient experience is expected to improve with reduced injection volume and dosing frequency, which may also lead to reduced injection site reactions.

[0080] ALP201 Physical and Chemical Properties ALP201 is a soluble Fc fusion protein with a molecular weight of approximately 160 kDa and is composed of two polypeptide chains covalently linked by two disulfide bonds, each of which contains 724 amino acids and is composed of three segments. The N-terminal region of the polypeptide, amino acids L1-A491, contains the catalytic enzyme TNSALP, the soluble portion of the human tissue-nonspecific alkaline phosphatase enzyme. Within the enzymatic region of each polypeptide chain, a single point mutation (E108M) was introduced to improve the enzymatic activity, and two N-linked glycosylation sites (N213Q and N286Q) were removed for process improvement. The second portion of the polypeptide, amino acids V492-K714, comprises the Fc portion of human immunoglobulin gamma 2 / 4 (IgG2 / 4), including the hinge, CH2 and CH3 domains. The C-terminal region of the polypeptide, amino acids D715-D724, contains 10 aspartic acids. This peptide sequence promotes 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 a 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. The 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 sites for zinc, one site for magnesium, and one site for calcium.

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

[0083] Pharmaceuticals ALP201 100mg / mL vial The ALP201 drug product (100 mg / mL) is a sterile, preservative-free, formulated liquid solution containing ALP201 and excipients in a single-dose 2 mL vial. The drug product does not contain any novel excipient(s) of animal or human origin. Each 2 mL vial typically contains 1.2 mL (overfill) of drug product to deliver 100 mg of ALP201 per vial. The drug product is filled in 2 mL type I clear glass vials with 13 mm chlorobutyl stoppers with aluminum seals. The quantitative composition of the ALP201 drug product is presented in Table 2. [Table 2]

[0084] In vivo testing ALP201 was evaluated using in vivo studies to characterize the pharmacology, PK / toxicokinetics (TK), local tolerability, and potential systemic toxicity. Pharmacology studies performed included repeat-dose studies in HPP mice (Akp2GW(- / -) mice) to identify the minimally effective dose of ALP201. Single-dose PK studies were performed in mice, rats, and monkeys to understand the PK properties of ALP201. Toxicity studies evaluated the systemic toxicity of SC ALP201 administered to rats and monkeys and included safety pharmacology endpoints (cardiovascular, respiratory, and nervous system function in monkey studies, and neurological function in rat studies), TK assessments, and recovery periods to evaluate reversibility of treatment-related effects.

[0085] A summary of the in vivo studies performed with ALP201 supporting its use in humans is provided in Table 3. [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 low TNSALP levels in HPP patients lead to elevated circulating PPi blood levels. Elevated pyrophosphate levels prevent proper bone mineralization and cause the abnormal bone symptoms observed in HPP patients. Therefore, we considered PPi as a natural substrate to be targeted at higher enzyme activity levels by engineered second-generation asfotase alpha molecules.

[0087] The substrate saturation curves for pyrophosphate hydrolysis by ALP201 and asfotase alfa are shown in Figure 1. ALP201 retains a similar Km value (47 mM) for pyrophosphate to that of asfotase alfa (53 mM), but ALP201 functions at a significantly higher turnover number (ALP201 has a kcat of 11,619 min -1 In contrast, asfotase alfa was 6,714 min -1 The similar Km values ​​for the wild-type TNSALP catalytic domain of ALP201 and asfotase alfa suggest that ALP201 is less likely to dangerously reduce circulating PPi levels than asfotase alfa when present at comparable serum alkaline phosphatase activity levels.

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

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

[0090] In vivo pharmacological properties Preclinical efficacy studies for ALP201 were conducted using Akp2GW(- / -) mice, an animal model of human HPP. Akp2GW(- / -) mice harbor the same HPP-inducing TNSALP mutation used in Akp2(- / -) mice previously used in the preclinical evaluation of asfotase alfa. Natural history studies conducted at Alexion during the development of Akp2GW(- / -) mice have revealed that homozygous TNSALP activity knockout Akp2GW(- / -) mice exhibit a bone mineralization and survival phenotype nearly identical to Akp2(- / -) mice (Akp2GW-NH-01-02).

[0091] In the efficacy studies used to evaluate the efficacy of ALP201 using the Akp2GW(- / -) mouse model, test articles were administered subcutaneously from postnatal day 1 to postnatal day 35. Outcomes reported in all studies included overall survival, weight gain, bone mineralization in hindlimb bones at day 36 (or at time of death if prior to end of study [EOS]), and EOS trough plasma enzyme activity levels (recorded on day 36 for the once-daily [qd] and once-weekly [q1w] treatment groups and on day 37 for the once-every-two-day [q2d] treatment group). Some studies determined femur and tibia lengths at EOS, as well as alkaline phosphatase activity in the femurs of mice.

[0092] In previous studies using Akp2(- / -) mice, asfotase alfa demonstrated efficacy in both bone mineralization and overall survival study endpoints at doses of 7-10 mg / kg / day relative to an active preparation of asfotase alfa, depending on the specific activity of the test article. The estimated minimal effective dose (MED) of asfotase alfa was defined as the dose that resulted in 85% of measurable mice in a group having normal bone mineralization scores by the end of the study or at the time of death if death occurred prior to 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 article.

[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 which the efficacy of ALP201 was compared to that observed in a positive control group receiving daily SC asfotase alfa.

[0094] In previous studies, asfotase alfa was administered SC at a maximally effective dose of 9.8 mg / kg / day on a qd schedule. The equivalent 4-MUP activity dose of ALP201 was administered SC in PBS at qd, q2d, and q1w dosing intervals. In one treatment group, the dose of ALP201 was log-halved after weaning of the mice on day 25. Subcutaneous administration of PBS on a qd schedule was used as a negative control. A list of these treatment groups can be found in Table 5.

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

[0096] Results of bone mineralization Bone mineralization outcomes on day 36 / 37 were determined by x-ray analysis of the hind limbs of treated Akp2GW(- / -) mice. X-ray visualization of hind limb bone mineralization was compared to baseline x-ray images on day 36 illustrating four classification categories: no effect, mild loss, moderate loss, and severe loss. A detailed description of these classifications can be found in Table 6. A blinded individual assigned a score to the images for each mouse and, upon completion, scores were tallied within individual treatment groups. [Table 6] The observed distribution of EOS hind paw bone mineralization index scores by treatment group can be seen in Table 7. [Table 7]

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

[0098] End-of-study trough plasma enzyme activity Trough plasma active TNSALP enzyme concentrations were determined by measuring TNSALP activity levels in plasma samples on days 36 / 37. Measured TNSALP activity was fitted to a standard curve of known enzyme activity and concentration to quantify active enzyme levels per unit volume of plasma. The distribution of EOS trough plasma active enzyme concentrations is shown in Figure 3. A summary of the mean EOS trough plasma active enzyme concentrations by treatment group is shown in Table 8. [Table 8-1] [Table 8-2]

