EA049823B1 - METHODS OF TREATMENT OF BONE TISSUE MINERALIZATION DISORDERS - Google Patents

Abstract

The invention provides compositions and methods for treating bone mineralization disorders such as hypophosphatasia (HPP). In some embodiments, the methods described herein are useful for treating adult subjects with HPP and/or treating or preventing bone fractures.

Description

Cross-reference to related applications

This application claims the benefit of U.S. Provisional Patent Application No. 63/074,917, filed September 4, 2020, the contents of which are incorporated herein by reference in its entirety.

State of the art

Hypophosphatasia (HPP) is a rare, serious genetic disorder caused by a loss-of-function mutation(s) in the gene encoding tissue-nonspecific alkaline phosphatase (TNALP). Hypophosphatasia is characterized by age-dependent, interdependent clinical manifestations resulting from a failure to mineralize bone matrix due to elevated concentrations of the TNALP substrate PPi. Increased levels of extracellular PPi inhibit bone mineralization by blocking the growth of hydroxyapatite crystals, causing a marked accumulation of unmineralized bone matrix. HPP is characterized by defective bone mineralization and impaired phosphate and calcium regulation as a direct result of TNALP deficiency, which can lead to progressive damage to vital organs along with other clinical sequelae including deformed and soft bones (rickets and osteomalacia), fractures and pseudofractures, pain, profound muscle weakness, respiratory failure (predominantly in infants), seizures (predominantly in infants), impaired renal function, impaired mobility, and dental abnormalities. In patients with perinatal/infantile onset HPP, the disease course is potentially fatal.

In patients with perinatal/infantile onset HPP, defects in bone mineralization with subsequent skeletal deformities and fractures and rachitic changes in the chest wall may result in failure of the chest wall to support normal respiratory function and may increase the risk of ventilator dependence and premature death. In the most severely affected patients, mortality ranges from 50% to 100%. In adults, HPP presents as recurrent and/or poorly healing fractures and chronic pain, resulting in severe impairment of quality of life and daily activities. Severe functional impairment is also common in adult patients with HPP, including mobility problems (gait and walking difficulties), muscle weakness and inability to perform daily activities, and dependence on mobility devices, all of which collectively greatly impact patients' quality of life. Adult patients with HPP are also frequently misdiagnosed for many years, and an accurate diagnosis of HPP is critical for appropriate treatment.

In patients who survive into adolescence and adulthood, long-term clinical consequences include recurrent and non-healing fractures, burden of orthopedic/dental surgeries, frailty, arthritis, inability to remove internal fixation devices (due to risk of recurrent fracture), pain, impaired mobility, and need for outpatient assistive devices (e.g., wheelchairs, wheeled walkers, and canes). Regardless of the age at which HPP first presents, the high disease burden can result in decreased health-related quality of life, which may further decline over the patient's lifetime. Signs and/or symptoms of HPP may also affect the mental and emotional health of children and adults with the disease, which is associated with significant use of healthcare resources.

Prior to asfotase alfa (STRENSIQ®, Alexion Pharmaceuticals, Inc. Boston, MA) receiving marketing authorization (in the European Union, Japan, Canada, the United States, etc.), clinical management of HPP was primarily supportive and aimed at resolving the symptoms (e.g., respiratory support, orthopedic intervention, and pain medication) of the disease but not the underlying pathophysiology. Thus, despite symptomatic treatment, most patients experienced significant complications (bone abnormalities and deformities, bone pain, fractures/pseudo-fractures, physical dysfunction, and/or respiratory distress).

Asfotase alfa is a human recombinant TNALP-Fc-deca-aspartate fusion protein produced by recombinant DNA technology using mammalian cell culture using Chinese hamster ovary (CHO) cells. It is a soluble glycoprotein of 726 amino acids that contains the catalytic domain of human TNALP, the Fc domain of human immunoglobulin (Ig) G1, and the deca-aspartate peptide domain used for bone targeting. Patients with childhood-onset HPP, including perinatal/infantile-onset and juvenile-onset (Table 1), are indicated for asfotase alfa, which has been approved in the United States for the long-term treatment of HPP.

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Table 1

Classifications of hypophosphatasia

Form of the disease Age at onset of first signs/symptoms Perinatal During intrauterine development Infant <6 months Childhood (juvenile) From > 6 months to < 18 years Adult >18 years old Form of the disease Age at onset of first signs/symptoms Only dental manifestations Dental hypophosphatasia

Some adult patients have a moderate bone mineralization phenotype that may not be properly diagnosed until later in life. Thus, there is a need for effective treatments for these patients with HPP and other bone mineralization disorders.

The essence of the present invention

In one aspect, a method is provided for treating a bone mineralization disorder in a human subject (e.g., an adult human subject) that exhibits a level of at least one biochemical, physical, quality of life, or bone metric that identifies the subject as being in need of treatment for a bone mineralization disorder. The method may include administering at least one dose of soluble alkaline phosphatase (sALP) to the subject in a first treatment phase of a treatment regimen; monitoring the subject during the first phase of treatment for changes in at least one biochemical, physical, quality of life, or bone metric and identifying an improvement (e.g., 1%, 2%, 3%, 4%, 5%, 10%, 15%, 20%, 25%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, or more) in at least one biochemical, physical, quality of life, or bone metric above a predetermined metric threshold. The subject may then enter the first non-treatment phase of the treatment regimen, during which the subject is not administered sALP.

The method may further comprise monitoring the subject during the first non-treatment phase for a period of time for changes in at least one biochemical, physical, quality of life, or bone metric; detecting a level of at least one biochemical, physical, quality of life, or bone metric below a predetermined threshold metric; and administering at least one dose of sALP to the subject in the second treatment phase of the treatment regimen. Any of the above steps may be repeated one or more times, such that the treatment regimen comprises multiple treatment and/or non-treatment phases.

A bone mineralization disorder may represent an alkaline phosphatase deficiency such as hypophosphatasia (HPP). HPP may represent juvenile-onset HPP. The subject may not have previously been diagnosed with HPP or may not be diagnosed with HPP until adulthood. The subject may be naïve (e.g., not previously administered sALP).

In another aspect, a method for treating an atypical femur fracture (AFF) in a human subject is provided, comprising administering to the subject a treatment regimen comprising at least one dose of sALP. sALP may be administered in an amount and for a time sufficient to recover from the AFF. The subject may have a level of at least one biochemical, physical, quality of life, or bone metric that allows the subject to be determined to be in need of treatment for the AFF. The method may further comprise monitoring the subject during the treatment regimen for changes in the at least one biochemical, physical, quality of life, or bone metric. The method may further comprise detecting an improvement in the at least one biochemical, physical, quality of life, or bone metric above a predetermined metric threshold and entering a non-treatment phase of the treatment regimen during which the subject is not administered sALP. The subject may have a bone mineralization disorder (e.g., alkaline phosphatase deficiency, e.g., HPP). Alternatively, the subject may not have a bone mineralization disorder (e.g., HPP) or may not have a previously diagnosed disorder. The HPP may be a juvenile-onset HPP. In some embodiments, the subject may not have a diagnosed HPP until adulthood. In some embodiments, the subject has previously been administered or is currently being administered a bisphosphonate.

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In some embodiments, the alkaline phosphatase is a tissue-non-specific alkaline phosphatase, a placental alkaline phosphatase, an intestinal alkaline phosphatase, an engineered alkaline phosphatase, a fusion protein comprising a fragment of an alkaline phosphatase, or a chimeric alkaline phosphatase. The alkaline phosphatase may comprise an amino acid sequence having at least 90%, 95%, 97%, or 99% sequence identity to the sequence of SEQ ID NO: 1. For example, the alkaline phosphatase may comprise or consist of the amino acid sequence of SEQ ID NO: 1.

The biochemical metric that can be used to monitor the subject can be selected from the group consisting of plasma pyrophosphate (PPi), pyridoxyl 5'phosphate (PLP), phosphoethanolamine (PEA), and alkaline phosphatase (ALP) levels, or a combination thereof.

The quality of life metric that can be used to monitor the subject can be selected from the group consisting of the EuroQol Five-Item Questionnaire (EQ-5D), the Child Health Questionnaire (CHAQ), the Pediatric Outcome Data Collection Instrument (PODCI), the Child Health Index-9D (CHU-9D), the Pediatric Quality of Life Instrument (PedsQL), the Lower Limb Function Scale (LEFS), the Short Form Health Questionnaire 36 (SF-36), and the Short Form Health Questionnaire 12 (SF-12), or a combination of these.

The physical metric that can be used to observe the subject can be selected from the group consisting of the six-minute walk test (6MWT), the Bruininks-Oseretsky Motor Skills Test 2nd Edition (BOT-2), the Bayley Scales of Infant and Toddler Development 3rd Edition (BSID-III), the muscle strength rating scale, the hand dynamometer (HHD) test, the Peabody Motor Development Scales 2nd Edition (PDMS-2), gait analysis, the Short Physical Performance Battery (SPPB), and the Timed Get Up and Go (TUG) test, or a combination of these. The SPPB may include the repeated chair stand test.

The physical metric indicator can be monitored using a wearable device, such as a wearable device that includes an accelerometer. The wearable device can be configured to be worn on the arm (e.g., wrist), leg, or torso of the subject. The wearable device can track one or more biometric readings of the subject. For example, the biometric readings can be selected from the number of steps per day, average heart rate, highest heart rate, a numeric value of the amount of physical activity, an average amount of time spent at different activity levels, a ratio of different activity levels, a duration of different activity levels, and a duration of total sleep time. The different activity levels can be selected from sedentary, light, moderate, and vigorous. The physical metric indicators can be monitored using the wearable device for a period of at least two weeks (e.g., during a treatment phase, or during a non-treatment phase, or during both phases).

During the off-treatment phase, the subject may not be administered sALP for a period of at least 1 month, such as at least 6 months, such as at least 1 year, or longer.

During the treatment phase, sALP may be administered at a dosage of about 1 mg/kg/week to about 12 mg/kg/week (e.g., about 2 mg/kg/week to about 9 mg/kg/week, such as about 2.5 mg/kg/week to about 6 mg/kg/week). In some embodiments, sALP is administered at a dosage of about 2.6 mg/kg/week, 3.6 mg/kg/week, or about 6 mg/kg/week.

The dose of sALP may be adjusted during treatment. For example, sALP may be administered at a dose of approximately 6 mg/kg/week and then tapered to approximately 3.6 mg/kg per week. For example, sALP may be administered at a dose of approximately 6 mg/kg/week for approximately 12 weeks and then tapered to approximately 3.6 mg/kg/week for approximately 24 weeks.

The methods described herein can be used to treat or reduce the risk of bone fracture in a subject. For example, the method can result in an increase in bone regeneration, resulting in the formation of new bone tissue at a control point in the subject compared to a control point in the subject before treatment. The increase in bone regeneration can be an increase in opacity. The increase in opacity can be determined using radiography or computed tomography (CT). The increase in bone regeneration can occur between two parts of a bone that have been separated by a fracture. The method can result in an average change in bone mineral density at a control point that is undetectable or does not exceed 0.01% eight months after administration of sALP compared to the bone mineral density at the control point in the subject before treatment.

In some embodiments, the method further comprises treating the subject with surgery. The surgery may comprise one or more of arthrodesis, bone removal, placement of bone graft material, and placement of metal structures.

- 3 049823 of a piece of hardware (such as a screw, rod, plate, metal frame, nail, pin or nut). The surgical procedure may be selected from a group consisting of knee, shoulder, hip, ankle, elbow, or wrist arthroscopy, fracture and meniscectomy, shoulder arthroscopy and decompression, carpal tunnel release, knee arthroscopy and chondroplasty, bearing implant removal, knee arthroscopy and ACL reconstruction, knee replacement, femoral neck fracture repair, atypical femoral fracture (AFF) repair, trochanteric fracture repair, skin/muscle/bone/fracture debridement, knee arthroscopy repair of one or both menisci, hip replacement, shoulder arthroscopy/distal clavicle resection, rotator cuff tendon repair, radius or ulna fracture repair, laminectomy, fracture repair ankle (bimalleolar type), shoulder arthroscopy and debridement, lumbar spine fusion, distal radius fracture repair, low back disc surgery, finger sheath dissection, ankle (fibula) fracture repair, femoral shaft fracture repair, and trochanteric fracture repair.

sALP may be administered once daily, once weekly, once every two weeks, or once monthly (e.g., during the treatment phase). For example, sALP may be administered twice weekly, three times weekly, four times weekly, five times weekly, six times weekly, or seven times weekly. During the treatment period, sALP may be administered over a treatment period of at least one month, at least two months, 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 ten months, at least 11 months, at least 12 months, or longer. The treatment-free period may last at least one month, at least two months, 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 ten months, at least 11 months, at least 12 months or longer.

sALP can be formulated in a pharmaceutical composition with at least one pharmaceutically acceptable carrier. The pharmaceutically acceptable carrier can include a saline solution. For example, the pharmaceutically acceptable carrier can include sodium chloride and sodium phosphate, (e.g., 150 mM sodium chloride and 25 mM sodium phosphate). The pharmaceutical composition can be administered subcutaneously, intramuscularly, intravenously, orally, nasally, sublingually, intrathecally, or intradermally.

In some embodiments of the methods described herein, the method results in an average increase in the Bruininks-Oseretsky Motor Skill Test 2nd Edition (BOT-2) running speed and agility score to about 9 or greater than about 9. The median overall BOT-2 running speed and agility score of a subject may be determined relative to the median overall BOT-2 running speed and agility score of an untreated subject with HPP. The overall median BOT-2 running speed and agility score of a subject may be determined from measurements selected from the group consisting of a balance beam stepover, a shuttle run, a two-legged lateral jump, and a single-legged lateral jump. The method can result in an average increase in the WOT-2 strength score to approximately 18 or greater than approximately 18. The median overall WOT-2 strength score of a subject can be determined relative to the median overall WOT-2 strength score of an untreated subject with HPP. The median overall WOT-2 strength score of a subject can be determined relative to the median overall WOT-2 strength score of a healthy subject. The overall median WOT-2 strength score of a subject can be determined from measurements selected from a group consisting of squats, lying V-crunches, standing long jump, wall sits, and push-ups.

In some embodiments of the methods described herein, the method may result in an improvement in walking ability compared to the walking ability of a subject selected from the group consisting of a healthy subject and an untreated subject with HPP. For example, the subject may increase the average number of daily steps by 100, 200, 300, 400, 500, 1000, 2000, 3000, 4000, 5000, 6000, 7000, 8000, 9000, 10000 or more per day. The subject may exhibit reduced dependence on a mobility assist device (e.g., a wheelchair, braces, crutches, or orthotics) after administration of sALP.

The methods described herein can result in an average decrease in the concentration of inorganic pyrophosphate (PPi) in a blood plasma sample from a subject relative to the concentration of PPi in a blood plasma sample from an untreated subject with HPP. The method can result in an average decrease in the concentration of pyridoxal-5'-phosphate (PLP) in a blood plasma sample from a subject relative to the concentration of PLP in a blood plasma sample from an untreated subject with HPP. The method can result in an average increase in the concentration of alkaline phospha

- 4,049,823 tase in a plasma sample from a subject relative to the alkaline phosphatase concentration in a plasma sample from an untreated subject with HPP.

In another aspect, the present invention provides a kit that includes a wearable device and an sALP. The wearable device may include an accelerometer. The wearable device may be configured to be worn on the wrist of a subject. The kit may further include a peripheral device (e.g., a smartphone). The peripheral device may be configured to run a software application (e.g., a mobile application, such as a smartphone application).

In another aspect, the present invention provides a method for monitoring a treatment regimen by means of sALP in a subject by detecting a change in at least one physical metric indicator based on biometric readings obtained using a wearable device. The biometric readings can be selected from the number of steps per day, the average heart rate, the highest heart rate, a numeric value of the amount of physical activity, an average amount of time spent at different activity levels, a ratio of different activity levels, a duration of different activity levels, and a duration of total sleep time. The different activity levels can be selected from sedentary, light, moderate, and vigorous. The physical metric indicators can be monitored using the wearable device for a period of at least two weeks (for example, during a treatment phase, or during a non-treatment phase, or during both phases).

In some embodiments of any of the above aspects, the dose is determined based on the actual body weight of the subject. In some embodiments of any of the above aspects, the dose is determined based on the ideal body weight of the subject.

Definitions

A term in its singular and plural form represents one specific noun or several specific nouns. For example, the phrase mammal cell means one or more mammal cells.

The term approximately means ±10% of the stated value. It is understood that all measurement results presented in this document are modified by the term approximately, regardless of whether this term is used explicitly or not, unless explicitly stated otherwise.

The term asfotase alfa (STRENSIQ®, Alexion Pharmaceuticals, Inc., Boston, MA) refers to a human recombinant TNALP-Fc-deca-aspartate fusion protein produced by recombinant DNA technology using mammalian cell culture using Chinese hamster ovary (CHO) cells. Asfotase alfa is a soluble fusion glycoprotein composed of 2 identical polypeptide chains. Each polypeptide chain contains the catalytic domain of human TNALP (Swiss-Prot, P05186), the Fc domain of human IgG1 (Swiss-Prot, P01857) (to facilitate purification), and a deca-aspartate peptide domain used for bone targeting. The sequence of asfotase alpha is set forth in SEQ ID NO: 1. The two polypeptide chains are linked by two interchain disulfide bonds in the hinge region. Asfotase alpha can be expressed in an engineered CHO cell line that retains endogenous folding, sorting, disulfide bridge formation, and N-glycosylation.

The term "bone targeting functional unit" means an amino acid sequence of at least 3 amino acid residues in length that has sufficient affinity for bone matrix such that the bone targeting functional unit, taken alone, has an in vivo binding affinity for bone matrix that is at least about 1 x 10 ^-5 M, or better (e.g., about 10 ^-6 M, about 10 ^-7 M, about 10 ^-8 M, about 10 ^-9 M, or better).

The term bone mineralization disorder refers to a disease or condition that affects the normal mineralization of bone. Illustrative bone mineralization disorders include HPP (e.g., perinatal HPP, infantile HPP, childhood HPP, and odontohypophosphatasia), HPP-like disease, calcium pyrophosphate deposition disorder (CPPD), familial hypophosphatemia, bone fracture, osteoporosis, sclerosteosis, chondrocalcinosis, tracheobronchomalacia, neurofibromatosis 1 (NF1), and craniosynostosis. Other bone mineralization disorders are known in the art. Bone mineralization disorder may result from a failure to mineralize bone matrix, such as due to elevated concentrations of the TNALP substrate PPi.

The terms Brief Pain Inventory-Short Form and BPI-SF, used interchangeably in this document, refer to a method for measuring pain in a patient, particularly a patient with a bone mineralization disorder such as HPP (e.g., patients approximately 13 years of age or older). The BPI-SF is a self-report measure of pain described by Cleeland & Ryan (Ann Acad Med Singapore, 23(2), 129-138; 1994), which

- 5 049823 paradise is incorporated herein by reference in its entirety. The BPI-SF is a questionnaire designed to assess the severity of pain and the impact of pain on daily functioning. The BPI-SF consists of 11 items that use a numeric rating scale to assess the severity of pain (4 items) and the impact of pain (7 items) in the 24 hours prior to questionnaire administration. The BPISF provides information about the intensity of pain and the extent to which pain interferes with the patient's daily functioning on a numeric rating scale from 0 (no pain) to 10 (severe pain or a great deal of interference due to pain); lower scores indicate better quality of life and less pain. For example, BPI-SF scores in adolescent and adult patients with HPP are the sum of the 11 pain ratings.

The terms Bruininks-Oseretsky Test of Motor Proficiency, 2nd Edition and BOT-2, as used herein, refer to the second edition of the standardized test of gross and fine motor skills in a patient with a bone mineralization disorder such as HPP (e.g., a child with HPP between approximately 5 and approximately 12 years of age, an adolescent with HPP between approximately 13 and approximately 17 years of age, or an adult with HPP greater than or equal to approximately 18 years of age). See Bruininks, R. H. (2005). Bruininks-Oseretsky Test of Motor Proficiency, (BOT-2). Minneapolis, MN: Pearson Assessment, which is incorporated herein by reference in its entirety. The BOT-2 is used individually to assess gross and fine motor skills in a range of patients. The WOT-2, for example, can be used to assess physical abnormalities and mobility limitations in patients with HPP, such as children with HPP between approximately 5 years of age and approximately 12 years of age, adolescents with HPP between approximately 13 years of age and approximately 17 years of age, or adults with HPP over approximately 18 years of age or older. The WOT-2 provides composite WOT-2 scores in the following illustrative areas: strength, running speed and agility, fine motor accuracy, fine motor coordination, manual dexterity, bilateral coordination, balance, and upper extremity coordination. For example, a total WOT-2 strength score can be determined by asking a patient to perform squats, lying V-crunches, standing broad jumps, wall sits, and push-ups. A total running speed and agility score can be determined by asking the patient to walk on a balance beam or perform a shuttle run, a two-legged lateral jump, or a one-legged lateral jump. Total scores for both the BOT-2 total strength and the BOT-2 running speed and agility range from 0 to 25, with a score of 10 to 25 considered representative of healthy subjects.

The term catalytically competent in the context of this document refers to sALP that hydrolyzes the bone mineralization inhibitor inorganic pyrophosphate (PPi) to inorganic phosphate (Pi), thereby reducing extracellular PPi concentrations. Thus, catalytically competent sALP improves skeletal mineralization in bone by regulating PPi concentrations.

The terms Child Health Questionnaire and CHAQ as used herein refer to a questionnaire used to assess the health status (eg, ability to perform activities of daily living (ADLs) and pain frequency) of a patient aged 1 to 19 years, such as a child, adolescent, and adult with a bone mineralization disorder such as HPP. For a description of the CHAQ, see Bruce & Fries (J. Rheumatol. 30(1): 167–178, 2003), which is incorporated herein by reference in its entirety. The CHAQ can be administered by interview or self-administered to children over 8 years of age. The CHAQ includes eight subscales to assess dressing/self-grooming, standing, eating, walking, hygiene, reaching, grip, and activities. The score range in each category is from 0 to 3, where a score of 0 indicates no difficulty, a score of 1 indicates some difficulty, and a score of 2 indicates some difficulty. a score of 2 indicates great difficulty; and a score of 3 indicates that the patient cannot perform the task. The CHAQ index can also be used to determine the presence and severity of pain.

The terms EuroQol Five-Item Questionnaire and EQ-5D as used herein refer to a questionnaire that is used to assess health status (e.g., mobility, self-care, ability to perform usual school, work, or household activities, ability to perform ADLs (e.g., dressing, toileting, and preparing food), experience of pain or discomfort, and anxiety or depression) in patients such as children with HPP between approximately 5 years and approximately 12 years of age, adolescents with HPP between 13 years and 17 years of age, or adults with HPP over approximately 18 years of age or older. For a description of the EQ-5D, see Reenan & Oppe (EQ-5D-3L User Guide Version 5.1, 2015), which is incorporated herein by reference in its entirety. The EQ-5D can be self-administered, completed by a clinician, or via an interview. The EQ-5D questionnaire includes the following five parameters characterizing the health status of a patient with HPP: mobility, self-care, ability to perform ADLs, frequency of pain or discomfort, and anxiety or depression. As described herein, the EQ-5D can be used in combination with at least one physical assessment, such as the 6MWT, to classify a patient with HPP as having the condition

- 6 049823 health level I, indicating no physiological problems, level II, indicating some physiological problems, level III, indicating severe physiological problems, or level IV, indicating the most severe physiological problems. The EQ-5D can also be used as part of an analysis to assess the transition of a patient with HPP from one health state to another health state, such as from health state IV to III, IV to II, IV to I, III to II, III to I, or II to I. The Child General Health Utility Index-9D (CHU-9D) can also be used to assess the health status of patients with HPP. For a description of the CHU-9D and EQ-5D indices, see Stevens (Appl Health Econ Health Policy. 9(3): 15769, 2011) and PCT publication no. WO 2018/191254, which are incorporated herein by reference in their entireties.

The term Fc means a region of a crystallizable fragment of an immunoglobulin, such as IgG1, IgG2, IgG3 or IgG4, comprising the CH2 and CH3 domains of the immunoglobulin heavy chain. Fc may also comprise any portion of the hinge region connecting the Fab and Fc regions. Fc may be obtained from any mammal, including a human, and may be post-translationally modified (e.g., by glycosylation). In a non-limiting example, Fc may be a region of a crystallizable fragment of human IgG1.

A fragment is understood to mean a portion of a polypeptide or nucleic acid molecule that preferably comprises 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 a reference nucleic acid molecule or reference polypeptide. A 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, 310, 320, 330, 340, 350, 360, 370, 380, 390, 400, 500, 600, 700, 800, 900, 1000, 1100, 1200, 1300, 1400, 1500, 1600, 1700, 1800, 1900, 2000, 2100 or more nucleotides, up to the entire length of the nucleic acid molecule, or 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 260, 270, 280, 290, 300, 400, 500, 600, 700 or more amino acid residues, up to the entire length of the polypeptide.

The terms hand dynamometry and FfflD, as used interchangeably herein, refer to a method of measuring the grip and muscle strength of a subject, particularly a subject with HPP (e.g., a subject approximately 13 years of age or older) or another bone mineralization disorder. A dynamometer can be used to assess the grip strength, knee flexion, knee extension, hip flexion, hip extension, and hip abduction of a subject with HPP. For example, knee flexion and extension, and hip flexion, extension, and hip abduction of a subject with HPP 13 years of age or older can be measured using a MICROFET2TM dynamometer, while the subject's grip strength can be measured using, for example, a JAMAR® grip dynamometer. Specifically, the examiner holds the dynamometer still and the subject applies maximum force to the dynamometer. Peak force data is collected in pounds and then converted to Newtons (N). Torque values are then calculated using the limb length value in Newton meters. The torque value can then be compared to that of, for example, a normal subject of approximately the same age, same gender, and/or same height, and expressed as a percentage to obtain the subject's HHD score.

The term health condition as used herein refers to the characterized physiological state of a patient with HPP (e.g., a child with HPP between approximately 5 years of age and approximately 12 years of age, an adolescent with HPP between approximately 13 years of age and approximately 17 years of age, or an adult with HPP greater than or equal to approximately 18 years of age or older) or other bone mineralization disorder. The patient's health status may be characterized using at least one physical status assessment selected from one or more of the following metrics: the Six-Minute Walk Test (6MWT), the Bruininks-Oseretsky Motor Skills Test 2nd Edition (BOT-2), the Bayley Scales of Infant and Toddler Development 3rd Edition (BSID-III), the Muscle Strength Rating Scale, the Hand Dynamometer (HHD), the Peabody Developmental Motor Scales 2nd Edition (PDMS-2), gait analysis, the Short Physical Performance Battery (SPPB), and the Timed Get Up and Go (TUG) test, and/or at least one quality of life assessment selected from one or more of the following metrics: the EuroQol Five-Item Questionnaire (EQ-5D), the Child Health Questionnaire (CHAQ), the Pediatric Outcome Data Collection Instrument (PODCI), the Child Health Unit-9D (CHU-9D), the Pediatric Quality of Life Scale (PedsQL), the Lower Extremity Function Scale (LEFS), the Short Form Health Questionnaire 36 (SF-36) and the Short Form Health Questionnaire 12 (SF-12). In particular, the patient's health status is characterized, for example, by means of the 6MWT in combination with the EQ-5D. After obtaining the results of at least one physical status assessment and at least one quality of life assessment selected from the above metric indicators, the patient can be defined as having a health status of level I, indicating the absence of problems with physical

- 7 049823 physiological state, level II indicating the presence of some problems with the physiological state, level III indicating the presence of serious problems with the physiological state, or level IV indicating the presence of the most serious problems with the physiological state. The metric(s) can be used to assess the transition of a patient from one health state to another health state after, for example, treatment with an sALP as described herein, for example, a transition from health state IV to III, IV to II, IV to I, III to II, III to I, or II to I after administration of an sALP.

The terms hypophosphatasia and HPP, as used herein, refer to a rare inherited disorder of the skeleton caused by, for example, one or more loss-of-function mutations in the ALPL (alkaline phosphatase, liver/bone/kidney) gene, which encodes TNALP. HPP may be further characterized as infantile HPP, childhood HPP, perinatal HPP (e.g., benign perinatal HPP or lethal perinatal HPP), odonto-HPP, adolescent HPP, or adult HPP. For example, the term childhood HPP describes a patient with HPP between approximately 5 years of age and approximately 12 years of age, the term adolescent HPP describes a patient with HPP between approximately 13 years of age and approximately 17 years of age, and the term adult HPP describes a patient with HPP who is approximately 18 years of age or older. The term adult HPP as used herein refers to a condition or phenotype characterized by the presence of one or more of the following: elevated levels of inorganic pyrophosphate (PPi) in the blood and/or urine, hypomineralization, hypercalciuria, one or more skeletal deformities, hypotonia, muscle weakness, rheumatoid complications, waddling gait, difficulty walking, bone pain, pain, bone fracture, calcium pyrophosphate dihydrate crystal deposition, pseudogout, arthritis, pyrophosphate arthropathy, chondrocalcinosis, calcific 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 PPi, PEA, or PLP 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, premature tooth loss, hypomineralization, pulmonary hypoplasia, respiratory failure, seizures, hypercalciuria, short stature, and growth retardation. The term childhood HPP as used herein refers to a condition or phenotype characterized by the presence of one or more of the following: elevated blood or urine PPi, PEA, or PLP levels; rickets, rib rickets, one or more skeletal deformities, hypotonia, muscle weakness, rheumatoid complications, arthritis, pseudogout, waddling gait, difficulty walking, bone pain, pain, premature tooth loss, hypomineralization, delayed motor development, seizures, hypercalciuria, short stature, bone fracture, pseudofracture, and growth retardation.

The terms Lower Extremity Function Scale and LEFS, as used interchangeably herein, refer to a method of measuring functional disability of the lower extremities in a patient, particularly a patient with HPP (e.g., patients approximately 13 years of age or older) or another bone mineralization disorder. The LEFS is a self-assessment instrument described in the work of Binkley et al. (Phys Ther. 79:371-83, 1999), which is incorporated herein by reference in its entirety. LEFS total scores range from 0 to 80, with higher scores indicating better lower extremity function. A change in LEFS score of approximately 9 units is considered a clinically significant change. A licensed physical therapist can administer the LEFS to a patient (e.g., a patient with HPP approximately 13 years of age or older) in an interview format. Higher LEFS scores indicate improved lower extremity function, including transitional movements (e.g., getting out of the bath or rolling over in bed), ambulation (e.g., walking or running on uneven surfaces), stair climbing, and squatting. LEFS can be used to assess functional impairment in one or both lower extremities in a patient, including the ability to monitor a patient over time and assess the effectiveness of asfotase alfa treatment.

The terms nucleic acid or nucleic acid molecule mean a polymeric molecule, such as RNA or DNA, comprising a sequence of two or more covalently linked, naturally occurring or modified nucleotides. A nucleic acid molecule may be, for example, single-stranded or double-stranded and may include modified or unmodified nucleotides or mixtures or combinations thereof. Also included are various salts, mixed salts, and free acid forms of nucleic acid molecules.

The terms Pediatric Outcome Data Collection Instrument and PODCI in the context of this document refer to a questionnaire used to assess general health status, pain frequency, and ability to perform ADLs in a patient under 19 years of age, particularly a patient with a chronic health condition such as a patient with HPP or other bone mineralization disorders. For a description of the PODCI, see Plint et al. (J. Pediatr. Orthop. 23(6): 788–790, 2003), which

- 8 049823 is incorporated herein by reference in its entirety. The questionnaire can be completed by the patient or by a parent/guardian of the patient who is familiar with the patient's condition. The eight scales derived from the PODCI are: 1) the Upper Limb and Physical Function Scale to measure difficulty performing activities of daily living such as self-care and learning; 2) the Ambulation and Basic Mobility Scale to measure difficulty performing standard movement and motor activities of daily living; 3) the Sports/Physical Function Scale to measure difficulty or limitations participating in more vigorous activities or sports; 4) the Pain/Comfort Scale to measure the level of pain experienced over the past week; 5) the Treatment Expectations Scale to measure long-term treatment expectations; 6) a happiness scale to measure overall satisfaction with one's appearance and feelings of similarity to friends and peers; 7) a symptom satisfaction scale to measure the patient's level of acceptance of current limitations if the condition is lifelong; and 8) a global ability scale, which is a general composite scale derived from the first four scales listed above. Standardized scores are derived from a series of questions in the PODCI and are converted to a 0-100 scale, with 0 representing a significant degree of disability and 100 representing the least degree of disability.

The term recombinant protein is known in the art. A recombinant protein may be a glycoprotein. For example, a recombinant protein or a recombinant protein variant produced in a CHO cell undergoes glycosylation, in which sugar residues are covalently attached to the protein, and is a glycoprotein. Briefly, the term recombinant protein may refer to a protein that can be produced using a cell culture system. The cells in the cell culture system may be derived, for example, from a mammalian cell, including a human cell, a CHO cell, an insect cell, a yeast cell, or a bacterial cell. In general, the cells in the cell culture contain an introduced nucleic acid encoding the recombinant protein of interest (such nucleic acid may be carried on a vector, such as a plasmid vector). The nucleic acid encoding the recombinant protein may also contain a heterologous promoter operably linked to the nucleic acid encoding the protein.

As used herein, the terms six-minute walk test and 6MWT refer to a physical assessment that is a standardized test to evaluate the walking ability of a patient with HPP (e.g., a child with HPP between approximately 5 years of age and approximately 12 years of age, an adolescent with HPP between approximately 13 years of age and approximately 17 years of age, or an adult with HPP greater than or equal to approximately 18 years of age) or another bone mineralization disorder. Specifically, walking ability refers to the patient's ability to lift and place each foot down one at a time. See the American Thoracic Society position statement: six-minute walk test recommendations (Amer. J. of Respiratory and Critical Care Medicine, 166(1): 111-7, 2002, which is incorporated herein by reference in its entirety). The 6MWT is determined by the distance (e.g., in meters) a patient walks on a flat, hard surface for a period of 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 gender), or the 6MWT distance of a healthy subject (e.g., a healthy subject of approximately the same age, height, and/or gender) and expressed as a percentage to determine the 6MWT value.

