Treatment of muscle weakness with alkaline phosphatase

Abstract

The present disclosure relates to the treatment of muscle weakness with alkaline phosphatase. The present disclosure features methods for treating or ameliorating at least one symptom in a subject having or susceptible to a muscle weakness disease, comprising administering to the subject a therapeutically effective amount of at least one recombinant polypeptide having alkaline phosphatase activity.

Description

[0001] This application is a divisional application of the invention patent filed on March 31, 2017, with application number 201780021666.7 (PCT / US2017 / 025618) and entitled "Treatment of Muscle Weakness with Alkaline Phosphatase". Background Technology

[0002] Hypophosphatase syndrome (HPP) is a rare, heritable skeletal disorder with an incidence of 1 in 100,000 births in its most severe form. When observed at birth, HPP is often fatal, with an infant mortality rate of approximately 70%. Severely affected individuals frequently die in infancy from respiratory failure due to progressive chest wall deformities. HPP can be caused by loss-of-function mutations in the gene encoding tissue-specific alkaline phosphatase (TNALP). HPP results in a range of pronounced symptoms and severity, from rickets (osteomalacia) to almost complete lack of bone mineralization in utero. Most patients exhibit skeletal changes, short stature, lower extremity pain, gait disturbances, and premature tooth loss. For example, infantile symptoms of HPP may include insufficient weight gain, the onset of rickets, impaired bone mineralization, progressive bone demineralization, rib fractures, and chest deformities, while childhood symptoms may include short stature and skeletal deformities such as bowlegs and enlarged wrists, knees, and ankles due to trumpet-shaped metaphysis. Muscle weakness (or hypotonia) is also an important symptom associated with HPP. Due to the physical impairment associated with HPP, patients with HPP often exhibit a reduced or absent ability to perform routine daily activities that healthy individuals could not perform without assistance.

[0003] In the absence of data, hypotonia in HPP has been asserted to be caused by PPi toxicity (Whyte, M.; J.). Bone Mineral Res. (January 2017) A paper shows that PPi can disrupt actin / myosin interactions in a bovine muscle model. Meat Science 84: 364-370 (2010) However, specific data on muscle weakness and PPi / ALP levels are not yet available. Early data involved skeletal changes (focusing on radiographic impressions of overall changes (RGI-C)) but failed to distinguish muscle weakness from phenotypic heterogeneity of HPP.

[0004] It is noteworthy that the treatment of HPP, particularly HPP-related outdoor injuries (such as muscle weakness) that persist for extended periods, is unknown. Therefore, there is a need for methods to treat muscle weakness associated with HPP or other conditions. Additionally, there is a need for methods to treat hypotonia or muscle weakness in human subjects caused by or related to elevated PPi and / or low alkaline phosphatase activity. Summary of the Invention

[0005] Muscle weakness has been reported as a symptom in some patients with HPP and other diseases or conditions. In HPP, elevated PPi concentrations are attributed to loss-of-function mutations in the gene ALPL, which encodes a tissue-nonspecific isoenzyme of alkaline phosphatase (TNALP; also known as liver / bone / kidney type ALP), an enzyme that is responsible for substrates such as inorganic pyrophosphate (PPi), phosphoethanolamine (PEA), and pyridoxal 5'-phosphate (PLP). This disclosure teaches a method for treating muscle weakness in subjects characterized by elevated pyrophosphate (PPi) concentrations and / or decreased alkaline phosphatase concentrations. The muscle weakness phenotype in HPP patients is often considered secondary and caused by defects in bone mineralization, which are considered a characteristic feature of HPP. Surprisingly, this disclosure teaches that muscle weakness in HPP may not be due to bone defects, as observed in wild-type (WT) mice and... AKP2 - / - No differences were observed in mouse muscles regarding the proportion of soleus muscle fiber types or the contractile properties of soleus or isolated EDL muscle. Instead, muscle weakness in HPP was found to be more correlated with elevated PPi concentrations, as reducing PPi through administration of asfotase alfa improved [the condition]. AKP2 - / - Mouse muscle grip strength. Therefore, subjects with myasthenia gravis characterized by elevated PPi concentrations, even without other symptoms of HPP or not yet diagnosed with HPP, can still be treated with Asfutex alpha. Methods for testing grip strength have been disclosed; see, for example, Whyte, M. et al., Bone 2016 Dec; 93: 125-138; Whyte, M. et al. JCI Insight 2016;27:87-102; Whyte, M. et al., Bone 2015 June; 75:229-39.

[0006] The present invention discloses (1) a method for identifying subjects (e.g., humans) suffering from or susceptible to myasthenia gravis and treating them with a recombinant polypeptide having alkaline phosphatase activity (such as TNALP, e.g., the sALP polypeptide of SEQ ID NO: 1 or a polypeptide variant having at least 95% sequence identity with the sequence of SEQ ID NO: 1, e.g., Aspheres Alpha), and (2) a method for treating such subjects with a recombinant polypeptide having alkaline phosphatase activity. Exemplary measures that can be used to assess the need for or efficacy of treatments using recombinant peptides with alkaline phosphatase activity include (1) plasma PPi and / or alkaline phosphatase concentrations, (2) the Bruininks-Oseretsky Motor Ability Test, Version 2 (BOT-2), (3) the Child Health Assessment Questionnaire (CHAQ), (4) the Pediatric Outcomes Data Collection Tool (PODCI), (5) the Bayley Infant Development Scale, Version 3 (BSID-III), (6) the Peabody Developmental Motor Scale, Version 2 (PDMS-2), (7) the Six-Minute Walk Test (6MWT), (8) muscle strength grades, and (9) the Handheld Force Measurement (HHD). The method further includes using one or more of the aforementioned indicators (e.g., plasma PPi concentration, alkaline phosphatase concentration, BOT-2, CHAQ, PODCI, BSID-III, PDMS-2, 6MWT, muscle strength grade, and HHD) alone or in any combination to evaluate the therapeutic efficacy of a recombinant peptide with alkaline phosphatase activity in subjects with or susceptible to muscular dystrophy, wherein improvement relative to a certain score or value indicates that the recombinant peptide with alkaline phosphatase activity is effective in treating muscular dystrophy. In one aspect, this disclosure provides a method for treating or improving muscle weakness in a subject suffering from or susceptible to muscular dystrophy, comprising administering to the subject a therapeutically effective amount of at least one recombinant polypeptide having alkaline phosphatase activity. In some embodiments, the subject has elevated concentrations of inorganic pyrophosphate (PPi) and / or low alkaline phosphatase activity or concentration. In one embodiment, the subject has elevated serum concentrations of inorganic pyrophosphate (PPi). In other embodiments, the subject has elevated concentrations of at least one alkaline phosphatase substrate (e.g., PPi, PLP, PEA, etc.). In another aspect, this disclosure also provides a method for identifying a subgroup of subjects who have or are susceptible to myasthenia gravis, wherein the subjects in the subgroup have elevated inorganic pyrophosphate (PPi) concentrations.

[0007] In some embodiments, the subject's muscles are not significantly different from those of a normal subject without the muscle weakness disease in at least one characteristic of such muscles. This characteristic may be selected from the proportion of muscle fiber types, fiber contractile properties, or other muscle properties known in the art. Such muscles may include any muscles of the subject, including, for example, skeletal or striated muscles, cardiac or smooth muscles. In some embodiments, such muscles include at least one type of arm and leg muscles, particularly at least one type of muscle selected from the soleus and extensor digitorum longus (EDL) muscles.

[0008] In some embodiments, the muscle weakness disease described herein is caused by elevated inorganic pyrophosphate (PPi) concentrations, such as PPi concentrations greater than about 4.5 μM. In one embodiment, the muscle weakness disease described herein is caused by elevated serum inorganic pyrophosphate (PPi) concentrations. For example, elevated PPi concentrations in samples (e.g., plasma samples) from infants or children (e.g., subjects younger than about 12 years of age) may be about 5.71 μM or greater; elevated PPi concentrations in samples (e.g., plasma samples) from adolescents (e.g., subjects aged about 13 to about 18 years of age) may be about 4.78 μM or greater; and elevated PPi concentrations in samples (e.g., plasma samples) from adults (e.g., subjects older than 18 years of age) may be about 5.82 μM or greater. In other embodiments, the muscle weakness disease described herein is caused by elevated concentrations of at least one alkaline phosphatase substrate (e.g., PPi, PLP, PEA, etc.). In some embodiments, elevated pyrophosphate (PPi) concentrations enhance the muscle weakness disease described herein in the subjects. In one implementation, the elevated serum PPi concentration enhances the myasthenia gravis as described herein in the subject. For example, the elevated serum inorganic PPi concentration enhancing myasthenia gravis may be, for example, about 5.71 μM or greater in samples (e.g., plasma samples) from infants or children (e.g., subjects younger than about 12 years of age), about 4.78 μM or greater in samples (e.g., plasma samples) from adolescents (e.g., subjects aged from about 13 to about 18 years of age), and about 5.82 μM or greater in samples (e.g., plasma samples) from adults (e.g., subjects older than 18 years of age).

[0009] In some implementations, muscle weakness is caused or aggravated by low alkaline phosphatase concentrations in the subject. For example, the concentration of low alkaline phosphatase in samples from subjects (e.g., plasma samples) may be, for example, about 90 U / L or less for subjects aged 0 to 14 days; about 134 U / L or less for subjects aged 15 days to less than 1 year; about 156 U / L or less for subjects aged about 1 year to less than 10 years; about 141 U / L or less for subjects aged about 10 years to less than about 13 years; about 62 U / L or less for female subjects aged about 13 years to less than about 15 years; about 127 U / L or less for male subjects aged about 13 years to less than about 15 years; about 54 U / L or less for female subjects aged about 15 years to less than about 17 years; about 89 U / L or less for male subjects aged about 15 years to less than about 17 years; about 48 U / L or less for female subjects aged about 17 years or older; or about 59 U / L for male subjects aged about 17 years or older. U / L or lower.

[0010] In other embodiments, elevated concentrations of at least one alkaline phosphatase substrate (e.g., PPi, PLP, PEA, etc.) enhance the muscle weakness disease described herein in the subject. The muscle weakness diseases described in this article include at least one of the following: such as hypophosphatase disease (HPP), calcium pyrophosphate dihydrate crystal deposition (CPPD), familial hypophosphatemia (such as autosomal dominant hypophosphatemic rickets (ADHR), autosomal recessive hypophosphatemic rickets, X-linked hypophosphatemic rickets, X-linked hypophosphatemia (XLH), etc.), or other diseases with a muscle weakness phenotype and elevated concentrations of at least one alkaline phosphatase substrate (e.g., PPi, PLP, PEA, etc.). In one embodiment, the muscle weakness disease described herein includes at least one of the following: for example, hypophospholipase disease (HPP), calcium pyrophosphate dihydrate crystal deposition (CPPD), familial hypophosphatemia (such as autosomal dominant hypophosphatemic rickets (ADHR), autosomal recessive hypophosphatemic rickets, X-linked hypophosphatemic rickets, X-linked hypophosphatemia (XLH), etc.), or other diseases having a muscle weakness phenotype and elevated concentrations (e.g., serum concentrations) of inorganic pyrophosphate (PPi).

[0011] In some embodiments, administration of at least one recombinant polypeptide with alkaline phosphatase activity (such as TNALP, for example, the sALP polypeptide of SEQ ID NO: 1 or a polypeptide variant having at least 95% sequence identity with the sequence of SEQ ID NO: 1, for example, Asphertex Alpha) reduces the concentration of PPi in a sample (e.g., a plasma sample) from the subject. For example, administering at least one recombinant polypeptide with alkaline phosphatase activity to a subject reduces the PPi concentration in a sample (e.g., a plasma sample) to less than about 5.71 μM for infants or children (e.g., plasma PPi concentrations of about 3.5 µM, about 4 µM, about 4.5 µM, about 5 µM, or about 5.5 µM, or plasma PPi concentrations in the range of about 3.5 µM to about 5.5 µM); less than about 4.78 μM for adolescents (e.g., plasma PPi concentrations of about 3.5 µM, about 4 µM, or about 4.5 µM, or plasma PPi concentrations in the range of about 3.5 µM to about 4.5 µM); or less than about 5.82 μM for adults (e.g., plasma PPi concentrations of about 3.5 µM, about 4 µM, about 4.5 µM, about 5 µM, or about 5.5 µM, or plasma PPi concentrations in the range of about 3.5 µM to about 5.5 µM).

[0012] In another aspect, this disclosure also provides a method comprising: (i) identifying a subject population suffering from or susceptible to myasthenia gravis; (ii) identifying a subject subgroup within the population in step (i), wherein: (a) the subjects in the subgroup have elevated concentrations of inorganic pyrophosphate (PPi); (b) enhancing the elevated concentrations of inorganic pyrophosphate (PPi) in the subjects of the subgroup to improve myasthenia gravis; or (c) both (a) and (b); and (iii) treating the subgroup in step (ii). In another aspect, this disclosure also provides a method comprising: (i) identifying a subject population suffering from or susceptible to muscular dystrophy; (ii) identifying a subject subgroup within the population in step (i), wherein: (a) the subjects in the subgroup have elevated concentrations of inorganic pyrophosphate (PPi); (b) enhancing the elevated concentrations of inorganic pyrophosphate (PPi) in the subjects of the subgroup to improve muscular dystrophy; or (c) both (a) and (b); and (iii) treating or improving at least one symptom of muscular dystrophy in the subjects of the subgroup in step (ii), comprising administering to the subjects a therapeutically effective amount of at least one recombinant polypeptide having alkaline phosphatase activity. In one embodiment, the subjects in the subgroup have elevated serum concentrations of inorganic pyrophosphate (PPi).

[0013] In some embodiments, the muscles of the subject in step (iii) described herein are not significantly different from the muscles of a normal subject without said type of muscle weakness in terms of at least one characteristic of the muscle. In one embodiment, at least one characteristic of the muscle includes, for example, fiber type ratio and / or fiber contractile properties. Such muscles may include any muscle of the subject, including, for example, skeletal or striated muscle, cardiac or smooth muscle. In some embodiments, such muscles include at least one type of arm and / or leg muscle, particularly at least one type of muscle selected from the soleus and extensor digitorum longus (EDL) muscles. In some embodiments, the method includes identifying subjects who have or are susceptible to muscle weakness and who have: elevated PPi concentration, elevated alkaline phosphatase concentration, reduced grip strength, for example, a mean BOT-2 strength score of less than 10, for example, a mean BOT-2 running speed and agility score of less than 5, for example, a mean CHAQ index score of greater than about 0.8, for example, a mean PODCI score of less than about 40, for example, a mean 6MWT of less than about 80% of the predicted 6MWT value, and / or a muscle strength grade of less than 5.

[0014] For example, elevated PPi concentrations in samples (e.g., plasma samples) from infants or children (e.g., subjects under about 12 years of age) may be about 5.71 μM or greater; elevated PPi concentrations in samples (e.g., plasma samples) from adolescents (e.g., subjects from about 13 to about 18 years of age) may be about 4.78 μM or greater; and elevated PPi concentrations in samples (e.g., plasma samples) from adults (e.g., subjects over about 18 years of age) may be about 5.82 μM or greater. Additionally, elevated alkaline phosphatase concentrations in samples from subjects (e.g., plasma samples) may be approximately 90 U / L or lower for subjects aged 0 to 14 days; approximately 134 U / L or lower for subjects aged 15 days to less than 1 year; approximately 156 U / L or lower for subjects aged approximately 1 year to less than 10 years; approximately 141 U / L or lower for subjects aged approximately 10 years to less than approximately 13 years; approximately 62 U / L or lower for female subjects aged approximately 13 years to less than approximately 15 years; approximately 127 U / L or lower for male subjects aged approximately 13 years to less than approximately 15 years; approximately 54 U / L or lower for female subjects aged approximately 15 years to less than approximately 17 years; approximately 89 U / L or lower for male subjects aged approximately 15 years to less than approximately 17 years; and approximately 48 U / L for female subjects aged approximately 17 years or older. U / L or lower; or for male subjects aged approximately 17 years or older, approximately 59 U / L or lower.

[0015] In some embodiments, the muscle weakness disease described herein is caused by elevated inorganic pyrophosphate (PPi) concentrations. In one embodiment, the muscle weakness disease described herein is caused by elevated serum inorganic pyrophosphate (PPi) concentrations. For example, elevated PPi concentrations in samples (e.g., plasma samples) from infants or children (e.g., subjects younger than about 12 years of age) may be about 5.71 μM or greater; elevated PPi concentrations in samples (e.g., plasma samples) from adolescents (e.g., subjects aged about 13 to about 18 years of age) may be about 4.78 μM or greater; and elevated PPi concentrations in samples (e.g., plasma samples) from adults (e.g., subjects older than about 18 years of age) may be about 5.82 μM. In other embodiments, the muscle weakness disease described herein is caused by elevated concentrations of at least one alkaline phosphatase substrate (e.g., PPi, PLP, PEA, etc.).

[0016] In some embodiments, elevated pyrophosphate (PPi) concentrations enhance the muscle weakness disease described herein in the subject during step (iii) described herein. In one embodiment, elevated serum inorganic pyrophosphate (PPi) concentrations enhance the muscle weakness disease described herein in the subject. For example, elevated PPi concentrations in samples (e.g., plasma samples) from infants or children (e.g., subjects younger than about 12 years of age) may be about 5.71 μM or greater; elevated PPi concentrations in samples (e.g., plasma samples) from adolescents (e.g., subjects aged about 13 to about 18 years of age) may be about 4.78 μM or greater; and elevated PPi concentrations in samples (e.g., plasma samples) from adults (e.g., subjects older than about 18 years of age) may be about 5.82 μM. In other embodiments, the subject’s muscle weakness disease as described herein is enhanced by elevated concentrations of at least one alkaline phosphatase substrate (e.g., PPi, PLP, PEA, etc.). The muscle weakness diseases described in this article for subgroup selection include at least one of the following: for example, hypophosphatase disease (HPP), calcium pyrophosphate dihydrate crystal deposition (CPPD), familial hypophosphatemia (such as autosomal dominant hypophosphatemic rickets (ADHR), autosomal recessive hypophosphatemic rickets, X-linked hypophosphatemic rickets, X-linked hypophosphatemia (XLH), etc.), or other diseases with a muscle weakness phenotype and elevated concentrations of at least one alkaline phosphatase substrate (e.g., PPi, PLP, PEA, etc.). In one embodiment, the muscle weakness disease described herein includes at least one of the following: for example, hypophospholipase disease (HPP), calcium pyrophosphate dihydrate crystal deposition (CPPD), familial hypophosphatemia (such as autosomal dominant hypophosphatemic rickets (ADHR), autosomal recessive hypophosphatemic rickets, X-linked hypophosphatemic rickets, X-linked hypophosphatemia (XLH), etc.), or other diseases having a muscle weakness phenotype and elevated concentrations (e.g., serum concentrations) of inorganic pyrophosphate (PPi). In some embodiments, administration of at least one recombinant polypeptide with alkaline phosphatase activity (such as TNALP, for example, the sALP polypeptide of SEQ ID NO: 1 or a polypeptide variant having at least 95% sequence identity with the sequence of SEQ ID NO: 1, for example, Asphertex Alpha) reduces the concentration of inorganic pyrophosphate (PPi) in a sample (e.g., a plasma sample) from said subject. For example, administering at least one recombinant polypeptide with alkaline phosphatase activity to a subject reduces the PPi concentration in a sample (e.g., a plasma sample) to less than about 5.71 μM for infants or children (e.g., plasma PPi concentrations of about 3.5 µM, about 4 µM, about 4.5 µM, about 5 µM, or about 5.5 µM, or plasma PPi concentrations in the range of about 3.5 µM to about 5.5 µM); less than about 4.78 μM for adolescents (e.g., plasma PPi concentrations of about 3.5 µM, about 4 µM, or about 4.5 µM, or plasma PPi concentrations in the range of about 3.5 µM to about 4.5 µM); or less than about 5.82 μM for adults (e.g., plasma PPi concentrations of about 3.5 µM, about 4 µM, about 4.5 µM, about 5 µM, or about 5.5 µM, or plasma PPi concentrations in the range of about 3.5 µM to about 5.5 µM).