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

[0100] Bone enzyme activity levels at the end of the study in treated Akp2GW(- / -) mice on day 36 / 37 Alkaline phosphatase activity levels in treated Akp2GW(- / -) mouse femoral tissue were determined in an ex vivo assay at the end of the HPP-MED-01 study using 4-MUP as a substrate. In this assay, only the mineralized portion of the femur was assayed for activity to avoid potential contamination with blood or highly perfused tissues that may still harbor high levels of ALP201 or asfotase alpha. The distribution of enzyme activity levels associated with treated Akp2GW(- / -) mouse femora is shown in Figure 4. Here, the data are presented in microunits of 4-MUP hydrolytic activity per mg of mineralized femoral tissue. [Table 9]

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

[0102] Survival outcomes of treated Akp2GW(- / -) mice All PBS-treated Akp2GW(- / -) 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 survival to 36 days in all treatment groups compared to PBS vehicle controls (Figure 5 and Table 10). All ALP201 treatment groups achieved EOS survival of at least 69%, with overall survival of 88% or greater in all qd and q2d interval treatment groups at doses >0.15 mg / kg / day. Survival curves for the ALP201 4.8 mg / kg q1w group and the asfotase alfa 9.8 mg / kg qd group showed very similar results. [Table 10]

[0103] Weight Results The mean body weights of Akp2GW(- / -) mice treated with ALP201 and asfotase alfa were consistently lower than those of their wild-type littermates treated with PBS in all groups. End-of-study body weights of all ALP201-treated groups were not statistically different from those of the asfotase alfa-treated groups.

[0104] Safety and pharmacological properties No independent safety pharmacology characterization studies were performed 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 increased heart rate (28% compared to control) only in the low dose group, with associated decreases in RR, PR, and QT intervals, but no significant changes in QRS duration. No biologically meaningful changes were observed in the mid- and high-dose groups. Thus, there was no dose-response relationship between the slight increases in heart rate and ALP201 dose. Because the magnitude of increases in heart rate were minor, these findings are not considered adverse. Furthermore, no ALP201-related findings were observed in ECG, blood pressure, or respiratory parameters evaluated in monkeys after 28 days of SC administration up to 20 mg / kg, the highest concentration evaluated in this study. ALP201 had no effect on behavioral indices or locomotor activity in rats after SC administration up to 30 mg / kg, the highest concentration evaluated in this study, once every 3 days for 28 days.

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

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

[0107] The mean bioavailability was 96%, 58%, and 78% in mice, rats, and monkeys, respectively. The time to reach maximum concentration (t max ) ranged from 17 to 48 h after administration, suggesting that absorption of ALP201 from the SC injection site is slow.

[0108] distribution After a single intravenous dose in mice, rats, and monkeys, the apparent volume of distribution of ALP201 ranged from 0.08 to 0.15 L / kg, values ​​much greater (>2-fold) than the plasma volume in these species, suggesting that ALP201 distributes beyond the intravascular compartment.

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

[0110] After IV administration, the concentration-time profile declined multi-exponentially. The CL was 0.0019 L / h / kg and the apparent terminal t1 / 2 was 48 hours (Table 11). d The time to reach the maximum concentration after SC administration (t max ) 48 hours after administration, suggesting that absorption from the SC injection site is relatively slow. Absolute bioavailability after SC administration was 96%.

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

[0112] When administered IV, the PK exposure of ALP201 (C max and AUC ∞ ) increased slightly less than dose-proportionally over the dose range tested, from 1 mg / kg to 27 mg / kg. d The mean CL values ​​ranged from 0.0012 to 0.0019 L / h / kg, and the mean V d Values ​​ranged from 0.12 to 0.15 L / kg. Similarly, the t1 / 2 of ALP201, which remained relatively similar across IV doses, averaged approximately 2 days. For SC administration, CL / F, V d / F, and t 1 / 2 Values ​​were similar to those estimated from the IV dose, with the exception of the 27 mg / kg SC group, which appeared to be an outlier. Mean t max The absorption rates ranged from 30 to 48 hours after administration, suggesting that absorption from the SC injection site was relatively slow. Absolute bioavailability after SC administration was 61% and 54% for the 2 and 27 mg / kg doses, respectively.

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

[0114] For IV administration, C max and AUC ∞ The increases in CL and V were nearly dose-proportional over the dose range tested, from 2 mg / kg to 20 mg / kg. d remained consistent across doses, with mean CL values ​​ranging from 0.0008 to 0.0011 L / h / kg, and mean V d The values ​​ranged from 0.08 to 0.10 L / kg. Similarly, the t values ​​of ALP201 remained relatively similar across doses. 1 / 2 The mean CL / F and V were approximately 3 days for SC administration. d / F, and t 1 / 2 Values ​​were consistent with those estimated after IV administration. Mean t max The absorption rates ranged from 17 to 19 hours after dosing, suggesting that absorption from the SC injection site was relatively slow. Absolute bioavailability was 69.6% and 86.9% for the 2 mg / kg and 20 mg / kg doses, respectively.

[0115] Repeated-dose toxicokinetic study with ALP201 SC dosing in rats for 4 weeks (1727-227): ALP201 was administered SC to rats at 2, 10, or 30 mg / kg / dose once every 3 days for 4 weeks. In addition, ALP201 was administered intravenously to rats at a single dose of 10 mg / kg to compare IV and SC route bioavailability. Pharmacokinetic parameters calculated using noncompartmental analysis based on ALP201 composite plasma concentrations after dosing on days 0 and 24 are shown in Table 14.

[0116] Systemic exposure to ALP201 appeared to be independent of sex after SC administration of ALP201 on days 0 and 24, and after a single IV bolus injection of ALP201 on day 0. After SC administration of ALP201 once every 3 days, the C max and AUC 0-72h The AUC values ​​increased with increasing dose in a slightly less than dose-proportional manner. 0-72h ) appeared to decrease after repeated SC administration. The decrease in exposure appeared to be dose-dependent, with a maximum decrease of up to 2-fold after repeated SC administration of 30 mg / kg ALP201. Subcutaneous bioavailability of ALP201 (AUC 0-72h The mean age (based on age) was approximately 44.2%. [Table 14-1] [Table 14-2]

[0117] SC dosing to monkeys for 4 weeks: ALP201 was administered SC to monkeys at 1, 5, or 20 mg / kg / dose every 3 days for 4 weeks. PK parameters calculated using noncompartmental analysis based on ALP201 plasma concentrations after dosing on days 1 and 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 days 0 and 24. After subcutaneous administration of ALP201 once every 3 days, the C max and AUC 0-72h The values ​​increased with increasing dose in a roughly dose-proportional manner. 0-72h values) appeared to increase after repeated subcutaneous administration of ALP201 on day 24.