The terms treat, cure and treatment mean providing medical control of a patient with the purpose of curing, alleviating the severity of, stabilizing, reducing the likelihood of or preventing the development of a disease state such as HPP (e.g., pediatric, adolescent or adult HPP) or other bone mineralization disorder, or one or more symptoms thereof and/or providing control of a patient who exhibits or may exhibit a disease state such as HPP or other bone mineralization disorder, for example, by administering a pharmaceutical composition (e.g., an sALP as described herein). The term includes active treatment, i.e., treatment specifically directed at providing an improvement in or associated with a cure for a disease, condition, disorder or event, and also includes etiologic treatment, i.e., treatment directed at eliminating the cause of the associated disease, condition, disorder or event. In addition, the term includes palliative treatment, i.e., treatment intended to relieve or reduce at least one symptom, but not to cure the disease, condition, disorder or event; symptomatic treatment, i.e. treatment aimed at the constitutional symptoms of the associated disease, pathological condition, disorder or phenomenon; preventive treatment, i.e. treatment aimed at minimizing or partially or completely suppressing the development of the associated disease, pathological condition

- 9 049823 disease, disorder or phenomenon, for example, in a patient who is not yet ill but who is susceptible to developing a specific disease, pathological condition, disorder or phenomenon, or is otherwise at risk of developing the same; and supportive care, i.e. treatment used in addition to another specific therapy, aimed at providing improvement in relation to the associated disease, pathological condition, disorder or phenomenon.

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

The terms sALP, soluble alkaline phosphatase, and extracellular domain of alkaline phosphatase are used interchangeably and mean soluble, non-membrane-bound alkaline phosphatase or a domain, biologically active fragment, or biologically active variant thereof. sALPs include, for example, alkaline phosphatase lacking the C-terminal GPI signal sequence and additional variants and analogs thereof that retain alkaline phosphatase activity, such as the ability to hydrolyze PPi or other natural or artificial substrates. This includes, unless otherwise noted, the TNALP, PALP, GALP, and IALP domain and a biologically active fragment or biologically active variant thereof. Mature sALP lacks the membrane GPI anchor and the signal peptide, which is cleaved during processing.

The terms isolated or purified mean separated from other naturally occurring associated components. Generally, a compound (e.g., a protein, polypeptide, nucleic acid, or small molecule), factor, cell, or other component is considered isolated if it is at least, for example, 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or even 99% by weight, free of proteins, antibodies, naturally occurring organic molecules, and other components with which it is naturally associated. In some cases, the purity of a component is at least 75%, 90%, or even 99% by weight.

The isolated component may be obtained by chemical synthesis, by isolating the factor from natural sources, or by producing the component in a recombinant host cell that does not naturally produce the component. Proteins and small molecules can be purified by one skilled in the art using standard techniques such as those described by Ausubel et al. (Current Protocols in Molecular Biology, John Wiley & Sons, New York, 2000). The component is preferably at least, for example, 2-, 5-, or 10-fold purer than the starting material, as measured by, for example, polyacrylamide gel electrophoresis, column chromatography, optical density, HPLC analysis, or Western blotting (Ausubel et al., 2000). Illustrative purification methods include column chromatography, filtration, immunoprecipitation, and magnetic bead immunoaffinity purification.

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

The term "pharmaceutical composition" means a composition comprising a polypeptide or nucleic acid molecule as described herein formulated with a pharmaceutically acceptable excipient, and includes compositions manufactured or marketed under the approval of a government regulatory agency as part of a therapeutic regimen for the treatment or prevention of a disease or event in a patient. Pharmaceutical compositions may be formulated, for example, for subcutaneous administration, intravenous administration (e.g., as a sterile, particle-embolic solution and in a solvent system suitable for intravenous administration), for oral administration (e.g., a tablet, capsule, caplet, gelcap, or syrup), or any other formulation described herein, for example, in unit dosage form.

The terms subject and patient are used interchangeably and mean a mammal, including but not limited to a human or non-human mammal such as a bovine, equine, canine, ovine or feline.

The term mammalian cell is known in the art and may refer to any cell of any mammal or obtained from any mammal, including, for example, a human, hamster, mouse, green monkey, rat, pig, cow, hamster or rabbit. The mammalian cell may be an immortalized cell, a differentiated cell or an undifferentiated cell.

The term therapeutically effective amount means the amount of the compound described in this document.

- 10 049823 ment of a polypeptide or nucleic acid molecule sufficient to substantially treat, prevent, delay, suppress or arrest any symptom of a disease or condition described herein, in particular HPP. A therapeutically effective amount of the composition described herein may depend on the severity of the disorder being treated, as well as the condition, weight and well-being of the subject, and can be determined by one of ordinary skill in the art taking into account such factors. A therapeutically effective amount of the composition described herein can be administered to a subject as a single dose or as multiple doses administered over a period of time.

In the context of this document, when a polypeptide or nucleic acid sequence is referred to as having at least X% sequence identity to a reference sequence, this means that at least X percent of the amino acid residues or nucleotides in the polypeptide or nucleic acid are identical to those in the reference sequence in an optimal sequence alignment. An optimal sequence alignment can be determined by a variety of 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 (Base Local Alignment Search Engine; 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 Dayhof, 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, or Megalign (DNASTAR). In addition, those skilled in the art will be able to determine appropriate parameters for measuring alignment, including any algorithms needed to achieve optimal alignment over the length of the sequences being compared.

As used herein, the term predetermined threshold refers to a metric value in a subject relative to a reference value (e.g., a metric value in a normal subject, such as a subject without a bone mineralization disorder (e.g., HPP), a subject with a disease treated with an sALP, or a subject with a disease that has not yet been treated with an sALP). The normal or treated subject may be of the same or similar age, weight, and/or gender. The predetermined threshold may refer to a metric value that is between the measured value in the subject with a bone mineralization disorder and the corresponding value in the normal subject. For example, if a subject with a bone mineralization disorder is determined to have a score of 50 steps on a 6MWT metric, and a normal subject has a score of 500 steps on this metric, the predetermined threshold may be a value that is in the range of 50 steps to 500 steps. The predetermined threshold may also be a metric value that is, for example, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100% or more lower than the value for a normal or treated subject. Alternatively, the predetermined threshold may be a metric score that is adjusted to reflect the subject's score after treatment with the sALP, such that the predetermined threshold becomes a metric score that is at least, for example, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100% greater than the subject's baseline metric score, for example, before treatment with the sALP.

The words preferred and preferably refer to embodiments of the disclosed polypeptides, compositions, and methods that may provide certain advantages under certain circumstances. However, other embodiments may also be preferred under the same or different circumstances. Moreover, the recitation of one or more preferred embodiments does not mean that other embodiments are not applicable, nor is it intended to exclude other embodiments from the scope of the present invention.

In the case of any method disclosed herein that includes separate steps, the steps may be performed in any acceptable order; also, if desired, any combination of two or more steps may be performed simultaneously.

The above summary of the present invention is not intended to describe each disclosed embodiment or each embodiment of the disclosed polypeptides, compositions, and methods. The following description demonstrates illustrative embodiments in more detail. At some points in this application, guidance may be given in the form of lists of examples that can be used in various combinations. In each case, the list provided serves only as a representative group and should not be interpreted as an exclusive list.

- 11 049823

All headings are provided for the convenience of the reader and should not be used to limit the meaning of the text following the heading unless otherwise indicated.

Other features and advantages of the present invention will be apparent from the detailed description, drawings and claims.

Brief description of graphic materials

Figure 1 shows a schematic representation of the study design described in Example 2. Group 1 is randomized to receive asfotase alfa at 6 mg/kg/week at baseline and continues at 6 mg/kg/week from week 12 until the end of treatment (week 36). Group 2 is randomized to receive asfotase alfa at 6 mg/kg/week at baseline and continues at 3.6 mg/kg/week from week 12 until the end of treatment (week 36). AA, asfotase alfa; HPP, hypophosphatasia; qw, once weekly.

Figures 2A–2D are graphs depicting physical function (Figures 2A–2C) and HRQoL scores (Figure 2D) among adults treated with asfotase alfa for childhood-onset HPP, according to data collected at baseline, 6 months, and 12 months. A) Six-minute walk test (6MWT), n=12, B) Chair rise test (part of SPPB, n=9, C) LEFS (n=9), D) SF-36v2 PCS scores (n=9); asterisks indicate statistical significance compared to baseline.

Figures 3A–3D show a set of graphs depicting the primary outcomes of physical function assessed by 6MWT distance (Figure 3A), TUG test time (Figure 3B), 4-minute walk speed (Figure 3C), and chair rise test (Figure 3D) among adults treated with asfotase alfa for childhood-onset HPP at baseline and after 12, 18, and 24 months of treatment. *p<0.05 compared with baseline. The lower and upper boundaries of the blue boxes represent the 25th and 75th percentiles, respectively. The horizontal black lines represent the median and the whiskers represent the maximum and minimum values. 6MWT (n=13); TUG test (n=7); 4-minute walk speed (n=9); chair rise test (n=8). 6MWT means 6-minute walk test; HPP means hypophosphatasia; IQR means interquartile range; SPPB means short physical function test battery; TUG means timed get up and go test.

Figures 4A–4D show a set of graphs depicting secondary outcome measures of patient-reported physical function and disease burden assessed by the LEFS (Figure 4A), SF-36v2 (Figures 4B and 4C), and the Pain Intensity Questionnaire (Figure 4D) among adults with childhood HPP treated with asfotase alfa for childhood-onset HPP at baseline, 12, 18, and 24 months of treatment. *p<0.05 compared with baseline. The lower and upper boundaries of the blue boxes represent the 25th and 75th percentiles, respectively. The horizontal black lines represent the median, and the whiskers represent the maximum and minimum values. LEFS score (n=10), PCS (n=9), MCS (n=9), Pain Intensity Questionnaire: baseline (n=13), 3 months (n=11), 6 months (n=11), 12 months (n=13), 24 months (n=5). HPP means hypophosphatasia; LEFS means lower extremity function scale; MCS means mental component summary; PCS means physical component summary; SF-36v2 means short form 36-item health questionnaire version 2.

Fig. 5 shows a graph showing weekly changes in the dose of asfotase alfa (mg/kg body weight).

Fig. 6 shows a graph depicting weekly changes in asfotase alfa dose (mg/kg ideal body weight).

Fig. 7 is a graph showing 6MWT data as in Fig. 3A up to 30 months.

Figures 8A and 8B are graphs showing the mean 4 m walk test time (Figure 8A) and mean speed (Figure 8B), as in Figure 3C, up to 30 months.

Fig. 9 is a graph showing data from the chair stand-up test as in Fig. 3D up to 30 months.

Fig. 10 is a graph showing the TUG test time data as in Fig. 3B up to 30 months.

Figures 11A and 11B show graphs showing grip strength of the dominant (Figure 11A) and non-dominant (Figure 11B) hand up to 30 months.

Figure 12 is a graph showing SF-36v2 (PCS) scores as in Figure 4B up to 30 months.

Fig. 13 is a graph showing pain levels as in Fig. 4D up to 30 months.

Figure 14 is a graph showing LEFS scores as in Figure 4A through 30 months.

Detailed description of the invention

Treatment options for bone mineralization disorders such as hypophosphatasia (HPP) and their symptoms are provided. HPP is a rare inherited skeletal disorder with an incidence of 1 in 100,000 live births for the most severe forms of the disease.

- 12 049823 HPP typically results from loss-of-function mutations in the gene encoding TNALP. HPP exhibits a wide spectrum of symptoms and severities, ranging from premature tooth loss to almost complete absence of bone mineralization during fetal development. The presentation of HPP varies markedly among individuals and also varies markedly with the age of the individuals. Many individuals with HPP present with skeletal changes, short stature, chronic pain, lower extremity pain, gait disturbance, and premature, atraumatic tooth loss. Because of loss-of-function mutations in endogenous TNALP, an individual with HPP requires restoration of native ALP activity to ensure normal bone matrix mineralization.

Because of the diversity of phenotypes and severity among subjects, different metrics may be used to assess or monitor disease status (e.g., before, during, and after treatment). In particular, some of these metrics are specific, such as those specific to infants or adults. For example, radiographic changes such as skeletal mineralization may be observed in infants as new bones grow during development. However, an adult with a moderate phenotype may exhibit normal or near-normal mineralization characteristics. Thus, a different metric may be needed to effectively monitor treatment effectiveness in these different patients. One such patient type includes adult patients (i.e., individuals aged 18 years and older) who experience decreased quality of life and/or chronic disability due to skeletal or muscle manifestations of the disease. Symptoms in adult patients include, for example, fractures and pseudofractures, chronic pain, muscle weakness, hypotonia, and/or low physical endurance. Sometimes, simple daily activities such as sitting, standing, or walking may be difficult for an adult patient. This document provides treatment options that include monitoring changes in biochemical, physical, and/or quality of life metrics to establish a treatment regimen for a patient with a bone mineralization disorder such as HPP, and in particular for an adult patient with HPP.

Treatment methods.

The methods described herein include treating a bone mineralization disorder in a human subject. The subject may have a level of at least one biochemical, physical, quality of life, or bone metric that identifies the subject as being in need of treating the bone mineralization disorder. The method may include administering at least one dose of sALP to the subject in a first treatment phase of a treatment regimen; monitoring the subject during the first treatment phase for changes in the at least one biochemical, physical, quality of life, or bone metric; and detecting an improvement in the at least one biochemical, physical, quality of life, or bone metric. The improvement may be above a predetermined threshold of the metric (e.g., an improvement of 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or more relative to the baseline value of the metric in the subject before treatment). If the subject shows improvement, the subject may then enter a first non-treatment phase of the treatment regimen during which the subject is not administered sALP. During the non-treatment phase, the method may further include monitoring the subject during the first non-treatment phase for a period of time for changes in at least one biochemical, physical, quality of life, or bone metric. If the subject experiences a decrease in the measured metric value, such as a decrease to or below a predetermined threshold (e.g., a decrease of 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or more of the metric value relative to the predetermined threshold), the treatment regimen may be restarted (e.g., the subject is administered at least one dose of sALP in a second or subsequent treatment phase of the treatment regimen). These steps may be repeated one or more times, with efficacy monitored (e.g., continuously or periodically) throughout the course of the treatment regimen. Although the predetermined threshold may be used as a cutoff value for assessing whether or not to administer sALP during a particular phase of the treatment regimen, one skilled in the art will appreciate that treatment may be continued or discontinued despite a decrease below or an increase above the predetermined threshold. For example, it may be desirable to resume dosing before the BOT-2 strength score decreases below a predetermined threshold to avoid a subject's metric score decreasing below the predetermined threshold (e.g., treatment may be restarted before the metric score reaches a value that is 1%, 2%, 5%, 10%, 15%, 20%, 25%, or 30% or more above the predetermined threshold).

Evaluation of treatment effectiveness.

Treatment of bone mineralization disorders (such as those seen in HPP, including, for example, perinatal HPP, infantile HPP, childhood HPP and odontohypophosphatasia and HPP-like disease)

- 13 049823 (CPPD and familial hypophosphatemia described herein) can be performed using alkaline phosphatase or a polypeptide having alkaline phosphatase activity (e.g. sALP, e.g. a polypeptide having the sequence of SEQ ID NO: 1, or a variant polypeptide having at least 95% sequence identity to the sequence of SEQ ID NO: 1, e.g. asfotase alfa). The methods described herein are also useful for diagnosing or monitoring a subject who has a bone mineralization disorder such as HPP or who is predisposed to developing one, determining a subject as having a bone mineralization disorder such as HPP or predisposed to developing one, or testing the effectiveness of a treatment for a bone mineralization disorder such as HPP. For example, a subject may be diagnosed as having a bone mineralization disorder such as HPP or as being predisposed to developing the same if the subject is characterized by the presence of certain characteristic biomarkers or metric score(s). The subject may be treated with an alkaline phosphatase or a polypeptide having alkaline phosphatase activity (e.g., an sALP, e.g., a polypeptide having the sequence of SEQ ID NO: 1, or a variant thereof with at least 85% sequence identity thereto), and the efficacy or effects of the treatment may be analyzed using the characteristic biomarkers or metric score(s). The predetermined metric score may include a characteristic predetermined threshold, wherein an increase above or a decrease below the threshold indicates the need to discontinue or initiate administration of the sALP.

Exemplary metrics useful for assessing the efficacy of treatment with an sALP (such as a TNALP, e.g., a fusion polypeptide comprising an sALP having the sequence of SEQ ID NO: 1 or a variant polypeptide having at least 95% sequence identity with the sequence of SEQ ID NO: 1, e.g., asfotase alpha) include biochemical metrics, physical metrics, quality of life metrics, and bone metrics. Biochemical metrics include monitoring of sALP substrates such as PPi, PEA, and PLP concentrations in serum, plasma, bone, muscle, or urine of a subject. Physical metrics include assessments such as the six-minute walk test (6MWT), Bruininks-Oseretsky Test of Motor Skills 2nd Edition (BOT-2), Bayley Scales of Infant and Toddler Development 3rd Edition (BSID-III), muscle strength rating scale, hand dynamometer (HHD) test, Peabody Developmental Motor Scales 2nd Edition (PDMS-2), gait analysis, timed get up and go (TUG) test, and the Short Physical Performance Battery (SPPB). The SPPB may include tests such as the repeated chair stand test. Methods may include tracking metrics such as decreasing dependence on assistive devices (e.g., wheelchairs, rolling walkers, and canes). Physical metrics can be monitored using a wearable device, such as a device that tracks steps (e.g., a pedometer), physical activity, heart rate (e.g., a heart rate monitor), and/or sleep patterns. Bone metrics include monitoring bone morphology, bone turnover, bone mineralization, and bone mineral density. Quality of life metrics include assessments such as the EuroQol Five-Item Questionnaire (EQ-5D), the Child Health Questionnaire (CHAQ), the Pediatric Outcomes Data Collection Instrument (PODCI), the Child Health Index-9D (CHU-9D), the Pediatric Quality of Life Scale (PedsQL), the Lower Limb Function Scale (LEFS), the Short Form 36 (SF-36), and the Short Form 12 (SF-12). The methods may further comprise using one or more of the described metrics (e.g., bone turnover, bone mineralization, bone mineral density, or plasma PPi and PLP concentrations, physical metrics, and quality of life metrics), alone or in combination, to assess the effectiveness of the treatment, wherein the improvements described herein demonstrate that the sALP is an effective treatment for the condition being treated. In addition, the method may further comprise discontinuing the treatment if the methods are effective in treating a subject in need thereof. Moreover, the methods may comprise varying the dosage and/or frequency of administration of the sALP (e.g., SEQ ID NO: 1; e.g., asfotase alfa) to determine an effective amount of sALP to administer to a subject in need thereof. If the subject fails to complete the treatment for a specified period of time as part of the treatment regimen, the subject may be observed at various time points to assess whether the treatment should be resumed or whether the subject should remain in a non-treatment phase. These metrics are described in further detail below.

Regeneration and mineralization of bone tissue.

A subject who has a bone mineralization disorder such as HPP, or who is predisposed to developing one, may be identified for treatment with alkaline phosphatase.

- 14 049823 tase or a polypeptide having alkaline phosphatase activity (e.g. sALP, e.g. a polypeptide characterized by the sequence under SEQ ID NO: 1, or a variant thereof with at least 85% sequence identity thereto) depending on the level of bone mineralization. For example, bone mineralization can be used as a metric for diagnosing a subject as having a bone mineralization disorder, such as HPP, or for testing the efficacy of the polypeptide described herein. In a subject with a bone mineralization disorder (e.g. HPP), administration of sALP results in improved bone regeneration in the subject following successful treatment.

Impairment of bone regeneration results in bone loss and includes decreased mineralization, which results in separation of two or more bones. Bone regeneration and decreased mineralization can be compared to a reference bone. Methods for identifying decreased bone regeneration and mineralization are standard and include non-invasive techniques such as radiography and computed tomography (CT). Typically, images of an appropriate area of the subject can be obtained before and at one or more time points after treatment with sALP and these images can be compared to assess the effectiveness of the treatment. Impairment of bone regeneration and/or mineralization can be determined by a decrease in opacity. Images can be obtained at any time during treatment with sALP and the time of their acquisition can be, for example, 1, 2, 3, 4, 5 or 6 days, weeks, months or years after the start of ERT treatment with sALP, or when a decrease in effectiveness is suspected. The impairment of bone regeneration and/or the decrease in bone mineralization in a subject may become detectable at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 weeks or months after the start of the sALP treatment period. The impairment of bone regeneration and/or the decrease in mineralization in a subject may, in some cases, persist for at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 weeks, months, or years after the start of the ERT treatment period with sALP. The improvement in bone regeneration and the increase in bone mineralization can be used to determine that the effectiveness of sALP is restored and the bone mineralization disorder (e.g., HPP) is effectively treated after therapy. If bone regeneration and mineralization increases to or above a predetermined threshold, sALP administration may be stopped. However, mineralization levels may be monitored periodically to determine whether administration should be restarted.

Bone mineral density (BMD).

A subject who has a bone mineralization disorder such as HPP, or who is predisposed to developing one, can be determined for treatment with alkaline phosphatase or a polypeptide having alkaline phosphatase activity (e.g., sALP, e.g., a polypeptide characterized by the sequence of SEQ ID NO: 1, or a variant thereof with at least 85% sequence identity thereto) depending on the level of BMD. A decrease in BMD (e.g., relative to a normal subject) can be used as a metric to diagnose a subject as having a bone mineralization disorder such as HPP.

The reduction in BMD can be used to monitor the effectiveness of sALP (e.g., the polypeptide with the sequence under SEQ ID NO: 1) during ERT. Methods for measuring BMD are known in the art and include, for example, bone biopsy, dual-energy X-ray absorptiometry (DXA or DEXA), peripheral quantitative CT (pQCT), high-resolution pQCT (HRpQCT), and quantitative ultrasound (QUS). Measurements can be made by any standard method, including Hounsfield CT measurement, and the results can be compared with a normative database or with a control subject. BMD is sometimes referred to as a Z-score or T-score. Pre-treatment BMD values may be measured at any time during ERT treatment with sALP and may be separated by time intervals such as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12 days, weeks, months, years after the start of ERT with sALP. The post-treatment BMD value may decrease by, for example, 0.01%, 0.05%, 0.1%, 0.5%, or 1%. The post-treatment BMD value may also remain unchanged or the change may not be detectable. An increase in bone BMD above a predetermined threshold may be used to determine that sALP efficacy is being restored and that a bone mineralization disorder (e.g., HPP) is being effectively treated following therapy. If recovery occurs, the subject may discontinue treatment and be periodically monitored for changes in BMD. If the BMD level decreases below a predetermined threshold, therapy can be restarted.

Plasma inorganic pyrophosphate (PPi) and alkaline phosphatase (ALP) concentrations.

A subject who has a bone mineralization disorder such as HPP, or who is predisposed to developing the same, can be identified for treatment with alkaline phosphatase or a polypeptide having alkaline phosphatase activity (e.g. sALP, e.g. the polypeptide having the sequence of SEQ ID NO: 1, or a variant thereof with at least 85% sequence identity thereto) by determining the concentrations of inorganic

- 15 049823 pyrophosphate (PPi) and/or alkaline phosphatase (ALP) in a sample, such as a blood plasma or urine sample, obtained from a subject. Any method known to those skilled in the art can be used to quantify the concentrations of PPi and/or ALP in a blood plasma sample or, alternatively, in a urine sample, as described in detail in Whyte et al., 1995 (J. Clin. Invest. 95(4): 1440-1445), which is incorporated herein by reference in its entirety. Methods for quantifying PPi concentrations in a blood 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, an alkaline phosphatase or a polypeptide having alkaline phosphatase activity (e.g., sALP, e.g., a polypeptide having the sequence of SEQ ID NO: 1, or a variant thereof with at least 85% sequence identity thereto) can be administered to a subject who has a bone mineralization disorder such as HPP, or who is predisposed to developing one, who has previously been determined to have PPi in blood plasma at a concentration of no more than about 6 μM (e.g., about 4.5 μM, about 5 μM, or about 5.5 μM, or PPi in blood plasma at a concentration in the range of about 4.5 μM to about 6 μM). For example, an alkaline phosphatase or a polypeptide having alkaline phosphatase activity is administered to, for example, an infant or child (e.g., a subject less than about 12 years of age) with a plasma PPi concentration of about 5.71 μM or greater; or an adolescent (e.g., a subject between about 13 and about 18 years of age) with a plasma PPi concentration of about 4.78 μM or greater.

In particular, alkaline phosphatase or a polypeptide having alkaline phosphatase activity is administered to an adult (e.g., a subject over approximately 18 years of age) with a plasma PPi concentration of approximately 5.82 μM or greater.

Additionally, an alkaline phosphatase or a polypeptide having alkaline phosphatase activity can be administered to a subject (e.g., a human) who has or is predisposed to developing a bone mineralization disorder such as HPP and who has previously been determined to have plasma ALP at a concentration of, for example, about 90 U/L or less in a subject aged 0 to 14 days; about 134 U/L or less in a subject aged 15 days to less than 1 year; about 156 U/L or less in a subject aged about 1 year to less than 10 years; about 141 U/L or less in a subject aged about 10 years to less than about 13 years; about 62 U/L or less in a female subject aged about 13 years to less than about 15 years; approximately 127 U/L or less for a male subject between approximately 13 years of age and less than approximately 15 years of age; approximately 54 U/L or less for a female subject between approximately 15 years of age and less than approximately 17 years of age; approximately 89 U/L or less for a male subject between approximately 15 years of age and less than approximately 17 years of age; approximately 48 U/L or less for a female subject approximately 17 years of age or older; or approximately 59 U/L or less for a male subject approximately 17 years of age or older.

The plasma PPi concentration and/or plasma ALP concentration in a subject (e.g., a human) that has or is predisposed to developing a bone mineralization disorder such as HPP can be compared to the plasma PPi concentration and/or plasma ALP concentration in a normal subject to determine the effect of treatment in a subject administered alkaline phosphatase or a polypeptide having alkaline phosphatase activity (e.g., sALP, e.g., a polypeptide characterized by the sequence of SEQ ID NO: 1, or a variant thereof with at least 85% sequence identity thereto). In particular, the alkaline phosphatase or the polypeptide having alkaline phosphatase activity can be administered for 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 longer than ten years, such as for the life of the subject). Alternatively, the methods can include determining the plasma PPi concentration and/or the plasma ALP concentration prior to administration of the alkaline phosphatase or the polypeptide having alkaline phosphatase activity to assess the effect of treatment with the alkaline phosphatase or the polypeptide having alkaline phosphatase activity in the subject.

These methods result in a decrease in the PPi concentration and/or an increase in the ALP concentration in a sample (e.g., a blood plasma sample) obtained from a subject (e.g., a human subject) that has or is predisposed to developing a bone mineralization disorder such as HPP. For example, treatment with alkaline phosphatase or a polypeptide having alkaline phosphatase activity (e.g., sALP, e.g., a polypeptide characterized by

- 16 049823 having the sequence of SEQ ID NO: 1, or a variant thereof with at least 85% sequence identity thereto), results in a decrease in the concentration of PPi in a sample (e.g., a blood plasma sample) obtained from a subject by 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%). Thus, following administration of alkaline phosphatase or a polypeptide having alkaline phosphatase activity, the subject exhibits PPi in the blood plasma at a concentration of, 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.

Similarly, treatment with alkaline phosphatase or a polypeptide having alkaline phosphatase activity results in an increase in the ALP concentration in a sample (e.g., a blood plasma sample) obtained from a subject (e.g., a human) who has a bone mineralization disorder such as HPP, or who is predisposed to developing one, by 30%, 35%, 40%, 45%, 50%, 55%, 60%, or greater than 60%, relative to the subject prior to administration of the alkaline phosphatase or polypeptide having alkaline phosphatase activity. For example, administration of alkaline phosphatase or a polypeptide having alkaline phosphatase activity increases the ALP concentration in a sample (e.g., a blood plasma sample) obtained from the subject to, for example, about 273 U/L or greater in the case of a subject between 0 and 14 days of age; approximately 518 U/L or greater for a subject 15 days to less than 1 year of age; approximately 369 U/L or greater for a subject 1 year to less than 10 years of age; approximately 460 U/L or greater for a subject 10 years to less than about 13 years of age; approximately 280 U/L or greater for a female subject 13 years to less than about 15 years of age; approximately 517 U/L or greater for a male subject 13 years to less than about 15 years of age; approximately 128 U/L or greater for a female subject 15 years to less than about 17 years of age; approximately 365 U/L or greater for a male subject 15 years to less than about 17 years of age; approximately 95 U/L or greater in the case of a female subject approximately 17 years of age or older; or approximately 164 U/L or greater in the case of a male subject approximately 17 years of age or older.

A decrease in the plasma PPi concentration and/or an increase in the ALP concentration in a subject (e.g., a human) who has a bone mineralization disorder such as HPP or who is predisposed to developing one may be maintained throughout the period of administration of an alkaline phosphatase or a polypeptide having alkaline phosphatase activity (e.g., sALP, e.g., a polypeptide characterized by the sequence of SEQ ID NO: 1, or a variant thereof with at least 85% sequence identity thereto). For example, the plasma PPi concentration decreases by about 25% and remains within ±10% of the decreased plasma PPi concentration during treatment with sALP, and/or the plasma ALP concentration increases by about 50% and remains within ±10% of the increased plasma ALP concentration during treatment with an alkaline phosphatase or a polypeptide having alkaline phosphatase activity.

Alternatively, if administration of an alkaline phosphatase or a polypeptide having alkaline phosphatase activity (e.g., an sALP, e.g., a polypeptide having the sequence of SEQ ID NO: 1, or a variant thereof with at least 85% sequence identity thereto) does not result in an average decrease in PPi concentrations in a blood plasma sample obtained from a subject (e.g., a human) who has a bone mineralization disorder such as HPP, or who is predisposed to developing one, by about 25% or more, the dosage and/or frequency of administration of the sALP can be varied to determine an effective amount of sALP for the subject. Similarly, if administration of the alkaline phosphatase or polypeptide having alkaline phosphatase activity does not result in an average increase in ALP concentrations in a blood plasma sample obtained from the subject of about 50% or greater, the dosage and/or frequency of administration of the alkaline phosphatase or polypeptide having alkaline phosphatase activity can be adjusted to determine an effective amount of the alkaline phosphatase or polypeptide having alkaline phosphatase activity for the subject. For example, the dosage of the alkaline phosphatase or polypeptide having alkaline phosphatase activity can be increased from, for example, about 0.5 mg/kg/week or about 3.5 mg/kg/week to about 3-6 mg/kg/week or about 6-9 mg/kg/week. If the desired PPi or ALP levels are achieved, the subject can be discontinued and periodically monitored for changes in PPi or ALP levels. For example, if PPi levels increase above and/or ALP levels decrease below a predetermined threshold, then therapy can be restarted.

Brief Physical Performance Battery (SPPB). The SPPB is designed to measure functional status and physical performance. It is a comprehensive measure

- 17 049823 which assesses walking speed, standing balance and performance of the sit-to-stand test (STS). The SPPB is calculated on the basis of 3 components: the ability to stand for a period of up to 10 seconds with 3 foot positions (feet parallel to each other, feet perpendicular and feet in sequence); time to complete a 3- or 4-metre walk and time to rise from a chair 5 times (i.e. the repeated chair stand test). The standing balance tests are scored based on the ability to maintain balance in each of these positions. Walking speed and STS tests are scored based on, firstly, the ability to perform the tasks and, secondly, on the time spent on each task. Each task is scored out of 4 points, with the scores for the 3 tests summed to give a maximum of 12 and a minimum of 0. A higher score indicates a higher level of function, while a lower score indicates a lower level of function. Lower SPPB scores have been shown to predict increased risk of falls, loss of ADL independence, decreased mobility, disability, poorer health, hospital readmission and length of stay, nursing home admission, and death. A 0.5 point change in SPPB is considered a small meaningful change, while a 1 point change in SPPB is considered a large meaningful change.

The chair stand test involves the activation of multiple lower limb muscles, primarily the knee extensor muscles (quadriceps femoris). The chair stand test has been used as a stand-alone assessment (e.g., STS exercise), which is widely used as a measure of lower limb strength.

The SPPB score in a subject who has a bone mineralization disorder such as HPP or who is predisposed to developing it can be used to assess the effectiveness of treatment with alkaline phosphatase or a polypeptide having alkaline phosphatase activity, such as sALP (e.g., a polypeptide characterized by the sequence of SEQ ID NO: 1, or a variant thereof with at least 85% sequence identity thereto), wherein improvements in the score in a certain test indicate that the alkaline phosphatase or polypeptide having alkaline phosphatase activity is effective for treating the inability to perform activities of daily living (ADL) and pain during their performance associated with a bone mineralization disorder such as HPP. In particular, a subject who has a bone mineralization disorder such as HPP or who is predisposed to developing it can be tested using the SPPB and to assess the effectiveness of treatment by administering sALP.

For example, if administration of an sALP described herein to a subject who has a bone mineralization disorder such as HPP or who is predisposed to developing one results in an average increase in the SPPB score to about 6 or greater than about 6, where the subject previously had an average SPPB score of less than about 6, then the sALP is effective in treating a bone mineralization disorder such as HPP. Alternatively, if administration of an sALP described herein does not result in an average increase in the SPPB score to about 6 or greater than about 6, then the dosage and frequency of administration of the sALP can be adjusted to determine an effective amount of sALP for a subject who has a bone mineralization disorder such as HPP or who is predisposed to developing one. For example, the dosage of sALP (e.g., a polypeptide having the sequence of SEQ ID NO: 1, or a variant thereof with at least 85% sequence identity thereto) can be increased, such as from about 0.5-3 mg/kg/week to about 3-6 mg/kg/week or from about 3-6 mg/kg/week to about 6-9 mg/kg/week. If SBBP increases above a predetermined threshold, the subject can discontinue treatment and be periodically monitored for changes in his SBBP score. If the SBBP score decreases below a predetermined threshold, therapy can be reinitiated.

Timed Get Up and Go (TUG) Test.

The timed get up and go (TUG) test measures the time in seconds it takes an individual to rise from a standard chair, walk a distance of 3 metres (approximately 10 feet), turn around, return to the chair, and sit down. This test requires that patients wear their usual shoes and use their usual walking aid (no aid, cane, walker). No physical assistance is provided. Patients begin the test with their back against the chair, their hands on the armrests, and their walking aid in their hands. They are instructed that when the word go is given, they should stand up and walk at a comfortable and safe pace to a line on the floor 3 metres away, turn around, return to the chair, and sit down again. Before timing, the patient completes the test once to familiarise themselves with the test. Either a stopwatch or a wristwatch with a second hand can be used to time the test. Normal healthy older adults typically complete the task in 10 seconds or less. Very weak or frail older adults with poor mobility may require 2 minutes or more. The score is

- 18 049823 >14 s, may indicate a high risk of falls. The TUG test can be performed by a blinded assessor.

