RU2774620C2 - Methods for treatment of hypophosphatasia (hpp) in adults and adolescents - Google Patents

Abstract

FIELD: medicine.

SUBSTANCE: group of inventions relates to methods for the treatment of hypophosphatasia (hereinafter – HPP) in a subject aged 18 or older, or from 13 and older, having at least one symptom of an adult form of HPP. Methods include the administration of soluble alkaline phosphatase (hereinafter – sALP) to a subject in a treatment scheme providing from 1mg/kg/week to 9 mg/kg/week of sALP during five years of treatment. sALP contains an amino acid sequence 95% sequence identical to an amino acid sequence SEQ ID NO: 1. Methods additionally include: 1) determination of a concentration of inorganic pyrophosphate (hereinafter – PPi) in blood of a subject, while the administration of sALP for five years leads to a stable statistically significant reduction in PPi concentration in blood of a subject by 1 mcM; or 2) determination of a concentration of pyridoxal-5’-phosphate (hereinafter – PLP) in blood of a subject, while the administration of sALP for five years leads to a stable statistically significant reduction in PLP concentration in blood of a subject by 100 ng/mg; or 3) determination of an indicator of a six-minute walk test (hereinafter – 6MWT) in a subject, at which the administration of sALP for at least five years leads to a stable statistically significant increase in a walking distance of a subject by 50 meters, according to the assessment using 6MWT.

EFFECT: claimed inventions provide long-term stable efficiency of hypophosphatasia treatment.

45 cl, 21 dwg, 21 tbl, 15 ex

Description

FIELD OF TECHNOLOGY

The disclosure provides methods for treating hypophosphatasia (HPP).

BACKGROUND OF THE INVENTION

Hypophosphatasia (HPP - HPP - hypophosphatasia) is a rare hereditary skeletal disease with an incidence of 1 case per 100,000 newborns for the most severe forms of the disease. The disorder results from mutations leading to loss of functionality in the gene encoding tissue nonspecific alkaline phosphatase (TNLAP - tissue nonspecific alkaline phosphatase). HFF is characterized by a significant spectrum of symptoms and severity, from rickets to an almost complete lack of bone mineralization in utero.

The manifestation of HPP varies among patients and varies with the age of the patient. Most patients with HFF present with skeletal changes, short stature, chronic pain, lower extremity pain, gait disturbance, and premature atraumatic tooth loss. For example, common symptoms in adults with HPP may include osteomalacia, poorly mineralized bones, increased levels of inorganic pyrophosphate (PPi - PPi inorganic pyrophosphate) in the blood and / or urine, pyridoxal-5'-phosphate (P5P - PLP pyridoxal 5'-phosphate) or phosphoethanolamine (PEA - PEA - phosphoethanolamine), hypomineralization, hypercalciuria, short stature, skeletal deformity, waddling gait, bone pain, bone fractures, calcium pyrophosphate dihydrate crystal deposition, arthritis, and pyrophosphate arthropathy. Common symptoms in adolescents with HPP may include elevated levels of nPF, PEA, or P5P in the blood or urine, osteomalacia, skeletal deformity, hypotonia, muscle weakness, rheumatoid complications, arthritis, pseudogout, waddling gait, difficulty walking, bone pain, pain, early tooth loss, hypomineralization, pulmonary hypoplasia, respiratory failure, seizures, hypercalciuria, short stature and growth retardation.

Due to the physical impairment associated with HPP, adult patients suffering from HPP often have a reduced ability to walk compared to healthy subjects (Weinstein RS, Whyte MP, Arch Intern Med. 1981;141(6): 727-731) . Adult patients with HPP may suffer from calcium pyrophosphate deposition disease (CPPD), pseudogout, chondrocalcinosis, calcified periarthritis, vertebral fractures, subtrochanteric fractures, metatarsal fractures, and/or pseudo-fractures (Coe JD, et al., J Bone Joint Surg Am 1986;68(7):981-990; Sutton, RAL, et al., J Bone Miner Res. 2012;27(5):987-994). Other symptoms such as rickets, rickety ribs, severe hypomineralization, developmental delay, motor retardation, insufficient weight gain, pulmonary hypoplasia, respiratory failure, respiratory failure, craniosynostosis (including complications of craniosynostosis), and seizures occur in young children or in children with HFF, but not in adults with HFF.

It is noteworthy that the efficacy and safety of treating HPP, in particular the specific physiological and physical disorders and reduced walking ability associated with forms of HPP in adults and adolescents, using therapeutic agents over a long period of time, is unknown. Thus, there is a need for methods that can be used to treat and monitor HPP in adults and adolescents over extended periods of time so that these patients can live with improved physical and physiological impairment and can regain appropriate levels of walking ability.

BRIEF DESCRIPTION OF THE INVENTION

Disclosed are (1) methods for identifying adults with HPP (e.g., adults with HPP at about 18 years of age or older, such as adults with HPP that started in childhood) and/or adolescents with HPP (e.g., adolescents with HPP aged from about 13 years to about 17 years) for treatment with soluble alkaline phosphatase (sALP - soluble alkaline phosphatase; for example, SEQ ID NO: 1) and (2) treatment of such patients with sALP. Symptoms of the adult form of HPP, as defined herein, include, for example, elevated levels of phosphoethanolamine (PEA) in the blood and/or urine, hypomineralization, hypercalciuria, skeletal deformity, waddling gait, bone pain, bone fracture, calcium pyrophosphate dihydrate crystal deposition, arthritis, pyrophosphate arthropathy, chondrocalcinosis, calcific periarthritis, and pseudofracture. Symptoms of the juvenile form of HPP as defined herein include, for example, elevated levels of nPF, PEA, or P5P in the blood or urine, osteomalacia, one or more skeletal deformities, hypotension, muscle weakness, rheumatoid complications, arthritis, pseudogout, waddling gait, difficulty walking, bone pain, pain, early loss of teeth, hypomineralization, pulmonary hypoplasia, respiratory failure, seizures, hypercalciuria, short stature and stunting.

Typical metrics useful in assessing the need for or efficacy of rAPF treatment (e.g., SEQ ID NO: 1) include (1) plasma concentrations of nPF and P5F, (2) 6-minute walk test (6MWT), (3) Bruinx test -Ozeretsky Motor Assessment 2nd Edition (BOT-2), (4) Handheld Dynamometer (HHD), (5) Lower Limb Function Scale (LEFS) and (6) Brief Pain Assessment Questionnaire (BPI-SF) . Additional tests and metrics may include the Tinetti Test - Performance Oriented Mobility Assessment (POMA; modified if necessary) and space-time gait analysis software and hardware such as GAITRITE® (Clinical Image Retrieval System Inc.). The methods further include using one or more of the described metrics (e.g., BOT-2, 6MWT, plasma concentrations of nPF and P5F, HHD, LEFS, or BPI-SF) alone or in combination to evaluate the efficacy of rAPF treatment (e.g., SEQ ID NO: 1) for a patient with HPP in which improvement relative to a certain indicator or value demonstrates that rAP is effective for the treatment of HPP. In addition, the level of ALP activity in a sample (eg, plasma sample) of a patient having HPP can also be used alone or in combination with one or each of these indicators to evaluate the effectiveness of rAPF treatment (eg, SEQ ID). NO: 1) patient. In addition, the methods further comprise altering the dosage and/or frequency of sAP (e.g., SEQ ID NO: 1), administering to determine an effective amount of sAP to administer to an adult with HPP (e.g., an adult with childhood-onset HPP), or adolescent with HFF.

In a first aspect, a method is described for treating HPP in a patient with childhood-onset HPP, about 18 years of age or older (e.g., a human), characterized by adult HPP symptoms, which comprises administering rAPF (e.g., SEQ ID NO: 1 ) to a patient on a treatment regimen providing from about 1 mg/kg/week to about 9 mg/kg/week, preferably 6 mg/kg/week, rAPF over a treatment period of at least two weeks (e.g., at least , three weeks, at least four weeks, at least five weeks, at least six weeks, at least seven weeks, at least eight weeks, at least nine weeks, at least ten weeks at least three months at least four months at least five months at least six months at least seven months at least eight months at least nine months, at least one year, at least two years, at least at least three years, at least four years, at least five years, at least six years, at least seven years, at least eight years, at least nine years, or at least at least ten years or during the life of the patient; in particular, at least six weeks). Preferably, the adult patient is one who has not been treated (ie, untreated) with respect to treatment with sAP. In particular, rAP contains an amino acid sequence having at least 95% sequence identity with the amino acid sequence shown in SEQ ID NO: 1 (eg, asfotase alpha). Administration of rAPF (e.g., SEQ ID NO: 1) results in one or more of the following: (i) a statistically significant decrease in the concentration of NPF in a patient's plasma sample of at least about 1 μM relative to the concentration of NPF in a plasma sample of a subject with HFF that started in childhood and was untreated at about 18 years of age or older; (ii) a statistically significant decrease in the concentration of P5P in a patient's plasma sample of at least about 100 ng/ml relative to the concentration of P5P in a plasma sample of a subject with HPP, onset in childhood, untreated at about 18 years of age or older; and (iii) a statistically significant increase in the patient's walking distance of at least 50 meters, as assessed by 6MWT, relative to the walking distance in 6 minutes in the patient who was not administered. In addition, there may be an increase in ALP activity in a sample (eg a plasma sample) following administration of sAP (eg, SEQ ID NO: 1).

As a result of the use of these methods, a decrease in the concentration of nPP in a patient's plasma sample (for example, a patient with HPP starting in childhood, not treated, about 18 years of age or older), a decrease in the concentration of P5P in a patient's plasma sample, and/or increasing the patient's walking distance over a treatment period of at least two weeks (e.g., at least three weeks, at least four weeks, at least five weeks, at least six weeks, at least seven weeks at least eight weeks at least nine weeks at least ten weeks at least three months at least four months at least five months at least six months, at least seven months, at least eight months, at least nine months, at least one year, at least two years, at least three years, at least four years , at least five years, at least six years, at least seven years, at least eight years, at least nine years, or at least ten years, or during the lifetime of the patient; in particular, at least six weeks).

Prior to administration of rAPF (e.g., SEQ ID NO: 1), a patient (e.g., a childhood-onset, untreated HPP patient, about 18 years of age or older) can be characterized as having a plasma nPF concentration of up to about 6 μM ( for example, about 3.5 µM, about 4 µM, about 4.5 µM, about 5 µM, or about 5.5 µM, or a plasma concentration of NPP ranging from about 3.5 µM to about 6 µM), a plasma P5P concentration of up to 1300 ng/ml (e.g. plasma P5F concentration about 200 ng/ml, 300 ng/ml, 400 ng/ml, 500 ng/ml, 600 ng/ml, 700 ng/ml, 800 ng/ml, 900 ng/ml ml, 1000 ng/ml, 1100 ng/ml, 1200 ng/ml, or 1300 ng/ml, or a plasma P5P concentration in the range of about 200 ng/ml to about 1300 ng/ml) and/or walking distance for six minutes about 350 meters or less (for example, walking distance for six minutes about 50 meters, 75 meters, 100 meters, 125 meters, 150 meters, 175 meters, 200 meters, 225 meters, 250 meters, 275 meters or 350 meters or walking distance within ten hundred minutes within a range of about 50 meters to about 350 meters). After administration of sAP during the treatment period, the decrease in the concentration of nPP in a plasma sample from a patient may be at least about 2 μM (for example, a patient has a concentration of nPP in plasma in the range from about 2 μM to about 5 μM after administration of rAP), a decrease in the concentration of P5P in the patient's sample is at least about 200 ng/mL (for example, the patient has a plasma concentration of P5P in the range from about 2 ng/mL to about 150 ng/mL after administration of rAP), and/or manifests an increase in walking distance within six minutes of at least 100 meters or more (for example, the patient demonstrates a walking distance within six minutes of about 420 meters or more after administration of rAP). The patient may also show reduced dependence on an assistive mobility device (eg, walker, wheelchair, orthodontic braces, crutches, and orthopedic appliances) and/or a reduction in fracture rate (eg, vertebral fracture, subtrochanteric fracture, or metatarsal fracture) after administration of rAPF (SEQ ID NO: 1). Treatment of patients with rAPF may be excluded for patients with, for example, serum calcium levels below the range of a healthy subject (e.g., about 8.5 to about 10.2 mg/dL), serum phosphate levels below the range of a healthy subject ( e.g., about 2.5 to about 4.5 mg/dl), serum vitamin D levels below the range of a healthy subject (e.g., about 20 ng/mL), serum creatinine above the range of a healthy subject (e.g., about 0.6 mg/dl to 1.2 mg/dl) and/or parathyroid hormone levels above the range of a healthy subject (eg, about 10 to about 65 pg/ml).

The second aspect relates to a method of treating HPP in a patient of about 13 years of age or older (e.g., a human), which comprises administering sAP (e.g., SEQ ID NO: 1) to the patient in a treatment regimen providing from about 1 mg/kg/week up to about 9 mg/kg/week, preferably 6 mg/kg/week, rAPF over a treatment period of at least two weeks (e.g., at least three weeks, at least four weeks, at least five weeks at least six weeks at least seven weeks at least eight weeks at least nine weeks at least ten weeks at least three months at least four months, at least five months, at least six months, at least seven months, at least eight months, at least nine months, at least one year, at least two years at least three years, at least four years, at least five years, at least six years, at least seven years, at least eight years, at least nine years, or at least ten years, or during the lifetime of the patient; in particular, at least six weeks). The patient may be one who has not been treated with respect to treatment with rAP. In particular, rAP contains an amino acid sequence having at least 95% sequence identity with the amino acid sequence shown in SEQ ID NO: 1 (eg, asfotase alpha). Administration of rAPF (e.g., SEQ ID NO: 1) results in one or more of the following: (i) a percent (%) change in a patient's predicted HHD score of about 5% or more (e.g., about 5%, 6%, 7 %, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, or 20% or more) relative to the predicted HHD % estimate in a patient prior to treatment with rAP; (ii) a change in the patient's LEFS score of about 3 or more (eg, about 3, 4, 5, 6, 7, 8, 9, or 10 or more) relative to the patient's LEFS score before treatment with rAPF; and (iii) a change in the patient's BPI-SF score of about -2 or more (eg, about -2, about -3, about -4, about -5, about -6, or about -7 or more) relative to the BPI score. -SF in a patient prior to treatment with rAP.

In the first or second aspect, rAPF (e.g., SEQ ID NO: 1) may be administered one or more times per day, week, month, or year (e.g., twice a week, three times a week, four times a week, five times a week, six times a week or seven times a week). In particular, rAP can be administered in multiple doses two days a week, three days a week, four days a week, five days a week, six days a week, or seven days a week. For example, rAPF (e.g., SEQ ID NO: 1) is administered at an initial dose of about 2.1 mg/kg/week to about 3.5 mg/kg/week (e.g., an initial dose of about 0.3 mg/kg/week). day to about 0.5 mg/kg/day rAP) and subsequently increased to a dose of about 6 mg/kg/week or more (for example, 9 mg/kg/week). In particular, the starting dose may be increased after a treatment period of at least six months, at least one year, at least two years, at least three years, or at least four years or more ( for example, at least five years, at least six years, at least seven years, at least eight years, at least nine years, at least ten years, or more than ten years, for example, throughout the life of the patient). In addition, rAPF (e.g., SEQ ID NO: 1) may be administered at a dosage of about 1.3 mg/kg/week, about 2.7 mg/kg/week, or about 6 mg/kg/week or more (e.g., about 9 mg/kg/week), for example, rAPF is administered at a dosage of about 2 mg/kg three times a week, about 3 mg/kg twice a week, about 3 mg/kg three times a week, or about 1 mg/kg six once a week. In addition, rAP can be administered once a day in a row or every other day. In addition, rAPF (e.g., SEQ ID NO: 1) may be administered in an amount that is therapeutically effective for treating at least one symptom of adult HPP (e.g., one or more of elevated blood levels of phosphoethanolamine (PEA) and /or urine, hypomineralization, hypercalciuria, skeletal deformity, waddling gait, bone pain, bone fracture (eg, spinal fracture, subtrochanteric fracture, or metatarsal fracture), calcium pyrophosphate dihydrate crystal deposits, arthritis, pyrophosphate arthropathy, chondrocalcinosis, calcified periarthritis, and pseudo-fracture).

In a first or second aspect, rAPF (eg, SEQ ID NO: 1) is administered in compositions containing at least one pharmaceutically acceptable carrier, diluent, or excipient such as saline or sodium chloride and sodium phosphate. For example, at least one pharmaceutically acceptable carrier, diluent, or excipient contains 150 mM sodium chloride and 25 mM sodium phosphate. In addition, the pharmaceutical composition can be administered subcutaneously, intramuscularly, intravenously, orally, nasally, sublingually, intrathecally, or intradermally. In particular, the pharmaceutical composition is administered subcutaneously.

The third aspect relates to rAPF containing an amino acid sequence having at least 95% sequence identity with the amino acid sequence of SEQ ID NO: 1 (for example, asfotase alfa) for the treatment of hypophosphatasia (HPP) in patients with HPP that began in childhood, not treated, aged about 18 years or older (for example, in humans). In particular, rABP is formulated to be administered to a patient in a treatment regimen providing from about 1 mg/kg/week to about 9 mg/kg/week, preferably 6 mg/kg/week, rABP over a treatment period of at least two weeks. (e.g. at least three weeks, at least four weeks, at least five weeks, at least six weeks, at least seven weeks, at least eight weeks, at least nine weeks, at least ten weeks, at least three months, at least four months, at least five months, at least six months, at least seven months, at least eight months at least nine months, at least one year, at least two years, at least three years, at least four years, at least five years, at least six years at least seven years, at least eight years, at least nine years, or at least ten years or during the life of the patient; in particular, at least six weeks). rAPF (e.g., SEQ ID NO: 1) is characterized in that administration of rAPF to a patient results in one or more of the following: nPF in a plasma sample from an untreated childhood onset HPP subject aged about 18 years or older; (ii) a statistically significant decrease in the concentration of P5P in a patient's plasma sample of at least about 100 ng/ml relative to the concentration of P5P in a plasma sample of a subject with HPP, onset in childhood, untreated at about 18 years of age or older; and (iii) a statistically significant increase in the patient's walking distance of at least 50 meters, as assessed by 6MWT, relative to the walking distance in 6 minutes in the patient who was not administered.

In a third aspect, rAP may be characterized by a decrease in the concentration of nPP in a patient's plasma sample (e.g., an untreated patient with childhood-onset HPP, about 18 years of age or older), a decrease in the concentration of P5P in a patient's plasma sample, and/or an increase in walking distance of the patient during a treatment period of at least two weeks (e.g., at least three weeks, at least four weeks, at least five weeks, at least six weeks, at least seven weeks, at least eight weeks, at least nine weeks, at least ten weeks, at least three months, at least four months, at least five months, at least six months at least seven months, at least eight months, at least nine months, at least one year, at least two years, at least three years, at least four years, for at least five years, about at least six years, at least seven years, at least eight years, at least nine years, or at least ten years, or during the lifetime of the patient; in particular, at least six weeks).

In a third aspect, rAPF (e.g., SEQ ID NO: 1) may be characterized in that, prior to rAPF administration, the patient (e.g., an untreated HPP patient of childhood onset, aged about 18 years or older) has a concentration of nPPF in plasma about 6 µM, plasma P5F concentration up to 1300 ng/ml and/or walking distance for six minutes about 350 meters or less. In particular, sAP is characterized in that the decrease in the concentration of nPP in a patient's plasma sample is at least about 2 μM (for example, the patient has a plasma concentration of nPP from about 2 μM to about 5 μM after administration of rAP), a decrease in the concentration of P5P in a patient sample is at least about 200 ng/mL (e.g., the patient has a plasma P5P concentration of about 2 ng/mL to about 150 ng/mL after administration of sAP) and/or administration of sAP during the treatment period , which is at least one year, leads to an increase in walking distance in six minutes, at least 100 meters or more (for example, the patient shows a walking distance in six minutes, which is about 420 meters or more after administration of rAP). The patient may also show reduced dependence on an assistive mobility device (eg, walker, wheelchair, orthodontic braces, crutches, and orthopedic appliances) and/or a reduction in fracture rate (eg, vertebral fracture, subtrochanteric fracture, or metatarsal fracture) after administration of rAPF (for example, SEQ ID NO:). one).

In a third aspect, rAPF (e.g., SEQ ID NO: 1) can be formulated to be administered one or more times per day, week, month, or year (e.g., twice a week, three times a week, four times a week, five once a week, six times a week, or seven times a week). In particular, rAP can be formulated to be administered in multiple doses two days a week, three days a week, four days a week, five days a week, six days a week, or seven days a week. For example, rAPF (e.g., SEQ ID NO: 1) is formulated to be administered at an initial dose of about 2.1 mg/kg/week to about 3.5 mg/kg/week (e.g., an initial dosage of about 0.3 mg/kg/week). kg/day to about 0.5 mg/kg/day rAP) and subsequently increased to a dose of about 6 mg/kg/week or more (eg, 9 mg/kg/week). In particular, the starting dose may be increased after a treatment period of at least six months, at least one year, at least two years, at least three years, or at least four years or more ( for example, at least five years, at least six years, at least seven years, at least eight years, at least nine years, at least ten years, or more than ten years, for example, throughout the life of the patient). In addition, rAPF (e.g., SEQ ID NO: 1) may be formulated to be administered at a dosage of about 1.3 mg/kg/week, about 2.7 mg/kg/week, or about 6 mg/kg/week or more ( e.g., about 9 mg/kg/week), e.g., rAPF is formulated to be administered at a dosage of about 2 mg/kg three times a week, about 3 mg/kg twice a week, about 3 mg/kg three times a week, or about 1 mg/kg six times a week. In addition, rAP can be prepared for administration once a day in a row or every other day. In addition, rAPF (e.g., SEQ ID NO: 1) can be formulated to be administered in an amount that is therapeutically effective for treating at least one symptom of the adult form of HPP (e.g., one or more of the elevated levels of phosphoethanolamine (PEA) in blood and/or urine, hypomineralization, hypercalciuria, skeletal deformity, waddling gait, bone pain, bone fracture (eg, spinal fracture, subtrochanteric fracture, or metatarsal fracture), calcium pyrophosphate dihydrate crystal deposits, arthritis, pyrophosphate arthropathy, chondrocalcinosis, calcified periarthritis and pseudofracture).

In a third aspect, rAPF (eg, SEQ ID NO: 1) can be formulated for administration in compositions containing at least one pharmaceutically acceptable carrier, diluent, or excipient such as saline or sodium chloride and sodium phosphate. For example, at least one pharmaceutically acceptable carrier, diluent, or excipient contains 150 mM sodium chloride and 25 mM sodium phosphate. In addition, the pharmaceutical composition may be formulated for subcutaneous, intramuscular, intravenous, oral, nasal, sublingual, intrathecal, or intradermal administration. In particular, the pharmaceutical composition is formulated for subcutaneous administration.

In any of the above aspects, a patient (e.g., an untreated childhood HPF patient, about 18 years of age or older) may be a patient who has not been diagnosed based on the presence of symptoms of perinatal, infant, or juvenile HPF, such as rickets, rickety ribs, severe hypomineralization, developmental delay, motor retardation, insufficient weight gain, pulmonary hypoplasia, respiratory failure, respiratory distress, craniosynostosis (including complications of craniosynostosis), and seizures

In any of the above aspects, the patient is a human. A patient (eg, a childhood-onset, untreated HPP patient, aged about 18 years or older) may not have ectopic calcification following rAPF administration (eg, SEQ ID NO: 1). For example, a patient does not show ectopic calcification after rAPF administration for at least one year, at least two years, at least three years, at least four years, at least five years, at least at least six years, at least seven years, at least eight years, at least nine years, or at least ten years or more, for example, throughout the life of the patient. In addition, a patient (e.g., a patient with HPP who is about 13 years of age or older, e.g., an untreated patient with HPP starting in childhood who has not been treated, who is about 18 years of age or older) may be tolerant of rAPF administration (e.g., SEQ ID NO: 1), such as the absence or reduction in the frequency of adverse events selected from the group consisting of erythema at the injection site, decreased hemoglobin, hyperthermia, pneumonia, upper respiratory tract infection, otitis media, vomiting, constipation, diarrhea, tooth loss , nasopharyngitis, rash, dental caries and irritability.

In any of the above aspects, rAPLP (e.g., SEQ ID NO: 1) contains or consists of the amino acid sequence of SEQ ID NO: 1. For example, rAPLP (e.g., SEQ ID NO: 1) is physiologically active against PEA, is competent to improve skeletal mineralization in bones and/or is a soluble extracellular domain of alkaline phosphatase.

In any of the above aspects, the administration of rAPF may result in a statistically significant change in the limit of the mean from baseline concentrations of nPP and/or P5P in a patient's plasma sample. The administration of _sAP can result in an AUC sec from about 2000 U x day/L to about 7000 U x day/L, where AUC _sec is the area under the concentration-time curve from zero to the time of the last observed concentration in the dosing interval. Administration of sAP can result in an AUC _ins from about 4,000 U x day/l to about 10,000 U x day/l, with AUC _ins equal to the area under the concentration-time curve from zero to infinity. The introduction of rAPF can lead to t _1/2 from about 3 days to about 8 days.

Definitions

When used in the context of this document, the use of nouns in the singular means "at least one" or "one or more" unless otherwise indicated. In addition, singular forms include plural forms unless the context clearly dictates otherwise.

When used in the context of this document, the term "about" refers to an amount that is ± 10% of the listed value, and preferably ± 5% of the listed value, or more preferably ± 2% of the listed value.

As used herein, the term "at least" refers to an amount that is ≤10% of the listed value, and preferably ≤5% of the listed value, or more preferably ≤2% of the listed value.

By "asphotase alfa" is meant a human TNAP fusion protein (hTNAP) prepared for the treatment of HPP. Asphotase alfa (STRENSIQ®, Alexion Pharmaceuticals, Inc.) is a fusion protein comprising a soluble glycoprotein of two identical polypeptide chains, wherein each polypeptide chain comprises amino acid residues 1-726 of SEQ ID NO: 1. The structure of each polypeptide chain includes the hTNAP catalytic domain , the human Fc domain of immunoglobulin G ₁ and the deca-aspartate peptide used as a bone targeting domain (structure hTNSAP - Fc-D ₁₀ ). The two polypeptide chains are covalently linked by two disulfide bonds. Asfotase alfa has been approved worldwide under the brand name STRENSIQ®, including in the US, Europe, Japan, Canada, Israel, Australia and Korea.

As used herein, the term "mean" refers to a numerical value expressing the mean or median of a set of data. The average value of the data set is calculated by dividing the sum of the values in the population by their number. The median of a data set is calculated by taking the middle value in a list of odd numbers, or by taking the average of the two data values in the middle in a list of even numbers.

As used herein, the term "bone-targeted component" refers to an amino acid sequence of 1 to 50 amino acid residues in length having sufficient affinity for bone matrix such that the bone-targeted component, in particular, has an in vivo binding affinity for bone matrix. , which is from about 10 ^-6 M to about 10 ^-15 M (for example, 10 ^-7 M, 10 ^-8 M, 10 ^-9 M, 10 ^-10 M, 10 ^-11 M, 10 ^-12 M, 10 ^{- 13} M, 10 ^-14 M or 10 ^-15 M).

The terms "Brief Pain Assessment Questionnaire" and "BPI-SF", used interchangeably herein, refer to a method of measuring the pain of patients, in particular patients with HPF (eg, patients aged about 13 years or older). BPI-SF is the pain self-assessment described in Cleeland & Ryan (Ann Acad Med Singapore, 23(2), 129-138; 1994), incorporated herein in its entirety by reference. The BPI-SF is a questionnaire designed to assess the severity of pain and its impact on daily functioning. The BPI-SF consists of 11 items that use a numeric rating scale to assess pain severity (4 units) and pain intervention (7 units) 24 hours prior to questionnaire submission. The BPI-SF questionnaire provides information on the intensity of pain and the extent to which pain affects the daily functioning of a patient (e.g., a HFF patient aged about 13 years or older) on a numerical scale from 0 (no pain) to 10 (severe pain or significant interference caused by pain); lower scores indicate improved quality of life and reduced pain. For example, the BPI-SF scores for adolescents and adults with HFF are a composite of 11 pain scores.

As used herein, the terms "Bruinks-Ozeretsky Motor Assessment Test 2nd Edition" or "BOT-2" refer to the second edition of a standardized gross and fine motor test in patients, for example, in adults who have HFF, such as in adults, with HFF that began in childhood. See Bruininks, R. H. (2005). Bruininks-Oseretsky Test of Motor Proficiency, (BOT-2). Minneapolis, MN: Pearson Assessment, which is incorporated herein in its entirety by reference. BOT-2 is given individually to assess gross and fine motor skills in a range of patients. BOT-2, for example, can be used to assess physical impairment and mobility restrictions in patients with HFF (eg, adults with HFF, such as adults with HFF onset in childhood). BOT-2 provides BOT-2 composite scores in the following sample areas: strength, running speed and agility, fine motor skill, fine motor integrity, manual dexterity, bilateral coordination, balance and upper limb coordination. For example, an overall BOT-2 strength score can be determined if the patient performs squats, straight leg raises while standing, standing long jumps, supported squats, and push-ups. An overall assessment of running speed and agility can be determined by having the patient jump over a balance beam or perform a shuttle run, a two-leg side jump, or a one-leg side jump. Both BOT-2 total strength and BOT-2 total running speed and total agility scores range from 0 to 25, with a score of 10 to 25 being considered representative of healthy subjects. Normative indicators of strength, running speed and maneuverability are 15±5. Adult grades are not normalized (do not use a rating scale) and therefore higher values represent better performance. Either the means or the median of the estimates can be used, with the median of the estimates being preferred for smaller sample sizes or smaller datasets.

As used herein, the term "catalytically competent" refers to sAP that hydrolyzes the bone mineralization inhibitor inorganic pyrophosphate (IPP) to form inorganic phosphate (P), thereby reducing extracellular concentrations of NP. Thus, catalytically competent rAP improves skeletal mineralization in bone by regulating the concentration of nPP.

By "extracellular domain" is meant any functional extracellular portion of a native protein, such as alkaline phosphatase. In particular, the signal peptide is absent in the extracellular domain.

By "Fc" is meant a fragment of a crystallizable region of an immunoglobulin, eg IgG-1, IgG-2, IgG-3, IgG-3 or IgG-4, including the immunoglobulin heavy chain CH2 and CH3 domains. The Fc may also include any portion of the hinge region connecting the Fab and Fc regions. The Fc may be any mammal, including humans, and may be post-translationally modified (eg, by glycosylation). In a non-limiting example, the Fc may be a fragment of the human IgG-1 crystallizable region having the amino acid sequence of SEQ ID NO: 20.

By "fragment" is meant a portion of a polypeptide or nucleic acid molecule which preferably contains at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 75%, 80%, 85% , 90%, 95%, 96%, 97%, 98%, 99% or more of the entire length of the reference nucleic acid molecule or polypeptide. The fragment may contain, for example, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 110, 120, 130 . 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 260, 270, 280, 290, 300, 400, 500, 600, 700 or more amino acid residues up to full length polypeptide. Typical rAP fragments have amino acid residues 18-498, 18-499, 18-500, 18-501, 18-502, 18-503, 18-504, 18-505, 18-506, 18-507, 18-508 18 -509, 18-510, 18-511, or 18-512 ALP (eg, SEQ ID NOs: 2-6) and may include additional C-terminal and/or N-terminal portions.

The terms "Handheld Dynamometry" and "HHD", used interchangeably herein, refer to a method for measuring grip strength and muscle strength in subjects, particularly HFF patients aged about 13 years or older. A dynamometer can be used to assess grip strength, knee flexion, knee extension, hip flexion, hip extension, and hip abduction in a subject with HFF. For example, knee flexion and extension, and hip lateral flexion, extension, and abduction in a subject with a HFF age of about 13 years or older can be measured using, for example, a MICROFET2TM Dynamometer, while the subject's grip strength can be measured using for example the JAMAR® Hand Dynamometer. Specifically, the administrator holds the dynamometer stationary while the subject applies maximum force to the dynamometer. Peak force data is collected in pounds and then converted to Newtons (N, N). Torque values are then calculated using limb length in Newton meters. The torque value can then be compared with that of, for example, a normal subject of about the same age, same sex, and/or same height, and expressed as a percentage to obtain an estimate of the subject's HHD.

The terms "hypophosphatasia" or "HPP" as used herein refer to a rare inherited skeletal disorder caused, for example, by one or more loss-of-function mutations in the ALPL (alkaline phosphatase, liver/bone/kidney) gene, which codes for a non-specific tissue alkaline phosphatase (TNSP). HPP can be further characterized as infantile HPP, childhood-onset HPF, perinatal HPF (e.g., benign perinatal HPF or lethal perinatal HPF), HPF that affects the teeth, juvenile HPF, or adult HPF. For example, "adult HPP" describes a patient with HPP who is 18 years of age or older. "Adolescent HFF" describes a patient with HFF between about 13 and about 17 years of age. The term "adult HPP" as used herein refers to a condition or phenotype characterized by the presence of one or more of the following symptoms: elevated blood and/or urine levels of inorganic pyrophosphate (nPP), phosphoethanolamine (PEA), or pyridoxal-5 ''-phosphate (P5P), hypomineralization, hypercalciuria, skeletal deformity, waddling gait, bone pain, bone fracture (eg, vertebral fracture, subtrochanteric fracture, or metatarsal fracture), calcium pyrophosphate dihydrate crystal deposits, arthritis, pyrophosphate arthropathy, chondrocalcinosis , calcified periarthritis and pseudofracture). The term "adolescent HPP" as used herein refers to a condition or phenotype characterized by the presence of one or more of the following: elevated levels of nPP, PEA, or P5P in the blood or urine, osteomalacia, one or more skeletal deformities, hypotonia, muscle weakness, rheumatoid complications, arthritis, pseudogout, waddling gait, difficulty walking, bone pain, pain, early tooth loss, hypomineralization, pulmonary hypoplasia, respiratory failure, seizures, hypercalciuria, short stature and growth retardation.

The terms "functional scale of the lower limb" and "LEFS", used interchangeably herein, refer to a method for measuring the dysfunction of the lower limbs of patients, in particular patients with HFF (for example, patients aged about 13 years or older). LEFS is a self-assessment scale described in Binkley et al. (Phys Ther. 79:371-83, 1999), incorporated herein by reference in its entirety. Total LEFS scores range from 0 to 80, with higher scores indicating better lower limb function. A change in LEFS score of 9 points is considered a clinically significant change. A licensed physical therapist may assign LEFS assessments to patients with HFF (eg, patients with HFF aged about 13 years or older) in an interview format. Higher LEFS scores indicate improved lower limb function, including transitional movements (eg, getting out of the bath or turning over in bed), locomotion (eg, walking or running on uneven ground), climbing stairs, and crouching. LEFS can be used to assess the functional impairment of one or both lower limbs of a patient with HPF, including the ability to follow the patient over time and assess the effectiveness of treatment with asphotase alfa.

By "untreated patient" or "untreated subject" is meant a patient or subject having childhood-onset HPP who has not previously been treated with an alkaline phosphatase or a polypeptide having alkaline phosphatase activity, such as sAP (e.g., TNAP, e.g., an rAPF polypeptide of SEQ ID NO: 1, or a polypeptide variant having at least 95% sequence identity with the sequence of SEQ ID NO: 1, e.g., asphotase alfa).

The terms "childhood-onset form of hypophosphatasia" and "childhood-onset HPF" refer to the onset of HPF symptoms in a subject before the age of 18 years.

The terms "polypeptide" and "protein" are used interchangeably and refer to any chain of two or more naturally occurring or non-naturally occurring amino acid residues, regardless of post-translational modification (e.g., glycosylation or phosphorylation), constituting all or part of the naturally occurring or non-naturally occurring the nature of the polypeptide or peptide as described herein.

By "pharmaceutically acceptable carrier, diluent, or excipient" is meant a carrier, diluent, or excipient, respectively, that is physiologically acceptable to the subject (eg, human) while maintaining the therapeutic properties of the pharmaceutical composition with which they are administered. One exemplary pharmaceutically acceptable carrier, diluent, or excipient is saline. For example, a pharmaceutically acceptable carrier, diluent, or excipient may contain sodium chloride (eg, 150 mM sodium chloride) and sodium phosphate (eg, 25 mM sodium phosphate). Other physiologically acceptable carriers, diluents or excipients and their preparations are known to the person skilled in the art.

By "pharmaceutical composition" is meant a composition containing a polypeptide (eg, compositions containing sAP, such as asphotase alfa) as described herein, formulated with at least one pharmaceutically acceptable carrier, diluent, or excipient. The pharmaceutical composition may be manufactured or sold with the approval of a government regulatory body as part of a therapeutic regimen for the treatment or prevention of a disease or complication in a patient. Pharmaceutical compositions can be prepared, for example, for subcutaneous administration, intravenous administration (for example, in the form of a sterile solution containing no particles capable of causing blockage of blood vessels and in a system of solvents suitable for intravenous use), for oral administration (for example, tablets, capsules, caplets, gelatin capsules or syrup), or any other preparation described herein, for example, in unit dosage form.

As used herein, the term "physical impairment" refers to a physiological condition such as bone fragility and muscle weakness associated with HPP that may limit or result in loss of, for example, the ability to walk, functional endurance, and the ability to perform basic self-care activities (ADL). ) in a patient (eg, an adult with HPP, such as an adult with HPP onset in childhood). In particular, physical impairments may limit or result in the loss of a patient's ability to perform ADLs, which are routine activities that healthy subjects perform daily without requiring assistance, such as functional mobility or movement (eg, walking), bathing and showering, dressing, independent eating, personal hygiene and care. As described herein, therapeutic compositions (e.g., compositions comprising rAPF, such as asfotase alfa) can be administered to a patient (e.g., an adult with HPP, e.g., an adult with childhood-onset HPP) to reduce the degree the severity and/or frequency of physical impairments associated with the HFF phenotype and/or to increase the patient's ability to walk (eg, the ability to walk, as measured by distance walked by the patient within six minutes).

As used herein, the term "physiologically active" refers to rAP (e.g., SEQ ID NO: 1) that hydrolyzes phosphoethanolamine (PEA), inorganic pyrophosphate (nPP), and pyridoxal 5'-phosphate (P5P) to form P, thereby reducing extracellular concentrations of PEA, NPF and P5P.