[0017] In some embodiments, administration of at least one recombinant polypeptide with alkaline phosphatase activity (such as TNALP, for example, the sALP polypeptide of SEQ ID NO: 1 or a polypeptide variant having at least 95% sequence identity with the sequence of SEQ ID NO: 1, for example, Asphertex Alpha) increases the concentration of alkaline phosphatase in a sample (e.g., a plasma sample) from the subject. For example, administration of at least one recombinant polypeptide with alkaline phosphatase activity increases the alkaline phosphatase concentration in samples (e.g., plasma samples) from subjects to, for example, about 273 U / L or greater for subjects aged 0 to 14 days; about 518 U / L or greater for subjects aged 15 days to less than 1 year; about 369 U / L or greater for subjects aged about 1 year to less than 10 years; about 460 U / L or greater for subjects aged about 10 years to less than about 13 years; about 280 U / L or greater for female subjects aged about 13 years to less than about 15 years; about 517 U / L or greater for male subjects aged about 13 years to less than about 15 years; about 128 U / L or greater for female subjects aged about 15 years to less than about 17 years; about 365 U / L or greater for male subjects aged about 15 years to less than about 17 years; and about 95 U / L for female subjects aged about 17 years or older. U / L or greater; or approximately 164 U / L or greater for male subjects aged approximately 17 years or older.

[0018] In some implementations, subjects may also exhibit reduced dependence on assistive mobility devices (e.g., walkers, wheelchairs, harnesses, canes, and orthotics) after administration of at least one recombinant peptide with alkaline phosphatase activity.

[0019] In any of the above aspects, prior to administration of at least one recombinant polypeptide with alkaline phosphatase activity, the subject is characterized by having a mean hand-held force measurement (HHD) value less than about 80% of the predicted HHD value (e.g., relative to a normal subject of about the same age, sex, and / or height), specifically, where the HHD value represents the subject's grip strength, knee flexion, knee extension, hip flexion, hip extension, or hip abduction. For example, administration of at least one recombinant polypeptide with alkaline phosphatase activity results in the subject's mean HHD value being about 50% or more of the predicted HHD value, for example, where the HHD value represents the subject's grip strength, knee flexion, knee extension, hip flexion, hip extension, or hip abduction.

[0020] In some embodiments, the at least one recombinant polypeptide with alkaline phosphatase activity described herein may be administered to the subject daily, twice weekly, once weekly, or even less frequently. In one embodiment, the at least one recombinant polypeptide with alkaline phosphatase activity described herein is administered to the subject daily. The at least one recombinant polypeptide with alkaline phosphatase activity described herein may be administered to the subject for a period of at least one week, two weeks, one month, three months, six months, one year, or longer, up to the subject's entire lifespan.

[0021] In some embodiments, the at least one recombinant polypeptide with alkaline phosphatase activity described herein is or can be administered via at least one route. Such routes include, for example, subcutaneous, intravenous, intramuscular, sublingual, intrathecal, intradermal, or other routes known in the art. In one embodiment, the at least one recombinant polypeptide with alkaline phosphatase activity described herein is administered subcutaneously.

[0022] In some embodiments, the at least one recombinant polypeptide with alkaline phosphatase activity described herein comprises at least one of the following: tissue nonspecific alkaline phosphatase (TNALP), placental alkaline phosphatase (PALP), germ cell alkaline phosphatase (GCALP), intestinal alkaline phosphatase (IALP), and functional fragments, fusions, or chimeric constructs thereof. In one embodiment, the at least one recombinant polypeptide with alkaline phosphatase activity described herein comprises at least one soluble fragment of TNALP, PALP, GCALP, and IALP. In one embodiment, the tissue nonspecific alkaline phosphatase (TNALP) described herein comprises or is composed of the amino acid sequence of amino acids 1-485 of SEQ ID NO: 1. In another embodiment, the tissue nonspecific alkaline phosphatase (TNALP) described herein comprises or is composed of the amino acid sequence of SEQ ID NO: 1.

[0023] In some embodiments, the at least one recombinant polypeptide with alkaline phosphatase activity described herein is a fusion protein. In one embodiment, the at least one recombinant polypeptide with alkaline phosphatase activity described herein comprises an immunoglobulin molecule. Such an immunoglobulin molecule may be, for example, a crystallizable fragment (Fc) of IgG or its full length or a fragment thereof, including but not limited to IgG1, IgG2, IgG3, IgG4, IgG5, IgG6, IgG7, IgG8, IgG9 ... 2 / 4 Or other IgG fusions. In one embodiment, the Fc described herein comprises the amino acid sequence of SEQ ID NO: 20.

[0024] In some embodiments, at least one recombinant polypeptide with alkaline phosphatase activity described herein comprises a negatively charged peptide. Such a negatively charged peptide may comprise at least one poly(glutamate) (polyE) or poly(aspartic acid) (polyD) peptide. For example, at least one recombinant polypeptide with alkaline phosphatase activity comprises 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or 16 consecutive acidic residues, particularly aspartic acid (D) or glutamate (E), such as D... 10 D 16 E 10 and E 16 At least one of the following. In some embodiments, at least one recombinant polypeptide having alkaline phosphatase activity includes E6, E7, E8, E9, E 10 E 11 E 12 E 13 E 14 E 15 E 16 D6, D7, D8, D9, D 10 D 11 D 12 D 13 D 14 D 15 Or D 16 For example, E6, E 10 D6 or D 10 .

[0025] In some embodiments, at least one recombinant polypeptide with alkaline phosphatase activity described herein comprises bone-targeting alkaline phosphatase comprising a polypeptide having the following structure: Z-sALP-Y-spacer-X-Wn-V, wherein sALP is the extracellular domain of alkaline phosphatase; V is absent or has an amino acid sequence of at least one amino acid; X is absent or has an amino acid sequence of at least one amino acid; Y is absent or has an amino acid sequence of at least one amino acid; Z is absent or has an amino acid sequence of at least one amino acid; and Wn is polyaspartic acid or polyglutamic acid, wherein n = 10 to 16.

[0026] In some embodiments, the spacer described herein comprises a fragment crystallizable region (Fc). In one embodiment, the Fc described herein comprises the amino acid sequence of SEQ ID NO: 20.

[0027] In some embodiments, at least one recombinant polypeptide with alkaline phosphatase activity described herein comprises sALP-Fc-D 10 The structure.

[0028] In one embodiment, at least one recombinant polypeptide having alkaline phosphatase activity described herein comprises a dimer comprising a monomer of the amino acid sequence of SEQ ID NO: 1.

[0029] In some embodiments, at least one recombinant polypeptide with alkaline phosphatase activity described herein is administered at a dose of about 0.1 mg / kg / day to about 20 mg / kg / day or an equivalent weekly dose. In one embodiment, at least one recombinant polypeptide with alkaline phosphatase activity described herein is administered at a dose of about 0.5 mg / kg / day to about 20 mg / kg / day or an equivalent weekly dose. In another embodiment, at least one recombinant polypeptide with alkaline phosphatase activity described herein is administered at a dose of about 0.5 mg / kg / day to about 10 mg / kg / day or an equivalent weekly dose. In yet another embodiment, at least one recombinant polypeptide with alkaline phosphatase activity described herein is administered at a dose of about 1 mg / kg / day to about 10 mg / kg / day or an equivalent weekly dose.

[0030] In some implementations, the subjects described herein are mammals (e.g., humans).

[0031] definition As used herein, unless otherwise stated, “an” means “at least one” or “one or more”. Furthermore, unless the context clearly specifies otherwise, the singular forms “an,” “a,” and “the” include multiple indicators.

[0032] As used herein, “about” means ±10% of the value, and preferably ±5% of the value, or more preferably ±2% of the value.

[0033] As used herein, “at least” means ≤ 10% of the value and preferably ≤ 5% of the value, or more preferably ≤ 2% of the value.

[0034] "Asfertes Alpha" refers to a human TNALP (hTNALP) fusion protein formulated for the treatment of HPP. Asfertes Alpha is a fusion protein of a soluble glycoprotein comprising two identical polypeptide chains, each containing amino acid residues 1-726 of SEQ ID NO: 1. The structure of each polypeptide chain includes the catalytic domain of hTNALP, the human immunoglobulin G1 Fc domain, and a deca-aspartic peptide (structure hTNALP-Fc-D) serving as a bone-targeting domain. 10 The two polypeptide chains are covalently linked by two disulfide bonds. Asphertex Alpha is available in the United States, Europe, Japan, Canada, Israel, Australia, and South Korea under the trade name STRESIQ. ®(Alexion Pharmaceuticals, Inc., New Haven, CT).

[0035] The terms “individual,” “subject,” and “patient” are used interchangeably and refer to any subject for whom a diagnosis, treatment, or therapy is desired, specifically a human being. Other subjects may include, for example, cattle, dogs, cats, guinea pigs, rabbits, rats, mice, horses, etc. As used herein, a “at-risk” subject or a subject “vulnerable” to a disease is a subject identified as having a risk of developing a disease, condition, or symptom associated with myasthenia gravis.

[0036] As used in this article, "mean" refers to the numerical value representing the average or median of a dataset. The mean of a dataset is calculated by dividing the sum of the values ​​in the dataset by their number. The median of a dataset is calculated by determining the middle value in an odd-numbered list of data or by determining the average of the two middle data values ​​in an even-numbered list.

[0037] The term "wildtype" or "wildtype sequence" used in this disclosure for TNALP or other genes or proteins refers to the typical form of such genes or proteins because it occurs in normal humans, non-human mammals, or other organisms in nature. In contrast to non-standard, "mutated" alleles or amino acid sequences / modifications / interactions, a wildtype sequence can refer to a standard "normal" allele at a gene locus or a standard "normal" primary amino acid sequence of a polypeptide or protein (optionally having standard "normal" post-translational modifications and / or inter-chain bonds and / or interactions between amino acid residues). "Mutated" alleles can vary considerably and, in the case of genetic drift within a population, can even become wild-type. It is now recognized that most or all gene loci (and less frequently, but still possible, for most polypeptide sequences) exist in multiple allele forms, with frequencies varying across the geographic range of the species, and a uniform wild-type may not necessarily exist. However, in general, the most prevalent allele or amino acid sequence—that is, the sequence or amino acid sequence with the highest frequency in normal human or other organisms—is considered wild-type in this disclosure.

[0038] Unless otherwise stated, the term "normal" as used for humans or other organisms in this specification means the absence of any disease (e.g., HPP), condition, and / or symptom or physiological consequence (e.g., muscle weakness) caused or associated with abnormal activity of the relevant gene or polypeptide / protein (which may be attributed to, for example, a defect or lack of the gene or protein product and / or a defect or loss of function of the gene or protein product). The most obvious example of a normal person is one who lacks muscle weakness or symptoms of muscle weakness and who lacks mutations or modifications to genes or proteins (e.g., the ALPL gene and ALP protein) that can cause HPP-related muscle weakness. In another case where ALP function is of interest, the scope of "normal" in this disclosure can be extended to include any person who does not have abnormal endogenous alkaline phosphatase activity (which can be tested, for example, by substrate (PPi, PEA, and PLP) levels and compared with the corresponding activities of other healthy or normal individuals).

[0039] As used herein, "elevated" or "increased" concentration means a concentration (e.g., PPi) in a subject who has or is susceptible to the muscular dystrophy described herein that is higher than that in a wild-type subject, another subject without the muscular dystrophy, the same subject at a time when the subject does not have the muscular dystrophy, or the same subject who should not have the muscular dystrophy. This "elevated concentration" refers to an elevated concentration within the subject described herein (including any cells, tissues, organs, or part of the subject). In one embodiment, this "elevated concentration" includes an elevated concentration in the subject's serum.

[0040] As used herein, the terms “Bayley Infant and Toddler Developmental Scales, 3rd Edition” or “BSID-III” refer to a standardized series of measurements used to assess a patient’s motor (fine and gross), language (receptive and expressive), and cognitive development. See Bayley, (2006). Bayley scales of infant and toddler development: administration manual. San AntonioTX: Harcourt Assessment, hereby incorporated in its entirety by reference. BSID-III measurements consist of a series of developmental play tasks to be administered to the patient. Raw scores of successful completion of the tasks are converted into scale scores. The scale scores are then used to determine patient performance compared to healthy, age-adjusted patients. BSID-III may also include a social-emotional adaptive behavior questionnaire completed by a parent / guardian to establish the patient's range of adaptive behaviors. For example, measurements used to determine BSID-III scores (e.g., BSID-III Gross Motor Function Scale scores) may include grasping, sensorimotor integration, motor planning and speed, visual tracking, extension, object grasping, object manipulation, functional hand skills, response to tactile information, limb and trunk movement, static positioning, dynamic movement, balance, and motor planning. These patient measurements are then converted into BSID-III scale scores ranging from 0 to 14 (e.g., BSID-III Gross Motor Function Scale scores), where scores of approximately 7 to approximately 13 are considered within the normal range for healthy patients.

[0041] As used herein, the term "bone-targeting moiety" refers to an amino acid sequence of 1 to 50 amino acid residues that has sufficient affinity for the bone matrix to target approximately 10 amino acid residues individually. -6 M to approximately 10 -15 M (for example, 10) -7 M, 10 -8 M, 10 -9 M, 10 -10 M, 10 -11 M, 10 -12 M, 10 -13 M, 10 -14 M or 10 -15 The bone matrix of M) has in vivo binding affinity.

[0042] As used herein, the term "Breeninkes-Osiris Motor Ability Tests Version 2" or "BOT-2" refers to the second version of a standardized test of gross and fine motor performance in patients, for example, aged approximately 4 to approximately 21 years. See also Bruininks, RH (2005).Bruininks-Oseretsky Test of Motor Proficiency, (BOT-2) .Minneapolis, MN: Pearson Assessment, the entire text of which is incorporated herein by reference. BOT-2 is administered alone to assess gross and fine motor skills in a range of patients. Specifically, BOT-2 can be used to assess physical impairment and mobility limitations in patients with HPP. BOT-2 provides a composite BOT-2 score for: strength, running speed and agility, fine motor precision, fine motor integration, hand dexterity, bilateral coordination, balance, and upper limb coordination. For example, BOT-2 strength scores can be determined by having patients perform sit-ups, V-sit-ups, standing long jumps, sit-and-reach exercises, and push-ups. Running speed and agility scores can be determined by having patients hop across a balance beam or perform shuttle runs, two-legged lateral jumps, or one-legged lateral jumps. Both BOT-2 strength and BOT-2 running speed and agility scores range from 0 to 25, with scores of approximately 10 to 20 considered representative of healthy patients.

[0043] As used herein, the term "Children's Health Assessment Questionnaire" or "CHAQ" refers to a questionnaire used to assess the health status (e.g., ability to perform activities of daily living (ADL) and experience pain) of patients aged 1 to 19 years (such as those with HPP). For a description of the CHAQ index, see [link to CHAQ questionnaire]. Bruce&Fries (J. Rheumatol. 30(1): 167-178, 2003) The entire text is hereby incorporated by reference. The CHAQ can be administered to children older than 8 years through interviews or self-reporting. The CHAQ consists of eight subscales for dressing / grooming, standing, eating, walking, hygiene, stretching, grasping, and activity. Scores in each category range from 0 to 3, where a score of 0 indicates no difficulty; a score of 1 indicates some difficulty; a score of 2 indicates great difficulty; and a score of 3 indicates the patient is unable to perform the activity. The CHAQ index can also be used to determine the presence and severity of pain.

[0044] "Extracellular domain" refers to any functional extracellular portion of a natural protein (such as alkaline phosphatase). In particular, extracellular domains lack signal peptides.

[0045] “Fc” refers to a fragment crystallizable region of an immunoglobulin (e.g., IgG-1, IgG-2, IgG-3, IgG-4), including the CH2 and CH3 domains of the immunoglobulin heavy chain. Fc may also include any portion of the hinge region connecting the Fab and Fc regions. Fc can be any mammal, including humans, and can be post-translational modified (e.g., by glycosylation). In a non-limiting example, Fc can be a fragment crystallizable region of human IgG-1 having the amino acid sequence of SEQ ID NO: 20.

[0046] "Fragment" refers to a portion of a polypeptide or nucleic acid molecule, preferably containing 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 polypeptide. The fragment may contain, for example, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 260, 270, 280, 290, 300, 400, 500, 600, 700 or more amino acid residues, up to the entire length of the polypeptide. Exemplary sALP fragments have amino acid residues 18-498, 18-499, 18-500, 18-501, 18-502, 18-503, 18-504, 18-505, 18-506, 18-507, 18-508, 18-509, 18-510, 18-511, or 18-512 of ALP (e.g., SEQ ID NO: 2-6), and may include additional C-terminal and / or N-terminal portions.

[0047] As used interchangeably in this document, the terms "handheld dynamometer" and "HHD" refer to methods for measuring the grip and muscle strength of a subject (particularly those with or susceptible to muscular dystrophy). A dynamometer can be used to assess a subject's (e.g., those with or susceptible to muscular dystrophy) grip strength, knee flexion, knee extension, hip flexion, hip extension, and hip abduction. For example, a MICROFET2, such as [example device], can be used. TM A dynamometer measures knee flexion and extension, as well as hip flexion, extension, and abduction, in subjects with or prone to muscular dystrophy. Simultaneously, a grip strength measurement may be performed using, for example, a Jamar Grip dynamometer. Specifically, the administrator keeps the dynamometer stationary, and the subject applies maximum force to the dynamometer. Peak force data are collected in pounds and then converted to Newtons (N). Torque values ​​are then calculated using limb length in N meters. The torque values ​​are then compared to, for example, values ​​of normal subjects of approximately the same age, sex, and / or height, and expressed as a percentage to generate the subject's HHD value.

[0048] As used herein, the term "hypophosphatase syndrome" or "HPP" refers to a rare, heritable skeletal disorder caused by one or more loss-of-function mutations in genes such as ALPL (alkaline phosphatase, liver / bone / kidney), which encode tissue-nonspecific alkaline phosphatase (TNALP). HPP can be further characterized as infantile HPP, childhood HPP, perinatal HPP (e.g., benign or lethal perinatal HPP), or dental HPP.

[0049] "Treatment-naïve patient" or "Treatment-naïve subject" means a patient or subject with the muscle weakness disease described herein who has never received treatment with alkaline phosphatase or a polypeptide with alkaline phosphatase activity (such as sALP (e.g., TNALP, such as the sALP polypeptide of SEQ ID NO: 1 or a polypeptide variant having at least 95% sequence identity with the sequence of SEQ ID NO: 1, e.g., Asphertex Alpha)).

[0050] As used herein, “pain” refers to physical discomfort or distress caused by the muscle weakness disorder described herein, such as muscle aches. Symptoms of pain may include, for example, aches, tightness, or stiffness. The severity of pain can vary from patient to patient (e.g., chronic pain or acute pain). In particular, chronic pain is defined as pain that lasts longer than three to six months or that extends beyond the expected healing period. Conversely, acute pain is defined as pain that typically lasts less than three to six months. As described herein, therapeutic compositions (e.g., including sALP, such as Aspheres Alpha) may be administered to patients suffering from pain (e.g., muscle aches) in an amount sufficient to reduce or at least partially reduce symptoms of pain (e.g., discomfort, aches, tightness, or stiffness) and its complications (e.g., fatigue, insomnia, weakened immune system, depression, anxiety, stress, irritability, or disability).

[0051] As described herein, 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 modifications (e.g., glycosylation or phosphorylation), constituting all or part of a naturally occurring or non-natural polypeptide or peptide.