The TUG score of a subject who has a bone mineralization disorder such as HPP or who is predisposed to developing it can be used to assess the effectiveness of treatment with an alkaline phosphatase or a polypeptide having alkaline phosphatase activity, such as sALP (e.g., a polypeptide characterized by the sequence of SEQ ID NO: 1, or a variant thereof with at least 85% sequence identity thereto), wherein improvements in the score in a certain test indicate that the alkaline phosphatase or the polypeptide having alkaline phosphatase activity is effective for treating a bone mineralization disorder such as HPP. In particular, a subject who has a bone mineralization disorder such as HPP or who is predisposed to developing it can be tested using the TUG test to assess the effectiveness of treatment by administering sALP.

For example, if administration of an sALP as described herein to a subject who has a bone mineralization disorder such as HPP or who is susceptible to developing one results in an average decrease in TUG score to about 14 or less than about 14, where the subject previously had an average TUG score of greater than about 14, then the sALP is effective in treating, for example, the inability to perform activities of daily living (ADL) and the presence of pain during their performance associated with a bone mineralization disorder such as HPP. Alternatively, if administration of an sALP as described herein does not result in an average decrease in TUG score to about 14 or less than about 14, then the dosage and frequency of administration of the sALP can be adjusted to determine an effective amount of sALP for a subject who has a bone mineralization disorder such as HPP or who is susceptible to developing one. For example, the dosage of sALP (e.g., a polypeptide having the sequence of SEQ ID NO: 1, or a variant thereof with at least 85% sequence identity thereto) can be increased, such as from about 0.5-3 mg/kg/week to about 3-6 mg/kg/week or from about 3-6 mg/kg/week to about 6-9 mg/kg/week. If the subject's TUG test result decreases below a predetermined threshold, the subject can discontinue treatment and be periodically monitored for changes in the TUG test score. If the TUG score increases above a predetermined threshold, the treatment can be reinitiated.

Bruininks-Oseretsky Test of Motor Proficiency, 2nd Edition (BOT-2). An illustrative Bruininks-Oseretsky Test of Motor Proficiency, 2nd Edition (BOT-2) is described in Bruininks, R.H. (2005). Bruininks-Oseretsky Test of Motor Proficiency, (BOT-2), Minneapolis, MN: Pearson Assessment, which is incorporated herein by reference in its entirety. In particular, the BOT-2 can be used to assess physical pathologies and mobility limitations in a subject who has or is predisposed to developing a bone mineralization disorder (e.g., HPP) to obtain a BOT-2 score for the subject.

The BOT-2 includes a number of tests to assess the physical pathologies of a subject, which can be administered, for example, using a battery containing these tests. The BOT-2 provides a composite BOT-2 score in the following areas: strength, running speed and agility, fine motor precision, fine motor coordination, manual dexterity, bilateral coordination, balance, and upper limb coordination. For example, to determine the BOT-2 strength score, a subject who has or is predisposed to developing a bone mineralization disorder such as HPP may perform squats, lying V-crunches, standing long jumps, wall sits, and/or push-ups. To determine the BOT-2 running speed and agility score, a subject who has or is susceptible to developing a bone mineral disorder such as HPP may walk on a balance beam and/or perform a shuttle run, a two-legged lateral jump, and/or a single-legged lateral jump. To determine the BOT-2 fine motor accuracy score, a subject who has or is susceptible to developing a bone mineral disorder such as HPP may cut out a circle and/or connect the dots. To determine the BOT-2 fine motor coordination score, a subject who has or is susceptible to developing a bone mineral disorder such as HPP may copy a star and/or copy a square. To determine the Manual Dexterity score, a subject who has or is susceptible to developing a bone mineral disorder such as HPP may pass pennies, sort cards, and/or line up blocks. To determine the BOT-2 Bilateral Coordination score, a subject who has or is susceptible to developing a bone mineral disorder such as HPP may tap his or her foot or finger and/or perform standing hops. To determine the BOT-2 Balance score, a subject who has or is susceptible to developing a bone mineral disorder such as HPP may walk forward in a line and/or stand on one foot on a balance beam. To determine the BOT-2 Coor

- 19 049823 Upper Limb Performance according to BOT-2 a subject who has a bone mineralization disorder such as HPP or who is predisposed to developing one can throw a ball at a target and/or catch a thrown ball.

A subject who has or is predisposed to developing a bone mineralization disorder such as HPP may undergo tests in one or more of the described domains (strength, running speed and agility, fine motor precision, fine motor coordination, manual dexterity, bilateral coordination, balance, and upper limb coordination) to obtain a BOT-2 score that is indicative of the subject's physical pathologies. In each BOT-2 domain (strength, running speed and agility, fine motor precision, fine motor coordination, manual dexterity, bilateral coordination, balance, and upper limb coordination), such a subject may undergo one or more tests to determine the subject's BOT-2 score, for example, to determine the BOT-2 strength score, a subject may perform one or more of the squats, lying V-crunches, standing broad jumps, wall sits, and push-ups. Thus, to determine the BOT-2 score (e.g., the BOT-2 strength score) in a subject who has or is predisposed to developing a bone mineralization disorder such as HPP (e.g., an HPP-like disorder), only one test (e.g., one test selected from the group of squats, lying V-curls, standing broad jump, wall sits, and push-ups) may be performed.

Each of the BOT-2 scores (strength, running speed and agility, fine motor precision, fine motor coordination, manual dexterity, bilateral coordination, balance, and upper limb coordination) in a subject who has or is predisposed to developing a bone mineral disorder such as HPP may be compared to the BOT-2 score in a subject without a bone mineral disorder such as HPP to, for example, make a comparison of the BOT-2 score at baseline. Each of the BOT-2 scores (e.g., strength, running speed and agility, fine motor accuracy, fine motor coordination, manual dexterity, bilateral coordination, balance, and upper limb coordination) of a subject who has or is susceptible to developing a bone mineralization disorder such as HPP may be compared with the BOT-2 score of other subjects who have or are susceptible to developing a bone mineralization disorder such as HPP to, for example, obtain a relative BOT-2 score for the subject.

BOT-2 scores (e.g., strength, running speed and agility, fine motor accuracy, fine motor coordination, manual dexterity, bilateral coordination, balance, and upper limb coordination) range from approximately 0 to approximately 25 or less, with a score of approximately 10 to approximately 20 considered representative of a healthy subject (e.g., a subject without a bone mineralization disorder such as HPP). A subject with an average BOT-2 score (e.g., strength, running speed and agility, fine motor accuracy, fine motor coordination, manual dexterity, bilateral coordination, balance, and upper limb coordination) of less than about 10 can be treated with alkaline phosphatase or a polypeptide having alkaline phosphatase activity, such as sALP, such as the polypeptide having the sequence of SEQ ID NO: 1, or a variant thereof with at least 85% sequence identity thereto.

For example, a subject who has a bone mineralization disorder such as HPP, or who is predisposed to developing one, with a BOT-2 strength 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 be treated with an sALP (e.g., a polypeptide having the sequence of SEQ ID NO: 1, or a variant thereof with at least 85% sequence identity thereto) for a period of time, up to and including for the life of the subject. Similarly, a subject who has a bone mineralization disorder such as HPP, or who is predisposed to developing one, with 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 be treated with an sALP (e.g., a polypeptide having the sequence of SEQ ID NO: 1, or a variant thereof with at least 85% sequence identity thereto) for a period of time, up to and including for the life of the subject.

The methods can result in an improvement in a BOT-2 score (e.g., a score for strength, running speed and agility, fine motor accuracy, fine motor coordination, manual dexterity, bilateral coordination, balance, and/or upper limb coordination) in a subject who has or is predisposed to developing a bone mineralization disorder such as HPP. For example, treatment with alkaline phosphatase or a polypeptide having alkaline phosphatase activity such as sALP (e.g., a polypeptide characterized by

- 20 049823 having the sequence of SEQ ID NO: 1, or a variant thereof with at least 85% sequence identity thereto), such as treatment with sALP over a period of time, can result in an average increase in BOT-2 strength score in the range of from about 10 to about 20 (e.g., about 10, about 11, about 12, about 13, about 14, about 15, about 16, about 17, about 18, about 19, or about 20). In addition, treatment with an sALP (e.g., a polypeptide having the sequence of SEQ ID NO: 1, or a variant thereof with at least 85% sequence identity thereto) can result in an average increase in BOT-2 running speed and agility score in the range of from about 5 to about 20 (e.g., about 5, about 6, about 7, about 8, about 9, about 10, about 11, about 12, about 13, about 14, about 15, about 16, about 17, about 18, about 19, or about 20).

An increase in a BOT-2 score (e.g., a score for strength, running speed and agility, fine motor accuracy, fine motor coordination, manual dexterity, bilateral coordination, balance, and/or upper limb coordination) may be maintained throughout the period of administration of alkaline phosphatase or a polypeptide having alkaline phosphatase activity, such as sALP (e.g., a polypeptide characterized by the sequence of SEQ ID NO: 1, or a variant thereof with at least 85% sequence identity thereto), such as over a period of time. Similarly, a reduction in muscle physical pathologies following administration of alkaline phosphatase or a polypeptide having alkaline phosphatase activity may be maintained throughout the period of administration of the alkaline phosphatase or a polypeptide having alkaline phosphatase activity.

The BOT-2 scores (scores for strength, running speed and agility, fine motor accuracy, fine motor coordination, manual dexterity, bilateral coordination, balance, and upper limb coordination) in a subject who has or is predisposed to developing a bone mineralization disorder such as HPP may be used alone or in combination with other metrics to assess the effectiveness of treatment with alkaline phosphatase or a polypeptide having alkaline phosphatase activity such as sALP (e.g., a polypeptide characterized by the sequence of SEQ ID NO: 1, or a variant thereof with at least 85% sequence identity thereto), whereby improvements in the score in a particular test indicate that the alkaline phosphatase or polypeptide having alkaline phosphatase activity is effective for the treatment of muscle pathologies associated with a bone mineralization disorder such as HPP. For example, if administration of an sALP described herein to a subject who has a bone mineralization disorder such as HPP, or who is predisposed to developing one, results in an average increase in the BOT-2 running speed and agility score of about 5 or greater than about 5, where the subject previously had an average BOT-2 running speed and agility score of less than about 5, then the sALP is considered effective in, for example, treating physical pathologies associated with a bone mineralization disorder such as HPP.

In addition, in each BOT-2 domain (strength, running speed and agility, fine motor precision, fine motor coordination, manual dexterity, bilateral coordination, balance, and upper limb coordination), a subject who has a bone mineralization disorder such as HPP or who is predisposed to developing one may complete one or more tests to determine the subject's BOT-2 score.

Alternatively, if administration of an alkaline phosphatase or a polypeptide having alkaline phosphatase activity, such as an sALP as described herein, does not result in an average increase in the BOT-2 running speed and agility score of greater than about 5, the dosage and/or frequency of administration can be adjusted to determine an effective amount of the alkaline phosphatase or polypeptide having alkaline phosphatase activity for a subject who has a bone mineralization disorder such as HPP, or who is predisposed to developing one. For example, the dosage of the sALP (e.g., a polypeptide characterized by the sequence of SEQ ID NO: 1, or a variant thereof with at least 85% sequence identity thereto) can be increased, such as from about 0.5-3 mg/kg/week to about 3-6 mg/kg/week, or from about 3-6 mg/kg/week to about 6-9 mg/kg/week. If a subject's BOT-2 score increases above a predetermined threshold, the subject may discontinue treatment and be monitored periodically for changes in his or her BOT-2 score. If the BOT-2 score decreases below a predetermined threshold, therapy may be reinitiated.

Short Form 36-Item Questionnaire (SF-36).

Health-related quality of life refers to functioning and well-being in the physical, mental, and social domains of life. The SF-36 consists of 8 multi-item scales (35 items),

- 21,049,823 assessing physical function (10 items), role limitations due to physical health problems (4 items), physical pain (2 items), general health (5 items), life activity (4 items), social functioning (2 items), role limitations due to emotional problems (3 items), and emotional well-being (5 items). These 8 scales can be combined into 2 summary scores: the PCS score and the mental component summary (MCS) score. Item 36, which asks about changes in health, is not included in the scale or summary scores.

The SF-36 is a self-administered questionnaire. Patients complete 1 of a range of options for each of 36 questions. The combination of item responses is then combined to calculate a score for each of the 8 scales listed. Scores for each dimension range from 0 to 100, with higher scores indicating better health. Body pain is also scored in the same way, with higher scores indicating less pain. The 2 summary measures (PCS and MCS) are scored differently from the 8 dimension scores. These scales are scored using norm-referenced methods. A score of 50 reflects the average score for these populations. Scores below 50 reflect below-average health, and scores above 50 reflect above-average health.

The SF-36 score of a subject who has or is predisposed to developing a bone mineralization disorder such as HPP can be used to assess the effectiveness of treatment with alkaline phosphatase or a polypeptide having alkaline phosphatase activity such as sALP (e.g., a polypeptide characterized by the sequence of SEQ ID NO: 1, or a variant thereof with at least 85% sequence identity thereto), whereby improvements in the score on a particular test indicate that the alkaline phosphatase or polypeptide having alkaline phosphatase activity is effective for treating the inability to perform activities of daily living (ADL) and pain associated with a bone mineralization disorder such as HPP. Specifically, a subject who has a bone mineralization disorder such as HPP or who is predisposed to developing one may be asked one or more questions in one or more of the eight scales to obtain an average SF-36 score and to assess the effectiveness of treatment with sALP.

For example, if administration of an sALP described herein to a subject who has a bone mineralization disorder such as HPP or who is susceptible to developing one results in an average increase in SF-36 score to about 50 or greater than about 50, where the subject previously had an average SF-36 score of less than about 50, then the sALP is effective in treating, for example, the inability to perform activities of daily living (ADL) and the presence of pain during their performance associated with a bone mineralization disorder such as HPP. Alternatively, if administration of an sALP described herein does not result in an average increase in SF-36 score to about 50 or greater than about 50, then the dosage and frequency of administration of the sALP can be adjusted to determine an effective amount of sALP for a subject who has a bone mineralization disorder such as HPP or who is susceptible to developing one. For example, the dosage of sALP (e.g., a polypeptide having the sequence of SEQ ID NO: 1, or a variant thereof with at least 85% sequence identity thereto) can be increased, such as from about 0.5-3 mg/kg/week to about 3-6 mg/kg/week or from about 3-6 mg/kg/week to about 6-9 mg/kg/week. If the subject's SF-36 score increases above a predetermined threshold, the subject can discontinue treatment and be periodically monitored for changes in his SF-36 score. If the SF-36 score decreases below a predetermined threshold, the therapy can be reinitiated.

Lower Extremity Function Scale (LEFS).

The LEFS refers to a method for measuring lower extremity functional disability in patients, particularly patients with HPP (e.g., patients approximately 13 years of age or older). The LEFS is a self-report measure described in Binkley et al. (Phys Ther. 79:371-83, 1999), which is incorporated by reference herein in its entirety. LEFS total scores range from 0 to 80, with higher scores indicating better lower extremity function. A change in LEFS score of approximately 9 units is considered a clinically significant change. The LEFS can be administered to patients with HPP (e.g., patients with HPP approximately 13 years of age or older) using an interview format. Higher LEFS scores indicate improved lower extremity function, including transitional movements (eg, getting out of the bath or rolling over in bed), ambulation (eg, walking or running on uneven surfaces), stair climbing, and squatting. The LEFS can be used to assess functional impairment in one or both lower extremities in a patient with HPP, including the ability to monitor the patient over time and assess the effectiveness of asfotase alfa treatment.

An increase in LEFS score in a subject with impaired bone mineralization

- 22 049823 nor, such as HPP, can be maintained for the entire period of administration of the alkaline phosphatase or the polypeptide having alkaline phosphatase activity (e.g. sALP, e.g. the polypeptide characterized by the sequence under SEQ ID NO: 1, or a variant thereof with at least 85% sequence identity thereto), for example, 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 at least eight years, at least nine years, or at least ten years, or for the life of the subject; in particular at least six weeks). For example, the LEFS score increases by at least 9 points and remains within ±10% of the LEFS score during treatment with alkaline phosphatase or a polypeptide having alkaline phosphatase activity.

Alternatively, if administration of an alkaline phosphatase or a polypeptide having alkaline phosphatase activity (e.g., sALP, e.g., a polypeptide having the sequence of SEQ ID NO: 1, or a variant thereof with at least 85% sequence identity thereto) does not result in an average increase in LEFS score of at least 9 points, then the dosage and/or frequency of administration of the alkaline phosphatase or polypeptide having alkaline phosphatase activity may be adjusted to determine an effective amount of the alkaline phosphatase or polypeptide having alkaline phosphatase activity for a subject who has a bone mineralization disorder such as HPP. For example, the dosage of alkaline phosphatase or a polypeptide having alkaline phosphatase activity may be increased from, for example, about 0.1-3 mg/kg/week or about 3-6 mg/kg/week to about 3-6 mg/kg/week or about 6-9 mg/kg/week. If the subject's LEFS score increases above a predetermined threshold, the subject may discontinue treatment and be monitored periodically for changes in his or her LEFS score. If the LEFS score decreases below a predetermined threshold, therapy may be reinitiated.

Child Health Assessment Questionnaire (CHAQ).

The Child Health Questionnaire (CHAQ) can be used to assess the health status of children with a bone mineral disorder such as HPP to obtain a child's CHAQ score as described by Brice & Fries (J. Rheumatol. 30(1): 167-178, 2003) and Klepper (Arthritis & Rheumatism, 49: S5-S14, 2003), which are incorporated herein by reference in their entireties. The CHAQ includes eight categories of questions on dressing/grooming, standing, eating, walking, hygiene, reaching, grip, and activities in which the parent or caregiver records the degree of difficulty the child with a bone mineral disorder such as HPP has in performing the relevant activities. The score range in each category is from 0 to 3, with a score of 0 indicating no difficulty; A score of 1 indicates some difficulty; a score of 2 indicates great difficulty; and a score of 3 indicates that the child cannot perform the task.

Children who have or are predisposed to developing a bone mineralization disorder such as HPP with a mean CHAQ score (e.g., indicating inability to perform activities of daily living (ADL) and/or pain during activities of daily living) of greater than about 0.8 (e.g., about 0.8, about 1, about 1.2, about 1.4, about 1.6, about 1.8, about 2.0, about 2.2, about 2.4, about 2.6, about 2.8, or about 3.0) can be treated by administering alkaline phosphatase or a polypeptide having alkaline phosphatase activity such as sALP (e.g., a polypeptide having the sequence of SEQ ID NO: 1, or a variant thereof with at least 85% sequence identity thereto). For example, children with a mean CHAQ score of greater than about 0.8 may be treated by administering alkaline phosphatase or a polypeptide having alkaline phosphatase activity, such as sALP (e.g., a polypeptide having the sequence of SEQ ID NO: 1, or a variant thereof with at least 85% sequence identity thereto) for a period of time, up to and including for the life of the subject. Moreover, a child who has a bone mineralization disorder such as HPP as disclosed herein, or who is predisposed to developing the same, may be asked one or more questions in one or more of eight categories (dressing/grooming, standing, eating, walking, hygiene, reaching, grip, and activities) to obtain an average CHAQ score, and if the average CHAQ score is greater than approximately 0.8, the child may be treated with alkaline phosphatase or a polypeptide having alkaline phosphatase activity such as sALP.

- 23 049823

The CHAQ score of a child who has or is predisposed to developing a bone mineralization disorder such as HPP as disclosed herein may be compared to the CHAQ score of children without such a bone mineralization disorder such as HPP to, for example, determine a standard deviation of the CHAQ score. Additionally, the CHAQ score of a child who has or is predisposed to developing a bone mineralization disorder such as HPP as disclosed herein may be compared to the CHAQ score of other children who have or are predisposed to developing a bone mineralization disorder such as HPP as disclosed herein to, for example, determine a standard deviation of the CHAQ score.

The methods may result in an improvement in the CHAQ score (e.g., indicating the inability to perform ADLs and/or the presence of pain when performing them) in a child who has a bone mineralization disorder, such as HPP, as disclosed herein, or who is predisposed to developing the same. For example, treatment with an sALP (e.g., a polypeptide characterized by the sequence of SEQ ID NO: 1, or a variant thereof with at least 85% sequence identity thereto), such as treatment with an sALP for a period of time, up to the entire life of the child, may result in an average decrease in the CHAQ score in the range of from about 0 to about 0.5 or less (e.g., about 0, about 0.1, about 0.2, about 0.4, or about 0.5) in children with an HPP-like disease.

A reduction in the CHAQ score in a child who has or is predisposed to developing a bone mineralization disorder such as HPP may be maintained throughout the period of administration of an alkaline phosphatase or a polypeptide having alkaline phosphatase activity such as sALP (e.g., a polypeptide having the sequence of SEQ ID NO: 1, or a variant thereof with at least 85% sequence identity thereto), such as for a period of time up to for the child's lifetime. Similarly, an increase in ADL and/or a reduction in pain in a child may be maintained throughout the period of administration of an sALP (e.g., a polypeptide having the sequence of SEQ ID NO: 1, or a variant thereof with at least 85% sequence identity thereto) for a period of time up to for the child's lifetime.

The CHAQ score of a child with or predisposed to developing a bone mineralization disorder such as HPP can be used to assess the effectiveness of treatment with alkaline phosphatase or a polypeptide having alkaline phosphatase activity such as sALP (e.g., a polypeptide characterized by the sequence of SEQ ID NO: 1, or a variant thereof with at least 85% sequence identity thereto), whereby improvements in the score on a particular test indicate that the alkaline phosphatase or polypeptide having alkaline phosphatase activity is effective in treating the inability to perform activities of daily living (ADL) and pain associated with a bone mineralization disorder such as HPP. Specifically, a child who has or is susceptible to developing a bone mineralization disorder such as HPP may be asked one or more questions in one or more of eight categories (dressing/grooming, standing, eating, walking, hygiene, reaching, grip, and activities) to obtain an average CHAQ score and to assess the effectiveness of treatment with an sALP. For example, if administration of an sALP described herein to a child who has or is susceptible to developing a bone mineralization disorder such as HPP results in an average decrease in the CHAQ score to approximately 0.5 or less, and the child previously had an average CHAQ score of greater than approximately 0.8, then the sALP is effective in treating, for example, the inability to perform activities of daily living (ADL) and pain with ADL associated with a bone mineralization disorder such as HPP. Alternatively, if administration of sALP does not result in a mean decrease in CHAQ score of about 0.5 or less, then the dosage and/or frequency of administration of sALP can be adjusted to determine an effective amount of sALP for a child who has or is predisposed to developing a bone mineralization disorder such as HPP. For example, the dosage of sALP (e.g., a polypeptide characterized by the sequence of SEQ ID NO: 1, or a variant thereof with at least 85% sequence identity thereto) can be increased, such as from about 0.5-3 mg/kg/week to about 3-6 mg/kg/week, or from about 3-6 mg/kg/week to about 6-9 mg/kg/week. If CHAQ increases above a predetermined threshold, then the subject can discontinue treatment and be periodically monitored for changes in his or her CHAQ score. If the CHAQ score decreases below a predetermined threshold, then therapy can be reinitiated.

Pediatric Outcomes Data Collection Instrument (PODCI). Certain subjects who

- 24 049823 have a bone mineralization disorder such as HPP or are predisposed to developing one, can be identified for treatment with alkaline phosphatase or a polypeptide having alkaline phosphatase activity such as sALP (e.g., a polypeptide characterized by the sequence of SEQ ID NO: 1, or a variant thereof with at least 85% sequence identity thereto) using the Pediatric Outcomes Data Collection Instrument (PODCI). The PODCI can be used to assess health status in children by obtaining a PODCI score for the subject as described in Plint et al. (J. Pediatr. Orthop. 23(6): 788-790, 2003). The PODCI includes eight categories of questions that can be answered by a subject who has a bone mineralization disorder such as HPP or who is predisposed to developing one, or by a parent/guardian of the subject. Categories that may be used to determine PODCI in a subject who has or is susceptible to developing a bone mineralization disorder such as HPP include the following: 1) the Upper Limb and Physical Function Scale to measure difficulty performing activities of daily living such as self-care and learning; 2) the Ambulation and Basic Mobility Scale to measure difficulty performing standard movement and motor activities of daily living; 3) the Sports/Physical Function Scale to measure difficulty or limitations participating in more vigorous activities or sports; 4) the Pain/Comfort Scale to measure the level of pain experienced over the past week; 5) the Treatment Expectations Scale to measure long-term expectations of treatment; 6) the Happiness Scale to measure overall satisfaction with one's appearance and feelings of belonging to friends and peers; 7) a symptom satisfaction scale to measure the subject's level of acceptance of the current limitations if the condition is lifelong; and 8) a global functional ability scale, which is a general composite scale calculated from the first four scales listed above. In each category, a standardized score is determined for a subject who has a bone mineralization disorder such as HPP or who is predisposed to developing one and then converted to a score on a scale from 0 to 100, where 0 represents a significant degree of disability and 100 represents the least degree of disability.

A subject who has or is predisposed to developing a bone mineralization disorder such as HPP, with an average PODCI score (e.g., indicating an inability to perform ADLs and/or pain when performing them) of less than about 40 (e.g., about 5, about 10, about 15, about 20, about 25, about 30, about 35, or about 39), can be treated by administering alkaline phosphatase or a polypeptide having alkaline phosphatase activity, such as sALP (e.g., a polypeptide characterized by the sequence of SEQ ID NO: 1, or a variant thereof with at least 85% sequence identity thereto). For example, a subject with an average PODCI score of less than 40 can be treated by administering sALP for a period of time, up to for the life of the subject. Moreover, a subject who has a bone mineralization disorder such as HPP or who is predisposed to developing the same may be asked one or more questions on one or more of the eight scales described above (e.g., the scale for assessing the ability to transfer and basic mobility, the ability to perform sports/physical functions, and pain/discomfort) to obtain an average PODCI score, and if the average PODCI score exceeds a value of less than 40, the subject may be treated with sALP.

The methods described herein may result in an increase in the PODCI score (e.g., indicating inability to perform ADLs and/or pain when performing them) in a subject who has a bone mineralization disorder such as HPP or who is predisposed to developing one. For example, treatment with an alkaline phosphatase or a polypeptide having alkaline phosphatase activity, such as sALP (e.g., a polypeptide having the sequence of SEQ ID NO: 1, or a variant thereof with at least 85% sequence identity thereto), such as treatment with sALP over a period of time, up to and including the lifetime of the subject, can result in an average increase in PODCI score in the range of from about 40 to about 50 (e.g., about 40, about 41, about 42, about 43, about 44, about 45, about 46, about 47, about 48, about 49, or about 50).

The increase in PODCI score may be maintained throughout the period of administration of alkaline phosphatase or a polypeptide having alkaline phosphatase activity such as sALP (e.g., a polypeptide having the sequence of SEQ ID NO: 1, or a variant thereof with at least 85% sequence identity thereto), such as for a period of time, up to and including for the lifetime of the subject having the bone mineralization disorder.

- 25 049823 tissue such as HPP, or which is predisposed to developing the same. Similarly, the increase in ADL and/or the reduction in pain may be maintained throughout the period of administration of the sALP (e.g., the polypeptide characterized by the sequence under SEQ ID NO: 1, or a variant thereof with at least 85% sequence identity thereto) for some period of time, up to the entire life of the subject.

The PODCI score of a subject who has or is predisposed to developing a bone mineralization disorder such as HPP can be used to assess the effectiveness of treatment with alkaline phosphatase or a polypeptide having alkaline phosphatase activity such as sALP (e.g., a polypeptide characterized by the sequence of SEQ ID NO: 1, or a variant thereof with at least 85% sequence identity thereto), wherein improvements in the score on a particular test indicate that the alkaline phosphatase or polypeptide having alkaline phosphatase activity is effective for treating the inability to perform activities of daily living (ADL) and pain associated with a bone mineralization disorder such as HPP. Specifically, a subject who has or is predisposed to developing a bone mineralization disorder such as HPP may be asked one or more questions on one or more of eight scales (upper limb and physical function scale, ambulation and basic mobility scale, sports/physical function scale, pain/comfort scale, treatment expectation scale, happiness scale, symptom satisfaction scale, and global function scale) to obtain an average PODCI score and to assess the effectiveness of treatment by administering sALP.

For example, if administration of an sALP described herein to a subject who has a bone mineralization disorder such as HPP or who is susceptible to developing one results in an average increase in PODCI score to about 40 or greater than about 40, where the subject previously had an average PODCI score of less than about 40, then the sALP is effective in treating, for example, the inability to perform activities of daily living (ADL) and the presence of pain during their performance associated with a bone mineralization disorder such as HPP. Alternatively, if administration of an sALP described herein does not result in an average increase in PODCI score to about 40 or greater than about 40, then the dosage and frequency of administration of the sALP can be adjusted to determine an effective amount of sALP for a subject who has a bone mineralization disorder such as HPP or who is susceptible to developing one. For example, the dosage of sALP (e.g., a polypeptide having the sequence of SEQ ID NO: 1, or a variant thereof with at least 85% sequence identity thereto) can be increased, such as from about 0.5-3 mg/kg/week to about 3-6 mg/kg/week or from about 3-6 mg/kg/week to about 6-9 mg/kg/week. If the subject's PODCI score increases above a predetermined threshold, the subject can discontinue treatment and be periodically monitored for changes in his or her PODCI score. If the PODCI score decreases below a predetermined threshold, the therapy can be reinitiated.

Bayley Scales of Infant and Toddler Development, 3rd edition (BSIDIII).

Another endpoint, the Bayley Scales of Infant and Toddler Development, 3rd Edition (BSID-III), can be used to assess the health status of a subject who has or is susceptible to developing a bone mineralization disorder such as HPP from birth to obtain a BSID-III score for the subject as described in Bayley. (2006). Bayley scales of infant and toddler development: administration manual. San Antonio, TX: Harcourt Assessment. The BSID-III provides a series of developmental play tasks that can be administered to a subject to determine a raw BSID-III score. For example, categories for determining a BSID-III score for a subject who has or is predisposed to developing a bone mineralization disorder such as HPP (e.g., infants approximately three years of age or younger with HPP) may include grasping, perceptual-motor coordination, motor planning and speed, visual tracking, reaching, grasping, manipulating, functional manual skills, responses to tactile information, limb and trunk movement, holding still, dynamic movement, balance, and motor planning. The BSID-III measurements are then converted to a normalized BSID-III score that can be used to determine the subject's performance relative to healthy subjects, adjusted for age. The normalized BSID-III score for a subject who has a bone mineralization disorder such as HPP or who is predisposed to developing one (e.g., a subject with HPP) can range from 0 to 14, with scores of approximately 7 to approximately 13 considered the normal range for a healthy subject.

- 26 049823

A subject who has, or is predisposed to developing, a bone mineralization disorder such as HPP may be tested in one or more of the categories described (grasp, perceptual-motor coordination, motor planning and speed, visual tracking, reaching, grasping, manipulation, functional hand skills, tactile responses, limb and trunk movement, holding still, dynamic movement, balance, and motor planning) at an infant age (e.g., approximately 3 years of age or less than 3 years of age) to obtain a BSID-III score indicating motor delay. A subject who has, or is predisposed to developing, a bone mineralization disorder such as HPP, with a mean BSID-III score in one or more of the described categories (grasp, perceptual-motor coordination, motor planning and speed, visual tracking, reaching, grasping, object manipulation, functional hand skills, tactile responses, limb and trunk movement, holding still, dynamic movement, balance, and motor planning) of less than about 2, at the age of an infant, may be treated by administering an sALP (e.g., a polypeptide having the sequence of SEQ ID NO: 1, or a variant thereof with at least 85% sequence identity thereto). In particular, a subject who has a bone mineralization disorder such as HPP, or who is predisposed to developing one, with a mean BSID-III score of less than about 2 as an infant may be treated with sALP for a period of time, up to and including for the life of the subject.

Use of the methods may result in an improvement in the average BSID-III score (e.g., indicative of motor developmental delay) in a subject who has or is predisposed to developing a bone mineralization disorder, such as HPP. For example, treatment with an sALP (e.g., a polypeptide characterized by the sequence of SEQ ID NO: 1, or a variant thereof with at least 85% sequence identity thereto), such as treatment with an sALP over a period of time, up to the lifetime of the subject, may result in an average increase in the BSID-III score to greater than about 5 (e.g., about 5, about 6, about 7, about 8, about 9, about 10, about 11, about 12, or about 13).

The increase in the BSID-III score may be maintained for the duration of administration of the alkaline phosphatase or a polypeptide having alkaline phosphatase activity, such as sALP (e.g., a polypeptide having the sequence of SEQ ID NO: 1, or a variant thereof with at least 85% sequence identity thereto), for a period of time, up to for the lifetime of the subject who has a bone mineralization disorder, such as HPP, or who is predisposed to developing it. Similarly, the increase in the level of motor development may be maintained for the duration of administration of the alkaline phosphatase or a polypeptide having alkaline phosphatase activity, such as sALP (e.g., a polypeptide having the sequence of SEQ ID NO: 1, or a variant thereof with at least 85% sequence identity thereto), for example, for a period of time, up to for the lifetime of the subject.

The BSID-III score of a subject who has or is predisposed to developing a bone mineralization disorder such as HPP can be used to assess the effectiveness of treatment with alkaline phosphatase or a polypeptide having alkaline phosphatase activity, such as sALP (e.g., a polypeptide characterized by the sequence of SEQ ID NO: 1, or a variant thereof with at least 85% sequence identity thereto), whereby improvements in the score in a particular test indicate that the alkaline phosphatase or polypeptide having alkaline phosphatase activity is effective for treating, for example, motor delay associated with a bone mineralization disorder such as HPP. In particular, a subject who has a bone mineralization disorder such as HPP, or who is predisposed to developing one, may be tested in one or more of the described categories (grasp, perceptual-motor coordination, motor planning and speed, visual tracking, reaching, grasping, manipulation, functional hand skills, tactile responses, limb and trunk movement, holding still, dynamic movement, balance, and motor planning) at an infant age (e.g., approximately three years of age or younger with HPP) to obtain a mean BSID-III score and to assess the effectiveness of treatment with sALP.