The terms "rCF", "soluble alkaline phosphatase", and "extracellular domain of alkaline phosphatase" are used interchangeably and refer to a soluble, non-membrane-bound alkaline phosphatase or domain, a biologically active fragment, or a biologically active variant thereof. rAPs include, for example, an alkaline phosphatase lacking a C-terminal glycolipid anchor (GPI signal sequence, e.g., polypeptides comprising or consisting of amino acid residues 18-502 of human TNAP (SEQ ID NO: 2, 3, 4, 5, or 6) ). In particular, TNAP may include, for example, a polypeptide comprising or consisting of amino acid residues 1-485 of SEQ ID NO: 1, such as asphotase alpha or a polypeptide variant having at least 95% sequence identity with amino acid residues 1-485 of SEQ ID NO: 1. In addition, rAPF includes, for example, human TNAP orthologues such as rhesus TNAP (SEQ ID NO: 7), rat TNAP (SEQ ID NO: 8), dog TNAP (SEQ ID NO: 9) , porcine TNAP (SEQ ID NO: 10), mouse TNAP (SEQ ID NO: 11), bovine TNAP (SEQ ID NO: 12-14) or feline TNAP (SEQ ID NO: 15). rAPF also includes soluble, non-membrane-bound forms of human PAP (e.g., polypeptides comprising or consisting of amino acid residues 18-502 of SEQ ID NO: 16 or 17), ALP (e.g., polypeptides comprising or consisting of amino acid residues 18-502 SEQ ID NO: 18), APLP (e.g., polypeptides comprising or consisting of amino acid residues 18-502 of SEQ ID NO: 19), and additional variants and analogs thereof that retain alkaline phosphatase activity, such as the ability to hydrolyze nPP. In particular, rAP lacks an N-terminal signal peptide (eg AA 1-17 SEQ ID NO: 2-6, 8, 11-13 or 15 or AA 1-25 SEQ ID NO: 7).

By "polypeptide sAP" is meant a polypeptide having the structure A-sAP-B, wherein sAP is as defined herein and each of A and B is absent or is an amino acid sequence of at least one amino acid (e.g., any fused rAPF polypeptide as described herein (e.g., an rAPF fusion polypeptide of SEQ ID NO: 1 or a polypeptide variant having at least 95% sequence identity with the sequence of SEQ ID NO: 1, eg, asphotase alfa).

By "signal peptide" is meant a short peptide (5-30 amino acids long) at the N-terminus of a polypeptide that directs the polypeptide to a secretory pathway (eg, to the extracellular space). The signal peptide is usually cleaved during secretion of the polypeptide. The signal sequence may direct the polypeptide to an intracellular compartment or organelle, such as the Golgi apparatus. The signal sequence can be identified by homology or biological activity in comparison with a peptide with a known function of targeting a specific region of the cell by the polypeptide. One skilled in the art can identify the signal peptide using readily available software (e.g., Sequence Analysis Software Package of the Genetics Computer Group, University of Wisconsin Biotechnology Center, 1710 University Avenue, Madison, Wis. 53705, BLAST, or PILEUP/PRETTYBOX programs) . The signal peptide may be one which, for example, is substantially identical to amino acid residues 1-17 of SEQ ID NO: 2-6 or amino acid residues 1-25 of SEQ ID NO: 7.

As used herein, when a polypeptide or nucleic acid sequence is referred to as having "at least X% sequence identity" with a control sequence, where "X" is a real number, this means that at least X percent of the residues amino acids or nucleotides in a polypeptide or nucleic acid are identical to those in a control sequence when the sequences are optimally aligned. Optimal sequence alignment can be determined by various methods known to those skilled in 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 are available using publicly available computer software such as "Best Fit" (Smith and Waterman, Advances in Applied Mathematics, 482-489, 1981), which is included in GeneMatcher Plus (Schwarz and Dayhoff, Atlas of Protein Sequence and Structure, Dayhoff, M.O., Ed. Pp. 353-358, 1979), BLAST, BLAST-2, BLAST-P, BLAST-N, BLAST-X, WU-BLAST-2, ALIGN, ALIGN-2, CLUSTAL, Megalign (DNASTAR), or other alignment software/hardware. In addition, those skilled in the art can determine suitable parameters for measuring alignment, including any algorithms necessary to achieve optimal length alignment of the compared sequences.

The terms "patient" or "subject" refer to a mammal, including, but not limited to, a human or non-human mammal such as a bull, horse, dog, sheep, or cat. Of particular interest are human patients.

"Parental administration", "parenteral administration" and other grammatically equivalent phrases used herein refer to routes of administration other than enteral and topical administration, usually by injection, and include, without limitation, subcutaneous, intradermal, intravenous, intranasal, intraocular, pulmonary, intramuscular, intraarterial, intrathecal, intracapsular, intraorbital, intracardiac, intrapulmonary, intraperitoneal, transtracheal, subcuticular, intraarticular, subcapsular, subarachnoid, intraspinal, epidural, intracerebral, intracranial, intracarotid and intrasternal injection and infusion.

As used herein, the terms "6 Minute Walk Test" and "6MWT" refer to a standardized test for assessing a patient's ability to walk (e.g., an adult with HFF, such as an adult with childhood-onset HFF), specifically the ability to patient to raise and lower each leg in turn. See the American Thoracic Society statement: recommendations for the six-minute walk test (American Journal of Respiratory and Critical Care Medicine, 166(1):111-7, 2002), incorporated herein in its entirety by reference. 6MWT is defined as the distance (eg, meters) a patient walks on a flat, hard surface for six minutes. The 6MWT distance can then be compared to the patient's 6MWT distance at baseline, the 6MWT distance of an untreated subject (e.g., an untreated subject of approximately the same age, height, and/or sex), or the healthy subject's 6MWT distance (e.g., a healthy subject of about the same age, height and/or sex) and expressed as a percentage to determine the 6MWT value.

By "therapeutically effective amount" is meant an amount of a polypeptide (e.g., rAPF, such as SEQ ID NO: 1) or nucleic acid molecule described herein that is sufficient to substantially improve, treat, prevent, delay, suppress, or terminate at least , a single symptom of HPP in adults (eg, elevated blood and/or urine levels of nPF, P5F, and PEA, hypomineralization, hypercalciuria, skeletal deformity, waggling gait, bone pain, bone fracture, calcium pyrophosphate dihydrate crystal deposits, arthritis, pyrophosphate arthropathy , chondrocalcinosis, calcifying periarthritis and pseudofracture). A therapeutically effective amount of the composition described herein may depend on the severity of the disorder being treated, as well as on the pathological condition, weight and general condition of the patient, and can be determined by a person skilled in the art taking into account such factors. A 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.

By "exposing", "treatment" or "therapy" is meant the medical management of a patient with the intent to cure, improve, stabilize, reduce the likelihood of, or prevent the adult form of HPP and/or treat a patient exhibiting or likely to have the adult form of HPP, for example, by administering pharmaceutical composition (eg, rAP, such as SEQ ID NO: 1). This term includes active treatment, i.e. treatment directed specifically at or associated with the treatment of a disease, condition, disorder or complication, and also includes etiotropic therapy, i.e. therapy aimed at eliminating the cause of an underlying disease, condition, disorder or complication. . In addition, this term includes palliative care, that is, treatment intended to alleviate or improve at least one symptom, and not to cure a disease, pathological condition, disorder or complication; symptomatic treatment, i.e. treatment aimed at constitutional symptoms associated with a disease, pathological condition, disorder or complication; prophylactic treatment, i.e. treatment aimed at minimizing or partially or completely inhibiting the development of a concomitant disease, condition, disorder or complication, for example, in a patient who is not yet ill but who is susceptible to, or otherwise threatened by, a particular disease , pathological condition, disorder or complication; and maintenance treatment, that is, treatment used to supplement other specific therapy aimed at improving a concomitant disease, pathological condition, disorder or complication.

Other features and advantages of the present disclosure will become apparent from the following detailed description, drawings and claims.

BRIEF DESCRIPTION OF GRAPHICS

On FIG. 1A-1B are graphs showing changes in the median concentrations of inorganic pyrophosphate (IPP; Fig. 1A) and pyridoxal-5'-phosphate (P5P; Fig. 1B) in plasma samples from adult patients with a form of hypophosphatasia (HPP) that began in childhood. , compared with baseline up to six months of treatment with asphotase alfa. Data are presented as a median (minimum, maximum). ^a The P-value compares the pooled alpha-asphotase group (adult patients with HPP receiving doses of 0.3/kg/day and 0.5 mg/kg/day of alpha-asphotase) with an untreated control group. Shows the number (n) of treated patients and untreated control subjects, estimated by the concentration of nPF and P5P in plasma.

Fig. 2 is a graph showing the mean concentration of NPF in plasma samples from adult patients with childhood-onset HPP treated with asfotase alfa over a 6 year treatment period. The upper limit of the norm and the lower limit of the normal concentration of nPF for healthy subjects over 18 years of age are shown. The control group started treatment with asphotase alfa six months after the combination treatment group. A treatment period is shown during which the dosage of alpha-asphotase was increased to 6 mg/kg/week by changing the protocol. ^a P < 0.0001 for change in median plasma nPF concentration from baseline at six months.

Fig. 3 is a graph showing the median concentration of P5F in plasma samples from adult patients with childhood-onset HPP treated with asphotase alfa for 6 treatments. The upper limit of normal and the lower limit of normal P5P concentrations are shown for healthy subjects over 18 years of age. The control group began treatment with asfotase alfa six months after the combination treatment group. A treatment period is shown during which alpha-asfotase was increased to a dosage of 6 mg/kg/week by changing the protocol. ^a P < 0.0001 for change in median plasma nPF concentration from baseline at six months.

Fig. 4 is a graph depicting the predicted percentage median distance of the Six Minute Walk Test (6MWT) of adult patients with childhood-onset HPP treated with alpha-asphotase over a 6 year treatment period. Median, minimum, maximum, and n values are shown for each time interval (baseline, 6 months, 1 year, 2 years, 3 years, 4 years, 5 years, and 6 years). The gray area represents the normal 6MWT distance range for healthy subjects of the same age and height. The control group started treatment with asphotase alfa six months after the combination treatment group. A treatment period is shown during which the dosage of alpha-asphotase was increased to 6 mg/kg/week by changing the protocol. Patients with ^a HFF treated with 0.3 mg/kg/day and 0.5 mg/kg/day asfotase alfa prior to the protocol change to 6 mg/kg/week were pooled throughout the treatment period.

On FIG. 5 is a graph showing the median scores of running speed and agility test according to the Bruincks-Ozeretsky Motor Ability Test, 2nd edition (BOT-2) for adult patients with childhood-onset HPP who were administered asphotase alfa over a period of treatment for 6 years. Median, minimum, maximum, and n values are shown for each time interval (baseline, 6 months, 1 year, 2 years, 3 years, 4 years, 5 years, and 6 years). The control group began treatment with asfotase alfa six months after the combination treatment group. Initially, HPF patients treated with 0.3 mg/kg/day and 0.5 mg/kg/day of asphotase alfa prior to the protocol change to 6 mg/kg/week were pooled throughout the treatment period.

On FIG. 6 is a graph showing the median BOT-2 potency scores for adult patients with childhood-onset HPF treated with asfotase alfa over a 6-year treatment period. Median, minimum, maximum, and n values are shown for each time interval (baseline, 6 months, 1 year, 2 years, 3 years, 4 years, 5 years, and 6 years). The control group began treatment with asfotase alfa six months after the combination treatment group. Initially, HPF patients treated with 0.3 mg/kg/day and 0.5 mg/kg/day of asphotase alfa prior to the protocol change to 6 mg/kg/week were pooled throughout the treatment period.

On FIG. Figure 7 is a diagram showing a study design for the administration of asphotase alfa to adolescents and adults with HPP over a five-year period, including an initial phase of asphotase alfa treatment (from the start time point) to six months and an extension phase of asphotase alfa treatment (from six months treatment up to five or more years of treatment). ^a Asfotase alfa dose adjusted every 3 months as weight changes; the maximum dose was 80 mg asfotase alfa unless, after consultation with a medical supervisor, the investigator approved a dose greater than 80 mg. ^b Dose of asfotase alfa was increased by protocol change in all patients. During the prolongation phase, all patients initially received daily doses of asfotase alfa 0.5 mg/kg/week for about 6 months to 1 year, which was subsequently changed to 1 mg/kg for 6 days/week.

On FIG. Figure 8 is a diagram showing the number of adolescent and adult patients with HPF at different stages of the study (screening, randomization, primary treatment period from the initial time point to six months of asphotase alfa treatment, the extension period of the open study from six months to one year of asfotase treatment, and the period , during which the dose of asfotase alfa was increased to 1 mg/kg/day, was administered 6 days/week until the fifth year of the study).

On FIG. 9A-B are graphs showing changes in median concentrations of pyridoxal-5'-phosphate (P5P; Fig. 9A) and inorganic pyrophosphate (NPP; Fig. 9B) in plasma samples from adolescent and adult HPP patients from baseline to six months of treatment. asphotase alpha. Data are presented as a median (minimum, maximum). ^a The P-value compares the pooled alpha-asphotase group (adults and adolescents with HPP receiving doses of 0.3/kg/day and 0.5 mg/kg/day of alpha-asphotase) with an untreated control group. Shows the number (n) of treated patients and untreated control subjects, estimated by the concentration of nPF and P5P in plasma.

On FIG. 10A-10B are graphs showing the median concentrations of P5P (FIG. 10A) and nPP (P and 10B) in plasma samples from adolescent and adult HPP patients treated with asfotase alfa over a five-year treatment period. Also shown are the upper limit of the norm and the lower limit of the normal concentration of nPF and P5F in healthy subjects. The control group began treatment with asfotase alfa six months after the combination treatment group. A treatment period is shown during which the dosage of asfotase alfa was increased to 6 mg/kg/week by changing the protocol.

On FIG. 11A-11B are graphs showing median 6MWT walking distance (FIG. 11A) and percentage predicted median 6MWT test distance (FIG. 11B) of adolescent and adult HPF patients treated with asfotase alfa over a 5-year treatment period. . Median, minimum, maximum, and n values are shown for each time interval (baseline, 6 months, 1 year, 2 years, 3 years, 4 years, and 5 years). The gray area represents the normal 6MWT distance range for healthy subjects of the same age and height. The control group began treatment with asfotase alfa six months after the combination treatment group. A treatment period is shown during which the dosage of asfotase alfa was increased to 6 mg/kg/week by changing the protocol.

On FIG. 12A-12B are graphs showing the correlation between distance traveled during 6MWT at screening and baseline time in adolescents with HPF (FIG. 12A) and adults with childhood-onset HPP (FIG. 12B).

On FIG. 13 is a graph showing the change in median total BOT-2 scores versus baseline time during BOT-2 running speed and agility tests in adolescent and adult HPP patients administered asphotase alfa over a 5 year treatment period. Median, minimum, maximum, and n values are shown for each time interval (baseline, 6 months, 1 year, 2 years, 3 years, 4 years, and 5 years). The control group began treatment with asfotase alfa six months after the combination treatment group. Initially, HPF patients treated with 0.3 mg/kg/day and 0.5 mg/kg/day of asphotase alfa prior to the protocol change to 6 mg/kg/week were pooled throughout the treatment period.

On FIG. 14 is a schematic diagram showing the dosing regimen of patients receiving asphotase alfa at various concentrations during phase 2a clinical trials.

On FIG. 15 is a graph showing the significant differences observed between least squares means (LSM) in NPF changes from baseline to pre-dose time 3 Week 9 during phase 2a clinical trials.

On FIG. 16 is a graph showing the significant differences observed between changes in LSM in P5F from baseline to pre-dose time 3 Week 9 during phase 2a clinical trials.

On FIG. 17A-17B are schematic diagrams showing the drug regimen of each dosage cohort (FIG. 17A) and a plot of pharmacokinetic, pharmacodynamic, and immunogenic evaluations (FIG. 17B).

On FIG. 18A-18B are graphs showing individual PK profiles of asphotase alfa activity (U/L) in each dosage cohort (0.5 mg/kg 3×/week, 2 mg/kg 3×/week, and 2 mg/kg 3× /week) every day after the initial dose on day 1 (Fig. 18A) and after multiple doses of the drug on day 61 (Fig. 18B).

On FIG. 19A-19C are graphs showing the mean±SD of asphotase alfa PK activity profiles (U/L) in each dosing cohort each day after the initial dose on day 1 (FIG. 20A), following multiple dosing between days 15-61 ( Fig. 19B) and after multiple dosing on day 61 (Fig. 19C).

On FIG. 20A-20B are graphs showing mean ± SD, dose-normalized asphotase alfa activity (U/L) in each dosing cohort each day after the initial dose on day 1 (FIG. 20A) and after multiple dosing on day 61 (FIG. 20B).

On FIG. 21 is a graph showing the difference in Day 61 scores from baseline (CBL) NPF as a function of Cav _{, cc} exposure quartiles of asphotase alpha. Median CBL nPF values are indicated by a line in the center of the rectangles. Rectangles mark the interquartile range (IQR). Confidence intervals correspond to 1.5 * IQR. Outliers are marked outside the confidence intervals with filled circles.

DETAILED DESCRIPTION OF THE INVENTION

We have found that asfotase alfa (SEQ ID NO: 1, STRENSIQ®, Alexion Pharmaceuticals, Inc.) can be effectively used to treat hypophosphatasia (HPP), its symptoms, and associated physical disorders in adults with HPP (eg, in adults with GPP who are about 18 years of age or older, such as adults who have HPF that started in childhood), or adolescents who have HPF (eg, adolescents who have HPF, aged about 13 to about 17 years) over an extended period of time (for example, at least one year, at least two years, at least three years, at least four years, at least five years, at least six years, at least seven years, at least eight years, at least nine years, at least ten years, or more than ten years, for example, throughout the life of the patient). In particular, asphotase alfa (SEQ ID NO: 1) can be administered to treat adults or adolescents with HPP who exhibit physical impairment (eg, bone or muscle weakness), decreased ability to walk compared to a healthy subject (eg, subject without HPP of approximately the same age, sex, and/or height), a decrease in alkaline phosphatase (AP) in a sample (e.g., plasma sample) of a relatively healthy subject (e.g., a subject without GPP of approximately the same age, sex, and/or height ), decreased grasping and muscle strength relative to a healthy subject (e.g., a HPP-free subject of approximately the same age, sex, and/or height), decreased functional ability in the lower limbs compared to a healthy subject (e.g., a HPP-free subject of approximately the same age, same sex, and/or height) or increased pain compared to a healthy subject (eg, a non-HPP subject of approximately the same age, same sex, and/or height). In addition, an adult or adolescent having HPP may be an untreated patient who has not previously been treated with asphotase alfa (SEQ ID NO: 1).

Described are methods of administering asfotase alfa (SEQ ID NO: 1) to an adult with HPP (e.g., an adult with HPP about 18 years of age or older, such as an adult with HPP that started in childhood) or an adolescent with HPP ( for example, adolescents with HPP between the ages of about 13 and about 17 years old) who show elevated concentrations of inorganic pyrophosphate (nPP) or pyridoxal-5'-phosphate (P5P) in a plasma sample from an adult or adolescent with HPP, relative to the concentrations of nPP or P5P in a plasma sample from an untreated adult or adolescent with HPP. Prior to administration of sAP, a patient with HPP may be characterized as having a plasma concentration of NPF up to about 6 μM and/or a P5P concentration of up to 1300 ng/mL. Also disclosed are methods of administering asphotase alfa (SEQ ID NO: 1) to an adult with HPP (e.g., an adult with HPP onset in childhood) or an adolescent with HPP (e.g., adolescents with HPP aged from about 13 years to about 17 years), resulting in improved walking ability in an adult with HFF. For example, asphotase alfa (SEQ ID NO: 1) can be administered to an adult or adolescent with HPP with reduced walking ability such that the patient is characterized as having a six minute walking distance of about 350 meters or less prior to administration of sAP. For example, in an adult with HPP, after administration of rAP, there is reduced dependence on an assistive mobility device such as a walker, wheelchair, orthopedic braces, crutches, and orthopedic devices.

Also described are methods of administering asfotase alfa (SEQ ID NO: 1) to an adult with HPF (e.g., an adult with childhood onset HPF) or an adolescent with HPF (e.g., adolescents with HPF aged from about 13 years to about 17 years), having scores on the Bruinks-Ozeretsky 2nd Edition (BOT-2) General Running Speed and Agility Test or Strength Test indicating physical impairment (e.g., a BOT-2 score of less than about 7 on one or more BOT-2 scores) regarding strength, running speed and agility, fine motor accuracy, fine motor integrity, manual dexterity, bilateral coordination, balance and upper limb coordination). For example, asphotase alfa (SEQ ID NO: 1) may be administered to an adult having HPP with BOT-2 running speed and agility scores of less than about 6. In addition, asphotase alfa (SEQ ID NO: 1) may be administered to an adult having GPP , with a BOT-2 strength score of less than about 12.

Also described are methods of administering asfotase alfa (SEQ ID NO: 1) to an adult with HPP (e.g., an adult with HPP onset in childhood) or an adolescent with HPP (e.g., adolescents with HPP aged about 13 to about 17 years) with a Handheld Dynamometer (HHD) score indicating decreased grasping and muscle strength (e.g., an HHD score of less than about 50, such as about 10, 20, 30, 40, or 50), a Lower Limb Function Scale (LEFS) score, indicative of reduced functional ability in the lower extremities (eg, an LEFS score of less than about 40, such as about 10, 20, 30, or 40), and/or a Brief Pain Rating Questionnaire (BPI-SF) score indicating the presence of pain (eg , a score of more than about 10, such as about 10, about 15, about 20, about 25, or about 30 or more).

In any of these ways, asphotase alfa (SEQ ID NO: 1) can be administered to an adult with HPP (e.g., an adult with childhood-onset HPP) or an adolescent with HPP over an extended period of time, such as at least one year, at least two years, at least three years, at least four years, at least five years, at least six years, at least seven years, at least eight years, at least nine years, at least ten years, or more than ten years, for example, throughout the life of the patient). In addition, given the results described herein using asphotase alfa, other rAPF (such as a polypeptide variant having at least 95% sequence identity with the sequence of SEQ ID NO: 1) can be used to treat an adult human with HPP ( e.g., an adult with childhood-onset HFF) for an extended period of time, e.g., at least one year, at least two years, at least three years, at least four years, at least five years, at least six years, at least seven years, at least eight years, at least nine years, at least ten years, or more than ten years, for example, within throughout the patient's life).

Methods of Treatment

This document provides methods for treating an adult with HPP (e.g., an adult with HPP who is about 18 years of age or older, such as an adult with HPP that started in childhood) or an adolescent with HPP (for example, adolescent with HFF, aged about 13 to about 17 years). Adults with HPP (eg, an adult with HPP that started in childhood) or adolescents with HPP (eg, adolescents with HPP between about 13 and about 17 years of age) may be treated with rAPF (such as TNAP, e.g., an rAPF fusion polypeptide, such as an rAPF fusion polypeptide of SEQ ID NO: 1, or a polypeptide variant having at least 95% sequence identity with SEQ ID NO: 1, e.g., asphotase alfa) range of ages, for example, from about 18 to about 20 years, from about 20 to about 25 years, from about 25 to about 30 years, from about 30 to about 35 years, from about 35 to about 40 years, from about 40 to about 45 years old, about 45 to about 50 years old, about 50 to about 55 years old, about 60 to about 65 years old, about 20 to about 30 years old, about 30 to about 40 years old, about 40 to about 50 years old , about 50 to about 60 years old, about 60 to about 70 years old, about 20 to about 65 years old, about 30 to about 65 years old t or older than 65 years.

Adults (for example, an adult with HFF who is about 18 years of age or older, such as an adult with HFF that started in childhood) or adolescents (for example, an adolescent with HFF who is between about 13 years of age and about 17 years of age) , can be diagnosed as having HPP prior to administration of sAP (such as TNAP, e.g., an rAPF fusion polypeptide such as an rAPF fusion polypeptide of SEQ ID NO: 1 or a polypeptide variant having at least 95% sequence identity with the sequence of SEQ ID NO: 1, for example, asfotase alfa). An adult with HPP (eg, an adult with HPP onset in childhood) or an adolescent with HPP may exhibit, for example, physical and walking impairments compared to an adult without HPP.

In addition, an adult with HFF (for example, an adult with HFF who is about 18 years of age or older, such as an adult with HFF that started in childhood) or an adolescent with HFF (for example, an adolescent with HFF who is aged about 13 years to about 17 years of age) may be an untreated patient who has not previously been treated with rAPF (such as TNAP, e.g., an rAPF fusion polypeptide, such as an rAPF fusion polypeptide of SEQ ID NO: 1 or a polypeptide variant , having at least 95% sequence identity with the sequence of SEQ ID NO: 1, for example, asfotase alpha). The method comprises administering rAPLP (such as TNAP, e.g., an rAPF fusion polypeptide, such as an rAPF fusion polypeptide of SEQ ID NO: 1 or a polypeptide variant having at least 95% sequence identity with the sequence of SEQ ID NO: 1, e.g., asphotase alfa ) an adult with HPP or an adolescent with HPP, e.g., administration of rAPF over a treatment period of at least one year (e.g., at least two years, at least three years, at least four years at least five years, at least six years, at least seven years, at least eight years, at least nine years, at least ten years, or more than ten years, for example, throughout the life of the patient).

In particular, rAPF, such as asfotase alfa, can be administered during a treatment period to an adult with HPP (e.g., an adult with HPP who is about 18 years of age or older, such as an adult with HPP that started in childhood) or an adolescent with HPP (e.g., an adolescent with HPP, aged about 13 years to about 17 years) who previously had a plasma NPF concentration of up to about 6 µM (eg, about 3.5 µM, about 4 µM, about 4.5 µM, about 5 µM or about 5.5 µM), plasma P5F concentration up to 1300 ng/ml (for example, plasma P5F concentration about 200 ng/ml, 300 ng/ml, 400 ng/ml, 500 ng/ml, 600ng/ml, 700ng/ml, 800ng/ml, 900ng/ml, 1000ng/ml, 1100ng/ml, 1200ng/ml or 1300ng/ml), walking distance for six minutes about 350 meters or less (for example, the walking distance for six minutes is about 50 meters, 75 meters, 100 meters, 125 meters, 150 meters, 175 meters, 200 meters, 225 meters, 250 meters, 275 meters or 350 meters), speed indicators ha and dexterity BOT-2 overall score less than about 6.0, BOT-2 overall strength score less than about 12, HHD score less than about 80% of predicted HHD score (e.g., about 30% , about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, or about 75% of the predicted HHD score), an LEFS score less than about 40 (e.g., an LEFS score of less than about 10, about 20, about 30, or about 40) and/or a BPI-SF score of less than about 12 (e.g., a BPI-SF score of less than about 0, about 1, about 2, about 3, about 4 , about 5, about 6, about 7, about 8, about 9 or about 10). In addition, plasma nPF concentration, plasma P5F concentration, six-minute walking distance, BOT-2 strength score, BOT-2 running speed and agility score, HHD score, LEFS score, and BPI-SF score in an adult or adolescent with HPP can be compared with plasma nPF concentration, plasma P5F concentration, six-minute walking distance, BOT-2 strength scores, BOT-2 running speed and agility scores, HHD score, LEFS score, and BPI-SF score, respectively, patient at the starting point in time to evaluate the effect in an adult or adolescent after treatment with rAP.

In addition, plasma nPF concentration, plasma P5F concentration, six-minute walking distance, BOT-2 strength score, BOT-2 running speed and agility score, HHD score, LEFS score, and BPI-SF score in an adult with HFF (e.g., an adult with HFF who is about 18 years of age or older, such as an adult with HFF that started in childhood) or an adolescent with HFF (e.g., an adolescent with HFF who is from about 13 years of age to about 17 years) can be compared with plasma nPF concentration, plasma P5F concentration, six minutes walking distance, BOT-2 strength score, BOT-2 running speed score and agility score, HHD score, LEFS score and BPI- score. SF in a healthy patient to determine the effect of treatment in adults or adolescents treated with rAP (eg, asphotase alfa). In particular, rAPF can be administered over a treatment period of at least one year (e.g., at least two years, at least three years, at least four years, at least five years, at least at least six years, at least seven years, at least eight years, at least nine years, at least ten years, or more than ten years, for example, throughout the life of the patient). Alternatively, the methods may include determining plasma nPF concentration, plasma P5F concentration, six minute walking distance, BOT-2 strength score, BOT-2 running speed and agility score, HHD score, LEFS score, and BPI-SF score prior to administration. sAP, such as asfotase alfa, as described herein, to evaluate the effect of treatment with sAP in an adult or adolescent.

In addition, each of the measures described (e.g., plasma nPF concentration, plasma P5F concentration, six-minute walking distance, BOT-2 strength score, BOT-2 running speed and agility score, HHD score, LEFS score, and BPI score -SF) can be used alone or in any combination to evaluate the effectiveness of treatment with rAPF (such as TNAP, for example, an rAPF fusion polypeptide of SEQ ID NO: 1 or a polypeptide variant having at least 95% sequence identity with the sequence of SEQ ID NO : 1, e.g., asfotase alfa) in an adult with HPP (e.g., an adult with HPP who is about 18 years of age or older, such as an adult with HPP onset in childhood) or in an adolescent with HPP (e.g. , in an adolescent with HFF, aged about 13 years to about 17 years), while improvements related to a particular score or score can be used to demonstrate the effect of treatment in a patient with HPP using rAP.

Hypophosphatasia in Adults and Adolescents

Adult patients with HFF (eg, adults with HFF who are about 18 years of age or older, such as adults with HFF that started in childhood) or adolescents with HFF (eg, adolescents with HFF who are aged about 13 to about 17 years of age) can be treated with rAPF (such as TNAP, e.g. an rAPF fusion polypeptide of SEQ ID NO: 1 or a polypeptide variant having at least 95% sequence identity with SEQ ID NO: 1, e.g. , asfotase alfa) for a period of at least two weeks (e.g., at least three weeks, at least four weeks, at least five weeks, at least six weeks, at least seven weeks at least eight weeks at least nine weeks at least ten weeks at least three months at least four months at least five months at least six months for at least seven months, at least eight months, at least nine months, at least one year, at least two years, at least three years, at least four years, at least five years, at least six years, at least seven years, at least eight years, at least nine years, or at least ten years, or during the lifetime of the patient; in particular, at least six weeks). In particular, asphotase alfa (STRENSIQ®) can be administered as described herein for the treatment of childhood-onset HPP in an adult patient (eg, an untreated patient). Accordingly, the methods are useful for alleviating one or more or all of the symptoms of HPP described herein, especially when an rAPF (such as TNAP, e.g., an rAPF fusion polypeptide of SEQ ID NO: 1 or a polypeptide variant having at least 95% identity sequence of SEQ ID NO: 1, e.g., asfotase alfa) are administered over a period of at least two weeks (e.g., at least three weeks, at least four weeks, at least five weeks, at least six weeks, at least seven weeks, at least eight weeks, at least nine weeks, at least ten weeks, at least three months, at least four months, at least at least five months, at least six months, at least seven months, at least eight months, at least nine months, at least one year, at least two years, at least , three years, at least four years, at least five years, at least six years, at least seven years, at least eight years). In particular, the treatment period is at least six weeks.

For example, the methods are useful for treating symptoms of adult HPP including, but not limited to, elevated blood and/or urine levels of phosphoethanolamine (PEA), hypomineralization, hypercalciuria, skeletal deformity, waddling gait, bone pain, bone fracture (e.g., spinal fracture). , subtrochanteric or metatarsal fracture), calcium pyrophosphate dihydrate crystal deposition, arthritis, pyrophosphate arthropathy, chondrocalcinosis, calcific periarthritis, and pseudofracture. These methods are also useful in the treatment of symptoms of HPP in adolescents, including, but not limited to, elevated levels of nPF, PEA, or P5P in the blood or urine, osteomalacia, one or more skeletal deformities, hypotension, muscle weakness, rheumatoid complications, arthritis, pseudogout, waddling gait, difficulty walking, bone pain, pain, early tooth loss, hypomineralization, pulmonary hypoplasia, respiratory failure, seizures, hypercalciuria, short stature and stunting.

Exemplary metrics useful for assessing the need for or effectiveness of treatment with sAP (such as TNAP, e.g., an rAPF fusion polypeptide of SEQ ID NO: 1, or a polypeptide variant having at least 95% sequence identity with the sequence of SEQ ID NO: 1, e.g. , asphotase alfa) include (1) plasma concentrations of nPF and/or P5F, (2) 6-minute walk test (6MWT), (3) Bruinx-Ozeretsky Motor Assessment Test 2nd edition (BOT-2), ( 4) Handheld Dynamometry (HHD), (5) Lower Limb Function Scale (LEFS), and (6) Brief Pain Rating Questionnaire (BPI-SF), which are described in more detail below.

Plasma concentrations of inorganic pyrophosphate (nPP) and pyridoxal-5'-phosphate (P5P)

Patients with HFF (eg, adults with HFF who are about 18 years of age or older, such as adults with HFF onset in childhood or adolescents with HFF between about 13 years of age and about 17 years of age) may be determined to be treatable with sAP (such as TNAP, e.g., an rAPF fusion polypeptide of SEQ ID NO: 1, or a polypeptide variant having at least 95% sequence identity with SEQ ID NO: 1, e.g., asphotase alfa), by determining the concentrations of NPF and or P5P in a sample, such as a plasma or urine sample from a patient. Any method known to those skilled in the art can be used to quantify the concentrations of nPP and P5P in a plasma sample or, alternatively, in a urine sample, as detailed in Whyte et al., 1995 (J. Clin. Invest. 95(4 ): 1440-1445), incorporated herein by reference in its entirety. Methods for quantifying the concentration of NPF in a plasma or urine sample are also described in Cheung et al., 1977 (Anal. Biochem. 83: 61-63), Cook et al., 1978 (Anal. Biochem. 91: 557-565), and Johnson et al, 1968 (Anal. Biochem. 26: 137-145), each of which is incorporated herein by reference in its entirety. In particular, the concentrations of nPP and P5P in a plasma sample can be used to assess ALP activity in a patient (e.g., an adult with HPP who is about 18 years of age or older, such as an adult with HPP that started in childhood, or in a teenager, having HFF, aged from about 13 years to about 17 years).

Compared with healthy subjects (e.g., healthy subjects of approximately the same age, same sex, and/or same height), patients with HPP typically have elevated plasma concentrations of nPF and P5P, e.g., an NPF concentration of up to about 6 µM (e.g. , about 3.5 µM, about 4 µM, about 4.5 µM, about 5 µM, or about 5.5 µM) and/or a P5P concentration up to about 1300 ng/mL (for example, a plasma P5P concentration of about 200 ng/mL , 300 ng/ml, 400 ng/ml, 500 ng/ml, 600 ng/ml, 700 ng/ml, 800 ng/ml, 900 ng/ml, 1000 ng/ml, 1100 ng/ml, 1200 ng/ml or 1300 ng/ml). The lower limit of normal for the plasma concentration of nPF in healthy adults is about 1 µM, and the upper limit of normal is about 5.9 µM. The lower limit of normal for P5P plasma concentrations in healthy adults is less than about 10 ng/ml, while the upper limit of normal is about 60 ng/ml. The lower limit of normal for plasma concentrations of nPF in healthy adolescents is less than about 0.75 µM, while the upper limit of normal is about 4.78 µM. The lower limit of normal for P5P plasma concentrations in healthy adolescents is less than about 5.74 ng/mL, while the upper limit of normal is about 61.15 ng/mL.

Patients with HPP (eg, adults with HPP aged about 18 years and older, such as adults with HPP that started in childhood, or adolescents with HPP, aged about 13 years to about 17 years) with elevated concentrations Plasma nPF and/or PLP may be treated with an rAPF (such as TNAP, e.g., an rAPF fusion polypeptide of SEQ ID NO: 1 or a polypeptide variant having at least 95% sequence identity with SEQ ID NO: 1 e.g., asphotase alfa), such as administering rAP during a treatment period of at least two weeks (e.g., at least three weeks, at least four weeks, at least five weeks, at least six weeks, at least seven weeks, at least eight weeks, at least nine weeks, at least ten weeks, at least three months, at least four months, at least five months for at least six months, at least seven months, at least eight months, at least nine months, at least one year, at least two years, at least three years, at least four years, at least five years, at least six years, at least seven years, at least eight years, at least nine years, or at least ten years, or during the lifetime of the patient; in particular, at least six weeks). For example, a patient with HPP (eg, an adult with HPP who is about 18 years of age or older, such as an adult with HPP that started in childhood, or an adolescent with HPP between about 13 years of age and about 17 years of age ) with an NPF concentration of up to about 6 µM can be treated with rAP during the treatment period. Similarly, a patient having a GPP with a P5P concentration of up to about 1300 ng/mL may be treated with rAP during the treatment period.

The methods result in a statistically significant decrease in the concentration of nPF and/or P5P in a sample (eg, plasma sample) of the patient. For example, treatment with rAPF results in a decrease in the concentration of NPF in a patient sample (eg, plasma sample) of about 1 µM, about 1.5 µM, about 2 µM, about 2.5 µM, or about 3 µM, or 25% or more. (e.g., 30%, 35%, 40%, 45%, 50%, 55%, 60%, or more than 60%) relative to the concentrations of NPF in a sample (e.g., plasma sample) of an untreated HPP patient (e.g., subject with HFF onset in childhood, around 18 years of age or older). Thus, the patient has a plasma concentration of NPF, for example, from about 2 μM to about 5 μM, from about 3 μM to about 5 μM, from about 2 μM to about 4 μM, or from about 2 μM to about 3 μM after administration. rCHF.

Similarly, treatment with rAPF results in a decrease in the concentration of P5P in a sample (eg, plasma sample) of the patient by about 100 ng/mL, 125 ng/mL, 150 ng/mL, 175 ng/mL, 200 ng/mL, 225 ng/mL. ml or 250 ng/ml or 50% or more (e.g. 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or more than 95%) relative to P5P concentrations in a sample (eg, plasma sample) of an untreated HPP patient (eg, a subject with childhood-onset HPP, about 18 years of age or older). Thus, the patient has a plasma concentration of P5P, for example, from about 2 ng/ml to about 150 ng/ml, from about 4 ng/ml to about 100 ng/ml, from about 10 ng/ml to about 75 ng/ml. ml or from about 1 ng/ml to about 50 ng/ml after administration of sAP.

Decreased plasma concentrations of nPP and/or P5F in a patient with HPP (eg, in an adult with HPP who is about 18 years of age or older, such as an adult with HPP onset in childhood, or in an adolescent with HPP in age from about 13 years to about 17 years) can be maintained throughout the period of administration of rAP. For example, plasma nPP concentration decreases by about 25% and remains at ± 10% of the reduced plasma nPF concentration during treatment with rAP and/or plasma P5F concentration decreases by about 50% and remains at ± 10% of the reduced plasma concentration. Plasma P5P concentrations during treatment with rAP.