[0052] "Pharmaceutically acceptable carrier" or "pharmaceutically acceptable excipient" means, respectively, at least one carrier or excipient that is physiologically acceptable to the patient being treated while retaining the therapeutic properties of the compound administered with it. An exemplary pharmaceutically acceptable carrier substance is physiological saline. For example, a pharmaceutically acceptable carrier may include sodium chloride (e.g., 150 mM sodium chloride) and sodium phosphate (e.g., 25 mM sodium phosphate). 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 (20th edition), A. Gennaro, Ed., 2000, Lippincott, Williams & Wilkins, Philadelphia, PA.

[0053] "Pharmaceutical composition" means a composition containing a polypeptide or nucleic acid molecule as described herein, formulated together with at least one pharmaceutically acceptable excipient, diluent, or carrier. Pharmaceutical compositions may be manufactured or marketed with the approval of a government regulatory authority as part of a treatment regimen for treating or preventing a patient's disease or event. Pharmaceutical compositions may be formulated, for example, in unit dosage forms, such as for subcutaneous administration, intravenous administration (e.g., as a sterile solution of a particulate suppository and a solvent system suitable for intravenous use), for oral administration (e.g., tablets, capsules, sachets, capsule tablets, or syrups), or any other formulation described herein. In one embodiment, the pharmaceutical composition of this disclosure is for subcutaneous administration or is formulated for subcutaneous administration.

[0054] As used herein, the term "physical impairment" refers to a physiological condition, such as osteomalacia and muscular dystrophy as described herein, that may limit or eliminate a patient's ability, for example, to walk, functional endurance, and to perform activities of daily living (ADL). In particular, physical impairment may limit or eliminate a patient's ability to perform ADL, which are routine daily activities performed by healthy patients without assistance, such as functional activities or transfers (e.g., walking), bathing and showering, dressing, self-feeding, and personal hygiene and grooming. As described herein, therapeutic compositions (e.g., including sALP, such as compositions from Aspheres Alpha) may be administered to patients to reduce the severity and / or frequency of physical impairment associated with muscle weakness.

[0055] As used herein, the term “Pediatric Outcome Data Collection Tool” or “PODCI” refers to a questionnaire used to assess the overall health status, pain incidence, and ability to perform activities of daily living (ADL) in patients under 19 years of age, particularly those with chronic health conditions such as HPP. For a description of the PODCI, see Plant et al. (…). J. Pediatr. Orthop. 23(6): 788-790, 2003(This text is incorporated herein by reference in its entirety.) The questionnaire can be completed by the patient or by the patient's parents / guardians who are familiar with the patient's condition. The eight scales generated by PODCI include the following: 1) Upper Limb and Physical Function Scale, which measures difficulties encountered in performing daily personal care and school activities; 2) Transfer and Basic Mobility Scale, which measures difficulties experienced in performing routine and motor activities in daily activities; 3) Motor / Physical Function Scale, which measures difficulties or limitations encountered when participating in more active activities or sports; 4) Pain / Comfort Scale, which measures the degree of pain experienced in the past week; 5) Treatment Expectation Scale, which measures long-term expectations of treatment; 6) Well-being Scale, which measures overall satisfaction with personal appearance and similarity with peers and others; 7) Symptom Satisfaction Scale, which measures the patient's acceptance of current limitations corresponding to a lifelong condition; and 8) Global Function Scale, which is a general combination scale calculated from the first four scales listed above. The standardized score is generated from a series of questions in the PODCI and converted into a 0 to 100 scale, where 0 represents significant disability and 100 represents less severe disability.

[0056] As used herein, the term "Peabody Developmental Motor Scale, 2nd Edition" or "PDMS-2" refers to the Early Childhood Motor Development Program, which provides an assessment of gross and fine motor skills in patients from birth through childhood (e.g., infancy and childhood). For a description of the PDMS-2 scale, see van Hartingsveldt et al. ( Occup. Ther. Int. 12(1): 1-13, 2005), which is incorporated herein by reference in its entirety. PDMS-2 consists of six subtests that measure relevant motor abilities in early development. The six subtests include the following: 1) a motor subtest to measure a patient’s ability to move from one place to another (measurements include crawling, walking, running, jumping and jumping forward); 2) a reflex subtest to measure a patient’s ability to respond automatically to environmental events; 3) a stationary subtest to measure a patient’s ability to maintain his or her body control and balance within his or her center of gravity; 4) an object manipulation subtest to measure a patient’s ability to manipulate objects, such as catching, throwing and kicking a ball; 5) a grasping subtest to measure a patient’s ability to use his or her hands, such as the ability to grasp an object with one hand and actions involving the controlled use of the fingers of both hands; and 6) a visual-motor integration subtest to measure a patient’s ability to use his or her visual perception skills to perform complex eye-hand coordination tasks, such as reaching and grasping objects, building and copying designs with blocks. The PDMS-2 measurements for each subtest were converted into PDMS-2 scores, such as the PDMS-2 exercise standard score ranging from 0 to 13, where the range for healthy patients was from about 7 to about 13.

[0057] The terms “sALP,” “soluble alkaline phosphatase,” and “extracellular domain of alkaline phosphatase” are used interchangeably and refer to a soluble, non-membrane-bound alkaline phosphatase or its domain, bioactive fragment, or bioactive variant thereof. sALP includes, for example, alkaline phosphatases lacking a C-terminal glycolipid anchor (GPI signal sequence, e.g., amino acid residues 18-502 of human TNALP or polypeptides thereof (SEQ ID NO: 2, 3, 4, 5, or 6)). Specifically, TNALP may include, for example, polypeptides comprising amino acid residues 1-485 of SEQ ID NO: 1 or polypeptides thereof, such as Aspheres alpha, or polypeptide variants having at least 95% sequence identity with amino acid residues 1-485 of SEQ ID NO: 1. sALP also includes, for example, mammalian orthologs of human TNALP, such as rhesus monkey TNALP (SEQ ID NO: 7), rat TNALP (SEQ ID NO: 8), canine TNALP (SEQ ID NO: 9), porcine TNALP (SEQ ID NO: 10), mouse TNALP (SEQ ID NO: 11), bovine TNALP (SEQ ID NO: 12-14), or cat TNALP (SEQ ID NO: 15). sALP also includes soluble, non-membrane-bound forms of human PALP (e.g., peptides comprising amino acid residues 18-502 of SEQ ID NO: 16 or 17, or peptides thereof), GCALP (e.g., peptides comprising amino acid residues 18-502 of SEQ ID NO: 18, or peptides thereof), and IALP (e.g., peptides comprising amino acid residues 18-502 of SEQ ID NO: 19, or peptides thereof), as well as other variants and analogs that retain alkaline phosphatase activity (e.g., the ability to hydrolyze PPi). In particular, sALP lacks the N-terminal signal peptide (e.g., aa 1-17 of SEQ ID NO: 2-6, 8, 11-13 or 15, or aa 1-25 of SEQ ID NO: 7).

[0058] "sALP polypeptide" means a polypeptide having the structure A-sALP-B, wherein sALP is as defined herein, and A and B are either absent or have an amino acid sequence of at least one amino acid. Exemplary sALP polypeptides have an amino acid sequence comprising amino acids 1-485 of SEQ ID NO: 1 or composed thereof. Other exemplary sALP polypeptides include any sALP fusion polypeptide described herein (e.g., the sALP fusion polypeptide of SEQ ID NO: 1 or a polypeptide variant having at least 95% sequence identity with the sequence of SEQ ID NO: 1, e.g., Asphertex Alpha).

[0059] A “signal peptide” refers to a short peptide (5-30 amino acids long) at the N-terminus of a polypeptide that directs the polypeptide toward a secretory pathway (e.g., extracellular space). Signal peptides are typically cleaved during polypeptide secretion. The signal sequence can direct the polypeptide toward an intracellular compartment or organelle, such as the Golgi apparatus. Signal sequences can be identified by homology or bioactivity as peptides with known functions of targeting specific regions of the cell. Those skilled in the art can identify signal peptides using readily available software (e.g., sequence analysis software packages from the Genetics Computing Group, University of Wisconsin Biotechnology Center, 1710 University Avenue, Madison, Wis. 53705, BLAST, or PILEUP / PRETTYBOX programs). A signal peptide can be, for example, a signal peptide substantially identical to amino acid residues 1-17 of SEQ ID NO: 2-6 or amino acid residues 1-25 of SEQ ID NO: 7.

[0060] As used herein, when a polypeptide or nucleic acid sequence is described as having "at least X% sequence identity" with a reference sequence, "X" is a real number meaning that, at optimal sequence alignment, at least X% of the amino acid residues or nucleotides in the polypeptide or nucleic acid are identical to those amino acid residues or nucleotides in the reference sequence. Optimal sequence alignment can be determined in various ways within the scope of the art, for example, using the Smith Waterman alignment algorithm (Smith et al.). J. Mol.Biol . 147:195-7, 1981) and BLAST (Basic Local Alignment Search Tool; Altschul et al., J. Mol. Biol.215: 403-10, 1990). These and other alignment algorithms are accessible using publicly available computer software, such as “BestFit” incorporated into GeneMatcher Plus (Schwarz and Dayhoff, Atlas of Protein Sequence and Structure, Dayhoff, MO editor, pp. 353-358, 1979) (Smith and Waterman, Advances in Applied Mathematics, 482-489, 1981), BLAST, BLAST-2, BLAST-P, BLAST-N, BLAST-X, WU-BLAST-2, ALIGN, ALIGN-2, CLUSTAL, Megalign (DNASTAR), or other software / hardware for alignment. Furthermore, those skilled in the art can determine the parameters suitable for measuring the alignment, including any algorithm required to achieve optimal alignment across the lengths of the compared sequences.

[0061] The terms “patient” and “subject” are used interchangeably and refer to mammals, including but not limited to human or non-human mammals such as cattle, horses, dogs, sheep or cats.

[0062] "Therapeutic effective amount" means that the amount of the polypeptide or nucleic acid molecule described herein is sufficient to significantly improve, treat, prevent, delay, inhibit, or prevent at least one symptom of HPP. The therapeutic effective amount of the composition described herein may depend on the severity of the condition being treated and the patient's symptoms, weight, and general condition, and may be determined by a person skilled in the art taking such factors into account. The therapeutic effective amount of the composition described herein may be administered to the patient in single or multiple doses over a period of time.

[0063] "Treatment" or "treatment" means, for example, the medical management of a patient intended (e.g., in a patient with HPP) to cure, improve, stabilize, reduce the likelihood of or prevent the development of myasthenia gravis by administering a pharmaceutical composition, and / or the management of a patient who exhibits or may have myasthenia gravis (e.g., in a patient with HPP).

[0064] This term includes active treatment, which is treatment directly and specifically aimed at improving or related to the cure of a disease, pathological symptom, condition, or event; and also includes etiological treatment, which is treatment aimed at eliminating the cause of the related disease, pathological symptom, condition, or event. Furthermore, this term includes palliative treatment, which is treatment designed to eliminate or improve at least one symptom rather than cure a disease, pathological symptom, condition, or event; symptomatic treatment, which is treatment for systemic symptoms of a related disease, pathological symptom, condition, or event; preventive treatment, which, for example, in patients who are not yet ill but are susceptible to or otherwise have a risk of a specific disease, pathological symptom, condition, or event, aims to minimize or partially or completely suppress the development of the related disease, pathological symptom, condition, or event; and supportive treatment, which is treatment used to complement another specific therapy aimed at improving a related disease, pathological symptom, condition, or event.

[0065] As used herein, “walking ability” refers to a patient’s (e.g., a patient with the muscle weakness disorder described herein) ability to lift and lower each foot sequentially. Walking ability can be assessed by testing, specifically, the six-minute walk test (6MWT). See the American Thoracic Society statement: Six-Minute Walk Test Guidelines ( American Journal of Respiratory and Critical Care Medicine , 166(1):111-7, 2002), and the full text is incorporated here by reference.

[0066] Other features and advantages of this disclosure will become apparent from the following detailed description, drawings and claims. Attached Figure Description

[0067] Figure 1 It shows that it comes from wild-type (WT) mice or AKP2 - / - A graph showing the percentage of myosin fiber types in all soleus muscle fibers of mice.

[0068] Figure 2A-2D From wild-type (WT) mice or AKP2 - / - A diagram showing the fiber size distribution in the soleus muscle of a dissected mouse. It illustrates the distribution of all fibers (…). Figure 2A Type I fiber Figure 2B Type IIa fiber () Figure 2C ) or type IIb fiber ( Figure 2D Different minimum dimensions (µm) 2 ) fiber percentage.

[0069] Figures 3A-3D It comes from wild-type (WT) mice or AKP2 - / -A diagram showing the contractile characteristics of the soleus muscle in mice. Muscle mass was compared between male and female mice. Figure 3A ),strength( Figure 3B ), force frequency ( Figure 3C ) and fatigue characteristics ( Figure 3D ).

[0070] Figures 4A-4D It comes from wild-type (WT) mice or AKP2 - / - A diagram illustrating the contractile properties of the extensor digitorum longus (EDL) muscle in mice. Muscle mass was compared between male and female mice. Figure 4A ),strength( Figure 4B ), force frequency ( Figure 4C ) and fatigue characteristics ( Figure 4D For fatigue and force frequency: n=3 per group. For capacity and specific force: n=6 per group.

[0071] Figures 5A-5B It comes from wild-type (WT) mice or AKP2 - / - Soleus muscle of mouse anatomy Figure 5A ) and extensor digitorum longus (EDL) ( Figure 5B A graph showing the contractile properties of muscle in relation to PPi concentration.

[0072] Figure 6 These were wild-type (WT) mice that received continuous aspartate alpha (Tx-Tx) treatment after day 35. AKP2 - / - Mice, or those who stopped treatment with Asftase Alpha (Tx-V) after day 35. AKP2 - / - A graph showing the grip strength of a mouse's forelimbs or hindlimbs. Detailed Implementation

[0073] Muscle weakness has been reported as one of several symptoms of HPP (Seshia et al., 1990). Archives of Disease in Childhood 65 (130-131). Besides HPP, other diseases or conditions can also cause muscle weakness. For example, magnesium deficiency can lead to muscle weakness in patients with calcium pyrophosphate deposition diseases (CPPD or CPDD) (Hahn et al., 2012 BMC). Gastroenterology12-19). Some muscle weakness disorders or conditions, such as HPP, CPPD, and familial hypophosphatemia (such as autosomal dominant hypophosphatemic rickets (ADHR), autosomal recessive hypophosphatemic rickets, X-linked hypophosphatemic rickets, X-linked hypophosphatemic rickets (XLH), etc.), are characterized by elevated pyrophosphate (PPi) concentrations in subjects with these disorders or conditions. In HPP, the elevated PPi concentration is attributed to a loss-of-function mutation in the gene ALPL, which encodes the tissue-nonspecific isoenzyme of alkaline phosphatase (TNALP; also known as liver / bone / kidney type ALP), an enzyme that produces substrates such as PPi, phosphoethanolamine (PEA), and pyridoxal 5'-phosphate (PLP). In CPPD, a deficiency of Mg, a cofactor for various phosphatases, results in higher levels of PPi, which is an essential precursor for the formation of CPPD crystals. Calcium pyrophosphate deposition can further lead to chronic inflammatory arthritis, hypophosphoesterase syndrome, hypomagnesemia, hyperparathyroidism with chondrocalcinosis, and acute attacks of "pseudogout".

[0074] This disclosure teaches a method for treating myasthenia gravis in subjects characterized by one or more of the following: elevated PPi concentration, decreased alkaline phosphatase concentration, for example, a mean BOT-2 strength score less than 10, for example, a mean BOT-2 running speed and agility score less than 5, for example, a mean CHAQ index score greater than about 0.8, or for example, a mean PODCI score less than about 40, for example, a mean 6MWT less than about 80% of the predicted 6MWT value (e.g., where the predicted 6MWT value is the 6MWT value of an age-matched and / or sex-matched normal subject), for example, a muscle strength grade less than 5, and / or a mean HHD value (e.g., a mean HHD muscle or grip strength value) less than about 50% of the predicted HHD value (e.g., where the predicted HHD value is the HHD value of an age-matched and / or sex-matched normal subject). Specifically, the subject has been identified as having or being susceptible to myasthenia gravis.

[0075] For example, methods are disclosed for identifying subjects (e.g., humans) suffering from or susceptible to muscle weakness disorders in order to treat them with recombinant peptides having alkaline phosphatase activity (such as TNALP, e.g., the sALP peptide of SEQ ID NO: 1 or a peptide variant having at least 95% sequence identity with the sequence of SEQ ID NO: 1, e.g., Aspheres Alpha), said patients being characterized by elevated PPi concentrations, decreased ALP concentrations, e.g., a mean BOT-2 strength score less than 10, e.g., a mean BOT-2 running speed and agility score less than 5, e.g., a mean CHAQ index score greater than about 0.8, e.g., a mean PODCI score less than about 40, e.g., a mean 6MWT less than about 80% of the predicted 6MWT value, e.g., a muscle strength grade less than 5, and / or an mean HHD value less than about 80% of the predicted HHD value (e.g., mean HHD muscle or grip strength value). For example, elevated PPi concentrations in samples (e.g., plasma samples) from infants or children (e.g., subjects under about 12 years of age) may be about 5.71 μM or greater; elevated PPi concentrations in samples (e.g., plasma samples) from adolescents (e.g., subjects from about 13 to about 18 years of age) may be about 4.78 μM or greater; and elevated PPi concentrations in samples (e.g., plasma samples) from adults (e.g., subjects over about 18 years of age) may be about 5.82 μM or greater. Specifically, the reduced ALP concentration in samples from subjects (e.g., plasma samples) may be, for example, about 90 U / L or less for subjects aged 0 to 14 days; about 134 U / L or less for subjects aged 15 days to less than 1 year; about 156 U / L or less for subjects aged about 1 year to less than 10 years; about 141 U / L or less for subjects aged about 10 years to less than about 13 years; about 62 U / L or less for female subjects aged about 13 years to less than about 15 years; about 127 U / L or less for male subjects aged about 13 years to less than about 15 years; about 54 U / L or less for female subjects aged about 15 years to less than about 17 years; about 89 U / L or less for male subjects aged about 15 years to less than about 17 years; about 48 U / L or less for female subjects aged about 17 years or older; or about 59 U / L for male subjects aged about 17 years or older. U / L or lower.

[0076] This disclosure provides a method for treating or improving muscle weakness in a subject who has or is susceptible to muscle weakness, comprising administering to the subject a therapeutically effective amount of at least one recombinant polypeptide having alkaline phosphatase activity. Specifically, the subject has been identified as having or being susceptible to muscle weakness.

[0077] This disclosure also provides a method for identifying a subgroup of subjects who have or are susceptible to muscle weakness, wherein the subjects in the subgroup have elevated PPi concentrations, decreased alkaline phosphatase concentrations, and / or reduced grip strength or muscle strength (e.g., as assessed using BOT-2, 6MWT, CHAQ, PODCI, muscle strength grades, and / or HHD).

[0078] The method is also described for: 1) identifying a subgroup of subjects who have or are susceptible to myasthenia gravis, wherein the subjects in the subgroup have elevated PPi concentrations, decreased ALP concentrations, and / or reduced grip strength or muscle strength; and 2) then treating or improving at least one symptom of myasthenia gravis in the subjects in the subgroup.

[0079] The method for identifying a subgroup of subjects who have or are susceptible to muscle weakness is also described, the subjects being characterized by elevated PPi concentration, decreased ALP concentration, and / or reduced grip strength or muscle strength.