[0119] Pharmacokinetics, pharmacodynamics, and immunogenicity in multiple dose studies 28-day toxicity study in rats with a 28-day recovery period The TK / ADA of ALP201 was evaluated in male and female rats after SC administration of 2, 10, or 30 mg / kg (10 / sex / dose group, 5 / sex / time point) every 3 days for 4 weeks (total of 10 doses) and a single IV dose of 10 mg / kg (total of 1 dose). Blood samples for TK analysis were collected from all animals over 72 hours starting on days 0 and 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, pooled gender results are included in Table 16. ALP201 exposure (C) on days 0 and 24 after SC administration of ALP201 once every 3 days (q3d) max and AUC 72h ) increased with increasing dose in a slightly less than dose-proportional manner. Systemic exposure on day 24 (C max and AUC 72h) appeared to decrease after repeated SC administration. The decrease in exposure appeared to be dose-dependent, with a maximum decrease of up to 60% after repeated SC administration of 30 mg / kg ALP201. The subcutaneous bioavailability of ALP201 was approximately 44.2%.

[0121] ADA responses were negative in all pre-dose samples. The incidence of ADA was 58% and 90% for post-dose ADA samples on D28 and D56, respectively. The reduced exposure (60%) on day 24 of the 30 mg / kg dose was likely due to an immunogenic response.

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

[0123] There were no gender differences in systemic exposure to ALP201 on days 0 and 24. Therefore, pooled gender results are included in Table 17. max and AUC 72h ) increased with increasing dose in a roughly dose-proportional manner. 72h values) appeared to increase after repeated subcutaneous administration of ALP201 on day 24, with the mean cumulative AUC 72h The rates were 2.12, 1.83, and 2.15 at 1, 5, and 20 mg / kg, respectively.

[0124] ADA responses were negative in pre-dose samples. The incidence of ADA was 23% and 92% for post-dose ADA samples on D28 and D56, respectively. Because ADA samples were not sampled on the TK harvest days after repeated dosing, a direct correlation between positive ADA responses and the concentration-time profile of systemic exposure could not be made, although 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 dosing regimens ALP201 Modeling Population Pharmacokinetic (Pop-PK) Model To predict human ALP201 exposure from mouse dose-response results, a Pop-PK model was developed to predict human PK parameter estimates using weight-based allometric scaling and pooling mouse, rat, and monkey PK data. Mouse PK data included a single C-value from a single-dose wild-type mouse study (HPP-PK-01) and two repeat-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, dose ranging studies in rats and cynomolgus monkeys, and a 4-week GLP toxicity study with repeat 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 allometric principles. Here, animal body weight was centered at 70 kg, and allometric exponents were fixed at 0.75 for clearance parameters and 1.0 for volume of distribution parameters. The effect of ADA on PK was evaluated at CL based on a 4-week TK, and ALP201 concentrations appeared to be reduced in ADA+ animals in rats, and to a lesser extent in monkeys. The current best Pop-PK model is a two-compartment model with loss of linearity. Testing of the effect of ADA+ on ALP201 concentrations was inconclusive. Estimated bioavailability in humans was approximately 75%, and the effective half-life calculated for humans was 7-9 days. Human PK simulations used variability estimated from the human asfotase alfa Pop-PK model and the mean (and standard deviation) adult baseline body weight of HPP patients enrolled in clinical trials evaluating asfotase alfa as a treatment for HPP.

[0127] Dose-response model E max The dose-response model was selected by testing a family of models. Most dose-response relationships were consistent with E max The current best dose-response characterization is E max+E0 (baseline) model. The efficacy endpoint bone mineralization was chosen because it was radiographically assessed, which is the same as the clinical definition of asfotase alfa efficacy used in the nonclinical dose-response evaluation. Bone mineralization after treatment with asfotase alfa or ALP201 from two efficacy studies (HPP-PoC-01 and HPP-MED-01) was plotted against dose (Figure 6). Asfotase alfa data from a previous efficacy study was included for comparison. The y-axis represents % normal, defined as the percentage of mice in the treatment group with a bone mineralization score of 4. The x-axis represents dose normalized to mg / kg / day. The dose resulting in normal mineralization in 85% of the treated population (ED85) was selected as the target effective dose.

[0128] Mouse to Human Dose Conversion and Proposed Starting Dose in Humans Allometric scaling was applied to the target effective dose (ED85) in mice to determine the human dose. The formula used to predict the human equivalent dose (HED, mg / kg / day) from the mouse ED85 was ED85 x (0.025 kg mouse / 70 kg human). 0.25 The HED was converted to a weekly flat dose of 45 mg / week. This will be used as the dose for FIH study 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 (both IV and SC).

[0129] Predicted human exposure and PK safety margin of ALP201 The Pop-PK model was used to simulate detailed PK profiles based on allometrically adjusted doses in humans (Figure 7). The simulated PK data were used to estimate the exposure metric C max and AUC were calculated.

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

[0131] Toxicity testing Nonclinical safety studies were conducted in rats and monkeys to evaluate local tolerability, systemic toxicity, and safety pharmacology parameters following SC administration of ALP201 for up to 28 days. A nonclinical safety evaluation of a single IV dose of ALP201 was also evaluated in a 28-day toxicity study in rats to characterize the bioavailability of SC ALP201.

[0132] Single-dose toxicity study No separate single-dose tolerability study was conducted for ALP201. However, ALP201 tolerability in rats and monkeys was evaluated as part of the single-dose PK study. ALP201 tolerability was evaluated by clinical observations, including injection site reactions and clinical pathology data. No notable ALP201-related injection site reactions, clinical observations, or clinical pathology observations were observed in the rat or monkey single-dose PK studies. In summary, single doses of ALP201 were well tolerated when administered via IV or SC at doses up to 27 mg / kg in rats and 20 mg / kg in monkeys. Doses for the ALP201 repeat-dose-finding (GLP) toxicity study were selected based on the tolerability and clinical pathology data obtained.

[0133] Repeated dose toxicity study Definitive or GLP-compliant 28-day toxicity studies were conducted in Sprague-Dawley rats and cynomolgus monkeys. In a 28-day rat toxicity study, administration of ALP201 via subcutaneous injection to male and female CD® rats every 3 days for 28 days (days 1, 4, 7, 10, 13, 16, 19, 22, 25, and 28) for a total of 10 doses was well tolerated up to 30 mg / kg / dose, the highest SC dose evaluated in the study. In addition, IV administration of 10 mg / kg ALP201 was also well tolerated after a single injection. No ALP201-related changes were noted in clinical observations, body weight, body weight gain, quantitative food intake, ophthalmological examination, functional observation global score, hematology, urinalysis, gross findings, and organ weights. At the end of the dosing phase, a dose-related increase in alkaline phosphatase (ALP) activity (expected pharmacological effect) was seen at ≥2 mg / kg / dose SC of ALP201, which fully resolved after a 28-day recovery period. Non-adverse microscopic changes were associated with injection treatment with reactive changes in the draining lymph nodes. No discernible ALP201-related changes in incidence, severity, or microscopic characteristics of changes were seen at the injection site or draining lymph nodes. Although less pronounced in the recovery group animals, the microscopic changes at the injection site and draining lymph nodes of recovered animals were similar to those noted in terminal necropsy animals. In conclusion, for subcutaneous administration, a dose level of 30 mg / kg / dose was considered to be the no observed adverse effect level (NOAEL), which was determined by a C of 43.9 μg / mL in males and females combined on day 24. max value, and AUC of 2120hr*μg / mL 0-72hr Equivalent to the value.