Alternatively, when administration of rAPF does not result in a decrease in nPP concentrations in a plasma sample from a patient with HPP (e.g., in an adult with HPP who is about 18 years of age or older, such as an adult with HPP that started in childhood, or in a teenager having HPP between about 13 and about 17 years of age) by about 25% or more, the dose and/or frequency of sAP administration can be adjusted to determine the effective amount of rAPK for a patient with HPP (e.g., an adult with GPP, around 18 years of age or older, such as an adult with childhood-onset HFF). Similarly, when sAP administration does not result in a decrease in P5P concentrations in a patient's plasma sample of about 50% or more, the dose and/or frequency of sAP administration can be modified to determine the effective amount of sAP for a patient with GPP (e.g., an adult with GPP). , about 18 years of age or older, such as an adult with HFF onset in childhood, or an adolescent with HFF between about 13 years of age and about 17 years of age). For example, the dose of rABP can be increased, for example, from about 2.1 mg/kg/week or about 3.5 mg/kg/week to about 6 mg/kg/week or about 9 mg/kg/week.

6 Minute Walk Test (6MWT)

Adults with HFF (eg, adults with HFF who are about 18 years of age or older, such as adults with HFF that started in childhood or an adolescent with HFF between about 13 and about 17 years of age) may be determined to be treatable with sAP (such as TNAP, e.g., an rAPF fusion polypeptide of SEQ ID NO: 1 or a polypeptide variant having at least 95% sequence identity with SEQ ID NO: 1, e.g., asphotase alfa) with using 6MWT. In particular, 6MWT can be used to assess the walking ability of an adult with HFF (e.g., an adult with HFF who is about 18 years of age or older, such as an adult with childhood-onset HFF), or an adolescent having HFF (e.g., an adolescent with HFF, aged from about 13 years to about 17 years) to obtain a 6MWT value for an adult or adolescent.

6MWT can be performed indoors or outdoors using a flat, straight, enclosed corridor (eg about 30 meters long) with a hard surface. A stopwatch or other timer may be used to track time, and a mechanical counter or other device may be used to determine the walking distance (eg, in meters) of a patient with HFF (eg, an adult with HFF who is about 18 years of age or older, such as an adult with HFF that started in childhood or an adolescent with HFF (for example, an adolescent with HFF between about 13 and about 17 years of age.) For example, the length of a corridor can be marked every three meters to determine the number meters walked by a patient with HPP, with the turning point at 30 meters and the baseline also marked The distance walked by the patient in six minutes can then be compared to the predicted number of meters walked, e.g., in an untreated subject of about the same age same sex and/or same height, and expressed as a percentage to obtain a patient's 6MWT value. p, an adult with HPP who is about 18 years of age or older, such as an adult with HPP onset in childhood, or an adolescent with HPP between about 13 and about 17 years of age) can be compared to a patient's 6MWT value at starting point in time. In addition, the 6MWT value in an adult or adolescent with HPP can be compared with the 6MWT value in a healthy patient.

Patients with HFF (eg, adults with HFF who are about 18 years of age or older, such as adults with HFF that started in childhood, or adolescents with HFF between about 13 and about 17 years of age) with 6MWT less than about 80% of the predicted 6MWT can be treated with an rAPF (such as TNAP, e.g., an rAPF fusion polypeptide of SEQ ID NO: 1 or a polypeptide variant having at least 95% sequence identity with the sequence of SEQ ID NO: 1, e.g., asfotase alfa), such as administering rAP during a treatment period of at least two weeks (e.g., at least three weeks, at least four weeks, at least five weeks, at least at least six weeks at least seven weeks at least eight weeks at least nine weeks at least ten weeks at least three months at least four months at least , five months at least six months, at least seven months, at least eight months, at least nine months, at least one year, at least two years, at least three years at least four years, at least five years, at least six years, at least seven years, at least eight years, at least nine years, or at least ten years , or over the lifetime of the patient; in particular, at least six weeks). For example, a HFF patient with a 6MWT less than about 80% of the predicted 6MWT (e.g., about 50%, about 55%, about 60%, about 65%, about 70%, or about 75% of the predicted 6MWT) may be treated with rAPLP (such as TNAP, e.g., an rAPF fusion polypeptide of SEQ ID NO: 1 or a polypeptide variant having at least 95% sequence identity with the sequence of SEQ ID NO: 1, e.g., asphotase alfa) for a period of time at least two weeks (e.g. at least three weeks, at least four weeks, at least five weeks, at least six weeks, at least seven weeks, at least eight weeks at least nine weeks, at least ten weeks, at least three months, at least four months, at least five months, at least six months, at least seven months, at least eight months, at least nine months, at least at least one year, at least two years, at least three years, at least four years, at least five years, at least six years, at least seven years, at least , eight years, at least nine years, or at least ten years, or during the lifetime of the patient; in particular, at least six weeks).

The methods may result in an improvement in the 6MWT value in a patient with HPP (e.g., an adult with HPP who is about 18 years of age or older, such as an adult with HPF that started in childhood, with HPF that started in childhood, or in a teenager having HFF, aged from about 13 years to about 17 years). For example, treatment with rAPLP (such as TNAP, e.g., an rAPF fusion polypeptide of SEQ ID NO: 1 or a polypeptide variant having at least 95% sequence identity with the sequence of SEQ ID NO: 1, e.g., asphotase alfa), such as treatment with rAP during a treatment period of at least two weeks (e.g., at least three weeks, at least four weeks, at least five weeks, at least six weeks, at least seven weeks at least eight weeks at least nine weeks at least ten weeks at least three months at least four months at least five months at least six months at least seven months, at least eight months, at least nine months, at least one year, at least two years, at least three years, at least four years, at least five years, at least six years, at least seven years at least eight years, at least nine years, or at least ten years, or during the lifetime of the patient; in particular, at least six weeks) can result in an increase in the 6MWT value to about 80% or more relative to the patient's predicted 6MWT value (for example, about 82%, about 84%, about 86%, about 88%, about 90% , about 92%, about 94%, about 96%, about 98% or more relative to the predicted value of 6MWT).

An increase in 6MWT in a patient with HPP (e.g., an adult with HPP who is about 18 years of age or older, such as an adult with HPP that started in childhood, or an adolescent with HPP who is about 13 years of age or years to about 17 years) can be maintained throughout the period of administration of sAP (such as TNAP, for example, an rAPF fusion polypeptide of SEQ ID NO: 1 or a polypeptide variant having at least 95% sequence identity with the sequence of SEQ ID NO: 1, e.g., asfotase alfa), e.g., over a period of at least two weeks (e.g., at least three weeks, at least four weeks, at least five weeks, at least six weeks, at least seven weeks at least eight weeks at least nine weeks at least ten weeks at least three months at least four months at least five months at least at least six months from at least eight months, at least eight months, at least nine months, at least one year, at least two years, at least three years, at least four years, at least five years, at least six years, at least seven years, at least eight years, at least nine years, or at least ten years, or during the lifetime of the patient; in particular, at least six weeks). For example, the 6MWT value increases to greater than about 80% of the patient's predicted 6MWT value and remains at +/- 10% of the increased 6MWT value during treatment with rAPF (eg, asphotase alfa).

Similarly, an increase in the ability to walk in a patient with HFF (e.g., an adult with HFF who is about 18 years of age or older, such as an adult with HFF onset in childhood, or an adolescent with HFF who is aged from about 13 years to about 17 years) can be maintained throughout the period of administration of rAP, for example, for a period of time of at least two weeks (for example, at least three weeks, at least four weeks, at least , five weeks, at least six weeks, at least seven weeks, at least eight weeks, at least nine weeks, at least ten weeks, at least three months, at least four months at least five months at least six months at least seven months at least eight months at least nine months at least one year at least two years, at least three years, at least four years, at least five years, at least six years, at least seven years, at least eight years, at least nine years, or at least ten years, or during the lifetime the patient; in particular, at least six weeks). For example, a patient with HPP exhibits less dependence on an assistive mobility device such as a walker, wheelchair, braces, crutches, or orthotics during treatment with rAPF.

Alternatively, if administration of rAPF does not result in an increase in 6MWT greater than 80% of the predicted 6MWT (e.g., an untreated HPP subject of approximately the same age, sex, and/or height) dosage and/or the frequency of rAPF administration can be modified to determine the effective amount of rAPF for a patient with HPP (e.g., an adult with HPP who is about 18 years of age or older, such as an adult with HPP that started in childhood, or an adolescent with HPP , aged from about 13 years to about 17 years). For example, the dosage of rAPLP (such as TNAP, e.g., an rAPF fusion polypeptide of SEQ ID NO: 1 or a polypeptide variant having at least 95% sequence identity with the sequence of SEQ ID NO: 1, e.g., asphotase alfa) can be increased, for example, from about 2.1 mg/kg/week or about 3.5 mg/kg/week to about 6 mg/kg/week or about 9 mg/kg/week.

Bruinx-Ozeretsky Motor Assessment Test, 2nd Edition (BOT-2)

Adults who have HFF (for example, an adult with HFF who is about 18 years of age or older, such as an adult who has HFF that started in childhood) or adolescents who have HFF (for example, adolescents who have HFF from about 13 years of age) years to about 17 years of age) can be identified for treatment with sAP (such as TNAP, for example, the rAPF fusion polypeptide of SEQ ID NO: 1 or a polypeptide variant having at least 95% sequence identity with the sequence of SEQ ID NO: 1, for example, asphotase alfa) using the BOT-2 running speed and agility tests and the BOT-2 strength test. In particular, the BOT-2 Speed and Agility Tests and the BOT-2 Strength Tests can be used to assess physical impairment and mobility limitations in an adult with HFF to obtain a BOT-2 Total Speed and Agility Score and/or a BOT-2 Total Strength Score. for an adult.

BOT-2 includes a range of tests to assess physical impairment in a patient with HFF (e.g., an adult with HFF who is about 18 years of age or older, such as an adult with HFF that started in childhood, or an adolescent with HFF , aged from about 13 years to about 17 years), which can be performed, for example, using a kit that includes tests. BOT-2 provides BOT-2 composite scores in the following areas: strength, running speed and agility, fine motor accuracy, fine motor integrity, manual dexterity, bilateral coordination, balance and upper limb coordination. For example, an adult or adolescent with HFF may perform squats, prone straight leg raises, standing long jumps, supported squats, and/or push-ups to determine a BOT-2 strength score; an adult or juvenile with HFF may walk the balance beam and/or shuttle, side-to-side jumps on two legs, and/or side-to-side jumps on one leg to determine running speed and BOT-2 agility; an adult or teen with HFF can cut a circle and/or connect dots to determine the BOT-2 fine motor accuracy score; an adult or teen with a GFF can copy an asterisk and/or copy a square to determine the BOT-2 fine motor integrity score; an adult or teenager with a GFF can pass coins, sort cards and/or string blocks to determine a manual dexterity score; an adult or adolescent with HFF may tap foot and toe and/or perform side-leg hopping to determine a BOT-2 score of bilateral coordination; an adult or adolescent with HFF may walk forward in a line and/or stand on one leg on a balance bar to determine a BOT-2 balance score; and an adult or adolescent with HFF can throw a ball at a target and/or catch a thrown ball to determine the BOT-2 upper limb coordination score. The BOT-2 score is the cumulative sum of each assessed area. In addition, the BOT-2 score used to assess a patient's fitness may be a baseline total score or a normative score.

An adult with HFF (for example, an adult with HFF who is about 18 years of age or older, such as an adult with HFF that started in childhood) or an adolescent who has HFF (for example, an adolescent with HFF who is about 18 years of age or older) 13 to about 17 years of age) can perform tests in one or more of the areas described (strength, running speed and agility, fine motor skills, fine motor integrity, manual dexterity, bilateral coordination, balance and upper limb coordination) to obtain a BOT-2 score, indicating physical impairment in an adult or adolescent. Within each BOT-2 area (strength, running speed and agility, fine motor skills, fine motor integrity, manual dexterity, bilateral coordination, balance, and upper limb coordination), an adult or adolescent with HFF may take one or more tests to determine the score. BOT-2 for an Adult or Adolescent, for example, an adult or adolescent may perform one or more of the Squat, Straight Leg Raise, Standing Long Jump, Supported Squat to determine BOT-2 strength. If necessary, only one test (for example, one test selected from the group consisting of squats, straight leg raises, standing long jumps, supported squats, and push-ups) may be performed to determine the BOT-2 score ( for example, BOT-2 strength score) of an adult or adolescent with HFF.

Each of the BOT-2 scores (strength, running speed and agility, fine motor accuracy, fine motor integrity, manual dexterity, bilateral coordination, balance, and upper limb coordination) of a patient with HFF (e.g., an adult with HFF, aged about 18 years of age or older, such as an adult with HPP onset in childhood or an adolescent with HPP between about 13 and about 17 years of age) can be compared with the BOT-2 score of patients without HPP (eg, an adult who does not aged about 18 years and older, or an adolescent with HFF, aged from about 13 years to about 17 years), for example, to determine the standard deviation of the BOT-2 score. Each of the BOT-2 scores (e.g., strength, running speed and agility, fine motor accuracy, fine motor integrity, manual dexterity, bilateral coordination, balance, and upper limb coordination) of a patient with HPP can be compared to the BOT-2 score of other patients , having GFF (eg, patients with GFF, approximately the same age, height and/or sex), for example, to determine the BOT-2 score for a patient with GFF.

BOT-2 scores (e.g., measures of strength, running speed and agility, fine motor skills, fine motor integrity, manual dexterity, bilateral coordination, balance, and upper limb coordination) range from 0 to about 25, or less, with a score greater than than about 10 is considered appropriate in healthy subjects (eg, patients without HPP). Patients with a BOT-2 score (e.g., measures of strength, running speed and agility, fine motor skills, fine motor integrity, manual dexterity, bilateral coordination, balance, and upper limb coordination) of less than 10 may be treated with rAPF (such as TNAP, e.g., an rAPF fusion polypeptide of SEQ ID NO: 1 or a polypeptide variant having at least 95% sequence identity with the sequence of SEQ ID NO: 1, e.g. weeks (e.g. at least three weeks, at least four weeks, at least five weeks, at least six weeks, at least seven weeks, at least eight weeks, at least nine weeks at least ten weeks at least three months at least four months at least five months at least six months at least seven months at least eightmonths, at least nine months, at least one year, at least two years, at least three years, at least four years, at least five years, at least six years at least seven years, at least eight years, at least nine years, or at least ten years, or during the lifetime of the patient; in particular, at least six weeks).

For example, a patient with HPP (eg, an adult with HPP who is about 18 years of age or older, such as an adult with HPP that started in childhood, or an adolescent with HPP between about 13 years of age and about 17 years of age ) and a BOT-2 running speed and agility score of less than 10 (e.g., about 0, about 1, about 2, about 3, about 4, about 5, about 6, about 7, about 8, about 9, or about 10) can pass treatment with rAPLP (such as TNAP, e.g., an rAPF fusion polypeptide of SEQ ID NO: 1 or a polypeptide variant having at least 95% sequence identity with the sequence of SEQ ID NO: 1, e.g., asphotase alfa) for a period of time at least two weeks (e.g. at least three weeks, at least four weeks, at least five weeks, at least six weeks, at least seven weeks, at least eight weeks, at least nine weeks, at least ten weeks, at least three months, alternating at least four months, at least five months, at least six months, at least seven months, at least eight months, at least nine months, at least one year, at least at least two years, at least three years, at least four years, at least five years, at least six years, at least seven years, at least eight years, at least , nine years or at least ten years, or during the lifetime of the patient; in particular, at least six weeks). Similarly, a patient with HFF (e.g., an adult with HFF who is about 18 years of age or older, such as an adult with HFF that started in childhood, or an adolescent with HFF between about 13 years of age and about 17 years of age). years) with a BOT-2 strength score less than 12 (e.g., about 0, about 1, about 2, about 3, about 4, about 5, about 6, about 7, about 8, about 9, about 10, about 11, about 12, about 13, or about 14) may then be treated with sAP (such as TNAP, e.g., an rAPF fusion polypeptide of SEQ ID NO: 1 or a polypeptide variant having at least 95% sequence identity with the sequence of SEQ ID NO: 1 , for example, asfotase alfa) for a period of at least two weeks (for example, at least three weeks, at least four weeks, at least five weeks, at least six weeks, at least , seven weeks, at least eight weeks, at least nine weeks, at least ten weeks, n at least three months, at least four months, at least five months, at least six months, at least seven months, at least eight months, at least nine months, at least one year, at least two years, at least three years, at least four years, at least five years, at least six years, at least seven years, at least at least eight years, at least nine years, or at least ten years, or during the lifetime of the patient; in particular, at least six weeks).

The methods result in an improvement in a BOT-2 score (e.g., strength, running speed and agility, fine motor skills, fine motor integrity, manual dexterity, bilateral coordination, balance, and/or upper limb coordination score) in a HFF patient (e.g., an adult with HFF, at about 18 years of age or older, such as an adult with HFF onset in childhood, or an adolescent with HFF between about 13 years of age and about 17 years of age). For example, treatment with rAPLP (such as TNAP, e.g., an rAPF fusion polypeptide of SEQ ID NO: 1 or a polypeptide variant having at least 95% sequence identity with the sequence of SEQ ID NO: 1, e.g., asphotase alfa), such as treatment with rAP during a treatment period of at least two weeks (e.g., at least three weeks, at least four weeks, at least five weeks, at least six weeks, at least seven weeks at least eight weeks at least nine weeks at least ten weeks at least three months at least four months at least five months at least six months at least seven months, at least eight months, at least nine months, at least one year, at least two years, at least three years, at least four years, at least five years, at least six years, at least seven years at least eight years, at least nine years, or at least ten years, or during the lifetime of the patient; in particular, at least six weeks) can lead to an increase in BOT-2 strength to about 10, to about 20 or more (for example, about 9, about 10, about 11, about 12, about 13, about 14, about 15, about 16, about 17, about 18, about 19, about 20, about 21, about 22, about 23, about 24 or about 25). In addition, treatment with sAPLP (such as TNAP, e.g., an rAPF fusion polypeptide of SEQ ID NO: 1, or a polypeptide variant having at least 95% sequence identity with the sequence of SEQ ID NO: 1, e.g., asphotase alfa) is as treatment with rAP during a treatment period of at least two weeks (e.g., at least three weeks, at least four weeks, at least five weeks, at least six weeks, at least seven weeks at least eight weeks at least nine weeks at least ten weeks at least three months at least four months at least five months at least six months, at least seven months, at least eight months, at least nine months, at least one year, at least two years, at least three years, at least four years at least five years, at least six years, at least seven years is at least eight years, at least nine years, or at least ten years, or during the lifetime of the patient; in particular, at least six weeks) can result in an increase in BOT-2 running speed and agility to about 9, to about 20 or more (e.g., about 9, about 10, about 11, about 12, about 13, about 14, about 15, about 16, about 17, about 18, about 19, about 20, about 21, about 22, about 23, about 24, or about 25).

Increases in BOT-2 (eg, strength, running speed, and agility, fine motor accuracy, fine motor integrity, manual dexterity, bilateral coordination, balance, and/or upper limb coordination) can be maintained throughout the duration of rAPF (such as TNAP) rAPF fusion polypeptide of SEQ ID NO: 1 or a polypeptide variant having at least 95% sequence identity with the sequence of SEQ ID NO: 1, for example, asphotase alfa), for example, over a period of time of at least two weeks (e.g. at least three weeks, at least four weeks, at least five weeks, at least six weeks, at least seven weeks, at least eight weeks, at least nine weeks, at least ten weeks, at least three months, at least four months, at least five months, at least six months, at least seven months, at least eight months at least nine months, at least one year, at least two years, at least three years, at least four years, at least five years, at least six years at least seven years, at least eight years, at least nine years, or at least ten years, or during the lifetime of the patient; in particular, at least six weeks). Similarly, mitigation of physical impairment may be maintained throughout the period of administration of sAP (such as TNAP, e.g., an rAPF fusion polypeptide of SEQ ID NO: 1, or a polypeptide variant having at least 95% sequence identity with the sequence of SEQ ID NO: 1, e.g. , asfotase alfa), e.g., over a period of at least two weeks (e.g., at least three weeks, at least four weeks, at least five weeks, at least six weeks, at least at least seven weeks at least eight weeks at least nine weeks at least ten weeks at least three months at least four months at least five months at least , six months, at least seven months, at least eight months, at least nine months, at least one year, at least two years, at least three years, at least four years, at least five years, at least six years, at least seven years, at least eight years, at least nine years, or at least ten years, or during the lifetime of the patient; in particular, at least six weeks).

BOT-2 scores (strength, running speed and agility, fine motor accuracy, fine motor integrity, manual dexterity, bilateral coordination, balance, and upper limb coordination) in a patient with HFF (e.g., an adult with HFF, aged about 18 years or older, such as an adult with HPP onset in childhood, or an adolescent with HPP between about 13 and about 17 years of age) can be used alone or in combination to assess the effectiveness of treatment with rAPF (such as in as TNAP, e.g., an rAPF fusion polypeptide of SEQ ID NO: 1 or a polypeptide variant having at least 95% sequence identity with the sequence of SEQ ID NO: 1, e.g. that rAP is effective in the treatment of physical disorders associated with HPP. For example, administering sAP to a patient with HPP (e.g., an adult with HPP who is about 18 years of age or older, such as an adult with HPP that started in childhood, or an adolescent with HPP who is from about 13 years of age to about 17 years of age) results in an increase in BOT-2 running speed and agility to about 9 or higher, where the patient previously had a BOT-2 running speed and agility score of less than about 7, thus rAPF is considered effective in, for example, treatment physical disorders associated with HFF. Alternatively, an increase of at least two or more points (e.g., 2, 3, 4, 5, 6, 7, 8, 9, or 10 points) compared to BOT-2 running speed and agility before treatment indicates efficacy (eg, in combination with a sustained high score for more than 1 year of treatment).

In addition, in each area of the BOT-2 (strength, running speed and agility, fine motor accuracy, fine motor integrity, manual dexterity, bilateral coordination, balance, and upper limb coordination), a patient with HFF (e.g., an adult with HFF, in about 18 years of age or older, such as an adult with HFF onset in childhood or an adolescent with HFF between the ages of about 13 and about 17 years old) may perform one or more tests to determine a BOT-2 score patient. For example, an adult or adolescent with HFF may perform one or more of squats, straight leg raises, standing long jumps, supported squats, and push-ups to determine a BOT-2 strength score and evaluate the effectiveness of treatment with rAPF. . An adult or adolescent with HFF may perform one or more of beam walking, shuttle running, bipedal lateral jumps, and/or single leg lateral jumps to determine a BOT-2 running speed and agility score and to evaluate the effectiveness of treatment using introduction of rAP. An adult or adolescent with HPP may cut a circle and/or connect the dots to determine the BOT-2 Fine Motor Accuracy Score and assess the effectiveness of treatment with rAPF. An adult or adolescent with HPP may copy the asterisk and/or copy the square to determine the BOT-2 Fine Motor Integrity Score and assess the effectiveness of treatment with rAPF. An adult or adolescent with HFF may perform one or more of coin passing, card sorting, and string blocks to determine a BOT-2 manual dexterity score and evaluate the effectiveness of treatment with rAPF. An adult or adolescent with HPP may tap his or her foot and finger and/or perform jumps to determine the BOT-2 bilateral coordination score and assess the effectiveness of treatment with rAPF. An adult or adolescent with HPP may walk forward in a line and/or stand on one leg on a balance beam to determine the BOT-2 balance score and evaluate the effectiveness of treatment with rAPF. An adult or adolescent with HPP can throw a ball at a target and/or catch a thrown ball to determine the BOT-2 upper limb coordination score and assess the effectiveness of treatment with rAPF.

Alternatively, if rAPF administration does not result in an increase in BOT-2 running speed and agility to greater than about 9 (e.g., an increase of at least 2 to 10 points (e.g., 2, 3, 4, 5, 6, 7, scores of 8, 9, or 10) compared to BOT-2 running speed and agility before sAP treatment), the dosage and/or frequency of sAP administration can be modified to determine the effective amount of sAP for a patient with HPP (e.g., an adult with HFF, at about 18 years of age or older, such as an adult with HFF onset in childhood, or an adolescent with HFF between about 13 years of age and about 17 years of age). For example, the dosage of rAPLP (such as TNAP, e.g., an rAPF fusion polypeptide of SEQ ID NO: 1 or a polypeptide variant having at least 95% sequence identity with the sequence of SEQ ID NO: 1, e.g., asphotase alfa) can be increased, for example, from about 2.1 mg/kg/week or about 3.5 mg/kg/week to about 6 mg/kg/week or about 9 mg/kg/week.

Handheld Dynamometer (HHD)

The grasping and muscle strength of a patient with HPP (e.g., an adult with HPP aged about 18 years or older, such as an adult with HPP onset in childhood, or an adolescent with HPP aged about 13 years to about 17 years) can be assessed using handheld dynometry (HHD). For example, knee flexion and extension, as well as hip flexion, extension, and hip abduction of a patient with HFF can be measured using, for example, a MICROFET2 ^™ dynamometer, while the grasping force of a patient with HFF can be measured using eg the JAMAR® gripper dynamometer. Specifically, the administrator holds the dynamometer stationary while the patient applies maximum force to the dynamometer. Peak force data is collected in pounds and then converted to Newtons (N, N). Torque values are then calculated using limb length in Newton meters. The torque value can then be compared to a torque value of, for example, a normal subject of about the same age, same sex, and/or same height, and expressed as a percentage to obtain an estimate of the subject's HHD.

A patient with HFF (eg, an adult with HFF who is about 18 years of age or older, such as an adult with HFF that started in childhood, or an adolescent with HFF between about 13 years of age and about 17 years of age) with an HHD score of less than about 80% of the predicted HHD score (e.g., relative to a normal subject of about the same age, same sex, and/or the same height) may be treated with rAPLP (such as TNAP, e.g., an rAPF SEQ ID fusion polypeptide NO: 1 or a polypeptide variant having at least 95% sequence identity with the sequence of SEQ ID NO: 1, for example, asfotase alfa). For example, a HPP patient with an HHD less than about 80% of predicted HHD may be treated with rAPF for a treatment period of at least two weeks (e.g., at least three weeks, at least four weeks at least five weeks, at least six weeks, at least seven weeks, at least eight weeks, at least nine weeks, at least ten weeks, at least three months, at least four months, at least five months, at least six months, at least seven months, at least eight months, at least nine months, at least one year, at least two years, at least three years, at least four years, at least five years, at least six years, at least seven years, at least eight years, at least at least nine years, or at least ten years, or over the life span of the patient enta; in particular, at least six weeks).

The methods may result in an improvement in HDD in a patient with HPP (e.g., an adult with HPP who is about 18 years of age or older, such as an adult with HPP that started in childhood, or an adolescent with HPP who is aged from about 13 years old to about 17 years old). For example, treatment with rAPLP (such as TNAP, e.g., an rAPF fusion polypeptide of SEQ ID NO: 1 or a polypeptide variant having at least 95% sequence identity with the sequence of SEQ ID NO: 1, e.g., asphotase alfa), such as treatment with rAP during a treatment period of at least two weeks (e.g., at least three weeks, at least four weeks, at least five weeks, at least six weeks, at least seven weeks at least eight weeks at least nine weeks at least ten weeks at least three months at least four months at least five months at least six months at least seven months, at least eight months, at least nine months, at least one year, at least two years, at least three years, at least four years, at least five years, at least six years, at least seven years at least eight years, at least nine years, or at least ten years, or during the lifetime of the patient; in particular, at least six weeks) can result in an increase in HDD score of about 5% or more relative to the predicted HDD score of the patient (for example, an increase of about 5%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45% or about 50%).

An increase in HDD in a patient with HFF (eg, an adult with HFF who is about 18 years of age or older, such as an adult with HFF onset in childhood or an adolescent with HFF who is about 13 years of age or years to about 17 years) can be maintained throughout the period of administration of sAP (such as TNAP, for example, an rAPF fusion polypeptide of SEQ ID NO: 1 or a polypeptide variant having at least 95% sequence identity with the sequence of SEQ ID NO: 1, e.g., asfotase alfa), e.g., over a period of at least two weeks (e.g., at least three weeks, at least four weeks, at least five weeks, at least six weeks, at least seven weeks at least eight weeks at least nine weeks at least ten weeks at least three months at least four months at least five months at least at least six months at least seven months at least eight months at least nine months at least one year at least two years at least three years at least four years at least five years, at least six years, at least seven years, at least eight years, at least nine years, or at least ten years, or during the lifetime of a patient having HPP; in particular, at least six weeks). For example, the HHD score increases by about 5% or more of the predicted HHD score and remains at ±10% of the increased HHD score during rAPF treatment. Alternatively, if administration of rAPF does not result in an increase in HHD of about 5% or more relative to predicted HHD (e.g., in a patient with HPP of about the same age, sex, and/or height), dosage and/or frequency of administration rAPF can be changed to determine the effective amount of rAPK for a patient with HPP. For example, the dose of rAPF may be increased, for example, from about 2.1 mg/kg/week or about 3.5 mg/kg/week to about 6 mg/kg/week or about 9 mg/kg/week.

Lower Limb Function Scale (LEFS)

Decreased functional capacity in the lower extremities in a patient with HPP (e.g., an adult with HPP who is about 18 years of age or older, such as an adult with HPP that started in childhood, or an adolescent with HPP who is about 13 years of age or years to about 17 years of age) can be assessed using the Lower Limb Function Scale (LEFS). LEFS can be used to assess the functional impairment of one or both lower limbs of a patient with HPF, including the ability to follow the patient over time and assess the effectiveness of treatment with asphotase alfa. A licensed physical therapist may administer LEFS to a patient with HFF (eg, a patient with HFF who is about 13 years of age or older) in an interview format.

A patient with HFF (eg, an adult with HFF who is about 18 years of age or older, such as an adult with HFF that started in childhood, or an adolescent with HFF between about 13 years of age and about 17 years of age) with an LEFS score of less than about 40 (e.g., an LEFS score of about 10, about 15, about 20, about 25, about 30, about 35, or about 40) may be treated with an rAPF (such as TNAP, e.g., an rAPF SEQ ID fusion polypeptide NO: 1 or a polypeptide variant having at least 95% sequence identity with the sequence of SEQ ID NO: 1, for example, asfotase alfa). For example, a HPP patient with an LEFS score of less than about 40 (e.g., an LEFS score of about 10, about 15, about 20, about 25, about 30, about 35, or about 40) may be treated with rAP during the treatment period. at least two weeks (e.g., at least three weeks, at least four weeks, at least five weeks, at least six weeks, at least seven weeks, at least eight weeks, at least nine weeks, at least ten weeks, at least three months, at least four months, at least five months, at least six months, at least seven months at least eight months, at least nine months, at least one year, at least two years, at least three years, at least four years, at least five years, at least six years, at least seven years, at least eight years, at least nine years, or, about at least ten years, or during the lifetime of the patient; in particular, at least six weeks).

The methods may result in an improvement in the LEFS score in a patient with HFF (e.g., an adult with HFF who is about 18 years of age or older, such as an adult with HFF onset in childhood, or an adolescent with HFF who is aged from about 13 years old to about 17 years old). For example, treatment with rAPLP (such as TNAP, e.g., an rAPF fusion polypeptide of SEQ ID NO: 1 or a polypeptide variant having at least 95% sequence identity with the sequence of SEQ ID NO: 1, e.g., asphotase alfa), such as treatment with rAP during a treatment period of at least two weeks (e.g., at least three weeks, at least four weeks, at least five weeks, at least six weeks, at least seven weeks at least eight weeks at least nine weeks at least ten weeks at least three months at least four months at least five months at least six months at least seven months, at least eight months, at least nine months, at least one year, at least two years, at least three years, at least four years, at least five years, at least six years, at least seven years at least eight years, at least nine years, or at least ten years, or during the lifetime of the patient; in particular, at least six weeks) can lead to an increase in the LEFS score of about 3 or more (for example, about 3, about 4, about 5, about 6, about 7, about 8, about 9, or about 10).

An increase in LEFS score in a patient with HFF (e.g., an adult with HFF who is about 18 years of age or older, such as an adult with HFF onset in childhood or an adolescent with HFF who is about 13 years of age or older) years to about 17 years) can be maintained throughout the period of administration of sAP (such as TNAP, for example, an rAPF fusion polypeptide of SEQ ID NO: 1 or a polypeptide variant having at least 95% sequence identity with the sequence of SEQ ID NO: 1, e.g., asfotase alfa), e.g., over a period of at least two weeks (e.g., at least three weeks, at least four weeks, at least five weeks, at least six weeks, at least seven weeks at least eight weeks at least nine weeks at least ten weeks at least three months at least four months at least five months at least at least six months at least seven months at least eight months at least nine months at least one year at least two years at least three years at least four years at least five years, at least six years, at least seven years, at least eight years, at least nine years, or at least ten years, or during the lifetime of a patient having HPP; in particular, at least six weeks). For example, the LEFS score may increase by about 3 or more (e.g., about 3, about 4, about 5, about 6, about 7, about 8, about 9, or about 10) and remains at ±10% of the increased LEFS during time of treatment with rAPF. Alternatively, when rAPF administration does not increase the LEFS score by about 3 or more (e.g., about 3, about 4, about 5, about 6, about 7, about 8, about 9, or about 10) dosage and/or frequency of rAPF administration may be modified to determine the effective amount of rAP for a patient with HPP. For example, the dose of rAPF may be increased, for example, from about 2.1 mg/kg/week or about 3.5 mg/kg/week to about 6 mg/kg/week or about 9 mg/kg/week.

Brief Pain Rating Questionnaire (BPI-SF)

Level of pain in a patient with HFF (e.g., an adult with HFF who is about 18 years of age or older, such as an adult with HFF onset in childhood, or an adolescent with HFF between about 13 years of age and about 17 years of age ) can be assessed using the Brief Pain Rating Questionnaire (BPI-SF). The BPI-SF is a questionnaire designed to assess the severity of pain and its impact on daily functioning. The BPI-SF consists of 11 items that use a numeric rating scale to assess pain severity (4 units) and pain intervention (7 units) 24 hours prior to questionnaire submission. The BPI-SF questionnaire provides information on the intensity of pain and the extent to which pain affects the daily functions of a patient with HFF (e.g., a patient with HFF who is about 13 years of age or older) on a numerical scale from 0 (no pain) to 10 (severe pain or significant interference caused by pain); lower scores indicate improved quality of life and reduced pain. For example, the BPI-SF scores for adolescents and adults with HFF are a composite of 11 pain scores.

A patient with HFF (eg, an adult with HFF who is about 18 years of age or older, such as an adult with HFF that started in childhood, or an adolescent with HFF between about 13 years of age and about 17 years of age) with a BPI-SF score greater than about 10 (e.g., a BPI-SF score of about 10, about 15, about 20, about 25, or about 30 or more) may be treated with rAPLP (such as TNAP, e.g., SEQ rAPF fusion polypeptide ID NO: 1 or a polypeptide variant having at least 95% sequence identity with the sequence of SEQ ID NO: 1, for example, asfotase alfa). For example, a HPP patient with a BPI-SF score of greater than about 10 (e.g., a BPI-SF score of about 10, about 15, about 20, about 25, or about 30 or more) may be treated with rAPF for a period treatment for at least two weeks (e.g., at least three weeks, at least four weeks, at least five weeks, at least six weeks, at least seven weeks, at least eight weeks at least nine weeks at least ten weeks at least three months at least four months at least five months at least six months at least seven months, at least eight months, at least nine months, at least one year, at least two years, at least three years, at least four years, at least five years at least six years, at least seven years, at least eight years, at least nine years, or alternatively at least ten years, or during the lifetime of the patient; in particular, at least six weeks).

The methods may result in an improvement in the BPI-SF score in a patient with HFF (e.g., in an adult with HFF who is about 18 years of age or older, such as an adult with HFF onset in childhood, or in an adolescent with HFF, between the ages of about 13 and about 17). For example, treatment with rAPLP (such as TNAP, e.g., an rAPF fusion polypeptide of SEQ ID NO: 1 or a polypeptide variant having at least 95% sequence identity with the sequence of SEQ ID NO: 1, e.g., asphotase alfa), such as treatment with rAP during a treatment period of at least two weeks (e.g., at least three weeks, at least four weeks, at least five weeks, at least six weeks, at least seven weeks at least eight weeks at least nine weeks at least ten weeks at least three months at least four months at least five months at least six months at least seven months, at least eight months, at least nine months, at least one year, at least two years, at least three years, at least four years, at least five years, at least six years, at least seven years at least eight years, at least nine years, or at least ten years, or during the lifetime of the patient; in particular, at least six weeks) can result in a decrease in BPI-SF by about - 2 or more (for example, by about - 2, about - 3, about - 4, about - 5, about - 6 or about - 7 or more).

A decrease in BPI-SF in a patient with HFF (e.g., an adult with HFF who is about 18 years of age or older, such as an adult with HFF onset in childhood, or an adolescent with HFF from the age of about 13 years to about 17 years) can be maintained throughout the period of administration of sAP (such as TNAP, for example, an rAPF fusion polypeptide of SEQ ID NO: 1 or a polypeptide variant having at least 95% sequence identity with the sequence of SEQ ID NO: 1, e.g., asfotase alfa), e.g., over a period of at least two weeks (e.g., at least three weeks, at least four weeks, at least five weeks, at least six weeks at least seven weeks, at least eight weeks, at least nine weeks, at least ten weeks, at least three months, at least four months, at least five months, at least six months, at least seven months at least eight months at least nine months at least one year at least two years at least three years at least four years at least five years, at least six years, at least seven years, at least eight years, at least nine years, or at least ten years, or during the lifetime of a patient having HPP; in particular, at least six weeks). For example, the BPI-SF score decreases by about -2 or more (e.g., about -2, about -3, about -4, about -5, about -6, or about -7 or more) and remains at ±10% relatively reduced BPI-SF during treatment with rAP.

Alternatively, if administration of rAPLP (such as TNAP, e.g., an rAPF fusion polypeptide of SEQ ID NO: 1 or a polypeptide variant having at least 95% sequence identity with SEQ ID NO: 1, e.g., asphotase alfa) does not result in decrease in BPI-SF by about - 2 or more (eg, about - 2, about - 3, about - 4, about - 5, about - 6 or about - 7 or more), the dosage and/or frequency of rAPF can be changed to determine the effective amount of rAP for a patient with HPP. For example, the dose of rAPF may be increased, for example, from about 2.1 mg/kg/week or about 3.5 mg/kg/week to about 6 mg/kg/week or about 9 mg/kg/week.

Alkaline phosphatase

Asfotase alfa is a human TNAP fusion polypeptide (hTNAP; SEQ ID NO: 1) formulated for the treatment of HPP. In particular, asphotase alfa (SEQ ID NO: 1) can be effectively used to treat hypophosphatasia (HPP), its symptoms and physical disorders associated with it, in an adult with HPP (for example, in an adult with HPP, aged about 18 years of age or older, such as an adult with HFF onset in childhood or an adolescent with HFF between the ages of about 13 years and about 17 years) for an extended period of time (e.g., at least six months, at least one year, at least two years, at least three years, at least four years, at least five years, at least six years, at least seven years, at least eight years, at least nine years, at least ten years, or more than ten years, for example, throughout the life of the patient).