[0080] Subgroups of subjects can be identified regardless of whether they have been previously diagnosed with hypophosphatase disease (HPP), calcium pyrophosphate deposition disease (CPPD), or familial hypophosphatemia (such as autosomal dominant hypophosphatemic rickets (ADHR), autosomal recessive hypophosphatemic rickets, X-linked hypophosphatemic rickets, X-linked hypophosphatemic rickets (XLH), etc.). For example, subgroups can be identified based on elevated PPi concentrations in these subjects. Elevated PPi concentrations can be caused by, for example, defects in signaling molecules that regulate production or degradation, mutations in genes encoding such signaling molecules, or other mechanisms affecting PPi stability. For instance, defects or mutations in signaling molecules can lead to overexpression of PPi or reduced degradation or hydrolysis of PPi. Patients with HPP have a defective or absent tissue-specific alkaline phosphatase that hydrolyzes PPi. Therefore, similar to HPP, elevated PPi concentrations may be present in other diseases attributed to alkaline phosphatase deficiency. Defects in signaling molecules also include defects in cofactors or other molecules that promote the function of signaling molecules. For example, in CPPD, the lack of magnesium, which is a cofactor for various phosphatases, leads to elevated PPi levels.

[0081] This document describes methods for identifying a subgroup of subjects exhibiting symptoms associated with muscular dystrophy or at risk of developing such symptoms. The identified subgroup may include subjects previously diagnosed with such muscular dystrophy or who are asymptomatic without a prior diagnosis. Muscular dystrophy in this disclosure includes, for example, HPP or HPP-related diseases, CPPD or CPPD-related diseases, familial hypophosphatemia (such as autosomal dominant hypophosphatemic rickets (ADHR), autosomal recessive hypophosphatemic rickets, X-linked hypophosphatemic rickets, X-linked hypophosphatemia (XLH), etc.), or any other muscular dystrophy with elevated PPi. Methods for identifying a subgroup of subjects include, for example, detecting elevated inorganic pyrophosphate (PPi) concentrations in such subjects.

[0082] Targeting myasthenia gravis This document provides methods for treating or improving at least one symptom in a subject who has or is susceptible to a muscle weakness disorder. Muscle weakness disorder, myopathy, or muscle weakness as described herein may include at least one symptom that causes or is attributed to muscle weakness, or any disease or condition associated therewith. The terms “muscle weakness,” “myopathy,” “muscle weakness,” or other similar expressions in this disclosure refer to a condition associated with a state of impaired muscle function, such as a lack or deficiency in muscle strength, compared to other subjects without such a condition or to the same subject at a point in time prior to having such a condition. Muscle weakness can be classified as having a condition with true or perceived muscle weakness. True muscle weakness may include a condition in which the force exerted by the muscles is less than expected. For example, true muscle weakness includes various skeletal muscle diseases, including muscular dystrophy and inflammatory myopathy. Exemplary diseases or conditions include neuromuscular junction disorders, such as myasthenia gravis. Muscle weakness may also be caused by low levels of potassium and other electrolytes within muscle cells, in which the force exerted by the muscles is less than expected. Perceived muscle weakness (or non-neuromuscular weakness) describes a condition in which a subject feels that they need to exert more effort than a normal person (i.e., compared to other subjects without this condition or the same subject at a point in time before having this condition) to exert a certain amount of force, but the actual muscle strength is normal, such as chronic fatigue syndrome.

[0083] In some cases, such as myasthenia gravis, muscle strength is normal at rest, but real weakness occurs after muscle exercise. The same is true for some cases of chronic fatigue syndrome, where objective post-exercise muscle weakness with a delayed recovery time has been measured and is characterized by some publicly defined features. These diseases or conditions are also included in the “muscle weakness disorders” section of this disclosure.

[0084] Muscle weakness can also be classified as "proximal" or "distal" based on the location of the muscles it affects. Proximal muscle weakness affects the muscles closest to the midline of the body, while distal muscle weakness affects muscles further down the limbs. Proximal muscle weakness can be seen in Cushing's syndrome and hyperthyroidism.

[0085] In practice, other types of muscle weakness exist. For example, neuromuscular fatigue can be classified as "central" or "peripheral" depending on its cause. Central muscle fatigue manifests as a generalized feeling of energy depletion, while peripheral muscle fatigue manifests as a localized, muscle-specific inability to function. Based on the following exemplary criteria, the severity of muscle weakness can be classified into different "grades": Level 0: No contraction or muscle movement.

[0086] Grade 1: Signs of contraction, but no movement at the joint.

[0087] Level 2: Eliminates joint movement under the influence of gravity.

[0088] Level 3: Motion that resists gravity, but not additional resistance.

[0089] Level 4: Movement that resists external resistance, with a lower intensity than usual.

[0090] Level 5: Normal intensity.

[0091] Hypophosphatase syndrome (HPP) and muscle weakness Hypophosphatase disorder (HPP) is a rare, inherited metabolic disorder caused by a loss-of-function mutation in the tissue nonspecific alkaline phosphatase (TNSALP) gene. The biochemical marker is lower than normal serum ALP activity (hypophosphatase disorder), which leads to elevated blood and / or urine levels of three phosphate substrates: inorganic pyrophosphate (PPi), phosphoethanolamine (PEA), and pyridoxal 5'-phosphate (PLP). TNSALP deficiency can lead to a range of sequelae, including premature loss of primary teeth, rickets, stunted growth, muscle weakness, impaired physical function, and pain. Muscle weakness or myopathy associated with HPP has been identified decades ago. For example, Seshia et al. (1990) reported three children with HPP who also experienced muscle pain, stiffness, and proximal lower limb muscle weakness occurring early in the course of the disease (two of whom still present). Interestingly, Seshia et al. (1990) found these symptoms “unexplained by bone damage” and “similar to osteomalacia.” Other signs and symptoms of HPP may include: chronic pain in muscles or joints, arthritis (in adults and children), pseudogout caused by calcium deposits in the joints, and inability to walk without assistive devices such as crutches, walkers, or wheelchairs. Recently, Asphertex Alpha, a recombinant bone-targeting human TNASLP (i.e., sALP-Fc-D) has been reported. 10 It reduces elevated inorganic pyrophosphate (PPi) levels in patients with HPP and improves bone mineralization, growth, and physical function. Children aged 5–12 years with HPP treated with Asphertex Alpha for more than three years showed improvements in muscle strength, measured by handheld dynamometer (HHD) and individual subtests (including strength and running speed / agility scale scores) of the Brunincus-Osiris Motor Ability Test Version 2 (BOT-2). As a result, their muscle function significantly increased, impacting their ability to perform daily living activities.

[0092] Asfutez Alpha can be used to treat, for example, perinatal HPP, infant HPP, pediatric HPP, and dental HPP. For example, patients with pediatric HPP (e.g., children aged approximately 5 to approximately 12 years with HPP) or infant HPP (e.g., infants approximately 3 years of age or younger) can be treated with sALP (such as TNALP, such as the sALP peptide of SEQ ID NO: 1 or a peptide having at least 95% sequence identity with the sequence of SEQ ID NO: 1, such as Asfutez Alpha) for at least one year (e.g., at least five years, at least six years, at least seven years, at least eight years, at least nine years, at least ten years, or more than ten years (e.g., the patient's lifetime)).

[0093] Because Asphertex Alpha treatment significantly improves bone mineralization in patients, it is uncertain whether its effect on muscle strength is solely a result of restored bone formation and subsequent recovery of skeletal muscle attachment / growth, or a direct therapeutic effect on the patient's muscles. As described in this article, we have found that Asphertex Alpha has a therapeutic effect on the muscles of patients (e.g., those with myasthenic diseases such as muscle weakness in patients with HPP).

[0094] Calcium pyrophosphate deposition disease (CPPD or CPDD) and muscle weakness Calcium pyrophosphate deposition disease (CPPD or CPDD), or calcium pyrophosphate dihydrate crystal deposition disease, is a metabolic arthropathy caused by the deposition of calcium pyrophosphate dihydrate crystals within and around joints, particularly articular cartilage and fibrocartilage. Although CPPD is often asymptomatic, with only radiographic changes observed (i.e., chondrocalcinosis), it can present with a variety of clinical manifestations, including acute (pseudogout) and chronic arthritis. The crystal deposits cause joint inflammation, which can lead to the breakdown of articular cartilage. The disease can take several different arthritis-related forms: osteoarthritis, chronic rheumatoid arthritis (RA)-like inflammatory arthritis, or an acute, painful inflammatory condition called pseudogout. The name pseudogout comes from the fact that it resembles another acute, painful condition called gout. The main difference lies in the type of crystal involved in the inflammation and damage. CPPD can involve almost any joint, although the knee, wrist, and hip are most frequently affected. This condition is the most common cause of secondary metabolic osteoarthritis. Patients with CPPD may experience significant morbidity due to acute attacks of pseudogout or pain as a symptom of chronic arthropathy. Treating symptomatic CPPD is important for preventing further extremity damage, but it cannot reverse the joint disease.

[0095] The exact mechanisms underlying the development of CPPD remain unclear. Patients exhibit increased adenosine triphosphate (ATP) breakdown, leading to increased inorganic pyrophosphate concentrations in the joints, whether due to aging, genetic factors, or both. Changes in the cartilage matrix may play a significant role in promoting the deposition of calcium pyrophosphate dihydrate crystals. Excessive activity of enzymes that break down triphosphates, such as nucleoside triphosphate pyrophosphate hydrolase, has been observed in the cartilage of patients with CPPD. Consequently, inorganic pyrophosphate can bind to calcium, leading to deposition in the cartilage and synovium. (See Beutler et al., 1993) Arthritis Rheum. 36(5):704-715). Hyaline cartilage is most commonly affected, but fibrocartilage, such as the meniscus cartilage of the knee, can also be involved. (Pritzker et al., 1988) Rheumatol. 15(5):828-835).

[0096] Other diseases and muscle weakness Similar to HPP and CPPD (or CPDD), other diseases or conditions may include at least one symptom of muscle weakness. Among these, some types of myasthenic diseases are characterized by elevated inorganic pyrophosphate (PPi) concentrations. These myasthenic diseases with elevated PPi concentrations are also targets for treatment with Asftatex alpha in this disclosure.

[0097] For example, familial hypophosphatemia (such as autosomal dominant hypophosphatemic rickets (ADHR), autosomal recessive hypophosphatemic rickets, X-linked hypophosphatemic rickets, X-linked hypophosphatemic rickets (XLH), etc.) typically presents with a muscle weakness phenotype. Hypophosphatemia or hypophosphatemic rickets is a form of rickets characterized by low serum phosphate levels and resistance to treatment with ultraviolet radiation or vitamin D ingestion. X-linked hypophosphatemia (XLH) is an dominant condition and accounts for more than 80% of all familial hypophosphatemia. XLH is considered a systemic condition resulting from a mutation in the phosphate-regulating gene (PHEX), which is homologous to endopeptidase on the X chromosome. XLH patients exhibit normal or low serum concentrations of 1,25-dihydroxyvitamin D3, suggesting inadequate formation of this vitamin D metabolite. The remaining 20% ​​of patients with familial hypophosphatemia have autosomal dominant hypophosphatemia, which is derived from acquired autosomal recessive hypophosphatemia rickets and hereditary hypophosphatemia rickets with hypercalciuria.

[0098] Treatment This article provides methods for treating or improving at least one symptom in subjects, children, adolescents, or adults who have or are susceptible to muscular dystrophy. Such treatment may include administration of an alkaline phosphatase or a polypeptide having alkaline phosphatase activity to reduce elevated PPi concentrations in such subjects. For example, soluble alkaline phosphatases (sALPs, such as TNALP, such as the sALP polypeptide of SEQ ID NO: 1 or a polypeptide variant having at least 95% sequence identity with the sequence of SEQ ID NO: 1, such as Asphertex Alpha) may be administered to children, adolescents, or adult subjects across a range of age groups.

[0099] Prior to administration of alkaline phosphatase or a peptide with alkaline phosphatase activity (e.g., sALP, such as TNALP, such as the sALP peptide of SEQ ID NO: 1, or a peptide variant having at least 95% sequence identity with the sequence of SEQ ID NO: 1, such as Aspheres Alpha), the subject may be diagnosed with a myasthenic disease (such as HPP, CPPD, familial hypophosphatemia, etc. as described herein). Additionally, subjects with or susceptible to myasthenic disease may be treatment-naïve subjects who have not previously received treatment with sALP (such as TNALP, such as the sALP peptide of SEQ ID NO: 1, or a peptide variant having at least 95% sequence identity with the sequence of SEQ ID NO: 1, such as Aspheres Alpha).

[0100] The method involves administering alkaline phosphatase or a polypeptide with alkaline phosphatase activity (e.g., sALP, such as TNALP, such as the sALP polypeptide of SEQ ID NO: 1, or a polypeptide variant having at least 95% sequence identity with the sequence of SEQ ID NO: 1, such as Asphertex Alpha) to subjects with or susceptible to myasthenia gravis over a period of time in single or multiple doses. Specifically, sALP, such as Asphertex Alpha, may be administered to subjects previously identified as having elevated inorganic pyrophosphate (PPi) concentrations or having at least one predetermined biomarker / score for muscle weakness (such as a mean BOT-2 strength score less than 10, a mean BOT-2 running speed and agility score less than 5, a mean CHAQ index score greater than about 0.8, and / or a mean PODCI score less than about 40, a mean 6MWT less than about 80% of the predicted 6MWT value, a muscle strength grade less than 5, and / or a mean HHD value (e.g., mean HHD muscle or grip strength value) less than about 80% of the predicted HHD value). For example, sALP can be administered to subjects previously identified as having a PPi concentration greater than about 5.71 μM for infants and children (e.g., subjects younger than about 12 years of age); greater than about 4.78 μM for adolescents (e.g., subjects aged from about 13 to about 18 years of age); or greater than about 5.82 μM for adults (e.g., subjects older than about 18 years of age). In other embodiments, the muscle weakness disease described herein is caused by an elevated concentration of at least one alkaline phosphatase substrate (e.g., PPi, PLP, PEA, etc.). Alternatively, prior to determining such scores (e.g., BOT-2 strength score, BOT-2 running speed and agility score, CHAQ index score, BSID-III scale score, PDMS-2 standard score, muscle strength score, 6MWT value, and / or HHD value), alkaline phosphatase or peptides with alkaline phosphatase activity (e.g., sALP, such as TNALP, such as the sALP peptide of SEQ ID NO: 1, or peptide variants having at least 95% sequence identity with the sequence of SEQ ID NO: 1, such as Aspheres Alpha) may be administered to subjects with or susceptible to muscle weakness disorders to allow for, for example, increased ADL activity, reduced pain, and / or improved motor development.

[0101] Additionally, each described score (e.g., BOT-2 strength score, BOT-2 running speed and agility score, CHAQ index score, BSID-III scale score, PDMS-2 standard score, 6MWT, 12-POMA-G, modified performance-oriented mobility assessment (mPOMA-G, such as that shown in Phillips et al., Bone Abstracts 4: P136, 2015) or HHD value) in subjects with or susceptible to the muscle weakness described herein can be used alone or in any combination to evaluate the therapeutic efficacy of sALP (such as TNALP, e.g., the sALP peptide of SEQ ID NO: 1 or a peptide variant having at least 95% sequence identity with the sequence of SEQ ID NO: 1, e.g., Asphertex Alpha), where improvement relative to a certain test score indicates that sALP is effective for treating this muscle weakness.

[0102] For example, when administration of alkaline phosphatase or a peptide with alkaline phosphatase activity (e.g., sALP, such as TNALP, such as the sALP peptide of SEQ ID NO: 1, or a peptide variant having at least 95% sequence identity with the sequence of SEQ ID NO: 1, such as Aspheres Alpha) to a subject with or susceptible to muscular dystrophy results in an average increase in the BOT-2 strength score to about 10 or greater than about 10, where the subject previously had an average BOT-2 strength score of less than about 10, then treatment with alkaline phosphatase or a peptide with alkaline phosphatase activity is effective in treating, for example, physical injuries associated with muscular dystrophy. Alternatively, when administration of sALP does not result in an average increase in the BOT-2 strength score to about 10 or greater than about 10, the dose and / or frequency of administration of alkaline phosphatase or a peptide with alkaline phosphatase activity may be varied to determine the effective amount of alkaline phosphatase or a peptide with alkaline phosphatase activity for the subject. For example, the dose of sALP (such as TNALP, such as the sALP peptide of SEQ ID NO: 1 or a peptide variant having at least 95% sequence identity with the sequence of SEQ ID NO: 1, such as Asphertex Alpha) may be increased from, for example, about 3 mg / kg / week to about 6 mg / kg / week or from about 6 mg / kg / week to about 9 mg / kg / week.

[0103] Additionally, when administration of alkaline phosphatase or a peptide with alkaline phosphatase activity (e.g., sALP, such as TNALP, such as the sALP peptide of SEQ ID NO: 1, or a peptide variant having at least 95% sequence identity with the sequence of SEQ ID NO: 1, such as Aspheres Alpha) to a subject with or susceptible to muscular dystrophy results in an improvement in the muscle strength grade classification of one or more subjects (e.g., from a previous, lower muscle strength grade to a muscle strength grade of 1, 2, 3, 4, or 5), wherein the subject previously had an average muscle strength grade of less than about 5, then treatment with alkaline phosphatase or a peptide with alkaline phosphatase activity is effective in treating, for example, physical injuries associated with muscular dystrophy. Alternatively, when administration of sALP does not result in an improvement in the muscle strength grade classification of one or more subjects from a previous, lower muscle strength grade, the dose and / or frequency of administration of alkaline phosphatase or a peptide with alkaline phosphatase activity may be varied (e.g., increased) to determine the effective amount of alkaline phosphatase or a peptide with alkaline phosphatase activity for the subject. For example, the dose of sALP (such as TNALP, such as the sALP peptide of SEQ ID NO: 1 or a peptide variant having at least 95% sequence identity with the sequence of SEQ ID NO: 1, such as Asphertex Alpha) may be increased from, for example, about 3 mg / kg / week to about 6 mg / kg / week or from about 6 mg / kg / week to about 9 mg / kg / week.

[0104] Biomarkers / endpoints for the diagnosis and / or treatment of myasthenia gravis In a preferred embodiment, muscular dystrophy (such as HPP, including, for example, perinatal HPP, infant HPP, childhood HPP, and dental hypophosphatase syndrome, CPPD, and familial hypophosphatemia as described herein) is treated with alkaline phosphatase or a polypeptide with alkaline phosphatase activity (e.g., sALP, such as TNALP, such as the sALP polypeptide of SEQ ID NO: 1 or a polypeptide variant having at least 95% sequence identity with the sequence of SEQ ID NO: 1, such as Aspheres Alpha). The methods described herein can also be used to diagnose subjects with or predisposed to muscular dystrophy, to identify subjects as members of a specific subgroup of subjects with or predisposed to muscular dystrophy, or to test the efficacy of treatment for muscular dystrophy. For example, if a subject exhibits certain characteristic biomarkers, such a subject can be diagnosed with or predisposed to muscular dystrophy. Subjects may be treated with alkaline phosphatase or a polypeptide with alkaline phosphatase activity (e.g., sALP, such as TNALP, such as the sALP polypeptide of SEQ ID NO: 1 or a polypeptide variant having at least 95% sequence identity with the sequence of SEQ ID NO: 1, such as Asphertex Alpha), and the efficacy or effect of treatment may be analyzed using certain characteristic biomarkers or endpoints. Such biomarkers may include, for example, elevated concentrations of inorganic pyrophosphate (PPi) and / or decreased alkaline phosphatase (ALP) in the subject's serum, bone or muscle tissue, or urine. Exemplary endpoints available in the methods for treating muscle weakness described herein may include: (1) the Brunincus-Osiris Motor Ability Test, Version 2 (BOT-2), (2) the Child Health Assessment Questionnaire (CHAQ), (3) the Pediatric Outcomes Data Collection Tool (PODCI), (4) the Bayley Infant Development Scale, Version 3 (BSID-III), (5) the Peabody Developmental Motor Scale, Version 2 (PDMS-2), (6) the Six-Minute Walk Test (6MWT), (7) the Muscle Strength Rating, and (8) the Handheld Force Measurement (HHD), which are described in further detail below.