For example, if administration of sALP to a child who has, or is predisposed to developing, a bone mineralization disorder such as HPP results in a mean increase in the BSID-III normed score to greater than approximately 5, the child

- 27 049823 previously had a mean BSID-III normed score of less than about 2 as an infant (e.g., approximately 3 years of age or less than 3 years of age), then sALP is effective in treating, for example, motor delay associated with an HPP-like disorder. Alternatively, if administration of sALP does not result in a mean increase in the BSID-III normed score to greater than about 5, the dosage and/or frequency of administration of sALP may be adjusted to determine an effective amount of sALP for a child who has, or is predisposed to developing, a bone mineralization disorder such as HPP. For example, the dosage of sALP (e.g., a polypeptide having the sequence of SEQ ID NO: 1, or a variant thereof with at least 85% sequence identity thereto) can be increased, such as from about 0.5-3 mg/kg/week to about 3-6 mg/kg/week or from about 3-6 mg/kg/week to about 6-9 mg/kg/week. If the subject's BSID-III score increases above a predetermined threshold, the subject can discontinue treatment and be periodically monitored for changes in his BSID-III score. If the BSID-III score decreases below a predetermined threshold, the therapy can be reinitiated.

Peabody Developmental Motor Scales-2nd Edition (PDMS-2). Another endpoint, the Peabody Developmental Motor Scales-2nd Edition (PDMS-2), can be used to assess the health status of a subject who has or is susceptible to developing a bone mineralization disorder such as HPP, from birth to obtain a PDMS-2 score for the subject, as described by van Hartingsveldt et al. (Occup. Ther. Int. 12(1): 1-13, 2005). The PDMS-2 includes six subtest categories to measure motor skills in a subject, such as a subject with HPP.

Specifically, for the PDMS-2, the following subtests may be used to measure performance: 1) a locomotor subtest to measure the subject's ability to move from one location to another (measurements include crawling, walking, running, hopping, and jumping forward); 2) a reflex assessment subtest to measure the subject's ability to respond automatically to external events; 3) a steady state subtest to measure the subject's ability to maintain control of the body's center of gravity and maintain balance; 4) an object manipulation subtest to measure the subject's ability to manipulate an object, such as catching, throwing, and kicking a ball; 5) a grasp subtest to measure the subject's ability to use his or her hands, such as the ability to hold an object with one hand and to perform actions that involve the controlled use of the fingers of both hands; and 6) a visual-motor coordination subtest to measure a subject's ability to use his or her visual perceptual skills to perform complex tasks that require eye-hand coordination, such as the ability to reach for and grasp an object, build with blocks, and copy structures. The PDMS-2 measurement results for one or more of these categories may be determined for a subject who has or is susceptible to developing a bone mineralization disorder such as HPP, and then converted to a PDMS-2 score, such as a standard PDMS-2 locomotor score that ranges from 0 to 13, with the range in healthy subjects (e.g., subjects without a bone mineralization disorder such as HPP) being from approximately 7 to approximately 13.

A subject who has or is predisposed to developing a bone mineralization disorder such as HPP, with a mean PDMS score (e.g., indicating motor delay) may be treated by administering an sALP (e.g., a polypeptide characterized by the sequence of SEQ ID NO: 1, or a variant thereof with at least 85% sequence identity thereto).

The methods described herein can result in an improvement in the PDMS-2 score (e.g., indicative of motor developmental delay) in a subject who has or is predisposed to developing a bone mineralization disorder, such as HPP. For example, treatment with alkaline phosphatase or a polypeptide having alkaline phosphatase activity, such as sALP (e.g., a polypeptide characterized by the sequence of SEQ ID NO: 1, or a variant thereof with at least 85% sequence identity thereto) can result in an average increase in the PDMS-2 score in the range of from about 7 to about 13 (e.g., about 7, about 8, about 9, about 10, about 11, about 12, or about 13).

The increase in PDMS-2 score may be maintained throughout the period of administration of alkaline phosphatase or a polypeptide having alkaline phosphatase activity, such as sALP (e.g., a polypeptide characterized by the sequence of SEQ ID NO: 1, or a variant thereof with at least 85% sequence identity thereto), for an extended period of time, such as for some period of time, up to and including the lifetime of the subject who has or is predisposed to developing a bone mineralization disorder such as HPP. Similarly, the increase in motor development may be maintained throughout the period of administration of sALP (e.g., a polypeptide characterized by the sequence

- 28 049823 under SEQ ID NO: 1, or a variant thereof with at least 85% sequence identity thereto), for a period of time, up to the entire life of a subject who has a bone mineralization disorder such as HPP, or who is predisposed to developing the same.

The PDMS-2 score of a subject who has or is predisposed to developing a bone mineralization disorder such as HPP can be used to assess the effectiveness of treatment with an alkaline phosphatase or a polypeptide having alkaline phosphatase activity, such as sALP (e.g., a polypeptide characterized by the sequence of SEQ ID NO: 1, or a variant thereof with at least 85% sequence identity thereto), whereby improvements in the score in a particular test indicate that the alkaline phosphatase or polypeptide having alkaline phosphatase activity is effective for treating, for example, motor delay associated with a bone mineralization disorder such as HPP. For example, a child who has a bone mineralization disorder such as HPP or who is predisposed to developing one could perform tests in one or more of the described categories (locomotor, reflex, steady state, object manipulation, grasp, and visual-motor) at approximately 5 years of age or less than 5 years of age to obtain an average PDMS-2 score and to assess the effectiveness of treatment with sALP.

For example, if administration of sALP to a child who has a bone mineralization disorder such as HPP or who is predisposed to developing one results in an average increase in the PDMS-2 standard score of approximately 7, where the child previously had an average PDMS-2 standard score of approximately 5, then sALP is effective in treating, for example, motor delay associated with an HPP-like disorder. Alternatively, if administration of sALP does not result in an average increase in the PDMS-2 standard score of approximately 7, the dosage and/or frequency of administration of sALP may be adjusted to determine an effective amount of sALP for the child. For example, the dosage of sALP (e.g., a polypeptide having the sequence of SEQ ID NO: 1, or a variant thereof with at least 85% sequence identity thereto) can be increased, such as from about 0.5-3 mg/kg/week to about 3-6 mg/kg/week or from about 3-6 mg/kg/week to about 6-9 mg/kg/week. If the subject's PDMS-2 score increases above a predetermined threshold, the subject can discontinue treatment and be periodically monitored for changes in his or her PDMS-2 score. If the PDMS-2 score decreases below a predetermined threshold, the therapy can be reinitiated.

Six-minute walk test (6MWT).

A subject having a bone mineralization disorder such as HPP can be determined for treatment with alkaline phosphatase or a polypeptide having alkaline phosphatase activity (e.g., sALP, e.g., a polypeptide having the sequence of SEQ ID NO: 1, or a variant thereof with at least 85% sequence identity thereto) using the 6MWT. In particular, the 6MWT can be used to assess the walking ability of an adult having a bone mineralization disorder such as HPP to obtain a 6MWT value for an adult. The 6MWT can be conducted indoors or outdoors using a level, straight, enclosed corridor (e.g., approximately 30 meters in length) with a hard surface. A stopwatch or other timer can be used to track the time, and a mechanical counter or other device can be used to determine the distance (e.g., in meters) that the subject having a bone mineralization disorder such as HPP walks. For example, the length of a corridor can be marked every three meters to determine the number of meters walked by a subject with a bone mineralization disorder such as HPP, with a turning point being at a distance of 30 meters and a starting line also being marked. The distance walked in six minutes by a subject with a bone mineralization disorder such as HPP can then be compared with the predicted number of meters walked by, for example, a normal subject of approximately the same age, the same gender and/or the same height, and expressed as a percentage to obtain the subject's 6MWT value. The 6MWT value of a subject with a bone mineralization disorder such as HPP can be compared with the subject's baseline 6MWT value. Additionally, the 6MWT value of a subject with a bone mineralization disorder such as HPP can be compared with the 6MWT value of a normal subject.

A subject having a bone mineralization disorder such as HPP with a mean 6MWT value that is less than about 80% of the predicted 6MWT value (e.g., relative to a normal subject of approximately the same age, same sex, and/or same height) can be treated with an alkaline phosphatase or a polypeptide having alkaline phosphatase activity (e.g., sALP, e.g., a polypeptide characterized by the sequence of SEQ ID NO: 1, or a variant thereof with at least 85% sequence identity thereto), such as by administering the alkaline phosphatase or polypeptide having

- 29 049823 alkaline phosphatase activity, 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 nine years, or at least ten years, or for the entire life of the subject; in particular at least six weeks). For example, a subject who has a bone mineralization disorder such as HPP with a mean 6MWT that is 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) can be treated with alkaline phosphatase or a polypeptide having alkaline phosphatase activity 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 nine years or at least ten years, or throughout the subject's life; in particular at least six weeks).

The methods may result in an improvement in the 6MWT value in a subject who has a bone mineralization disorder such as HPP. For example, treatment with an alkaline phosphatase or a polypeptide having an alkaline phosphatase activity (e.g., sALP, e.g., a polypeptide characterized by the sequence of SEQ ID NO: 1 or a variant thereof with at least 85% sequence identity thereto), such as treatment with an alkaline phosphatase or a polypeptide having an alkaline phosphatase activity, 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 nine years, or at least ten years, or for the entire life of the subject; in particular at least six weeks), can result in an average increase in the 6MWT value to about 80% or more of the predicted 6MWT value in the subject (e.g., about 82%, about 84%, about 86%, about 88%, about 90%, about 92%, about 94%, about 96%, about 98% or more of the predicted 6MWT value).

The increase in 6MWT in a subject having a bone mineralization disorder such as HPP may be maintained for the duration of administration of the alkaline phosphatase or a polypeptide having alkaline phosphatase activity (e.g., sALP, e.g., a polypeptide having the sequence of SEQ ID NO: 1, or a variant thereof with at least 85% sequence identity thereto), such as 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 nine years, or at least ten years, or for the life of the subject; in particular at least six weeks). For example, the 6MWT value increases to greater than about 80% of the predicted 6MWT value in a subject who has a bone mineralization disorder such as HPP and remains within ±10% of the increased 6MWT value during treatment with alkaline phosphatase or a polypeptide having alkaline phosphatase activity.

Similarly, improving the walking ability of a subject with a mineral impairment

- 30 049823 bone tissue lysis, such as HPP, can be maintained for the entire period of administration of alkaline phosphatase or a polypeptide having alkaline phosphatase activity, such as 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 nine years, or at least ten years, or for the entire life of the subject; in particular, at least six weeks). For example, a subject who has a bone mineralization disorder such as HPP demonstrates reduced dependence on a mobility assistive device such as a walker, wheelchair, brace, crutch, or orthotic during treatment with sALP.

Alternatively, if administration of an alkaline phosphatase or a polypeptide having alkaline phosphatase activity (e.g., sALP, e.g., a polypeptide having the sequence of SEQ ID NO: 1, or a variant thereof with at least 85% sequence identity thereto) does not result in an average increase in 6MWT to greater than 80% of the predicted 6MWT value (e.g., relative to a normal subject of approximately the same age, gender, and/or height), the dosage and/or frequency of administration of the alkaline phosphatase or polypeptide having alkaline phosphatase activity can be varied to determine an effective amount of alkaline phosphatase or a polypeptide having alkaline phosphatase activity for a subject who has a bone mineralization disorder such as HPP. For example, the dosage of alkaline phosphatase or a polypeptide having alkaline phosphatase activity can be increased from, for example, about 0.1-3 mg/kg/week or about 3-6 mg/kg/week to about 3-6 mg/kg/week or about 6-9 mg/kg/week. If the subject's 6MWT score increases above a predetermined threshold, the subject can be discontinued and monitored periodically for changes in his or her 6MWT score. If the 6MWT score decreases below a predetermined threshold, therapy can be reinitiated.

Hand Held Dynamometry (HHD).

The grip and muscle strength of a subject who has or is susceptible to developing a bone mineralization disorder such as HPP can be assessed using hand-held dynamometry (HHD). For example, knee flexion and extension, as well as hip flexion, extension, and hip abduction in a subject who has or is susceptible to developing a bone mineralization disorder such as HPP can be measured using a MICROFET2™ dynamometer, while the subject's grip strength can be measured using, for example, a Jamar grip dynamometer. Specifically, the examiner holds the dynamometer still and the subject applies maximal force to the dynamometer. Peak force data is collected in pounds and then converted to newtons (N). Torque values are then calculated using the limb length in Newton meters. The torque value can then be compared with the torque value of, for example, a normal subject of approximately the same age, same gender, and/or same height, and expressed as a percentage value to obtain the subject's HHD value.

A subject having a bone mineralization disorder such as HPP with a mean HHD of less than about 80% of the predicted HHD (e.g., relative to a normal subject of approximately the same age, same sex, and/or same height) can be treated with an alkaline phosphatase or a polypeptide having alkaline phosphatase activity (e.g., sALP, e.g., a polypeptide having the sequence of SEQ ID NO: 1, or a variant thereof with at least 85% sequence identity thereto), such as by administering the alkaline phosphatase or polypeptide having alkaline phosphatase activity 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 nine years, or at least ten years, or for the entire life of the subject; in particular, at least six weeks). For example, a subject who has a bone mineralization disorder such as HPP with a mean HHD of less than about

- 31 049823 approximately 80% of the predicted HHD value (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 value) can be treated with alkaline phosphatase or a polypeptide having alkaline phosphatase activity 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 nine years or at least ten years, or throughout the subject's life; in particular at least six weeks).

The methods may result in an improvement in the HHD value in a subject who has a bone mineralization disorder such as HPP. For example, treatment with an alkaline phosphatase or a polypeptide having an alkaline phosphatase activity (e.g., sALP, e.g., a polypeptide characterized by the sequence of SEQ ID NO: 1, or a variant thereof with at least 85% sequence identity thereto), such as treatment with an alkaline phosphatase or a polypeptide having an alkaline phosphatase activity, 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 nine years, or at least ten years, or for the entire life of the subject; in particular at least six weeks), can result in an average increase in the HHD value to about 80% or more of the predicted HHD value in the subject (e.g., about 83%, about 85%, about 87%, about 90%, about 93%, about 95%, about 97%, or about 100% or about 100% of the predicted HHD value).

The increase in HHD in a subject having a bone mineralization disorder such as HPP may be maintained for the duration of administration of the alkaline phosphatase or a polypeptide having alkaline phosphatase activity (e.g., sALP, e.g., a polypeptide having the sequence of SEQ ID NO: 1, or a variant thereof with at least 85% sequence identity thereto), such as 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 at least seven years, at least eight years, at least nine years, or at least ten years, or for the life of the subject; in particular at least six weeks). For example, the HHD value increases to greater than about 80% of the predicted HHD value in a subject who has a bone mineralization disorder such as HPP, and remains within ±10% of the increased HHD value during treatment with alkaline phosphatase or a polypeptide having alkaline phosphatase activity.

Alternatively, if administration of an alkaline phosphatase or a polypeptide having alkaline phosphatase activity (e.g., sALP, e.g., a polypeptide having the sequence of SEQ ID NO: 1, or a variant thereof with at least 85% sequence identity thereto) does not result in an average increase in HHD to greater than 80% of the predicted HHD (e.g., in a subject having a bone mineralization disorder such as HPP, of approximately the same age, gender, and/or height), the dosage and/or frequency of administration of the alkaline phosphatase or polypeptide having alkaline phosphatase activity can be varied to determine an effective amount of the alkaline phosphatase or polypeptide having alkaline phosphatase activity for a subject having a bone mineralization disorder such as HPP.

For example, the dosage of alkaline phosphatase or a polypeptide with alkaline phosphatase activity

- 32 049823 phosphatase, may be increased from, for example, about 0.1-3 mg/kg/week or about 3-6 mg/kg/week to about 3-6 mg/kg/week or about 6-9 mg/kg/week. If the subject's HHD score increases above a predetermined threshold, the subject may discontinue treatment and be monitored periodically for changes in his HHD score. If the HHD score decreases below a predetermined threshold, therapy may be reinitiated.

Wearable device.

The methods described herein can be performed in combination with a wearable device. The wearable device can be used to monitor biometric or biochemical readings during a treatment regimen. For example, the wearable device can be used to monitor the effectiveness of a treatment and help determine whether to discontinue administration of an sALP after effectiveness has been achieved or to restart it if effectiveness is not maintained, based on changes in biometric or biochemical readings.

A wearable device is a device comprising at least one sensor (e.g., one or more, e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10 or more sensors) and configured to be worn on the body of a subject undergoing treatment, e.g., on an arm, leg or torso of the subject. The wearable device can be worn on the wrist of the subject. The sensor can be any sensor known from the prior art and can be, for example, a position or motion sensor, such as an accelerometer. In some cases, one or more sensors may be selected from a group consisting of a motion sensor, an orientation sensor, an accelerometer, a gyroscope, a microelectromechanical systems (MEMS) sensor (e.g., a MEMS-based accelerometer), a G-sensor, a tilt sensor, a rotation sensor, a pressure sensor, a temperature sensor, a humidity sensor, an electromyography (EMG) sensor, a light detection sensor such as a LiDAR sensor, an EIM sensor, and combinations thereof.

The wearable device may comprise a chemical sensor for detecting biochemical biomarkers (e.g., PPi, PLP, PEA) in a subject.

The wearable device may be configured to establish a baseline measurement result of biometric readings selected from steps, gait, activity, ballistocardiography, heart rate, pulse rate volume, relative systolic volume, respiratory rate, rotation, balance, pressure, relative humidity, body composition, temperature, pulse transit time, blood pressure, pulse saturation and blood pressure. The sensors and readings described in this document can be used to monitor one or more biometric readings selected from the number of steps per day, average heart rate, highest heart rate, a numeric value of the amount of physical activity, an average amount of time spent at different activity levels, a ratio of different activity levels, a duration of different activity levels and a duration of total sleep time. Different activity levels can be selected from sedentary, light, moderate and vigorous. Readings can be measured for any predetermined amount of time during the treatment regimen, such as 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 2 weeks, 3 weeks, 4 weeks, 2 months, 3 months, 4 months, 5 months, 6 months, 7 months, 8 months, 9 months, 10 months, 11 months, 12 months or longer.

The wearable device may be part of a kit that includes a wearable device and an sALP. The wearable device may include an accelerometer. The wearable device may be configured to be worn on the subject's wrist. The kit may further include a peripheral device (e.g., a smartphone). The peripheral device may be configured to run a software application (e.g., a mobile application).

The wearable device can be used in a method for monitoring a treatment regimen by means of sALP in a subject by detecting a change in at least one physical metric indicator based on biometric readings obtained using the wearable device. The biometric readings can be selected from the number of steps per day, the average heart rate, the highest heart rate, a numeric value of the amount of physical activity, an average amount of time spent at different activity levels, a ratio of different activity levels, a duration of different activity levels, and a duration of total sleep time. The different activity levels can be selected from sedentary, light, moderate, and vigorous. The physical metric(s) indicator(s) can be monitored using the wearable device for a period of at least two weeks or more (e.g., during a treatment phase, or during a non-treatment phase, or during both phases).

In combination with the methods described herein, a subject may use a wearable device to monitor one or more of the biometric readings described herein (e.g., steps per day, average heart rate, physical activity numeric value) and may continue to wear the device for a predetermined period of time (e.g., two weeks or more). The subject may then begin a treatment regimen with

- 33 049823 using sALP (such as TNALP, e.g., a fusion polypeptide comprising sALP, having the sequence of SEQ ID NO: 1, or a variant polypeptide described herein, such as a variant having at least 95% sequence identity to the sequence of SEQ ID NO: 1, e.g., asfotase alfa) for the treatment of a bone mineralization disorder such as HPP. sALP can be administered during a first phase of a treatment regimen, and biometric readings can be monitored daily over time. If the biometric readings indicate an improvement in the subject's condition, such as an increase above a predetermined threshold (e.g., a certain number of steps per day or average heart rate), it can be determined that the treatment is or has been effective in improving or restoring normal physical condition in the subject. The subject can then enter a no-treatment phase of the treatment regimen in which sALP is not administered. The subject may continue to wear the wearable device to monitor the biometric readings. If the biometric readings indicate a deterioration in the subject's condition, such as a decrease in the value of the measured metric (e.g., a decrease in the value below a predetermined threshold), then it may be determined that the subject should re-enter the treatment phase of the treatment regimen, in which the sALP-based therapy is reintroduced.

The wearable device may be implemented as a system that further includes a peripheral electronic device, such as a smartphone, or a cloud database. Since the data is continuously monitored and measured by the wearable device, the data collected by the device may be transmitted to the peripheral device and stored on the peripheral device or in a cloud storage environment. The peripheral device may be configured to run a software application (e.g., a smartphone application) that provides the subject with navigation information for the purpose of tracking changes in the subject's physiological readings during the course of treatment (e.g., improvement over time). The data may also be transmitted to a third party, such as a physician administering the sALP-based therapy, for the purpose of monitoring the subject's treatment progress.

Treatment of bone fracture.

In some embodiments of the method, sALP (such as TNALP, e.g., a fusion polypeptide comprising sALP having the sequence of SEQ ID NO: 1, or a polypeptide variant described herein, such as a variant having at least 95% sequence identity to the sequence of SEQ ID NO: 1, e.g., asfotase alfa) is administered to the patient in an amount that is therapeutically effective for treating the bone manifestations of a bone mineralization disorder, e.g., HPP. For example, a subject may have, for example, spinal curvature, kyphosis, osteoporosis, weakening of the spinal bones, thinning of the ribs, rotation of the vertebrae, wedging of the vertebrae, erosion of the vertebrae, rotational or angular deformity of the spine, dural ectasia, incomplete mineralization of bone tissue, elevated levels of phosphoethanolamine (PEA) in the blood and/or urine, hypomineralization, hypercalciuria, short stature, skeletal deformity, waddling gait, bone pain, bone fracture, weakness, and calcium pyrophosphate dihydrate crystal deposition.

In some embodiments of the method, sALP (such as TNALP, e.g., a fusion polypeptide comprising sALP of SEQ ID NO: 1 or a variant polypeptide having at least 80%, 85%, 90%, 95%, 97%, 99%, or 100% sequence identity to SEQ ID NO: 1, e.g., asfotase alfa) is administered to the patient in an amount that is therapeutically effective to treat at least one symptom of dystrophic scoliosis, e.g., neurofibromatosis (NF1)-associated dystrophic scoliosis, e.g., shorter and sharper curves than those observed in non-dystrophic scoliosis, bone abnormalities including rib thinning, weakening of vertebral bones, significant rotation of vertebrae, wedging of vertebrae, and erosion of vertebrae by cerebrospinal fluid.

In one embodiment of the method, sALP (such as TNALP, e.g., a fusion polypeptide comprising sALP, having the sequence of SEQ ID NO: 1, or a variant polypeptide having at least 80%, 85%, 90%, 95%, 97%, 99%, or 100% sequence identity with the sequence of SEQ ID NO: 1, e.g., asfotase alfa) is administered to a patient in an amount that is therapeutically effective to induce bone regeneration. Examples of inducing bone regeneration include treating a bone fracture and arthrodesis, the surgical fusion of two joints. For example, the method is useful for providing a spinal fusion. Spinal fusion surgery can be used to eliminate bone-on-bone friction and/or restore nerve compression that causes pain and other symptoms. Bone fusion surgery can also be used on areas such as fingers, ankles and feet.

In some embodiments of the method, an sALP (such as TNALP, e.g., a fusion polypeptide comprising sALP, with the sequence of SEQ ID NO: 1, or a polypeptide variant described herein, such as a variant having at least 95% sequence identity to the sequence of SEQ ID NO: 1, e.g., asfotase alpha) is administered

- 34 049823 to a patient who has undergone orthopedic surgery. The surgery may involve the use of at least one implant or hardware placed during the surgery. The patient may have a disease or disorder or may have one or more bone-related conditions such as spinal curvature, kyphosis, osteoporosis, weakened spine bones, thinning ribs, vertebral rotation, vertebral wedging, vertebral erosion, rotational or angular deformity of the spine, dural ectasia, incomplete bone mineralization, elevated phosphoethanolamine (PEA) levels in the blood and/or urine, hypomineralization, hypercalciuria, short stature, skeletal deformity, waddling gait, bone pain, bone fracture, weakness, and calcium pyrophosphate dihydrate crystal deposition. In some embodiments, a patient has had a metal implant placed in a bone that is not functioning. The patient may have had one or more orthopedic surgeries (e.g., revision surgeries) in which the implant fixation failed. For example, the patient may have or may need a craniofacial implant, a dental implant, a spinal implant, a replacement joint part for the hip, knee, shoulder, spine, elbow, or wrist joints, or a bone fixation material such as a nail, screw, pin, nut, rod, and plate. A patient may also require revision surgery if there has been a failure of a craniofacial implant, dental implant, spinal implant, hip, knee, shoulder, spine, elbow or wrist joint replacement, or bone fixation material such as a nail, screw, pin, nut, rod and plate.

Orthopedic surgeries that may involve the use of an implant or hardware include, for example, surgery of the hand or upper limb, shoulder, elbow, joint (e.g., total joint reconstruction, e.g., arthroplasty), foot, ankle, spine, surgery related to musculoskeletal cancer, surgery related to sports injuries, surgery for trauma, etc. Other common orthopedic surgeries that may involve the use of hardware or an implant include, for example, arthroscopy of the knee, shoulder, hip, ankle, elbow, or wrist, fracture and meniscectomy, arthroscopy and decompression of the shoulder, carpal tunnel release, arthroscopy and chondroplasty of the knee, removal of abutment implant, arthroscopy of the knee and reconstruction of the anterior cruciate ligament, replacement knee, hip fracture repair, atypical hip fracture repair, trochanteric fracture repair, skin/muscle/bone/fracture debridement, single or both meniscus repair after knee arthroscopy, hip replacement, shoulder arthroscopy/distal clavicle resection, rotator cuff tendon repair, radius or ulna fracture repair, laminectomy, ankle fracture repair (bicalleolar type), shoulder arthroscopy and debridement, lumbar spine fusion, distal radius fracture repair, low back disc surgery, finger tendon sheath release, ankle (fibula) fracture repair, femoral shaft fracture repair, and trochanteric fracture repair. A patient who has undergone any of the above orthopaedic procedures may be treated with sALP in accordance with the methods described herein to improve bone regeneration or to improve implant or hardware retention.

sALP can be used to restore bone tissue after injury or to eliminate a bone tissue defect that may result from surgery or disease. Damaged or fragmented bone tissue can be reconstructed using an implant or a metal structure and supplemented by the introduction of sALP before, during or after surgery to eliminate the defect. The implant or metal structure in combination with the introduction of sALP can additionally provide the necessary mechanical support for the remaining parts of bone fragments at the site of injury and at the same time allow blood and bone-forming cells from adjacent tissues to penetrate into the implant to improve regeneration. For example, the methods described in this document can be used to eliminate bone defects in the cranial vault after neurosurgery and injury, in reconstructions of the bone floor of the orbit and jaw bones. The methods described in this document can also be used in orthopedics and in spine surgery, as well as in fixation of fragmented parts of bone and cosmetic surgeries. In the presence of tubular bones that are weakened due to disease, or in the case of loss of parts of the cortical bone substance, an implant can be used to strengthen the tubular bones and close the holes where the cortical bone substance has been lost.

sALP can be used to repair an atypical fracture, such as an atypical femur fracture (AFF), which is a transverse fracture of the femoral shaft. AFF may be due to the use (e.g., long-term use) of bisphosphonates, such as for the treatment of

- 35 049823 osteoporosis. sALP can be used to treat AFF in a subject (e.g., an adult subject) with HPP (e.g., childhood-onset HPP) or without HPP. In an alternative embodiment, sALP is used to treat NF1 and NF1-associated skeletal manifestations/complications such as tibial dysplasia, pseudoarthrosis, scalloped vertebrae, scoliosis, and others. Treatment of NF1, NF1-associated skeletal manifestations/complications, and/or other poorly healing fractures can be performed separately from or in addition to treatment of AFF. sALP (such as TNALP, e.g., a fusion polypeptide comprising sALP having the sequence of SEQ ID NO: 1, or a variant polypeptide described herein, such as a variant having at least 95% sequence identity to the sequence of SEQ ID NO: 1, e.g., asfotase alfa) can be administered to a subject, e.g., at a dosage of about 6 mg/kg/week. Administration of sALP can result in a transient increase in one or more metabolites or biomarkers (e.g., bone turnover markers), such as parathyroid hormone, procollagen type I N-propeptide (PINP), osteocalcin, and bone alkaline phosphatase (BAP). The transient increase can persist for at least, e.g., 1 week, 2 weeks, weeks, 1 month, 2 months, 3 months, 4 months, 5 months, or 6 months or more. After a temporary increase, the biomarker level may decrease back to baseline. This may indicate bone turnover and facilitation of bone remodeling in the subject. The fracture may be monitored for regeneration over a period of time (e.g., 1 week, 2 weeks, 3 weeks, 1 month, 2 months, 3 months, 4 months, 5 months, or 6 months or longer) until sufficient regeneration is observed to terminate treatment. Once the fracture has healed, a lower dosage may be administered (e.g., 3 mg/kg/week) or dosing may be administered less frequently (e.g., bimonthly or monthly, such as at a dosage of 0.5 mg/kg to 50 mg/kg). The subject may undergo physical therapy after treatment to resume normal activity levels. In some cases, the subject may continue therapy for an extended period of time (e.g., lifelong) to minimize recurrence of the fracture or adverse effects associated with the antibody-drug.

The methods described herein may also be used in relation to bones of reduced quality (e.g., affected by osteoporosis) or in revision surgeries (e.g., they may be used to replace previously installed implants). sALP (such as TNALP, e.g., a fusion polypeptide comprising sALP, having the sequence of SEQ ID NO: 1, or a variant polypeptide described herein, such as a variant having at least 95% sequence identity with the sequence of SEQ ID NO: 1, e.g., asfotase alpha) may be used, for example, in osteosynthesis for internal stabilization and/or fusion of bones, such as fractured bones (broken bones), either in combination with mechanical stabilizing devices, such as metal plates, pins, rods, or wires, or alone.

The methods described herein may also be used to treat a bone defect in a patient with a fracture that requires compression. In particular, sALP may be administered with an implant that may be used to provide compression fixation to the patient.

Bones that can be treated according to the methods described herein include, for example, a subarticular fracture, a defect of the spine or vertebra, or a defect of the radius, ulna, fibula, clavicle, humerus, pelvis, femur, patella, tibia, talus, calcaneus, navicular, cuneiform, metatarsal, metacarpal, phalanges, scapula, malleolus, maxilla or mandible. The implant or hardware can be placed near the bone defect (e.g., by placing the implant so that it contacts the intraosseous space of the bone). sALP (such as TNALP, for example, a fusion polypeptide comprising sALP, having the sequence of SEQ ID NO: 1, or a variant of the polypeptide described herein, such as a variant having at least 95% sequence identity with the sequence of SEQ ID NO: 1, for example, asfotase alpha) can be administered at any time before, during or after the introduction of the implant or hardware. sALP can be applied as a coating to the implant or hardware.

The methods of treatment of the present invention also include maxillomandibular or craniofacial fixation, temporary fixation for restoration of a bone defect in a staged reconstruction, joint or shoulder fixation, patellar fixation, or spinal fixation. For example, in spinal surgeries, the implant can be placed in a pedicle, used to anchor an intervertebral device, used to anchor plates for fusion and spacer replacement, used in osteoporotic vertebrae, or placed near the spinous processes of adjacent vertebrae. The method can also include placement of a rod, pin, nail, mesh, or bone plate near a bone defect. sALP can be administered at any time before, during, or after surgery. sALP can be applied in

- 36 049823 as a coating for any implant or any metal structure.

Specific bone defects that can be treated using the methods described herein include, for example, any area of bone deficiency such as a void, gap, notch, or other discontinuity in bone tissue. The bone defect can be caused by, for example, disease or injury. The implants or hardware described herein can be used, for example, in the correction of periodontal defects, in craniofacial or maxillofacial surgery or reconstruction, in cosmetic surgery, in hand surgery, in joint reconstruction, in fracture repair, in orthopedic surgical procedures, and in spinal fusion. The implants can also be used, for example, in osteosynthesis for internal stabilization and/or fusion of bones, such as fractured bones (broken bones), or in combination with other mechanical devices such as washers, metal plates, pins, rods, or wires, or alone. For example, the implants can be used with a washer to provide compression fixation of bone tissue defects and bone fractures. In particular, these methods are applicable to the treatment of defects or fractures of large bones. Non-limiting examples of bone fractures include, for example, stable fractures, transverse fractures, oblique fractures, spiral fractures, comminuted fractures, and open and displaced fractures. Illustrative large bones that may require fracture fixation include, for example, the femur, tibia, fibula, humerus, ulna, radius, ribs, innominate bone (pelvic bone), and sternum. For each of these surgeries, sALP may be administered at any time before, during, or after surgery.

Surgical intervention.

The administration of sALP (such as TNALP, e.g., a fusion polypeptide comprising sALP, having the sequence of SEQ ID NO: 1, or a variant polypeptide having at least 95% sequence identity with the sequence of SEQ ID NO: 1, e.g., asfotase alpha) in any of the disclosed methods may occur in combination with a physical intervention, such as a surgical intervention. Surgery may be used in the case of arthrodesis, bone removal, placement of bone graft material, or reduction of a broken bone. Surgery may involve the use of instruments (e.g., implantation of hardware, such as screws, rods, plates, and metal frames) to facilitate the healing of bone tissue. Surgery may include traditional open surgery, such as open neck surgery or open back surgery, minimally invasive surgery, or a combination thereof.

Alkaline phosphatase.

Asfotase alfa is a human TNALP fusion polypeptide (hTNALP; SEQ ID NO: 1) fusion polypeptide. In particular, asfotase alfa (SEQ ID NO: 1) can be effectively used to treat at least one symptom of a bone mineralization disorder, such as HPP.

Given the results described herein, treatment methods are not limited to the administration of a specific alkaline phosphatase (ALP) or a nucleic acid sequence encoding ALP. Alkaline phosphatases comprise a group of enzymes that catalyze the cleavage of a phosphate moiety (e.g., hydrolysis of pyrophosphate, PPi). There are four known mammalian alkaline phosphatase (ALP) isozymes: tissue-nonspecific alkaline phosphatase (TNALP; further described below), placental alkaline phosphatase (PLALP) (e.g., accession numbers P05187, NP_112603, and NP_001623), fetal alkaline phosphatase (GALP) (e.g., accession number P10696), and intestinal alkaline phosphatase (IALP) (e.g., accession numbers P09923 and NP_001622). In addition to the exemplary ALPs discussed herein, any polypeptide having an identical or similar catalytic site structure and/or enzymatic activity of an ALP (e.g., as an sALP or a fusion polypeptide comprising an sALP, as defined herein) can be used in the method disclosed herein. Conjugates for delivery to bone tissue comprising sALP are further described in PCT Publication Nos. WO 2005/103263 and WO 2008/138131.