Given the results described herein, treatments are not limited to administering a particular alkaline phosphatase (AP) or nucleic acid sequence encoding AP. Alkaline phosphatases encompass a group of enzymes that catalyze the cleavage of the phosphate moiety (eg pyrophosphate hydrolysis, NPP). There are four known mammalian alkaline phosphatase (AP) isoenzymes: tissue non-specific alkaline phosphatase (TNAP; detailed below), placental alkaline phosphatase (APAP) (for example, accession numbers P05187, NP_112603 and NP_001623), germ cell alkaline phosphatase (GAP) (for example , registration number P10696) and intestinal alkaline phosphatase (ALP) (for example, registration numbers P09923 and NP_001622). In addition to the exemplary ALP described above, any polypeptide having an identical or similar catalytic site structure and/or ALP enzymatic activity can be used (e.g., as an rAPP or an rAPF fusion polypeptide as defined herein) to treat patients with HFF, such as adults with HFF (for example, adults with HFF who are about 18 years of age or older, such as adults with HFF that started in childhood) or adolescents with HFF (for example, adolescents with HFF, between the ages of about 13 and about 17). Bone delivery conjugates containing sAP are further described in International Publication Nos: WO 2005/103263 and WO 2008/138131.

TNAP that can be used in accordance with the methods described herein include, for example, human TNAP (accession numbers NP_000469, AAI10910, AAH90861, AAH66116, AAH21289 and AAI26166); Rhesus TNSCHF (registration number XP_01109717); TNSAP rat (registration number NP_037191); TNSP dog (registration number AAF64516); porcine TNAP (accession number AAN64273), mouse (accession number NP_031457), bovine TNATF (accession numbers NP_789828, NP_776412, AAM 8209 and AAC33858), and feline TNATF (accession number NP_001036028). In particular, TNAP may be a recombinant human TNAP (e.g., SEQ ID NO: 1, asphotase alfa; see US Pat. adults with HFF (for example, adults with HFF who are about 18 years of age or older, such as adults with HFF that started in childhood) or adolescents with HFF (for example, adolescents with HFF who are from about 13 years of age up to about 17 years of age). The TNAP may also be one that has at least about 95% sequence identity with respect to the polypeptide or nucleic acid sequence of the above TNAP.

Soluble Alkaline Phosphatases

ALP that can be used in the methods described herein include soluble (eg, extracellular or non-membrane-bound) forms of any of the alkaline phosphatases described herein. sAP can be, for example, a soluble form of non-tissue-specific human alkaline phosphatase (human TNAP (hTNAP)). The methods are not limited to a particular sAP and may include any sAP that is physiologically active against, for example, phosphoethanolamine (PEA), inorganic pyrophosphate (nPP) and pyridoxal-5'-phosphate (P5P). In particular, rAP is catalytically competent to enhance skeletal bone mineralization. The methods further include nucleic acids encoding rAPF described herein, which can be used to treat the conditions described herein, for example, HPP, for example, adults with HPP (for example, adults with HPP, aged about 18 years or older, such as adults with HFF onset in childhood) or adolescents with HFF (eg, adolescents with HFF between about 13 and about 17 years of age).

TNAP is a membrane-bound protein anchored by a glycolipid moiety at the C-terminus (Swiss-Prot, P05186). This glycolipid anchor (GPI) is added post-translationally after the removal of the hydrophobic C-terminal end, which serves both as a temporary membrane anchor and as a signal for GPI addition. Although the GPI anchor is located in the cell membrane, the remaining portions of the TNAP are extracellular. In particular, TNAP (e.g., human TNAP (hTNAP)) can be modified to replace the first amino acid of the hydrophobic C-terminal sequence (alanine) with a stop codon, thereby producing a modified hTNAP that contains all amino acid residues of the native form of the attached TNAP and does not has a GPI membrane anchor. One of skill in the art will appreciate that the position of the GPI membrane anchor will vary between different APs and may include, for example, the last 10, 12, 14, 16, 18, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 32, 34, 36, 38, 40, 45, 50 or more amino acid residues at the C-terminus of the polypeptide. Recombinant rAPF may include, for example, amino acids 1 to 502 (18 to 502 when secreted), amino acids 1 to 501 (18 to 501 when secreted), amino acids 1 to 504 (18 to 504 when secreted), amino acids from 1 to 505 (18-505 when secreted) or amino acids from 1 to 502. Thus, the C-terminus of native ALP can be shortened by several amino acids without affecting ALP activity.

In addition to the C-terminal GPI anchor, TNAP also has an N-terminal signal peptide sequence. The N-terminal signal peptide is present in the synthesized protein when it is synthesized, but is cleaved from TNAP after translocation into the ER. rAPs include both secreted (ie, lacking an N-terminal signal) and non-secreted (ie, having an N-terminal signal) forms thereof. One skilled in the art will appreciate that the position of the N-terminal signal peptide will vary between different alkaline phosphatases and may include, for example, the first 5, 8, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 , 20, 21, 22, 23, 24, 25, 27, 30 or more amino acid residues at the N-terminus of the polypeptide. One skilled in the art can predict the position of the signal sequence cleavage site, for example, using an appropriate computer algorithm such as that described in Bendtsen et al. (J. Mol. Biol. 340(4):783-795, 2004) and is available on the Internet at www.cbs.dtu.dk/services/SignalP/.

The methods can also be implemented using rAPP consensus sequences derived from the extracellular domain of AP isozymes (eg, TNAP, APLP, APLP, APLP, etc.). Thus, similar to the rTNAP discussed above, this disclosure also provides other soluble human ALP isozymes, i.e. without a peptide signal, preferably containing the extracellular domain of AP. rAPP also includes polypeptide sequences that satisfy the consensus sequence derived from the extracellular domain of AP of human ALP isozymes and mammalian (human, mouse, rat, bovine, cat, and dog) orthologues of ALP or the consensus sequence derived from the extracellular domain of ALP only of mammalian (human) TNAP orthologues. , mice, rats, cows, cats and dogs). rAPs also include those that satisfy similar consensus sequences derived from various combinations of these TNAP orthologues or human AP isozymes. Such consensus sequences are given, for example, in WO 2008/138131.

sAP according to these methods may include not only the wild-type sAP sequence described above, but any polypeptide having at least 50% (e.g., 55%, 60%, 65%, 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more) sequence identity relative to these alkaline phosphatases (e.g., SEQ ID NO: 1-24; e.g., an rAPF fusion polypeptide of SEQ ID NO: 1 or a polypeptide variant having at least 95% sequence identity with SEQ ID NO: 1, e.g., asphotase alfa) . Examples of mutations that can be introduced into the ALP sequence are described in US Publication No. 2013/0323244, which is incorporated herein by reference in its entirety. sAP may optionally be glycosylated at any suitable one or more amino acid residues. In addition, rAPF may be at least 50% (e.g., 55%, 60%, 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86 %, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more) sequence identity to any of the described rAPs herein (such as TNAP, for example, an rAPF fusion polypeptide of SEQ ID NO: 1, or a polypeptide variant having at least 95% sequence identity with the sequence of SEQ ID NO: 1, for example, asfotase alpha). rAPLP can have 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more insertions, deletions, or substitutions relative to any of the rAPLP described herein (such as TNAP, e.g., rAPLP fusion polypeptide SEQ ID NO: 1 or a polypeptide variant having at least 95% sequence identity with the sequence of SEQ ID NO: 1, for example, asfotase alfa).

rAPF Fusion Polypeptides

Any of the sAP and linkers described herein can be combined into an sAPP polypeptide, for example, an sAPP A-rAPP-B polypeptide, where each of A and B is absent or is an amino acid sequence of at least one amino acid ( such as TNAP, for example, an rAPF polypeptide of SEQ ID NO: 1 or a polypeptide variant having at least 95% sequence identity with the sequence of SEQ ID NO: 1, for example, asfotase alfa). If present, A and/or B may be any linker described herein. Some polypeptides of rAP have no A, no B, or no A and B. The rAPF polypeptides of the invention may optionally contain an Fc region to produce an rAPF fusion polypeptide as described herein. The rAPF polypeptide may optionally contain a bone-targeting moiety, as described herein. In some polypeptides, an rAPF linker, eg, a flexible linker, can be inserted between the bone-targeting moiety and the rAPF, eg, as a dipeptide sequence (eg, leucine-lysine or aspartic acid-isoleucine). Other examples of Fc regions, linkers, and bone targeting moieties are described below.

Any of the sAP, linkers, and Fc regions described herein can be combined into a fusion polypeptide, e.g., a recombinant fusion polypeptide that contains the structure Z-sAP-Y-spacer-XW _n -V, ZW _n -X-spacer -Y-rAPLP-V, Z-rAPLP-YW _n -X-spacer-V, and ZW _n -X-rAPLP-Y-spacer-V (such as TNAP, e.g. having at least 95% sequence identity with the sequence of SEQ ID NO: 1, for example, asfotase alpha). In particular, the structure may be Z-rAP-Y-spacer-XW _n -V or ZW _n -X-spacer-Y-rAP-V. sAP can be full-length or functional fragments of AP, such as the soluble extracellular domain of AP, as described herein (eg, TNAP, APLP, APLP, and APLP). Any of X, Y, Z and V and/or the spacer may be absent or have an amino acid sequence of at least one amino acid. W _n may be a bone-targeting fragment, e.g., having a series of consecutive Asp or Glu residues, where n=1 to 50, e.g., n=3-30, e.g., 5-15, e.g., 1, 2, 3, 4 , 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22 , 23, 24, 25, 26, 27, 28, 29 , 30, 31, 32, 33, 34, 35, 36, 36, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50. The bone-targeting fragment, if present, may be located anywhere on the fusion polypeptide, for example, at the N-terminus or near the N-terminus or at the C-terminus and/or in the region of the linker. For example, the bone-targeting fragment is at the C-terminal end. rAPF polypeptides and fusion polypeptides may also lack a bone-targeting moiety.

The rAPF fusion polypeptides of the invention may have the structure hTNAP-Fc-D ₁₀ . In particular, rAPF fusion polypeptides may comprise the amino acid sequence of SEQ ID NO: 1 or a polypeptide variant having at least 95% sequence identity with the sequence of SEQ ID NO: 1, such as asphotase alfa.

Suitable spacers include, but are not limited to, Fc-containing polypeptides and hydrophilic and flexible polypeptides capable of attenuating the repulsive forces caused by the presence of a highly negatively charged terminal peptide (eg, W _n ). For example, rAPF may be a fusion polypeptide containing an immunoglobulin Fc region on an N-terminal or C-terminal domain. The immunoglobulin molecule has a structure that is well known in the art. It contains two light chains (~23 kDa each) and two heavy chains (~50-70 kDa each) connected by interchain disulfide bonds. Immunoglobulins are readily cleaved proteolytically (eg, by papain digestion) into Fab (containing the light chain and heavy chain VH and CH1 domains) and Fc (containing the heavy chain CH2 and CH3 domains along with adjacent sequences). Suitable Fc fragments as described herein include the Fc fragment of any immunoglobulin molecule, including IgG, IgM, IgA, IgD, or IgE, and their various subclasses (e.g., IgG-1, IgG-2, IgG-3, IgG-4 , IgA-1, IgA-2) from any mammal (eg human). For example, the Fc fragment is human IgG-1. Fc fragments of the invention may contain, for example, heavy chain CH2 and CH3 domains and any part of the hinge region. The Fc region may optionally be glycosylated at any suitable one or more amino acid residues known to those skilled in the art. In particular, the Fc fragment of the fusion polypeptide has the amino acid sequence of SEQ ID NO: 20, or has at least 50% (e.g., 55%, 60%, 65%, 70%, 75%, 80%, 81%, 82% , 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99 % or more) sequence identity relative to SEQ ID NO: 20. Modified, e.g., not naturally occurring, Fc regions can be used in the methods of the invention, e.g., as described in International Patent Application Publication No. WO2005/007809, which is incorporated herein. description by reference. The Fc fragment described herein may have 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 50 or more insertions, deletions or substitutions relative to any of the Fc fragments described herein.

The fused sAP polypeptides described herein (such as TNAP, e.g., an sAP polypeptide of SEQ ID NO: 1, or a polypeptide variant having at least 95% sequence identity with the sequence of SEQ ID NO: 1, e.g., asphotase alfa) can contain a peptide linker region between the Fc fragment. Additionally, a peptide linker region can be inserted between the Fc fragment and the optional bone-targeting fragment. The linker region can be of any sequence and length, which allows rAP to remain biologically active, for example, not subject to spatial complications. Typical linker lengths are from 1 to 200 amino acid residues, e.g. -50, 51-55, 56-60, 61-65, 66-70, 71-75, 76-80, 81-85, 86-90, 91-95, 96-100, 101-110, 111-120 , 121-130, 131-140, 141-150, 151-160, 161-170, 171-180, 181-190 or 191-200 amino acid residues. For example, linkers contain or consist of flexible portions, such as regions without significant fixed secondary or tertiary structure. Typical flexible linkers are glycine-rich linkers, for example, containing at least 50%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or even 100% glycine residues. Linkers may also contain, for example, serine residues. In some cases, the amino acid sequence of the linkers consists only of glycine and serine residues. The linker may optionally be glycosylated at any suitable one or more amino acid residues. In addition, the linker, as described herein, may contain any other sequence or fragment attached covalently or non-covalently. The linker may also be absent, with the Fc fragment and sAP fused together directly without intermediate residues. Certain Fc-rAPF or rAPF-Fc fusion polypeptides may be considered, according to this disclosure, to either 1) have no linker or 2) have a linker that corresponds to a portion of the rAPF. For example, an Fc fused directly to hsTNAP (1-502) can be considered, for example, as having no linker, with hsTNAP being amino acids 1-502, or as having a 17 amino acid linker, with hsTNAP (18-502) .

Additional amino acid residues may be introduced into the polypeptide in accordance with the cloning strategy used to obtain fused polypeptides. For example, additional amino acid residues do not provide an additional GPI attachment signal to maintain the polypeptide in soluble form. In addition, any such additional amino acid residues, when incorporated into a polypeptide of the invention, do not form a cleavage site for host cell endoproteases. The likelihood that a constructed sequence will be cleaved by host cell endoproteases can be predicted as described, for example, by Ikezawa (Biol. Pharm. Bull. 25:409-417, 2002).

sAP and sAP fusion polypeptides of the invention (such as TNAP, e.g., an sAP polypeptide of SEQ ID NO: 1, or a polypeptide variant having at least 95% sequence identity with the sequence of SEQ ID NO: 1, e.g., asphotase alfa) can be associated into dimers or tetramers. For example, two rAPF-Fc monomers can be covalently linked via two disulfide bonds located at the hinge regions of the Fc fragments. Additionally, a polypeptide or fusion polypeptide of the invention (eg, an sAP polypeptide or fusion polypeptide) may be glycosylated or pegylated.

Production of Nucleic Acids and Polypeptides

Nucleic acids encoding sAP and rAPF fusion polypeptides (such as TNAP, e.g. rAPF fusion polypeptide of SEQ ID NO: 1 or a polypeptide variant having at least 95% sequence identity with SEQ ID NO: 1, e.g. asphotase alfa), can be obtained by any method known in the art. Typically, a nucleic acid encoding the desired fusion polypeptide is obtained using molecular cloning techniques and is typically placed into a vector such as a plasmid or virus. The vector is used to transform the nucleic acid into a host cell suitable for expression of the fusion polypeptide. Representative methods are disclosed, for example, in Maniatis et al. (Cold Springs Harbor Laboratory, 1989). Many cell types can be used as suitable host cells, but mammalian cells are preferred because they are capable of providing appropriate post-translational modifications. Host cells may include, for example, a Chinese hamster ovary (CHO) cell, an L cell, a C127 cell, a 3T3 cell, a BHK cell, a COS-7 cell, or any other suitable host cell known in the art. For example, the host cell is a Chinese Hamster Ovary (CHO) cell (eg, CHO-DG44 cell).

sAP and sAP fused polypeptides (such as TNAP, e.g., an sAP fusion polypeptide of SEQ ID NO: 1 or a polypeptide variant having at least 95% sequence identity with the sequence of SEQ ID NO: 1, e.g., asphotase alfa) can be prepared in any conditions suitable for the expression of the rAPF polypeptide in the host cell. Such conditions include appropriate selection of media prepared with components such as buffer, bicarbonate and/or HEPES, ions such as chloride, phosphate, calcium, sodium, potassium, magnesium, iron, carbon sources such as simple sugars, amino acids, potentially lipids, nucleotides, vitamins and growth factors such as insulin; conventional commercial media such as alpha-MEM, DMEM, Ham's-F12 and IMDM supplemented with 2-4 mM L-glutamine and 5% fetal bovine serum; conventional commercially available animal protein-free media such as Hyclone™ SFM4CHO, Sigma CHO DHFR-, Cambrex POWER™ CHO CD supplemented with 2-4 mM L-glutamine. These media are preferably prepared without thymidine, hypoxanthine, and L-glycine to maintain selective pressure to ensure stable expression of the protein product.

Pharmaceutical compositions and formulations

A composition that can be used in the methods described herein (e.g., including sAP and an rAPF fusion polypeptide such as TNAP, e.g., an rAPP polypeptide of SEQ ID NO: 1 or a polypeptide variant having at least 95% sequence identity with the sequence of SEQ ID NO: 1, eg asfotase alfa) can be administered by various methods known in the art. As will be appreciated by one of skill in the art, the route of administration and/or mode of administration will vary depending on the desired results. The route of administration may depend on a variety of factors such as environment and therapeutic goals. In particular, the polypeptides and fusion polypeptides described herein may be administered by any route known in the art, such as subcutaneously (e.g., by subcutaneous injection), intravenously, orally, nasally, intramuscularly, sublingually, intrathecally, or intradermally. . For example, pharmaceutical compositions that can be used in the methods described herein may be in the form of a liquid, solution, suspension, pill, capsule, tablet, gelatin capsule, powder, gel, ointment, cream, spray, mist, vapor, aerosol, or phytosomes.

Dosage

Any amount of a pharmaceutical composition (e.g., including sAP or an rAPF fusion polypeptide such as TNAP, e.g., an rAPP polypeptide of SEQ ID NO: 1 or a polypeptide variant having at least 95% sequence identity with the sequence of SEQ ID NO: 1, e.g., asphotase alpha) can be administered to adults with HPP (eg, adults with HPP who are about 18 years of age or older, such as adults with HPP that started in childhood) or adolescents with HPP (eg, adolescents with HPP at between the ages of about 13 years and about 17 years). Dosages will depend on many factors including the route of administration and the age of the patient. For example, the rAPF polypeptides (such as TNAP, e.g., an rAPF polypeptide of SEQ ID NO: 1, or a polypeptide variant having at least 95% sequence identity with the sequence of SEQ ID NO: 1, e.g., asphotase alfa) described herein may be administered to a patient with HPP, such as an adult with HPP (for example, an adult with HPP who is about 18 years of age or older, such as an adult with HPP that started in childhood) or an adolescent with HPP (for example, a teenager having HFF, aged from about 13 years to about 17 years) as individual doses, for example, from 0.01 mg/kg to 500 mg/kg of the patient (for example, from 0.05 mg/kg to 500 mg/kg , from 0.1 mg/kg to 20 mg/kg, from 5 mg/kg to 500 mg/kg, from 0.1 mg/kg to 100 mg/kg, from 10 mg/kg to 100 mg/kg, from 0.1 mg/kg to 50 mg/kg, 0.5 mg/kg to 25 mg/kg, 1.0 mg/kg to 10 mg/kg, 1.5 mg/kg to 5 mg/kg or 2.0 mg/kg to 3.0 mg/kg) or 1 µg/kg to 1000 µg/kg (e.g. 5 µg/kg to 1000 µg/kg, 1 m kg/kg to 750 µg/kg, 5 µg/kg to 750 µg/kg) kg, 10 µg/kg to 750 µg/kg, 1 µg/kg to 500 µg/kg, 5 µg/kg to 500 mcg/kg, 10 mcg/kg to 500 mcg/kg, 1 mcg/kg to 100 mcg/kg, 5 mcg/kg to 100 mcg/kg, 10 mcg/kg to 100 mcg/kg, from 1 mcg/kg to 50 mcg/kg, 5 mcg/kg to 50 mcg/kg, or 10 mcg/kg to 50 mcg/kg patient).

Typical doses of rAPF 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 1000 µg/kg. In particular, compositions (e.g., containing rAPHF (such as TNAP, e.g., an rAPF polypeptide of SEQ ID NO: 1 or a polypeptide variant having at least 95% sequence identity with the sequence of SEQ ID NO: 1, e.g., asphotase alfa)) in according to this disclosure may be administered to patients at doses 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, sAP formulations (such as TNAP, eg sAP polypeptide SEQ ID NO: 1 or a polypeptide variant having at least 95% sequence identity with SEQ ID NO: 1, eg asfotase alfa) can be administered to patients on a weekly basis. dose, for example, from about 0.5 mg/kg/week to about 140 mg/kg/week, for example, 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 (eg, about 6 or about 9 mg/kg/week). In particular, sAP (such as TNAP, eg sAP polypeptide of SEQ ID NO: 1 or a polypeptide variant having at least 95% sequence identity with the sequence of SEQ ID NO: 1, eg asfotase alfa) may be administered at a dosage of 2 mg kg three times a week (total dose 6 mg/kg/week), 1 mg/kg six times a week (total dose 6 mg/kg/week), 3 mg/kg three times a week (total dose 9 mg/week). kg/week), 0.5 mg/kg three times a week (total dose 1.5 mg/kg/week) or 9.3 mg/kg three times a week (total dose 28 mg/kg/week). The dosage will be adapted by the attending physician in accordance with generally accepted factors such as the degree of disease and other parameters of the patient with HPP, such as an adult with HPP (for example, an adult with HPP, aged about 18 years or older, for example, an adult with HFF onset in childhood) or an adolescent who has HFF (eg, an adolescent with HFF between about 13 and about 17 years of age).

Dosages of compositions containing sAP and sAP fused polypeptides (such as TNAP, e.g., an sAP polypeptide of SEQ ID NO: 1, or a polypeptide variant having at least 95% sequence identity with SEQ ID NO: 1, e.g., asphotase alfa), can be implemented in schemes of single or multiple doses of the drug. Doses may be administered, for example, every hour, every other hour, once a day, twice a day, twice a week, three times a week, four times a week, five times a week, six times a week, weekly, once every two weeks, monthly, every two months or once a year. Alternatively, doses can be administered, for example, two, three times, four times, five times, six times, seven times, eight times, nine times, 10 times, 11 times, or 12 times a day. In particular, the dosage regimen is to take the drug once a week. The duration of the drug 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, weeks, months, or even the remaining life span of a patient with HFF, such as an adult with HFF (for example, an adult with HFF, aged about 18 years of age or older, such as an adult with HFF onset in childhood) or an adolescent with HFF (eg, an adolescent with HFF between about 13 and about 17 years of age). The amount, frequency, and duration of dosage will be adapted by the attending physician in accordance with generally accepted factors such as the degree of disease and other parameters of the patient with HPP, such as an adult with HPP (for example, an adult with HPP, aged about 18 years or older, for example, an adult with HPF onset in childhood) or an adolescent with HPF (eg, an adolescent with HPF between about 13 and about 17 years of age).

For example, sAP or an sAP fusion polypeptide (such as TNAP, e.g., an sAP polypeptide of SEQ ID NO: 1 or a polypeptide variant having at least 95% sequence identity with the sequence of SEQ ID NO: 1, e.g., asphotase alfa) can be Prepared in the form of a solution for injection, which is a clear, colorless to slightly yellow aqueous solution, pH 7.4. sAP or an rAPP polypeptide (such as TNAP, e.g., an rAPF polypeptide of SEQ ID NO: 1, or a polypeptide variant having at least 95% sequence identity with the sequence of SEQ ID NO: 1, e.g., asphotase alfa), can be prepared at a concentration 12 mg/0.3 ml, 18 mg/0.45 ml, 28 m/0.7 ml, 40 mg/1 ml or 80 mg/0.8 ml. In particular, the composition can be prepared as a 40 mg/ml solution for injection, with each ml of solution containing 40 mg of sAP or sAP polypeptide (for example, each vial contains 0.3 ml of solution and 12 mg of sAP (40 mg/ml) , each vial contains 0.45 ml of solution and 18 mg of rABF (40 mg/ml), each vial contains 0.7 ml of solution and 28 mg of rABF (40 mg/ml), or each vial contains 1.0 ml of solution and 40 mg asfotase alfa (40 mg/ml)). sAP or an rAPP polypeptide (such as TNAP, e.g., an rAPP polypeptide of SEQ ID NO: 1 or a polypeptide variant having at least 95% sequence identity with the sequence of SEQ ID NO: 1, e.g., asphotase alfa) can be prepared as solution for injection at a concentration of 100 mg / ml, while each 1 ml of the solution contains 100 mg of sAP or sAP polypeptide (for example, each vial contains 0.8 ml of the solution and 80 mg of asphotase alfa (100 mg / ml)). The volume of rAPF injection administered to the patient may be, for example, 0.15 ml, 0.18 ml, 0.20 ml, 0.23 ml, 0.25 ml, 0.28 ml, 0.30 ml, 0.33 ml, 0.35 ml, 0.38 ml, 0.40 ml, 0.43 ml, 0.45 ml, 0.48 ml, 0.50 ml, 0.63 ml, 0.75 ml, 0.88 ml or 1.00 ml.

For example, the recommended dosage of sAP or an sAP fusion polypeptide ((such as TNAP, e.g., an sAP polypeptide of SEQ ID NO: 1 or a polypeptide variant having at least 95% sequence identity with the sequence of SEQ ID NO: 1, e.g., asphotase alfa ) is 2 mg/kg body weight administered subcutaneously three times a week, or a regimen of 1 mg/kg body weight administered subcutaneously six times a week.Additional dosage information is provided below (Table 1). weighing 40 kg, who was administered a dose of 6 mg/kg/week, would receive an injection of 80 mg rAPF in 0.8 ml three times a week or 40 mg rAPF in 1.00 ml six times a week, while a patient weighing 50 kg dosed at 6 mg/kg/week would receive an injection of 50 mg rAP in 0.05 ml six times a week.

Table 1. DOSAGE OF ASPHOTASE ALPHA

Body weight (kg) With injections 3 times a week With injections 6 times a week Administered dose Injection volume Type of vessel used for injection Enter
May dose Enter
my volume Vessel type used
retractable for injection 3 6 mg 0.15 ml 0.3 ml four 8 mg 0.20 ml 0.3 ml 5 10 mg 0.25 ml 0.3 ml 6 12 mg 0.30 ml 0.3 ml 6 mg 0.15 ml 0.3 ml 7 14 mg 0.35 ml 0.45 ml 7 mg 0.18 ml 0.3 ml eight 16 mg 0.40 ml 0.45 ml 8 mg 0.20 ml 0.3 ml 9 18 mg 0.45 ml 0.45 ml 9 mg 0.23 ml 0.3 ml ten 20 mg 0.50 ml 0.7 ml 10 mg 0.25 ml 0.3 ml eleven 22 mg 0.55 ml 0.7 ml 11 mg 0.28 ml 0.3 ml 12 24 mg 0.60 ml 0.7 ml 12 mg 0.30 ml 0.3 ml 13 26 mg 0.65 ml 0.7 ml 13 mg 0.33 ml 0.45 ml fourteen 28 mg 0.70 ml 0.7 ml 14 mg 0.35 ml 0.45 ml fifteen 30 mg 0.75 ml 1 ml 15 mg 0.38 ml 0.45 ml 16 32 mg 0.80 ml 1 ml 16 mg 0.40 ml 0.45 ml 17 34 mg 0.85 ml 1 ml 17 mg 0.43 ml 0.45 ml eighteen 36 mg 0.90 ml 1 ml 18 mg 0.45 ml 0.45 ml 19 38 mg 0.95 ml 1 ml 19 mg 0.48 ml 0.7 ml twenty 40 mg 1.00 ml 1 ml 20 mg 0.50 ml 0.7 ml 25 50 mg 0.50 ml 0.8 ml 25 mg 0.63 ml 0.7 ml thirty 60 mg 0.60 ml 0.8 ml 30 mg 0.75 ml 1 ml 35 70 mg 0.70 ml 0.8 ml 35 mg 0.88 ml 1 ml 40 80 mg 0.80 ml 0.8 ml 40 mg 1.00 ml 1 ml fifty 50 mg 0.50 ml 0.8 ml 60 60 mg 0.60 ml 0.8 ml 70 70 mg 0.70 ml 0.8 ml 80 80 mg 0.80 ml 0.8 ml 90 90 mg 0.90 mg 0.8 ml (x2) 100 100 mg 1.00 ml 0.8 ml (x2)

Preparations

Compositions containing sAP and rAPP fusion polypeptides (such as TNAP, e.g., an rAPP polypeptide of SEQ ID NO: 1, or a polypeptide variant having at least 95% sequence identity with SEQ ID NO: 1, e.g., asphotase alfa) may be prepared according to standard methods. The pharmaceutical composition is well known in the art and is further described in, for example, Gennaro (2000) Remington: The Science and Practice of Pharmacy, 20th Edition, Lippincott, Williams & Wilkins (ISBN: ^0683306472 ); Ansel et al. (1999) Pharmaceutical Dosage Forms and Drug Delivery Systems, 7th Edition, Lippincott Williams & Wilkins Publishers (ISBN: ^0683305727 ); and Kibbe (2000) Handbook of Pharmaceutical Excipients American Pharmaceutical Association, 3rd Edition (ISBN: ^091733096X ). For example, an sAP composition (such as TNAP, e.g., an rAPF polypeptide of SEQ ID NO: 1, or a polypeptide variant having at least 95% sequence identity with the sequence of SEQ ID NO: 1, e.g., asphotase alfa) can be prepared, for example, in the form of a buffer solution in a suitable concentration and suitable for storage at a temperature of 2-8°C (for example, 4°C). The composition can also be prepared for storage at temperatures below 0°C (eg - 20°C or - 80°C). The composition can be further prepared for storage up to 2 years (e.g., one month, two months, three months, four months, five months, six months, seven months, eight months, nine months, 10 months, 11 months, 1 year, 1½ years or 2 years) at 2-8°C (e.g. 4°C). Thus, the compositions described herein can be storage stable for at least 1 year at 2-8°C (eg, 4°C).

Compositions containing sAP and rAPF fusion polypeptides (such as TNAP, e.g., an rAPP polypeptide of SEQ ID NO: 1, or a polypeptide variant having at least 95% sequence identity with the sequence of SEQ ID NO: 1, e.g., asphotase alfa) may be presented in various forms. These forms include, for example, liquid, semi-solid and solid dosage forms such as liquid solutions (eg solutions for injection and infusion), dispersions or suspensions, tablets, pills, powders, liposomes and suppositories. The preferred form depends in part on the intended route of administration and therapeutic application.

For example, compositions intended for systemic or topical delivery may be in the form of injectable or infusion solutions. Accordingly, compositions (such as TNAP, e.g., an rAPF polypeptide of SEQ ID NO: 1, or a polypeptide variant having at least 95% sequence identity with the sequence of SEQ ID NO: 1, e.g., asfotase alfa) can be formulated for parenteral administration. (eg, by subcutaneous, intravenous, intraperitoneal, or intramuscular injection).

Compositions containing sAP and rAPF fusion polypeptides (such as TNAP, e.g., an rAPF fusion polypeptide of SEQ ID NO: 1 or a polypeptide variant having at least 95% sequence identity with SEQ ID NO: 1, e.g., asphotase alfa) may be prepared as a solution, microemulsion, dispersion, liposome, or other ordered structure suitable for stable storage at high concentration. Sterile injectable solutions can be prepared by adding the composition described herein in the required amount in a suitable solvent with one or a combination of the ingredients listed above, as needed, followed by filter sterilization. Typically, dispersions are prepared by incorporating the composition described herein into a sterile vehicle that contains the basic dispersion medium and the required other ingredients from those listed above. In the case of sterile powders for the preparation of sterile injectable solutions, methods of preparation include vacuum drying and freeze drying, which allow the powder of the composition described herein, plus any additional desired ingredient (see below), to be obtained from its pre-sterile filtered solution. Suitable fluidity of the solution can be maintained, for example, by using a coating such as lecithin, by maintaining the required particle size in the case of a dispersion, and by using surfactants. Prolonged absorption of injectable compositions can be achieved by including in the composition a reagent that delays absorption, such as monostearate and gelatin salts.

The compositions described herein can also be formulated as immunoliposome compositions. Such preparations can be prepared by methods known in the art, such as, for example, those described in Epstein et al. (1985) Proc Natl Acad Sci USA 82:3688; Hwang et al. (1980) Proc Natl Acad Sci USA 77:4030; and U.S. Patent Nos. 4,485,045 and 4,544,545. Liposomes with extended circulation time are disclosed in, for example, US Pat. No. 5,013,556.

Compositions containing sAP and sAP fused polypeptides (such as TNAP, e.g., an sAP polypeptide of SEQ ID NO: 1 or a polypeptide variant having at least 95% sequence identity with the sequence of SEQ ID NO: 1, e.g., asphotase alfa), also may be formulated with a carrier that will protect the composition (eg, rAPF polypeptide or rAPF fusion polypeptide) from rapid release, eg, as a controlled release formulation, including implants and microencapsulated delivery systems. Biodegradable, biocompatible polymers such as ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters, and polylactic acid can be used. Many methods for the preparation of such preparations are known in the art. See, for example, JR Robinson (1978) Sustained and Controlled Release Drug Delivery Systems, Marcel Dekker, Inc., New York.

When the compositions are to be used in combination with a second active agent, the compositions may be formulated with the second agent, or the compositions may be prepared separately from the second agent formulation. For example, the respective pharmaceutical compositions may be mixed, eg just prior to administration, and administered together, or may be administered separately, eg at the same or different times.

Carriers/fillers

Formulations containing sAP or an rAPP fusion polypeptide (such as TNAP, e.g., an rAPP polypeptide of SEQ ID NO: 1, or a polypeptide variant having at least 95% sequence identity with SEQ ID NO: 1, e.g., asphotase alfa) can be given to patients with HFF, such as an adult with HFF (eg, an adult with HFF who is about 18 years of age or older, such as an adult with HFF that started in childhood), or an adolescent with HFF (eg, a teenager having HPP, aged from about 13 years to about 17 years) in combination with pharmaceutically acceptable sterile aqueous or non-aqueous diluents, suspensions or emulsions. Examples of non-aqueous solvents are propylene glycol, polyethylene glycol, vegetable oil, fish oil, and injectable organic esters. Aqueous vehicles include water, hydroalcoholic solutions, emulsions, or suspensions, including saline and buffered medical parenteral vehicles, including sodium chloride solution, dextrose Ringer's solution, dextrose plus sodium chloride, lactose-containing Ringer's solution, or fixed oils. For example, a pharmaceutically acceptable carrier may comprise sodium chloride and/or sodium phosphate, wherein the composition contains, for example, about 150 mM sodium chloride and/or about 25 mM sodium phosphate, pH 7.4.

Intravenous vehicles may contain fluid and nutrient replenishers, electrolyte replenishers such as those based on Ringer's dextrose, and the like. Pharmaceutically acceptable salts may also be included, for example mineral acid salts such as hydrochlorides, hydrobromides, phosphates, sulfates and the like; and organic acid salts such as acetates, propionates, malonates, benzoates and the like. In addition, adjuvants such as wetting or emulsifying agents, a pH buffering agent, and the like may be present in such carriers. A detailed discussion of pharmaceutically acceptable carriers is available in Remington's Pharmaceutical Sciences (Mack Pub. Co., N.J. 1991).

The following examples are for illustrative purposes, and should not be construed as limiting this disclosure. These studies describe the administration of asphotase alfa (SEQ ID NO: 1) to adults with HPP, such as adults with childhood-onset HPP, around 18 years of age or older, or adolescents with HPP, analyzing the symptoms of HPP in these patients after the administration of asfotase alfa and associated physical disturbances for a long period of time.

Example 1 Treatment of Childhood Onset Hypophosphatasia (HPP) Patients with Asphotase Alfa

Adolescents and adults with hypophosphatasia (HPP) aged from about 12 to about 66 years participated in a study to determine the efficacy, safety and tolerability of treatment with soluble alkaline phosphatase (sAP) (asfotase alfa; SEQ ID NO: 1). A subpopulation analysis was performed on 12 adults with childhood onset hypophosphatasia (HPP), aged from about 18 to about 66 years, to examine the biochemical and functional improvements resulting from alpha-asphotase treatment. At baseline, for 6 months, 9 adults with HPP were treated with asfotase alfa (treatment group) and 3 adults with HPP were not treated with asfotase alfa (control group). The mean age of onset of HFF symptoms in the control group was 3 years, with a minimum age of 0.8 years and a maximum age of 4 years, while the mean age of onset of HFF symptoms in the treatment group was 2 years, with a minimum age of 0.1 years and a maximum age of 4 years (Table 2, dosage groups (2.1 mg/kg/week and 3.5 mg/kg/week) were combined for all results). Data are presented as median (min, max) unless otherwise noted. 2 untreated adults were women (67%) and 7 treated adults were women (78%).

Table 2. Baseline scores in patients with childhood-onset HPP prior to treatment with asphotase alfa

Index Initial distribution into groups Total
(N=12) 5 Control
(n=3) Combined treatment
(n=9) Turn on age, years 21 (13, 58) 55 (14, 66) 53 (13, 66) Turn on age, years 54 (26, 58) 56 (45, 66) 55 (26, 66) Age category at inclusion, n (%)
Adults (age ≥18 years) 3 (100) 9 (100) 12 (100) Age of symptom onset, years (min, max) 3 (0.8, 4) 2 (0.1, 13) 2.5 (0.1, 13(10)) Age category at onset of symptoms, n (%) <18 years 3 (100) 9 (100) 12 (100) Women, n (%) 2 (67) 7 (78) 9 (75)

HPP patients selected for treatment with asphotase alfa had serum ALP levels below the age-adjusted normal range, plasma pyridoxal-5'-phosphate (P5P) concentrations at least twice the upper limit of normal, and evidence of osteopenia or osteomalacia on skeletal radiographs and/or bone biopsies. Patients were excluded from the study if serum calcium or phosphate levels were below normal levels, serum vitamin D levels were less than 20 ng/mL, serum creatinine or parathyroid hormone levels were above the upper limit of normal, or medical conditions or other extenuating circumstances that may significantly affect patient compliance with the study protocol.