[0105] Plasma inorganic pyrophosphate (PPi) and alkaline phosphatase (ALP) concentrations By determining the concentrations of inorganic pyrophosphate (PPi) and / or alkaline phosphatase (ALP) in samples from patients (such as plasma or urine samples), subjects with or susceptible to myasthenia gravis can be identified for treatment with alkaline phosphatase or peptides with alkaline phosphatase activity (e.g., sALP, such as TNALP, such as the sALP peptide of SEQ ID NO: 1, or a peptide variant having at least 95% sequence identity with the sequence of SEQ ID NO: 1, e.g., Aspheres alpha). Any method known to those skilled in the art for quantifying PPi and / or ALP concentrations in plasma samples or alternatively urine samples, as described in detail in Whyte et al., 1995 (J. Clin. Invest. 95(4): 1440–1445), is hereby incorporated by reference in its entirety. Methods for quantifying PPi concentrations in plasma or urine samples 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.

[0106] Specifically, alkaline phosphatase or a polypeptide with alkaline phosphatase activity (e.g., sALP, such as TNALP, such as the sALP polypeptide of SEQ ID NO: 1 or a polypeptide variant having at least 95% sequence identity with the sequence of SEQ ID NO: 1, such as Aspheres Alpha) may be administered to subjects (e.g., humans) who have or are susceptible to myasthenia gravis and have previously been determined to have a plasma PPi concentration of up to about 6 µM (e.g., about 4.5 µM, about 5 µM, or about 5.5 µM, or a plasma PPi concentration in the range of about 4.5 µM to about 6 µM). For example, alkaline phosphatase or a polypeptide with alkaline phosphatase activity may be administered to, for example, infants or children (e.g., subjects under about 12 years of age) with a plasma PPi concentration of about 5.71 μM or greater; adolescents (e.g., subjects from about 13 to about 18 years of age) with a plasma PPi concentration of about 4.78 μM or greater; or adults (e.g., subjects older than about 18 years of age) with a plasma PPi concentration of about 5.82 μM or greater. Additionally, alkaline phosphatase or peptides with alkaline phosphatase activity may be administered to subjects (e.g., humans) with pre-determined activity levels, for example, approximately 90 U / L or less for subjects aged 0 to 14 days; approximately 134 U / L or less for subjects aged 15 days to less than 1 year; approximately 156 U / L or less for subjects aged approximately 1 year to less than 10 years; approximately 141 U / L or less for subjects aged approximately 10 years to less than approximately 13 years; approximately 62 U / L or less for female subjects aged approximately 13 years to less than approximately 15 years; approximately 127 U / L or less for male subjects aged approximately 13 years to less than approximately 15 years; approximately 54 U / L or less for female subjects aged approximately 15 years to less than approximately 17 years; approximately 89 U / L or less for male subjects aged approximately 15 years to less than approximately 17 years; approximately 48 U / L or less for female subjects aged approximately 17 years or older; or approximately 59 U / L for male subjects aged approximately 17 years or older. Plasma ALP concentration of U / L or lower.

[0107] Plasma PPi and / or plasma ALP concentrations in subjects with or susceptible to myasthenia gravis (e.g., humans) can be compared with those in normal subjects to determine the therapeutic effect of administering alkaline phosphatase or a peptide with alkaline phosphatase activity (e.g., sALP, such as TNALP, such as the sALP peptide of SEQ ID NO: 1, or a peptide variant having at least 95% sequence identity with the sequence of SEQ ID NO: 1, such as Aspheres Alpha). Specifically, the alkaline phosphatase or the peptide with alkaline phosphatase activity may be administered for a treatment period of at least one year (e.g., at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, at least nine, at least ten years, or longer than ten years, such as a patient's lifetime). Alternatively, the method may include determining plasma PPi and / or plasma ALP concentrations prior to administration of alkaline phosphatase or a peptide with alkaline phosphatase activity to assess the efficacy in subjects treated with alkaline phosphatase or a peptide with alkaline phosphatase activity.

[0108] The method results in a decrease in PPi concentration and / or an increase in ALP concentration in samples (e.g., plasma samples) from subjects (e.g., humans) who have or are susceptible to myasthenia gravis. For example, treatment with alkaline phosphatase or a polypeptide with alkaline phosphatase activity (e.g., sALP, such as TNALP, such as the sALP polypeptide of SEQ ID NO: 1 or a polypeptide variant having at least 95% sequence identity with the sequence of SEQ ID NO: 1, such as Asphertex Alpha) results in a decrease in PPi concentration of about 1 μM, about 1.5 μM, about 2 μM, about 2.5 μM or about 3 μM or 25% or greater (e.g., 30%, 35%, 40%, 45%, 50%, 55%, 60% or more than 60%) in samples (e.g., plasma samples) from patients. Therefore, after administration of alkaline phosphatase or a peptide with alkaline phosphatase activity, subjects exhibit plasma PPi concentrations of, for example, about 2 μM to about 5 μM, about 3 μM to about 5 μM, about 2 μM to about 4 μM, or about 2 μM to about 3 μM.

[0109] Similarly, treatment with alkaline phosphatase or a peptide with alkaline phosphatase activity resulted in an increase of 30%, 35%, 40%, 45%, 50%, 55%, 60%, or more than 60% in ALP concentrations in samples (e.g., plasma samples) from subjects who had or were susceptible to muscular dystrophy. For example, administration of alkaline phosphatase or a peptide with alkaline phosphatase activity increases the ALP concentration in samples (e.g., plasma samples) from subjects to, for example, about 273 U / L or greater for subjects aged 0 to 14 days; about 518 U / L or greater for subjects aged 15 days to less than 1 year; about 369 U / L or greater for subjects aged about 1 year to less than 10 years; about 460 U / L or greater for subjects aged about 10 years to less than about 13 years; about 280 U / L or greater for female subjects aged about 13 years to less than about 15 years; about 517 U / L or greater for male subjects aged about 13 years to less than about 15 years; about 128 U / L or greater for female subjects aged about 15 years to less than about 17 years; about 365 U / L or greater for male subjects aged about 15 years to less than about 17 years; and about 95 U / L for female subjects aged about 17 years or older. U / L or greater; or approximately 164 U / L or greater for male subjects aged approximately 17 years or older.

[0110] A decrease in plasma PPi and / or an increase in ALP concentration in subjects (e.g., humans) with or susceptible to myasthenia gravis can be maintained throughout administration of alkaline phosphatase or a peptide with alkaline phosphatase activity (e.g., sALP, such as TNALP, such as the sALP peptide of SEQ ID NO: 1, or a peptide variant having at least 95% sequence identity with the sequence of SEQ ID NO: 1, such as Asphertex Alpha). For example, a decrease in plasma PPi of about 25% and maintenance at ±10% of the decreased plasma PPi concentration during treatment with sALP, and / or an increase in plasma ALP concentration of about 50% and maintenance at ±10% of the increased plasma ALP concentration during treatment with alkaline phosphatase or a peptide with alkaline phosphatase activity.

[0111] Alternatively, when administration of alkaline phosphatase or a polypeptide with alkaline phosphatase activity (e.g., sALP, such as TNALP, such as the sALP polypeptide of SEQ ID NO: 1 or a polypeptide variant having at least 95% sequence identity with the sequence of SEQ ID NO: 1, such as Aspheres Alpha) does not result in an average reduction of about 25% or greater in the PPi concentration in plasma samples from subjects (e.g., humans) suffering from or susceptible to myasthenia gravis, the dose and / or frequency of sALP administration may be varied to determine the effective amount of sALP for the subject. Similarly, when administration of alkaline phosphatase or a polypeptide with alkaline phosphatase activity does not result in an average increase of about 50% or greater in the ALP concentration in plasma samples from the subject, the dose and / or frequency of alkaline phosphatase or a polypeptide with alkaline phosphatase activity may be varied to determine the effective amount of alkaline phosphatase or a polypeptide with alkaline phosphatase activity for the subject. For example, the dose of alkaline phosphatase or a polypeptide with alkaline phosphatase activity may be increased from, for example, about 2.1 mg / kg / week or about 3.5 mg / kg / week to about 6 mg / kg / week or about 9 mg / kg / week.

[0112] Bruinx-OSIRES Athletic Performance Test Version 2 (BOT-2) The exemplary Bruininks-Osiris Tests of Motor Ability Version 2 (BOT-2) is described in Bruininks, RH (2005). . Bruininks-Oseretsky Test of Motor Proficiency, (BOT-2), Minneapolis, MN: Pearson Assessment, the entire text of which is incorporated herein by reference. In particular, BOT-2 can be used to assess physical impairment and mobility limitations in subjects who have or are susceptible to muscular dystrophy (e.g., HPP) to generate a BOT-2 score for the subject.

[0113] BOT-2 includes a series of tests to assess a subject's physical impairment, which can be performed using kits, for example, that include the tests. BOT-2 provides a composite BOT-2 score in the following areas: strength, running speed and agility, fine motor precision, fine motor integration, hand dexterity, bilateral coordination, balance, and upper limb coordination. For example, subjects with or susceptible to muscular dystrophy can perform sit-ups, V-sit-ups, standing long jumps, sit-and-reach, and / or push-ups to determine a BOT-2 strength score; subjects with or susceptible to muscular dystrophy can perform hopping across a balance beam or shuttle running, two-leg lateral jumps, and / or one-leg lateral jumps to determine a BOT-2 running speed and agility score; subjects with or susceptible to muscular dystrophy can cut out a circle and / or connect a point to determine a BOT-2 fine motor precision score; subjects with or susceptible to muscular dystrophy can copy a star and / or copy a square to determine a BOT-2 fine motor precision score. The OT-2 fine motor integration score; subjects with or susceptible to muscular dystrophy can transfer pennies, sorting cards, and / or string blocks to determine hand dexterity scores; subjects with or susceptible to muscular dystrophy can tap their feet and fingers and / or jump to determine BOT-2 bilateral coordination scores; subjects with or susceptible to muscular dystrophy can walk forward in a line and / or stand on a balance beam on one leg to determine BOT-2 balance scores; and subjects with or susceptible to muscular dystrophy can throw a ball toward a target and / or catch the thrown ball to determine BOT-2 upper limb coordination scores.

[0114] Subjects with or predisposed to muscular dystrophy (e.g., HPP) may be tested in one or more of the following aspects (strength, running speed and agility, fine motor precision, fine motor integration, hand dexterity, bilateral coordination, balance, and upper limb coordination) to generate a BOT-2 score indicative of physical impairment. Within each BOT-2 aspect (strength, running speed and agility, fine motor precision, fine motor integration, hand dexterity, bilateral coordination, balance, and upper limb coordination), such subjects may undergo one or more tests to determine their BOT-2 score. For example, subjects may perform one or more of sit-ups, V-ups, standing long jump, sit-up, and push-ups to determine a BOT-2 strength score. Therefore, only one test (e.g., a test selected from the group consisting of sit-ups, V-ups, standing long jump, sit-up, and push-ups) may be performed to determine a subject with or predisposed to muscular dystrophy (e.g., HPP) for their BOT-2 score (e.g., BOT-2 strength score).

[0115] Each BOT-2 score (intensity, running speed and agility, fine motor precision, fine motor integration, hand dexterity, bilateral coordination, balance, and upper limb coordination) of a subject with or susceptible to muscular dystrophy (e.g., HPP) can be compared with the BOT-2 scores of a subject without muscular dystrophy (e.g., HPP) to, for example, determine the standard deviation of the BOT-2 scores. Each BOT-2 score (e.g., intensity, running speed and agility, fine motor precision, fine motor integration, hand dexterity, bilateral coordination, balance, and upper limb coordination) of a subject with or susceptible to muscular dystrophy (e.g., HPP) can be compared with the BOT-2 scores of other subjects with or susceptible to muscular dystrophy (e.g., HPP) to, for example, determine the mean BOT-2 score of the subjects.

[0116] BOT-2 scores (e.g., strength, running speed and agility, fine motor precision, fine motor integration, hand dexterity, bilateral coordination, balance, and upper limb coordination scores) range from about 0 to equal to or less than about 25, where scores of about 10 to about 20 are considered representative of healthy subjects (e.g., subjects without muscle weakness disease (e.g., HPP)). Subjects with a mean BOT-2 score (e.g., strength, running speed and agility, fine motor precision, fine motor integration, hand dexterity, bilateral coordination, balance, and upper limb coordination scores) of less than about 10 may be treated with alkaline phosphatase or peptides with alkaline phosphatase activity (e.g., sALP, such as TNALP, such as the sALP peptide of SEQ ID NO: 1 or a peptide variant having at least 95% sequence identity with the sequence of SEQ ID NO: 1, such as Aspheres Alpha).

[0117] For example, subjects with or susceptible to muscular dystrophy who have a BOT-2 strength score 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) may be treated with sALP (such as TNALP, such as the sALP peptide of SEQ ID NO: 1 or a peptide variant having at least 95% sequence identity with the sequence of SEQ ID NO: 1, such as Aspheres Alpha) for a period of time, up to the patient's lifetime. Similarly, subjects with or susceptible to muscular dystrophy who have a BOT-2 running speed and agility score 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) may be treated with sALP (such as TNALP, such as the sALP peptide of SEQ ID NO: 1 or a peptide variant having at least 95% sequence identity with the sequence of SEQ ID NO: 1, such as Aspheres Alpha) for a period of time, up to the patient's lifetime.

[0118] The method can result in improved BOT-2 scores (e.g., strength, running speed and agility, fine motor precision, fine motor integration, hand dexterity, bilateral coordination, balance, and / or upper limb coordination scores) in subjects with or susceptible to muscle weakness disorders (e.g., HPP). For example, treatment with alkaline phosphatase or peptides with alkaline phosphatase activity, such as sALP (e.g., TNALP, such as the sALP peptide of SEQ ID NO: 1 or a peptide variant having at least 95% sequence identity with the sequence of SEQ ID NO: 1, e.g., Aspheres Alpha), such as treatment with sALP for a period of time, can result in an average increase in BOT-2 strength scores of 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). Additionally, treatment with sALP (such as TNALP, for example, the sALP peptide of SEQ ID NO: 1 or a peptide variant having at least 95% sequence identity with the sequence of SEQ ID NO: 1, for example, Aspheres Alpha) resulted in an average increase in BOT-2 running speed and agility scores of approximately 5 to approximately 20 (e.g., approximately 5, approximately 6, approximately 7, approximately 8, approximately 9, approximately 10, approximately 11, approximately 12, approximately 13, approximately 14, approximately 15, approximately 16, approximately 17, approximately 18, approximately 19 or approximately 20).

[0119] Increases in BOT-2 scores (e.g., scores for strength, running speed and agility, fine motor precision, fine motor integration, hand dexterity, bilateral coordination, balance, and / or upper limb coordination) can be maintained for a period of time, for example, throughout administration of alkaline phosphatase or peptides with alkaline phosphatase activity, such as sALP (e.g., TNALP, such as the sALP peptide of SEQ ID NO: 1 or a peptide variant having at least 95% sequence identity with the sequence of SEQ ID NO: 1, e.g., Aspheres Alpha). Similarly, reductions in physical damage to muscles following administration of alkaline phosphatase or peptides with alkaline phosphatase activity can be maintained throughout administration of alkaline phosphatase or peptides with alkaline phosphatase activity.

[0120] BOT-2 scores (strength, running speed and agility, fine motor precision, fine motor integration, hand dexterity, bilateral coordination, balance, and upper limb coordination scores) in subjects with or predisposed to muscular dystrophy (such as HPP) can be used alone or in combination with other endpoints to assess the therapeutic efficacy of alkaline phosphatase or peptides with alkaline phosphatase activity, such as sALP (e.g., TNALP, such as the sALP peptide of SEQ ID NO: 1 or a peptide variant having at least 95% sequence identity with the sequence of SEQ ID NO: 1, e.g., Aspheres Alpha), where improvement relative to a certain test score indicates that alkaline phosphatase or peptides with alkaline phosphatase activity are effective in treating muscle damage associated with muscular dystrophy. For example, when administration of sALP to subjects with or susceptible to myasthenia gravis results in an average increase in BOT-2 running speed and agility scores of about 5 or greater than about 5, where the subjects previously had an average BOT-2 running speed and agility score of less than about 5, then sALP is considered effective, for example, in treating physical injuries associated with myasthenia gravis.

[0121] In addition, within each BOT-2 aspect (intensity, running speed and agility, fine motor precision, fine motor integration, hand dexterity, bilateral coordination, balance, and upper limb coordination), subjects with or predisposed to muscular dystrophy (e.g., HPP, CPPD, familial hypophosphatemia, etc., as described herein) may undergo one or more tests to determine their BOT-2 score. For example, subjects with or predisposed to muscular dystrophy may perform one or more of the following to determine their BOT-2 intensity score and assess the therapeutic efficacy of sALP administration: sit-ups, V-sit-ups, standing long jump, sit-up, and push-ups. Subjects with or predisposed to muscular dystrophy may perform one or more of the following to determine their BOT-2 running speed and agility score and assess the therapeutic efficacy of sALP administration: subjects with or predisposed to muscular dystrophy may perform one or more of the following to determine their BOT-2 fine motor precision score and assess the therapeutic efficacy of sALP administration: subjects with or predisposed to muscular dystrophy may cut a circle and / or connect a point to determine their BOT-2 fine motor precision score and assess the therapeutic efficacy of sALP administration. Subjects with or susceptible to muscular dystrophy may replicate a star and / or a square to determine their BOT-2 fine motor integration score and assess the efficacy of sALP administration. Subjects with or susceptible to muscular dystrophy may perform one or more of the following exercises to determine their BOT-2 hand dexterity score and assess the efficacy of sALP administration. Subjects with or susceptible to muscular dystrophy may tap their feet and fingers and / or perform jumps to determine their BOT-2 bilateral coordination score and assess the efficacy of sALP administration. Subjects with or susceptible to muscular dystrophy may walk forward in a line and / or stand on a balance beam on one leg to determine their BOT-2 balance score and assess the efficacy of sALP administration. Subjects with or susceptible to muscular dystrophy may throw a ball toward a target and / or catch the thrown ball to determine their BOT-2 upper limb coordination score and assess the efficacy of sALP administration.

[0122] Alternatively, when administration of alkaline phosphatase or a peptide with alkaline phosphatase activity (such as sALP) does not result in an average increase in BOT-2 running speed and agility scores to greater than about 5, the dosage and / or frequency of administration may be varied to determine the effective amount of alkaline phosphatase or a peptide with alkaline phosphatase activity for subjects with or susceptible to muscle weakness disorders (e.g., HPP). For example, the dosage of sALP (such as TNALP, such as the sALP peptide of SEQ ID NO: 1 or a peptide variant having at least 95% sequence identity with the sequence of SEQ ID NO: 1, such as Asphertex Alpha) may be increased from, for example, about 3 mg / kg / week to about 6 mg / kg / week or from about 6 mg / kg / week to about 9 mg / kg / week.

[0123] Child Health Assessment Questionnaire (CHAQ) The Child Health Assessment Questionnaire (CHAQ) can be applied to assess the health status of children with muscular dystrophy (e.g., HPP), thereby generating a CHAQ index score for the child, such as that provided by Bruce & Fries. J. Rheumatol . 30(1):167-178, 2003) and Klepper ( Arthritis & Rheumatism As described in (49: S5–S14, 2003), the entire text of which is incorporated herein by reference. The CHAQ comprises eight categories of questions for dressing / grooming, standing, eating, walking, hygiene, stretching, grasping, and activity, in which parents or guardians record the degree of difficulty a child with a muscular dystrophy (e.g., HPP) experiences in performing the corresponding activity. Scores in each category range from 0 to 3, where a score of 0 indicates no difficulty; a score of 1 indicates some difficulty; a score of 2 indicates great difficulty; and a score of 3 indicates the child is unable to perform the activity.