TNALPs that can be used in accordance with the methods described herein include, for example, human TNALPs (Accession Numbers NP_000469, AAI10910, AAH90861, AAH66116, AAH21289, and AAI26166), rhesus monkey TNALPs (Accession Number XP_01109717), rat TNALPs (Accession Number NP_037191), dog TNALPs (Accession Number AAF64516), pig TNALPs (Accession Number AAN64273), mouse (Accession Number NP_031457), cow TNALPs (Accession Numbers NP_789828, NP_776412, AAM 8209, and AAC33858), and cat TNALPs (Accession Number NP_001036028). In particular, the TNALP can be a recombinant human TNALP (e.g., SEQ ID NO: 1, asfotase alpha; e.g., U.S. Patent Nos. 7,763,712 and 7,960,529) used in the method disclosed herein. The TNALP can also be a sequence that exhibits at least about 95% identity to a polypeptide sequence or a sequence

- 37,049,823 nucleic acids of the above-mentioned TNALPs.

Soluble alkaline phosphatases.

ALPs that can be used in the methods described herein include soluble (e.g., extracellular or non-membrane-bound) forms of any of the alkaline phosphatases described herein. The sALP can be, for example, a soluble form of human tissue-non-specific alkaline phosphatase (human TNALP (hTNALP)). The methods are not limited to a particular sALP and can include any sALP that is physiologically active, such as with respect to phosphoethanolamine (PEA), inorganic pyrophosphate (PPi), and pyridoxal 5'phosphate (PLP). In particular, sALP has the catalytic ability to improve the mineralization of skeletal structure in bone. The methods further include nucleic acids encoding the sALPs described herein that can be used in a method described herein.

TNALP is a membrane-bound protein anchored by a glycolipid functional unit at the C-terminus (Swiss-Prot, P05186). This glycolipid anchor (GPI) is added post-translationally after removal of the hydrophobic C-terminus, which serves both as a transient membrane anchor and as a signal for GPI addition. While the GPI anchor is located in the cell membrane, the remaining parts of TNALP are extracellular. In particular, TNALP (e.g., human TNALP (hTNALP)) can be engineered to replace the first amino acid of the hydrophobic C-terminal sequence (alanine) with a stop codon, thereby producing an engineered hTNALP that contains all the amino acid residues of the native, membrane-anchored form of TNALP and lacks the membrane GPI anchor. One skilled in the art will appreciate that the position of the membrane GPI anchor will vary in different ALPs 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 sTNALP may include, for example, amino acids 1-502 (18-502 during secretion), amino acids 1-501 (18-501 during secretion), amino acids 1-504 (18-504 during secretion), amino acids 1-505 (18-505 during secretion), or amino acids 1-502. Thus, the C-terminus of natural ALP can be shortened by removing certain amino acids without affecting ALP activity.

In addition to the C-terminal GPI anchor, TNALP also contains an N-terminal signal peptide sequence. The N-terminal signal peptide is present on the synthesized protein during its synthesis, but is cleaved from TNALP after translocation to the ER. sALPs include both secreted (i.e., lacking an N-terminal signal) and nonsecreted (i.e., having an N-terminal signal) forms. It will be appreciated by one skilled in the art that the position of the N-terminal signal peptide will vary in different alkaline phosphatases and may comprise, 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 available on the website at www.cbs.dtu.dk/services/SignalP/.

The methods can also be performed using sALP consensus sequences derived from the extracellular domain of ALP isozymes (e.g., TNALP, PALP, GCALP, IALP, etc.). Thus, by analogy with the sTNALP discussed above, the present invention also provides other soluble human ALP isozymes, i.e., without a signal peptide, preferably comprising an extracellular domain of ALP. sALPs also include polypeptide sequences that satisfy the requirements of a consensus sequence derived from the extracellular domain of ALP of human ALP isozymes and mammalian TNALP orthologs (human, mouse, rat, cow, cat, and dog), or a consensus sequence derived from the extracellular domain of ALP of mammalian TNALP orthologs (human, mouse, rat, cow, cat, and dog) only. sALPs also include those that satisfy the requirements of similar consensus sequences derived from different combinations of these TNALP orthologs or human ALP isozymes. Such consensus sequences are provided, for example, in WO 2008/138131.

sALPs of the present method may comprise not only the sequence of the wild-type sALPs described above, but also 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 to these alkaline phosphatases (e.g., a fusion polypeptide comprising sALP with the sequence of SEQ ID NO: 1 or a variant polypeptide having at least 95% sequence identity to the sequence of SEQ ID NO: 1, such as asfotase alpha). Examples of mutations that can be introduced into the ALP sequence are described in U.S. Patent Publication No. 2013/0323244. sALP can optionally be glycosylated at any suitable one or more amino acid residues. Additionally, sALP can be characterized by 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%

- 38 049 823 or more) sequence identity to any of the sALPs described herein (such as TNALP, e.g., a fusion polypeptide comprising sALP with the sequence of SEQ ID NO: 1 or a polypeptide variant having at least 95% sequence identity to the sequence of SEQ ID NO: 1, e.g., asfotase alpha). The sALP may comprise 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more additions, deletions or substitutions relative to any of the sALPs described herein (such as TNALP, e.g., a fusion polypeptide comprising sALP with the sequence of SEQ ID NO: 1 or a polypeptide variant having at least 95% sequence identity to the sequence of SEQ ID NO: 1, e.g., asfotase alpha).

Fusion polypeptides containing sALP.

Any of the sALPs (such as TNALP, e.g., a fusion polypeptide comprising sALP having the sequence of SEQ ID NO: 1 or a polypeptide variant having at least 95% sequence identity to the sequence of SEQ ID NO: 1, e.g., asfotase alpha), linkers, spacers (e.g., Fc regions), and bone targeting functional units described herein can be combined in a fusion polypeptide that includes the structures Z-sALP-Y-spacer-X-Wn-V, Z-Wn-X-spacer-Y-sALP-V, Z-sALP-Y-Wn-Xspacer-V, and Z-Wn-X-sALP-Y-spacer-V. In particular, the structure of the fusion polypeptide comprising sALP may be Z-sALP-Y-spacer-X-Wn-V or Z-Wn-X-spacer-Y-sALP-V. The sALP in the sALP fusion polypeptide may be full-length ALP or functional fragments of ALP, such as the soluble extracellular domain of ALP as described herein (e.g., TNALP, PALP, GCALP, and IALP).

Any of X, Y, Z and V and/or the spacer may be absent, or a linker region comprising an amino acid sequence of at least one amino acid may be absent. For example, X, Y, Z and V may be a dipeptide sequence (e.g., leucine-lysine or aspartic acid-isoleucine), such as a two-residue linker at position Y (e.g., leucine-lysine) or a two-residue linker at position X (e.g., aspartic acid-isoleucine). For example, fusion polypeptides comprising sALP may have the structure of hTNALP-Fc-D10 (e.g., a fusion polypeptide comprising sALP comprising the amino acid sequence of SEQ ID NO: 1 or a polypeptide variant having at least 95% sequence identity to the sequence of SEQ ID NO: 1, e.g., asfotase alpha).

The linker region can have any sequence and length that allows sALP to remain biologically active, such as being free of steric hindrance. Illustrative linker lengths are from 1 to 200 amino acid residues, such as 1-5, 6-10, 11-15, 16-20, 21-25, 26-30, 31-35, 36-40, 41-45, 46-50, 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 include or consist of flexible portions, such as regions without significant fixed secondary or tertiary structure.

Exemplary flexible linkers are glycine-rich linkers, such as those comprising at least 50%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or even 100% glycine residues. Linkers may also comprise, 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. Additionally, a linker described herein may include any other sequence or moiety attached by a covalent or non-covalent bond. A linker may also be absent if the spacer (e.g., Fc region) and sALP are fused together directly, without intervening residues.

Useful spacers include, but are not limited to, polypeptides comprising an Fc region. For example, sALP can be a fusion polypeptide comprising an immunoglobulin Fc region at the N-terminal or C-terminal domain. The immunoglobulin molecule has a structure well known in the art. It comprises two light chains (~23 kDa each) and two heavy chains (~50-70 kDa each) linked by interchain disulfide bonds. Immunoglobulins are readily cleaved proteolytically (e.g., by papain cleavage) into Fab (containing the light chain and the VH and CH1 domains of the heavy chain) and Fc (containing the CH2 and CH3 domains of the heavy chain along with adjacent sequences). Suitable Fc fragments described herein include an Fc fragment of any immunoglobulin molecule, including IgG, IgM, IgA, IgD, or IgE, as well as their various subclasses (e.g., IgG-1, IgG-2, IgG-3, IgG-4, IgA-1, IgA-2) from any mammal (e.g., a human). For example, the Fc fragment is human IgG-1. Fc fragments can comprise, for example, the CH2 and CH3 domains of the heavy chain and any portion of the hinge region. The Fc region can optionally be glycosylated at any suitable one or more amino acid residues that are known to those skilled in the art. Fusion polypeptides comprising sALP described herein, such as those described in WO 2005/007809, may include engineered, e.g., non-naturally occurring, Fc regions. The Fc region described herein may be characterized by the presence of 1, 2, 3, 4, 5,

- 39 049823

6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 50 or more additions, deletions or substitutions relative to any of the Fc regions described herein.

Wn may be a functional unit that provides bone targeting, such as one characterized by a series of consecutive aspartate (D) or glutamate (E) residues, wherein n=1-50, such as n=3-30, such as 5-15, such as 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 functional unit, if present, can be located anywhere in the fusion polypeptide, such as at or near the N-terminus or C-terminus and/or in the linker region. For example, the bone targeting functional unit can be present at the C-terminus of a fusion polypeptide comprising sALP. sALP and fusion polypeptides can also be lacking a bone targeting functional unit.

Additional amino acid residues may be introduced into the polypeptide in accordance with the cloning strategy used to produce the fusion polypeptides. For example, additional amino acid residues do not provide an additional GPI-anchoring signal, maintaining the polypeptide in a soluble form. Moreover, any such additional amino acid residues, when incorporated into the polypeptide by these methods, do not provide a cleavage site for host cell endoproteases. The likelihood that the engineered sequence will be cleaved by host cell endoproteases can be predicted as described, for example, by Ikezawa (Biol. Pharm. Bull. 25:409-417, 2002).

Fusion polypeptides comprising sALP (such as TNALP, e.g., a fusion polypeptide comprising sALP of SEQ ID NO: 1 or a polypeptide variant having at least 95% sequence identity to SEQ ID NO: 1, e.g., asfotase alpha) can be associated into dimers or tetramers. For example, two sALP-Fc monomers can be covalently linked by two disulfide bonds located in the hinge regions of the Fc fragments. Additionally, a fusion polypeptide comprising sALP (e.g., sALP or a fusion polypeptide comprising sALP) can be glycosylated or PEGylated.

Obtaining nucleic acids and polypeptides.

Nucleic acids encoding sALP (such as TNALP, e.g., a fusion polypeptide comprising sALP having the sequence of SEQ ID NO: 1 or a variant polypeptide having at least 95% sequence identity with the sequence of SEQ ID NO: 1, e.g., asfotase alpha) can be prepared by any method known in the art. Typically, a nucleic acid encoding the desired fusion polypeptide is prepared using molecular cloning techniques and is typically placed in a vector, such as a plasmid or virus. The vector is used to transform a host cell suitable for expression of the fusion polypeptide with the nucleic acid. 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, although mammalian cells are preferred because they are capable of providing appropriate post-translational modifications. The 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 cells known in the art. For example, the host cell is a Chinese hamster ovary (CHO) cell (e.g., a CHO-DG44 cell).

sALPs (such as TNALP, for example a fusion polypeptide comprising sALP having the sequence of SEQ ID NO: 1 or a variant polypeptide having at least 95% sequence identity to the sequence of SEQ ID NO: 1, for example asfotase alpha) can be produced under any conditions suitable for efficient expression of the polypeptide, which is sALP, in the host cell. Such conditions include an appropriate choice of medium prepared using 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; standard commercially available media such as alpha-MEM, DMEM, Ham's F12 and IMDM supplemented with 2-4 mM L-glutamine and 5% fetal bovine serum; standard commercially available media without animal protein such as Hyclone™ SFM4CHO, Sigma CHO DHFR-, Cambrex POWER™ CHO CD supplemented with 2-4 mM L-glutamine. These media should be prepared without thymidine, hypoxanthine and L-glycine to maintain selective pressure, ensuring stable expression of the protein product.

Treating a patient with sALP.

The patient to whom the sALP is administered may be of any age. In some specific embodiments, the subject may be an adult over about 18 years of age. The patient may suffer from at least one symptom of a bone mineralization disorder, such as HPP. The patient may be in need of induced bone regeneration and/or mineralization. For example, the patient may suffer from one or more bone fractures or pseudofractures. The patient

- 40 049823 may be characterized by poor muscle tone or hypotonia and may have difficulty performing daily activities such as sitting, standing, or walking. The patient may represent a patient who has not been diagnosed with HPP and/or has never received an sALP-based therapy.

Administration and dosage.

In any of the disclosed methods, any amount of a pharmaceutical composition (e.g., comprising an sALP or a fusion polypeptide comprising an sALP, such as TNALP, e.g., a polypeptide that is an sALP, having the sequence of SEQ ID NO: 1, or a polypeptide variant described herein, such as a variant having at least 80%, 85%, 90%, 95%, 97%, 99%, or 100% sequence identity to the sequence of SEQ ID NO: 1, e.g., asfotase alfa) can be administered to a patient. Dosages will depend on many factors, including the route of administration and the age of the patient. Typically, the amount of a composition (e.g., sALP or a fusion polypeptide comprising sALP, such as TNALP, e.g., a polypeptide comprising sALP, having the sequence of SEQ ID NO: 1 or a variant polypeptide having at least 95% sequence identity to the sequence of SEQ ID NO: 1, e.g., asfotase alfa) contained in a single dose will be an amount effective to treat a patient as described herein without inducing significant toxicity.

For example, in any of the disclosed methods, an sALP (such as TNALP, e.g., a polypeptide that is an sALP having the sequence of SEQ ID NO: 1 or a polypeptide variant having at least 95% sequence identity to the sequence of SEQ ID NO: 1, e.g., asfotase alfa) described herein can be administered to a patient in individual doses ranging from, e.g., 0.01 mg/kg to 500 mg/kg (e.g., 0.05 mg/kg to 500 mg/kg, 0.1 mg/kg to 20 mg/kg, 5 mg/kg to 500 mg/kg, 0.1 mg/kg to 100 mg/kg, 10 mg/kg to 100 mg/kg, 0.1 mg/kg to 50 mg/kg, 0.5 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, such as about 2.1 mg/kg, 2.2 mg/kg, 2.3 mg/kg, 2.4 mg/kg, 2.5 mg/kg, 2.6 mg/kg, 2.7 mg/kg, 2.8 mg/kg, 2.9 mg/kg, 3.0 mg/kg, 3.1 mg/kg, 3.2 mg/kg, 3.3 mg/kg, 3.4 mg/kg, 3.5 mg/kg, 3.6 mg/kg, 3.7 mg/kg, 3.8 mg/kg, 3.9 mg/kg, 4.0 mg/kg) or 1 μg/kg to 1000 μg/kg (e.g. about 5 µg/kg to 1000 µg/kg, from 1 µg/kg to 750 µg/kg, from 5 µg/kg to 750 µg/kg, from 10 µg/kg to 750 µg/kg, from 1 µg/kg to 500 µg/kg, from 5 µg/kg to 500 µg/kg, from 10 µg/kg to 500 µg/kg, from 1 µg/kg to 100 µg/kg, from 5 µg/kg to 100 µg/kg, from 10 µg/kg to 100 µg/kg, from 1 µg/kg to 50 µg/kg, from 5 µg/kg to 50 µg/kg or from 10 µg/kg to 50 µg/kg). Any of the above doses may be administered once daily. Any of the above doses may be administered once weekly.

Illustrative doses of sALP include, for example, 0.01, 0.05, 0.1, 0.5, 1, 2, 2.5, 5, 10, 20, 25, 50, 100, 125, 150, 200, 250, or 500 mg/kg; or 1, 2, 2.5, 5, 10, 20, 25, 50, 100, 125, 150, 200, 250, 500, 750, 900, or 1000 mcg/kg. For all doses or ranges specified herein, the term approximately may be used to modify these doses by ±10% of the stated values or range ends. In particular, compositions (e.g., comprising an sALP (such as TNALP, e.g., a polypeptide comprising an sALP, having the sequence of SEQ ID NO: 1 or a variant polypeptide having at least 95% sequence identity to the sequence of SEQ ID NO: 1, e.g., asfotase alfa)) according to the present invention can be administered to a patient in 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, sALP-based compositions (such as TNALP, e.g., a polypeptide that is sALP, having the sequence of SEQ ID NO: 1, or a variant polypeptide having at least 95% sequence identity to the sequence of SEQ ID NO: 1, e.g., asfotase alfa) can be administered to a patient once a week at a dosage ranging from, e.g., about 0.5 mg/kg/week to about 140 mg/kg/week, e.g., about 0.8 mg/kg/week to about 50 mg/kg/week, or about 1 mg/kg/week to about 10 mg/kg/week (e.g., about 3, or about 6, or about 9 mg/kg/week). In particular, sALP (such as TNALP, e.g., a polypeptide that is sALP, having the sequence of SEQ ID NO: 1 or a variant polypeptide having at least 95% sequence identity to the sequence of SEQ ID NO: 1, e.g., asfotase alfa) can be administered at a dosage of 2 mg/kg three times a week (total dose of about 6 mg/kg/week), 1 mg/kg six times a week (total dose of about 6 mg/kg/week), 3 mg/kg three times a week (total dose of 9 mg/kg/week), 0.5 mg/kg three times a week (total dose of 1.5 mg/kg/week), or 9.3 mg/kg three times a week (total dose of about 28 mg/kg/week). The dosage will be adapted by the treating physician in accordance with generally accepted factors, such as, for example, the degree of impairment of bone mineralization, such as the need for bone regeneration, and various patient parameters.

Dosages of pharmaceutical compositions comprising sALP (such as TNALP, for example, a polypeptide representing sALP, with the sequence under SEQ ID NO: 1 or a variant of the polypeptide

- 41 049823 yes, characterized by at least 95% sequence identity with the sequence of SEQ ID NO: 1, for example, asfotase alfa), can be provided according to regimens with single or multiple dosing. Doses can be administered, for example, once an hour, once every two hours, once a day, once every two days, twice a week, three times a week, four times a week, five times a week, six times a week, once a week, once every two weeks, once a month, once every two months, or once a year. Alternatively, the doses can be administered, for example, twice, 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 dosing regimen provides for administration three times a week. The duration of the dosing regimen may 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 or months. The dosing regimen may be started before, during or after a physical intervention such as surgery. The number, frequency and duration of dosing will be adapted by the treating physician in accordance with generally accepted factors such as the degree of bone mineralization disorder and various patient parameters.

For example, sALP or a fusion polypeptide comprising sALP (such as TNALP, e.g., a polypeptide comprising sALP, having the sequence of SEQ ID NO: 1, or a variant polypeptide having at least 95% sequence identity to the sequence of SEQ ID NO: 1, e.g., asfotase alfa) can be formulated as an injection solution that is a clear, colorless to slightly yellow aqueous solution, pH 7.4. sALP (such as TNALP, e.g. a polypeptide which is sALP, having the sequence of SEQ ID NO: 1 or a polypeptide variant having at least 95% sequence identity to the sequence of SEQ ID NO: 1, e.g. asfotase alpha) can be formulated at a concentration of, e.g., 12 mg/0.3 ml, 18 mg/0.45 ml, 28 mg/0.7 ml, 40 mg/1 ml, or 80 mg/0.8 ml. In particular, the composition can be formulated as a 40 mg/mL injection solution, wherein each mL of solution contains 40 mg sALP (e.g., each vial contains 0.3 mL of solution and 12 mg sALP (40 mg/mL), each vial contains 0.45 mL of solution and 18 mg sALP (40 mg/mL), each vial contains 0.7 mL of solution and 28 mg sALP (40 mg/mL), or each vial contains 1.0 mL of solution and 40 mg asfotase alfa (40 mg/mL)). sALP (such as TNALP, e.g. a polypeptide which is sALP, having the sequence of SEQ ID NO: 1 or a variant polypeptide having at least 95% sequence identity to the sequence of SEQ ID NO: 1, e.g. asfotase alfa) can be formulated as a 100 mg/mL injection solution, wherein each 1 mL of solution contains 100 mg of sALP or a polypeptide which is sALP (e.g. each vial contains 0.8 mL of solution and 80 mg of asfotase alfa (100 mg/mL)).

For example, the recommended dosage of an sALP (such as TNALP, e.g., a polypeptide that is an sALP having the sequence of SEQ ID NO: 1, or a polypeptide variant having at least 95% sequence identity to the sequence of SEQ ID NO: 1, e.g., asfotase alfa) is 2 mg/kg body weight administered subcutaneously three times per week, or a dosing regimen of 1 mg/kg body weight administered subcutaneously six times per week. Additional dosing information is provided below (Tables 2-1, 2-2, and 2-3).

In some embodiments described herein, the dose may be determined based on the actual body weight of the subject. In some embodiments, the dose may be determined based on the ideal body weight of the subject. The actual body weight refers to the true weight of the subject. The ideal body weight refers to the estimated body weight of the subject, which is an adjusted weight that takes into account the physical variables of the subject, such as age, gender, height, body size, wrist size, etc. Suitable formulas for calculating the ideal body weight are known in the art, such as the formula of G. J. Hamwi, the formula of B. J. Devine, the formula of J. D. Robinson, and the formula of D. R. Miller.

- 42 049823

Table 2-1

Administration of asfotase alfa doses of 2 mg/kg three times per week

Body weight (kg)* Injection dose Volume for injection Vial configuration 3 6 mg 0.15 ml 18 mg/0.45 ml 4 8 mg 0.2 ml 18 mg/0.45 ml 5 10 mg 0.25 ml 18 mg/0.45 ml 6 12 mg 0.3 ml 18 mg/0.45 ml 7 14 mg 0.35 ml 18 mg/0.45 ml 8 16 mg 0.4 ml 18 mg/0.45 ml 9 18 mg 0.45 ml 18 mg/0.45 ml 10 20 mg 0.5 ml 28 mg/0.7 ml 15 30 mg 0.75 ml 40 mg/ml 20 40 mg 1 ml 40 mg/ml 25 50 mg 1.25 ml Two vials of 28 mg/0.7 ml 30 60 mg 1.5 ml Two vials of 40 mg/ml 35 70 mg 1.75 ml Two vials of 40 mg/ml 40 80 mg 0.8 ml 80 mg/0.8 ml 50 100 mg 1 ml Two vials of 80 mg/0.8 ml 60 120 mg 1.2 ml** Two vials of 80 mg/0.8 ml 70 140 mg 1.4 ml** Two vials of 80 mg/0.8 ml 80 160 mg 1.6 ml** Two vials of 80 mg/0.8 ml

Table 2-2

Administration of asfotase alfa doses of 1 mg/kg six times per week

Body weight (kg)* Injection dose Volume for injection Vial configuration 3 3 mg 0.08 ml 18 mg/0.45 ml 4 4 mg 0.1 ml 18 mg/0.45 ml 5 5 mg 0.13 ml 18 mg/0.45 ml 6 6 mg 0.15 ml 18 mg/0.45 ml 7 7 mg 0.18 ml 18 mg/0.45 ml 8 8 mg 0.2 ml 18 mg/0.45 ml 9 9 mg 0.23 ml 18 mg/0.45 ml 10 10 mg 0.25 ml 18 mg/0.45 ml 15 15 mg 0.38 ml 18 mg/0.45 ml 20 20 mg 0.5 ml 28 mg/0.7 ml 25 25 mg 0.63 ml 28 mg/0.7 ml 30 30 mg 0.75 ml 40 mg/ml 35 35 mg 0.88 ml 40 mg/ml 40 40 mg 1 ml 40 mg/ml 50 50 mg 0.5 ml 80 mg/0.8 ml 60 60 mg 0.6 ml 80 mg/0.8 ml 70 70 mg 0.7 ml 80 mg/0.8 ml 80 80 mg 0.8 ml 80 mg/0.8 ml 90 90 mg 0.9 ml Two vials of 80 mg/0.8 ml 100 100 mg 1 ml Two vials of 80 mg/0.8 ml

Table 2-3

Administration of asfotase alfa doses of 3 mg/kg three times weekly (only if HPP is initiated in perinatal/infant age)

Body weight (kg)** Injection dose Volume for injection Vial configuration 3 9 mg 0.23 ml 18 mg/0.45 ml 4 12 mg 0.3 ml 18 mg/0.45 ml 5 15 mg 0.38 ml 18 mg/0.45 ml 6 18 mg 0.45 ml 18 mg/0.45 ml 7 21 mg 0.53 ml 28 mg/0.7 ml 8 24 mg 0.6 ml 28 mg/0.7 ml 9 27 mg 0.68 ml 28 mg/0.7 ml 10 30 mg 0.75 ml 40 mg/ml 15 45 mg 1.13 ml*** Two vials of 28 mg/0.7 ml 20 60 mg 1.5 ml*** Two vials of 40 mg/ml 25 75 mg 1.88 ml*** Two vials of 40 mg/ml

In some cases, administration of an sALP (such as TNALP, e.g., a polypeptide comprising sALP, having the sequence of SEQ ID NO: 1, or a variant polypeptide having at least 95% sequence identity to the sequence of SEQ ID NO: 1, e.g., asfotase alpha) according to the present invention may result in one or

- 43 049823 several of the following effects: increased bone regeneration in the patient and/or a change in bone mineral density that is undetectable or increased.

In any of the aspects disclosed herein, the patient is characterized by tolerability to the administration of sALP (such as TNALP, e.g., a polypeptide that is sALP, having the sequence of SEQ ID NO: 1 or a variant polypeptide having at least 95% sequence identity to the sequence of SEQ ID NO: 1, e.g., asfotase alfa), such as the absence or reduction in the incidence of adverse events selected from the group consisting of erythema at the injection site, decreased hemoglobin, fever, pneumonia, upper respiratory tract infection, otitis media, vomiting, constipation, diarrhea, tooth loss, nasopharyngitis, rash, dental caries, and irritability.

In any of the disclosed aspects, an sALP (such as TNALP, e.g., a polypeptide that is an sALP, having the sequence of SEQ ID NO: 1, or a polypeptide variant having at least 95% sequence identity to the sequence of SEQ ID NO: 1, e.g., asfotase alpha) is formulated in a pharmaceutical composition with at least one pharmaceutically acceptable carrier, such as a saline solution (e.g., sodium chloride and sodium phosphate). For example, the at least one pharmaceutically acceptable carrier may comprise 150 mM sodium chloride and 25 mM sodium phosphate.

In any of the above aspects, the sALP (such as TNALP, e.g. a polypeptide that is sALP, having the sequence of SEQ ID NO: 1 or a polypeptide variant having at least 95% sequence identity to the sequence of SEQ ID NO: 1, e.g. asfotase alpha) is physiologically active with respect to PEA, PPi and PLP, is catalytically competent to provide improved mineralization of skeletal structure in bone and/or is a soluble extracellular domain of alkaline phosphatase.

Compositions.

Pharmaceutical compositions comprising sALP (such as TNALP, e.g. a polypeptide which is sALP, having the sequence of SEQ ID NO: 1 or a polypeptide variant having at least 95% sequence identity to the sequence of SEQ ID NO: 1, e.g. asfotase alfa) can be formulated according to standard methods. The preparation of pharmaceutical formulations is a well-established 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, 3 ^rd Edition (ISBN: 091733096X). For example, a composition based on sALP (such as TNALP, e.g., a polypeptide that is sALP, having the sequence of SEQ ID NO: 1 or a variant polypeptide having at least 95% sequence identity to the sequence of SEQ ID NO: 1, e.g., asfotase alpha) can be formulated, for example, as a buffer solution at a suitable concentration, and be suitable for storage at 2-8°C (e.g., 4°C). The composition can also be formulated for storage at a temperature below 0°C (e.g., -20°C or -80°C). The composition may further be formulated for storage for 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, _{1 1/2} ^years , or 2 years) at 2-8°C (e.g., 4°C). Thus, the compositions disclosed herein may be stable when stored for at least 1 year at 2-8°C (e.g., 4°C).

Pharmaceutical compositions comprising sALP (such as TNALP, e.g., a polypeptide that is sALP, having the sequence of SEQ ID NO: 1 or a polypeptide variant having at least 95% sequence identity to the sequence of SEQ ID NO: 1, e.g., asfotase alfa) can be presented in a variety of forms. These forms include, for example, liquid, semi-solid and solid dosage forms, such as liquid solutions (e.g., injection and infusion solutions), dispersions or suspensions, tablets, pills, powders, liposomes and suppositories. The preferred form depends in part on the intended route of administration and the area of therapeutic use.

For example, compositions intended for systemic or local delivery may be presented in the form of injection or infusion solutions. Accordingly, compositions comprising sALP (such as TNALP, e.g., a polypeptide that is sALP, having the sequence 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) may be formulated for administration by parenteral route (e.g., by subcutaneous, intravenous, intraperitoneal or intramuscular injection).

Pharmaceutical compositions comprising sALP (such as TNALP, for example, a fusion polypeptide comprising sALP, having the sequence of SEQ ID NO: 1 or a polypeptide variant characterized by at least 95% sequence identity to the sequence of SEQ ID

- 44 049823

NO: 1, such as asfotase alfa), can be formulated in the form of a solution, microemulsion, dispersion, liposome or other ordered structure suitable for stable storage at high concentration. Sterile injectable solutions can be prepared by incorporating the composition described herein in the required amount in an appropriate solvent with one or a combination of ingredients listed above, if necessary, followed by filtered sterilization. Typically, dispersions are prepared by incorporating the composition described herein in a sterile carrier medium that contains a 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, the methods of preparation include vacuum drying and lyophilization, which allow obtaining a powder from the composition described herein plus any additional required ingredient (see herein) from a previously filtered to sterile condition solution thereof. Proper fluidity of the solution can be maintained, for example, by using an emulsifying agent such as lecithin, by maintaining the required particle size in the case of dispersion, and by using surfactants. Prolonged absorption of injection compositions can be ensured by including in the composition a reagent that delays absorption, for example, monostearate salts and gelatin.

The pharmaceutical compositions described herein can also be formulated into immunoliposome compositions. Such formulations 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 times are disclosed, for example, in U.S. Patent No. 5,013,556.

Pharmaceutical compositions comprising sALP (such as TNALP, e.g., a polypeptide comprising sALP of SEQ ID NO: 1 or a polypeptide variant having at least 95% sequence identity to SEQ ID NO: 1, e.g., asfotase alfa) may also be formulated with a carrier that will provide protection of the composition (e.g., a polypeptide comprising sALP or a fusion polypeptide comprising sALP) from rapid release, such as in 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 may be used. Many methods for preparing such formulations are known in the art. See, e.g., J.R. Robinson (1978) Sustained and Controlled Release Drug Delivery Systems, Marcel Dekker, Inc., New York.

If it is necessary to use the compositions in combination with a second active agent, the compositions can be formulated together with the second agent or the compositions can be formulated separately from the composition with the second agent. For example, the corresponding pharmaceutical compositions can be mixed, for example immediately before administration, and administered together or can be administered separately, for example at the same time or at different times.

Carriers/carrier environments.

Preparations comprising sALP (such as TNALP, for example, a polypeptide that is sALP, having the sequence of SEQ ID NO: 1 or a variant polypeptide having at least 95% sequence identity with the sequence of SEQ ID NO: 1, for example, asfotase alfa) can be provided to a patient, such as a patient with at least one symptom of a bone mineralization disorder (e.g., HPP), in combination with pharmaceutically acceptable sterile aqueous or non-aqueous solvents, suspensions or emulsions. Examples of non-aqueous solvents are propylene glycol, polyethylene glycol, vegetable oil, fish oil and organic esters for injection. Aqueous carriers include water, hydroalcoholic solutions, emulsions or suspensions, including saline solutions and buffered medical parenteral carrier media, including sodium chloride solution, Ringer's dextrose solution, dextrose plus sodium chloride solution, Ringer's solution containing lactose, or fixed oils. For example, a pharmaceutically acceptable carrier may contain sodium chloride and/or sodium phosphate, wherein the composition will include, for example, about 150 mM sodium chloride and/or about 25 mM sodium phosphate, pH 7.4.

Carrier media for intravenous administration may include fluid and nutrient replenishing agents, electrolyte replenishing agents such as those based on Ringer's dextrose solution, and the like. These may include pharmaceutically acceptable salts, such as salts of inorganic acids such as hydrochlorides, hydrobromides, phosphates, sulfates, and the like; and salts of organic acids such as acetates, propionates, malonates, benzoates, and the like. Additionally, such carrier media may contain auxiliary substances such as wetting or emulsifying agents, buffering agents for maintaining pH, and the like. A detailed discussion of pharmaceutically acceptable carriers is available in Remington's Pharmaceutical Sciences (Mack Pub. Co., N.J. 1991).

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Timing of the treatment phase.

The methods described herein include administering an sALP-based therapy during a treatment regimen that can include a treatment phase (e.g., one or more treatment phases) that includes administering the sALP to a subject in need thereof, and a non-treatment phase (e.g., one or more non-treatment phases) in which the sALP is discontinued. For example, the subject can be administered sALP during a first treatment phase that lasts for at least 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 2 weeks, 3 weeks, 4 weeks, 2 months, 3 months, 4 months, 5 months, 6 months, 12 months, 2 years, 3 years, 4 years, 5 years, 6 years, 7 years, 8 years, 9 years, 10 years, or longer. During the first treatment phase, the subject can be administered sALP, for example, once or more times per day, week, month, or year. Periodically, during the first phase of treatment, the subject may be monitored by at least one biochemical, physical, quality of life, or bone metric, such as described herein. When the measured metric value reaches a predetermined threshold (e.g., an improvement of at least about 1%, 2%, 3%, 4%, 5%, 10%, 20%, 30%, 40%, 50%, or more, or an improvement of, for example, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 97%, 99%, or 100% of the corresponding value in a normal subject), then it may be determined that the subject may discontinue therapy and may enter a non-treatment phase of the treatment regimen. The untreated phase may last at least 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 2 weeks, 3 weeks, 4 weeks, 2 months, 3 months, 4 months, 5 months, 6 months, 12 months, 2 years, 3 years, 4 years, 5 years, 6 years, 7 years, 8 years, 9 years, 10 years, or longer.