At the initial stage of treatment with asfotase alfa for 6 months, 4 adults received asfotase alfa at a dosage of 0.3 mg/kg/day (2.1 mg/kg/week), and 5 adults received asfotase alfa at a dosage of 0.5 mg/kg /day (3.5 mg/kg/week) by subcutaneous injection. 3 patients in the initial control group did not receive asphotase alfa treatment. After the initial phase of the study, patients in both the treatment group and the control group (total 12 patients) were treated with asfotase alfa. Asfotase alfa was administered subcutaneously at an initial dose of 0.5 mg/kg/day (3.5 mg/kg/week), which was increased after 1 year to 1 mg/kg/day, subcutaneously, once a day for 6 days ( 6 mg/kg/week). The dosage of asfotase alfa was maintained at 6 mg/kg/week during the remainder of the prolongation phase of treatment.

The parameters used during the prolongation phase of treatment to assess the treatment of adult patients with HPP with asphotase alfa included: AP; 2) 6-minute walking test (6MWT) to assess walking ability; and 3) scores on the Bruinks-Ozeretsky Motor Test, 2nd edition (BOT-2), to assess physical ability.

Example 2 Statistically Significant Decrease in Median Plasma Concentrations of Inorganic Pyrophosphate (NPP) and P5P (P5P) Following Administration of Asphotase Alfa

ALP activity in plasma samples from adults with HPP was assessed by quantifying the concentrations of the ALP substrates nPP and P5P as described in Whyte et al., 1995 (J. Clin. Invest. 95(4): 1440-1445), incorporated herein by reference in its entirety. The median concentrations of nPP and P5P in plasma samples of adults in the control group and adults treated with asfotase alfa were above the normal range at baseline (Table 3). Similarly, ALP activity values were relatively low for the control group (21.0 U/L) and for the treatment group (18.0 U/L) compared to the lower limit of normal (LNL) values of ALP activity adjusted for age and sex ( 31 U/L for ages 18 to less than 50 years or 35 U/L for ages 50 to less than 70 years).

Table 3 Measurements of plasma alkaline phosphatase (AP) activity and concentrations of inorganic pyrophosphate (NPP) and pyridoxal-5'-phosphate (P5P) in adults with childhood-onset HPP prior to treatment with asphotase alfa. All data are presented as median (minimum, maximum) unless otherwise noted. ^a Initial treatment groups are pooled for analysis. ^b Control and treatment adults are pooled because all adults are treated in the extension phase. NPN, lower limit of normal.

Index Initial distribution into groups Total
(N=12) ^b Control without treatment
(n=3) Combined treatment
(n=9) ^a ALP, U/l
NNP (taking into account age and gender)
18 to <50 years: 31 (M), 31 (F)
≥50 to <70 years: 35 (M), 35 (F) 21.0
(18.0, 26.0) 18.0
(18.0, 35.0) 18.0
(18.0, 35.0) NPF, µM
Normal range 13-18 years old: <0.75-4.78
>18 years old: 1.00-5.82 5.9
(5.2, 6.6) 5.5
(2.2, 8.2) 5.7
(2.2, 8.2) P5F, ng/mL Normal range: 5-18 years: 5.7-61.2
>18 years old: 2.8-26.7 199.0
(196.0, 474.0) 267.0
(28.8, 1270.0) 233.0
(28.8, 1270.0)

The median plasma nPF concentration was 5.9 μM (minimum plasma nPF concentration 5.2 μM; maximum nPF plasma concentration 6.6 μM) for the control group at baseline and 5.5 μM (minimum plasma nPF concentration 2, 2 μM; maximum plasma concentration of nPF 8.2 μM) for the treatment group at baseline (Table 3). Plasma nPF concentration decreased after 6 months of treatment with asfotase alfa, as evidenced by a statistically significant change in median from baseline of -3.0 for patients treated with asfotase alfa compared with a change of -0.3 for controls , untreated (Fig. 1A). The P-value for change in median NPF concentration from baseline was 0.0364 based on the Mann-Whitney exact U test when comparing the treatment group with the control group. Analysis of plasma concentrations of NPF after 6 months of treatment with asphotase alfa for two different dosage groups showed a median change from baseline of -2.870 for patients treated with 0.3 mg/kg/day asfotase alfa, and - 2,990 for patients treated with asphotase alfa 0.5 mg/kg/day (Table 4, P-value based on Mann-Whitney exact U-test, comparing each treatment group with the control group; estimate and exact confidence interval obtained Hodges-Lehmann-Sen method to shift the position in the distribution between the treatment group and the control group).

Table 4. Statistical analysis of NPF concentrations in adults with childhood-onset HPP after treatment with asphotase alfa for 6 months

Statistical indicators Control Group (n=3) Asfotase Alpha 0.3 mg/kg/
day (n=4) Asfotase Alpha 0.5 mg/kg/day (n=5) Asphotase Alpha Combined (n=9) n 3 four 5 9 Average (SD) -0.060 (1.0193) -2.790 (1.4903) -2.258 (1.4739) -2.494 (1.4134) 95% CI (-2.592, 2.472) (-5.161, -0.419) (-4.088, -0.428) (-3.581, -1.408) Median -0.340 -2,870 -2,990 -2,990 Min, Max -0.91, 1.07 -4.40, -1.02 -3.18, 0.32 -4.40, 0.32 P value 0.0571 0.1429 0.0364 Grade -2.925 -2.270 -2.640 95% CI ^b (BUT, BUT) (-4.250, 1.230) (-4.250, -0.110)

The median plasma P5F concentration was 199.0 ng/mL (minimum plasma P5F concentration 196.0 ng/mL; maximum P5F plasma concentration 474.0 ng/mL) for the control group at baseline and 267.0 ng/mL (minimum plasma P5F concentration 28.8 ng/ml; maximum plasma P5F concentration 1270.0 ng/ml) for the treatment group at baseline (Table 3). Plasma P5P concentration decreased after 6 months of treatment with asphotase alfa, as evidenced by a statistically significant change in median from baseline of -254.5 for patients treated with asfotase alfa compared to a change of 140.0 for controls ( Fig. 1B). The P-value for change in median P5F concentration from baseline was 0.0364 based on the exact Mann-Whitney U test when comparing the treatment group with the control group. Analysis of plasma P5P concentration after 6 months of treatment with asphotase alfa for two different dosage groups showed a median change from baseline of -298.60 for patients treated with 0.3 mg/kg/day asfotase alfa , and - 254.50 for patients treated with asfotase alfa 0.5 mg/kg/day (Table 5, P value based on Mann-Whitney exact U-test, comparing each treatment group with the control group; score and exact confidence interval obtained by the Hodges-Lehmann-Sen method for shifting the position in the distribution between the treatment group and the control group).

Table 5. Statistical analysis of P5F concentrations in adults with childhood-onset HPP after treatment with asfotase alfa for 6 months

Statistical indicators Control Group (N=3) Asfotase Alpha 0.3 mg/kg/day (N=4) Asfotase Alpha 0.5 mg/kg/day (N=5) Asphotase Alpha Combined (N=9) n 3 four 5 9 Average (SD) 123.60 (231.037) -301.30 (186.482) -383.72 (492.195) -347.09 (368.857) 95% CI (-450.33, 697.53) (-598.03, -4.57) (-994.86, 227.42) (-630.62, -63.56) Median 140.00 -298.60 -254.50 -254.50 Min, Max -115.2, 346.0 -486.4, -121.6 -1236.2, -17.2 -1236.2, -17.2 P value 0.0571 0.1429 0.0364 Grade -419.40 -394.50 -394.50 95% CI (BUT, BUT) (-1582.20, 98.00) (-1121.00, -6.40)

^a P value based on the exact Mann-Whitney U-test, comparing each treatment group with the control group; ^b estimate and exact confidence interval obtained by the Hodges-Lehmann-Sen method for shifting the position in the distribution between the treatment group and the control group.

For all subsequent measurements, plasma concentrations of nPF and P5F were pooled for the treatment and control groups at each respective time point (6 months, 1 year, 2 years, 3 years, 4 years, 5 years, and 6 years of asfotase alfa treatment) because the control group received asfotase alfa treatment after an initial phase of 6 months. The decrease in plasma nPF concentration was maintained throughout the prolongation phase of asphotase alfa treatment (FIG. 2). The median plasma nPF concentration decreased from 5.53 µM (minimum plasma nPF concentration 2.15 µM; maximum nPF plasma concentration 8.20 µM) to 3.30 µM (minimum plasma nPF concentration 0.75 µM; maximum plasma concentration plasma NPF 5.02 μM) after 6 months of treatment with asphotase alfa. Median NPF concentration was 3.74 µM at 1 year (minimum plasma nPF concentration 1.62 µM; maximum plasma nPF concentration 5.40 µM); median nPF concentration was 2.49 μM after 2 years (minimum plasma nPF concentration 1.32 μM; maximum plasma nPF concentration 10.90 μM); the median concentration of nPF was 2.09 μM after 3 years (minimum plasma nPF concentration 1.24 μM; maximum plasma nPF concentration 2.94 μM); the median concentration of NPF was 2.49 μM after 4 years (minimum plasma concentration of NPF 1.09 μM; maximum concentration of NPF in plasma 4.52 μM); median nPF concentration was 2.2 µM at 5 years (minimum plasma nPF concentration 0.92 µM; maximum plasma nPF concentration 5.36 µM); and the median nPF concentration was 2.60 µM at 5 years (minimum plasma nPF concentration 1.95 µM; maximum plasma nPF concentration 4.84 µM). The sample size was 9 adults with childhood-onset HFF for 6 months, 12 adults with childhood-onset HFF for 1 to 5 years, and 3 adults with childhood-onset HFF for 6 years.

Similarly, the reduction in plasma P5F concentration was maintained throughout the prolongation phase of asfotase alfa treatment (FIG. 3). The median plasma P5F concentration decreased from 267.0 ng/mL (minimum plasma P5F concentration 28.8 ng/mL; maximum plasma P5F concentration 1270.0 ng/mL) to 21.6 ng/mL (minimum plasma P5F concentration in plasma 2.5 ng/ml; maximum plasma concentration of P5F 141.0 ng/ml) after 6 months of treatment with asfotase alfa. The median P5F concentration was 65.1 ng/ml at 1 year (minimum plasma P5F concentration 15.9 ng/ml; maximum plasma P5F concentration 367.0 ng/ml); median P5F concentration was 20.1 ng/mL at 2 years (minimum plasma P5F concentration 2.7 ng/mL; maximum plasma P5F concentration 45.7 ng/mL); median P5F concentration was 14.0 ng/mL at 3 years (minimum plasma P5F concentration 2.5 ng/mL; maximum plasma P5F concentration 36.2 ng/mL); median P5F concentration was 12.0 ng/mL at 4 years (minimum plasma P5F concentration 2.5 ng/mL; maximum plasma P5F concentration 38.4 ng/mL); median P5F concentration was 7.8 ng/mL at 5 years (minimum plasma P5F concentration 2.5 ng/mL; maximum plasma P5F concentration 29.5 ng/mL); and the median P5F concentration was 6.0 ng/ml at 6 years (minimum plasma P5F concentration 4.7 ng/ml; maximum plasma P5F concentration 15.1 ng/ml). The sample size was 9 adults with childhood-onset HFF for 6 months and 1 year, 12 adults with childhood-onset HFF for ages 2 to 5 years, and 3 adults with childhood-onset HFF for six years.

Example 3 Improvement in Physical and Walking Ability in Adult Patients with Childhood-Onset HPF Using the 6 Minute Walk Test (6MWT)

Physical and walking ability in childhood-onset HFF patients was assessed using the 6-minute walking test (6MWT). Patients treated with asfotase alfa improved their average walking distance from baseline of 315.0 meters in 6 minutes (minimum distance 223.0, maximum distance 540.0) to an average walking distance of 421.5 meters in 6 minutes after 1 year of treatment with asfotase alfa (minimum distance 204.0, maximum distance 640.0). In contrast, the untreated control group showed no improvement from an average walking distance of 401.0 meters in 6 minutes at baseline (minimum distance 6.0, maximum distance 440.0) to an average walking distance of 355. 0 meters for 6 minutes after 6 months of treatment with asphotase alfa (minimum distance 13.0, maximum distance 420.0).

The mean change from baseline after 1 year of treatment with asfotase alfa was 88.0 meters walked in 6 minutes, while the mean change from baseline in the control group was 20.0 meters walked in 6 minutes. 6 minutes after 6 months without treatment (Table 6). This change also includes 3 patients in the control group who were unable to walk for 6 minutes at the start of the study. The mean change from baseline in the 6MWT test for the treated group was 17.6% with an increase from 62.8% predicted 6MWT (minimum predicted 6MWT 42.4%, maximum predicted 6MWT 101.3%) at baseline to 85.0% predicted 6MWT (minimum predicted 6MWT 31.1%, maximum predicted 6MWT 121.3%) after 1 year of treatment with asphotase alfa. In contrast, the mean change from baseline in the 6MWT test for the untreated control group was -6.2 with a decrease from 85.8% predicted 6MWT (minimum predicted 6MWT 79.1%, maximum predicted 6MWT 92 .4%) at baseline to 79.6% predicted 6MWT (minimum predicted 6MWT 70.4%, maximum predicted 6MWT 88.7%) after 1 year of treatment with asphotase alfa. In addition, the change from baseline in the predicted percentage of 6MWT at 6 months between controls and pediatric onset HPF treated with asfotase alfa was statistically significant (p=0.04). Thus, after 1 year of treatment with asphotase, patients with HFF showed a statistically significant improvement in walking ability, while the walking ability of patients in the control group worsened or did not improve.

Table 6 Distance Walked in Six Minutes in Adults with Childhood-Onset HPP After Treatment with Asphotase Alfa for 6 Months

Control without treatment Combined treatment meters
(n=3) % Predicted (n=2) Meters ^a (n=9) % predicted ^b (n=8) Baseline 401.0
(6.0, 440.0) 85.8
(79.1, 92.4) 315.0
(223.0, 540.0) 62.8
(42.4, 101.3) Follow-up (control: 6 months; treatment: 1 year) 355.0
(13.0, 420.0) 79.6
(70.4, 88.7) 421.5
(204.0, 640.0) 85.0
(31.1, 121.3) Mean change from baseline -20.0
(-46.0, 7.0) -6.2
(-8.7, -3.7) 88.0
(-123.0, 197.0) 17.6
(-23.1, 29.8)

^a n=12 for data for year 1; ^b n=10 for year 1 data and n=9 for change from baseline data.

For 6MWT values after baseline, 6MWT values were pooled for the treatment group and control group at each respective time point (1 year, 2 years, 3 years, 4 years, 5 years and 6 years of asfotase alfa treatment) as the control group received treatment asphotase alfa after an initial phase of 6 months. The change in 6MWT represents an increase from 62.9% of predicted 6MWT (minimum predicted 6MWT 42.4%, maximum predicted 6MWT 101.3%; n=8) at baseline to 80.8% predicted 6MWT (minimum predicted 6MWT value 46.0%, maximum predicted 6MWT value 109.0%; n=8) after 6 months of treatment with asphotase alfa (FIG. 4). The increase in 6MWT was maintained throughout treatment with asfotase alfa at 85% of predicted 6MWT (minimum predicted 6MWT 31.1%; maximum predicted 6MWT 121.3%; n=10) at 1 year; 91.8% of 6MWT predicted (minimum 6MWT predicted 63.3%; maximum 6MWT predicted 120.8%; n=9) after 2 years; 93.5% of 6MWT predicted (minimum 6MWT predicted 64.7%; maximum 6MWT predicted 121.6%; n=8) after 3 years; 91.7% of 6MWT predicted (minimum 6MWT predicted 59.0%; maximum 6MWT predicted 133.4%; n=8) after 4 years; and 85.1% relative to the predicted 6MWT value (minimum predicted 6MWT 61.5%; maximum predicted 6MWT 137.4%; n=8) after 5 years. Thus, the 6MWT value in patients with HPP treated with asphotase alfa improved from 76% of the predicted 6MWT value at baseline to 80.8% of the predicted 6MWT value, which is within the normal range of healthy subjects of the same age. and growth within 6 months. The improvement in 6MWT was maintained over 4 years of treatment with asphotase alfa.

Example 4 Improvement in Physical Ability in Adult Patients with Childhood-Onset HPF as Assessed by the Bruinx-Ozeretsky Motor Test, 2nd Edition (BOT-2)

Physical ability and impairment in patients with HFF was assessed using the Bruynks-Ozeretsky Motor Test for Running Speed, Agility and Strength, 2nd Edition (BOT-2). BOT-2 tests to assess running speed and agility in patients with HFF included 50-foot shuttle runs, lateral steps over the balance beam, and lateral hops on one or two legs. The BOT-2 tests for assessing the strength of patients with HFF included squats, straight leg raises in the prone position, standing long jumps, supported squats, and push-ups. BOT-2 overall speed and agility and BOT-2 overall strength were then determined from the sum of BOT-2 test scores for each time interval (baseline, 6 months, 1 year, 2 years, 3 years, 4 years 5 years and 6 years). For BOT-2 scores after baseline, BOT-2 scores were pooled for treatment and control groups at each respective time point (6 months, 1 year, 2 years, 3 years, 4 years, 5 years, and 6 years of treatment with asphotase alfa). ) as the control group received asfotase alfa treatment after an initial phase of 6 months.

Overall BOT-2 running speed and agility improved after 6 months of treatment with asfotase alfa compared to median BOT-2 overall running speed and agility (minimum BOT-2 score 0; maximum BOT-2 score 27; n=9) at baseline to 10.5 (minimum BOT-2 5; maximum BOT-2 27; n=8) at 6 months (FIG. 5). The improvement in median BOT-2 overall running speed and agility was maintained throughout the prolongation phase of asfotase alfa treatment, with a median BOT-2 overall running speed and agility score of 9.0 (minimum BOT-2 score of 4; maximum score of 35 ; n=11) after 1 year; median overall BOT-2 running speed and agility was 10.0 (minimum BOT-2 score 3; maximum score 33; n=11) at 2 years; median overall BOT-2 running speed and agility was 10.0 (minimum BOT-2 score 4; maximum score 30; n=10) at 3 years; median overall BOT-2 running speed and agility score was 9 (minimum BOT-2 score 4; maximum score 31; n=11) at 4 years; and median overall BOT-2 running speed and agility was 8.0 (minimum BOT-2 score 0; maximum score 35; n=9) at 5 years. Overall BOT-2 strength scores improved after 6 months of treatment with asfotase alfa from a median total BOT-2 strength score of 12 (minimum BOT-2 score 2; maximum score 30; n=5) at baseline to 14 (minimum score BOT-2 8; maximum score 35; n=5) at 6 months (Fig. 6).

Example 5 Improvement in Bone Mineralization in Adult Patients with Childhood-Onset HPF After Administration of Asphotase Alpha

Dual-energy X-ray absorptiometry (DXA) was performed on the femoral neck, lumbar spine, and whole body at baseline and 6 months for untreated control patients, and at baseline and 1 year for patients treated with asphotase alpha. Treated adult patients with childhood-onset HFF showed an increase from baseline in bone mineral density (BMD) in the lumbar spine after 6 months of asfotase alfa administration. A statistically significant increase in BMD in the lumbar spine was observed after six months of treatment with asphotase alfa (Table 7).

Table 7. Bone Mineral Density of the Lumbar Spine in Adults with Childhood-Onset HPP at Baseline and After Treatment with Asphotase Alfa for 12 Months

Control, no treatment (n=3) Combined treatment (n=9) Bone Mineral Density of the Lumbar Spine (g/cm ² ) Baseline 1.0947 (0.39910) 1.1853 (0.22806) Follow-up (6 months) 1.1127 (0.37064) 1.2128 (0.21789) Mean change from baseline 0.0180 (0.02858) 0.0262 (0.01861) 95% confidence interval for mean change from baseline (-0.053, 0.089) (0.011, 0.042)

Bone biopsy was also performed at baseline and 6 months for untreated controls, and at baseline and 1 year for asphotase alfa treated patients to determine osteoid volume and thickness (Table 8) and delay time of mineralization (Table 9). A statistically significant decrease in osteoid volume/bone volume (%) and osteoid thickness (µm) was observed after one year of treatment with asphotase alfa. This testing was significantly more invasive than DXA testing.

Table 8 Osteoid Volume/Bone Volume and Osteoid Thickness in Adults with Childhood-Onset HPF at Baseline and After Treatment with Asfotase alfa for 12 Months

Control, no treatment (n=3) Combined treatment (n=9) Osteoid volume/bone volume (%) Baseline 9.2 (4.1) 7.6 (4.2) Follow-up (control: 6 months; treatment: 1 year) 9.4 (3.9) 5.4 (4.5) Mean change from baseline 0.2 (3.2) -3.8 (-0.5) 95% confidence interval for mean change from baseline (-7.7, 8.1) (-3.8, -0.5) Osteoid thickness (µm) Baseline 11.2 (0.6) 10.2 (5.1) Follow-up (control: 6 months; treatment: 1 year) 9.0 (1.4) 8.3 (4.8) Mean change from baseline -2.2 (2.0) -1.8 (1.9) 95% confidence interval for mean change from baseline (-7.1, 2.8) (-3.3, -0.4)

At baseline, the mineralization delay time was increased for all HPF patients, although this included a greater degree of inter-patient variability. Such variability is expected for this type of histomorphometric measurement and study. After treatment with asfotase alfa for 1 year, mineralization delay time was reduced in the treated group with a mean change from baseline of -696 days compared to the untreated control group with a mean change from baseline of 65 days in 6 months. These results indicate that the administration of asphotase alfa to adult patients with childhood-onset HPP promotes bone mineralization.

Table 9. Time delay of mineralization in adults with childhood-onset HPP at baseline and after treatment with asphotase alfa for 12 months

Control, no treatment (n=3) Combined treatment
(n=9) Mineralization delay time (days) Baseline 456 (261), 2 964 (1212), 6 Follow-up (control: 6 months; treatment: 1 year) 369 (264), 3 436 (486), 7 Mean change from baseline 65 (270), 2 -696 (1069), 6 95% confidence interval for mean change from baseline (-1506.1460) (-1818, 426)

Example 6 Asphotase Alpha Treatment Study Design for Adolescents and Adults with HPF

Adolescents and adults with HPP aged from about 13 years to about 65 years participated in a study to determine the efficacy, safety and tolerability of treatment with rAP (asfotase alfa; Fig. 7). During initial treatment with asfotase alfa for 6 months, a total of 19 patients were randomized to receive alfa asfotase 0.3 mg/kg/day (2.1 mg/kg/week; n=7) by subcutaneous injection. , 0.5 mg/kg/day (3.5 mg/kg/week; n=6) by subcutaneous injection or no treatment (n=6) in the control group (Fig. 8). After baseline, patients in both the treated and control groups were treated with asphotase alfa at 0.5 mg/kg/day (3.5 mg/kg/week) subcutaneously for 6 months. . After the first 6 months of the extension phase, the dose was increased to 1 mg/kg/day administered 6 days/week (6 mg/kg/week) for all patients as per protocol amendment. Study inclusion and exclusion criteria are described in Table 10. Patient demographics and baseline characteristics for adolescents and adults with HFF, including HFF-related medical history, are presented in Table 11.

Table 10. Main inclusion and exclusion criteria for the study of adolescents and adults with HPP treated with asfotase alfa

Key Inclusion Criteria Main Exclusion Criteria age ≥13 and ≤65 years
Pre-established clinical diagnosis of HFF with onset at any age based on:
Low level of serum alkaline phosphatase, taking into account the age of the patient
High P5P pyridoxal 5''-phosphate (≥2 × upper limit of normal) ^a
Osteopenia or osteomalacia on skeletal x-ray ^b
Osteomalacia on bone biopsy (Z-score of mineralization delay time ≥+2) ^c Low serum calcium or phosphate
Low serum vitamin D 25 (OH) (<20 ng/mL) ^d
High serum creatinine or parathyroid hormone (PTH) or treatment with PTH for 6 months
Use of bisphosphonates within 2 years of study entry (for any time period) or for >2 years at any time ^e

^a No vitamin B6 supplementation ≥1 week prior to _P5F determination;

^b Osteomalacia confirmed by bone biopsy;

^c Treatment was not interrupted prior to biopsy, but all patients were confirmed to have osteomalacia by biopsy;

^d Patients not screened due to low levels may be retested after supplementation at the discretion of the investigator;

^e Patients with approved bisphosphonate prior use must have serum C-telopeptide and urinary N-telopeptide or deoxypyridinoline levels within normal or elevated levels.

Table 11. Demographic and baseline characteristics of adolescents and adults with HPF treated with asphotase alfa

Total ^a
(N=19) The main period of treatment, distribution into groups Control group
(n=6) Asphotase alfa ^b treated group
(n=13) Demographics Age at power on, years, median (min, max) 53 (13, 66) 21 (13, 58) 55 (14, 66) Age category when turned on
Adolescents (ages 13 - <18 years)
Adults (age ≥18 years) 6 (32)
13 (68) 3 (50)
3 (50) 3 (23)
10 (77) Age of symptom onset, years, median (min, max) 2.0 (0.36) 0.9 (0.2, 4) 2.0 (0.36) Age category of onset of symptoms, n (%)
<18 years old
≥18 years old 18 (95)
1 (5) ^s 6 (100)
0 (0) 12 (92)
1 (8) ^s Women, n (%) 12 (63) 2 (33) 10 (77) Caucasoids 18 (95) 5 (83) 13 (100) Medical history related to HFF Patients with fractures, n (%)
Number of fractures, median (min, max) 18 (95)
6.0 (1, 30) 6 (100)
5.0 (1, 8) 12 (92)
9.5 (1.30) Severity of bone pain, n (%)
Activity restriction
Requires painkillers 18 (95)
16 (84) 5 (83)
5 (83) 13 (100)
11 (85) Muscular manifestations, n (%)
Weakness
Pain 17 (90)
14 (74) 5 (83)
4 (67) 12 (92)
10 (77) Articular manifestations, n (%)
Pain
Swelling 17 (90)
7 (37) 5 (83)
2 (33) 12 (92)
5 (39) Abnormal gait, n (%) 15 (79) 4 (67) 11 (85) Auxiliary devices for movement, n (%) 5 (26) 2 (33) 3 (23) Craniosynostosis, n (%) 3 (16) 0 3 (23) Premature loss of milk teeth, n (%) 16 (84) 5 (83) 11 (85) Loss of molars, n (%)
Remaining molars, middle (min, max) 8 (42)
24 (0.30) 1 (17)
26 (0.28) 7 (54)
23 (0.30) Hypercalcemia, n (%) 6 (32) 3 (50) 3 (23) Hyperphosphatemia, n (%) 6 (32) 2 (33) 4 (31) Gout, n (%) 5 (26) 2 (33) 3 (23) Kidney stones, n (%) 4 (21) 2 (33) 2 (15)

^a Pooled as all patients were treated in the extension phase;

^b Nitrotase alfa-treated groups pooled for analysis;

^c One patient who was initially categorized as having adult HFF was subsequently identified as having signs and symptoms of childhood-onset HFF.

ALP=alkaline phosphatase; HPP=hypophosphatasia.

The metrics used during the extension phase of treatment to assess treatment of adolescents and adults with HPP with asphotase alfa included: activity of alkaline phosphatase; 2) 6 Minute Walk Test (6MWT) scores to assess walking ability; (3) scores on the Bruinx-Ozeretsky Motor Assessment Test, 2nd Edition (BOT-2), for assessing physical ability; 4) Dynamometry using Handheld Devices (HHD) to assess strength; 5) Lower Limb Function Scale (LEFS) to assess impairments; and 6) the Brief Pain Rating Questionnaire (BPI-SF) to assess the level of pain.

Efficacy analyzes were performed on the full analysis set, which included all randomized patients; All patients were on the treatment they were randomized to. Group comparisons were made between the pooled group treated with asfotase alfa and the control group at baseline. Analyzes were also conducted on the duration of exposure to asphotase alfa up to 5 years; for patients in the control group, treatment with asfotase alfa began 6 months later at the beginning of the extension phase of treatment. Regarding the outcome of changes in plasma concentrations of nPF and P5F from baseline to month 6, comparisons between the asphotase alfa-treated pooled group and the control group were performed using the exact Mann-Whitney U-test for each parameter using a two-tailed alpha 0.05; missing values were calculated using the last observation taken. For 6MWT, the percentage predicted value (%), defined as a percentage of the normal predicted walking distance based on age, sex, and height, was calculated if the patient walked a full 6 minutes at baseline.

Example 7 Decrease in Median APF and P5P Concentrations Following Administration of Asphotase Alpha to Adults and Adolescents with HPP

ALP activity in plasma samples from adults and adolescents with HPP was assessed by quantifying the concentrations of the ALP substrates nPP and P5P as described in Whyte et al., 1995 (J. Clin. Invest. 95(4): 1440-1445 ). After 6 months, there was a greater change in plasma concentrations of both nPP (-2.2 µM) and P5P (-254.5 ng/mL) in adolescents and adults with HPP treated with asfotase alfa relative to controls ( Fig. 9A and 9B, respectively). The between-group difference was statistically significant for changes in plasma P5F (P=0.0285), but not for changes in plasma nPF (P=0.0715). Post-hoc sensitivity analyzes that excluded one patient from the control group with a high baseline nPF concentration (12.1 μM) showed a statistically significant decrease in plasma nPF concentration at 6 months in the asfotase alfa-treated group, compared with the control group (P=0.0044). Decreases in median plasma concentrations of nPF and P5F in adolescent and adult patients with HPP were maintained during five years of treatment with asphotase alfa (FIGS. 10A and 10B, respectively).

Example 8 Improvement in Physical and Walking Ability in Adult Patients and Adolescents with HPF by the 6 Minute Walk Test (6MWT)

Physical and walking ability in adults and adolescents with HFF was assessed using the 6-minute walking test (6MWT). For 6MWT values after baseline, 6MWT values were pooled for the treated and control groups at each respective time point (1 year, 2 years, 3 years, 4 years, and 5 years of treatment with asfotase alfa) because the control group was reoriented and started treatment with asfotase alfa after an initial phase of 6 months. The original control group began treatment with asfotase alfa six months after the treated group. The baseline score for all analyzes was the last score before patients received their first dose of asphotase alfa. The predicted value of 6MWT was calculated only if the patient walked for all six minutes. Three patients originally assigned to the control group were not included in the predicted percentage analysis because these patients were unable to walk for 6 minutes at baseline due to physical and/or cognitive impairment. When measured at baseline, 5 of 19 patients used assistive devices for mobility (n=3, group, asfotase alfa treatment group; n=2, control group).

Adolescent and adult HPP patients treated with asfotase alfa improved their performance from 355 meters (minimum distance 10 meters; maximum distance 620 meters) at the start of treatment to 450 meters (minimum distance 280 meters; maximum distance 707 meters) after six months of treatment with asfotase alfa (FIG. 11A). Similarly, the median predicted percent distance traveled 6MWT increased from 76% predicted at baseline (minimum 42%, maximum 101%) to 85% (minimum 29%, maximum 109%) after six months of treatment with asphotase alfa (Fig. .11B). The increase in distance traveled at 6MWT and predicted % distance traveled at 6MWT were maintained over five years of treatment with asphotase alfa. After two years of treatment with asphotase alfa, all five patients using assistive mobility devices improved their performance: one moved from a wheelchair to crutches, one from a walker to a cane, one from a wheeled walker to independent mobility, and two from canes to independent movement.

A post hoc analysis was also performed to assess the outcome of 6MWT in adolescent patients with HPF (n=4) and adults with childhood-onset HPP (n=9) before and after treatment with asphotase alfa. Retest reliability of 6MWT was assessed by calculating Pearson's correlation coefficient (r) between 6MWT distances traveled at screening time points relative to baseline, and two-tailed P values were calculated (exact null hypothesis test: r=0). The reliability of the analysis was studied separately for adolescents from about 13 to about 17 years of age and adults over about 18 years of age. Pearson's correlation coefficients (r) between distance traveled at screening times relative to baseline were 0.81 (P=0.1250) for adolescent patients with HPF (Fig. 12A) and 0.94 (P=0.0001) for adult patients with childhood-onset HPF (Fig. 12B). The minimum clinically significant difference (MCD) for 6MWT of adolescent HFF patients (n=4) and adults with childhood-onset HFF (n=9) was calculated using distribution-based methods applied to 6MWT data at screening and baseline data. In the current post hoc analysis, methods based on the standard error of measurement (SEM) and 1/3 standard deviation (SD) of the distribution were used to estimate the IPPC. The most conservative IPPC estimates were 43 meters for adolescents (SOI method; n=4) and 31 meters for adults (SOI method; n=9) with childhood-onset HPP who completed 6MWT at screening time and for whom baseline data were obtained. data.

Example 9 Improvement in Physical Ability in Adolescent and Adult HFF Patients as Assessed by the Bruinx-Ozeretsky Motor Test, 2nd Edition (BOT-2)

Physical ability and impairment in adolescents and adults with HFF was assessed using the Bruynks-Ozeretsky Motor Test for Running Speed, Agility and Strength, 2nd edition (BOT-2). BOT-2 tests to assess running speed and agility in patients with HFF included 50-foot shuttle runs, lateral steps over the balance beam, and lateral hops on one or two legs. The BOT-2 tests for assessing the strength of patients with HFF included squats, straight leg raises in the prone position, standing long jumps, supported squats, and push-ups. BOT-2 overall speed and agility and BOT-2 overall strength were then determined from the sum of BOT-2 test scores for each time interval (baseline, 6 months, 1 year, 2 years, 3 years, 4 years and 5 years). For BOT-2 scores after baseline, BOT-2 scores were pooled for treatment and control groups at each respective time point (6 months, 1 year, 2 years, 3 years, 4 years, and 5 years of treatment with asfotase alfa). ) as the control group received asfotase alfa treatment after an initial phase of 6 months. The median BOT-2 total running speed and agility for adults and adolescents with HFF was 6.5 (minimum 0, maximum 39) at baseline and increased by 4.0 (minimum 5, maximum 18) after 5 years of treatment with asphotase alpha (Fig. 13). The median total BOT-2 strength score was 13.5 (minimum 0, maximum 33) at the start of treatment and increased by 3.5 (minimum 9, maximum 9) after five years of treatment with asphotase alfa (Fig. 13).

Example 10 Increased Grasp and Muscle Strength in Adolescent and Adult HPF Patients Assessed by Handheld Dynamometry (HHD)

Grip and muscle strength in adolescents and adults with HFF was assessed using Handheld Device Dynamometry (HHD). The following bilateral muscle groups were tested by HHD: grasping group, knee flexors, knee extensors, hip flexors, hip extensors, and hip abductors. Strength was represented in pounds. The measurements were taken in pounds (force), converted to newtons (N), and the torque was calculated in newton meters (NM), taking into account the length of the limb. Torque was used to determine % predicted HHD based on age, sex, and weight in patients under 16 years of age, and force was used to compare % predicted HHD in adults.

Median values for % predicted HHD hip abductors for adolescent and adult patients with HFF (n=13) were 45.60% at baseline (minimum 9.1%, maximum 126.2%). An increase in strength in the proximal thigh muscles was observed in asphotase alfa-treated HPP patients after 24 weeks of treatment with asfotase alfa. The change in median % predicted value (dominant side) from baseline to week 24 was 11.10% (minimum - 25.7%, maximum 20.4%) in the combination treatment with asfotase alfa compared with 6.70 % (minimum - 7.1%, maximum 12.0%) in the control group, in the case of hip abduction. The change in median % predicted value (dominant side) from baseline to week 24 was 7.95% (minimum 12.6%, maximum 19.5%) in the asfotase alfa combination treatment group compared to 1.90% (minimum 15.0%, maximum 11.2%) in the control group, in case of hip extension.

Adolescent and adult patients with HPP treated with asfotase alfa showed improvement in hip abduction after treatment with asfotase alfa at 48 weeks (median change - 11.00% of predicted value (minimum - 15.0, maximum - 30.5 %)), 96 weeks (change in median - 11.95% of predicted value (minimum - 14.0%, maximum 47.2%)), 144 weeks (change in median 17.05% of predicted value (minimum - 6.7% , maximum 55.5%)), 192 weeks (change in median 19.45% of predicted value (minimum - 24.2%, maximum 94.7%)), and 240 weeks (change in median 9.80% of predicted value (minimum - 7.2%, maximum 155.2%)). Adolescent and adult patients with HPP treated with asfotase alfa showed improvement in hip extension after treatment with asfotase alfa at 24 weeks (median change - 6.20% predicted value (minimum - 12.6, maximum 19.5% )), 48 weeks (change in median - 4.90% of predicted value (minimum 1.7, maximum - 27.7%)), 96 weeks (change in median 6.75% of predicted value (minimum - 24.1%, maximum 31.2%)), 144 weeks (median change 15.40% of the predicted value (minimum - 15.6%, maximum 45.2%)), 192 weeks (median change 17.10% of the predicted value (minimum - 3, 2%, maximum 50.2%)) and 240 weeks (median change 12.40% of predicted value (minimum - 9.2%, maximum 45.1%)). Adolescent and adult patients with HPP treated with asfotase alfa showed improvement in hip abduction after 240 weeks of treatment with asfotase alfa. Adolescents and adults with HPF had a median change in knee extension of 16.05% predicted (minimum 7.1%, maximum 40.7%) and knee flexion 14.30% predicted (minimum 2.1%, maximum 68, 0%)).

Example 11 Improvements in Functional Impairment in the Lower Limbs in Adolescent and Adult Patients with HPF as Evaluated by the Lower Limb Function Scale (LEFS)

Functional impairment in the lower limbs in adolescents and adults with HFF was assessed using the Lower Limb Function Scale (LEFS). A licensed physical therapist assessed LEFS in patients with HFF in an interview format. Total LEFS scores range from 0 to 80, with higher scores indicating better lower limb function. Baseline median and mean LEFS scores were much lower in the control group than in the combined asfotase alfa treatment group. There was an improvement in LEFS score in the combined asphotase alfa-treated group in adolescent and adult HPF patients compared to the untreated control group (Table 12). Clinically significant improvement (≥ 9 points increase) from baseline up to week 24 was observed in 1 of 5 patients (20.0%) in the control group compared to 4 of 13 (30.8%) treated patients with HFF with asphotase alfa.

Table 12. Improvement in Functional Impairment Scores on the Lower Limb Function Scale (LEFS) in HPF Patients from Baseline to Week 24 of Asphotase Alfa Treatment

Visit Parameter Control Group (N=6) Asfotase Alpha 0.3 mg/kg (N=7) Asfotase Alpha 0.5 mg/kg (N=6) Asphotase Alpha Combined (N=13/19(20)) Baseline n 5 7 6 13 Average (SD) 35.8 (4.66) 40.0 (25.04) 41.7 (15.62) 40.8 (20.39) Median 35.0 28.0 36.0 35.0 Min, Max 30, 41 17, 78 29, 70 17, 78 Week 24 n four 7 6 13 Average (SD) 42.0 (12.73) 52.3 (21.05) 44.8 (21.07) 48.8 (20.53) Median 37.5 58.0 47.5 53.0 Min, Max 33, 60 22, 78 22, 77 22, 78 Change From Baseline to Week 24 n four 7 6 13 Average (SD) 5.5 (13.70) 12.3 (17.13) 3.2 (13.09) 8.1 (15.51) Median 2.0 5.0 5.5 5.0 Min, Max -7, 25 -2, 40 -15, 23 -15, 40 p* value - - - 0.7248

*P value based on exact Mann-Whitney U-test comparing treated group with control group.