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

[0135] In a 28-day monkey toxicity study, ALP201 administered via SC injection to male and female cynomolgus monkeys at doses of 1 mg / kg, 5 mg / kg, or 20 mg / kg every 3 days for 28 days (days 1, 4, 7, 10, 13, 16, 19, 22, 25, and 28, a total of 10 injections) was well tolerated up to 20 mg / kg / dose, the highest dose evaluated in the study. No ALP201-related changes were noted in injection site reactions (skin scoring), body weight, body weight gain, qualitative food intake, ophthalmologic examination, manual respiration rate, indirect blood pressure, qualitative electrocardiogram examination, hematology, urinalysis, gross findings, or organ weights. At the end of the dosing phase, a dose-related increase in alkaline phosphatase (ALP) activity (expected pharmacological effect) was observed, which was fully (at 1 mg / kg / dose) or nearly fully (at ≥ 5 mg / kg / dose) reversed after a 28-day recovery period. Non-detrimental ALP201-related microscopic changes were limited to the injection site and included minimal to mild degeneration / necrosis, calcification, and mixed cell inflammation / infiltration. Partial reversal of degeneration / necrosis and mixed cell inflammation / infiltration at the injection site(s) was observed in the recovery groups, while minimal to mild calcification was seen in recovered males at ≥ 1 mg / kg / dose and females at 20 mg / kg / dose. Increased heart rate was observed in all animals at 1 mg / kg / dose and in some animals at ≥ 5 mg / kg / dose. These increases in heart rate are not considered to be adverse because of the inconsistencies in whole body exposure data, as well as the fact that heart rate and ECG values ​​remained within the normal range of biological variation in monkeys of this age. In conclusion, the dose level of 20 mg / kg / dose is considered to be the no observed adverse effect level (NOAEL), which corresponds to a C of 254 μg / mL in males and females combined, respectively, on day 24. max value, and mean AUC of 15400hr*μg / mL 0-72hr Equivalent to the value.

[0136] Local Tolerance Study Separate nonclinical studies were not conducted to evaluate local tolerability, although injection site evaluations were performed for SC ALP201 in general toxicity studies in rats and monkeys, and for IV ALP201 in general toxicity studies in rats. Subcutaneous and IV administration of ALP201 was well tolerated locally in monkeys, with no adverse findings observed at the injection site.

[0137] Summary of nonclinical observations related to clinical trials of ALP201 Safety assessment No ALP201-related changes were noted in clinical observations, body weight, body weight gain, quantitative food intake, ophthalmology, Comprehensive Neural Network (CNS) observations, cardiovascular endpoints, respiratory endpoints, hematology, urinalysis, gross findings, or organ weights in the 28-day toxicity study. At the end of the dosing phase, a significant dose-related increase in alkaline phosphatase (ALP) activity was noted in rats or monkeys, which is the expected pharmacological effect. No notable systemic organ toxicity was observed in rats or monkeys at any of the doses 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 the injection site, with minimal to mild degeneration / necrosis seen in males and females. Degeneration / necrosis and mixed cell inflammation / infiltration at the injection site(s) were less prevalent and severe in the recovery group, or were characterized by chronicity (calcification), indicating partial recovery. Non-adverse microscopic changes in rat studies were limited to injection treatment with reactive changes in the draining lymph nodes. No discernible ALP201-related changes in incidence, severity, or microscopic characteristics of changes were seen at the injection site or draining lymph nodes. Although less pronounced in the recovery group animals, the microscopic changes at the injection site and draining lymph nodes in recovery animals were similar to those shown in terminal necropsy animals.

[0139] The observed exposures at the NOAEL of 20 mg / kg / dose based on the GLP SC monkey toxicity study were approximately 113-fold (IV) and 83-fold (SC) higher than the predicted AUC exposures for the proposed human starting single dose of 15 mg IV or 15 mg qw x 3 SC (Table 18). Given the margin of safety and the lack of systemic toxicity or local tolerability findings in the 28-day rat and monkey toxicity studies with ALP201, the human safety risk for the 15 mg starting dose is very low.

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

[0141] In a rat toxicity study, ADA responses were positive in 9 of 20 animals (6 males, 3 females) in the control group, 10 of 19 animals (6 males, 4 females) at 2 mg / kg / dose, 12 of 20 animals (7 males, 5 females) at 10 mg / kg / dose, and 12 of 20 animals (6 males, 6 females) at 30 mg / kg / dose administered SC on PK day 28. In addition, ADA responses were positive in 14 of 20 animals (8 males; 6 females) administered a single dose of 10 mg / kg IV ALP201 on PK day 28. Although ADA may have contributed to the apparent reduction in systemic exposure at 30 mg / kg / dose on day 24, the positive ADA responses did not appear to consistently affect the composite plasma concentration-time profiles of the 2 and 10 mg / kg / dose SC groups on day 24. According to the investigations performed, the positive ADA responses in control animals are unlikely to be due to incorrect dosing of ALP201. A dose-dependent increase in ALP, the expected pharmacological effect of ALP201, was observed in the ≥2 mg / kg SC treated groups before terminal necropsy, which was reversed during recovery necropsy. The ALP results in control animals were similar both before terminal necropsy and before recovery necropsy. Furthermore, there were no in-life findings related to ALP201 during the dosing and recovery phases. Thus, taken together, the ADA positive responses observed in control animals on PK days 28 and 56 (study days 29 and 57) are likely mainly due to contamination during blood collection and will not affect the study.

[0142] In the monkey toxicity study, anti-ALP201 antibody responses were negative in all pre-dose samples on day 0. ADA responses were positive on day 28 in 4 of 10 animals at 1 mg / kg / dose, 6 of 10 animals at 5 mg / kg / dose, and 5 of 10 animals at 20 mg / kg / dose. Because ADA samples were not sampled on the TK collection days after repeated dosing, a direct correlation between positive ADA responses and the concentration-time profile of systemic exposure could not be made, although the concentration-time profile of some animals at ≦5 mg / kg appeared to be affected by anti-ALP201 antibodies. Animals affected by anti-ALP201 antibodies included 3 animals (2 males and 1 female) at 1 mg / kg / dose and 3 animals (3 females) at 5 mg / kg / dose, and for these animals, the days that affected the concentration-time profile ranged from day 18 to day 24.