Periodically, during the non-treatment phase, the subject may be monitored by at least one biochemical, physical, quality of life, or bone metric, such as described herein. When the measured metric value decreases to or below a predetermined threshold (e.g., a decrease of 1%, 2%, 3%, 4%, 5%, 10%, 20%, 30%, 40%, 50%, or more, or a decrease of, for example, 90%, 80%, 70%, 60%, 50%, or less of the corresponding value in a normal subject), then it may be determined that the subject should re-enter the treatment phase of the treatment regimen, in which sALP is again administered. If the subject does not experience a decrease below the predetermined threshold, then the subject may remain in the non-treatment phase of the treatment regimen. It will be appreciated that the duration of each treatment or non-treatment phase may vary for any given patient depending on a number of factors, such as the metric used and/or the predetermined threshold. Thus, the duration of the phases may be determined empirically and may differ from each other or coincide with each other. For example, the first treatment phase may be longer or shorter than the second treatment phase.

Examples

The following examples are intended for illustrative purposes only and are not intended to limit the present invention in any way.

Example 1: Dose-ranging clinical study in adults.

Asfotase alfa was administered at a dose of 3.5 mg/kg in a dose-ranging study that included 6 adolescent and 13 adult patients with HPP aged >13 to <65 years at study entry. Thirteen patients were randomized to receive asfotase alfa (7 patients to receive 2.1 mg/kg/qw and 6 patients to receive 3.5 mg/kg/qw) and 6 patients were randomized to an untreated control group. All patients in the combination treatment group (2.1 mg/kg/qw and 3.5 mg/kg/qw) experienced approximately 2-fold greater reductions in plasma PPi levels compared with the control group after 24 weeks of treatment (mean change -2,100 μM compared with -1,052 μM in control patients); however, this difference was not statistically significant (p=0.0715) because a decrease in plasma PPi was observed in the control patient who received high dose vitamin D and had an elevated PPi value at baseline (12.1 μM). Statistical significance was demonstrated when this patient was excluded from the full analysis set (FAS) (p=0.0044). After the first 24 weeks of treatment, all patients received a total weekly dose of 3.5 mg/kg for an additional 24 weeks. The mean (SD) change in plasma PPi from baseline to last total exposure for all patients at the 3.5 mg/kg/week dose was -1.859 μM (2.8071).

Of the 13 patients who received asfotase alfa, 10 patients were adults (the majority [76.9%] were >50 years of age). In adult patients (n=5) who received a total weekly dose of 3.5 mg/kg, a decrease in plasma PPi levels (mean change -2,258 μM) was observed after 24 weeks of treatment. Asfotase alfa was generally well tolerated by all 19 patients in this study.

Example 2. Clinical study involving adults with functional pathologies.

Hypophosphatasia has a wide spectrum of clinical manifestations in adults. Adult patients with HPP are often misdiagnosed for many years, with little or no treatment available.

- 46 049823 An accurate diagnosis of HPP is crucial (Hogler et al., BMC Musculoskelet Disord. 20:80, 2019). As a result, inappropriate therapies (e.g., high-dose vitamin D, excessive calcium supplementation, parathyroid hormone analogs and (bisphosphonates)) may be ineffective or even worsen clinical conditions and signs and symptoms (Conti et al., Clin Cases Miner Bone Metab.14:230-234, 2017). In addition, severe functional pathologies are often present in patients with HPP, including mobility problems (gait and walking difficulties), muscle weakness and inability to perform ADLs, which together greatly affect the patients' quality of life. Long-term clinical consequences include recurrent and non-healing fractures, burden of orthopedic/dental surgeries, frailty, arthritis, inability to remove internal fixation devices (due to risk of recurrent fracture), pain, and need for outpatient assistive devices (eg, wheelchairs, wheeled walkers, and canes) (Kishnani et al., Annual Meeting of the Endocrine Society; 2019; New Orleans). Regardless of the age at which HPP first presents, high disease burden can result in decreased health-related quality of life, which may further decline over the course of the patient's life (Szabo et al., Orphanet J Rare Dis. 14:85, 2019; Weber et al., Metab.: clin. and exper. 65(10):1522–1530, 2016). Signs and/or symptoms of HPP may also affect the mental and emotional health of children and adults with the disease, which is associated with significant healthcare resource utilization (Colazo JM, Ni JR, Dahir KM, Simmons JH. Neurological symptoms in Hypophosphatasia. Osteoporos Int. 2019; 30(2):469-480; Jenkins-Jones et al., Annual Meeting of the Amer. Soc. Bone and Mineral Res.; 2018; Montreal, QC, Canada).

Adult patients with a high disease burden (i.e., childhood-onset HPP with delayed diagnosis) often present with multiple lower extremity fractures (typically femoral or metatarsal fractures or pseudofractures; uni-/bilateral, lateral or medial subtrochanteric shaft) with poor healing, which can cause severe pain, tenderness, limited mobility, and poor quality of life (Genest et al., Osteoporos Int. 29:1815–1825, 2018; Marini et al., Clin Cases Miner Bone Metab. 14:324–328, 2017). Patients have also reported chronic muscle pain, decreased muscle strength and performance related to muscle weakness, and non-fracture bone pain (Conti et al., Clin Cases Miner Bone Metab. 14:230-234, 2017).

To date, the focus has been on the diagnosis and treatment of pediatric patients. In fact, there is a significant gap in data on functional outcomes in adult patients, including patients with childhood-onset HPP who were likely not diagnosed until serious complications occurred or were not considered suitable for an ERT such as asfotase alfa. This study will provide additional data on the effect of asfotase alfa in adult patients with childhood-onset HPP who have significant physical functional limitations due to their disease. This study will also examine the effects of a lower-dose asfotase alfa regimen following a 6 mg/kg/week loading dose regimen.

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Table 3

Goals and Endpoints

Goals Endpoints Primary (evaluate ONLY in patients in Group 1 [patients who received asfotase alfa 6 mg/kg/qw for a total of 36 weeks]) To evaluate the effect of asfotase alfa on plasma PPi concentrations at week 36. Change in plasma PPi concentration values from baseline to week 36 Key secondary (evaluate in patients in Group 1 [patients who received asfotase alfa 6 mg/kg/qw for a total of 36 weeks)] or Group 2 [patients who received asfotase alfa 6 mg/kg/qw for 12 weeks followed by 3.6 mg/kg/qw for 24 weeks] as specified) To evaluate the effect of asfotase alfa on plasma PPi concentration values in group 2 at week 36. Change in plasma PPi concentration values in group 2 from baseline to week 36 To evaluate the effect of asfotase alfa on the SF-36 PCS score in groups 1 and 2 at week 36 Change in PCS SF-36 score from baseline to week 36 in groups 1 and 2 To evaluate the effect of asfotase alfa on the repeated chair stand test (SPPB component) in groups 1 and 2 at week 36 Change in chair stand test (SPPB component) from baseline to week 36 in groups 1 and 2

- 48 049823

Remaining secondary (to be assessed in patients in Group 1 [patients who received asfotase alfa 6 mg/kg/qw for a total of 36 weeks)] or Group 2 [patients who received asfotase alfa 6 mg/kg/qw for 12 weeks followed by 3.6 mg/kg/qw for 24 weeks] as specified) To evaluate the effect of asfotase alfa on physical function and pain in groups 1 and 2 at week 36 Change from baseline to week 36 in groups 1 and 2 for the following: - TUG test results - BPI score - question 3 (worst pain) - LEFS score To evaluate the effect of asfotase alfa on the repeated chair stand test (SPPB component) in groups 1 and 2 at week 24 Change in chair stand test (SPPB component) from baseline to week 24 in groups 1 and 2 To evaluate the effect of asfotase alfa on the SF-36 PCS score in groups 1 and 2 at week 24 Change in PCS SF-36 score from baseline to week 24 in groups 1 and 2 Safety (assess in all patients) To evaluate the safety, tolerability and immunogenicity potential of asfotase alfa over a period of up to 36 weeks Incidence of TEAEs, TESAEs, and AESIs (IARs, including hypersensitivity reactions and ISRs); lipodystrophy; ectopic calcifications; immunogenicity (e.g., ADA results); physical examination findings; vital signs and clinical laboratory findings Exploratory (evaluate in patients in Group 1 [patients who received asfotase alfa 6 mg/kg/qw for a total of 36 weeks)] or Group 2 [patients who received asfotase alfa 6 mg/kg/qw for 12 weeks followed by 3.6 mg/kg/qw for 24 weeks] as specified) To assess the activity of asfotase alpha in the blood serum in groups 1 and 2 at weeks Change in serum asfotase alpha activity from baseline to

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12, 24 and 36 weeks 12, 24 and 36 in groups 1 and 2 To evaluate the effect of asfotase alfa on plasma PL and PLP levels in groups 1 and 2 at weeks 12, 24 and 36 Change in PLP to PL ratio from baseline to weeks 12, 24, and 36 in groups 1 and 2 To evaluate the effect of asfotase alfa on plasma PPi concentrations in groups 1 and 2 at week 12 Change in plasma PPi levels in groups 1 and 2 from baseline to week 12 To evaluate the effect of asfotase alfa on the repeated chair stand test (SPPB component) in groups 1 and 2 at week 12 Change in chair stand test (SPPB component) from baseline to week 12 in groups 1 and 2 To evaluate the effect of asfotase alfa on the SF-36 PCS score in groups 1 and 2 at week 12 Change in PCS SF-36 score from baseline to week 12 in groups 1 and 2 To evaluate the effect of asfotase alfa on physical function and pain in groups 1 and 2 at weeks 12 and 24 Change from baseline to week 12 and week 24 in groups 1 and 2 for the following: - TUG test results - BPI score - question 3 (worst pain) - LEFS score To evaluate the effect of asfotase alfa on bone quality/microarchitecture in groups 1 and 2 at week 36. Change from baseline to week 36 in HR-pQCT (total, volumetric trabecular and cortical BMD, cortical thickness, stiffness, trabecular number, trabecular separation, and trabecular thickness) To evaluate the effect of asfotase alfa on markers of bone turnover in groups 1 and 2 at weeks 12 and 36 Change in osteocalcin, P1NP and sCTX levels from baseline to weeks 12 and 36 in groups 1 and 2 To evaluate the effect of asfotase alfa on health-related quality of life in groups 1 and 2 at weeks 12, 24 and 36 Change in SF-36 total score from baseline to weeks 12, 24, and 36 in groups 1 and 2 Evaluate the proportion of regenerated The proportion of regenerated fractures by fractures or pseudofractures in patients with delayed regeneration ³ fractures or pseudofractures of the lower limb(s) in groups 1 and 2 at week 36 radiological assessment data in patients with delayed regeneration of active fractures or pseudofractures at week 36 in groups 1 and 2 To evaluate the time to fracture healing in patients with delayed healing of ³ fractures or pseudofractures of the lower limb(s) in groups 1 and 2 at weeks 12, 24 and 36 Time to fracture union/regeneration according to radiographic assessment at weeks 12, 24 and 36 in groups 1 and 2 To evaluate the effect of asfotase alfa on PGI-I in groups 1 and 2 at week 36. In groups 1 and 2: - PGI-I score at week 36 - Proportion of patients reporting very much better and much better status at week 36 on the PGI-I questionnaire

^a Delayed healing is defined as an active fracture or pseudofracture with no evidence of healing for at least 3 months prior to screening.

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Abbreviations: ADA=anti-drug antibody; AESI=adverse event of special interest; BMD=bone mineral density; BPI=short-form pain inventory; HR-pQCT=high-resolution peripheral quantitative computed tomography; IAR=injection-related reaction; ISR=injection site reaction; LEFS=lower limb function scale; P1NP=procollagen type I N-terminal propeptide; PCS=physical component summary; PGI-I=patient global impression of improvement; PL=pyridoxal; PLP=pyridoxal-5'-phosphate; PPi=inorganic pyrophosphate; qw=pa3 per week; sCTX=serum C-telopeptide-cross-linked collagen type I; SF-36=short form 36-item questionnaire; SPPB=short physical function battery; TEAE=treatment-emergent adverse event; TESAE=treatment-emergent serious adverse event; TUG=timed get up and go test.

General design.

Patients who meet the inclusion criteria for the study will be randomly assigned in a 1:1 ratio to either Arm 1 (n=25) or Arm 2 (n=25). All patients will start asfotase alfa at 6 mg/kg/qw at week 1. At week 13, patients in Arm 1 will continue to receive asfotase alfa at 6 mg/kg/qw for 24 weeks and patients in Arm 2 will start asfotase alfa at 3.6 mg/kg/qw for 24 weeks. Specific dosing times will be specified in the study procedural manual. Approximately 36 (75%) patients are expected to complete the study.

Number of patients.

Approximately 50 patients will be randomized (25 patients in group 1 and 25 patients in group 2) in multiple geographic regions.

Groups and duration of treatment.

Asfotase alfa will be supplied as a sterile aqueous solution in 80 mg vials (100 mg/mL strength) and will be administered subcutaneously at a total weekly dose of 6 mg/kg (2 mg/kg 3 times per week) or a total weekly dose of 3.6 mg/kg (1.2 mg/kg 3 times per week). If more frequent dosing is indicated (e.g. based on weight or tolerability issues due to an injection-related reaction or injection site reaction), the patient may switch to a 6 times per week dosing schedule.

The total duration of the study is expected to be approximately 50 weeks: up to 10 weeks of screening period, 36 weeks of treatment period and 30 days of follow-up via telephone calls.

Scheme.

The flow chart of the study design is shown in Fig. 1.

Table 4

Visit ¹

Week(s)

Time

Schedule of events

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intervals (in days) A A Meeting the inclusion criteria for the study Informed consent X Inclusion/exclusion X X Lab tests for screening X Anamnesis of patient A ⁴ X Demographic data ⁵ X Diagnosis of NRR ⁶ X Analysis of mutations of the HPP gene ⁷ X Renal pathology X

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I (eGFR) Randomization Randomization X Research implementation Results of physical examination ⁸ X X X X X X X Vital signs 9 X X X X X X X X X Body weight ¹⁰ X X X X X X X X X Blood serum analysis for pregnancy ¹¹ X X X X X X X X X Urine test for pregnancy ¹¹ X X X Administration of study drug ¹² Asfotase a alpha X X X X X X X X X X X X Safety assessments/laboratory analyses ¹³

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X-ray imaging ¹⁴ X X X X X X Chemistry ¹⁵ X X X X X X X 25- hydroxyvitamins hD X X X X X X Ophthalmological examination ¹⁶ X X X X Ultrasound examination of the kidneys X X X X RTN X X X X X HRpQCT ¹⁷ X X X Tryptase A in serious cases Y1AK ¹⁸ X X X X X X X X X X X X Review and assessment of adverse events "continuous observation" Biomarker assays Analysis X X X X

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markers in bone tissue renewal (osteocalcin, P1NP, sCTX) PK/PD/ADA ¹⁹ tests PD (PPi, PL and PLP) X X X X X X X RK X X X X X X X ADA X X X X X X X Results evaluations BPI X X X X X SF-36 X X X X X SPPB X X X X X TUG test results X X X X X LEFS X X X X X PGI-I X X Other Making phone calls within 30- X

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daytime period Review of concomitant medications/non-pharmacological therapies and procedures "continuous observation" Overview of the application of auxiliary X devices "continuous observation" Diary and patient "continuous observation"

Abbreviations: ADA=anti-drug antibody; ALP=alkaline phosphatase; BP1=short-form pain questionnaire; eGFR=estimated glomerular filtration rate; EOS=end of study; ET=early discontinuation; HPP=hypophosphatasia; HRpQCT=high-resolution peripheral quantitative tomography; IAR=injection related reaction; LEFS=lower limb function score; NA=not applicable; P1NP=procollagen type I N-terminal propeptide; PD=pharmacodynamics; PGI-I=patient global impression of improvement; PK=pharmacokinetics; PL=pyridoxal; PLP=pyridoxal-5'-phosphate; PPi=inorganic pyrophosphate; PTH=parathyroid hormone; sCTX=serum C-telopeptide-cross-linked collagen type 1; SF-36=short form 36-item questionnaire; SPPB=short physical performance battery; TUG=timed walk test; ULN=upper limit of normal.

1. Visits will take place at the study site, with some visits to administer doses to the patient at home.

2. If a patient discontinues the study before Week 36, all assessments, if possible, except study drug administration should be performed at this visit. To simplify scheduling of required study assessments at each visit, the study visit may be shortened or extended as needed, provided that the specified order of assessments regarding laboratory sample collection (e.g., PK/PD/ADA, serum pregnancy testing) and vital sign assessment is followed. The order of assessments will be specified in the Study Procedures Manual.

3. A follow-up telephone call will occur 30 days after the last dose of study drug (week 36). For patients who discontinued the study before week 36, a follow-up telephone call will be made.

- 56 049823 follow-up after 30 days (unless the patient withdraws consent to participate in the study). Female patients of childbearing potential will be followed for information on birth control and pregnancy. Any reported pregnancy in female patients or female partners of male patients will be followed until the pregnancy outcome is known.

4. The patient history includes a general patient history and a HPP-specific patient history, including details of childhood-onset disease. Details of adult tooth loss will be assessed as part of the patient history. A complete patient history will be collected at baseline, and patients will be interviewed to determine if there has been any change in the patient history since the previous visit during subsequent study clinic visits.

5. Demographic data will be collected depending on the region and may include date of birth, age, gender, ethnicity and race.

6. The clinical diagnosis of childhood-onset HPP is based on signs and symptoms consistent with HPP, supported by the following:

a. Documented mutation(s) in the ALPL gene from a certified laboratory;

b. Serum ALP level is below age- and sex-adjusted normal range, PLP is greater than 2xULN at screening.

7. If the results of the HPP gene mutation analysis are not already available in the patient's medical records, it should be performed within the first 2 weeks of the screening period.

8. The physical examination will include assessment of general appearance; skin; head, ear, eye, nose, and throat; neck; lymph nodes; chest; heart; abdomen; extremities; central nervous system; musculoskeletal system. A full physical examination will be performed at Screening and Baseline visits. Abbreviated physical examinations (based on the patient's signs and symptoms) will be performed at the time points specified in the activity schedule (Table 4). All physical examinations (full and abbreviated) will include weighing (using a calibrated scale) and inspection of asfotase alfa injection sites for potential injection site reaction(s). Physical examinations at Screening and Baseline visits will also include growth assessment.

9. Vital signs include blood pressure, heart rate, respiratory rate, and temperature (either temple, oral, or axillary). On in-clinic dosing days, vital signs should be measured 10 minutes before study drug injection. Please ensure that the method of measuring patient temperature is consistent throughout the study.

10. On dosing days in the clinic, body weight will need to be measured before dosing to confirm the correct dose.

11. Urine and serum pregnancy tests are required only for female patients of childbearing potential. Serum pregnancy tests are required at the times specified in the schedule of activities (Table 4) and will be performed during study visits to the clinic. Urine pregnancy tests, performed at the times specified in the schedule of activities, will be performed by female patients at home.

12. The study drug will be administered in the clinic at the times specified in the schedule of events (Table 4). All other dose administrations may be performed in the clinic or at home at the discretion of the investigator.

13. All blood samples for laboratory assessments of clinical biochemistry should be collected prior to administration of the study drug.

14. All patients will undergo radiographic examination to assess fracture(s) or pseudofracture(s) of the lower limbs during the screening period. During the study, radiographic examinations are required only for patients with documented fracture(s) or pseudofracture(s) at randomization or for patients with suspected new fracture(s) during the study.

15. The list of parameters included in clinical biochemistry is described below.

16. A complete ophthalmological examination will be performed at the times specified in the schedule of activities. The examination will assess for papilledema and signs of ectopic calcification and will include assessments of visual acuity; adjacent structures and slit lamp biomicroscopy of the anterior chamber, lens, conjunctiva, cornea and fundus. The ophthalmological examination should be performed by a blinded qualified ophthalmologist. Centres will be provided with ophthalmological worksheets required to complete the complete examination. Ophthalmological examinations may be performed by a qualified optometrist (e.g., Doctor of Optometry) provided that the optometrist is supervised by an ophthalmologist.

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17. All patients will have access to high-resolution peripheral quantitative computed tomography. If patients are willing and able to undergo this imaging assessment, travel arrangements will be made to study centers that have this imaging capability. Also, if a patient has had HR-pQCT performed within 3 months of the screening visit, it can be considered as a baseline assessment.

18. A serum tryptase sample will be collected prior to dosing on Day 1. Samples will only be collected if the patient does not currently have an active allergic reaction and will only be analyzed if the patient experiences a subsequent IAR. For acute or severe IARs (e.g., with signs and symptoms of hypersensitivity, regardless of the time from study drug administration to onset), additional blood and urine samples should be collected to evaluate the reaction as described in this document. For systemic hypersensitivity reactions, additional blood and urine samples should be collected to evaluate the reaction.

19. A serum sample for PK/PD/ADA analysis should be obtained 15-30 minutes prior to administration of asfotase alfa doses.

Administration of asfotase alfa according to the treatment regimen described in this document is expected to provide significant benefit to adult patients with childhood-onset HPP with functional impairment due to chronic disease.

Scientific rationale for the study design.

The scientific rationale for each aspect of the study design is discussed below.

This study is conducted in adult patients with childhood-onset HPP with a high disease burden (as evidenced by specific study inclusion criteria) in a population for which functional outcome data are insufficient.

The asfotase alfa dose of 6 mg/kg is the officially recommended dose with an established efficacy and safety profile.

The asfotase alfa dose of 3.6 mg/kg is similar to the dose (3.5 mg/kg) evaluated in a dose-ranging study that included 6 adolescent and 13 adult patients with HPP aged >13 to <65 years at study entry. In this study, the 3.6 mg/kg dose will be evaluated in a larger cohort of adult patients with childhood-onset HPP with a high disease burden (defined by specific study entry criteria).

The functional assessments (eg, 36-item Short Form [SF-36], Physical Component Summary [PCS], and Chair Stand-Up Test [a component of the Short Physical Performance Battery (SPPB)]) to be used in this study are validated subjective and objective outcome measures that have been assessed in conditions/diseases that have been reported to impair functional mobility and affect ADLs (eg, rheumatoid arthritis, osteoarthritis, chronic musculoskeletal pain, and X-linked hypophosphatemia).

Cut-off scores for key secondary functional endpoints, which are validated subjective and objective outcome measures (SF-36 PCS score < 40 and chair stand-up test [SPPB component] time > 13 s), were determined based on a comprehensive review of cumulative data reported in both conditions/diseases that impair functional mobility or ADL and in the healthy population.

Administration of doses of asfotase alfa.

Asfotase alfa at a dose of 3.6 mg/kg/qw will be administered to patients in Cohort 2 for 24 weeks (week 13 to week 36) after 6.0 mg/kg/qw for the first 12 weeks. Dose reduction to 3.6 mg/kg/qw is expected to maintain the reductions in plasma PPi levels from week 13 to week 36.

Asfotase alfa was administered daily at a dose of 3.5 mg/kg in Study ENB-009-10 (e.g., 0.5 mg/kg 7 times per week). In the present study, the asfotase alfa dose of 3.6 mg/kg was chosen to allow for convenient dosing 3 times per week (i.e., 1.2 mg/kg 3 times per week). It should be noted that there may be instances where more frequent dosing is indicated (i.e., 6 times per week).

Patient type and disease characteristics.

The clinical diagnosis of childhood-onset HPP is based on signs and symptoms consistent with HPP, supported by the following:

a) documented mutation(s) in the ALPL gene from a certified laboratory;

serum alkaline phosphatase (ALP) level below the age- and sex-adjusted normal range AND pyridoxal-5'-phosphate (PLP) level more than 2x the upper limit of normal at screening;

- 58 049823 body mass index <35 kg/ ^m2 at baseline;

functional pathology as evidenced by a repeated chair stand test (SPPB component) time of >13 s and a SF-36 PCS score of <40 at screening. A maximum of 4 patients who fail the repeated chair stand test (SPPB component) will be eligible for inclusion in the study;

patients must have a history of at least 1 non-vertebral fracture (or pseudofracture) sustained without evidence of major trauma;

patients with current unhealed fracture(s) or pseudofracture(s) (e.g. femur, tibia, fibula, metatarsals) of the lower limb(s) should have documentation of these fractures for at least 3 months prior to screening (with or without surgical intervention);

the administered(s) investigational(s) medicinal product(s);

Asfotase alfa will be supplied as a sterile aqueous solution in 80 mg vials (100 mg/mL) with an extraction volume of 0.8 mL. Further details are provided in Table 5.

Table 5

Investigational drug administered

Product under study Product Name: Asfotase alpha Dosage form: Liquid Single dose: Group 1: 6 mg/kg/qw for 36 weeks Group 2: 6 mg/kg/qw for 12 weeks, then 3.6 mg/kg/qw for 24 weeks Route of administration: SC Description of physical properties: A sterile, clear to slightly opalescent, colourless to pale yellow aqueous solution containing 100 mg/ml asfotase alfa, 25 mM sodium phosphate, 150 mM sodium chloride, pH 7.4, in a single-use glass vial Manufacturer: Alexion Pharmaceuticals, Inc.

Abbreviations: SC=subcutaneous; qw=pa3 per week. Preparation and Administration of Study Drug Patients randomized to Group 1 or Group 2 will begin with three times weekly dosing. The number of vials used to prepare asfotase alfa SC injections will be determined based on patient weight (Table 6 and Table 8).

If a patient receiving 6 mg/kg/qw weighs >90 kg, the patient should be converted to a 6 times weekly dosing schedule (Table 7). If more frequent dosing is indicated (e.g. based on weight or tolerability issues due to IAR/ISR as assessed by the investigator), the patient should be converted to a 6 times weekly dosing schedule (Table 7 and Table 9). Additional dose adjustments may also be considered for lack of efficacy or safety reasons. The specified dose volume may be withdrawn from the drug vial using a sterile, single-use, 1 mL injection syringe. Study drug should be administered SC using aseptic technique.

Table 6

Weight-based dosing for asfotase administration

alpha 6 mg/kg/week (2 mg/kg three once a week) Body weight (kg) Injection dose (mg) Volume for injection (ml) Vial configuration 40 80 0.80 80 mg/0.8 ml 50 100 1,00 Two (2) vials of 80 mg/0.8 ml 60 120 1.20 ^a Two (2) vials of 80 mg/0.8 ml 70 140 1.40 ^a Two (2) vials of 80 mg/0.8 ml 80 160 1.60 ^a Two (2) vials of 80 mg/0.8 ml

^a When obtaining an injection volume greater than 1 mL, divide the volume equally between 2 syringes and administer 2 injections into 2 separate injection sites approximately 1 inch (2.54 cm) apart.

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Table 7

Weight-based dosing for asfotase alfa 6 mg/kg/week (1 mg/kg six times per week)

Body weight (kg) Injection dose (mg) Volume for injection (ml) Vial configuration 40 40 0,40 80 mg/0.8 ml 50 50 0,50 80 mg/0.8 ml 60 60 0,60 80 mg/0.8 ml 70 70 0.70 80 mg/0.8 ml 80 80 0.80 80 mg/0.8 ml 90 90 0.90 Two (2) vials of 80 mg/0.8 ml 100 100 1,00 Two (2) vials of 80 mg/0.8 ml 110 110 1.10 ^a Two (2) vials of 80 mg/0.8 ml 120 120 1.20 ^a Two (2) vials of 80 mg/0.8 ml 130 130 1.30 ^a Two (2) vials of 80 mg/0.8 ml

^a When obtaining an injection volume greater than 1 mL, divide the volume equally between 2 syringes and administer 2 injections into 2 separate injection sites approximately 1 inch (2.54 cm) apart.

Table 8

Weight-based dosing for asfotase alfa 3.6 mg/kg/week (1.2 mg/kg three times per week)

Body weight (kg) Injection dose (mg) Volume for injection (ml) Vial configuration 40 48 0.48 80 mg/0.8 ml 50 60 0,60 80 mg/0.8 ml 60 72 0.72 80 mg/0.8 ml 70 84 0.84 Two (2) vials of 80 mg/0.8 ml 80 96 0.96 Two (2) vials of 80 mg/0.8 ml 90 108 1.08 ^a Two (2) vials of 80 mg/0.8 ml 100 120 1.20 ^a Two (2) vials of 80 mg/0.8 ml 110 132 1.32 ^a Two (2) vials of 80 mg/0.8 ml 120 144 1.44 ^a Two (2) vials of 80 mg/0.8 ml 130 156 1.56 ^a Two (2) vials of 80 mg/0.8 ml

^a When obtaining an injection volume greater than 1 mL, divide the volume equally between 2 syringes and administer 2 injections into 2 separate injection sites approximately 1 inch (2.54 cm) apart.

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Table 9

Weight-based dosing for asfotase alfa 3.6 mg/kg/week (0.6 mg/kg six times per week)____________

Body weight (kg) Injection dose (mg) Volume for injection (ml) Vial configuration 40 24 0.24 80 mg/0.8 ml 50 30 0,30 80 mg/0.8 ml 60 36 0.36 80 mg/0.8 ml 70 42 0.42 80 mg/0.8 ml 80 48 0.48 80 mg/0.8 ml 90 54 0.54 80 mg/0.8 ml 100 60 0,60 80 mg/0.8 ml 110 66 0.66 80 mg/0.8 ml 120 72 0.72 80 mg/0.8 ml 130 78 0.78 80 mg/0.8 ml

Dose modification.

After the first 12 weeks of treatment, patients in Group 2 will be crossed over to a 3.6 mg/kg/qw regimen. Dose modifications may be permitted if a patient experiences worsening of HPP-related signs and symptoms compared to pre-treatment baseline after dose reduction to 3.6 mg/kg/qw, with dose increases to 6 mg/kg/qw permitted.

Performance evaluations.

Inorganic pyrophosphate in blood plasma.

Hypophosphatasia is characterized by age-dependent interrelated clinical manifestations resulting from the failure to mineralize bone matrix due to elevated concentrations of the TNALP substrate PPi. Elevated extracellular PPi inhibits bone mineralization by blocking hydroxyapatite crystal formation, causing marked accumulation of unmineralized bone matrix, resulting in osteomalacia (softening of bone) in patients of all ages, skeletal deformities of rickets (abnormally mineralized bone, dysmorphic tubular bones and ribs), and subsequent growth abnormalities in infants and children. Administration of asfotase alpha is expected to hydrolyze PPi, releasing Pi to combine with calcium, thereby promoting hydroxyapatite crystal growth and bone mineralization, and restoring a normal skeletal phenotype.

Blood samples for measurement of plasma PPi levels can be collected at various time points, such as those listed in Table 4, to assess potential substrates for asfotase alpha activity.

Brief Physical Functioning Test Battery.

The SPPB was developed to measure functional status and physical performance. It is a composite measure that assesses gait speed, standing balance, and performance on the sit-to-stand test (STS). The SPPB is calculated based on three components: the ability to stand for up to 10 seconds with three foot positions (feet parallel, feet perpendicular, and feet in sequence); the time to complete a 3- or 4-meter walk; and the time to rise from a chair 5 times (e.g., the repeated chair stand test). The standing balance tests are scored based on the ability to maintain balance in each of these positions. Gait speed and the STS tests are scored based on the ability to perform the tasks and the time spent on each task. Each task is scored out of 4 points, with the scores for the 3 tests summed to give a maximum of 12 and a minimum of 0. A higher score indicates a higher level of function, while a lower score indicates a lower level of function. Lower SPPB scores have been shown to predict increased risk of falls, loss of ADL independence, decreased mobility, disability, poorer health outcomes, hospital readmission and length of stay, nursing home admission, and death. A 0.5 point change in SPPB is considered a small meaningful change, while a 1 point change in SPPB is considered a large meaningful change (Treacy et al., J Physiother. 64(1):61, 2018).

The chair stand test involves the activation of multiple muscles of the lower limb, primarily the knee extensor muscles (quadriceps femoris) (Bohannon et al. Isokinet Exerc Sci. 18(4):235–240, 2010). The chair stand test was used as a stand-alone

- 61 049823 assessment (e.g. STS exercise), which is widely used as a measure of lower extremity strength. There are several variations of the STS test; however, the SPPB component, which is the repeated chair stand test, will be used in this study.

The SPPB can be conducted at various time points, such as those indicated in Table 4, by a blinded evaluator.

A short form of the 36-item questionnaire.

Health-related quality of life refers to functioning and well-being in the physical, mental, and social domains of life. The SF-36 consists of eight multi-item scales (35 items) assessing physical function (10 items), role limitations due to physical health problems (4 items), physical pain (2 items), general health (5 items), life activity (4 items), social functioning (2 items), role limitations due to emotional problems (3 items), and emotional well-being (5 items). These eight scales can be combined into two summary scores: the PCS score and the mental component summary (MCS) score. The 36th item, concerning change in health status, is not included in the scale or summary scores (Farivar et al., Health Qual Life Outcomes 5:54, 2007).

The SF-36 is a self-administered questionnaire. Patients complete 1 of a range of options for each of 36 questions. The combination of item responses is then combined to calculate a score for each of the 8 scales listed. Scores for each dimension range from 0 to 100, with higher scores indicating better health. Body pain is also scored in the same way, with higher scores indicating less pain. The 2 summary scores (PCS and MCS) are scored differently from the 8 dimension scores. These scales are scored using norm-referenced methods. A score of 50 reflects the average score for these populations. Scores below 50 reflect below average health, and scores above 50 reflect above average health (Stewart et al., Aust J Physiother. 53:208, 2007). The SF-36 questionnaire can be completed at various time points, such as those indicated in Table 4.

Timed test get up and go.

The Timed Get Up and Go (TUG) test measures in seconds the time it takes an individual to rise from a standard chair, walk a distance of 3 m (approximately 10 ft), turn around, return to the chair, and sit down. Designed to be administered in a healthcare setting, this clinical test requires that patients wear their usual shoes and use their usual walking aid (no aid, cane, walker). No physical assistance is provided. Patients begin the test with their back against the chair, their hands on the armrests, and their walking aid in hand. They are instructed that when the word go is given, they should stand up and walk at a comfortable and safe pace to a line on the floor 3 m away, turn around, return to the chair, and sit down again. Before timing, the patient completes the test once to familiarize themselves with the test. A stopwatch or a wristwatch with a second hand can be used to measure the test time. Normal, healthy older adults typically complete the task in 10 s or less. Very frail or weak older adults with poor mobility may require 2 min or more. A score of > 14 s has been shown to indicate a high risk of falls (Lysack C. et al., Household and Neighborhood Safety, Mobility. 2 ^nd ed. St. Elsevier; 2010). The TUG test will be administered by a blinded assessor at various time points, such as those shown in Table 4.

Score for the brief questionnaire for assessing the severity of pain syndrome - question 3 (the most severe pain).

The Brief Pain Inventory (BPI), formerly known as the Brief Pain Questionnaire, is a self-administered questionnaire that was originally developed to assess cancer pain. It is now also used as a general pain questionnaire for other chronic pain conditions. It is available in a short (9-item) and a long (17-item) form. The short form of the BPI is more commonly used and is the one that is referred to when the BPI is mentioned in research.