[0124] Children with or susceptible to myasthenia gravis who have an average CHAQ score 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) (e.g., a disability indicating activities of daily living (ADL) and / or pain) can be treated with alkaline phosphatase or peptides with alkaline phosphatase activity, such as sALP (e.g., TNALP, such as the sALP peptide of SEQ ID NO: 1 or a peptide variant having at least 95% sequence identity with the sequence of SEQ ID NO: 1, e.g., Aspheres Alpha). For example, children with a mean CHAQ score greater than about 0.8 can be treated with alkaline phosphatase or a polypeptide with alkaline phosphatase activity, such as sALP (e.g., TNALP, such as the sALP polypeptide of SEQ ID NO: 1 or a polypeptide variant having at least 95% sequence identity with the sequence of SEQ ID NO: 1, such as Aspheres Alpha) for a period of time, up to the patient's lifetime. Furthermore, children with or susceptible to the muscle weakness disorders disclosed herein can be asked one or more questions in one or more of the eight categories (dressing / grooming, standing, eating, walking, hygiene, stretching, grasping, and activity) to obtain a mean CHAQ score, and if the mean CHAQ score is greater than about 0.8, the child can be treated with alkaline phosphatase or a polypeptide with alkaline phosphatase activity, such as sALP.

[0125] The CHAQ score of a child who has or is susceptible to the muscular dystrophy disclosed herein can be compared with the CHAQ score of a child who does not have the muscular dystrophy to determine, for example, the standard deviation of the CHAQ score. Alternatively, the CHAQ score of a child who has or is susceptible to the muscular dystrophy disclosed herein can be compared with the CHAQ score of other children who have or are susceptible to the muscular dystrophy disclosed herein to determine, for example, the standard deviation of the CHAQ score.

[0126] The methods described herein can result in improved CHAQ index scores (e.g., disabilities indicating ADL and / or pain) in children with or susceptible to the muscle weakness disorders disclosed herein. For example, treatment with sALP (such as TNALP, e.g., the sALP peptide of SEQ ID NO: 1 or a peptide variant having at least 95% sequence identity with the sequence of SEQ ID NO: 1, e.g., Aspheres alpha) for a period of time, up to the child's lifetime, can result in an average reduction in CHAQ index scores in children with HPP to about 0 to equal to or less than about 0.5 (e.g., about 0, about 0.1, about 0.2, about 0.4, or about 0.5).

[0127] A reduction in the CHAQ score in children with or predisposed to muscular dystrophy (e.g., HPP) may persist for some time, for example, throughout the child's life, throughout administration of alkaline phosphatase or peptides with alkaline phosphatase activity, such as sALP (e.g., TNALP, such as the sALP peptide of SEQ ID NO: 1 or a peptide variant having at least 95% sequence identity with the sequence of SEQ ID NO: 1, e.g., Aspheres Alpha). Similarly, an increase in ADL and / or a reduction in pain in the child may persist for some time, for example, throughout the child's life, throughout administration of sALP (e.g., TNALP, such as the sALP peptide of SEQ ID NO: 1 or a peptide variant having at least 95% sequence identity with the sequence of SEQ ID NO: 1, e.g., Aspheres Alpha).

[0128] The CHAQ score of children with or susceptible to muscular dystrophy (e.g., HPP) can be used to assess the therapeutic efficacy of alkaline phosphatase or peptides with alkaline phosphatase activity, such as sALP (e.g., TNALP, such as the sALP peptide of SEQ ID NO: 1 or a peptide variant having at least 95% sequence identity with the sequence of SEQ ID NO: 1, e.g., Aspheres Alpha), where improvement relative to a certain test score indicates that the alkaline phosphatase or peptide with alkaline phosphatase activity is effective in treating disabilities such as activities of daily living (ADL) and / or pain associated with muscular dystrophy. Specifically, children with or susceptible to muscular dystrophy may be asked one or more questions in one or more of the eight categories (dressing / grooming, standing, eating, walking, hygiene, stretching, grasping, and activity) to derive an average CHAQ score and assess the therapeutic efficacy of sALP administration. For example, when administration of sALP to a child with or susceptible to muscular dystrophy results in a mean reduction in CHAQ score to or less than about 0.5, wherein the child previously had a mean CHAQ score greater than about 0.8, then sALP is effective in treating disabilities such as activities of daily living (ADL) and pain associated with muscular dystrophy. Alternatively, when administration of sALP does not result in a mean reduction in CHAQ score to or less than about 0.5, the dose and / or frequency of sALP administration may be varied to determine the effective amount of sALP for a child with or susceptible to muscular dystrophy. For example, the dose of sALP (such as TNALP, such as the sALP peptide of SEQ ID NO: 1 or a peptide variant having at least 95% sequence identity with the sequence of SEQ ID NO: 1, such as Aspheres alpha) may be increased from, for example, about 3 mg / kg / week to about 6 mg / kg / week or from about 6 mg / kg / week to about 9 mg / kg / week.

[0129] Pediatric Outcomes Data Collection Tool (PODCI) The Pediatric Outcome Data Collection Tool (PODCI) can be used to identify certain subjects with or predisposed to myasthenic disorders (e.g., HPP) for treatment with alkaline phosphatase or peptides with alkaline phosphatase activity, such as sALP (e.g., TNALP, such as the sALP peptide of SEQ ID NO: 1 or a peptide variant having at least 95% sequence identity with the sequence of SEQ ID NO: 1, e.g., Aspheres alpha). PODCI can be administered to assess the child's health status to generate a patient's PODCI score, as shown in Plat et al. J. Pediatr Orthop. 23(6)As described in (788-790, 2003). PODCI includes eight categories of questions, which can be completed by a subject who has or is susceptible to a muscle weakness disorder (e.g., HPP) or by the subject's parent / guardian. The categories of PODCIs that can be used to identify subjects with or susceptible to muscular dystrophy include the following: 1) Upper Limb and Physical Function Scale, which measures difficulties encountered in performing daily personal care and school activities; 2) Transfer and Basic Mobility Scale, which measures difficulties experienced in performing routine and motor activities in daily activities; 3) Motor / Physical Function Scale, which measures difficulties or limitations encountered when participating in more active activities or motor activities; 4) Pain / Comfort Scale, which measures the degree of pain experienced in the past week; 5) Treatment Expectation Scale, which measures long-term expectations of treatment; 6) Well-being Scale, which measures overall satisfaction with one's personal appearance and similarity with peers and others; 7) Symptom Satisfaction Scale, which measures the patient's acceptance of current limitations corresponding to a lifelong condition; and 8) Global Function Scale, which is a general combination scale calculated from the first four scales listed above. Within each category, standardized scores are determined for subjects with or susceptible to muscular dystrophy and are then converted to a 0-100 scale, where 0 represents significant disability and 100 represents less severe disability.

[0130] Subjects with or predisposed to muscular dystrophy (e.g., HPP) who have a mean PODCI score of less than about 40 (e.g., about 5, about 10, about 15, about 20, about 25, about 30, about 35, or about 39) (e.g., a disability indicating ADL and / or pain) can be treated with administration of alkaline phosphatase or peptides with alkaline phosphatase activity, such as sALP (e.g., TNALP, such as the sALP peptide of SEQ ID NO: 1 or a peptide variant having at least 95% sequence identity with the sequence of SEQ ID NO: 1, e.g., Aspheres alpha). For example, subjects with a mean PODCI score of less than 40 can be treated with sALP for a period of time, up to the patient's lifetime. Furthermore, subjects with or predisposed to muscular dystrophy may be asked one or more questions from one or more of the eight scales mentioned above (e.g., transfer and basic mobility, motor / physical function, and pain / comfort scales) to arrive at a mean PODCI score, and if the mean PODCI score is greater than or less than 40, the patient can be treated with sALP.

[0131] The methods described herein can result in an increase in PODCI scores (e.g., disabilities indicating ADL and / or pain) in subjects who have or are susceptible to muscular dystrophy. For example, treatment with alkaline phosphatase or peptides with alkaline phosphatase activity, such as sALP (e.g., TNALP, such as the sALP peptide of SEQ ID NO: 1 or a peptide variant having at least 95% sequence identity with the sequence of SEQ ID NO: 1, e.g., Aspheres alpha), such as treatment with sALP for a period of time, up to the lifetime of the subject, can result in an average increase in PODCI scores of 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).

[0132] An increase in PODCI score may be maintained for a period of time, for example, throughout the lifetime of a subject with or susceptible to myasthenia gravis, through administration of alkaline phosphatase or peptides with alkaline phosphatase activity, such as sALP (e.g., TNALP, such as the sALP peptide of SEQ ID NO: 1 or a peptide variant having at least 95% sequence identity with the sequence of SEQ ID NO: 1, e.g., Aspheres Alpha). Similarly, an increase in ADL and / or a reduction in pain may be maintained for a period of time, for example, throughout the lifetime of a subject, through administration of sALP (e.g., TNALP, such as the sALP peptide of SEQ ID NO: 1 or a peptide variant having at least 95% sequence identity with the sequence of SEQ ID NO: 1, e.g., Aspheres Alpha).

[0133] The PODCI score of subjects with or predisposed to muscular dystrophy (e.g., HPP) can be used to assess the therapeutic efficacy of alkaline phosphatase or peptides with alkaline phosphatase activity, such as sALP (e.g., TNALP, such as the sALP peptide of SEQ ID NO: 1 or a peptide variant having at least 95% sequence identity with the sequence of SEQ ID NO: 1, e.g., Aspheres Alpha), where improvement relative to a certain test score indicates that the alkaline phosphatase or peptide with alkaline phosphatase activity is effective in treating disabilities such as activities of daily living (ADL) and / or pain associated with muscular dystrophy. Specifically, subjects with or predisposed to muscular dystrophy may be asked one or more questions from one or more of eight scales (Upper Limb and Body Function Scale, Transfer and Basic Mobility Scale, Motor / Body Function Scale, Pain / Comfort Scale, Treatment Expectations Scale, Well-being Scale, Symptom Satisfaction Scale, and Global Function Scale) to derive an average PODCI score and assess the therapeutic efficacy of sALP administration.

[0134] For example, when administration of sALP to a subject with or susceptible to muscular dystrophy results in an average increase in the PODCI score to about 40 or greater than about 40, wherein the subject previously had an average PODCI score of less than about 40, then sALP is effective in treating disabilities such as activities of daily living (ADL) and pain associated with muscular dystrophy. Alternatively, when administration of sALP does not result in an average increase in the PODCI score to about 40 or greater than about 40, the dose and frequency of sALP administration may be varied to determine the effective amount of sALP for a subject with or susceptible to muscular dystrophy. For example, the dose of sALP (such as TNALP, such as the sALP peptide of SEQ ID NO: 1 or a peptide variant having at least 95% sequence identity with the sequence of SEQ ID NO: 1, such as Aspherates Alpha) may be increased from, for example, about 3 mg / kg / week to about 6 mg / kg / week or from about 6 mg / kg / week to about 9 mg / kg / week.

[0135] Bayley Infant and Toddler Development Scales, 3rd Edition (BSID-III) Another endpoint can be applied, the Bayley Infant Development Scale, 3rd Edition (BSID-III), to assess subjects with or predisposed to muscular dystrophy (e.g., HPP) from their birth health status, thereby generating a subject's BSID-III score, as Bayley (2006). Bayley scales of infant and toddler development: administration manual As described in *San Antonio, TX: Harcourt Assessment*, BSID-III comprises a series of developmental play tasks administered to subjects to determine an initial BSID-III score. For example, categories used to determine a BSID-III score for subjects with or predisposed to muscular dystrophy (e.g., infants approximately 3 years of age or younger with HPP) may include grasping, sensorimotor integration, motor planning and speed, visual tracking, extension, object grasping, object manipulation, functional hand skills, response to tactile information, limb and trunk movement, static positioning, dynamic movement, balance, and motor planning. The BSID-III measurements are then converted to a scaled BSID-III score, which can be used to determine patient performance compared to healthy, age-adjusted patients. BSID-III scale scores for subjects with or predisposed to muscular dystrophy (e.g., patients with HPP) range from 0 to 14, with scores of approximately 7 to approximately 13 considered within the normal range for healthy subjects.

[0136] As infants (e.g., about 3 years of age or less), subjects with or susceptible to muscular dystrophy may be tested in one or more of the categories (grasping, sensorimotor integration, motor planning and speed, visual tracking, extension, object grasping, object manipulation, functional hand skills, response to tactile information, limb and trunk movement, static positioning, dynamic movement, balance, and motor planning) to generate a BSID-III score indicative of delayed motor development. As infants, subjects with or susceptible to muscular dystrophy whose mean BSID-III score is less than about 2 in one or more of the categories (grasping, sensorimotor integration, motor planning and speed, visual tracking, extension, object grasping, object manipulation, functional hand skills, response to tactile information, limb and trunk movement, static positioning, dynamic movement, balance, and motor planning) may be treated with administration of sALP (such as TNALP, e.g., the sALP peptide of SEQ ID NO: 1 or a peptide variant having at least 95% sequence identity with the sequence of SEQ ID NO: 1, e.g., Asphertex Alpha). In particular, infants with or susceptible to myasthenia gravis who have an average BSID-III score of less than about 2 can be treated with sALP for a period of time, up to the course of their lives.

[0137] The method can result in improved mean BSID-III scores (e.g., indicating delayed motor development) in subjects with or predisposed to muscular dystrophy. For example, treatment with sALP (such as TNALP, such as the sALP peptide of SEQ ID NO: 1 or a peptide variant having at least 95% sequence identity with the sequence of SEQ ID NO: 1, such as Aspheres alpha) for a period of time, up to the lifetime of the subject, can result in an average increase in 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).

[0138] An increase in BSID-III score may be maintained for a period of time throughout administration of alkaline phosphatase or peptides with alkaline phosphatase activity, such as sALP (e.g., TNALP, such as the sALP peptide of SEQ ID NO: 1 or a peptide variant having at least 95% sequence identity with the sequence of SEQ ID NO: 1, e.g., Aspheres Alpha), extending into the lifetime of a subject with or susceptible to muscular dystrophy. Similarly, an increase in motor development may be maintained for a period of time throughout administration of alkaline phosphatase or peptides with alkaline phosphatase activity, such as sALP (e.g., TNALP, such as the sALP peptide of SEQ ID NO: 1 or a peptide variant having at least 95% sequence identity with the sequence of SEQ ID NO: 1, e.g., Aspheres Alpha).

[0139] The BSID-III score of subjects with or predisposed to muscular dystrophy (e.g., HPP) can be used to assess the therapeutic efficacy of alkaline phosphatase or peptides with alkaline phosphatase activity, such as sALP (e.g., TNALP, such as the sALP peptide of SEQ ID NO: 1 or a peptide variant having at least 95% sequence identity with the sequence of SEQ ID NO: 1, e.g., Aspheresi alpha), where improvement relative to a certain test score indicates that alkaline phosphatase or peptides with alkaline phosphatase activity are effective for treating, for example, delayed motor development associated with muscular dystrophy. Specifically, as infants (e.g., approximately 3 years of age or younger, with HPP), subjects with or predisposed to muscular dystrophy can be tested in one or more of the categories (grasping, sensorimotor integration, motor planning and speed, visual tracking, extension, object grasping, object manipulation, functional hand skills, response to tactile information, limb and trunk movement, static positioning, dynamic movement, balance, and motor planning) to derive an average BSID-III score and assess the therapeutic efficacy of sALP administration.

[0140] For example, when administration of sALP to a child with or susceptible to muscular dystrophy results in an average increase in BSID-III score to greater than about 5, wherein the child, as an infant (e.g., about 3 years of age or less), previously had an average BSID-III score of less than about 2, then sALP is effective in treating, for example, delayed motor development associated with HPP. Alternatively, when administration of sALP does not result in an average increase in BSID-III score to greater than about 5, the dose and / or frequency of sALP administration may be varied to determine the effective amount of sALP for a child with or susceptible to muscular dystrophy. For example, the dose of sALP (such as TNALP, such as the sALP peptide of SEQ ID NO: 1 or a peptide variant having at least 95% sequence identity with the sequence of SEQ ID NO: 1, such as Aspheres alpha) may be increased from, for example, about 3 mg / kg / week to about 6 mg / kg / week or from about 6 mg / kg / week to about 9 mg / kg / week.

[0141] Peabody Developmental Motor Scale, 2nd Edition (PDMS-2) Another endpoint can be applied, the Peabody Developmental Motor Scale, 2nd Edition (PDMS-2), to assess whether a person has or is susceptible to a muscle weakness disorder (e.g., HPP The subjects' PDMS-2 scores are generated from their birth health status, as described by van Hartingsveldt et al. (Occup. Ther. Int. 12(1): 1-13, 2005). PDMS-2 includes six categories of subtests to measure the motor skills of subjects (such as those with HPP).

[0142] Specifically, PDMS-2 measurements can be determined from the following subtests: 1) a motor subtest to measure a subject’s ability to move from one place to another (measurements include crawling, walking, running, jumping, and forward jumping); 2) a reflex subtest to measure a subject’s ability to react automatically to environmental events; 3) a stationary subtest to measure a subject’s ability to maintain body control and balance within the center of gravity; 4) an object manipulation subtest to measure a subject’s ability to manipulate objects, such as catching, throwing, and kicking a ball; 5) a grasping subtest to measure a subject’s ability to use his or her hands, such as the ability to grasp an object with one hand and actions involving the controlled use of the fingers of both hands; and 6) a visual-motor integration subtest to measure a subject’s ability to use his or her visual perception skills to perform complex eye-hand coordination tasks, such as reaching and grasping objects, building and replicating designs with blocks. PDMS-2 measurements for one or more of these categories can be determined for subjects who have or are susceptible to muscle weakness disorders (e.g., HPP) and then converted into PDMS-2 scores (such as the PDMS-2 Exercise Criterion Score) ranging from 0 to 13, with healthy subjects (e.g., subjects without muscle weakness disorders) ranging from about 7 to about 13.

[0143] Subjects with or susceptible to myasthenia gravis who have an average PDMS score (e.g., indicating delayed motor development) can be treated with sALP (such as TNALP, such as the sALP peptide of SEQ ID NO: 1 or a peptide variant having at least 95% sequence identity with the sequence of SEQ ID NO: 1, such as Aspheres Alpha).

[0144] The methods described herein can lead to improved PDMS-2 scores (e.g., indicating delayed motor development) in subjects with or susceptible to myasthenia gravis. For example, treatment with alkaline phosphatase or peptides with alkaline phosphatase activity, such as sALP (e.g., TNALP, such as the sALP peptide of SEQ ID NO: 1 or a peptide variant having at least 95% sequence identity with the sequence of SEQ ID NO: 1, e.g., Aspherex alpha), can result in an average increase in PDMS-2 scores of about 7 to about 13 (e.g., about 7, about 8, about 9, about 10, about 11, about 12, or about 13).

[0145] An increase in PDMS-2 score can be sustained for a prolonged period, such as a time, throughout the lifetime of a subject with or susceptible to muscular dystrophy, through administration of alkaline phosphatase or peptides with alkaline phosphatase activity, such as sALP (e.g., TNALP, such as the sALP peptide of SEQ ID NO: 1 or a peptide variant having at least 95% sequence identity with the sequence of SEQ ID NO: 1, e.g., Asphertex Alpha). Similarly, an increase in motor development can be sustained for a time, throughout the lifetime of a subject with or susceptible to muscular dystrophy, through administration of sALP (e.g., TNALP, such as the sALP peptide of SEQ ID NO: 1 or a peptide variant having at least 95% sequence identity with the sequence of SEQ ID NO: 1, e.g., Asphertex Alpha).

[0146] The PDMS-2 score of subjects with or predisposed to muscular dystrophy (e.g., HPP) can be used to assess the therapeutic efficacy of alkaline phosphatase or peptides with alkaline phosphatase activity, such as sALP (e.g., TNALP, such as the sALP peptide of SEQ ID NO: 1 or a peptide variant having at least 95% sequence identity with the sequence of SEQ ID NO: 1, e.g., Aspheres Alpha), where improvement relative to a certain test score indicates that the alkaline phosphatase or peptide with alkaline phosphatase activity is effective in treating, for example, delayed motor development associated with muscular dystrophy. For example, children aged approximately 5 years or younger with or predisposed to muscular dystrophy can be tested in one or more of the categories (motor, reflex, rest, object manipulation, grasping, and visual-motor) to obtain an average PDMS-2 score and assess the therapeutic efficacy of sALP administration.