[0143] Example 2 The pharmacokinetic profile of asfotase alfa in multiple species suggests that frequent dosing is required due to a combination of reduced absolute bioavailability and reduced half-life. The catalytic domain of human TNSALP is a highly glycosylated molecule. The presence of non-sialylated glycans 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, but incorporates the removal of two non-essential N-linked glycans, a change in the human Fc isotype to IgG2 / 4, and numerous process improvements in the expression of the molecule that result in higher TSAC incorporation. In this report, we present the pharmacokinetic parameters of ALP201 as determined in a single-dose study in male C57BL / 6 mice administered intravenously and subcutaneously. We also report the PK parameters of multiple lots of ALP201 expressed and purified at various levels of TSAC incorporation to help determine which PK parameters were most strongly affected by clearance of nonsialylated glycans.

[0145] Materials and Methods animal lineage To maintain consistency with these studies and previous pharmacokinetic studies performed on asfotase alfa, the present studies were performed using male C57BL / 6 mice that were approximately 11-12 weeks old at the time of dosing. Information on the test molecules is shown in Table 19. [Table 19]

[0146] Animal dosing and blood sampling Animals aged 11-12 weeks were randomized by body weight and assigned to four groups. A dose of 4 mg / kg of ALP201 was administered to 16 mice / group by intravenous delivery (IV) (group 1) or subcutaneously (SC) (group 2) on day 0. An equivalent adjusted volume of PBS was administered to 4 mice / group by IV (group 3) or SC (group 4) on day 0. IV administration was performed via the tail vein. A SC bolus was administered in the upper scapular region of the shoulder.

[0147] A semi-continuous sampling design was used with 4 cohorts per group, n=4 per cohort, plus one additional mouse per group. In the Alexion study, each mouse was bled three times, including a terminal bleed. Time points (hours post-dose) by cohort are shown in Table 20.

[0148] For non-terminal blood draws, 100 mL of whole blood was collected by submandibular collection into pre-coated lithium heparinized tubes, and at least 50 μL of plasma was collected from each animal at each time point. At terminal blood draws, as much blood as possible was collected by cardiac puncture into lithium heparinized collection tubes. Blood was kept at 4°C, and plasma was processed by centrifugation as soon as possible after collection. Each plasma sample was divided into two equal volume aliquots, which were then flash frozen in CO2 / ethanol and stored at -80°C. [Table 20] [Table 21] [Table 22]

[0149] For lots 2, 3, and 4, groups of 32 mice were divided into 8 cohorts of 4 animals / time point per route of administration. For the IV administration group, each cohort was bled twice (first time point was survival draw, second time point was terminal) at approximately 0.5, 1, 3, 8, 12, 21, 26, 32, 45, 49, 72, 96, 120, 192, 264, and 336 hours post-dose. For the SC administration group, each cohort was bled twice (first survival draw, second time point was terminal) at approximately 1, 3, 6, 8, 12, 21, 26, 32, 45, 49, 72, 96, 120, 192, 264, and 336 hours post-dose.

[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 can be easily detected by fluorometer. Quantification of activity was performed via an enzyme activity standard curve.

[0151] In-house PK study sample analysis Quantification of activity was performed via an enzyme activity standard curve generated using serial dilutions of asfotase alpha protein reference standards of known activity on the same plate in the 4-MUP assay. Thawed plasma samples were diluted 100-2,000-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 active ALP201 enzyme concentrations. The final protein standard range on each plate was 4-80 ng / mL.

[0152] The diluted samples were quantified as follows: All diluted samples were brought to 37°C before the start of the assay reaction by adding 4-MUP to the protein samples to give 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 on a plate reader maintained at 37°C, with the plate read every 40 seconds for a total of 20 minutes. The reaction rates of the plasma and standard samples were calculated by linear regression of the reaction slope in units of relative fluorescence units (RFU) per minute using Microsoft Excel. The reaction rates of the standard samples were used to generate a linear standard curve in units of RFU / min / U. Comparison of the reaction rates measured in the plasma samples was compared to the standard curve to determine the plasma activity of ALP201 in U / mL. The sample activity in units / mL was converted to mass units in mg / L by dividing the sample activity by the specific activity (units U / mg) of the protein sample tested. Samples were run twice in duplicate to pool data for each independent sampling point.

[0153] PK study sample analysis The same 4-MUP hydrolysis assay was performed. On the day of the assay, standards, quality controls (QCs), dilution controls (DCs), and blanks were diluted 250-fold in assay buffer. Additionally, standards, QCs, DCs, and blanks were diluted 2-fold in the substrate 4-methylumbelliferyl phosphate (4-MUP) for a total minimum required dilution (MRD) of 500-fold. DCs were diluted 200-fold in mouse plasma before performing the MRD. ALP201 was quantified based on the fluorescent product methylumbelliferone resulting from hydrolysis of the 4-MUP substrate used in the assay. Plates were placed in a plate reader and read once every 60 seconds at 37°C for 25 minutes with kinetic 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 of fluorescence intensity / min) was determined from a best-fit straight line through the data. Blanks were not included in the curve fit. Results are reported in mg / L. Values ​​were corrected for differences in specific activity between the test articles and protein activity standards.

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

[0155] The following noncompartmental PK parameters were calculated: area under the concentration-time curve from time 0 (dose) to the last detectable concentration (AUC t ), the dose-normalized area under the concentration-time curve from time 0 (administration) to the last detectable concentration (AUC t / dose), the area under the concentration-time curve (AUC inf ), the area under the dose-normalized concentration-time curve (AUC inf / dose), maximum observed plasma concentration (C max ), and dose-normalized maximum observed plasma concentration (C max / dose), time to peak observed plasma concentration (Tmax ), terminal elimination half-life (t 1 / 2 ), total clearance (CL), apparent volume of distribution (V d ), and bioavailability (F). Actual sample collection times were used to calculate PK parameters. Actual doses were used to calculate dose-normalized PK parameters.

[0156] Results and Discussion Pharmacokinetic parameters of ALP201 after IV and SC administration in mice The PK of ALP201 was evaluated following a single IV or SC administration to male C57BL / 6 mice. Sixteen mice were dosed per group and blood was collected from cohorts of four mice per group at 0.25, 1, 6, 24, 48, 72, 96, 120, 192, 264, 336, and 480 hours post-dose.

[0157] ALP201 concentrations in plasma were determined using an enzyme activity assay that measures the alkaline phosphatase catalytic activity of ALP201, and the ALP201 enzyme activity was then converted to mass units (mg / L) for reporting purposes.

[0158] Mean active ALP201 plasma concentration versus time profiles following IV and SC administration are presented on semi-log scale in Figures 8A and 8B. A summary of ALP201 PK parameters is provided in Tables 7 and 11 above.