During treatment with asphotase alfa, 14 of 18 patients (77.8%) with baseline LEFS scores experienced an increase (improvement) in total LEFS score from baseline to five years of treatment with asfotase alfa. In addition, 8 of 18 patients (44.4%) demonstrated a clinically significant improvement (≥ 9 points) in their LEFS score at their final assessment compared to baseline LEFS score. The mean change from baseline for all 18 patients to the last total exposure to asphotase alfa was 7.5 (SD 13.33).

Example 12 Pain in Adolescent and Adult Patients with HFF Assessed Using the Brief Pain Assessment Questionnaire (BPI-SF)

Pain in adolescents and adults with HFF was assessed using the Brief Pain Assessment Questionnaire (BPI-SF). The BPI-SF scores for adolescents and adults with HFF are a composite of 11 pain scores. There was no mean increase from baseline to week 24 in the combined BPI-SF in adolescents and adults with HFF (Table 13). Changes in mean and median from baseline to week 24 in BPI-SF scores were similar in asfotase alfa-treated adolescents and adults with HPF and controls.

Table 13 No Changes in Brief Pain Assessment Questionnaire (BPI-SF) Scores in HPF Patients from Baseline to Week 24 of Asfotase Alpha Treatment

Visit Parameter Control Group (N=6) Asfotase Alpha 0.3 mg/kg (N=7) Asfotase Alpha 0.5 mg/kg (N=6) Asphotase Alpha Combined (N=13/19) Baseline n 6 7 6 13 Average (SD) 12.3 (7.17) 10.3 (10.78) 17.8 (8.18) 13.8 (10.07) Median 12.0 8.0 20.0 15.0 Min, Max 5, 25 0.30 3, 25 0.30 Week 24 n four 7 6 13 Average (SD) 10.3 (6.60) 6.0 (7.16) 15.2 (8.82) 10.2 (8.98) Median 10.0 3.0 16.0 7.0 Min, Max 3, 18 0.21 4, 26 0.26 Change From Baseline to Week 24 n four 7 6 13 Average (SD) -4.0 (6.00) -4.3 (7.04) -2.7 (6.38) -3.5 (6.51) Median -3.0 -3.0 -0.5 -2.0 Min, Max -11, 1 -17, 3 -11, 4 -17, 4 p* value - - - 0.7315

*P value based on exact Mann-Whitney U-test comparing treated group with control group.

During treatment with asphotase alfa for five years, BPISF rates in adolescents and adults with HPP were consistent with those observed during the initial 24-week treatment period. At 96 weeks (N=19) and 144 weeks (N=17) of treatment with asfotase alfa, the median change was -4.0 (range: -13.11) and -4.0 (range: -24.9) for Adolescents and adults with HPP treated with asphotase alfa and controls, respectively. At 192 weeks (N=15) and 240 weeks (N=16) of treatment with asphotase alfa, the median change was -3.0 (range: -20.8) and -3.5 (range: -20.5) for adolescents and adults with HFF and controls, respectively. Thus, adolescents and adults with HPF did not show an increase in pain after treatment with asphotase alfa for about 5 years.

Example 13 Tolerability of Long Term Treatment with Asphotase Alpha

Treatment with asfotase alfa was generally well tolerated in adolescents and adults with HPP, with the majority of adverse events (AEs) consisting of injection site reactions (IRRs) and arthralgia. Safety assessments included ongoing monitoring of adverse events (AEs), including RMI and injection-related reactions (IRS). RMI were localized at the injection site and occurred at any time after the start of treatment with asfotase alfa. RSI included systemic signs, symptoms, or manifestations (eg, generalized urticaria or pruritus, hypotension, or respiratory distress) that occurred within 3 hours of asphotase alfa administration.

No deaths occurred during treatment with asphotase alfa. Only one patient discontinued participation due to injection site hypersensitivity and anaphylactoid reaction (1 episode each). All patients experienced at least one treatment-related adverse event (AEAE) during the study; most of the AEs were mild (864/1145 (75%)) or moderate (229/1145 (20%)) intensity. NJVL, representing arthralgia, erythema at the injection site, pain in the extremity, back pain, hematoma at the injection site, bone pain, discoloration at the injection site, ophthalmic calcifications, musculoskeletal pain, peripheral edema, dizziness, foot fracture, injection site reaction, joint swelling, upper respiratory infections, headache, injection site pain, nasopharyngitis, cough, weakness, injection site atrophy, injection site itching, pain during procedure, sinusitis, fatigue, injection site swelling , hypertrophy at the injection site, nausea, oropharyngeal pain, osteoarthritis, paresthesia, and post-traumatic pain were reported in more than three patients with HFF (Table 14).

Table 14. Treatment-Emergent Adverse Events (AEEP) Occurring in More Than Three HPF Patients During Asfotase Alfa Treatment

NYVL (preferred term) Patients, n (%)
N=19 Arthralgia 13 (68) Erythema at the injection site 13 (68) Pain in the limb 12 (63) Backache 10 (53) Hematoma at the injection site 10 (53) Pain in the bones 9 (47) Color change at the injection site 9 (47) Ophthalmic calcifications ^a 9 (47) Musculoskeletal pain 8 (42) peripheral edema 8 (42) Dizziness 7 (37) foot fracture 7 (37) Reaction at the injection site 7 (37) joint swelling 7 (37) Upper respiratory tract infection 7 (37) Headache 6 (32) Pain at the injection site 6 (32) Nasopharyngitis 6 (32) Cough 5 (26) Weakness 5 (26) Atrophy at the injection site 5 (26) Itching at the injection site 5 (26) Pain during the procedure 5 (26) Sinusitis 5 (26) Fatigue 4 (21) Swelling of the injection site 4 (21) injection site hypertrophy 4 (21) Nausea 4 (21) Pain in the oropharynx 4 (21) Osteoarthritis 4 (21) paresthesia 4 (21) Post-traumatic pain 4 (21)

^a NEEP includes the preferred terms "ocular deposits" and "conjunctival deposits".

The most common AEs were RMI (385/1145 (34%)), which occurred in all patients; The most common RMI (≥5% RMI) were erythema (121/385 (31%)), discoloration (66/385 (17%)), RMI without further specification (56/385 (15%)), hematoma ( 33/385 (9%)), pain (26/385 (7%)) and itching (20/385 (5%)). A total of two patients experienced AELI that were classified as RSI representing hypersensitivity (oral hypoesthesia and chills in one patient; anaphylactoid reaction in one patient), which were considered moderate in intensity. A total of 29 serious treatment-emergent AEs (SAEs) were reported in 9 patients, and 8 SAEs reported in two patients were assessed by the investigator as being related to asfotase alfa administration (oral hypoaesthesia, chills, pain in extremities, and headache in one patient). patient and hypersensitivity reaction and anaphylactoid reaction in one patient). In general, administration of asfotase alfa was well tolerated by adolescents and adults with HPP.

Example 14 Pharmacodynamic Results of a Phase 2a, Randomized, Multicentre, Open-Label Dosing Trial of Asphotase Alpha in Adults with Childhood Onset HPP

In studies in patients aged 6-12 years, as well as in adolescents and adults with HPF, asfotase alfa reduced circulating levels of nPF and P5F and improved functional outcomes. Asfotase alfa has been approved in the United States, Europe, and Japan for the treatment of HPP in patients of all ages with childhood-onset HPP at the recommended dose of 2.0 mg/kg 3 times a week or 1.0 mg/kg 6 times a week . In the United States, this dose may be increased to 3.0 mg/kg 3 times a week if the attending physician considers that the effectiveness is insufficient.

The pharmacodynamics, pharmacokinetics, and safety/tolerability of asfotase alfa was evaluated in adults with childhood-onset HPP at 3 doses over a 6-fold range of the currently recommended dose of 2.0 mg/kg 3 times weekly. Pharmacodynamics and safety/tolerability were measured.

Eligible patients included men and non-pregnant women ≥18 years of age with childhood-onset HPF and a diagnosis of HPF as evidenced by a documented history of HPF-related skeletal abnormalities and one or more of the following: a documented mutation(s) ) TNAP gene (ALPL), serum alkaline phosphatase activity below the age-adjusted normal range, and P5F above the upper limit of normal at screening (historical results for P5F can be used to determine patient eligibility; plasma P5F criterion is not applicable for patients treated with pyridoxine) , plasma NPF ≥3.9 μM at screening and no treatment with asphotase alfa within 3 years of study entry.

The study design and treatment was a 13-week, multicenter, randomized, open-label phase 2a dose-response study. After screening, sampling for substrate analysis was allowed in the initial preparatory period. Patients were then randomized 1:1:1 to a single dose of asfotase alfa (0.5, 2.0, or 3.0 mg/kg) administered subcutaneously (SC) during Week 1. Patients did not receive asfotase alfa during Week 2. Asfotase alfa was then administered from Weeks 3 to 9 at 0.5, 2.0, or 3.0 mg/kg SC 3 times a week (equivalent to 1.5, 6.0, or 9.0 mg/kg/week) ( Fig. 14).

Pharmacodynamic outcome measurements were changes in plasma nPF (primary) and P5F (secondary) from baseline to pre-dose time 3 (residual level) at Week 9, with baseline defined as the mean of all scores prior to the first SC injection with asfotase alpha. Blood samples for measurement of plasma NPF and P5F were collected at screening, preparatory, Week 1, Week 9 and Week 13 in the clinical setting. Blood samples during Weeks 2-8 and Weeks 10-12 could be collected in a clinical setting or at home. Other secondary outcome measures include safety/tolerability and pharmacokinetic parameters. The results of pharmacodynamics and safety/tolerability are presented.

Primary hypotheses tested whether differences in plasma NPF change between cohorts were different from zero when measured predose 3 Weeks 9. To control for repeatability, a fixed-sequence testing procedure was used to first determine the difference between the 3.0 and 0.5 mg/kg cohorts and, only if it was statistically significant, then the difference between the 2.0 and 0.5 mg/kg cohorts was determined. A two-tailed 5% error of the first kind (P<0.05) was used in the significance test. The primary efficacy end point was reached if the null hypothesis was rejected for both comparisons at a testing significance level of 0.05 (P<0.05 for both comparisons). Primary analysis was performed using a Restricted Maximum Likelihood (REML) based mixed repeated measures model to estimate change from baseline to week 9 (up to the time point before the 3rd dose; day 61). This model used results from pre-dose samples taken at Week 4 (Day 22), Week 5 (Day 29), Week 7 (Day 43), Week 8 (Day 50), Week 9 (Day 57), and Week 9 (Day 61). The pre-dose time point of Week 3 (Day 15) was not included as it was obtained after a 2-week elimination period after a single dose and it was expected that at this point values would be similar to those at baseline. Following the protocol, pharmacodynamic evaluations were not obtained at Week 6. The analysis included covariates for the fixed, categorical effect of the visit, treatment cohort, baseline NPF level, gender, baseline weight group (≥ median versus < median), and study drug lot prescription. The unstructured covariance framework was used to model intra-individual errors, and the Kenward-Roger approximation was used to estimate the degrees of freedom of the denominator.

Of the 35 patients examined, 27 were included in the study and completed the study. Demographic and baseline disease characteristics are summarized in Table 15. Among the three dose cohorts, the median age was 45.0 years (range: 18-77) and the majority of patients were Caucasoid (96%) and female (59%).

Table 15. Demographic and Baseline Characteristics of the GFF

Cohort AA 0.5 mg/kg (n=8) Cohort AA 2.0 mg/kg (n=10) Cohort AA 3.0 mg/kg (n=9) Total AA (N=27) Age at inclusion, years, median (range) 44.5 (18-64) 42.5 (24-77) 55.0 (24-69) 45.0 (18-77) Gender, n (%) 3 (38) 4 (40) 4 (44) 11 (41) Men 5 (63) 6 (60) 5 (56) 16 (59) Race, n (%) Caucasoids 7 (88) 10 (100) 9 (100) 26 (96) mixed 1 (13) 0 0 fourteen) Height, cm, mean (SD) 161 (14) 163(7) 163 (9) 163 (10) Weight, kg, average (SD) 89 (24) 73(13) 85(19) 82 (20) HFF form, n (%) ^a perinatal or infant 0 1 (10) 1 (11) 2 (7) Children's 3 (38) 5 (50) 4 (44) 12 (44) Perinatal, infant or child 5 (63) 4 (40) 4 (44) 13 (48) Mutation of the ALPL gene sequence, n(%) ^b 7 (88) 9 (90) 9 (100) 25 (93) Gene mutation category, n (%) homozygous 0 1 (10) 0 fourteen) Heterozygous 2 (25) 3 (30) 4 (44) 9 (33) Complex heterozygous 5 (63) 5 (50) 5 (56) 15 (56) Undefined 1 (13) 1 (10) 0 2 (7) History of high P5F used to diagnose HPP at screening, n (%) 7 (88) 9 (90) 8 (89) 24 (89) Baseline NPF, μM, mean (SD) ^s 5.4 (1.6) 5.3 (1.2) 5.0 (0.9) 5.2 (1.2) Baseline P5F, ng/ml, mean (SD) ^d 309.5 (349.2) 382.1 (385.9) 229.5 (321.4) 309.7 (347.0) Baseline ALP, U/l, mean (SD) ^e 20.1 (4.2) 20.4 (6.6) 25.6 (20.5) 22.0 (12.5)

^a Based on categorical selection based on age at first signs/symptoms;

^b Gene mutation analysis was performed as part of a central laboratory study;

^c Normal range of NPF levels: 0.75-4.78 µM for patients aged 13-18 years and 1.00-5.82 for patients aged >18 years;

^d Normal P5F range: 5.74–61.15 ng/mL for patients aged 6–18 years and 2.81–26.70 ng/mL for patients >18 years of age;

^e Normal ALP range: 60–270 U/L for male patients aged 16–19 years, 40–120 U/L for male patients aged >19 years, and 40–120 U/L for female patients aged aged >15 years. For analysis, values below the LCL of 18 U/L were set to 18 U/L.

AA=asphotase alpha; ALP=alkaline phosphatase; HPP= hypophosphatasia; TNSAP=tissue non-specific alkaline phosphatase; P5P=pyridoxal 5'-phosphate; NPP=inorganic pyrophosphate; SD=standard deviation.

Significant differences were observed between changes in the mean limit (LSM) of nPF from baseline to the time point before the 3rd dose at Week 9 (Table 16 and Fig. 15). Significant differences were also observed between baseline LSM P5Fot changes up to the time point before the 3rd dose at Week 9 (Table 16 and Fig. 16).

Table 16 Mean Change in NPF and P5F from Baseline to Dose 3 at Week 9

Cohort AA 0.5 mg/kg (n=8) ^a Cohort AA 2.0 mg/kg (n=10) ^a Cohort AA 3.0 mg/kg (n=9) ^a NPF, µM Baseline, mean (SD) 5.4 (1.6) 5.3 (1.2) 5.0 (0.9) Before 3rd dose Week 9, mean (SD) ^b 3.1 (0.7) 1.9 (0.6) 1.1 (0.3) ^s LSM, change from baseline (OS) ^d -2.6 (0.2) -3.8 (0.2) -4.5 (0.2) P5F, n/ml Baseline, mean (SD) 309.5 (349.2) 382.1 (385.9) 229.5 (321.4) Before 3rd dose Week 9, mean (SD) ^b 40.7 (45.8) 13.0 (9.5) 4.8 (4.4) LSM, change from baseline (OS) ^d -304.0 (0.4) -333.4 (8.1) -338.0 (8.5)

^a Doses are administered 3 times a week during the multiple dosing period;

^b Prior to the 3rd dose of Week 9, the sample was collected on Day 61;

^c There was no result in one patient;

^d Primary analysis using a mixed repeated measures model based on the constrained maximum likelihood method.

AA=asphotase alpha; CI=confidence interval; LSM=Limit Mean; P5P=pyridoxal 5'-phosphate; NPP=inorganic pyrophosphate; SD=standard deviation; OS=error of the mean.

In terms of safety and tolerability, treatment-related adverse events (AEs) seen in ≥5 patients overall are presented in Table 17. For example, injection site reactions (137 events in 21 patients) were more frequent in the 2.0 cohorts and 3.0 mg/kg. Lipodystrophy was observed (2 cases in 1 patient in the 2.0 mg/kg cohort). Ectopic calcifications (4 cases in 4 patients; 2 each in the 2.0 and 3.0 mg/kg cohorts) were observed in the kidneys (2 patients; 1 each in the 2.0 and 3.0 mg/kg cohorts), in the eyes ( 1 patient in the 2.0 mg/kg cohort) and liver (1 patient in the 3.0 mg/kg cohort). Hypersensitivity reactions have also been observed (4 events in 1 patient in the 2.0 mg/kg cohort). Overall, 99% (480/485) of AELI were mild or moderate in severity. No serious AEs were reported and no patients were withdrawn from the study due to AEs.

Table 17 Treatment-Occurring Adverse Events in ≥5 Patients

Cohort AA 0.5 mg/kg (n=8) ^a Cohort AA 2.0 mg/kg (n=10) ^a Cohort AA 3.0 mg/kg (n=9) ^a Total (N=27) NJVL Phenomena, n Patients, n (%) Phenomena, n Patients, n (%) Phenomena, n Patients, n (%) Phenomena, n Patients, n (%) Pain in the limb 17 4 (50) eleven 5 (50) ten 4 (44) 38 13 (48) Arthralgia 13 2 (25) 12 4 (40) fifteen 5 (56) 40 11 (41) Headache ten 3 (38) 9 3 (30) 7 5 (56) 26 11 (41) Fatigue 9 3 (38) eleven 4 (40) 5 3 (33) 25 10 (37) Erythema at the injection site four 2 (25) four 3 (30) four 4 (44) 12 9 (33) Reaction at the injection site four 1 (13) 21 3 (30) 68 4 (44) 93 8 (30) Backache 13 4 (50) four 1 (10) 2 2 (22) 19 7 (26) Pain in the bones 3 2 (25) 3 3 (30) 3 2 (22) 9 7 (26) Erythema four 2 (25) 7 3 (30) 2 1 (11) 13 6 (22) Pain at the injection site 2 2 (25) 2 1 (10) four 3 (33) eight 6 (22) Nausea 3 2 (25) 2 1 (10) 2 2 (22) 7 5 (19) Upper respiratory tract infection one 1 (13) 3 3 (30) one 1 (11) 5 5 (19)

^a Doses are administered 3 times per week during the multiple dosing period.

AA=Asphotase Alpha.

Adult patients with childhood-onset HPP treated with asfotase alfa at the recommended dose or higher (2.0 or 3.0 mg/kg) had a statistically significant decrease from baseline in nPF and P5F values obtained before the introduction of the 3rd dose of Week 9 compared with patients who received doses lower than the recommended dose (0.5 mg / kg). Injection site reactions were more frequent with increasing doses. Otherwise, asfotase alfa was well tolerated at all doses.

Example 15 Population Pharmacokinetic and Pharmacokinetic-Pharmacodynamic Analysis of Asphotase Alpha in Adult Patients with Childhood Onset HPP in Study AA-HPP-208

We calculated the pharmacokinetic (PK) parameters of asphotase alfa using non-compartmental analysis (NCA) to describe the PK of asphotase alfa in adult patients with childhood-onset HPP. We assessed population PK parameters, including typical values and random inter-individual and residual variations, and identified individual-specific covariate factors (eg, weight) that predict unexplained random variability in population PK analysis. We have described a population pharmacokinetic-pharmacodynamic (PKPD) relationship between asphotase alfa exposure and NPF response. In addition, we assessed population parameters of FC-nPF, including typical values and random inter-individual and residual variations. In addition, we modeled the exposure-response (ER) relationship between FA-nPP in adult patients with childhood-onset HPP treated with asphotase alfa to support the recommended dose of 6 mg/kg/week. Finally, we described a population association of FCPD between exposure to asphotase alfa and pyridoxal 50-phosphate (P5P) response in patients who did not receive vitamin B6 supplements. This includes describing the PK-P5F dose or ER and assessing typical values and random inter-individual and residual variations.

The phase 2a study (protocol AA-HPP-208) was a randomized parallel group design of three asfotase alfa multiple dosing escalation regimens in 27 HPP patients over a period of about 13 weeks (FIGS. 17A-17B). The study consisted of a screening period, a single dosing period (3 weeks), and a multiple dosing treatment period (10 weeks). The study consisted of a single dose of 0.5, 2.0, or 3.0 mg/kg asfotase alfa followed by multiple doses of equal amounts 3 times per week starting 2 weeks later. Twenty-seven subjects (about 9 per cohort) were enrolled in the study, and extensive sampling of FA, nPP, and P5P was performed after single and multiple dosing with selective sampling during the accumulation phase.

Individual and mean PK profiles were obtained for all three cohorts. Separate PK profiles indicated accumulation of asphotase alfa after a single dosing on day 1 (FIG. 18A) until several subsequent dosing events on day 61 (FIG. 18B). The PK profiles (mean ± SD) from both single and multiple dosing showed that asfotase alfa exhibited a linear PK profile with increased exposure over the tested dose range of 0.5 to 3 mg/kg (FIGS. 19A and 19C). Steady state was reached at about day 29 (or after 4 weeks of multiple dosing) when asphotase alpha levels consistently plateaued across all three dosing regimens (FIG. 19B). Dose-normalized PK profiles (mean ± SD) showed that asphotase alfa PK increased approximately dose-proportional (or 2-fold proportional band) from 0.5 mg/kg to 3.0 mg/kg (Figs. 20A-20B ).

Population PK data, including concentration observations, dosing history, timing of events, and covariate factors (age, height, weight, sex, and race) were acquired and formatted for analysis. The FK parameter values were estimated using the NCA module in PhoenixWinNonlin v 7.0 (Certara, Princeton, NJ, USA). The dataset for NCA was derived from the NONMEM® FKFD population dataset. PK parameters derived from concentration results were used to assess dose proportionality of asphotase alfa. Population-based analysis of PKFD for repeated measures endpoints was performed using non-linear mixed effects modeling using a qualified installation of Non-linear Mixed Effects Modeling (NONMEM®) software, version 7.3 (ICON Development Solutions, Hanover, MD).

Previous modeling work has suggested that asphotase alfa has a multi-compartment distribution, so initial modeling was performed using a two-compartment model with first-order absorption parameterized in terms of clearance (CL), central volume of distribution (V ₂ ), peripheral volume distribution ( V ₃ ), intercompartmental clearance (Q), absolute bioavailability (F), absorption rate constant (k _a ), and subcutaneous absorption delay time (ALAG) (ADVAN13) with corresponding random distributions of effects. Model selection was based on various fit criteria, including diagnostic correlation plots, similarity of at least 2 significant digits, reliability of parameter estimates, accuracy of parameter estimates, correlation between model parameter estimation errors

<0.95, and AIC, considering the minimum value of the objective function and the number of estimated parameters. The final model parameter estimates were presented with a measure of estimation uncertainty, including asymptotic standard errors (obtained from the NONMEM®$COVARIANCE step).

Preliminary modeling of asphotase alfa was also guided by covariant modeling. The full model was built with care to avoid correlation or collinearity in the predictors. The model parameters were evaluated and any remaining trends were assessed by plotting all covariate effects. Conclusions about the clinical significance of the parameters were based on the obtained estimates of the parameters of the full model and indicators of the accuracy of the estimate. Individual PK parameters were also assessed and the mean concentration over the steady state dosing interval ( _{Cav, ss} ) was calculated. A predictive validation model evaluation step was performed to assess the quality of the final model and parameters.

To analyze the PA-nPP data, the plasma NPF response was plotted in terms of change from baseline and summarized by exposure quartiles. Frequencies of pre-dose nPF concentrations from Day 61 of the study below the lower limit of normal values of nPF (1.33 μM) (LNP) were tabulated by interquartile range of Cmean _{, cc,} and treatment cohorts. Individual values of Cav _{, ss} asphotase alpha ranging from 253 to 6800 U/L were calculated and used to plot and quantify this ER ratio.

The Asphotase alfa PDK dataset was compiled from 27 patients, resulting in a total of 1140 serum asphotase alfa concentrations and 1293 plasma nPF concentrations over the entire study period. There were 198 observations below the limit of quantitation, representing more than 17% of the total number of observations of serum asfotase alfa. The study population consisted of 11 males and 16 females, aged 18 to 77 years and weighing 48.2 to 121.3 kg. Most of the patients were Caucasian (96.3%). Height, sex, age, and race were included in the data set and summarized in a tabular format for study completeness, but were not considered as potential covariates in population models. Lot information used in the analyzes included lot potency, total sialic acid content (TSAC), and lot size.

NCA analysis showed that the relatively long half-life and frequency of dosing resulted in marked accumulation at all dose levels. Extensive individual variability was observed in the concentration versus time profiles in each cohort, although all profiles appeared to show a mono-exponential decline when observed on a semi-log plot. Serum levels (maximum concentration in the dosing interval (C _max ), area under the concentration-time curve from zero to the time of the last observed concentration in the dosing interval (AUC _sec ) and area under the concentration-time curve from time to zero to infinity (AUC _infinity) )) increased with increasing dose and between single and multiple doses. Due to limited concentrations above the lower detection limit in the 0.5 mg/kg cohort, AUC _est , observed clearance after subcutaneous administration (CL/F), observed volume of distribution after subcutaneous administration (V _z /F ) and terminal half-life (t _1/2 ) have not been evaluated. The geometric mean estimates for CL/F and V _z /F were 18.8 L/day and 99.1 L for the 2.0 mg/kg cohort, 22.1 L/day, and 150 L for the 3.0 mg/kg cohort, respectively. . The t _1/2 estimates had geometric mean values ranging from 3.66 to 5.54 day ^-1 . The median time to peak concentration in the dosing interval assessments (T _max ) ranged from 1.91 to 3.0 and from 0.747 to 1.16 days in the single and multiple dosing cohorts, respectively. Dose proportionality in PK asfotase alfa was assessed using a logarithmic form of the degree model, suitable for C _max values for single and multiple dosing and for AUC _max and AUC _pos for single dosing.

The results were inconclusive for all cases, except for C _max single dosing, where disproportionality was found. For the parameter _Cmax single dosing, the ratio of threshold dose to proportionality deviation was 7.85. The maximum dose proportional ratios for these parameters ranged from 1.37 to 1.69 compared to the maximum dose ratio of 18.2 in the data set.

Our previous modeling work suggested that asphotase alfa has a multi-compartment distribution, so initial modeling was done using a two-compartment first-order absorbance model with appropriate random effect distributions. The full covariance model from the previous analysis included predetermined effects of allometrically weighted weight, TSAC, and drug-neutralizing antibodies (NAbs) on CL; allometrically weighted weight for V ₂ , V ₃ , and Q, and production lot size as a covariate for F and k _a since several lot sizes were included. The production lots for the current analysis only included a 20K lot size, so the observed drug potency for each lot was also included in the model as a direct dose factor, but this was not an estimated covariate effect. Typical estimates (95% CI) of PK model parameters for reference covariate effects (70 kg, 1.7 mol/mol, no anti-drug antibodies, 20K lot size) were 17.9 (13.7, 23.4) L/ day, 36.6 (0.876, 1530) L, 95.7 (18.5, 496) L, 3490 (6.47, 1.88e+06) L/day and 1.10 (0.603, 2.02) day ^-1 , for CL/F, observed (subcutaneous) central volume of distribution (V ₂ /F ), observed (subcutaneous) peripheral volume of distribution (V ₃ /F), observed (subcutaneous) intercompartmental clearance (Q/F) and constant single dose absorption rate (kaSD), respectively. These results were consistent with those of the NCA and were consistent to some extent with previous analyses.

The effect of covariant factors on the PK of asphotase alfa has also been investigated. The expected impact of covariates on CL/F, V ₂ /F, V ₃ /F and Q/F was illustrated by calculating _{Cav, ss} at the values corresponding to each predictor that was different from the null effect. Between the extremes of body weight (48.2 and 121.3 kg), TSAC (1.4 and 1.6 mol/mol), and in the presence or absence of anti-drug antibodies, the predicted median C _{av, ss} ranged from 1750 U/L to 2769 U/L when the normalized value for TSAC was 1.7. Unexplained random variability (% CV) was reduced for CL/F (coefficient of variation 43.9% (CV%)) in the final model compared to the baseline CL/F variance estimate (48.8 CV%). The clinical relevance of this finding is that dose adjustment for known covariates will not significantly reduce response variability.

The final population PK model provided a good description of the PK data. The fit criteria showed that the final model was consistent with the observed data and that no bias was found. The model evaluation results provided evidence that both the fixed and random effects components of the final model also reflected the observed data.

Plasma NPF responses tended to be higher with increases in serum asphotase alfa, although significant overlap was observed across exposure quartiles. The frequency of this NPF response plateaued to 66.7% at concentrations above the median Cav _{, ss} of asphotase alfa 2030 U/L, indicating that the increase in median _{Cav, ss} over 2030 U/L does not appear to be has a further effect on increasing the NPF response rate.

Asfotase alfa PK parameters were calculated using NCA and are shown in Table 18. The relatively long half-life and dosing frequency resulted in a marked accumulation at all dose levels, however AUC _besc , CL/F, V _z /F , and t _1/2 did not were evaluated in the 0.5 mg/kg cohort due to the low number of concentrations above the lower detection limit.

Table 18. Summary table of NCA parameters

Parameter Asfotase Alpha 0.5 mg/kg (N=8) Asfotase Alpha 2.0 mg/kg (N=10) Asfotase Alpha 3.0 mg/kg (N=9) Day 1 Day 61 Day 1 Day 61 Day 1 Day 61 C _max (U/l) 218 (24.1) 743 (63.6) 617 (51.8) 1900 (53.6) 915 (35.3) 3600 (43.7) T _max (day) 1.91 (0.485, 3.00) 0.747 (0.00, 2.46) 2.34 (1.49, 5.04) 1.16 (0.00, 2.49) 3.00 (1.50, 6.04) 0.990 (0.254, 1.33) AUC _sec (U × day / l) 588 (77.0) BUT 3310 (60.6) BUT 5620 (63.3) BUT AUC _besk (U × day/L) BUT BUT 5350 (45.6) BUT 9030 (40.3) BUT t _1/2 (day) BUT 5.12 (10.2) 3.66 (16.4) 4.62 (28.0) 4.69 (17.9) 5.54 (18.3) λ _z (day ^-1 ) BUT 0.135 (10.2) 0.189 (16.4) 0.150 (28.0) 0.148 (17.9) 0.125 (18.3) CL/F (U/L) BUT BUT 18.8 (37.2) BUT 22.1 (41.2) BUT _Vz /F (l) BUT BUT 99.1 (25.9) BUT 150 (36.2) BUT C-R BUT 4.15 (35.3) BUT 3.14 (65.5) BUT 4.68 (33.6)

Geometric mean and geometric CV% were obtained for all parameters except t _max where the median and range were reported.

λ _z =terminal excretion rate constant; AUC _ins = area under the concentration-time curve from zero to infinity; AUC _sec = area under the concentration-time curve zero until the time of the last observed concentration in the dosing interval; CL/F=apparent clearance after subcutaneous administration; C _max =maximum concentration in the dosing interval; CR=ratio of concentration 48 hours after first dose to pre-dose concentration 61 days after multiple dosing; NO=not evaluated; t _1/2 =final half-life; t _max = time of maximum concentration in the dosing interval; V _z /F=apparent volume of distribution after subcutaneous dosing.

Accumulation plots and plots of mean asfotase alfa serum concentration versus time demonstrated an increase in serum levels with increasing doses, although most concentrations in the 0.5 mg/kg cohort had data that were below the limit of quantification (BLQ) post-dose on the 1st day. Extensive individual variability was observed in the concentration versus time profiles in each cohort, although all profiles appeared to show a mono-exponential decline when observed on a semi-log plot.

Serum levels (C _max , AUC _last , and AUC _besc ) increased with dose and between single and multiple doses. Due to the insufficient number of concentrations above the lower limit of detection in the 0.5 mg/kg cohort, AUC _ines , CL/F, V _z /F, and t _½ were not evaluated. The geometric mean estimates for CL/F and V _z /F were 18.8 L/day and 99.1 L for the 2.0 mg/kg cohort, 22.1 L/day, and 150 L for the 3.0 mg/kg cohort, respectively. . t _½ indicators had geometric mean values ranging from 3.66 to 5.54 days. Median t _{max values} ranged from 1.91 to 3.0 days and from 0.747 to 1.16 days in the single and multiple dosing cohorts, respectively.

Given the irregular time between successive doses (i.e. dosing 3 times a week results in 2 intervals of 2 days and 1 interval of 3 days for 7 days), the rate of accumulation cannot be directly assessed by standard methods. The accumulation approximation at 3x/week dosing was evaluated by calculating the ratio of the concentration 48 hours post-first dose to the pre-dose concentration on Day 61 after multiple dosing. The geometric mean values of these ratios ranged from 3.14 to 4.68 in 3 cohorts. The complete population model of FC is presented in Table 19.

Table 19. Complete Model: Population Model FC 30502

Parameter FK (Unit) NONMEM parameter Index 95% CI* CL/F (l/day) exp(θ ₁ ) 17.9 (13.7, 23.4) V2/F (l) exp( _θ2 ) 36.6 (0.876, 1530) V3/F (l) exp(θ ₃ ) 95.7 (18.5, 496) Q/F (L/day) exp(θ ₄ ) 3490 (6.47, 1.88e+06) ka _CO (day ^-1 ) exp(θ ₅ ) 1.10 (0.603, 2.02) ALAG (day) exp(θ ₆ ) 0.0771 (0/00939, 0.634) ka _honey (day ^-1 ) exp(θ ₇ ) 2.09 (1.14, 3.82) CL/F ~ Allometric Exponent _θ8 0.776 FIX CL/F~ADA+/Nab- exp(θ ₉ ) 1.11 FIX CL/F~TSAC θ ₁₀ -1.08 FIX IIVvarCL/F (ω ² _CL/F ) Ω _1,1 (η ₁ ) 0.176 (%CV=43.9) (0.00, 0.472) IIVcovCL/F, V ₂ /F (ω _CL/F , ω _V2/F ) Ω _2.1 0.0253 (-0.126, 0.176) IIVvarV ₂ /F (ω ² _V2/F ) Ω _2,2 (η ₂ ) 0.108 (%CV=33.7) (0.00, 1.13) IIVcovCL/F, V ₃ /F (ω _CL/F , ω _V3/F ) Ω _3.1 0.229 (-0.00757, 0.465) IIVcovV ₂ /F, V ₃ /F (ω _V2/F , ω _V3/F ) Ω _3.2 -0.0295 (-0.502, 0.443) IIVvarV ₃ /F (ω ³ _V3/F ) Ω _3.3 (η ₃ ) 0.405 (%CV=70.6) (0.00, 1.07) IIVcovCL/F, ka (ω _CL/F ,ω _ka ) Ω _4.1 0.101 (-0.118, 0.320) IIVcovV ₂ /F, ka (ω _V2/F ,ω _ka ) Ω _4.2 0.175 (-0.527, 0.876) IIVcovV ₃ /F, ka (ω _V3/F ,ω _ka ) Ω _4.3 0.104 (-0.136, 0.344) IIVvar k _a (ω ⁴ _ka ) Ω _4.4 (η ₄ ) 0.372 (%CV=67.1) (0.00, 0.891) Σ _1.1,additive (ε ₁ ) Σ _1.1,additive (ε ₁ ) 0.0488 (SD=0.2221) (0.0435, 0.0541)

*95% CI derived from standard error rates obtained in the NONMEM $COVARIANCE step;

IIV=interindividual variability, CV=coefficient of variation, SD=standard deviation

The population PK of asphotase alfa has been described in adult patients with childhood-onset HPP using a two-compartment first-order absorption model with absorption delay time. Typical population parameters (95% CI) 17.9 (13.7, 23.4) L/h, 36.6 (0.876, 1530) L, 95.7 (18.5, 496) L, 3490 (6 .47, 1.88e+06) l/day and 1.10 (0.603, 2.02) day ^-1 were estimated for CL/F, V ₂ /F, V ₃ /F, Q/F and ka _co respectively . CL/F and V _z /F scores were consistent between NCA and population analysis.

Individual covariate factors that predicted unexplained random population variability included the effects of allometrically measured weight on CL/F, V ₂ /F, V ₃ /F and Q/F, as well as the effect of antibody status and TSAC on CL/F. The effect of a single dose over multiple doses was also considered for k _a .

The relationship of population FCFD between serum asfotase alpha and NPF response was tabulated as predicted Cav _{, ss} quantiles and examined graphically. Plasma nPF response tended to be higher with increasing dose and serum asphotase alfa, although there was significant overlap in responses across exposure quartiles (FIG. 21 and Table 20).

Table 20. PA-nPF Response: Serum Cav _{, ss} (U/L) Relative to Change in nPF Response (µM) from Baseline on Study Day 61

C _rms (U/l) N Average Median Min Max [253,935] 7 -2.44 -2.94 -5.64 0.192 (9.35, 2.03e+03] 7 -2.89 -2.35 -4.99 -0.938 (2.03e+03, 2.63e+03] 6 -4.14 -4.62 -5.46 -1.85 (2.63e+03, 6.8e+03] 7 -3.83 -3.64 -5.03 -2.87

N=number of patients, Cmrr=interquartile range Cmd _{, ss} within which all changes from baseline nPF were summed, Cmd _{, ss} = mean steady state concentration of asphotase alfa calculated using individual empirical Bayesian estimates CL=F and taking into account the last doses (activity units) administered to each patient, which were administered 3 times during the 7-day dosing interval.

The NPF response rate plateaued to 66.7% at concentrations above the median Cav _{, cc} asfotase alfa 2030 U/l (Table 21).

Table 21. NPF < LNR by Quartile C _{av, ss} : Frequency of NPF < Lower Limit of Normal (1.33 μM) on Study Day 61, by Exposure Quartile

C _rms (U/l) N Frequency (%) [253,935] 7 0.0 (9.35, 2.03e+03] 7 14.3 (2.03e+03, 2.63e+03] 6 66.7 (2.63e+03, 6.8e+03] 7 66.7

N=number of patients, Cmrr=interquartile range Cmd _{, ss} within which all changes from baseline nPF were summed, Cmd _{, ss} = mean steady state concentration of asphotase alfa calculated using individual empirical Bayesian estimates CL=F and taking into account the last doses (activity units) administered to each patient, which were administered 3 times during the 7-day dosing interval. Frequency calculated using pre-dose nPF concentration on Study Day 61.