[0147] For example, when administration of sALP to a child with or susceptible to myasthenia gravis results in an average increase in PDMS-2 standard score to about 7, wherein the child previously had an average PDMS-2 standard score of about 5, then sALP is effective in treating, for example, delayed motor development associated with HPP. Alternatively, when administration of sALP does not result in an average increase in PDMS-2 standard score to about 7, the dose and / or frequency of sALP administration may be varied to determine an effective amount of sALP for the child. For example, the dose of sALP (such as TNALP, such as the sALP peptide of SEQ ID NO: 1 or a peptide variant having at least 95% sequence identity with the sequence of SEQ ID NO: 1, such as Aspheres alpha) may be increased from, for example, about 3 mg / kg / week to about 6 mg / kg / week or from about 6 mg / kg / week to about 9 mg / kg / week.

[0148] Six-minute walk test (6MWT) The 6MWT can be used to identify subjects with muscular dystrophy for treatment with alkaline phosphatase or peptides with alkaline phosphatase activity (e.g., sALP, such as TNALP, such as the sALP peptide of SEQ ID NO: 1, or a peptide variant having at least 95% sequence identity with the sequence of SEQ ID NO: 1, such as Aspheres alpha). Specifically, the 6MWT can be used to assess the walking ability of adults with muscular dystrophy to generate a 6MWT value for said adults. The 6MWT can be performed indoors or outdoors using a flat, straight, enclosed corridor (e.g., approximately 30 meters long) 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 walked (e.g., in meters) by the subject with muscular dystrophy. For example, the length of the corridor can be marked every three meters to determine the number of meters walked by the subject with muscular dystrophy, with the turnaround point at 30 meters, and a starting line also marked. The distance walked by a subject with myasthenia gravis in six minutes can then be compared to the predicted number of meters walked by a normal subject of approximately the same age, sex, and / or height, and expressed as a percentage to generate the subject's 6MWT value. The 6MWT value of a subject with myasthenia gravis can be compared to the subject's 6MWT value at baseline. Additionally, the 6MWT value of a subject with myasthenia gravis can be compared to the 6MWT value of a normal subject.

[0149] Approximately 80% of subjects with myasthenia gravis whose mean 6MWT is less than the predicted 6MWT value (e.g., relative to normal subjects of approximately the same age, sex, and / or height) can be treated with alkaline phosphatase or a peptide with alkaline phosphatase activity (e.g., sALP, such as TNALP, such as the sALP peptide of SEQ ID NO: 1 or a peptide variant having at least 95% sequence identity with the sequence of SEQ ID NO: 1, such as Aspheres Alpha), such as by administering alkaline phosphatase or a peptide with alkaline phosphatase activity for a period of at least two weeks (e.g., at least three weeks, at least four weeks, at least five weeks, at least six weeks, at least seven weeks, at least eight weeks, at least nine weeks, at least ten weeks, at least three months, at least four months, at least five months, at least six months, 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 the patient's lifetime; particularly at least six weeks). For example, subjects with myasthenia gravis whose mean 6MWT is less than approximately 80% of the predicted 6MWT value (e.g., approximately 50%, 55%, 60%, 65%, 70%, or 75% of the predicted 6MWT value) can be treated with alkaline phosphatase or a peptide with alkaline phosphatase activity for a period of at least two weeks (e.g., at least three weeks, at least four weeks, at least five weeks, at least six weeks, at least seven weeks, at least eight weeks, at least nine weeks, at least ten weeks, at least three months, at least four months, at least five months, at least six months, 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 the patient's lifetime; particularly at least six weeks).

[0150] The method can lead to an improvement in the 6MWT value in subjects with myasthenia gravis. For example, using alkaline phosphatase or a peptide with alkaline phosphatase activity (e.g., sALP, such as TNALP, e.g., the sALP peptide of SEQ ID NO: 1 or with SEQ ID NO: 1). Treatment with a peptide variant of 1 that has at least 95% sequence identity, such as Aspheres Alpha, such as treatment with alkaline phosphatase or a peptide having alkaline phosphatase activity for a period of at least two weeks (e.g., at least three weeks, at least four weeks, at least five weeks, at least six weeks, at least seven weeks, at least eight weeks, at least nine weeks, at least ten weeks, at least three months, at least four months, at least five months, at least six months, 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 the patient's lifetime; particularly at least six weeks) can result in an average increase in 6MWT value to about 80% or more of the patient's predicted 6MWT value (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).

[0151] An increase in the 6MWT value in subjects with myasthenia gravis can be maintained throughout the course of treatment with alkaline phosphatase or a polypeptide with alkaline phosphatase activity (e.g., sALP, such as TNALP, such as the sALP polypeptide of SEQ ID NO: 1 or a polypeptide variant having at least 95% sequence identity with the sequence of SEQ ID NO: 1, such as Aspheres Alpha). This can be maintained for at least two weeks (e.g., at least three weeks, at least four weeks, at least five weeks, at least six weeks, at least seven weeks, at least eight weeks, at least nine weeks, at least ten weeks, at least three months, at least four months, at least five months, at least six months, at least seven months, at least eight months, at least nine months, at least one year, at least two years, at least three years, at least four years, at least five years, at least six years, at least seven years, at least eight years, at least nine years or at least ten years or the patient's lifetime; particularly at least six weeks). For example, the 6MWT value increased to approximately 80% greater than the predicted 6MWT value for subjects with myasthenia gravis, and remained at ±10% of the increased 6MWT value during treatment with alkaline phosphatase or peptides with alkaline phosphatase activity.

[0152] Similarly, improvements in walking ability in subjects with muscular dystrophy can be sustained throughout the course of treatment with alkaline phosphatase or peptides with alkaline phosphatase activity for at least two weeks (e.g., at least three weeks, at least four weeks, at least five weeks, at least six weeks, at least seven weeks, at least eight weeks, at least nine weeks, at least ten weeks, at least three months, at least four months, at least five months, at least six months, at least seven months, at least eight months, at least nine months, at least one year, at least two years, at least three years, at least four years, at least five years, at least six years, at least seven years, at least eight years, at least nine years, or at least ten years, or a patient's lifetime; particularly at least six weeks). For example, subjects with muscular dystrophy exhibited reduced dependence on assistive mobility devices (such as walkers, wheelchairs, harnesses, canes, or orthotics) during sALP treatment.

[0153] Alternatively, when administration of alkaline phosphatase or a peptide with alkaline phosphatase activity (e.g., sALP, such as TNALP, such as the sALP peptide of SEQ ID NO: 1, or a peptide variant having at least 95% sequence identity with the sequence of SEQ ID NO: 1, such as Aspheres Alpha) does not result in an average increase in 6MWT values ​​to greater than (e.g., about 80% of the predicted 6MWT values ​​in normal subjects of the same age, sex, and / or height), the dose and / or frequency of administration of alkaline phosphatase or a peptide with alkaline phosphatase activity may be altered to determine the effective amount of alkaline phosphatase or a peptide with alkaline phosphatase activity for subjects with myasthenia gravis. For example, the dose of alkaline phosphatase or a peptide with alkaline phosphatase activity may be increased from, for example, about 2.1 mg / kg / week or about 3.5 mg / kg / week to about 6 mg / kg / week or about 9 mg / kg / week.

[0154] Handheld force measurement method (HHD) Handheld force measurement (HHD) can be used to assess the grip and muscle strength of subjects with or prone to muscle weakness. For example, the MICROFET2 can be used. TM A dynamometer measures knee flexion and extension, as well as hip flexion, extension, and abduction, in subjects with or prone to muscular dystrophy. A grip strength measurement may also be performed using, for example, a Jamar Grip dynamometer. Specifically, the dynamometer is kept stationary by an administrator, and the subject applies maximum force to the dynamometer. Peak force data are collected in pounds and then converted to Newtons (N). Torque values ​​are then calculated using limb length in N meters. These torque values ​​are then compared to those of normal subjects, for example, of approximately the same age, sex, and / or height, and expressed as a percentage to generate the subject's HHD value.

[0155] Subjects with myasthenia gravis whose average HHD value is less than the predicted HHD value (e.g., relative to about 80% of normal subjects of the same age, sex, and / or height) can be treated with alkaline phosphatase or a peptide with alkaline phosphatase activity (e.g., sALP, such as TNALP, such as the sALP peptide of SEQ ID NO: 1 or a peptide variant having at least 95% sequence identity with the sequence of SEQ ID NO: 1, such as Aspheres Alpha), such as by administering alkaline phosphatase or a peptide with alkaline phosphatase activity for a period of at least two weeks (e.g., at least three weeks, at least four weeks, at least five weeks, at least six weeks, at least seven weeks, at least eight weeks, at least nine weeks, at least ten weeks, at least three months, at least four months, at least five months, at least six months, 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 the patient's lifetime; particularly at least six weeks). For example, subjects with myasthenia gravis whose mean HHD is less than about 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) may be treated with alkaline phosphatase or a peptide with alkaline phosphatase activity for a period of at least two weeks (e.g., at least three weeks, at least four weeks, at least five weeks, at least six weeks, at least seven weeks, at least eight weeks, at least nine weeks, at least ten weeks, at least three months, at least four months, at least five months, at least six months, 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 the patient's lifetime; particularly at least six weeks).

[0156] The method can lead to improved HHD values ​​in subjects with myasthenia gravis. For example, using alkaline phosphatase or peptides with alkaline phosphatase activity (e.g., sALP, such as TNALP, e.g., the sALP peptide of SEQ ID NO: 1 or with SEQ ID NO: 1). Treatment with a peptide variant of 1 having at least 95% sequence identity, such as Aspheres alpha, such as treatment with alkaline phosphatase or a peptide having alkaline phosphatase activity for a period of at least two weeks (e.g., at least three weeks, at least four weeks, at least five weeks, at least six weeks, at least seven weeks, at least eight weeks, at least nine weeks, at least ten weeks, at least three months, at least four months, at least five months, at least six months, 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 the patient's lifetime; particularly at least six weeks) can result in an average increase in HHD values ​​to about 80% or more of the patient's predicted HHD value (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).

[0157] An increase in HHD values ​​in subjects with muscular dystrophy can be maintained throughout the course of treatment with alkaline phosphatase or a peptide with alkaline phosphatase activity (e.g., sALP, such as TNALP, such as the sALP peptide of SEQ ID NO: 1, or a peptide variant having at least 95% sequence identity with the sequence of SEQ ID NO: 1, such as Aspheres Alpha). This can be maintained for at least two weeks (e.g., at least three weeks, at least four weeks, at least five weeks, at least six weeks, at least seven weeks, at least eight weeks, at least nine weeks, at least ten weeks, at least three months, at least four months, at least five months, at least six months, at least seven months, at least eight months, at least nine months, at least one year, at least two years, at least three years, at least four years, at least five years, at least six years, at least seven years, at least eight years, at least nine years, or at least ten years, or the patient's lifetime; particularly at least six weeks). For example, an increase in HHD values ​​to approximately 80% greater than the predicted HHD value for a subject with muscular dystrophy, and maintenance at ±10% of the increased HHD value during treatment with alkaline phosphatase or a peptide with alkaline phosphatase activity.

[0158] Alternatively, when administration of alkaline phosphatase or a peptide with alkaline phosphatase activity (e.g., sALP, such as TNALP, such as the sALP peptide of SEQ ID NO: 1, or a peptide variant having at least 95% sequence identity with the sequence of SEQ ID NO: 1, such as Aspheres Alpha) does not result in an average increase in HHD values ​​greater than (e.g., about 80% of the predicted HHD values ​​for subjects with myasthenia gravis of the same age, sex, and / or height), the dose and / or frequency of administration of alkaline phosphatase or a peptide with alkaline phosphatase activity may be varied to determine the effective amount of alkaline phosphatase or a peptide with alkaline phosphatase activity for subjects with myasthenia gravis. For example, the dose of alkaline phosphatase or a peptide with alkaline phosphatase activity may be increased from, for example, about 2.1 mg / kg / week or about 3.5 mg / kg / week to about 6 mg / kg / week or about 9 mg / kg / week.

[0159] alkaline phosphatase Asfutez Alpha is a human TNALP (hTNALP; SEQ ID NO: 1) fusion protein formulated for the treatment of HPP. In particular, Asfutez Alpha (SEQ ID NO: 1) is effective for the treatment of hypophospholipase syndrome, its symptoms and associated physical impairment in subjects with or susceptible to myasthenia gravis for extended periods of time (e.g., at least one day, at least one week, at least two weeks, at least three weeks, at least one month, at least three months, at least six months, at least one year, at least two years, at least three years, at least four years, at least five years, at least six years, at least seven years, at least eight years, at least nine years, at least ten years, or more than ten years (e.g., the lifetime of the subject)).

[0160] In light of the results described herein, this disclosure is not limited to specific alkaline phosphatases (ALPs) or nucleic acid sequences encoding ALPs. Alkaline phosphatases comprise a group of enzymes that catalyze the partial cleavage of phosphate groups (e.g., hydrolysis of pyrophosphate, PPi). Four known mammalian alkaline phosphatase (ALP) isoenzymes exist: tissue nonspecific alkaline phosphatase (TNALP; further described below), placental alkaline phosphatase (PLALP) (e.g., accession numbers P05187, NP_112603, and NP_001623), germ cell 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 above, this disclosure also provides any polypeptide having the same or similar ALP catalytic site structure and / or enzymatic activity for the treatment of subjects with or susceptible to muscular dystrophy. Bone delivery conjugates including sALP are further described in PCT Publications: WO 2005 / 103263 and WO 2008 / 138131.

[0161] TNALPs that can be used according to 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); porcine TNALPs (accession number AAN64273), mouse TNALPs (accession number NP_031457), bovine TNALPs (accession numbers NP_789828, NP_776412, AAM8209, and AAC33858), and feline TNALPs (accession number NP_001036028). In particular, the TNALP can be recombinant human TNALPs (e.g., SEQ ID NO: 1, Asftes Alpha (see U.S. Patent Nos. 7,763,712 and 7,960,529, all of which are incorporated herein by reference) is used to treat subjects who have or are susceptible to muscular dystrophy. TNALP may also be a TNALP whose polypeptide or nucleic acid sequence exhibits at least approximately 95% sequence identity with the aforementioned TNALP.

[0162] Soluble alkaline phosphatase The ALPs of the present invention include soluble (e.g., extracellular or non-membrane-bound) forms of any alkaline phosphatase described herein. The sALPs of the present invention may be, for example, soluble forms of human tissue-specific alkaline phosphatase (human TNALP (hTNALP)). This disclosure is not limited to specific sALPs and may include any sALP polypeptide that is physiologically active for, for example, phosphoethanolamine (PEA), inorganic pyrophosphate (PPi), and pyridoxal 5'-phosphate (PLP). In particular, the sALPs of the present invention have catalytic capabilities to improve bone mineralization in bone. This disclosure further includes nucleic acids encoding the sALPs described herein, which may be used to treat the muscle weakness symptoms described herein, including, for example, HPP, CPPD, familial hypophosphatemia (such as autosomal dominant hypophosphatemic rickets (ADHR), autosomal recessive hypophosphatemic rickets, X-linked hypophosphatemic rickets, X-linked hypophosphatemia (XLH), etc.).

[0163] TNALP is a membrane-bound protein (Swiss-Prot, P05186) anchored at its C-terminus by a glycolipid moiety. This glycolipid anchor (GPI) is added posttranslatively after the removal of the hydrophobic C-terminus, serving as both a temporary membrane anchor and a signal for GPI addition. While the GPI anchor is localized within the cell membrane, the remainder of TNALP is extracellular. Specifically, TNALP (e.g., human TNALP (hTNALP)) can be engineered to replace the first amino acid (alanine) of the hydrophobic C-terminal sequence with a stop codon, resulting in engineered hTNALP containing all amino acid residues of the naturally anchored form of TNALP but lacking the GPI membrane anchor. Those skilled in the art will understand that the position of the GPI membrane 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 to 502 (18 to 502 during secretion), amino acids 1 to 501 (18 to 501 during secretion), amino acids 1 to 504 (18 to 504 during secretion), amino acids 1 to 505 (18-505 during secretion), or amino acids 1 to 502. Therefore, the C-terminus of native ALPs can be truncated by certain amino acids without affecting ALP activity.

[0164] In addition to the C-terminal GPI anchor, TNALP also has an N-terminal signal peptide sequence. The N-terminal signal peptide is present on the synthesized protein during synthesis but is cleaved from the TNALP after translocation to the ER. The sALP of the present invention includes both secreted (i.e., lacking the N-terminal signal) and non-secreted (i.e., having the N-terminal signal) forms. Those skilled in the art will understand that the position of the N-terminal signal peptide will vary in different alkaline phosphatases and may include, for example, the first 5, 8, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 27, 30 or more amino acid residues at the N-terminus of the polypeptide. Those skilled in the art can, for example, through descriptions such as those in Bendtsen et al. (… J. Mol. Biol. In 340(4):783-795, 2004), and on the website www.cbs.dtu.dk / services / SignalP / , appropriate computer algorithms are available to predict the location of the breakpoint in the signal sequence.

[0165] This invention also includes sALP concordant sequences derived from the extracellular domains of ALP isoenzymes (e.g., TNALP, PALP, GCALP, IALP, etc.). Therefore, similar to sTNALP discussed above, this disclosure also provides other soluble human ALP isoenzymes, i.e., those without peptide signals, preferably containing the extracellular domains of ALP. The sALPs of this invention also include polypeptide sequences satisfying concordant sequences of the extracellular domains of ALP derived from human ALP isoenzymes and mammalian TNALP orthologs (human, mouse, rat, bovine, cat, and dog) or concordant sequences of the extracellular domains of ALP only from mammalian TNALP orthologs (human, mouse, rat, bovine, cat, and dog). The sALPs of this invention also include those satisfying similar concordant sequences of various combinations derived from these TNALP orthologs or human ALP isoenzymes. For example, such concordant sequences are given in WO 2008 / 138131.

[0166] The sALP of the present invention may include not only the wild-type sequence of the sALP 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 with these alkaline phosphatases (e.g., SEQ ID NO: 1-24; e.g., the sALP polypeptide of SEQ ID NO: 1 or a polypeptide variant having at least 95% sequence identity with the sequence of SEQ ID NO: 1, e.g., Asphertex Alpha). Examples of mutations that may be introduced into the ALP sequence are described in U.S. Publication No. 2013 / 0323244, the entire contents of which are incorporated herein by reference. The sALP may optionally be glycosylated at any suitable one or more amino acid residues. Furthermore, sALP may have at least 50% (e.g., 55%, 60%, 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more) sequence identity with any sALP described herein (such as TNALP, such as the sALP peptide of SEQ ID NO: 1 or a peptide variant with at least 95% sequence identity with the sequence of SEQ ID NO: 1, such as Asphertex Alpha). With respect to any sALP described herein (such as TNALP, such as the sALP peptide of SEQ ID NO: 1 or a peptide variant with at least 95% sequence identity with the sequence of SEQ ID NO: 1, such as Asphertex Alpha), sALP may have 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more additions, deletions or substitutions.

[0167] sALP fusion peptide Any sALP and linker described herein may be combined in a sALP peptide (e.g., a sALP peptide of A-sALP-B) wherein A and B are either absent or contain at least one amino acid sequence (such as TNALP, e.g., the sALP peptide of SEQ ID NO: 1 or a peptide variant having at least 95% sequence identity with the sequence of SEQ ID NO: 1, e.g., Asphertex Alpha). When present, A and / or B may be any linker described herein. In some sALP peptides, A is absent, B is absent, or both A and B are absent. As described herein, the sALP peptides of the present invention may optionally include an Fc region to provide a sALP fusion peptide. As described herein, the sALP peptide may optionally include a bone-targeting moiety. In some sALP peptides, a linker, e.g., a flexible linker, may be included between the bone-targeting moiety and the sALP, such as a dipeptide sequence (e.g., leucine-lysine or aspartic-isoleucine). Other exemplary Fc regions, linkers, and bone-targeting moieties are described below.