[0159] The plasma activity profiles of individual mice in some of the IV groups would suggest that a portion of the dose may have been delivered subcutaneously, which may account for the relatively high variability observed at early time points in the IV dosing profile. If this occurred, the C calculated for the IV dosing group would be max The values ​​of t may also be artificially low. After IV administration, the concentration-time profile declined multi-exponentially (Figures 8A and 8B). The CL was 0.0019 L / h / kg, and the apparent terminal t was 48 hours. After SC administration, the time to reach the maximum concentration (t max) 48 hours after administration, suggesting that absorption from the SC injection site is relatively slow. Absolute bioavailability after SC administration was 96%.

[0160] Pharmacokinetic parameters of ALP201 lots with various total sialic acid contents in mice Three separate lots of ALP201 purified with measured TSAC values, i.e., 5.9, 5.0, and 3.2 moles of sialic acid per mole of protein monomer, were administered to 32 mice by IV and SC dosing, with blood collected from cohorts of four mice per treatment group at 0.5, 1, 3, 8, 12, 21, 26, 32, 45, 49, 72, 96, 120, 192, 264, and 336 hours post-dose for IV dosing; and 1, 3, 6, 8, 12, 21, 26, 32, 45, 49, 72, 96, 120, 192, 264, and 336 hours post-dose for SC dosing, with each mouse bled twice.

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

[0162] The mean active ALP201 plasma concentration vs. time profiles following IV and SC administration per protein lot are presented on semi-log scale in Figures 9A and 9B, 10A and 10B, and 11A and 11B. A collection of plasma concentration vs. time profiles following IV administration at various TSAC values ​​is shown in Figure 12. A collection of plasma concentration vs. time profiles following SC administration is shown in Figure 13. Tabulated summaries of the mean PK parameters and mean dose-normalized PK parameters following IV and SC administration can be found in Tables 21 and 22, 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 moles of sialic acid per common N-linked glycan site, and the PK profiles of all lots (both IV and SC administration) were similar in shape and T max and t 1 / 2 The values ​​were also similar.

[0164] Increasing the TSAC value slightly decreased clearance and increased C clearance by both IV and SC routes. max and AUC increased, and the volume of distribution decreased. All of these changes are consistent with the idea that lower TSAC values ​​are associated with higher levels of immature N-linked glycans, which may be rapidly removed by cellular receptors such as ASGPR, leading to higher clearance of protein samples and increased C max The changes in these parameters are relatively small, with a maximum difference of 2-fold.

[0165] Comparison of PK parameters of ALP201 in mice with those of asfotase alfa in mice As a second generation alkaline phosphatase ERT, ALP201 was designed to build on and improve upon the PK parameters of asfotase alfa 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] The entire PK data set collected for ALP201 in male C57BL / 6 mice demonstrated that ALP201 has an excellent in vivo half-life (t 1 / 2The results showed that ALP201 exhibited significantly lower clearance in mice than asfotase alfa in all samples tested.

[0167] Because the release specification of asfotase alfa limits the molecule to a TSAC value of 1.2-3.0 mol / mol and the molecule has six N-linked glycosylation sites per monomer, the sialic acid incorporation of asfotase alfa is only 0.20-0.50 moles of sialic acid per glycan. This indicates 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 of asfotase alfa compared to ALP201.

[0168] All of these samples of ALP201 are much more bioavailable than asfotase alfa, with the smallest bioavailable sample of ALP201 being approximately twice the observed bioavailability of asfotase alfa (72.1% vs. 38.8%). Differences in the human IgG Fc domain isotype between the two constructs may be responsible for this observation. We have previously demonstrated that the IgG2 / 4 Fc domain of ALP201 does not bind to a panel of common Fcg receptors, whereas the IgG1 Fc domain of asfotase alfa binds strongly 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 bioavailability and exposure.

[0169] conclusion The findings of these studies suggest that ALP201 has a higher bioavailability, dose-normalized C maxThe results show that ALP201 significantly improves the dose-corrected pharmacokinetic profile in mice compared to asfotase alfa after IV or SC administration, with dose-normalized exposures. Additional PK studies using ALP201 samples with various levels of TSAC uptake demonstrated that C increases with increasing TSAC levels. max and in vivo exposure was slightly increased. max , and bioavailability appeared to be relatively unaffected by changes in TSAC values.

[0170] Example 3 Summary of single-dose PK study with ALP201 As part of the asfotase alfa nonclinical 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 studies. [Table 26]

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

[0172] Conclusions based on single-dose PK studies ALP201 was designed to improve upon the properties of the approved product, asfotase alfa, 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 asfotase alfa. This modification was designed to improve the pharmacokinetic properties of ALP201, including systemic PK exposure, half-life, and bioavailability.

[0173] Preclinical data from three species (mouse, rat, and monkey) support evidence that these objectives have been achieved. Compared to asfotase alfa, ALP201 demonstrated significantly improved PK exposure (>10-fold), absolute bioavailability (2-fold), and half-life (2-fold) following IV / SC administration in three species. Preclinical population PK modeling also anticipates that the improved PK properties of ALP201 will be extrapolated to humans, which would facilitate easier patient treatment through smaller injection volumes, less frequent dosing, and lower total annual doses, thereby reducing the treatment burden on patients.

[0174] Non-clinical safety studies Summary of Toxicology Studies Supporting First-in-Human Studies No separate single-dose toxicity / tolerability study was conducted for ALP201. The ALP201 doses in the repeat dose-finding (GLP) toxicity studies were selected based on data obtained from single-dose IV and SC PK studies of ALP201 in rats and monkeys. The tolerability of ALP201 was evaluated by clinical observations, including injection site reactions and clinical pathology data, in single-dose IV and SC PK studies in rats and monkeys. In summary, ALP201 was well tolerated when administered via IV or SC in single doses up to 27 mg / kg in rats and 20 mg / kg in monkeys.

[0175] Definitive GLP-compliant 28-day toxicity studies of ALP201 in cynomolgus monkeys and Sprague-Dawley rats are ongoing. The in-life portions, including recovery phases, of the rat and monkey studies are completed and reports are being prepared. Although audited draft reports of these studies are not available, all data from both studies, except for the ADA data, are available and a summary of the findings is provided 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 meaningful treatment-related adverse effects from any of the toxicity endpoints evaluated in both studies, including safety pharmacology endpoints. [Table 27-1] [Table 27-2]

[0176] Preclinical safety strategy for ALP201 in late-stage clinical development ALP201 is an improved ERT compared to the ERT product asfotase alfa (STRENSIQ®) sold by Alexion. The human TNSALP catalytic domain of ALP201 contains rationally designed changes at three positions to confer higher enzymatic activity. The Fc portion of ALP201 is human immunoglobulin gamma 2 / 4 (IgG2 / 4), whereas the Fc portion of asfotase alfa is human immunoglobulin IgG1. The C-terminal regions of ALP201 and asfotase alfa that are targeted to bone are identical.