The median predicted value of C _{av, ss} for treatment with 2 mg/kg × 3 days/week in adult patients with AA-HPP-208 (1810 U/L) approaches this cut-off concentration, while the rate of patients achieving an NPF response (20%) consistent with previous predictions for the adult group (17.6%) at this dose. Taken together, these results support a dosing regimen of 2 mg/kg x 3 days/week in adult patients with childhood-onset HFF.

These data from adult patients with childhood-onset HFF support the efficacy of a 2 mg/kg x 3 days/week dosing regimen in adolescent HFF patients. New available dose range and PK/PD data from study AA-HPP-208 show effective dosing in adult patients with HPP ≥ 18 years of age, a previously unexplored group of patients, and confirm that adult patients with childhood-onset HPP benefit from treatment with asphotase alfa.

In addition, there are significant differences in the ability to validate the therapeutic efficacy of asfotase alfa in pediatric patients compared with adult patients with childhood-onset HPP. For example, in pediatric patients, the efficacy of asfotase alfa can be determined using the radiographic change scale (RGI-C) to assess skeletal changes in pediatric patients. The RGI-C score is limited for use in HFF because it can only be used to assess people who have exposed bone growth plates (eg, pediatric patients). Therefore, the RGI-C scale cannot be used to assess efficacy in adult patients with childhood-onset HPF. However, the PK/PD/potency results from the AA-HPP-208 study support the therapeutic efficacy of asphotase alfa in these adult patients.

Finally, prior to the AA-HPP-208 study, there was no evidence as to whether treatment with asfotase alfa would be well tolerated by adult patients with childhood-onset HPP. These data confirm that although the side effects of asphotase alfa treatment in children differ between adolescent and adult patients, asfotase alfa treatment was well tolerated by both groups with minimal side effects.

OTHER MODES FOR IMPLEMENTING THE INVENTION

All publications, patents, and patent applications referred to in the foregoing description are incorporated by reference to the same extent as if each individual publication, patent, or patent application were specifically and individually identified as being incorporated by reference in its entirety. Various modifications and variations of the described methods, pharmaceutical compositions and kits according to the invention will be apparent to those skilled in the art without departing from the scope and essence of the claimed invention. Although the disclosure has been described in connection with specific embodiments of the invention, it should be understood that further modifications may be made to it and that the claimed invention should not be limited to such specific embodiments of the invention.

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SEQUENCE LIST

<110> Alexion Pharmaceuticals, Inc.