The first optional item is a control question about the respondent's pain during the day. The questionnaire then consists of graphical pain diagrams: 4 items about pain intensity (worst pain, least pain, average pain, pain at the moment), 2 items about pain medication or drug, and 1 item about the impact of pain with 7 subitems (general activity, mood, ability to walk, normal walking, relationships with others, sleep, enjoyment of life).

The BPI provides 2 main scores: the pain severity score and the pain impact score. The pain severity score is calculated from 4 items concerning the intensity of pain. Each item is rated from 0, i.e. no pain, to 10, i.e. the pain is as severe as you can imagine; and the item contributes the same amount to the total score, which ranges from 0 to 40. The pain impact score corresponds to the pain impact item. The 7 sub-items are rated from 0, i.e. has no impact, to 10, i.e. has the greatest impact.

- 62 049823 cialis impact; and the item contributes the same amount to the total score, which ranges from 0 to 70. The first item, the pain graphs (painful and most painful areas), and the items about pain treatment or medication (list of treatments and degree of relief) are not included in the scoring. Completing the BPI takes approximately 5 min (Poquet et al., J Physiother. 62:52, 2016). The BPI can be completed at various time points, such as those shown in Table 4.

Lower limb function scale score.

The Lower Extremity Function Scale (LEFS) is a patient-reported measure of functional status in the presence of lower extremity musculoskeletal problems. The LEFS consists of 20 items with scores ranging from 0 (extremely difficult/impossible to perform activity) to 4 (no difficulty). A total score can be obtained by summing the individual item scores. A maximum score of 80 indicates no functional limitation, and a minimum score of 0 indicates extreme limitation (Mehta et al., J orthopaed. and sports phys. ther. 46:200–216, 2016). The LEFS can be completed at various time points, such as those shown in Table 4.

Radiological assessment of fracture regeneration.

This study will evaluate the proportion of fractures or pseudofractures that heal in patients with delayed fracture or pseudofracture healing of the lower extremity(ies) using radiographic evaluation. Radiographic bone regeneration is defined as the observation of evidence of cortical fusiform callus and/or near-complete fracture line obliteration. The radiographic tibial fracture union (RUST) score uses these radiographic features to assess regeneration. RUST and modified RUST (mRUST) are based on scores assigned to callus formation and fracture line visibility in each of 4 visible cortical areas on 2 radiographs. An mRUST score of 1 is assigned if there is no callus; a score of 2 is assigned if callus is present; A score of 3 is assigned when fusiform callus is present, and a score of 4 is assigned when bone is remodeled and a fracture is not visible (Leow et al., Bone Joint Res. 5:116–121, 2016; Litrenta et al., J Orthop Trauma 29:516–520, 2015; Whelan et al., J Trauma. 68:629–632, 2010). An mRUST score of 3 will be considered as evidence of regeneration in this study.

Radiographs will be evaluated by a blinded panel of experts (e.g., radiologists, orthopedic surgeons). Radiographic imaging may be performed at various time points, such as those listed in Table 4.

Use of assistive devices.

Hypophosphatasia is associated with a high burden of fractures and orthopedic/dental surgery, pain, mobility impairment, need for assistive devices for walking, decreased functional status, and ADL abnormalities in children and adults (Berkseth et al., Bone. 54:21–27, 2013; Coe et al., J. Bone Joint Surg. Surg. American volume. 68:981–990, 1986; Weber et al., Metabolism: clinical and experimental. 65:1522–1530, 2016). This study will evaluate the reasons for assistive device use, including pain, weakness, fatigue, balance, fear of falling, or fear of fracture. Information on the types of devices used (e.g., crutches, wheelchair dependent [all the time], wheelchair dependent [part of the time], walker, cane, leg brace, ramp, bath/shower modification, grab bars/rails, or other assistive devices) can also be collected. Assistive device use can be monitored continuously throughout the study.

Peripheral high-resolution quantitative computed tomography.

High-resolution peripheral quantitative computed tomography (HR-pQCT) is a noninvasive, low-dose technique for assessing bone microarchitecture and volumetric bone mineral density (BMD) in the cortical and trabecular regions of the distal radius and distal tibia. Its application in a clinical study has helped to understand differences in bone structure across a wide range of bone metabolism disorders, fracture risk, and bone response to various osteoporosis therapies (Cheung et al., Current oseteopor. reports. 11:136–146, 2013). Microarchitecture analysis using this methodology provides a better understanding of potential trabecular and cortical bone damage due to disease-related components. High-resolution peripheral quantitative CT will be available for all patients. HR-pQCT can be performed to assess total, volumetric trabecular and cortical BMD, cortical thickness, stiffness, trabecular number, trabecular separation, and trabecular thickness, for example, at the time points listed in Table 4.

Patient's overall impression of improvement.

The Patient Global Impression of Improvement (PGI-I) is a 1-item questionnaire used to assess the patient's overall perception of their condition relative to baseline.

- 63 049823 level. The patient is asked to rate the expected change in his/her condition in response to therapy or intervention (Yalcin et al., Am J Obstet Gynecol. 189:98-101, 2003). The PGI-I assessment can be performed at various time points, such as the time points listed in Table 4.

Safety assessments.

Patient history and demographic information.

Demographic information will include date of birth, age, gender, ethnicity, and race. Patient history will include age at onset of earliest signs or symptoms of HPP, as well as specific signs or symptoms at diagnosis and study entry, including skeletal, neurologic, constitutional/metabolic, muscular, dental, respiratory, renal, and rheumatic diseases. Patient history and demographic information may be obtained at various time points, such as the time points listed in Table 4.

Physical examination.

The physical examination will include assessment of the general appearance; skin; head, ear, eye, nose, and throat; neck; lymph nodes; chest; heart; abdomen; extremities; central nervous system; musculoskeletal system. An abbreviated physical examination will include assessment of the patient's signs and symptoms. All physical examinations (complete and abbreviated) will include weighing (using a calibrated scale) and inspection of the asfotase alfa injection sites for potential reaction(s). Complete and abbreviated physical examinations may be performed at various time points, such as those listed in Table 4.

Vital signs.

Vital signs include systolic and diastolic blood pressure (millimeters of mercury [mmHg]), heart rate (beats/minute), respiratory rate (breaths/minute), and temperature (degrees Celsius [°C] or degrees Fahrenheit [°F]). On in-clinic dosing days, vital signs may be measured 10 minutes before study drug administration. Vital signs may be measured at various time points, such as those listed in Table 4.

Pharmacodynamics.

Samples can be collected 15-30 min prior to asfotase alfa dosing to measure plasma PPi and PLP to assess potential substrates for asfotase alfa activity. Pyridoxal (PL) can be measured independently and can be reported as the PLP/PL ratio. Pyridoxal 5'-phosphate is a substrate for asfotase alfa and results in PL formation through dephosphorylating activity. Thus, the PLP/PL ratio is expected to be higher prior to treatment and lower during asfotase alfa treatment because PLP is dephosphorylated to PL (Akiyama et al., Mol Genet Metab. 125:174-180, 2018). Changes in PL reflect asfotase alfa activity when assayed as a ratio to PLP. The actual date and time (24-hour format) of each sample collection will be recorded.

Blood samples for measurement of PPi, PLP and PL in plasma can be collected at various time points, such as the time points shown in Table 4.

Biomarkers.

Hypophosphatasia is characterized by interdependent clinical manifestations resulting from the failure to mineralize bone matrix due to elevated concentrations of TNALP substrates, including PPi and PLP. Failure to mineralize bone matrix results in osteomalacia (softening of the bones). Bone turnover markers such as osteocalcin, procollagen type I N-terminal propeptide (P1NP), and serum C-telopeptide-cross-linked type I collagen (sCTX) are a collection of protein or protein-derived biomarkers released during bone remodeling by osteoblast or osteoclast activity. Bone turnover markers rapidly respond to changes in bone physiology; therefore, they are useful in determining patient response to and compliance with therapies for metabolic bone disorders (Greenblatt et al., Clin Chem. 63:464–474, 2017).

Blood samples for analysis of bone turnover markers (osteocalcin, P1NP, and sCTX) can be collected at various time points, such as the time points shown in Table 4.

Efficiency analyses.

Primary analysis.

The primary endpoint of this study is the change in plasma PPi concentrations in Group 1 from baseline to week 36. The primary analysis for the primary endpoint can be performed using an MMRM model appropriate for treatment groups and visits to estimate the change at each time point after baseline. The primary hypothesis tests the within-group change from baseline to week 36.

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Secondary analyses.

Key secondary objectives.

The change in plasma PPi concentration from baseline to week 36 in group 2, and the change in chair stand-up test score (SPPB component) from baseline to week 36, and the SF-36 PCS score in groups 1 and 2 can be analyzed in the same way as for PPi (primary analysis).

Subgroup analysis can be performed for patients with any fracture type at randomization (fracture or pseudofracture) (yes or no).

Key secondary endpoints can be ordered and tested sequentially to control for overall type 1 error rates in the following order: (1) PPi for group 2, (2) SF-36 PCS for group 1, (3) SF-36 PCS for group 2, (4) Chair Stand Up Test (SPPB component) for group 1, and (5) Chair Stand Up Test (SPPB component) for group 2.

Other secondary points.

The following endpoints can be analyzed in groups 1 and 2 in the same manner as for the change in plasma PPi concentration from baseline to week 36 (the key secondary endpoint):

change in chair stand repetition test (SPPB component) results from baseline to week 24;

change in PCS SF-36 score from baseline to week 24;

A statistical analysis similar to that performed for the primary endpoint will be performed for the following endpoints:

change in TUG test from baseline to week 36;

change in BPI from baseline to week 36;

change in LEFS scores from baseline to week 36. Exploratory analyses.

For changes from baseline to weeks 12, 24, and 36 for exploratory endpoints, the analysis can be performed in the same manner as for changes from baseline to week 36 for PPi.

The proportion of regenerated fractures or pseudofractures at week 36 can be summarized using count and percentage. Time to fracture union or regeneration can be analyzed using the Kaplan-Meier method.

To assess the strength, the change in PPi level from week 12 to week 36 can be obtained. The ratio of PLP to PL can be determined.

The asfotase alpha RK profile can provide individual serum asfotase alpha activity versus time data and actual blood sampling times, with observed activity versus time and change plotted. Summary information on serum alpha asfotase activity can be presented using descriptive statistics, with mean activity versus time profiles and comparisons between the 2 treatment groups.

Example 3: Physical function and HRQoL in adults treated with asfotase alfa for childhood-onset HPP.

Data from adults (>18 years) with childhood-onset HPP who received asfotase alfa for at least 12 months were collected in routine clinical practice at the Institute of Orthopaedics, Julius-Maximilians-University Würzburg (NCT03418389). Physical function was assessed using the Short Physical Functioning Battery (SPPB), the Lower Limb Function Scale (LEFS), and the 6-Minute Walk Test (6MWT); pain was assessed using the PainQuest instrument. The 36-item Short Form Health Questionnaire version 2 (SF-36v2) was used to assess HRQoL. The change in scores for each instrument from baseline to 12 months was analyzed. Safety issues were also recorded during this period.

In 14 included patients (11 women, 3 men), the median (minimum, maximum) age at the start of asfotase alfa treatment was 53 (20, 78) years for women and 46 (19, 57) years for men. The median (minimum, maximum) height was 160 (135, 167) cm in women and 176 (170, 180) cm in men. All patients had heterozygous mutations in the ALPL gene. In 12 patients with 6MWT data, the median (minimum, maximum) walking distance significantly increased from 272 (0, 760) m to 301 (0, 605) m after 12 months of treatment (Fig. 2A). Median (minimum, maximum) scores on the Chair Rise Test, part of the SPPB, significantly improved from 22 seconds at baseline to 13 seconds after 12 months of treatment (n=9; Fig. 2B). Significant improvements in LEFS and SF-36v2 Physical Component Summary scores were observed after 12 months of treatment (n=9; Figs. 2C and 2D). Changes in grip strength (n=12) or pain (n=8) were variable over 12 months of treatment. The safety profile of asfotase alfa in this population was similar to that reported in previous clinical trials. Although only a small number of patients were evaluated, this real-world treatment cohort of adults demonstrated improvements in physical functioning and HRWoL with asfotase alfa treatment over six or 12 months.

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Example 4. Wearable device for treatment monitoring.

A phase 4 clinical trial may be conducted in conjunction with a wearable device that is designed to be worn on the wrist of each subject receiving treatment. The wearable device includes an accelerometer and may be used to track the subject's steps and physical activity. The wearable device may be used to monitor biometric readings during the treatment regimen and may be used to monitor the effectiveness of the treatment and to help determine whether to discontinue sALP administration once effectiveness is achieved or to restart it if effectiveness is not maintained, based on changes in biometric readings.

The wearable device may be implemented as a system that further includes a peripheral electronic device, such as a smartphone, or a cloud database. Since the data is continuously monitored and measured by the wearable device, the data collected by the device may be transmitted to the peripheral device and stored on the peripheral device or in a cloud storage environment. The peripheral device may be configured to run a software application (e.g., a smartphone application) that provides the subject with navigation information for the purpose of tracking changes in the subject's physiological readings during the course of treatment (e.g., improvement over time). The data may also be transmitted to a third party, such as a physician administering the sALP-based therapy, for the purpose of monitoring the subject's treatment progress.

Study endpoints.

Illustrative endpoints and their rationale are listed in the table below.

Table 10

Endpoints for wearable devices

Proposed endpoint Justification Change from the initial average value of the amounts of average daily activity • Provide an objective measure of overall physical activity and ambulation over time The ratio of the amounts of average daily activity recorded during the most active 30 minutes of a subject's day between specified intervals and the baseline • Provide an objective estimate of an individual’s maximum physical exercise potential over time Change from baseline in average daily time spent in different activity levels (sedentary, light, moderate, and vigorous) during a sleep-deprived period • Provide an objective estimate of the distribution of an individual’s activity across various intensity states over time Change from baseline in average daily activity to sedentary state ratio • Provide an objective measure of an individual’s overall physical activity and ambulation over time Change in average daily duration of activity periods compared to baseline at different activity intensity thresholds • Provide an objective estimate of the distribution of an individual’s activity across various intensity states over time Change from baseline in average daily total sleep time • Provide an objective measure of changes in an individual’s sleep duration over time Change from baseline in average activity (numbers per minute) during sleep • Provide an objective measure of sleep quality and sleep disturbances over time

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The relationship between proposed endpoints and the clinical outcomes of interest.

For patients with HPP, particularly older and more mobile patients, especially adult patients, treatment goals include improving functional status (changes in endurance, strength, and gait), reducing fatigue, and reducing joint pain. Digital endpoints are designed to measure cumulative activity levels that are expected to change over time in response to improvements in the stated treatment goals. Various studies have demonstrated an association between accelerometer-derived measures and pain symptoms in specific populations. Similarly, accelerometer measures have been shown to correlate with functional measures such as the six-minute walk test (6MWT).

Device variant - Actigraph Insight Watch.

Accelerometric monitors are commercially available. The Actigraph Insight Watch is a wrist-worn, 510(k) Class II medical device that contains a three-axis accelerometer. The battery and memory on the device allow for continuous data collection and storage on the device for 30 days at a default sampling rate of 32 Hz. Data transfer between the device and the cloud is accomplished via the Centrepoint Data Hub, which securely transfers data via Bluetooth or USB from the device to the Centrepoint Cloud. Data stored in the Centrepoint Cloud can be uploaded by study staff directly through the Centrepoint web portal or retrieved via a web API (Application Programming Interface) with appropriate security credentials. A set of accelerometer endpoints are calculated by Actigraph using published and publicly available algorithms, including energy expenditure, METs, physical activity, active periods, sedentary periods, sleep latency, total sleep time, wakes after sleep onset, and sleep efficiency. In addition, raw 3-axis accelerometer data is available via a web API, allowing the development of alternative endpoints and statistics not calculated by Actigraph. The Actigraph Insight watch has standard watch hardware, allowing participants or study investigators to replace the provided wristband based on their preference in style or material. The device is designed to mask any recorded data from study participants and only display the time of day, date, and remaining battery life to avoid biasing wearer behavior. Additionally, the use of such a device could include periodic data collection to more effectively track long-term bone growth over weeks and months.

Benefits of the Actigraph Insight Watch include:

Insight Watch is a device that provides participants with the option of using a commercial wearable device;

the battery life and memory on the device are sufficient for the expected data collection time, minimizing the burden on participants associated with transmitting data during the study;

raw device data is available via API;

The data flow procedures are well proven, secure and offer multiple export methods;

Actigraph devices can be used to collect data.

Example 5. Treatment of an adult patient with HPP with lower limb pain.

An adult male subject has an elevated inorganic pyrophosphate (PPi) concentration of approximately 5.82 μM and a mean BOT-2 strength score of less than 10. The subject is 24 years old and has lower extremity pain and gait disturbance. The subject may be diagnosed with HPP and may be eligible for treatment. The subject may undergo radiographic examination and bone mineral density testing, both of which may demonstrate decreased bone mineralization in the legs.

A pharmaceutical formulation comprising the polypeptide of SEQ ID NO: 1 may be formulated at a concentration of 0.1 mg/mL. The formulation may be administered subcutaneously to a subject once weekly for 8 weeks at a dosage of 3.5 mg/kg/week. The subject may be assessed for treatment efficacy after 8 weeks of the treatment regimen. The subject may notice a decrease in bone pain and a normalization of gait. The subject may undergo subsequent radiographic examinations and bone mineral density tests, which may demonstrate normalization of bone mineralization relative to the pre-treatment situation. The subject's PPi concentration may decrease to below 5 μM and their BOT-2 strength score may improve to 12, indicating a therapeutic effect obtained using the polypeptide. The subject then discontinues treatment.

Six months after stopping treatment, the subject begins to experience lower extremity pain and gait disturbance. The PPi concentration increases to approximately 5.82 μM and there is a mean BOT-2 strength score of less than 10. The subject then enters a phase of the treatment regimen in which sALP is reintroduced. After one month of the second phase of treatment, lower extremity pain and gait disturbance resolve.

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Example 6. Treatment of a female adolescent with tooth loss and chronic pain.

An adolescent female subject has an elevated inorganic pyrophosphate (PPi) concentration of approximately 4.78 μM and a mean 6MWT value that is less than approximately 70% of the predicted 6MWT value. The subject is 14 years old and suffers from tooth loss and chronic pain. The subject may be diagnosed with HPP and may be eligible for treatment. The subject may undergo radiographic examination and bone mineral density testing, both of which may demonstrate bone mineralization in her teeth and femurs.

The subject is fitted with a wearable device designed to be worn on the wrist. The wearable device monitors her physical activity over a period of two weeks. On average, she walks 3,000 steps per day and has two periods per day of at least 5 minutes, during which her heart rate increases by at least 10%. She then begins treatment with high-dose or low-dose asfotase alfa.

A pharmaceutical formulation comprising the polypeptide of SEQ ID NO: 1 may be formulated at a concentration of 0.5 mg/mL. The formulation may be administered subcutaneously to a subject once weekly for 4 weeks at a dosage of 2.5 mg/kg/week. The subject may be assessed for treatment efficacy after 4 weeks of the treatment regimen. The subject may notice a reduction in chronic pain. The subject may undergo subsequent radiographic examinations and bone mineral density tests, which may demonstrate normalization of her femur mineralization relative to pre-treatment. The subject's PPi concentration may decrease to less than 4 μM and her 6MWT score improves to approximately 85% of the predicted value, indicating a therapeutic effect obtained using the polypeptide. The wearable device is worn for two weeks (weeks 3 and 4 of the treatment regimen) and shows that her steps per day can increase to approximately 8,000 steps per day and 8 periods per day of at least 5 min duration, with her heart rate increasing by at least 10%. The subject can wear the wearable device for one or more two-week periods and then be assessed. It can be determined that she can stop administering sALP and she can be monitored periodically for any signs of recurrence of symptoms.

Example 7. Bone turnover and mineral metabolism in adult patients with childhood-onset HPP treated with asfotase alfa.

We assessed changes in bone turnover markers in adults with childhood-onset HPP treated with asfotase alfa. We conducted an observational study of patients aged >18 years (y) with childhood-onset HPP treated with asfotase alfa for 12 months (mo) during standard of care at a single center. Data were obtained from 14 patients aged 19-78 y (median 52 y) with combined heterozygous ALPL mutations and were assessed for skeletal (at least 1 fracture) and other HPP history. At baseline, serum pyridoxal-5'-phosphate (PLP) concentrations median 286 ng/mL and decreased significantly (n=12, P=0.005) after 12 mo of treatment to 8.5 ng/mL (normal: 5-30). There were significant reductions in the urinary phosphoethanolamine (PEA)/creatinine ratio (n=11, P=0.008) with a decrease from 54.9 mmol/mol creatinine at baseline to 22.4 mmol/mol creatinine at 12 min (normal: 2.3-11.3). The transient increase in parathyroid hormone levels of 1-84 at 3 min of treatment was not accompanied by changes in calcium or phosphate levels.

A significant transient increase in procollagen type I N-propeptide (PINP) at 3 min and osteocalcin at 3 min and 6 min was observed; both returned to near baseline levels by 12 min. Numerical increases in collagen type I N-telopeptide (NTx) and tartrate-resistant acid phosphatase 5b (TRAP 5b) were not statistically significant.

The first systematic evaluation of bone turnover markers during treatment with asfotase alfa shows that, in addition to significantly reducing PLP and PEA and improving bone mineralization, asfotase alfa promotes bone remodeling in adults with childhood-onset HPP.

Example 8. Treatment of an adult patient with HPP with an atypical femur fracture (AFF).

An adult male subject (age 52) with childhood-onset HPP has AFF but no other bone mineralization defects. The subject can be treated with asfotase alfa, given at a dose of 6 mg/kg/week or a lower dose, for three months. The subject can be monitored monthly for fracture healing. After three months, if moderate but not complete healing of the fracture is observed, treatment can be continued for an additional three months at the same dose. AFF can be monitored during treatment until resolution. Once resolution, a lower dose should be tried. Generally, therapy is recommended for the life of the patient to minimize any potential problems associated with drug antibodies.

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Example 9. Treatment of an adult patient with AFF.

An adult female subject (45 years of age) without HPP who is being treated with a bisphosphonate has AFF. The subject can be treated with asfotase alfa, administered at a dose of 6 mg/kg/week, for six months. The subject can be monitored monthly for fracture healing. After three months, if complete healing of the fracture is observed, therapy can be stopped early. The patient can be prescribed three months of physical therapy and can resume normal physical activities six months after the injury.

Example 10. Physical function and health-related quality of life in adults treated with asfotase alfa for childhood-onset HPP.

Materials and methods.

Study design.

This was an observational, retrospective and prospective, single-center study in adults (aged >18 years) diagnosed with childhood-onset HPP who were treated with asfotase alfa in routine clinical practice for at least 30 months.

Study population.

Adults with childhood-onset HPP (confirmed by low alkaline phosphatase and/or alkaline phosphatase gene mutation (ALPL) and clinical signs and/or symptoms consistent with HPP) who had been receiving asfotase alfa for at least 30 months were included. All subjects provided informed consent prior to study entry. Treatment for HPP and asfotase alfa were administered according to clinical practice, with continued physiotherapy and manual therapy similar to previously established individual requirements. The initial dose of asfotase alfa was determined by the treating physician. The recommended dosing regimen for asfotase alfa is subcutaneous injection of either 2 mg/kg body weight 3 times weekly or 1 mg/kg body weight 6 times weekly.

Data collection.

Data on demographics, age at HPP initiation, and HPP-related events were collected retrospectively from electronic and printed medical records for the lookback period corresponding to the time from study entry to the earliest documented date of HPP diagnosis (or birth, if applicable). Data collected both retrospectively and prospectively included clinical symptoms, physical function assessment, HRQoL assessments, adverse events (AEs), and asfotase alfa treatment information. Data collection was based on available information on assessments routinely performed in this patient population at the study center (i.e., no out-of-standard-of-care studies were performed). Baseline clinical assessments were typically recorded 6 months before initiation of enzyme replacement therapy. Follow-up assessments, including data collection, were routinely scheduled at 3, 6, 12, 18, 24, and 30 months of treatment.

Detailed description of procedures.

Brief Physical Performance Battery (SPPB).

The SPPB is a summary performance measure consisting of a 4 m walking speed at a usual pace, a timed repeated chair stand test, and 3 standing balance tests of increasing difficulty (not yet performed). Each performance measure was assigned a categorical score ranging from 0 (failure to perform) to 4 (optimal performance). A composite score, ranging from 0 (poorest performers) to 12 (best performers), was calculated by summing the scores of the 3 components.

Walking speed for 4 meters.

The 4-meter walking speed test is part of the SPPB. The participant also had to be able to walk unaided without the use of a cane or walker. Walking speed was measured indoors on a level surface marked at the start and 4 meters from the start. The patient was instructed to walk at his or her normal pace from the start mark, passing the 4-meter mark without hesitation. The stopwatch was started when the examiner said start and stopped when the first foot crossed the finish line. This test was repeated 2 more times. The faster result of the two tests was used to calculate the walking speed.

Chair Rise Test.

The chair rise test was a method of measuring strength and endurance. A straight-backed chair without armrests (seat height 43 cm = 17 inches) was used. During the test, the participant was seated in the middle of the chair with the legs bent at 90° and the arms crossed in front of the chest. The feet were kept flat on the floor. The stopwatch was started when the examiner said start. The participant rose to a standing position and sat back down completely as quickly as possible. This procedure was repeated five times. The clock was stopped when the participant reached the final standing position.

6-minute walk test.

The 6MWT was conducted to assess the maximum distance a person can walk in 6

- 69 049823 minute period. The walking distance was to be 30 m in length. The length of the corridor was marked every meter. The turning points were marked with a cone or something else. The starting line, which marked the beginning and end of each 60-meter circle, was marked on the floor using brightly colored tape.

Wrist dynamometer test.

Grip strength was measured for the dominant and non-dominant hand using a hand dynamometer. A total of three procedures were performed for each right and left hand. The pause between measurements was 20 s. The test was performed with the participant sitting on a chair without armrests. First, grip strength was measured for the dominant hand. The elbow was bent at an angle of 90°, and the wrist was in a comfortable position (approximately 0-30° of wrist extension). Any contraindications to the test were noted.

Variables.

Physical function.

Patients' physical function was assessed using validated qualitative and quantitative measures. The primary outcome measures were the 6-minute walk test (6MWT), the timed stand and go (TUG) test, the Short Physical Performance Battery (SPPB; a summary measure of performance consisting of the balance test, 4-m usual walking speed test, and the repeated chair stand test), and the hand dynamometer test (grip strength). The Lower Extremity Function Scale (LEFS) was a secondary physical function measure. If applicable, whether the patient required an assistive device (eg, crutches or walker) to perform these assessments was documented.

Health-related quality of life and pain.

HRQoL was assessed as a secondary outcome using the Short Form 36-Item Health Survey version 2 (SF-36v2). Pain prevalence was assessed using the following five categories: never, rarely, sometimes, often, or always. If pain was present, its intensity was quantified using a 10-item Likert scale. Information on patients' use of pain medication was also collected.

Injection site reactions and safety reporting.

All patients were advised to monitor injection sites at all times and to document any local or systemic observations they may have made themselves in relation to injections. All serious and non-serious AEs that were reported by the patient and/or that were identified in response to open-ended questions from the treating physician or that were identified during observation, physical examination or other study procedures were documented in the study protocols/patient source documents.

Data analysis.

SPSS Statistics 25 statistical software (SPSS Inc., an IBM company, Chicago, IL, USA) was used for data analysis. Quantitative variables were presented as medians, means with interquartile range (IQR) or means with standard deviation (SD). They were tested for normal distribution using the Shapiro-Wilk test. Because of significant deviations from normal distribution, subsequent analyses were performed using nonparametric methods. Comparisons between baseline and 3, 6, 12, 18, 24, and 30 months of treatment for each variable were assessed using the Wilcoxon matched-pairs test; only patients with data for both time points compared were included in the statistical analysis. Absolute and percentage frequencies are presented for qualitative variables. All tests were two-sided with a significance level of 5%. Because this analysis was primarily descriptive, alpha correction for multiple testing was not applied. Presentation of longitudinal results included only those patients for whom data were available for all 4 time points.

Results.

At the start of therapy, 21 patients received a weekly median dose (mg/kg BW) of asfotase alfa of 6.0 mg, range 3.0 mg - 6.0 mg (mean mg/kg BW 5.4 ± 1.1). This corresponds to a weekly median dose of 6.3 mg/kg ideal body weight (iBW). Most patients (n = 19) received asfotase alfa injections 3 days per week, two patients received injections 4 days per week. Thirteen patients received a total of 6 syringes per week, 4 patients received 3 syringes, 2 patients received 9 syringes, and 1 patient received 2 or 4 syringes, respectively. The median body weight for asfotase alfa dose calculation was 65.0 kg, range 46.0 - 123.0 kg, mean weight was 73.2 ± 21.0 kg. During the treatment period, there were no more than 5 changes in the weekly dose of asfotase alfa per patient. Seventeen patients had at least 1 change, an additional change occurred in 9 patients, 3 patients had 3 changes, 2 patients had 2 changes, and 1 patient had 5 changes in the weekly dose of asfotase alfa. Details of the changes in the weekly dose of asfotase alfa (mg/kg BW) are presented in Fig. 5. Changes in the weekly dose of asfo

- 70 049823 alpha tazy in mg/kg iBW are presented in Fig. 6. Dose adjustments were made in accordance with best clinical practice at the discretion of the treating physician. Actual body weight (BW) refers to the true weight of the subject. Ideal body weight (iBW) refers to the estimated body weight of the subject, which is an adjusted weight that takes into account the subject's physical variables such as age, gender, height, body size, wrist size, etc.

Demographic data and clinical characteristics of patients at baseline.

A total of 14 patients (11 women, 3 men; mean (SD) age 51 (17) years, range 19–78 years (Table 11)) were included in the analysis. All patients were unrelated, had coexisting heterozygous ALPL mutations, and had documented skeletal manifestations of the disease (including history of at least one fracture) in addition to other HPP-related manifestations. Twelve patients were started on an asfotase alfa dose of approximately 6.0 mg/kg/week; two patients who were concerned about potential adverse effects were started on a dose of 3.0 mg/kg/week. All patients received the weekly dose over three days of injection. Dosing was adjusted as clinically indicated during 30 months of treatment.

Table 11 Demographic data and clinical characteristics of patients at baseline

Characteristics Patients (N=14) Gender, η (%) Women 11 (79) Age at baseline, years Mean (SD) 51(17) Range 19-78 Average height, cm (SD) Men 175 (5) Women 157(9) Average weight, kg (SD) Men 98(18) Women 73 (22) Average BMI, kg/ ^m2 (SD) Men 32(7)

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Women 30(8) Combined heterozygous mutations ALPL, η (%) 14(100) Mean ALP activity at baseline, IU/L (SD) ^a Men 14(4) Women 18(7) Mean PLP activity at baseline, ng/ml (SD) ^b 451 (390) Mean PEA/creatinine ratio at baseline, mmol/mol creatinine ⁰ 76 (59) Menopause status (women only) Postmenopause (n, %) ^d 5(45) History of NRR-related manifestations, η (%) ^e Dental 14(100) Fractures 14(100) Muscular 14(100) Neurological 6(43) Kidney 3(21) Rheumatic 3(21) Skeletal 14(100) Pain 14(100)

ALP alkaline phosphatase; ALPL=reH alkaline phosphatase; BM1=body mass index; HPP=hypophosphatasia; PEA=phosphoethanolamine; PLP=pyridoxal-5'-phosphate; SD=standard deviation.

^a Reference ranges for ALP: 53-128 IU/L (men), 42-98 IU/L (women).

^b Reference range for PLP: 5–30 ng/mL ^c Reference range for PEA/creatinine ratio: 2.3–11.3 mmol/mol creatinine; data were not available for three patients.

^d Calculated as proportion of female patients; data were not available for one patient.

^e Calculated as the proportion of patients for whom data were available.

'Excluding fractures. Diagnoses related to skeletal structure included bowing of the tubular bones, congenital deformed feet, craniosynostosis, scoliosis and kyphosis.

Physical function.

6-minute walk test.

Of the 14 patients in this study, 13 had 6MWT assessments at each time point. At baseline, the median (IQR) distance walked was 267 (0-368) m (mean [SD], 237 [223] m), which increased to 320 (234-469) m after 12 months of treatment (mean [SD], 335 [159] m) (Fig. 3A). The change in median 6MWT distance walked from baseline to 12 months was 53 m (p=0.023), representing a 20% improvement. The median changes from baseline to 3 months and from baseline to 6 months of treatment were -7 m and 39 m, respectively. The change in 6MWT distance from baseline to 18 and 24 months was not significantly different (p=0.221 and p=0.060, respectively). The median distance was similar at 12 and 24 months (p=0.115) and at 30 months (p=0.133) (Fig. 7).

Seven of the evaluable patients required assistive devices to complete the 6MWT at baseline (three patients used crutches, four used a walker). Two of these patients were able to complete the test unaided later in the study; one patient was able to complete the test unaided at 3 months, and the other patient was able to complete the 12-month assessment without assistive devices. None of the patients who walked unaided at baseline required assistance at any other time point during the study.

Timed test get up and go.

A total of nine patients were able to complete the TUG test at all time points. The median (IQR) TUG test completion time was 14.4 (10.7-28.9) s at baseline and decreased to 11.3 (9.4-16.9) s after 12 months of treatment (Fig. 3B). The change in median test completion time from baseline to 12 months of treatment corresponded to an improvement of 22% and was statistically significant (p=0.008). The median (IQR) test completion time at 3 months and 6

- 72 049 823 months of treatment were 13.6 (10.9-20.3) s and 12.2 (10.1-17.8) s, respectively; this change was statistically significant when compared at baseline and after 6 months of treatment (p=0.021). One patient required an assistive device to complete this test at baseline, but no longer required it after 3 months of treatment.

Significant differences in TUG duration were observed from baseline to 12, 18, and 24 months (p=0.008, p=0.018, and p=0.013, respectively). Early improvements in median TUG completion time at 12 months remained stable from 12 to 24 months of treatment (p=0.382). Duration at 30 months was similar to that at 24 months (Fig. 10).

Brief Physical Functioning Test Battery.