[0177] Findings from ALP201 and selected asfotase alfa studies include the following: 1. Toxicity findings from the GLP 28-day toxicity study of ALP201 in rats and monkeys are very similar (Table 31) 2. Toxicity findings from toxicity studies of ALP201 and asfotase alfa of the same duration in monkeys and rats (4 weeks / 28 days) were very similar (Tables 32 and 33, respectively). 3. Toxicity findings from the complete nonclinical safety package of asfotase alfa were limited to transient local tolerability findings in rats, with no other notable adverse asfotase alfa treatment-related findings.

[0178] Long-term / 6-month general toxicity study strategy for ALP201 A single-species long-term toxicity testing strategy is supported by the following observations: 4. In a 28-day GLP toxicity study in rats and monkeys, no notable systemic toxicity and / or local tolerability findings were observed after 10 repeated doses of ALP201 administered SC once every 3 days (Q3D), however, systemic exposure of ALP201 was significantly higher than that of asfotase alfa. A summary of systemic exposure is shown in Tables 31 and 32. 5. Toxicity findings from 28-day GLP toxicity studies of ALP201 in rats and monkeys were very similar. A summary of findings from these studies is shown in Table 28. 6. When toxicological findings from short-term general toxicity studies are similar, a single species long-term general toxicity study is usually sufficient. 7. Rats are the preferred species for long-term toxicity studies with ALP201. In addition, if only one species is used to conduct long-term general toxicity studies, rats are the preferred species. [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 regimens ALP201 Modeling Data used in the current model To perform dose extrapolation from mice (a disease model species) to humans, adequate PK analysis is required to simulate human exposure from allometrically adjusted doses. Limited mouse PK data, a single C-terminal PK profile from a single-dose wild-type mouse study (HPP-PK-01) and two repeat-dose Akp2GW(- / -) mouse efficacy studies (HPP-PoC-01 and HPP-MED-01), are available. トラフ Measured values ​​(D36 / D37) were available. To generate a robust PK characterization of ALP201, the mouse data was augmented with PK data from dose-ranging studies in rats and cynomolgus monkeys. Table 30 shows the data analyzed to date (47 animals and 387 PK concentrations) using a population pharmacokinetic (Pop-PK) modeling approach. Efficacy (bone mineralization) data from two Akp2GW(- / -) mouse efficacy studies (HPP-PoC-01 and HPP-MED-01) were used for dose-response modeling. [Table 30]

[0180] Current Pop-PK model The current Pop-PK model was developed using the NONMEM software program, version 7.2 (ICON solutions), which simultaneously analyzed PK data from three animal species, taking into account species differences in PK kinetics with body weight based on allometric principles. Here, animal body weight was centered at 70 kg, and allometric exponents were fixed at 0.75 for clearance parameters and 1.0 for volume of distribution parameters. IV and SC data were also fitted simultaneously to estimate extravascular parameters, e.g., absolute bioavailability after SC injection. The current model is a two-compartment model with loss of linearity, and the model parameter estimates are shown in Table 31. [Table 31]

[0181] Standard values ​​or mean parameter estimates of PK parameters were estimated well. Variability of CL (BSV) was within acceptable limits and well estimated, whereas variability of Ka and F was large (86% and 76%, respectively) and not well estimated. Shrinkage is an assessment of how well the data informed the estimates of PK parameters, with values ​​<30% being good. Given the high shrinkage values ​​of Ka and F (40% and 59%, respectively), additional data from a 4-week GLP toxicity study should improve the estimates of Ka and F parameters. Overall, the current Pop-PK model should provide reasonable dose simulations given the good mean parameter estimates. However, the variability region may be overly wide.

[0182] Current dose-response models E max The dose-response model was selected by testing a family of models. Most dose-response relationships were consistent with E max The current best dose-response characterization is E max+E0 (baseline) model. The efficacy endpoint bone mineralization was chosen because it is a radiographic assessment and is the same as the clinical definition of asfotase alfa efficacy used in non-clinical dose-response evaluations. Bone mineralization after treatment with asfotase alfa or ALP201 from two efficacy studies (HPP-PoC-01 and HPP-MED-01) was plotted against dose (Figure 16). Asfotase alfa data from a previous efficacy study, ALP-PT-12, was included for comparison. The y-axis represents % normal, defined as the percentage of mice in the treatment group with a bone mineralization score of 4. The x-axis represents dose normalized to mg / kg / day. The dose resulting in normal mineralization in 85% of the treated population (ED85) was selected as the target effective dose.

[0183] Mouse to Human Dose Conversion and Proposed Starting Dose in Humans Allometric 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 the mouse ED85 is ED85 x (0.025 kg mouse / 70 kg human). 0.25 The HED translates to a weekly fixed dose of 45 mg / week, which will be used as the starting dose for the MAD arm of the FIH study. After considering the results from the NOAEL and MABEL methods, the maximum recommended starting dose (MRSD) for the SAD arm was selected. It was decided to select the test dose that produced the minimum on-target pharmacological response and convert it to a fixed human dose (5 mg).

[0184] Other embodiments The foregoing detailed description and examples have been presented for clarity of understanding only, and no unnecessary limitations should be understood therefrom. The present disclosure is not limited to the exact details shown and described, and variations obvious to those skilled in the art are intended to be included within the scope defined by the claims.

[0185] Unless otherwise indicated, all numbers expressing quantities of ingredients, molecular weights, and the like used in the specification and claims should be understood in all instances to be modified by the term "about." Accordingly, unless specifically indicated to the contrary, the numerical parameters set forth in the specification and claims are approximations that may vary depending upon the desired properties sought to be obtained. At the very least, and not as an attempt to limit the doctrine of equivalents to the scope of the claims, each numerical parameter should at least be construed in light of the number of reported significant digits by applying ordinary rounding techniques.

[0186] Notwithstanding that the numerical ranges and parameters setting forth the broad scope of the present disclosure are approximations, the numerical values ​​set forth in the specific examples are reported as precisely as possible, however, all numerical values ​​inherently contain certain ranges necessarily resulting from the standard deviation found in their corresponding testing measurements.