<120> METHODS OF TREATMENT OF HYPOPHOSPHATASIA (HPP) IN ADULTS AND

TEENAGERS

<130> 50694-069WO4

<150> US 62/643,953

<151> 2018-03-16

<150> US 62/502,255

<151> 2017-05-05

<150> US 62/480,213

<151> 2017-03-31

<160> 20

<170> PatentIn version 3.5

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<213> Homo sapiens

<400> 4

Met Ile Ser Pro Phe Leu Val Leu Ala Ile Gly Thr Cys Leu Thr Asn

1 5 10 15

Ser Leu Val Pro Glu Lys Glu Lys Asp Pro Lys Tyr Trp Arg Asp Gln

20 25 30

Ala Gln Glu Thr Leu Lys Tyr Ala Leu Glu Leu Gln Lys Leu Asn Thr

35 40 45

Asn Val Ala Lys Asn Val Ile Met Phe Leu Gly Asp Gly Met Gly Val

50 55 60

Ser Thr Val Thr Ala Ala Arg Ile Leu Lys Gly Gln Leu His His Asn

65 70 75 80

Pro Gly Glu Glu Thr Arg Leu Glu Met Asp Lys Phe Pro Phe Val Ala

85 90 95

Leu Ser Lys Thr Tyr Asn Thr Asn Ala Gln Val Pro Asp Ser Ala Gly

100 105 110

Thr Ala Thr Ala Tyr Leu Cys Gly Val Lys Ala Asn Glu Gly Thr Val

115 120 125

Gly Val Ser Ala Ala Thr Glu Arg Ser Arg Cys Asn Thr Thr Gln Gly

130 135 140

Asn Glu Val Thr Ser Ile Leu Arg Trp Ala Lys Asp Ala Gly Lys Ser

145 150 155 160

Val Gly Ile Val Thr Thr Thr Arg Val Asn His Ala Thr Pro Ser Ala

165 170 175

Ala Tyr Ala His Ser Ala Asp Arg Asp Trp Tyr Ser Asp Asn Glu Met

180 185 190

Pro Pro Glu Ala Leu Ser Gln Gly Cys Lys Asp Ile Ala Tyr Gln Leu

195 200 205

Met His Asn Ile Arg Asp Ile Asp Val Ile Met Gly Gly Gly Arg Lys

210 215 220

Tyr Met Tyr Pro Lys Asn Lys Thr Asp Val Glu Tyr Glu Ser Asp Glu

225 230 235 240

Lys Ala Arg Gly Thr Arg Leu Asp Gly Leu Asp Leu Val Asp Thr Trp

245 250 255

Lys Ser Phe Lys Pro Arg Tyr Lys His Ser His Phe Ile Trp Asn Arg

260 265 270

Thr Glu Leu Leu Thr Leu Asp Pro His Asn Val Asp Tyr Leu Leu Gly

275 280 285

Leu Phe Glu Pro Gly Asp Met Gln Tyr Glu Leu Asn Arg Asn Asn Val

290 295 300

Thr Asp Pro Ser Leu Ser Glu Met Val Val Val Ala Ile Gln Ile Leu

305 310 315 320

Arg Lys Asn Pro Lys Gly Phe Phe Leu Leu Val Glu Gly Gly Arg Ile

325 330 335

Asp His Gly His His Glu Gly Lys Ala Lys Gln Ala Leu His Glu Ala

340 345 350

Val Glu Met Asp Arg Ala Ile Gly Gln Ala Gly Ser Leu Thr Ser Ser

355 360 365

Glu Asp Thr Leu Thr Val Val Thr Ala Asp His Ser His Val Phe Thr

370 375 380

Phe Gly Gly Tyr Thr Pro Arg Gly Asn Ser Ile Phe Gly Leu Ala Pro

385 390 395 400

Met Leu Ser Asp Thr Asp Lys Lys Pro Phe Thr Ala Ile Leu Tyr Gly

405 410 415

Asn Gly Pro Gly Tyr Lys Val Val Gly Gly Glu Arg Glu Asn Val Ser

420 425 430

Met Val Asp Tyr Ala His Asn Asn Tyr Gln Ala Gln Ser Ala Val Pro

435 440 445

Leu Arg His Glu Thr His Gly Gly Glu Asp Val Ala Val Phe Ser Lys

450 455 460

Gly Pro Met Ala His Leu Leu His Gly Val His Glu Gln Asn Tyr Val

465 470 475 480

Pro His Val Met Ala Tyr Ala Ala Cys Ile Gly Ala Asn Leu Gly His

485 490 495

Cys Ala Pro Ala Ser Ser Ala Gly Ser Leu Ala Ala Gly Pro Leu Leu

500 505 510

Leu Ala Leu Ala Leu Tyr Pro Leu Ser Val Leu Phe

515 520

<210> 5

<211> 524

<212> Protein

<213> Homo sapiens

<400> 5

Met Ile Ser Pro Phe Leu Val Leu Ala Ile Gly Thr Cys Leu Thr Asn

1 5 10 15

Ser Leu Val Pro Glu Lys Glu Lys Asp Pro Lys Tyr Trp Arg Asp Gln

20 25 30

Ala Gln Glu Thr Leu Lys Tyr Ala Leu Glu Leu Gln Lys Leu Asn Thr

35 40 45

Asn Val Ala Lys Asn Val Ile Met Phe Leu Gly Asp Gly Met Gly Val

50 55 60

Ser Thr Val Thr Ala Ala Arg Ile Leu Lys Gly Gln Leu His His Asn

65 70 75 80

Pro Gly Glu Glu Thr Arg Leu Glu Met Asp Lys Phe Pro Phe Val Ala

85 90 95

Leu Ser Lys Thr Tyr Asn Thr Asn Ala Gln Val Pro Asp Ser Ala Gly

100 105 110

Thr Ala Thr Ala Tyr Leu Cys Gly Val Lys Ala Asn Glu Gly Thr Val

115 120 125

Gly Val Ser Ala Ala Thr Glu Arg Ser Arg Cys Asn Thr Thr Gln Gly

130 135 140

Asn Glu Val Thr Ser Ile Leu Arg Trp Ala Lys Asp Ala Gly Lys Ser

145 150 155 160

Val Gly Ile Val Thr Thr Thr Arg Val Asn His Ala Thr Pro Ser Ala

165 170 175

Ala Tyr Ala His Ser Ala Asp Arg Asp Trp Tyr Ser Asp Asn Glu Met

180 185 190

Pro Pro Glu Ala Leu Ser Gln Gly Cys Lys Asp Ile Ala Tyr Gln Leu

195 200 205

Met His Asn Ile Arg Asp Ile Asp Val Ile Met Gly Gly Gly Arg Lys

210 215 220

Tyr Met Tyr Pro Lys Asn Lys Thr Asp Val Glu Tyr Glu Ser Asp Glu

225 230 235 240

Lys Ala Arg Gly Thr Arg Leu Asp Gly Leu Asp Leu Val Asp Thr Trp

245 250 255

Lys Ser Phe Lys Pro Arg Tyr Lys His Ser His Phe Ile Trp Asn Arg

260 265 270

Thr Glu Leu Leu Thr Leu Asp Pro His Asn Val Asp Tyr Leu Leu Gly

275 280 285

Leu Phe Glu Pro Gly Asp Met Gln Tyr Glu Leu Asn Arg Asn Asn Val

290 295 300

Thr Asp Pro Ser Leu Ser Glu Met Val Val Val Ala Ile Gln Ile Leu

305 310 315 320

Arg Lys Asn Pro Lys Gly Phe Phe Leu Leu Val Glu Gly Gly Arg Ile

325 330 335

Asp His Gly His His Glu Gly Lys Ala Lys Gln Ala Leu His Glu Ala

340 345 350

Val Glu Met Asp Arg Ala Ile Gly Gln Ala Gly Ser Leu Thr Ser Ser

355 360 365

Glu Asp Thr Leu Thr Val Val Thr Ala Asp His Ser His Val Phe Thr

370 375 380

Phe Gly Gly Tyr Thr Pro Arg Gly Asn Ser Ile Phe Gly Leu Ala Pro

385 390 395 400

Met Leu Ser Asp Thr Asp Lys Lys Pro Phe Thr Ala Ile Leu Tyr Gly

405 410 415

Asn Gly Pro Gly Tyr Lys Val Val Gly Gly Glu Arg Glu Asn Val Ser

420 425 430

Met Val Asp Tyr Ala His Asn Asn Tyr Gln Ala Gln Ser Ala Val Pro

435 440 445

Leu Arg His Glu Thr His Gly Gly Glu Asp Val Ala Val Phe Ser Lys

450 455 460

Gly Pro Met Ala His Leu Leu His Gly Val His Glu Gln Asn Tyr Val

465 470 475 480

Pro His Val Met Ala Tyr Ala Ala Cys Ile Gly Ala Asn Leu Gly His

485 490 495

Cys Ala Pro Ala Ser Ser Ala Gly Ser Leu Ala Ala Gly Pro Leu Leu

500 505 510

Leu Ala Leu Ala Leu Tyr Pro Leu Ser Val Leu Phe

515 520

<210> 6

<211> 524

<212> Protein

<213> Homo sapiens

<400> 6

Met Ile Ser Pro Phe Leu Val Leu Ala Ile Gly Thr Cys Leu Thr Asn

1 5 10 15

Ser Leu Val Pro Glu Lys Glu Lys Asp Pro Lys Tyr Trp Arg Asp Gln

20 25 30

Ala Gln Glu Thr Leu Lys Tyr Ala Leu Glu Leu Gln Lys Leu Asn Thr

35 40 45

Asn Val Ala Lys Asn Val Ile Met Phe Leu Gly Asp Gly Met Gly Val

50 55 60

Ser Thr Val Thr Ala Ala Arg Ile Leu Lys Gly Gln Leu His His Asn

65 70 75 80

Pro Gly Glu Glu Thr Arg Leu Glu Met Asp Lys Phe Pro Phe Val Ala

85 90 95

Leu Ser Lys Thr Tyr Asn Thr Asn Ala Gln Val Pro Asp Ser Ala Gly

100 105 110

Thr Ala Thr Ala Tyr Leu Cys Gly Val Lys Ala Asn Glu Gly Thr Val

115 120 125

Gly Val Ser Ala Ala Thr Glu Arg Ser Arg Cys Asn Thr Thr Gln Gly

130 135 140

Asn Glu Val Thr Ser Ile Leu Arg Trp Ala Lys Asp Ala Gly Lys Ser

145 150 155 160

Val Gly Ile Val Thr Thr Thr Arg Val Asn His Ala Thr Pro Ser Ala

165 170 175

Ala Tyr Ala His Ser Ala Asp Arg Asp Trp Tyr Ser Asp Asn Glu Met

180 185 190

Pro Pro Glu Ala Leu Ser Gln Gly Cys Lys Asp Ile Ala Tyr Gln Leu

195 200 205

Met His Asn Ile Arg Asp Ile Asp Val Ile Met Gly Gly Gly Arg Lys

210 215 220

Tyr Met Tyr Pro Lys Asn Lys Thr Asp Val Glu Tyr Glu Ser Asp Glu

225 230 235 240

Lys Ala Arg Gly Thr Arg Leu Asp Gly Leu Asp Leu Val Asp Thr Trp

245 250 255

Lys Ser Phe Lys Pro Arg His Lys His Ser His Phe Ile Trp Asn Arg

260 265 270

Thr Glu Leu Leu Thr Leu Asp Pro His Asn Val Asp Tyr Leu Leu Gly

275 280 285

Leu Phe Glu Pro Gly Asp Met Gln Tyr Glu Leu Asn Arg Asn Asn Val

290 295 300

Thr Asp Pro Ser Leu Ser Glu Met Val Val Val Ala Ile Gln Ile Leu

305 310 315 320

Arg Lys Asn Pro Lys Gly Phe Phe Leu Leu Val Glu Gly Gly Arg Ile

325 330 335

Asp His Gly His His Glu Gly Lys Ala Lys Gln Ala Leu His Glu Ala

340 345 350

Val Glu Met Asp Arg Ala Ile Gly Gln Ala Gly Ser Leu Thr Ser Ser

355 360 365

Glu Asp Thr Leu Thr Val Val Thr Ala Asp His Ser His Val Phe Thr

370 375 380

Phe Gly Gly Tyr Thr Pro Arg Gly Asn Ser Ile Phe Gly Leu Ala Pro

385 390 395 400

Met Leu Ser Asp Thr Asp Lys Lys Pro Phe Thr Ala Ile Leu Tyr Gly

405 410 415

Asn Gly Pro Gly Tyr Lys Val Val Gly Gly Glu Arg Glu Asn Val Ser

420 425 430

Met Val Asp Tyr Ala His Asn Asn Tyr Gln Ala Gln Ser Ala Val Pro

435 440 445

Leu Arg His Glu Thr His Gly Gly Glu Asp Val Ala Val Phe Ser Lys

450 455 460

Gly Pro Met Ala His Leu Leu His Gly Val His Glu Gln Asn Tyr Val

465 470 475 480

Pro His Val Met Ala Tyr Ala Ala Cys Ile Gly Ala Asn Leu Gly His

485 490 495

Cys Ala Pro Ala Ser Ser Ala Gly Ser Leu Ala Ala Gly Pro Leu Leu

500 505 510

Leu Ala Leu Ala Leu Tyr Pro Leu Ser Val Leu Phe

515 520

<210> 7

<211> 652

<212> Protein

<213> Macaca mulatta

<400> 7

Met Pro Thr Val Lys Thr Lys Gln Glu Ser His Ala Gly Ser Gly Ser

1 5 10 15

Gly Pro Arg Leu Ala Glu Arg Lys Gly Arg Val Gly Ala Ala Arg Arg

20 25 30

Gln Ser Pro Arg Ala Pro Gly Gly Gly Leu Pro Gly Pro Arg Ser Gly

35 40 45

Pro Ala Ala Ala Phe Ile Arg Arg Arg Gly Arg Trp Pro Gly Pro Arg

50 55 60

Cys Ala Pro Ala Thr Pro Arg Pro Arg Ser Arg Leu Cys Ala Pro Thr

65 70 75 80

Arg Leu Cys Leu Asp Glu Pro Ser Ser Val Leu Cys Ala Gly Leu Glu

85 90 95

His Gln Leu Thr Ser Asp His Cys Gln Pro Thr Pro Ser His Pro Arg

100 105 110

Arg Ser His Leu Trp Ala Ser Gly Ile Lys Gln Val Leu Gly Cys Thr

115 120 125

Met Ile Ser Pro Phe Leu Val Leu Ala Ile Gly Thr Cys Leu Thr Asn

130 135 140

Ser Leu Val Pro Glu Lys Glu Lys Asp Pro Lys Tyr Trp Arg Asp Gln

145 150 155 160

Ala Gln Glu Thr Leu Lys Tyr Ala Leu Glu Leu Gln Lys Leu Asn Thr

165 170 175

Asn Val Ala Lys Asn Val Ile Met Phe Leu Gly Asp Gly Met Gly Val

180 185 190

Ser Thr Val Thr Ala Thr Arg Ile Leu Lys Gly Gln Leu His His Asn

195 200 205

Pro Gly Glu Glu Thr Arg Leu Glu Met Asp Lys Phe Pro Phe Val Ala

210 215 220

Leu Ser Lys Thr Tyr Asn Thr Asn Ala Gln Val Pro Asp Ser Ala Gly

225 230 235 240

Thr Ala Thr Ala Tyr Leu Cys Gly Val Lys Ala Asn Glu Gly Thr Val

245 250 255

Gly Val Ser Ala Ala Thr Glu Arg Ser Arg Cys Asn Thr Thr Gln Gly

260 265 270

Asn Glu Val Thr Ser Ile Leu Arg Trp Ala Lys Asp Ala Gly Lys Ser

275 280 285

Val Gly Ile Val Thr Thr Thr Arg Val Asn His Ala Thr Pro Ser Ala

290 295 300

Ala Tyr Ala His Ser Ala Asp Arg Asp Trp Tyr Ser Asp Asn Glu Met

305 310 315 320

Pro Pro Glu Ala Leu Ser Gln Gly Cys Lys Asp Ile Ala Tyr Gln Leu

325 330 335

Val His Asn Ile Arg Asp Ile Asp Val Ile Met Gly Gly Gly Arg Lys

340 345 350

Tyr Met Tyr Pro Lys Asn Lys Thr Asp Val Glu Tyr Glu Ile Asp Glu

355 360 365

Lys Ala Arg Gly Thr Arg Leu Asp Gly Leu Asp Leu Val Asn Ile Trp

370 375 380

Lys Ser Phe Lys Pro Arg His Lys His Ser His Phe Ile Trp Asn Arg

385 390 395 400

Thr Glu Leu Leu Thr Leu Asp Pro His Asn Val Asp Tyr Leu Leu Gly

405 410 415

Leu Phe Glu Pro Gly Asp Met Glu Tyr Glu Leu Asn Arg Asn Asn Val

420 425 430

Thr Asp Pro Ser Leu Ser Glu Met Val Val Val Ala Ile Gln Ile Leu

435 440 445

Arg Lys Asn Pro Lys Gly Phe Phe Leu Leu Val Glu Gly Gly Arg Ile

450 455 460

Asp His Gly His His Glu Gly Lys Ala Lys Gln Ala Leu His Glu Ala

465 470 475 480

Val Glu Met Asp Arg Ala Ile Gly Gln Ala Gly Ser Met Thr Ser Leu

485 490 495

Glu Asp Thr Leu Thr Val Val Thr Ala Asp His Ser His Val Phe Thr

500 505 510

Phe Gly Gly Tyr Thr Pro Arg Gly Asn Ser Ile Phe Gly Leu Ala Pro

515 520 525

Met Leu Ser Asp Thr Asp Lys Lys Pro Phe Thr Ala Ile Leu Tyr Gly

530 535 540

Asn Gly Pro Gly Tyr Lys Val Val Gly Gly Glu Arg Glu Asn Val Ser

545 550 555 560

Met Val Asp Tyr Ala His Asn Asn Tyr Gln Ala Gln Ser Ala Val Pro

565 570 575

Leu Arg His Glu Thr His Gly Gly Glu Asp Val Ala Val Phe Ser Lys

580 585 590

Gly Pro Met Ala His Leu Leu His Gly Val His Glu Gln Asn Tyr Ile

595 600 605

Pro His Val Met Ala Tyr Ala Ala Cys Ile Gly Ala Asn Leu Asp His

610 615 620

Cys Ala Pro Ala Ser Ser Ala Gly Ser Leu Ala Ala Gly Pro Leu Leu

625 630 635 640

Leu Pro Leu Ala Leu Phe Pro Leu Ser Ile Leu Phe

645 650

<210> 8

<211> 524

<212> Protein

<213> Rattus norvegicus

<400> 8

Met Ile Leu Pro Phe Leu Val Leu Ala Ile Gly Pro Cys Leu Thr Asn

1 5 10 15

Ser Phe Val Pro Glu Lys Glu Lys Asp Pro Ser Tyr Trp Arg Gln Gln

20 25 30

Ala Gln Glu Thr Leu Lys Asn Ala Leu Lys Leu Gln Lys Leu Asn Thr

35 40 45

Asn Val Ala Lys Asn Ile Ile Met Phe Leu Gly Asp Gly Met Gly Val

50 55 60

Ser Thr Val Thr Ala Ala Arg Ile Leu Lys Gly Gln Leu His His Asn

65 70 75 80

Thr Gly Glu Glu Thr Arg Leu Glu Met Asp Lys Phe Pro Phe Val Ala

85 90 95

Leu Ser Lys Thr Tyr Asn Thr Asn Ala Gln Val Pro Asp Ser Ala Gly

100 105 110

Thr Ala Thr Ala Tyr Leu Cys Gly Val Lys Ala Asn Glu Gly Thr Val

115 120 125

Gly Val Ser Ala Ala Thr Glu Arg Thr Arg Cys Asn Thr Thr Gln Gly

130 135 140

Asn Glu Val Thr Ser Ile Leu Arg Trp Ala Lys Asp Ala Gly Lys Ser

145 150 155 160

Val Gly Ile Val Thr Thr Thr Arg Val Asn His Ala Thr Pro Ser Ala

165 170 175

Ala Tyr Ala His Ser Ala Asp Arg Asp Trp Tyr Ser Asp Asn Glu Met

180 185 190

Arg Pro Glu Ala Leu Ser Gln Gly Cys Lys Asp Ile Ala Tyr Gln Leu

195 200 205

Met His Asn Ile Lys Asp Ile Asp Val Ile Met Gly Gly Gly Arg Lys

210 215 220

Tyr Met Tyr Pro Lys Asn Arg Thr Asp Val Glu Tyr Glu Leu Asp Glu

225 230 235 240

Lys Ala Arg Gly Thr Arg Leu Asp Gly Leu Asp Leu Ile Ser Ile Trp

245 250 255

Lys Ser Phe Lys Pro Arg His Lys His Ser His Tyr Val Trp Asn Arg

260 265 270

Thr Glu Leu Leu Ala Leu Asp Pro Ser Arg Val Asp Tyr Leu Leu Gly

275 280 285

Leu Phe Glu Pro Gly Asp Met Gln Tyr Glu Leu Asn Arg Asn Asn Leu

290 295 300

Thr Asp Pro Ser Leu Ser Glu Met Val Glu Val Ala Leu Arg Ile Leu

305 310 315 320

Thr Lys Asn Pro Lys Gly Phe Phe Leu Leu Val Glu Gly Gly Arg Ile

325 330 335

Asp His Gly His His Glu Gly Lys Ala Lys Gln Ala Leu His Glu Ala

340 345 350

Val Glu Met Asp Glu Ala Ile Gly Lys Ala Gly Thr Met Thr Ser Gln

355 360 365

Lys Asp Thr Leu Thr Val Val Thr Ala Asp His Ser His Val Phe Thr

370 375 380

Phe Gly Gly Tyr Thr Pro Arg Gly Asn Ser Ile Phe Gly Leu Ala Pro

385 390 395 400

Met Val Ser Asp Thr Asp Lys Lys Pro Phe Thr Ala Ile Leu Tyr Gly

405 410 415

Asn Gly Pro Gly Tyr Lys Val Val Asp Gly Glu Arg Glu Asn Val Ser

420 425 430

Met Val Asp Tyr Ala His Asn Asn Tyr Gln Ala Gln Ser Ala Val Pro

435 440 445

Leu Arg His Glu Thr His Gly Gly Glu Asp Val Ala Val Phe Ala Lys

450 455 460

Gly Pro Met Ala His Leu Leu His Gly Val His Glu Gln Asn Tyr Ile

465 470 475 480

Pro His Val Met Ala Tyr Ala Ser Cys Ile Gly Ala Asn Leu Asp His

485 490 495

Cys Ala Trp Ala Ser Ser Ala Ser Ser Pro Ser Pro Gly Ala Leu Leu

500 505 510

Leu Pro Leu Ala Leu Phe Pro Leu Arg Thr Leu Phe

515 520

<210> 9

<211> 502

<212> Protein

<213> Canis lupus familiaris

<400> 9

Glu Lys Asp Pro Lys Tyr Trp Arg Asp Gln Ala Gln Gln Thr Leu Lys

1 5 10 15

Tyr Ala Leu Arg Leu Gln Asn Leu Asn Thr Asn Val Ala Lys Asn Val

20 25 30

Ile Met Phe Leu Gly Asp Gly Met Gly Val Ser Thr Val Thr Ala Thr

35 40 45

Arg Ile Leu Lys Gly Gln Leu His His Asn Pro Gly Glu Glu Thr Arg

50 55 60

Leu Glu Met Asp Lys Phe Pro Tyr Val Ala Leu Ser Lys Thr Tyr Asn

65 70 75 80

Thr Asn Ala Gln Val Pro Asp Ser Ala Gly Thr Ala Thr Ala Tyr Leu

85 90 95

Cys Gly Val Lys Ala Asn Glu Gly Thr Val Gly Val Ser Ala Ala Thr

100 105 110

Gln Arg Thr His Cys Asn Thr Thr Gln Gly Asn Glu Val Thr Ser Ile

115 120 125

Leu Arg Trp Ala Lys Asp Ala Gly Lys Ser Val Gly Ile Val Thr Thr

130 135 140

Thr Arg Val Asn His Ala Thr Pro Ser Ala Ala Tyr Ala His Ser Ala

145 150 155 160

Asp Arg Asp Trp Tyr Ser Asp Asn Glu Met Pro Pro Glu Ala Leu Ser

165 170 175

Gln Gly Cys Lys Asp Ile Ala Tyr Gln Leu Met His Asn Val Lys Asp

180 185 190

Ile Glu Val Ile Met Gly Gly Gly Arg Lys Tyr Met Phe Pro Lys Asn

195 200 205

Arg Thr Asp Val Glu Tyr Glu Met Asp Glu Lys Ser Thr Gly Ala Arg

210 215 220

Leu Asp Gly Leu Asn Leu Ile Asp Ile Trp Lys Asn Phe Lys Pro Arg

225 230 235 240

His Lys His Ser His Tyr Val Trp Asn Arg Thr Glu Leu Leu Ala Leu

245 250 255

Asp Pro Tyr Thr Val Asp Tyr Leu Leu Gly Leu Phe Asp Pro Gly Asp

260 265 270

Met Gln Tyr Glu Leu Asn Arg Asn Asn Val Thr Asp Pro Ser Leu Ser

275 280 285

Glu Met Val Glu Ile Ala Ile Lys Ile Leu Ser Lys Lys Pro Arg Gly

290 295 300

Phe Phe Leu Leu Val Glu Gly Gly Arg Ile Asp His Gly His His Glu

305 310 315 320

Gly Lys Ala Lys Gln Ala Leu His Glu Ala Val Glu Met Asp Arg Ala

325 330 335

Ile Gly Lys Ala Gly Val Met Thr Ser Leu Glu Asp Thr Leu Thr Val

340 345 350

Val Thr Ala Asp His Ser His Val Phe Thr Phe Gly Gly Tyr Thr Pro

355 360 365

Arg Gly Asn Ser Ile Phe Gly Leu Ala Pro Met Val Ser Asp Thr Asp

370 375 380

Lys Lys Pro Phe Thr Ala Ile Leu Tyr Gly Asn Gly Pro Gly Tyr Lys

385 390 395 400

Val Val Gly Gly Glu Arg Glu Asn Val Ser Met Val Asp Tyr Ala His

405 410 415

Asn Asn Tyr Gln Ala Gln Ser Ala Val Pro Leu Arg His Glu Thr His

420 425 430

Gly Gly Glu Asp Val Ala Val Phe Ala Lys Gly Pro Met Ala His Leu

435 440 445

Leu His Gly Val His Glu Gln Asn Tyr Ile Pro His Val Met Ala Tyr

450 455 460

Ala Ala Cys Ile Gly Ala Asn Gln Asp His Cys Ala Ser Ala Ser Ser

465 470 475 480

Ala Gly Gly Pro Ser Pro Gly Pro Leu Leu Leu Leu Leu Ala Leu Leu

485 490 495

Pro Val Gly Ile Leu Phe

500

<210> 10

<211> 252

<212> Protein

<213> Sus scrofa

<400> 10

Ala Glu Leu Leu Ala Leu Asp Pro His Thr Val Asp Tyr Leu Leu Gly

1 5 10 15

Leu Phe Glu Pro Gly Asp Met Gln Tyr Glu Leu Asn Arg Asn Asn Val

20 25 30

Thr Asp Pro Ser Leu Ser Glu Met Val Glu Met Ala Ile Arg Ile Leu

35 40 45

Ile Lys Asn Pro Lys Gly Phe Phe Leu Leu Val Glu Gly Gly Arg Ile

50 55 60

Asp His Gly His His Glu Gly Lys Ala Lys Gln Ala Leu His Glu Ala

65 70 75 80

Val Glu Met Asp Arg Ala Ile Glu Gln Ala Gly Ser Met Thr Ser Val

85 90 95

Glu Asp Thr Leu Thr Val Val Thr Ala Asp His Ser His Val Phe Thr

100 105 110

Phe Gly Gly Tyr Thr Pro Arg Gly Asn Ser Ile Phe Gly Leu Ala Pro

115 120 125

Met Val Ser Asp Thr Asp Lys Lys Pro Phe Thr Ala Ile Leu Tyr Gly

130 135 140

Asn Gly Pro Gly Tyr Lys Val Val Gly Gly Glu Arg Glu Asn Val Ser

145 150 155 160

Met Val Asp Tyr Ala His Asp Asn Tyr Gln Ala Gln Ser Ala Val Pro

165 170 175

Leu Arg His Glu Thr His Gly Gly Glu Asp Val Ala Ile Phe Ala Arg

180 185 190

Gly Pro Met Ala His Leu Leu His Gly Val His Glu Gln Asn Tyr Ile

195 200 205

Pro His Val Met Ala Tyr Ala Ala Cys Val Gly Ala Asn Arg Asp His

210 215 220

Cys Ala Ser Ala Ser Ser Ser Gly Ser Pro Ser Pro Gly Pro Leu Leu

225 230 235 240

Leu Leu Leu Ala Leu Leu Pro Leu Gly Ile Leu Phe

245 250

<210> 11

<211> 524

<212> Protein

<213> Mus musculus

<400> 11

Met Ile Ser Pro Phe Leu Val Leu Ala Ile Gly Thr Cys Leu Thr Asn

1 5 10 15

Ser Phe Val Pro Glu Lys Glu Arg Asp Pro Ser Tyr Trp Arg Gln Gln

20 25 30

Ala Gln Glu Thr Leu Lys Asn Ala Leu Lys Leu Gln Lys Leu Asn Thr

35 40 45

Asn Val Ala Lys Asn Val Ile Met Phe Leu Gly Asp Gly Met Gly Val

50 55 60

Ser Thr Val Thr Ala Ala Arg Ile Leu Lys Gly Gln Leu His His Asn

65 70 75 80

Thr Gly Glu Glu Thr Arg Leu Glu Met Asp Lys Phe Pro Phe Val Ala

85 90 95

Leu Ser Lys Thr Tyr Asn Thr Asn Ala Gln Val Pro Asp Ser Ala Gly

100 105 110

Thr Ala Thr Ala Tyr Leu Cys Gly Val Lys Ala Asn Glu Gly Thr Val

115 120 125

Gly Val Ser Ala Ala Thr Glu Arg Thr Arg Cys Asn Thr Thr Gln Gly

130 135 140

Asn Glu Val Thr Ser Ile Leu Arg Trp Ala Lys Asp Ala Gly Lys Ser

145 150 155 160

Val Gly Ile Val Thr Thr Thr Arg Val Asn His Ala Thr Pro Ser Ala

165 170 175

Ala Tyr Ala His Ser Ala Asp Arg Asp Trp Tyr Ser Asp Asn Glu Met

180 185 190

Pro Pro Glu Ala Leu Ser Gln Gly Cys Lys Asp Ile Ala Tyr Gln Leu

195 200 205

Met His Asn Ile Lys Asp Ile Asp Val Ile Met Gly Gly Gly Arg Lys

210 215 220

Tyr Met Tyr Pro Lys Asn Arg Thr Asp Val Glu Tyr Glu Leu Asp Glu

225 230 235 240

Lys Ala Arg Gly Thr Arg Leu Asp Gly Leu Asp Leu Ile Ser Ile Trp

245 250 255

Lys Ser Phe Lys Pro Arg His Lys His Ser His Tyr Val Trp Asn Arg

260 265 270

Thr Glu Leu Leu Ala Leu Asp Pro Ser Arg Val Asp Tyr Leu Leu Gly

275 280 285

Leu Phe Glu Pro Gly Asp Met Gln Tyr Glu Leu Asn Arg Asn Asn Leu

290 295 300

Thr Asp Pro Ser Leu Ser Glu Met Val Glu Val Ala Leu Arg Ile Leu

305 310 315 320

Thr Lys Asn Leu Lys Gly Phe Phe Leu Leu Val Glu Gly Gly Arg Ile

325 330 335

Asp His Gly His His Glu Gly Lys Ala Lys Gln Ala Leu His Glu Ala

340 345 350

Val Glu Met Asp Gln Ala Ile Gly Lys Ala Gly Ala Met Thr Ser Gln

355 360 365

Lys Asp Thr Leu Thr Val Val Thr Ala Asp His Ser His Val Phe Thr

370 375 380

Phe Gly Gly Tyr Thr Pro Arg Gly Asn Ser Ile Phe Gly Leu Ala Pro

385 390 395 400

Met Val Ser Asp Thr Asp Lys Lys Pro Phe Thr Ala Ile Leu Tyr Gly

405 410 415

Asn Gly Pro Gly Tyr Lys Val Val Asp Gly Glu Arg Glu Asn Val Ser

420 425 430

Met Val Asp Tyr Ala His Asn Asn Tyr Gln Ala Gln Ser Ala Val Pro

435 440 445

Leu Arg His Glu Thr His Gly Gly Glu Asp Val Ala Val Phe Ala Lys

450 455 460

Gly Pro Met Ala His Leu Leu His Gly Val His Glu Gln Asn Tyr Ile

465 470 475 480

Pro His Val Met Ala Tyr Ala Ser Cys Ile Gly Ala Asn Leu Asp His

485 490 495

Cys Ala Trp Ala Gly Ser Gly Ser Ala Pro Ser Pro Gly Ala Leu Leu

500 505 510

Leu Pro Leu Ala Val Leu Ser Leu Arg Thr Leu Phe

515 520

<210> 12

<211> 524

<212> Protein

<213> Bos taurus

<400> 12

Met Ile Ser Pro Phe Leu Leu Leu Ala Ile Gly Thr Cys Phe Ala Ser

1 5 10 15

Ser Leu Val Pro Glu Lys Glu Lys Asp Pro Lys Tyr Trp Arg Asp Gln

20 25 30

Ala Gln Gln Thr Leu Lys Asn Ala Leu Arg Leu Gln Thr Leu Asn Thr

35 40 45

Asn Val Ala Lys Asn Val Ile Met Phe Leu Gly Asp Gly Met Gly Val

50 55 60

Ser Thr Val Thr Ala Ala Arg Ile Leu Lys Gly Gln Leu His His Ser

65 70 75 80

Pro Gly Glu Glu Thr Lys Leu Glu Met Asp Lys Phe Pro Tyr Val Ala

85 90 95

Leu Ser Lys Thr Tyr Asn Thr Asn Ala Gln Val Pro Asp Ser Ala Gly

100 105 110

Thr Ala Thr Ala Tyr Leu Cys Gly Val Lys Ala Asn Glu Gly Thr Val

115 120 125

Gly Val Ser Ala Ala Thr Gln Arg Ser Gln Cys Asn Thr Thr Gln Gly

130 135 140

Asn Glu Val Thr Ser Ile Leu Arg Trp Ala Lys Asp Ala Gly Lys Ser

145 150 155 160

Val Gly Ile Val Thr Thr Thr Arg Val Asn His Ala Thr Pro Ser Ala

165 170 175

Ser Tyr Ala His Ser Ala Asp Arg Asp Trp Tyr Ser Asp Asn Glu Met

180 185 190

Pro Pro Glu Ala Leu Ser Gln Gly Cys Lys Asp Ile Ala Tyr Gln Leu

195 200 205

Met His Asn Ile Lys Asp Ile Glu Val Ile Met Gly Gly Gly Arg Lys

210 215 220

Tyr Met Phe Pro Lys Asn Arg Thr Asp Val Glu Tyr Glu Leu Asp Glu

225 230 235 240

Lys Ala Arg Gly Thr Arg Leu Asp Gly Leu Asn Leu Ile Asp Ile Trp

245 250 255

Lys Ser Phe Lys Pro Lys His Lys His Ser His Tyr Val Trp Asn Arg

260 265 270

Thr Asp Leu Leu Ala Leu Asp Pro His Ser Val Asp Tyr Leu Leu Gly

275 280 285

Leu Phe Glu Pro Gly Asp Met Gln Tyr Glu Leu Asn Arg Asn Asn Ala

290 295 300

Thr Asp Pro Ser Leu Ser Glu Met Val Glu Met Ala Ile Arg Ile Leu

305 310 315 320

Asn Lys Asn Pro Lys Gly Phe Phe Leu Leu Val Glu Gly Gly Arg Ile

325 330 335

Asp His Gly His His Glu Gly Lys Ala Lys Gln Ala Leu His Glu Ala

340 345 350

Val Glu Met Asp Gln Ala Ile Gly Gln Ala Gly Ala Met Thr Ser Val

355 360 365

Glu Asp Thr Leu Thr Val Val Thr Ala Asp His Ser His Val Phe Thr

370 375 380

Phe Gly Gly Tyr Thr Pro Arg Gly Asn Ser Ile Phe Gly Leu Ala Pro

385 390 395 400

Met Val Ser Asp Thr Asp Lys Lys Pro Phe Thr Ala Ile Leu Tyr Gly

405 410 415

Asn Gly Pro Gly Tyr Lys Val Val Gly Gly Glu Arg Glu Asn Val Ser

420 425 430

Met Val Asp Tyr Ala His Asn Asn Tyr Gln Ala Gln Ser Ala Val Pro

435 440 445

Leu Arg His Glu Thr His Gly Gly Glu Asp Val Ala Val Phe Ala Lys

450 455 460

Gly Pro Met Ala His Leu Leu His Gly Val His Glu Gln Asn Tyr Ile

465 470 475 480

Pro His Val Met Ala Tyr Ala Ala Cys Ile Gly Ala Asn Arg Asp His

485 490 495

Cys Ala Ser Ala Ser Ser Ser Gly Ser Pro Ser Pro Gly Pro Leu Leu

500 505 510

Leu Leu Leu Ala Leu Leu Pro Leu Gly Ser Leu Phe

515 520

<210> 13

<211> 524

<212> Protein

<213> Bos taurus

<400> 13

Met Ile Ser Pro Phe Leu Leu Leu Ala Ile Gly Thr Cys Phe Ala Ser

1 5 10 15

Ser Leu Val Pro Glu Lys Glu Lys Asp Pro Lys Tyr Trp Arg Asp Gln

20 25 30

Ala Gln Gln Thr Leu Lys Asn Ala Leu Arg Leu Gln Thr Leu Asn Thr

35 40 45

Asn Val Ala Lys Asn Val Ile Met Phe Leu Gly Asp Gly Met Gly Val

50 55 60

Ser Thr Val Thr Ala Ala Arg Ile Leu Lys Gly Gln Leu His His Ser

65 70 75 80

Pro Gly Glu Glu Thr Lys Leu Glu Met Asp Lys Phe Pro Tyr Val Ala

85 90 95

Leu Ser Lys Thr Tyr Asn Thr Asn Ala Gln Val Pro Asp Ser Ala Gly

100 105 110

Thr Ala Thr Ala Tyr Leu Cys Gly Val Lys Ala Asn Glu Gly Thr Val

115 120 125

Gly Val Ser Ala Ala Thr Gln Arg Ser Gln Cys Asn Thr Thr Gln Gly

130 135 140

Asn Glu Val Thr Ser Ile Leu Arg Trp Ala Lys Asp Ala Gly Lys Ser

145 150 155 160

Val Gly Ile Val Thr Thr Thr Arg Val Asn His Ala Thr Pro Ser Ala

165 170 175

Ser Tyr Ala His Ser Ala Asp Arg Asp Trp Tyr Ser Asp Asn Glu Met

180 185 190

Pro Pro Glu Ala Leu Ser Gln Gly Cys Lys Asp Ile Ala Tyr Gln Leu

195 200 205

Met His Asn Ile Lys Asp Ile Glu Val Ile Met Gly Gly Gly Arg Lys

210 215 220

Tyr Met Phe Pro Lys Asn Arg Thr Asp Val Glu Tyr Glu Leu Asp Glu

225 230 235 240

Lys Ala Arg Gly Thr Arg Leu Asp Gly Leu Asn Leu Ile Asp Ile Trp

245 250 255

Lys Ser Phe Lys Pro Lys His Lys His Ser His Tyr Val Trp Asn Arg

260 265 270

Thr Asp Leu Leu Ala Leu Asp Pro His Ser Val Asp Tyr Leu Leu Gly

275 280 285

Leu Phe Glu Pro Gly Asp Met Gln Tyr Glu Leu Asn Arg Asn Asn Ala

290 295 300

Thr Asp Pro Ser Leu Ser Glu Met Val Glu Met Ala Ile Arg Ile Leu

305 310 315 320

Asn Lys Asn Pro Lys Gly Phe Phe Leu Leu Val Glu Gly Gly Arg Ile

325 330 335

Asp His Gly His His Glu Gly Lys Ala Lys Gln Ala Leu His Glu Ala

340 345 350

Val Glu Met Asp Gln Ala Ile Gly Gln Ala Gly Ala Met Thr Ser Val

355 360 365

Glu Asp Thr Leu Thr Val Val Thr Ala Asp His Ser His Val Phe Thr

370 375 380

Phe Gly Gly Tyr Thr Pro Arg Gly Asn Ser Ile Phe Gly Leu Ala Pro

385 390 395 400

Met Val Ser Asp Thr Asp Lys Lys Pro Phe Thr Ala Ile Leu Tyr Gly

405 410 415

Asn Gly Pro Gly Tyr Lys Val Val Gly Gly Glu Arg Glu Asn Val Ser

420 425 430

Met Val Asp Tyr Ala His Asn Asn Tyr Gln Ala Gln Ser Ala Val Pro

435 440 445

Leu Arg His Glu Thr His Gly Gly Glu Asp Val Ala Val Phe Ala Lys

450 455 460

Gly Pro Met Ala His Leu Leu His Gly Val His Glu Gln Asn Tyr Ile

465 470 475 480

Pro His Val Met Ala Tyr Ala Ala Cys Ile Gly Ala Asn Arg Asp His

485 490 495

Cys Ala Ser Ala Ser Ser Ser Gly Ser Pro Ser Pro Gly Pro Leu Leu

500 505 510

Leu Leu Leu Ala Leu Leu Pro Leu Gly Ser Leu Phe

515 520

<210> 14

<211> 124

<212> Protein

<213> Bos taurus

<400> 14

Asp Pro Lys Tyr Trp Arg Asp Gln Ala Gln Gln Thr Leu Lys Asn Ala

1 5 10 15

Leu Gly Leu Gln Lys Leu Asn Thr Lys Val Ala Lys Asn Val Ile Leu

20 25 30

Phe Leu Gly Asp Gly Met Gly Val Ser Thr Val Thr Ala Ala Arg Ile

35 40 45

Leu Lys Gly Gln Leu His His Asn Pro Gly Glu Glu Thr Arg Leu Glu

50 55 60

Met Asp Lys Phe Pro Phe Val Ala Leu Ser Lys Thr Tyr Asn Thr Asn

65 70 75 80

Ala Gln Val Pro Asp Ser Ala Gly Thr Ala Pro His Pro Val Arg Val

85 90 95

Lys Ala Met Arg Ala Pro Trp Gly Glu Pro His Gln Arg Gln Cys Asn

100 105 110

Thr Arg Arg Ala Thr Ser Thr His Leu Leu Ala Gly

115 120

<210> 15

<211> 524

<212> Protein

<213> Felis catus

<400> 15

Met Ile Ser Pro Phe Leu Val Leu Ala Ile Gly Thr Cys Leu Thr Asn

1 5 10 15

Ser Leu Val Pro Glu Lys Glu Lys Asp Pro Lys Tyr Trp Arg Asp Gln

20 25 30

Ala Gln Gln Thr Leu Lys Asn Ala Leu Arg Leu Gln Lys Leu Asn Thr

35 40 45

Asn Val Val Lys Asn Val Ile Met Phe Leu Gly Asp Gly Met Gly Val

50 55 60

Ser Thr Val Thr Ala Ala Arg Ile Leu Lys Gly Gln Leu His His Asn

65 70 75 80

Pro Gly Glu Glu Thr Arg Leu Glu Met Asp Lys Phe Pro Tyr Val Ala

85 90 95

Leu Ser Lys Thr Tyr Asn Thr Asn Ala Gln Val Pro Asp Ser Ala Gly

100 105 110

Thr Ala Thr Ala Tyr Leu Cys Gly Val Lys Ala Asn Glu Gly Thr Val

115 120 125

Gly Val Ser Ala Ala Thr Gln Arg Thr Gln Cys Asn Thr Thr Gln Gly

130 135 140

Asn Glu Val Thr Ser Ile Leu Arg Trp Ala Lys Asp Ser Gly Lys Ser

145 150 155 160

Val Gly Ile Val Thr Thr Thr Arg Val Asn His Ala Thr Pro Ser Ala

165 170 175

Ala Tyr Ala His Ser Ala Asp Arg Asp Trp Tyr Ser Asp Asn Glu Met

180 185 190

Pro Pro Glu Ala Leu Ser Gln Gly Cys Lys Asp Ile Ala Tyr Gln Leu

195 200 205

Met His Asn Val Arg Asp Ile Glu Val Ile Met Gly Gly Gly Arg Lys

210 215 220

Tyr Met Phe Pro Lys Asn Arg Thr Asp Val Glu Tyr Glu Met Asp Glu

225 230 235 240

Lys Ala Arg Gly Thr Arg Leu Asp Gly Leu Asn Leu Val Asp Ile Trp

245 250 255

Lys Ser Phe Lys Pro Arg His Lys His Ser His Tyr Val Trp Asn Arg

260 265 270

Thr Glu Leu Leu Thr Leu Asp Pro Tyr Gly Val Asp Tyr Leu Leu Gly

275 280 285

Leu Phe Glu Pro Gly Asp Met Gln Tyr Glu Leu Asn Arg Asn Ser Thr

290 295 300

Thr Asp Pro Ser Leu Ser Glu Met Val Glu Ile Ala Ile Lys Ile Leu

305 310 315 320

Ser Lys Asn Pro Lys Gly Phe Phe Leu Leu Val Glu Gly Gly Arg Ile

325 330 335

Asp His Gly His His Glu Gly Lys Ala Lys Gln Ala Leu His Glu Ala

340 345 350

Val Glu Met Asp Gln Ala Ile Gly Arg Ala Gly Ala Met Thr Ser Val

355 360 365

Glu Asp Thr Leu Thr Ile Val Thr Ala Asp His Ser His Val Phe Thr

370 375 380

Phe Gly Gly Tyr Thr Pro Arg Gly Asn Ser Ile Phe Gly Leu Ala Pro

385 390 395 400

Met Val Ser Asp Thr Asp Lys Lys Pro Phe Thr Ser Ile Leu Tyr Gly

405 410 415

Asn Gly Pro Gly Tyr Lys Val Val Gly Gly Glu Arg Glu Asn Val Ser

420 425 430

Met Val Asp Tyr Ala His Asn Asn Tyr Gln Ala Gln Ser Ala Val Pro

435 440 445

Leu Arg His Glu Thr His Gly Gly Glu Asp Val Ala Val Phe Ala Lys

450 455 460

Gly Pro Met Ala His Leu Leu His Gly Val His Glu Gln Asn Tyr Ile

465 470 475 480

Pro His Val Met Ala Tyr Ala Ala Cys Ile Gly Ala Asn Leu Asp His

485 490 495

Cys Ala Ser Ala Ser Ser Ala Gly Gly Pro Ser Pro Gly Pro Leu Phe

500 505 510

Leu Leu Leu Ala Leu Pro Ser Leu Gly Ile Leu Phe

515 520

<210> 16

<211> 532

<212> Protein

<213> Homo sapiens

<400> 16

Met Gln Gly Pro Trp Val Leu Leu Leu Leu Gly Leu Arg Leu Gln Leu

1 5 10 15

Ser Leu Gly Ile Ile Pro Val Glu Glu Glu Asn Pro Asp Phe Trp Asn

20 25 30

Arg Gln Ala Ala Glu Ala Leu Gly Ala Ala Lys Lys Leu Gln Pro Ala

35 40 45

Gln Thr Ala Ala Lys Asn Leu Ile Ile Phe Leu Gly Asp Gly Met Gly

50 55 60

Val Ser Thr Val Thr Ala Ala Arg Ile Leu Lys Gly Gln Lys Lys Asp

65 70 75 80

Lys Leu Gly Pro Glu Thr Phe Leu Ala Met Asp Arg Phe Pro Tyr Val

85 90 95

Ala Leu Ser Lys Thr Tyr Ser Val Asp Lys His Val Pro Asp Ser Gly

100 105 110

Ala Thr Ala Thr Ala Tyr Leu Cys Gly Val Lys Gly Asn Phe Gln Thr

115 120 125

Ile Gly Leu Ser Ala Ala Ala Arg Phe Asn Gln Cys Asn Thr Thr Arg

130 135 140

Gly Asn Glu Val Ile Ser Val Met Asn Arg Ala Lys Lys Ala Gly Lys

145 150 155 160

Ser Val Gly Val Val Thr Thr Thr Thr Arg Val Gln His Ala Ser Pro Ala

165 170 175

Gly Ala Tyr Ala His Thr Val Asn Arg Asn Trp Tyr Ser Asp Ala Asp

180 185 190

Val Pro Ala Ser Ala Arg Gln Glu Gly Cys Gln Asp Ile Ala Thr Gln

195 200 205

Leu Ile Ser Asn Met Asp Ile Asp Val Ile Leu Gly Gly Gly Arg Lys

210 215 220

Tyr Met Phe Pro Met Gly Thr Pro Asp Pro Glu Tyr Pro Asp Asp Tyr

225 230 235 240

Ser Gln Gly Gly Thr Arg Leu Asp Gly Lys Asn Leu Val Gln Glu Trp

245 250 255

Leu Ala Lys His Gln Gly Ala Arg Tyr Val Trp Asn Arg Thr Glu Leu

260 265 270

Leu Gln Ala Ser Leu Asp Pro Ser Val Thr His Leu Met Gly Leu Phe

275 280 285

Glu Pro Gly Asp Met Lys Tyr Glu Ile His Arg Asp Ser Thr Leu Asp

290 295 300

Pro Ser Leu Met Glu Met Thr Glu Ala Ala Leu Leu Leu Leu Ser Arg

305 310 315 320

Asn Pro Arg Gly Phe Phe Leu Phe Val Glu Gly Gly Arg Ile Asp His

325 330 335

Gly His His Glu Ser Arg Ala Tyr Arg Ala Leu Thr Glu Thr Ile Met

340 345 350

Phe Asp Asp Ala Ile Glu Arg Ala Gly Gln Leu Thr Ser Glu Glu Asp

355 360 365

Thr Leu Ser Leu Val Thr Ala Asp His Ser His Val Phe Ser Phe Gly

370 375 380

Gly Tyr Pro Leu Arg Gly Ser Ser Ile Phe Gly Leu Ala Pro Gly Lys

385 390 395 400

Ala Arg Asp Arg Lys Ala Tyr Thr Val Leu Leu Tyr Gly Asn Gly Pro

405 410 415

Gly Tyr Val Leu Lys Asp Gly Ala Arg Pro Asp Val Thr Glu Ser Glu

420 425 430

Ser Gly Ser Pro Glu Tyr Arg Gln Gln Ser Ala Val Pro Leu Asp Gly

435 440 445

Glu Thr His Ala Gly Glu Asp Val Ala Val Phe Ala Arg Gly Pro Gln

450 455 460

Ala His Leu Val His Gly Val Gln Glu Gln Thr Phe Ile Ala His Val

465 470 475 480

Met Ala Phe Ala Ala Cys Leu Glu Pro Tyr Thr Ala Cys Asp Leu Ala

485 490 495

Pro Arg Ala Gly Thr Thr Asp Ala Ala His Pro Gly Pro Ser Val Val

500 505 510

Pro Ala Leu Leu Pro Leu Leu Ala Gly Thr Leu Leu Leu Leu Gly Thr

515 520 525

Ala Thr Ala Pro

530

<210> 17

<211> 535

<212> Protein

<213> Homo sapiens

<400> 17

Met Leu Gly Pro Cys Met Leu Leu Leu Leu Leu Leu Leu Gly Leu Arg

1 5 10 15

Leu Gln Leu Ser Leu Gly Ile Ile Pro Val Glu Glu Glu Asn Pro Asp

20 25 30

Phe Trp Asn Arg Glu Ala Ala Glu Ala Leu Gly Ala Ala Lys Lys Leu

35 40 45

Gln Pro Ala Gln Thr Ala Ala Lys Asn Leu Ile Ile Phe Leu Gly Asp

50 55 60

Gly Met Gly Val Ser Thr Val Thr Ala Ala Arg Ile Leu Lys Gly Gln

65 70 75 80

Lys Lys Asp Lys Leu Gly Pro Glu Ile Pro Leu Ala Met Asp Arg Phe

85 90 95

Pro Tyr Val Ala Leu Ser Lys Thr Tyr Asn Val Asp Lys His Val Pro

100 105 110

Asp Ser Gly Ala Thr Ala Thr Ala Tyr Leu Cys Gly Val Lys Gly Asn

115 120 125

Phe Gln Thr Ile Gly Leu Ser Ala Ala Ala Arg Phe Asn Gln Cys Asn

130 135 140

Thr Thr Arg Gly Asn Glu Val Ile Ser Val Met Asn Arg Ala Lys Lys

145 150 155 160

Ala Gly Lys Ser Val Gly Val Val Thr Thr Thr Arg Val Gln His Ala

165 170 175

Ser Pro Ala Gly Thr Tyr Ala His Thr Val Asn Arg Asn Trp Tyr Ser

180 185 190

Asp Ala Asp Val Pro Ala Ser Ala Arg Gln Glu Gly Cys Gln Asp Ile

195 200 205

Ala Thr Gln Leu Ile Ser Asn Met Asp Ile Asp Val Ile Leu Gly Gly

210 215 220

Gly Arg Lys Tyr Met Phe Arg Met Gly Thr Pro Asp Pro Glu Tyr Pro

225 230 235 240

Asp Asp Tyr Ser Gln Gly Gly Thr Arg Leu Asp Gly Lys Asn Leu Val

245 250 255

Gln Glu Trp Leu Ala Lys Arg Gln Gly Ala Arg Tyr Val Trp Asn Arg

260 265 270

Thr Glu Leu Met Gln Ala Ser Leu Asp Pro Ser Val Thr His Leu Met

275 280 285

Gly Leu Phe Glu Pro Gly Asp Met Lys Tyr Glu Ile His Arg Asp Ser

290 295 300

Thr Leu Asp Pro Ser Leu Met Glu Met Thr Glu Ala Ala Leu Arg Leu

305 310 315 320

Leu Ser Arg Asn Pro Arg Gly Phe Phe Leu Phe Val Glu Gly Gly Arg

325 330 335

Ile Asp His Gly His His Glu Ser Arg Ala Tyr Arg Ala Leu Thr Glu

340 345 350

Thr Ile Met Phe Asp Asp Ala Ile Glu Arg Ala Gly Gln Leu Thr Ser

355 360 365

Glu Glu Asp Thr Leu Ser Leu Val Thr Ala Asp His Ser His Val Phe

370 375 380

Ser Phe Gly Gly Tyr Pro Leu Arg Gly Ser Ser Ile Phe Gly Leu Ala

385 390 395 400

Pro Gly Lys Ala Arg Asp Arg Lys Ala Tyr Thr Val Leu Leu Tyr Gly

405 410 415

Asn Gly Pro Gly Tyr Val Leu Lys Asp Gly Ala Arg Pro Asp Val Thr

420 425 430

Glu Ser Glu Ser Gly Ser Pro Glu Tyr Arg Gln Gln Ser Ala Val Pro

435 440 445

Leu Asp Glu Glu Thr His Ala Gly Glu Asp Val Ala Val Phe Ala Arg

450 455 460

Gly Pro Gln Ala His Leu Val His Gly Val Gln Glu Gln Thr Phe Ile

465 470 475 480

Ala His Val Met Ala Phe Ala Ala Cys Leu Glu Pro Tyr Thr Ala Cys

485 490 495

Asp Leu Ala Pro Pro Ala Gly Thr Thr Asp Ala Ala His Pro Gly Arg

500 505 510

Ser Val Val Pro Ala Leu Leu Pro Leu Leu Ala Gly Thr Leu Leu Leu

515 520 525

Leu Glu Thr Ala Thr Ala Pro

530 535

<210> 18

<211> 532

<212> Protein

<213> Homo sapiens

<400> 18

Met Gln Gly Pro Trp Val Leu Leu Leu Leu Gly Leu Arg Leu Gln Leu

1 5 10 15

Ser Leu Gly Ile Ile Pro Val Glu Glu Glu Asn Pro Asp Phe Trp Asn

20 25 30

Arg Gln Ala Ala Glu Ala Leu Gly Ala Ala Lys Lys Leu Gln Pro Ala

35 40 45

Gln Thr Ala Ala Lys Asn Leu Ile Ile Phe Leu Gly Asp Gly Met Gly

50 55 60

Val Ser Thr Val Thr Ala Ala Arg Ile Leu Lys Gly Gln Lys Lys Asp

65 70 75 80

Lys Leu Gly Pro Glu Thr Phe Leu Ala Met Asp Arg Phe Pro Tyr Val

85 90 95

Ala Leu Ser Lys Thr Tyr Ser Val Asp Lys His Val Pro Asp Ser Gly

100 105 110

Ala Thr Ala Thr Ala Tyr Leu Cys Gly Val Lys Gly Asn Phe Gln Thr

115 120 125

Ile Gly Leu Ser Ala Ala Ala Arg Phe Asn Gln Cys Asn Thr Thr Arg

130 135 140

Gly Asn Glu Val Ile Ser Val Met Asn Arg Ala Lys Lys Ala Gly Lys

145 150 155 160

Ser Val Gly Val Val Thr Thr Thr Thr Arg Val Gln His Ala Ser Pro Ala

165 170 175

Gly Ala Tyr Ala His Thr Val Asn Arg Asn Trp Tyr Ser Asp Ala Asp

180 185 190

Val Pro Ala Ser Ala Arg Gln Glu Gly Cys Gln Asp Ile Ala Thr Gln

195 200 205

Leu Ile Ser Asn Met Asp Ile Asp Val Ile Leu Gly Gly Gly Arg Lys

210 215 220

Tyr Met Phe Pro Met Gly Thr Pro Asp Pro Glu Tyr Pro Asp Asp Tyr

225 230 235 240

Ser Gln Gly Gly Thr Arg Leu Asp Gly Lys Asn Leu Val Gln Glu Trp

245 250 255

Leu Ala Lys His Gln Gly Ala Arg Tyr Val Trp Asn Arg Thr Glu Leu

260 265 270

Leu Gln Ala Ser Leu Asp Pro Ser Val Thr His Leu Met Gly Leu Phe

275 280 285

Glu Pro Gly Asp Met Lys Tyr Glu Ile His Arg Asp Ser Thr Leu Asp

290 295 300

Pro Ser Leu Met Glu Met Thr Glu Ala Ala Leu Leu Leu Leu Ser Arg

305 310 315 320

Asn Pro Arg Gly Phe Phe Leu Phe Val Glu Gly Gly Arg Ile Asp His

325 330 335

Gly His His Glu Ser Arg Ala Tyr Arg Ala Leu Thr Glu Thr Ile Met

340 345 350

Phe Asp Asp Ala Ile Glu Arg Ala Gly Gln Leu Thr Ser Glu Glu Asp

355 360 365

Thr Leu Ser Leu Val Thr Ala Asp His Ser His Val Phe Ser Phe Gly

370 375 380

Gly Tyr Pro Leu Arg Gly Ser Ser Ile Phe Gly Leu Ala Pro Gly Lys

385 390 395 400

Ala Arg Asp Arg Lys Ala Tyr Thr Val Leu Leu Tyr Gly Asn Gly Pro

405 410 415

Gly Tyr Val Leu Lys Asp Gly Ala Arg Pro Asp Val Thr Glu Ser Glu

420 425 430

Ser Gly Ser Pro Glu Tyr Arg Gln Gln Ser Ala Val Pro Leu Asp Gly

435 440 445

Glu Thr His Ala Gly Glu Asp Val Ala Val Phe Ala Arg Gly Pro Gln

450 455 460

Ala His Leu Val His Gly Val Gln Glu Gln Thr Phe Ile Ala His Val

465 470 475 480

Met Ala Phe Ala Ala Cys Leu Glu Pro Tyr Thr Ala Cys Asp Leu Ala

485 490 495

Pro Arg Ala Gly Thr Thr Asp Ala Ala His Pro Gly Pro Ser Val Val

500 505 510

Pro Ala Leu Leu Pro Leu Leu Ala Gly Thr Leu Leu Leu Leu Gly Thr

515 520 525

Ala Thr Ala Pro

530

<210> 19

<211> 528

<212> Protein

<213> Homo sapiens

<400> 19

Met Gln Gly Pro Trp Val Leu Leu Leu Leu Gly Leu Arg Leu Gln Leu

1 5 10 15

Ser Leu Gly Val Ile Pro Ala Glu Glu Glu Asn Pro Ala Phe Trp Asn

20 25 30

Arg Gln Ala Ala Glu Ala Leu Asp Ala Ala Lys Lys Leu Gln Pro Ile

35 40 45

Gln Lys Val Ala Lys Asn Leu Ile Leu Phe Leu Gly Asp Gly Leu Gly

50 55 60

Val Pro Thr Val Thr Ala Thr Arg Ile Leu Lys Gly Gln Lys Asn Gly

65 70 75 80

Lys Leu Gly Pro Glu Thr Pro Leu Ala Met Asp Arg Phe Pro Tyr Leu

85 90 95

Ala Leu Ser Lys Thr Tyr Asn Val Asp Arg Gln Val Pro Asp Ser Ala

100 105 110

Ala Thr Ala Thr Ala Tyr Leu Cys Gly Val Lys Ala Asn Phe Gln Thr

115 120 125

Ile Gly Leu Ser Ala Ala Ala Arg Phe Asn Gln Cys Asn Thr Thr Arg

130 135 140

Gly Asn Glu Val Ile Ser Val Met Asn Arg Ala Lys Gln Ala Gly Lys

145 150 155 160

Ser Val Gly Val Val Thr Thr Thr Thr Arg Val Gln His Ala Ser Pro Ala

165 170 175

Gly Thr Tyr Ala His Thr Val Asn Arg Asn Trp Tyr Ser Asp Ala Asp

180 185 190

Met Pro Ala Ser Ala Arg Gln Glu Gly Cys Gln Asp Ile Ala Thr Gln

195 200 205

Leu Ile Ser Asn Met Asp Ile Asp Val Ile Leu Gly Gly Gly Arg Lys

210 215 220

Tyr Met Phe Pro Met Gly Thr Pro Asp Pro Glu Tyr Pro Ala Asp Ala

225 230 235 240

Ser Gln Asn Gly Ile Arg Leu Asp Gly Lys Asn Leu Val Gln Glu Trp

245 250 255

Leu Ala Lys His Gln Gly Ala Trp Tyr Val Trp Asn Arg Thr Glu Leu

260 265 270

Met Gln Ala Ser Leu Asp Gln Ser Val Thr His Leu Met Gly Leu Phe

275 280 285

Glu Pro Gly Asp Thr Lys Tyr Glu Ile His Arg Asp Pro Thr Leu Asp

290 295 300

Pro Ser Leu Met Glu Met Thr Glu Ala Ala Leu Arg Leu Leu Ser Arg

305 310 315 320

Asn Pro Arg Gly Phe Tyr Leu Phe Val Glu Gly Gly Arg Ile Asp His

325 330 335

Gly His His Glu Gly Val Ala Tyr Gln Ala Leu Thr Glu Ala Val Met

340 345 350

Phe Asp Asp Ala Ile Glu Arg Ala Gly Gln Leu Thr Ser Glu Glu Asp

355 360 365

Thr Leu Thr Leu Val Thr Ala Asp His Ser His Val Phe Ser Phe Gly

370 375 380

Gly Tyr Thr Leu Arg Gly Ser Ser Ile Phe Gly Leu Ala Pro Ser Lys

385 390 395 400

Ala Gln Asp Ser Lys Ala Tyr Thr Ser Ile Leu Tyr Gly Asn Gly Pro

405 410 415

Gly Tyr Val Phe Asn Ser Gly Val Arg Pro Asp Val Asn Glu Ser Glu

420 425 430

Ser Gly Ser Pro Asp Tyr Gln Gln Gln Ala Ala Val Pro Leu Ser Ser

435 440 445

Glu Thr His Gly Gly Glu Asp Val Ala Val Phe Ala Arg Gly Pro Gln

450 455 460

Ala His Leu Val His Gly Val Gln Glu Gln Ser Phe Val Ala His Val

465 470 475 480

Met Ala Phe Ala Ala Cys Leu Glu Pro Tyr Thr Ala Cys Asp Leu Ala

485 490 495

Pro Pro Ala Cys Thr Thr Asp Ala Ala His Pro Val Ala Ala Ser Leu

500 505 510

Pro Leu Leu Ala Gly Thr Leu Leu Leu Leu Gly Ala Ser Ala Ala Pro

515 520 525

<210> 20

<211> 227

<212> Protein

<213> Faux Sequence

<220>

<223> Synthetic Construction

<400> 20

Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly

1 5 10 15

Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met

20 25 30

Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His

35 40 45

Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val

50 55 60

His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr

65 70 75 80

Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly

85 90 95

Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile

100 105 110

Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val

115 120 125

Tyr Thr Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser

130 135 140

Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu

145 150 155 160

Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro

165 170 175

Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val

180 185 190

Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met

195 200 205

His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser

210 215 220

Pro Gly Lys

225

<---

Claims (56)

1. A method of treating hypophosphatasia (HPP) in a subject 18 years of age or older having at least one symptom of adult HPP, the method comprising subcutaneous administration of soluble alkaline phosphatase (sALP) to the subject in a regimen providing from 1 mg /kg/week to 9 mg/kg/week of sALP for a treatment period of at least five years, wherein the sALP contains an amino acid sequence having at least 95% sequence identity with the amino acid sequence of SEQ ID NO: 1, and wherein the method further comprises one or more of the following: (i) determining the concentration of inorganic pyrophosphate (PPi) in the blood of the subject, while the introduction of sALP for at least five years leads to a stable statistically significant decrease in the concentration of PPi in the blood of the subject, at least 1 μm; (ii) determining the concentration of pyridoxal-5'-phosphate (PLP) in the subject's blood, wherein administration of sALP for at least five years results in a sustained, statistically significant decrease in the subject's blood PLP concentration of at least 100 ng/mg ; and (iii) determining a subject's 6 Minute Walk Test (6MWT) score in which administration of sALP for at least five years results in a sustained statistically significant increase in the subject's walking distance of at least 50 meters as assessed by 6MWT. 2. The method according to claim 1, wherein the treatment period is at least six years, at least seven years, at least eight years, at least nine years, or at least ten years, or equal to the lifespan of the subject. 3. The method according to claim 1 or 2, wherein the decrease in the concentration of PPi in the subject's blood is at least 2 μM. 4. The method according to any one of paragraphs. 1-3, in which before the introduction of sALP the concentration of PPi in the blood of the subject is 6 μm. 5. The method according to any one of paragraphs. 1-4, in which the decrease in the concentration of PPi in the blood of the subject is from 2 μm to 5 μm after administration of sALP. 6. The method according to any one of paragraphs. 1-5, in which the decrease in the concentration of PLP in the blood of the subject is at least 200 ng/ml. 7. The method according to any one of paragraphs. 1-6, in which, prior to administration of sALP, the concentration of PLP in the subject's blood is up to 1300 ng/mL. 8. The method according to any one of paragraphs. 1-7, in which the decrease in the concentration of PLP in the blood of the subject is from 2 ng/ml to 150 ng/ml after administration of sALP. 9. The method according to any one of paragraphs. 1-8, wherein administration of sALP results in an increase in walking distance in six minutes of at least 100 meters or more. 10. The method according to any one of paragraphs. 1-9, in which the subject's walking distance in six minutes is 350 meters or less before sALP administration. 11. The method according to any one of paragraphs. 1-10, wherein the subject's walking distance for six minutes is 420 meters or more after sALP administration. 12. The method according to any one of paragraphs. 1-11 in which the subject exhibits reduced dependence on an assistive device for mobility following sALP administration. 13. The method of claim 12 wherein the assistive mobility device is at least one device selected from the group consisting of walkers, wheelchairs, orthodontic braces, crutches, and orthotics. 14. The method according to any one of paragraphs. 1-13, in which the subject has a reduction in the incidence of fractures after administration of sALP. 15. A method of treating hypophosphatasia (HPP) in a subject 13 years of age or older, said method comprising subcutaneous administration of soluble alkaline phosphatase (sALP) to the subject in a regimen providing 1 mg/kg/week to 9 mg/kg/week sALP over a treatment period of at least five years, wherein the sALP comprises an amino acid sequence having at least 95% sequence identity with the amino acid sequence of SEQ ID NO: 1, and wherein the method further comprises one or more of the following: (i) determining a subject's Portable Device Dynamometry (HHD) score in which administration of sALP for at least five years results in a stable percentage (%) change in the subject's predicted HHD score of 5% or more relative to the % predicted score HHD subject without sALP injection; (ii) determining a subject's Lower Limb Functionality Scale (LEFS) score, wherein administration of sALP for at least five years results in a sustained change in the subject's LEFS score of 3 or more relative to the subject's LEFS score without sALP administration; and (iii) determining a Subject's Brief Pain Score Questionnaire (BPI-SF) score in which administration of sALP for at least five years results in a sustained change in the subject's BPI-SF score of -2 or more relative to the BPI- score. SF in a subject without sALP administration. 16. A method of treating hypophosphatasia (HPP) in a subject 13 years of age or older, said method comprising subcutaneous administration of soluble alkaline phosphatase (sALP) to the subject in a treatment regimen providing 1 mg/kg/week to 9 mg/kg/week sALP over a treatment period of at least five years, wherein the sALP comprises an amino acid sequence having at least 95% sequence identity with the amino acid sequence of SEQ ID NO: 1, and wherein the method further comprises one or more of the following: (i) determining the blood concentration of PPi in the subject, wherein the administration of sALP for at least five years results in a sustained decrease in the concentration of PPi in the blood of the subject by 25% or more relative to the concentrations of PPi in the blood of the subject without administration of sALP; (ii) determining the concentration of PLP in the blood of the subject, wherein the introduction of sALP for at least five years leads to a steady decrease in the concentration of PLP in the blood of the subject by 50% or more relative to the concentrations of PLP in the blood of the subject without administration of sALP; and (iii) determining the subject's 6MWT value, wherein administration of sALP for at least five years results in a sustained increase in the subject's 6MWT value to 80% or more than 80% of the subject's predicted 6MWT value compared to the subject's 6MWT value without administration sALP. 17. The method according to any one of paragraphs. 1-16, in which sALP is administered one or more times a day, a week, or a month. 18. The method of claim 17, wherein the sALP is administered at least twice a week, three times a week, four times a week, five times a week, six times a week, or seven times a week. 19. The method according to any one of paragraphs. 1-18 wherein sALP is administered in multiple doses two days a week, three days a week, four days a week, five days a week, six days a week, or seven days a week. 20. The method according to any one of paragraphs. 1-19, in which sALP is administered at an initial dosage of 2.1 mg/kg/week to 3.5 mg/kg/week and then increased to a dose of 6 mg/kg/week. 21. The method of claim 20 wherein sALP is administered at an initial dosage of 0.3 mg/kg/day to 0.5 mg/kg/day sALP. 22. The method according to any one of paragraphs. 1-21, in which sALP is administered at a dosage of 1.3 mg/kg/week, 2.7 mg/kg/week, or 6 mg/kg/week. 23. The method according to any one of paragraphs. 1-22, in which sALP is administered at a dosage of 2 mg/kg three times a week, 3 mg/kg twice a week, 3 mg/kg three times a week, or 1 mg/kg six times a week. 24. The method of claim 23, wherein the sALP is administered once a day every day or every other day. 25. The method according to any one of paragraphs. 1-24, in which sALP contains the amino acid sequence of SEQ ID NO: 1. 26. The method of claim 25, wherein the sALP consists of the amino acid sequence of SEQ ID NO: 1. 27. The method according to any one of paragraphs. 1-26 wherein sALP is administered in an amount that is therapeutically effective for treating at least one symptom of adult HPP. 28. The method of claim 27, wherein at least one symptom of adult HPP includes one or more of elevated blood and/or urine levels of phosphoethanolamine (PEA), hypomineralization, hypercalciuria, skeletal deformity, waddling gait, bone pain , bone fracture, calcium pyrophosphate dihydrate crystal deposits, arthritis, pyrophosphate arthropathy, chondrocalcinosis, calcific periarthritis, and pseudofracture. 29. The method according to any one of paragraphs. 1-28, in which the subject does not experience ectopic calcification after administration of sALP. 30. The method of claim 29, wherein the subject does not experience ectopic calcification after administration of sALP for at least one year, at least two years, at least three years, at least four years, at least five years, at least six years, at least seven years, at least eight years, at least nine years, or at least ten years or more. 31. The method according to any one of paragraphs. 1-30, in which the subject shows tolerance to the introduction of sALP. 32. The method of claim 31, wherein tolerability includes the absence or reduction in the incidence of an adverse event selected from the group consisting of injection site erythema, decreased hemoglobin, hyperthermia, pneumonia, upper respiratory tract infection, otitis media, vomiting, constipation, diarrhea, tooth loss, nasopharyngitis, rash, dental caries and irritability. 33. The method according to any one of paragraphs. 1-32 wherein sALP is administered in compositions containing at least one pharmaceutically acceptable carrier, diluent or excipient. 34. The method of claim 33 wherein at least one pharmaceutically acceptable carrier, diluent or excipient is saline. 35. The method according to p. 33, in which at least one pharmaceutically acceptable carrier, diluent or excipient contains sodium chloride and sodium phosphate. 36. The method according to p. 35, in which at least one pharmaceutically acceptable carrier, diluent or excipient contains 150 mm sodium chloride and 25 mm sodium phosphate. 37. The method according to any one of paragraphs. 1-36, in which sALP is physiologically active against PEA, PPi and PLP. 38. The method according to any one of paragraphs. 1-37, in which sALP has the catalytic ability to enhance skeletal bone mineralization. 39. The method according to any one of paragraphs. 1-38, in which sALP is a soluble extracellular domain of alkaline phosphatase. 40. The method according to any one of paragraphs. 1-39, in which the subject is a human. 41. The method according to any one of paragraphs. 1-40, in which administration of sALP results in one or both of the following: (i) a statistically significant change in least squares mean from baseline in the concentration of PPi in the subject's plasma sample; or (ii) a statistically significant change in least squares mean from baseline to the PLP concentration in the subject's plasma sample. 42. The method according to any one of paragraphs. 1-41, in which administration of _sALP results in an AUC after 2,000 U (U) × day/L to 7,000 U × day/L, where AUC _after is equal to the area under the concentration-time curve from zero to the time of the last observed concentration in the interval dosing. 43. The method according to any one of paragraphs. 1-42, in which the introduction of sALP leads to AUC _ins from 4.000 U × day/L to 10.000 U × day/L, and AUC _insq is equal to the area under the concentration-time curve from zero to infinity. 44. The method according to any one of paragraphs. 1-43, in which the introduction of sALP leads to t _1/2 from 3 days to 8 days. 45. The method of any one of claims 1-44, wherein the treatment regimen provides 6 mg/kg/week of sALP.

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