A total of 10 patients completed the 4-m gait speed test, the feet-up-and-down components of the SPPB sequentially at baseline and follow-up visits; 9 patients completed the chair stand-up test at all time points. The median (IQR) 4-m gait speed was 0.8 (0.7–1.1) m/s at baseline and 1.1 (0.8–1.4) m/s after 12 months of treatment (Fig. 3C). The change in median usual gait speed was significant when compared at baseline to after 12 months of treatment (p=0.007), representing a 38% improvement. After 3 months and 6 months of treatment, the median (IQR) gait speed was 1.1 (0.7–1.2) m/s and 1.0 (0.8–1.3) m/s, respectively.

Significant differences in walking speed were observed from baseline to 12 months (p=0.007) and 24 months (p=0.017), while the change from baseline to 18 months was not significantly different (p=0.173). The slight decrease in median walking speed from 12 to 24 months was not statistically significant (p=0.610). Median walking speed significantly increased at 30 months (p=0.012) compared with baseline (Figures 8A and 8B).

Four patients required an assistive device to complete this assessment at baseline; two of these patients performed the task without assistance at 3 months of treatment, and the other patient no longer required an assistive device to complete the test after 6 months of treatment. The median (IQR) time to complete the repeated chair rise test was 22.2 (16.2–30.8) s at baseline and 13.0 (10.8–22.4) s after 12 months of treatment (Fig. 3D). Changes from baseline were significant at 3, 6, and 12 months of treatment (all p=0.008), representing a 41% improvement.

Significant differences in chair rise time were observed from baseline to 12, 18, and 24 months (p=0.008, p=0.012, and p=0.008, respectively). Early improvements in median chair rise time at 12 months remained stable from 12 to 24 months of treatment (p=0.398). Chair rise time at 30 months was similar to that at 24 months.

At baseline, all patients were able to maintain a standing position with feet perpendicular to each other for 10 s. One patient was unable to perform this task after 3 months of treatment, but was able to perform it after 6 months and 12 months of treatment.

At baseline, eight patients were able to maintain a perpendicular footstand for 10 s. Two patients who were unable to perform this task were able to perform it after 12 months of treatment. One patient who was unable to perform the task at baseline was unable to perform it after 12 months of treatment. The 30-month data are presented in Fig. 9.

Additional physical therapy.

The effects of additional physical therapy were also investigated. Data were available for 16 patients; of these, 15 patients received physical therapy, 8 patients received massage, 5 patients received manual therapy, 4 patients received thermotherapy, and 2 patients received lymphatic drainage (see Table 12).

Regarding walking ability, 13 out of 21 patients (61.9%) required walking assistance, and 8 patients (38.1%) could walk without assistance at baseline.

At month 3, 8 patients (38.1%) required assistance with walking, and 11 patients (52.4%) were able to walk without assistance. Data were missing for 2 patients.

At month 6 and month 12, 6 patients (28.6%) required assistance with walking, and 14 patients (66.7%) were able to walk without assistance. Data were missing for 1 patient.

At month 18, 4 patients (19.0%) required assistance with walking, and 12 patients (57.1%) were able to walk without assistance. Data were missing for 5 patients.

At month 24, 5 patients (23.8%) required assistance with walking, and 11 patients (52.4%) were able to walk without assistance. Data were missing for 5 patients.

At month 30, 5 patients (23.8%) required assistance with walking, and 9 patients (42.9%) were able to walk without assistance. Data were missing for 7 patients. A summary of the assistive devices used is presented in Table 12.

Fourteen of the 21 patients reported exercising; 4 patients reported exercising rarely, 8 patients reported exercising at least once a week, and 2 patients reported exercising at least twice a week.

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Table 12

Assistive devices used for walking__________

Visit Initial level Month 3 Month 6 Month 12 Month 18 Month 24 Month 30 Device N % N % N % N % N % N % N % Crutches 8 38.1 4 19,0 4 19,0 2 9.5 1 4.8 1 4.8 1 4.8 Walkers 1 4.8 1 4.8 1 4.8 1 4.8 0 0 2 9.5 1 4.8 Wheelchair 4 19,0 3 14.3 1 4.8 3 14.3 3 14.3 1 4,5 2 9.5 Cane 0 0 0 0 0 28.6 0 0 0 0 1 4.8 1 4.8 Total 13 61.9 8 38.1 6 0 6 28.6 4 19,0 5 23.8 5 23, 8 Absent 8 38.1 13 61.9 15 71.4 15 71.4 17 81,0 16 76.2 16 76, 2

Grip strength (dominant hand).

Grip strength measurements were available for 12 of 14 patients at all time points. Median (IQR) grip strength was 22.7 (11.6-25.3) kg at baseline and 22.5 (14.3-25.4) kg at 12 months of treatment. At 3 months and 6 months of treatment, median (IQR) grip strength was 22.8 (12.4-25.1) kg and 22.7 (13.6-25.7) kg, respectively. Although changes were significant at 6 months (p=0.031) and 12 months of treatment (p=0.046) compared with baseline, the change represented an improvement of only 1%. The effect size was small, so the changes are unlikely to be clinically significant.

Statistically significant change in dominant hand grip strength could be observed when comparing from baseline to month 6 (p=0.033), to month 18 (p=0.008), to month 24 (p=0.015), and to month 30 (p=0.028). The difference was not statistically significant at months 3 and 23.

All patients showed an increase in grip strength in the dominant hand, with 10 patients at months 3 and 12, 13 patients at months 6 and 18, 11 patients at month 24, and 10 patients again at month 30. A worsening of grip strength was observed in 9 patients at months 3 and 12, 6 patients at month 6, 2 patients at month 18, 4 patients at month 24, and 3 patients at month 30 (Figs. 11A and 11B).

Lower Extremity Function Scale (LEFS).

Ten of the 14 patients included in this study had LEFS data for all time points. At baseline, the median (IQR) LEFS score was 24 (17-54) points, which increased to 53 (36-65) points after 12 months of treatment (Fig. 4A). The change in score from baseline to 12 months of treatment was significant (p=0.002) and corresponded to an improvement of 121%, indicating an improvement in the ability to perform activities of daily living. The median (IQR) LEFS score was 44 (31-56) points after 3 months of treatment and 52 (36-63) points after 6 months of treatment (p=0.009 and p=0.010, respectively, compared with baseline).

Significant differences in LEFS scores were observed from baseline to 12, 18, and 24 months (p=0.002, p=0.004, and p=0.004, respectively). Early improvements in median LEFS scores at 12 months remained stable from 12 to 24 months of treatment (p=0.813).

The median as well as the mean LEFS total score significantly increased at all visits compared with baseline. The increase in LEFS total score was significant at all time points tested (3, 6, 12, 18, 24, and 30 months) compared with baseline (Wilcoxon signed rank test, p=0.005, p=0.001, p=0.000, p=0.002, p=0.002, and p=0.006, respectively) (Fig. 14).

Health-related quality of life and pain.

Short Form 36-Item Health Status Questionnaire Version 2.

A total of 9 patients completed the SF-36v2 at all four time points. At baseline, the median (IQR) physical component summary (PCS) score was 26 (21-31), which increased to 33 (26-45) at 12 months of treatment (p=0.010) (Fig. 4B); an improvement of 27%. Changes were also significant from baseline to 3 months (p=0.028). The median (IQR) mental component summary (MCS) score was 53 (33-60) at baseline and 56 (39-60) at 12 months of treatment (Fig. 4C); an improvement of 5%. No statistically significant changes were observed for the MCS score at any time point compared with baseline.

Significant differences in PCS scores were observed from baseline to 12, 18, and 24 months (p=0.010, p=0.003, and p=0.003, respectively). Early improvements in Sf-36v2 PCS scores at 12 months remained stable from 12 to 24 months of treatment (p=0.422). The 30-month PCS scores are presented in Fig. 12 and were similar to those at 24 months.

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No significant differences in MCS scores were observed from baseline to 12, 18, and 24 months (p=1.000, p=0.131, and p=0.424). Median SF-36v2 MCS scores were similar at 12 and 24 months of treatment.

Pain.

Information on the prevalence of pain by category (never, rarely, sometimes, often, or constantly) was available for all 14 patients at baseline, for 12 patients at 3 months and 6 months, and for 13 patients at 12 months. With the exception of one patient at baseline, all patients reported having pain at any given time point. At baseline, 9/14 patients (64%) reported constant or frequent pain; this proportion decreased to 3/12 patients (25%) at 6 months and 5/13 patients (38%) at 12 months. Pain intensity data were available for 13 patients at baseline, 11 patients at 3 months and 6 months, and 13 patients at 12 months; when pain was present, its intensity was quantified using a 10-point Likert scale (1=least pain, 10=worst possible pain). The median (IQR) pain intensity at baseline was 6 (4–8.25) points, which decreased to 5 (4–6) points after 12 months of treatment (Fig. 4D); an improvement of 17%. After 3 months and 6 months of treatment, the median (IQR) pain intensity was 5 (4–7) and 4 (3.5–5.75) points, respectively. The changes in median pain intensity from baseline after 3 months and 12 months of treatment were not statistically significant; however, a significant decrease in pain intensity from baseline was observed after 6 months of treatment (p=0.036).

Twelve patients had data on analgesic medication at baseline; all were using an analgesic medication before starting treatment with asfotase alfa. Eight of these patients were using analgesic medication daily, and six patients were using a combination of analgesic medications. After 6 months of treatment, two of these patients were able to discontinue their analgesic medication; one of these patients has not used an analgesic medication for 12 months. Over the course of the study, four patients reduced their analgesic medication use from daily to as-needed.

A statistically significant reduction in pain was observed at 18 months (p=0.011); however, the reduction in pain at 12 months (p=0.139) and 24 months (p=0.380) was not statistically significant. Changes in pain varied from 12 to 24 months.

After 30 months, the number of patients reporting pain was noted to decrease over time for almost all pain sites except the forearm (Fig. 13).

Injection site reactions and safety reporting.

A retrospective assessment of available patient records and longitudinal data from photo documentation of injection sites and injection site reactions showed that half of the patients (n=7) self-administered asfotase alfa injections and half of them had the drug administered by their partner or friend.

Although patients were instructed to alternate between four subcutaneous injection sites, including the abdomen, thigh, upper arm, and buttock, only one patient consistently used all injection sites. Eight patients used three different sites; three patients used two sites; and two patients tolerated injections at only one injection site (one abdomen, one buttock).

A total of 11 of 14 patients reported injection site redness and/or tenderness of varying intensity and duration at some point during the first 3 months of treatment; this increased to 13 patients by 12 months of treatment. The injection sites affected were the abdomen (n=12), thighs (n=4), and upper arm (n=3). Comparison of available photographs in the study revealed that five patients had subtle initial signs of soft tissue stretching during the first 3 months of treatment, including a protrusion of subcutaneous fat, consistent with lipohypertrophy; no bulky fat masses were palpated, but rather skin laxity, consistent with subcutaneous fat degeneration providing inadequate anchorage to the overlying skin. Of the 11 women included in this study, such changes were visible on the abdomen in nine of these patients at 12 months of treatment; All of these patients had extensive abdominal adipose tissue prior to treatment. None of these soft tissue distensions improved with time; however, these findings did not lead to interruption or discontinuation of treatment. In the two nonobese women (one of whom did not inject into the abdomen), no tissue distension was observed at any of the injection sites. No corresponding tissue distension was observed in the men, although two of them had extensive abdominal adipose tissue.

In addition to injection site reactions, a total of 46 AEs were reported in patients treated with asfotase alfa for 12 months; all patients experienced at least one AE. The majority of these events (n = 33) were not related or were only marginally related to asfotase alfa treatment; rather, they were related to the underlying disease and/or

- 75 049 823 comorbidities such as degenerative spinal disease, low back pain/lumbago, osteoarthritis of the knee, myogelosis (muscle tension/stiffness), severe trochanteric pain syndrome and skin irritation. The 13 AEs reported as possibly related to asfotase alfa treatment were fatigue (n=2), weight gain (n=2), headache (n=2), back pain, pain increased, decreased ability to perform daily activities, stress fracture, increased intraocular pressure, small bowel obstruction and skin irritation (n=1 each).

Discussion.

The results of this real-world cohort study demonstrate that asfotase alfa is effective in improving physical functioning and HRQoL in adults with childhood-onset HPP. The study also highlights the relevance of specific assessment tools to evaluate treatment effectiveness and monitor patient improvement over time.

There were more women (n=16) than men (n=5) in this 30-month analysis. The mean age of women was 52.0 years and the mean age of men was 38.4 years. The mean BMI was 30.0 kg/ ^m2 , range 18.0-50.5 kg/ ^m2 at baseline. There was no significant change between baseline and V3, V6, or V12 and V18, but there was a significant increase in BMI when comparing V0 with V24 and V30 with p=0.003 in each case. In addition, a significant increase in weight could be noted when comparing V0 with V24 and with V30 with p=0.002 in each case.

When assessing the physical constitution, significant changes were found in the phase angle (significant increase at V18 with p=0.002), body cell mass nutrition (significant decrease at V12 with p=0.044 and at V18 with p=0.023), muscle mass nutrition (significant increase at V18 with p=0.034) and fat mass nutrition (significant increase at V18 with p=0.039, V24 with p=0.028 and V30 with p=0.037).

At 6MWT, the observed changes in walking distance were not statistically significant at VI8 and V30, but the change was statistically significant at all other visits (V3, p=0.012; V6, p=0.004; V12, p=0.002, V24, p=0.019). Regarding the use of assistive devices at VI8 and V24 (after 18 and 24 months), the number of patients requiring assistive devices decreased; 3 patients (18.8%) out of 16 required assistive devices; no patient worsened during the study.

Eleven patients had an improvement in 4 m walking speed at V3, 13 patients had an improvement at V6 (they had a faster speed), 14 patients had an improvement at V12, 8 patients had an improvement at V18, 10 patients had an improvement at V24 and 9 patients had an improvement at V30 compared to baseline. Median walking speed was significantly increased at all visits except V18: V3 (p=0.021), V6 (p=0.008), V12 (p=0.001), V24 (p=0.010) and V30 (p=0.012).

In the chair rise test, almost all patients showed improvement; rise time was shorter at all visits compared with baseline, except for 1 patient at V3, whose condition worsened. The results were statistically significant when comparing the median time at baseline with the median time at all visits V3, V6, V12, V18, V24 and V30 with p=0.002, p=0.001, p=0.001, p=0.008, p=0.005 and p=0.008, respectively.

The reduction in TUG duration was statistically significant at all visits V3, V6, V12, V18, V24, and V30 compared with baseline with p=0.004, p=0.002, p=0.001, p=0.012, p=0.008, and p=0.028, respectively. Eleven patients improved (decreased time) at V3, 13 patients improved at V6, 14 patients improved at V12, 8 patients improved at V18, 9 patients improved at V24, and 8 patients improved at V30 compared with baseline. TUG duration was longer at V3 in 3 patients and at V6, V24, and V30 in 1 patient.

Statistically significant change in grip strength in the dominant hand could be seen when comparing from baseline to V6 (p=0.033), to V18 (p=0.008), to V24 (p=0.015), and to V30 (p=0.028). At V3 and V12, the difference was not statistically significant. Improvement in grip strength in the non-dominant hand was only statistically significant when comparing baseline to V24 with p=0.011.

Data related to self-reported global health perception were not consistent but varied across visits. For example, at V3, compared with baseline, self-reported global health perception could be improved by 12 patients, had no change in 5 patients, and worsened by 1 patient. At V18, eight patients improved, 3 patients felt no change, and 2 patients reported worsening health. At V30, 6 patients improved, 3 patients felt no change, and 4 patients reported worsening health.

At all visits, a quantitative improvement in the physical functioning item was observed compared to visit 0, and this improvement was statistically significant with p=0.002, p=0.001, p=0.001, p=0.005, and p=0.005, respectively.

Again, quantitative improvements were observed in the physical role functioning item at V6, V12, and V18 compared to visit 0, and these improvements were statistically significant (p=0.009, p=0.036, p=0.016, respectively).

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The improvement in median physical pain subscale score was statistically significant when comparing baseline with V3, V6, V12, and V18 with p=0.044, p=0.030, p=0.002, and p=0.027, respectively.

A significant positive change (improvement) in health perception could be observed between visit 0 and V3, V6, V18, V24 and V30 with p=0.013, p=0.001, p=0.004, p=0.011 and p=0.023 respectively, but not at V12.

Improvement in vital activity could be observed in 14, 15, 13, 11 and 8 patients at the corresponding visit V compared to baseline. This improvement in vital activity was statistically significant when comparing baseline with visits V3, V6, V12, V18 and V24 with p=0.006, p=0.001, p=0.013, p=0.005 and p=0.036, but not at V30.

Significant improvement could be demonstrated between visit 0 and V6, V12 and V18 (p=0.002, p=0.014, p=0.007). At these visits, 12 of 17 patients (V6), 12 of 18 patients (V12) and 10 of 14 patients (V18) reported improvement in social functioning.

There was a statistically significant improvement in mental health, which was recorded by 13 of 18 patients at V6 and V12 and 11 of 14 patients at V18 compared with baseline (p=0.024, p=0.014 and p=0.048, respectively).

There was a significant improvement in physical component generalization scores from baseline at all visits (p=0.003, p=0.006, p=0.002, p=0.003, and p=0.001). In contrast, the change in mental component generalization scores was not statistically significant at any visit.

The median as well as the mean LEFS total score significantly increased at all visits compared to baseline. The increase in LEFS total score was significant at all time points tested compared to baseline (p=0.005, p=0.001, p=0.000, p=0.002, p=0.002, and p=0.006).

Individual Pain Questionnaire: The reduction in pain score was statistically significant (p=0.015 and p=0.027) when comparing baseline with V6 and V18. In addition, it could be noted that the number of patients reporting pain decreased over time for almost all pain sites except the forearm.

Almost all patients took analgesics throughout the study, and took up to 4 different analgesics.

The results presented here showed that asfotase alfa was effective in improving physical functioning as well as QoL in the majority of patients in the study.

Physical function and HRQoL scores in this study population before initiating asfotase alfa treatment indicate a high disease burden in adult patients with childhood-onset HPP compared with a population of age-matched healthy adults. For example, the mean ±SD 6MWT distance achieved by patients at baseline in this study was significantly shorter than that observed in a population of 444 healthy adults (237 ± 223 m versus 571 ± 90 m, respectively). Similarly, the median TUG time at baseline was longer than that observed in a cohort of healthy adults aged 50 to 59 years (14.4 s versus 9.9 s, respectively); as was median grip strength (22.7 kg in this study at baseline versus 27 kg [women] and 46 kg [men] in a healthy North American population)

(America and Europe of similar age). The median SF-36v2 PCS score at baseline was also significantly lower than that observed in the general population of the country (26 versus 49, respectively); however, the median MCS scores at baseline in these populations did not differ significantly.

Among the primary outcome measures of physical function (6MWT, TUG test, SPPB, and grip strength), statistically significant improvements were observed in both the 6MWT and TUG, as well as in two of the three components of the SPPB, from baseline to, in some cases, 12 months of treatment, demonstrating the effectiveness of asfotase alfa in improving the functional health of adult patients with childhood-onset HPP. The minimal clinically important difference (MCID) in adults with HPP was established for the 6MWT (31 m); in this study, the median 6MWT distance increased by a clinically important 39 m at 6 months and by 53 m at 12 months of treatment compared with baseline. Although the improvements observed at 6 months were not significant, statistically significant improvements were noted at 12 months. Similarly, normal walking speed increased during treatment, and this improvement reached statistical significance after 12 months of treatment. In addition, improvements in physical function were observed for the TUG test time and the chair rise test. The time to complete these tests was significantly reduced after 6 months of treatment for the TUG test and after 3 months of treatment for the repeated chair rise test.

Grip strength has been recommended as an index of muscle strength in pediatric patients with HPP and has been used previously in pediatric cohort studies; however, there is limited evidence for its use as an index in adult populations and its utility is uncertain. The changes in grip strength observed with treatment in this study were not clinically

- 77 049823 significant and not as significant as the improvements in hip extensor and abductor strength that were reported in a clinical trial of patients treated with asfotase alfa. Muscle strength assessments in these patients are likely to be systematically biased by a variety of confounding factors, including pain and bone and joint problems. Therefore, it remains to be determined how best to measure muscle strength and performance in these patients.

Statistically significant improvements were also observed in the LEFS, a secondary outcome measure of physical function in this study. The LEFS score increased significantly after just 3 months of treatment with asfotase alfa and remained stable at all follow-up time points compared to baseline. These increases were significantly greater than the 9-point MCID calculated by Binkley et al. for a lower extremity musculoskeletal dysfunction population and patients observed in a clinical trial population of adults treated with asfotase alfa for a similar period of time, and demonstrated an improvement in patients’ ability to perform daily activities.

Improvements in physical well-being were also reflected in significant improvements in secondary HRQoL outcome measures. Improvements in the median SF-36v2 PCS score over the course of the study were statistically significant at 3 months and 12 months of asfotase alfa treatment compared with baseline. This indicates an overall improvement in physical health, a comprehensive assessment of the domains of physical functioning, physical role functioning, bodily pain, and general health. Although the median MCS score (which covers measures of vital activity, social functioning, emotional role functioning, and mental health) was also slightly increased at all time points compared with baseline, this was not significant. Given that few studies have described the mental and emotional impact of HPP in adult patients, the similarity in MCS scores observed in the general population and in this study cohort at baseline suggests that the SF-36v2 may not be an accurate measure of mental well-being in patients with HPP.

The negative impact of pain on HRQoL is well known. All patients included in this study experienced pain; however, although the median pain intensity reported in the cohort significantly decreased after 6 months of treatment with asfotase alfa, pain intensity increased again after 12 months of treatment. Given that significant improvements in physical function were observed during this time period, it is possible that this is due to patients becoming more active during the course of their treatment. A 5-year study of the efficacy of asfotase alfa in adult and adolescent patients with HPP found that greater reductions in pain levels were observed after 5 years of treatment; therefore, it is also possible that the follow-up period in this interim analysis was too short to observe any meaningful reduction in pain.

Patient perception of overall pain may also change over time, and this assessment tool may not be specific enough to monitor consistent changes in HPP-related pain in adults.

The incidence of AEs and safety events in the present study was similar to that seen in clinical trials in which asfotase alfa was well tolerated; no new safety signals emerged from the present study. Injection site reactions, particularly injection site erythema and tenderness, appear to be common. Soft tissue changes at abdominal injection sites, including lipodystrophy with skin laxity resembling lipohypertrophy, are also common and appear to be more prevalent in women with extensive abdominal adipose tissue.

Taken together, these results indicate that asfotase alfa is an effective treatment for improving physical functioning and HRQoL in adult patients with HPP and demonstrates a favorable safety profile.

Several of the physical function and HRQoL measures used in this study have been recommended for treatment follow-up in adults with HPP, including the 6MWT and SF-36v2. The results of this study suggest that the specific physical function and HRQoL measures used in this analysis, including the 6MWT, TUG, SPPB Chair Rise Test, LEFS, and the PCS component of the SF-36v2, can and should be used to measure the effectiveness of asfotase alfa, some as early as three months after treatment initiation. Among these measures, LEFS had the greatest percentage improvement from baseline to 12 months of treatment in this patient population, suggesting that LEFS may be the most appropriate scale to assess treatment effectiveness on functional outcomes in adults. These tests should be considered as part of standard practice by treating physicians for monitoring early treatment response in adults with HPP, particularly because they are practical and easy to perform in a clinical setting with minimal technical equipment and training. These measures may also be useful for monitoring long-term treatment efficacy, such as monitoring patients treated for more than 12 months.

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In addition to these data on validated physical function and HRQoL assessments, all 14 patients included in this study notably had a history of dental pathology. This highlights the importance of good dental networks in communities to ensure that metabolic bone diseases (including HPP) are recognised early and appropriate follow-up treatment can be implemented. This approach is particularly important given that dentists may often be among the first healthcare professionals in the clinical pathway of patients with HPP and other metabolic bone diseases.

In addition, 53% of patients included in this study had the c.571G>A mutation in the ALPL gene. Although careful evaluation of family history did not reveal consanguinity between participants, this could not be excluded since a haplotyping approach was not used. However, a similar proportion of individuals (55%) were found to carry this mutation in a comparable HPP cohort, confirming that this mutation is common in patients of European descent with HPP. These results provide a starting point for how to monitor the treatment of these patients, in particular what can be expected in terms of treatment efficacy and clinical improvement, and what to focus on to reduce adverse events.

Conclusion.

These data from a real-world cohort of adult patients with childhood-onset HPP suggest that asfotase alfa may be effective in improving physical functioning and HRQoL, and detail the relevance and suitability of specific practical assessment tools, including the 6MWT, the chair stand-up test component of the SPPB, the TUG test, LEFS, and the PCS component of the SF-36v2, to assess the effectiveness of asfotase alfa and monitor improvements in patients over time.

A 30-month analysis of this non-interventional study assessed data from routine use of asfotase alfa in patients with childhood-onset HPP in routine clinical practice. These clinical trial results assessed efficacy and safety.

This study suggests that patients treated with asfotase alfa showed an improvement in physical functioning (6MWT, 4m walk test speed, chair rise test, TUG, dominant hand grip strength). There was also an improvement in QoL measured by the SF36 questionnaire and LEFS, which was statistically significant. Moreover, pain levels and analgesic consumption were reduced. Finally, it was noted that the dose of asfotase alfa could be reduced in most patients.

In general, the results of physical functioning tests as well as QoL questionnaires provide evidence of the effectiveness of treatment. Moreover, these assessment tools can also be used to determine the timing of treatment, such as when treatment is needed or when treatment regimens need to be modified to improve the effect or to reduce the dose of the treatment without losing the improvement.

Example 11. Observation of treatment of an adult patient with HPP with lower limb pain.

An adult male subject has an elevated inorganic pyrophosphate (PPi) concentration of approximately 5.82 μM, a mean BOT-2 strength score of less than 10, and a TUG test score of approximately 14.4. The subject is 46 years old and has lower extremity pain and gait disturbance. The subject may be diagnosed with HPP and may be eligible for treatment. The subject may undergo radiographic examination and bone mineral density testing, both of which may demonstrate decreased bone mineralization in the legs.

A pharmaceutical formulation comprising the polypeptide of SEQ ID NO: 1 may be formulated at a concentration of 0.1 mg/mL. The formulation may be administered subcutaneously to a subject once weekly for 12 weeks at a dosage of 6 mg/kg/week. The subject may be assessed for treatment efficacy after 12 weeks of the treatment regimen. The subject may notice a decrease in bone pain and a normalization of gait. The subject may undergo subsequent radiographic examinations and bone mineral density tests, which may demonstrate normalization of bone mineralization relative to the pre-treatment situation. The subject's PPi concentration may decrease to below 5 μM, their BOT-2 strength score may improve to 12, and their TUG score may decrease to 11.3, indicating a therapeutic effect obtained using the polypeptide. The subject then discontinues treatment or, alternatively, receives treatment if and when symptoms recur.

Other embodiments

The foregoing detailed description and examples are given for clarity of understanding only. No unjustified conclusions about any limitations should be drawn from them. The present invention is not limited to the detailed data presented and described, since the present invention defined by the claims includes variations obvious to a person skilled in the art.

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Claims (21)

Unless otherwise indicated, all numbers expressing quantities of components, molecular weight values, etc. used in the present specification and claims are to be understood as being modified in all instances by the term "approximate." Accordingly, unless otherwise indicated, the numerical parameters set forth in the specification and claims are approximations that may vary depending on the desired properties sought to be achieved by the present invention. At the very least, and not as an attempt to limit the doctrine of equivalents to the scope of the claims, each numerical parameter should at least be construed in light of the number of reported significant digits and by applying standard rounding techniques. Although the numerical ranges and parameters defining the broad scope of the present invention are approximate values, the numerical values indicated in the specific examples are presented as precisely as possible. However, all numerical values by definition provide a range that is inevitably obtained as a result of the standard deviation determined in their respective measurements during testing. The entire disclosures of all patents, patent applications, including provisional patent applications, publications, including patent publications and non-patent publications, and electronically available materials (including, for example, nucleotide sequence representations in, for example, GenBank and RefSeq, and amino acid sequence representations in, for example, SwissProt, PIR, PRF, PDB, and translated sequences from annotated coding regions in GenBank and RefSeq) mentioned herein are incorporated by reference. The foregoing detailed description and examples are provided for clarity of understanding only. No undue limitation should be drawn from them. The present invention is not limited to the detailed information provided and described, since variations obvious to those skilled in the art are included in the embodiments defined by the claims. CLAUSE OF INVENTION 1. A method for treating a bone mineralization disorder in a human subject, wherein the subject exhibits a level of at least one physical metric indicator that identifies the subject as being in need of treatment for a bone mineralization disorder, the method comprising: (a) administering at least one dose of soluble alkaline phosphatase (sALP) having at least 90% sequence identity to the sequence of SEQ ID NO: 1 to a subject in a first treatment phase of the treatment regimen; (b) periodically or continuously monitoring the subject during a first treatment phase for changes in average daily activity for at least one physical metric, wherein the physical metric is monitored using a wearable device; and (c) detecting an improvement in average daily activity for at least one physical metric above a predetermined metric threshold and entering a first non-treatment phase of the treatment regimen during which the subject is not administered sALP. 2. The method according to item 1, additionally comprising: (d) periodically or continuously monitoring the subject during the first phase without treatment for a period of time for changes in average daily activity on at least one physical metric; (e) identifying a level of average daily activity for at least one physical metric indicator below a predetermined metric indicator threshold; (f) administering at least one dose of sALP to the subject in a second treatment phase of the treatment regimen; and (g) repeating steps (a)-(0) one or more times. 3. The method according to claim 1 or 2, wherein the subject is an adult. 4. The method according to any one of claims 1 to 3, wherein the bone mineralization disorder is an alkaline phosphatase deficiency. 5. The method of claim 4, wherein the alkaline phosphatase deficiency is hypophosphatasia (HPP), in particular wherein the HPP is juvenile-onset HPP. 6. The method according to any one of claims 1-5, wherein the method allows for treating or reducing the risk of a bone fracture in a subject. 7. The method according to any one of claims 1 to 6, wherein the alkaline phosphatase is tissue-non-specific alkaline phosphatase, placental alkaline phosphatase, intestinal alkaline phosphatase, engineered alkaline phosphatase, a fusion protein containing a fragment of alkaline phosphatase, or a chimeric alkaline phosphatase. 8. The method according to any one of claims 1 to 7, wherein the alkaline phosphatase comprises an amino acid sequence characterized by at least 95, 97 or 99% sequence identity with the sequence under SEQ ID NO: 1. - 80 049823 9. The method of any one of claims 1-8, further comprising monitoring the subject during the first phase of treatment for changes in at least one biochemical metric, quality of life metric, or bone tissue. 10. The method of claim 9, wherein the biochemical metric is selected from the group consisting of plasma pyrophosphate (PPi), pyridoxyl-5'-phosphate (PLP), phosphoethanolamine (PEA), and alkaline phosphatase (ALP) levels, and/or the quality of life metric is selected from the group consisting of the EuroQol Five-Item Questionnaire (EQ-5D), the Child Health Assessment Questionnaire (CHAQ), the Pediatric Outcome Data Collection Instrument (PODCI), the Child Health Unit-9D (CHU-9D), the Pediatric Quality of Life Scale (PedsQL), the Lower Limb Function Scale (LEFS), the Short Form Health Questionnaire 36 (SF-36), and the Short Form Health Questionnaire 12 (SF-12), and/or the physical metric is selected from the group consisting of the Bruininks-Oseretsky Motor Skills Test 2nd Edition (BOT-2), Bayley Scales of Infant and Toddler Development-3rd Edition (BSID-III), Muscle Strength Scale, Hand Dynamometer Test (HHD), Peabody Motor Development Scales-2nd Edition (PDMS-2), Gait Analysis, Short Physical Performance Battery (SPPB), and Timed Get Up and Go (TUG) Test. 11. The method of claim 10, wherein the SPPB includes a repeated chair stand test. 12. The method according to any one of claims 1-11, wherein the wearable device comprises an accelerometer. 13. The method according to claim 12, wherein the wearable device is designed to be worn on the arm, leg or torso of the subject. 14. The method according to claim 12 or 13, wherein the wearable device tracks one or more biometric readings selected from a change from a baseline average value of the amounts of average daily activity, a ratio of the amounts of average daily activity recorded during the most active 30 minutes of the subject's day between certain intervals and a baseline level, a change from a baseline level of the average daily time spent on various levels of activity during a period without sleep, a change from a baseline level of the average daily ratio of activity to sedentary state, a change from a baseline level of the average daily duration of periods of activity compared to a baseline level at various thresholds of activity intensity, a change from a baseline level of the average daily duration of total sleep time, a change from a baseline level of average activity during sleep. 15. The method of claim 14, wherein the different activity levels are selected from sedentary, light, moderate and vigorous. 16. The method according to any one of claims 12 to 15, wherein the average daily activity for at least one physical metric is monitored using a wearable device over a period of at least two weeks during step (b). 17. The method according to any one of claims 1 to 16, wherein in step (c) the subject is not administered sALP for a period of at least 1 month. 18. The method of any one of claims 1 to 17, wherein sALP is administered at a dosage of from about 1 to about 12 mg/kg/week. 19. The method of claim 18, wherein sALP is administered at a dosage of about 6 mg/kg/week and then the dosage is reduced to a value of about 3.6 mg/kg/week, in particular sALP is administered at a dosage of about 6 mg/kg/week for about 12 weeks and then the dosage is reduced to a value of about 3.6 mg/kg/week for about 24 weeks. 20. The method of any one of claims 1-19, further comprising treating the subject with a surgical intervention comprising one or more of arthrodesis, bone removal, placement of bone graft material, and placement of a metal structure, wherein the metal structure is a screw, rod, plate, metal frame, nail, pin, or nut. 21. The method of claim 20, wherein the at least one surgical intervention is selected from the group consisting of arthroscopy of the knee, shoulder, hip, ankle, elbow, or wrist, fracture and meniscectomy, arthroscopy and shoulder decompression, carpal tunnel release, arthroscopy and chondroplasty of the knee, removal of abutment implant, knee arthroscopy and anterior cruciate ligament reconstruction, knee replacement, hip fracture repair, atypical hip fracture repair, trochanteric fracture repair, skin/muscle/bone/fracture debridement, repair of one or both menisci after knee arthroscopy, hip replacement, shoulder arthroscopy/distal clavicle resection, rotator cuff tendon repair, radius or ulna fracture repair, laminectomy, ankle fracture repair (bimalleolar type), shoulder arthroscopy and debridement, lumbar spine fusion, distal radius fracture repair, low back disc surgery, finger sheath dissection, ankle (fibula) fracture repair, femoral shaft fracture repair, and trochanteric fracture repair. -