[0187] The complete disclosures 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 amino acid sequence submissions in, for example, SwissProt, PIR, PRF, PDB, and translations from the annotated coding regions of GenBank and RefSeq) described herein are incorporated by reference. The foregoing detailed description and examples are presented for clarity of understanding only. No unnecessary limitations should be understood therefrom. The present disclosure is not strictly limited to the details shown and described, and variations obvious to one skilled in the art are intended to be 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 to SEQ ID NO:5 and at least one mutation selected from E108M, N213Q, and N286Q compared to SEQ ID NO:1, and a pharma- ceutically acceptable carrier comprising one or more of phosphate, proline, and sucrose. (Item 2) 2. The pharmaceutical composition of item 1, wherein the polypeptide has at least 85%, 90%, 95%, 97%, or 99% sequence identity to SEQ ID NO:5. (Item 3) 3. The pharmaceutical composition according to item 2, wherein the polypeptide comprises or consists of the sequence of SEQ ID NO:5. (Item 4) 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) 5. The pharmaceutical composition according to any one of items 1 to 4, wherein the composition comprises about 1 mM to about 100 mM of phosphate. (Item 6) 6. The pharmaceutical composition according to item 5, wherein the composition comprises about 5 mM to about 20 mM of phosphate. (Item 7) 7. The pharmaceutical composition of claim 6, wherein the composition comprises about 10 mM phosphate. (Item 8) 8. The pharmaceutical composition according to any one of items 1 to 7, wherein the phosphate is sodium phosphate. (Item 9) 9. The pharmaceutical composition according to any one of items 1 to 8, wherein the composition comprises about 1 mM to about 500 mM proline. (Item 10) 10. The pharmaceutical composition according to item 9, wherein the composition comprises about 50 mM to about 200 mM proline. (Item 11) 11. The pharmaceutical composition of claim 10, wherein the composition comprises about 140 mM proline. (Item 12) 12. The pharmaceutical composition according to any one of items 1 to 11, wherein the composition comprises about 1 mM to about 500 mM sucrose. (Item 13) 13. The pharmaceutical composition according to item 12, wherein the composition comprises about 50 mM to about 200 mM sucrose. (Item 14) 14. The pharmaceutical composition of claim 13, wherein the composition comprises about 140 mM sucrose. (Item 15) 15. The pharmaceutical composition according to any one of items 6 to 14, wherein the composition comprises about 10 mM phosphate, about 140 mM proline, and about 140 mM sucrose. (Item 16) 16. The pharmaceutical composition according to any one of items 1 to 15, wherein the composition comprises about 0.01% to about 0.5% polyoxyethylene (20) sorbitan monooleate. (Item 17) 17. The pharmaceutical composition according to item 16, wherein the composition comprises about 0.01% to about 0.1% polyoxyethylene (20) sorbitan monooleate. (Item 18) 18. The pharmaceutical composition according to claim 17, wherein the composition comprises about 0.05% polyoxyethylene (20) sorbitan monooleate. (Item 19) 19. The pharmaceutical composition of claim 18, wherein the composition comprises about 140 mM proline, about 140 mM sucrose, and about 0.05% polyoxyethylene (20) sorbitan monooleate. (Item 20) 20. The pharmaceutical composition according to any one of items 1 to 19, wherein the polypeptide comprises a total sialic acid content (TSAC) of about 1.0 mol / mol to about 6.0 mol / mol. (Item 21) 21. The pharmaceutical composition according to item 20, wherein the TSAC is from about 3.0 mol / mol to about 6.0 mol / mol. (Item 22) 22. The pharmaceutical composition of claim 21, wherein the TSAC is about 3.2 mol / mol, 5.0 mol / mol, or about 5.9 mol / mol. (Item 23) 23. The pharmaceutical composition according to any one of items 1 to 22, wherein the composition comprises a pH of about 7.3. (Item 24) 24. The pharmaceutical composition according to any one of items 1 to 23, wherein the composition is prepared at a concentration of about 0.1 mg / mL to about 200 mg / mL. (Item 25) 25. The pharmaceutical composition according to any one of items 1 to 24, wherein the composition is prepared in a volume of about 0.1 mL to about 50 mL. (Item 26) 26. The pharmaceutical composition according to item 24 or 25, wherein the composition is prepared at a concentration of about 100 mg / mL in a volume of about 1 mL. (Item 27) 24. A vial comprising the pharmaceutical composition according to any one of items 1 to 23. (Item 28) 28. The vial according to item 27, wherein the composition is prepared in a volume of about 0.1 mL to about 50 mL. (Item 29) 29. The vial according to item 28, wherein the composition is prepared in a volume of about 10 mL. (Item 30) 30. The vial according to any one of items 27 to 29, wherein the composition is prepared at a concentration of about 0.1 mg / mL to about 200 mg / mL. (Item 31) 31. The vial according to item 30, wherein the composition is prepared at a concentration of about 100 mg / mL. (Item 32) 32. The vial according to any one of items 27 to 31, wherein the composition is prepared at a concentration of about 100 mg / mL in a volume of about 1 mL. (Item 33) 27. A method of treating a bone mineralization disorder in a patient in need thereof, comprising administering to the patient a dose of the pharmaceutical composition according to any one of items 1 to 26. (Item 34) 34. The method of claim 33, wherein the bone mineralization disorder is selected from hypophosphatasia (HPP), bone fracture, osteoporosis, sclerosteosis, chondrocalcinosis, hypotonia, Duchenne muscular dystrophy, tracheobronchomalacia, stroke, neurofibromatosis, and craniosynostosis. (Item 35) 35. The method of claim 33 or 34, wherein the polypeptide is administered in an amount and for a duration sufficient to treat the bone mineralization disorder. (Item 36) 36. The method according to any one of items 33 to 35, wherein the treating promotes bone formation in the patient. (Item 37) 37. The method according to any one of items 33 to 36, wherein the polypeptide is administered at a dosage of about 0.01 mg / kg to about 20 mg / kg. (Item 38) Item 38. The method of item 37, wherein the polypeptide is administered at a dosage of about 0.1 mg / kg to about 10 mg / kg. (Item 39) 39. The method according to any one of items 33 to 38, wherein the polypeptide is administered once a day, once a week, once a month, or once a year. (Item 40) 40. The method according to any one of items 33 to 39, wherein about 10 to about 100 mg of the polypeptide is administered per week. (Item 41) 41. The method of item 40, wherein about 15 to about 90 mg of the polypeptide is administered per week. (Item 42) 42. The method of claim 41, wherein about 15 mg, about 30 mg, about 45 mg, or about 90 mg of the polypeptide is administered per week. (Item 43) 43. The method according to any one of items 33 to 42, wherein the polypeptide is administered for at least one day, one week, one month, one year, or more. (Item 44) 44. The method according to any one of items 33 to 43, wherein the composition is administered subcutaneously, intravenously, intramuscularly, sublingually, intrathecally, or intradermally. (Item 45) 45. The method of claim 44, wherein the composition is administered subcutaneously or intravenously. (Item 46) 46. ​​The method according to any one of items 33 to 45, wherein the patient is a human. (Item 47) 47. The method of claim 46, wherein the human is a neonate, a child, an adolescent, or an adult. (Item 48) The method provides an AUC of about 50 μg × hr / mL to about 4000 μg × hr / mL in the patient's blood. 0-168h 48. The method according to any one of Items 33 to 47, wherein (Item 49) The method provides an AUC of about 1000 μg × hr / mL to about 3000 μg × hr / mL in the patient's blood. 0-168h 49. The method according to claim 48, wherein (Item 50) The method provides a C of about 0.5 μg / mL to about 25 μg / mL in the patient's blood. max 49. The method according to any one of items 33 to 48, wherein (Item 51) The method provides a C of about 0.6 μg / mL to about 20 μg / mL in the patient's blood. max 50. The method according to claim 49, wherein

Claims

[Claim 1] The invention described in the specification.

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