[0168] Any sALP, linker, and Fc region described herein may be present in fusion peptides (e.g., recombinant fusion peptides that include the structure Z-sALP-Y-spacer-XW). n -V、ZW n -X-spacer-Y-sALP-V, Z-sALP-YW n -X-spacer-V and ZW n -X-sALP-Y-spacer-V (such as TNALP, e.g., the sALP polypeptide of SEQ ID NO: 1 or a polypeptide variant having at least 95% sequence identity with the sequence of SEQ ID NO: 1, e.g., Asphertex Alpha)). In particular, the structure can be a combination of Z-sALP-Y-spacer-XW. n -V or ZW n -X-spacer-Y-sALP-V. sALP can be the full-length or functional fragment of ALP, such as the soluble extracellular domain of ALP, as described herein (e.g., TNALP, PALP, GCALP, and IALP). Any one of X, Y, Z, and V and / or the spacer may be absent or may be an amino acid sequence containing at least one amino acid. W nIt can be a bone-targeting portion, for example, having a series of consecutive Asp or Glu residues, where n = 1 to 50, for example, n = 3-30, for example, 5-15, 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, 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 portion (if present) can be located at any position on the fusion polypeptide, for example, at or near the N-terminus or C-terminus, and / or in the linker region. For example, the bone-targeting portion is at the C-terminus. sALP peptides and fusion peptides may also exclude the bone-targeting component.

[0169] The sALP fusion peptide of the present invention may have the structure hTNALP-Fc-D 10 In particular, the sALP fusion peptide may include the amino acid sequence of SEQ ID NO: 1 or a peptide variant having at least 95% sequence identity with the sequence of SEQ ID NO: 1, such as Asphertex Alpha.

[0170] Available spacers include, but are not limited to, peptides containing Fc, and peptides capable of mitigating the effects of highly negatively charged terminals (e.g., W). nThe presence of a repulsive force induced by the presence of a hydrophilic and flexible polypeptide. For example, sALP can be a fusion polypeptide that includes the Fc region of an immunoglobulin at its N-terminal or C-terminal domain. Immunoglobulin molecules have a structure well known in the art. They consist of two light chains (approximately 23 kD each) and two heavy chains (approximately 50-70 kD each) linked by interchain disulfide bonds. Immunoglobulins are readily proteolytically cleaved (e.g., by papain) into Fab (containing the VH and CH1 domains of the light and heavy chains) and Fc (containing the CH2 and CH3 domains of the heavy chain, along with adjacent sequences). The Fc fragments available as described herein include Fc fragments of any immunoglobulin molecule from any mammal (e.g., human), including IgG, IgM, IgA, IgD, or IgE, and their various subclasses (e.g., IgG-1, IgG-2, IgG-3, IgG-4, IgA-1, IgA-2). For example, the Fc fragment is human IgG-1. The Fc fragment of the present invention may include, for example, the CH2 and CH3 domains of the heavy chain and any portion of the hinge region. The Fc region may optionally be glycosylated at any suitable one or more amino acid residues known to those skilled in the art. In particular, the Fc fragment of the fusion polypeptide has the amino acid sequence of SEQ ID NO: 20, or has at least 50% (e.g., 55%, 60%, 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more) sequence identity with SEQ ID NO: 20. Engineered, e.g., non-naturally occurring Fc regions may be used in the methods of the present invention, for example, as described in International Application Publication No. WO2005 / 007809, which is hereby incorporated by reference. Relative to any Fc segment described herein, an Fc segment as described herein may have 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 50 or more additions, deletions or replacements.

[0171] The sALP fusion peptides described herein (such as TNALP, e.g., the sALP peptide of SEQ ID NO: 1, or peptide variants having at least 95% sequence identity with the sequence of SEQ ID NO: 1, e.g., Asphertex Alpha) may include a peptide linker region between Fc fragments. Furthermore, the peptide linker region may be included between the Fc fragment and an optional bone-targeting portion. The linker region may have any sequence and length that allows sALP to remain biologically active, e.g., unhindered by steric hindrance. Exemplary linker lengths are between 1 and 200 amino acid residues, for example, 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, the linker includes or is composed of flexible portions (e.g., regions without significantly fixed secondary or tertiary structures). Exemplary flexible linkers are glycine-rich linkers (e.g., containing at least 50%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or even 100% glycine residues). Linkers may also contain, for example, serine residues. In some cases, the amino acid sequence of the linker consists only of glycine and serine residues. The linker may optionally be glycosylated at any suitable one or more amino acid residues. Additionally, linkers as described herein may include any other sequence or portion covalently or non-covalently linked. A linker may also be absent, where the Fc fragment and sALP are directly fused together without intermediate residues. According to this disclosure, certain Fc-sALP or sALP-Fc fusion peptides may be considered as 1) lacking a linker, or 2) having a linker corresponding to a portion of sALP. For example, an Fc directly fused with hsTNALP (1-502) may be considered, for example, lacking a linker, where hsTNALP is amino acid 1-502, or a linker having 17 amino acids, where hsTNALP (18-502).

[0172] Depending on the cloning strategy used to generate the fusion peptide, additional amino acid residues may be introduced into the peptide. For example, these additional amino acid residues do not provide additional GPI anchoring signals to maintain the peptide in a soluble form. Furthermore, any such additional amino acid residues, when incorporated into the peptide of the present invention, do not provide cleavage sites for endonucleases of the host cell. The likelihood that the designed sequence will be cleaved by endonucleases of the host cell can be predicted, for example, as described by Ikezawa (Biol. Pharm. Bull. 25:409-417, 2002).

[0173] The sALP and sALP fusion peptides of the present invention (such as TNALP, for example, the sALP peptide of SEQ ID NO: 1 or a peptide variant having at least 95% sequence identity with the sequence of SEQ ID NO: 1, for example, Asphertex Alpha) can be associated with dimers or tetramers. For example, two sALP-Fc monomers can be covalently linked by two disulfide bonds located in the hinge region of the Fc fragment. Additionally, the peptides or fusion peptides of the present invention (e.g., sALP peptides or fusion peptides) can be glycosylated or polyethylene glycol-modified.

[0174] Production of nucleic acids and peptides Nucleic acids encoding the sALP and sALP fusion peptides of the present invention (such as TNALP, e.g., the sALP peptide of SEQ ID NO: 1 or a peptide variant having at least 95% sequence identity with the sequence of SEQ ID NO: 1, e.g., Asphertex Alpha) can be produced by any method known in the art. Typically, nucleic acids encoding the desired fusion peptide are generated using molecular cloning methods and are usually placed in a vector (such as a plasmid or virus). The vector is used to convert the nucleic acid into a host cell suitable for expressing the fusion peptide. 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 conferring appropriate post-translational modifications. Host cells of the present invention may include, for example, Chinese hamster ovary (CHO) cells, L cells, C127 cells, 3T3 cells, BHK cells, COS-7 cells, or any other suitable host cell known in the art. For example, the host cell is a Chinese hamster ovary (CHO) cell (e.g., CHO-DG44 cells).

[0175] sALP and sALP fusion peptides (such as TNALP, e.g., the sALP peptide of SEQ ID NO: 1 or peptide variants having at least 95% sequence identity with the sequence of SEQ ID NO: 1, e.g., Asphertex Alpha) can be generated under any conditions suitable for achieving sALP peptide expression in host cells. Such conditions include appropriately selected media prepared with components such as buffers, bicarbonate and / or HEPES, ions (e.g., chloride, phosphate, calcium, sodium, potassium, magnesium, iron), carbon sources (e.g., monosaccharides, amino acids, potential lipids, nucleotides), vitamins, and growth factors (e.g., insulin); conventional commercially available culture media (e.g., α-MEM, DMEM, Ham's-F12, and IMDM supplemented with 2–4 mM L-glutamine and 5% fetal bovine serum); and conventional commercially available animal protein-free culture media (e.g., Hyclone™ SFM4CHO, Sigma CHO DHFR-, Cambrex POWER™ CHO CD supplemented with 2–4 mM L-glutamine). Ideally, these culture media should be prepared without the use of thymidine, hypoxanthine, and L-glycine to maintain selective pressure, thereby allowing stable protein-product expression.

[0176] Pharmaceutical compositions and formulations The compositions of the present invention (e.g., comprising sALP or sALP fusion peptides, such as TNALP, for example the sALP peptide of SEQ ID NO: 1 or a peptide variant having at least 95% sequence identity with the sequence of SEQ ID NO: 1, such as Aspheres Alpha) can be administered by a variety of methods known in the art. As those skilled in the art will understand, the route and / or mode of administration will vary depending on the desired outcome. The route of administration can depend on a variety of factors, such as environment and therapeutic purpose. In particular, the peptides and fusion peptides described herein can be administered by any route known in the art, such as subcutaneous (e.g., by subcutaneous injection), intravenous, oral, nasal, intramuscular, sublingual, intrathecal, or intradermal. For example, the pharmaceutical compositions of the present invention can be in the form of liquids, solutions, suspensions, pills, capsules, tablets, capsule tablets, powders, gels, ointments, creams, nebulae, mists, atomized vapors, aerosols, or phospholipid complexes.

[0177] dose Any amount of the pharmaceutical composition (e.g., comprising sALP or sALP fusion peptides, such as TNALP, such as the sALP peptide of SEQ ID NO: 1, or a peptide variant having at least 95% sequence identity with the sequence of SEQ ID NO: 1, such as Aspheres alpha) may be administered to a subject who has or is susceptible to myasthenia gravis. The dosage will depend on many factors, including the administration modality and the patient’s age. Generally, the amount of composition (e.g., sALP or sALP fusion peptides, such as TNALP, such as the sALP peptide of SEQ ID NO: 1, or a peptide variant having at least 95% sequence identity with the sequence of SEQ ID NO: 1, such as Aspheres alpha) contained in a single dose will be an amount that is effective in treating the condition described herein (e.g., HPP) without causing significant toxicity.

[0178] For example, the sALP peptides described herein (such as TNALP, e.g., the sALP peptide of SEQ ID NO: 1 or peptide variants having at least 95% sequence identity with the sequence of SEQ ID NO: 1, e.g., Aspherex alpha) may range, for example, from 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) or from 1 μg / kg to 1,000 μg / kg (e.g., 5 μg / kg to 1,000 μg / kg, 1 A single dose of 5 μg / kg to 750 μg / kg, 10 μg / kg to 750 μg / kg, 1 μg / kg to 500 μg / kg, 5 μg / kg to 500 μg / kg, 10 μg / kg to 500 μg / kg, 1 μg / kg to 100 μg / kg, 5 μg / kg to 100 μg / kg, 10 μg / kg to 100 μg / kg, 1 μg / kg to 50 μg / kg, 5 μg / kg to 50 μg / kg or 10 μg / kg to 50 μg / kg may be administered to subjects with or susceptible to myasthenia gravis.

[0179] Exemplary doses of sALP include, for example, 0.01, 0.05, 0.1, 0.5, 1, 2, 2.5, 5, 10, 20, 25, 50, 100, 125, 150, 200, 250, or 500 mg / kg; or 1, 2, 2.5, 5, 10, 20, 25, 50, 100, 125, 150, 200, 250, 500, 750, 900, or 1,000 μg / kg. For all doses or ranges described herein, the term “about” can be used to modify these doses to ±10% of the endpoints of the stated values ​​or ranges. In particular, compositions according to this disclosure (e.g., including sALP (such as TNALP, such as the sALP peptide of SEQ ID NO: 1 or a peptide variant having at least 95% sequence identity with the sequence of SEQ ID NO: 1, such as Aspheres Alpha)) can be administered to patients at doses ranging from about 0.001 mg / kg / day to about 500 mg / kg / day, about 0.01 mg / kg / day to about 100 mg / kg / day, or about 0.01 mg / kg / day to about 20 mg / kg / day). For example, sALP compositions (such as TNALP, such as the sALP peptide of SEQ ID NO: 1 or a peptide variant having at least 95% sequence identity with the sequence of SEQ ID NO: 1, such as Asphertex Alpha) can be administered to patients at weekly doses ranging from about 0.5 mg / kg / week to about 140 mg / kg / week, such as about 0.8 mg / kg / week to about 50 mg / kg / week, or about 1 mg / kg / week to about 10 mg / kg / week (such as about 6 or about 9 mg / kg / week). Specifically, sALP (such as TNALP, e.g., the sALP peptide of SEQ ID NO: 1 or a peptide variant having at least 95% sequence identity with the sequence of SEQ ID NO: 1, e.g., Aspherex alpha) can be administered at doses of 2 mg / kg three times a week (total dose 6 mg / kg / week), 1 mg / kg six times a week (total dose 6 mg / kg / week), 3 mg / kg three times a week (total dose 9 mg / kg / week), 0.5 mg / kg three times a week (total dose 1.5 mg / kg / week), or 9.3 mg / kg three times a week (total dose 28 mg / kg / week). The dosage will be adjusted by the clinician based on routine factors such as the severity of the disease and different parameters from subjects with or susceptible to muscular dystrophy.

[0180] Compositions comprising sALP and sALP fusion peptides (such as TNALP, e.g., the sALP peptide of SEQ ID NO: 1 or a peptide variant having at least 95% sequence identity with the sequence of SEQ ID NO: 1, e.g., Asphertex Alpha) may be provided in single-dose or multi-dose regimens. Dosage may be administered, for example, hourly, every two hours, daily, every two days, twice weekly, three times weekly, four times weekly, five times weekly, six times weekly, weekly, every two weeks, monthly, every two months, or annually. Alternatively, dosage may be administered daily, for example, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 times. In particular, the dosing regimen is once weekly. The duration of the dosing regimen can be, for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 days, weeks, months, or even the remaining lifespan of a subject with or predisposed to muscular dystrophy. The dosage, frequency, and duration will be adjusted by the clinician based on routine factors such as the severity of the disease and different parameters from subjects with or predisposed to muscular dystrophy.

[0181] For example, sALP or sALP fusion peptides (such as TNALP, such as the sALP peptide of SEQ ID NO: 1 or a peptide variant having at least 95% sequence identity with the sequence of SEQ ID NO: 1, such as Asphertex Alpha) can be formulated into an injectable solution, which is a clear, colorless to slightly yellow aqueous solution with a pH of 7.4. sALP or sALP peptides (such as TNALP, such as the sALP peptide of SEQ ID NO: 1 or a peptide variant having at least 95% sequence identity with the sequence of SEQ ID NO: 1, such as Asphertex Alpha) can be formulated into concentrations of 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. Specifically, the composition can be formulated as a 40 mg / ml injection solution, wherein each ml of solution contains 40 mg sALP or sALP peptide (e.g., each vial contains 0.3 ml solution and 12 mg sALP (40 mg / ml), each vial contains 0.45 ml solution and 18 mg sALP (40 mg / ml), each vial contains 0.7 ml solution and 28 mg sALP (40 mg / ml), or each vial contains 1.0 ml solution and 40 mg Aspheres Alpha (40 mg / ml)). sALP or sALP peptides (such as TNALP, e.g., the sALP peptide of SEQ ID NO: 1 or a peptide variant having at least 95% sequence identity with the sequence of SEQ ID NO: 1, e.g., Aspherex Alpha) can be formulated into an injectable solution at a concentration of 100 mg / ml, wherein each 1 ml solution contains 100 mg of sALP or sALP peptide (e.g., each vial contains 0.8 ml solution and 80 mg of Aspherex Alpha (100 mg / ml)).

[0182] For example, the recommended dose of sALP or sALP fusion peptides (such as TNALP, e.g., the sALP peptide of SEQ ID NO: 1 or a peptide variant having at least 95% sequence identity with the sequence of SEQ ID NO: 1, e.g., Aspherex alpha) is 2 mg / kg body weight subcutaneously three times a week, or 1 mg / kg body weight subcutaneously six times a week. Additional dosage information is provided below (Table 1).

[0183] Table 1. Dosage of Aspherex Alpha preparation Compositions comprising sALP and sALP fusion peptides (such as TNALP, for example, the sALP peptide of SEQ ID NO: 1 or a peptide variant having at least 95% sequence identity with the sequence of SEQ ID NO: 1, for example, Asphertex Alpha) can be formulated according to standard methods. Drug formulation is a long-established field, and is further described in, for example, Gennaro (2000) "Remington: The Science and Practice of Pharmacy," 20th edition, Lippincott, Williams & Wilkins (ISBN: 0683306472); Ansel et al. (1999) "Pharmaceutical Dosage Forms and Drug Delivery Systems," 7th edition, Lippincott Williams & Wilkins Publishers (ISBN: 0683305727); and Kibbe (2000) "Handbook of Pharmaceutical Excipients American Pharmaceutical Association," 3rd edition (ISBN: 091733096X). For example, sALP compositions (such as TNALP, such as the sALP peptide of SEQ ID NO: 1 or a peptide variant having at least 95% sequence identity with the sequence of SEQ ID NO: 1, such as Asphertex Alpha) can be formulated as buffer solutions of appropriate concentrations suitable for storage at 2°C–8°C (e.g., 4°C). The compositions can also be formulated for storage at temperatures below 0°C (e.g., -20°C or -80°C). The compositions can be further formulated for storage at 2°C–8°C (e.g., 4°C) for up to 2 years (e.g., 1 month, 2 months, 3 months, 4 months, 5 months, 6 months, 7 months, 8 months, 9 months, 10 months, 11 months, 1 year, 1.5 years, or 2 years). Therefore, the compositions described herein are stable at 2°C–8°C (e.g., 4°C) for at least 1 year of storage.

[0184] Compositions comprising sALP and sALP fusion peptides (such as TNALP, e.g., the sALP peptide of SEQ ID NO: 1 or a peptide variant having at least 95% sequence identity with the sequence of SEQ ID NO: 1, e.g., Aspherex Alpha) can be in a variety of forms. These forms include, for example, liquid, semi-solid, and solid dosage forms, such as liquid solutions (e.g., injectable and infusionable solutions), dispersions or suspensions, tablets, pills, powders, liposomes, and suppositories. Preferred forms depend in part on the intended mode of administration and therapeutic application.

[0185] For example, compositions intended for systemic or local delivery may be in the form of injectable or infusionable solutions. Thus, compositions (such as TNALP, e.g., the sALP polypeptide of SEQ ID NO: 1, or polypeptide variants having at least 95% sequence identity with the sequence of SEQ ID NO: 1, e.g., Aspheresa alpha) may be formulated for administration via parenteral routes (e.g., subcutaneous, intravenous, intraperitoneal, or intramuscular injection). As used herein, “parenteral administration,” “administered via parenteral route,” and other grammatically equivalent phrases refer to routes of administration other than enteral and local administration, typically by injection, and including but not limited to subcutaneous, intradermal, intravenous, intranasal, intraocular, pulmonary, intramuscular, intraarticular, intrathecal, intracapsular, intrasacral, intraorbital, intracardiac, intradermal, intrapulmonary, intraperitoneal, tracheal, subepidermal, intra-articular, subcapsular, subarachnoid, spinal, epidural, intracerebral, intracranial, carotid, and intrasternal injections and infusions).

[0186] Compositions including sALP and sALP fusion peptides (such as TNALP, e.g., the sALP peptide of SEQ ID NO: 1 or a peptide variant having at least 95% sequence identity with the sequence of SEQ ID NO: 1, e.g., Asphertex Alpha) can be formulated into solutions, microemulsions, dispersions, liposomes, or other ordered structures suitable for stable storage at high concentrations. Sterile injectable solutions can be prepared by combining a desired amount of the composition described herein with one or a combination of the ingredients listed above (if necessary) in a suitable solvent, followed by sterile filtration. Generally, dispersions are prepared by incorporating the composition described herein into a sterile medium containing a basic dispersion medium and desired additional components from those listed above. In the case of sterile powders used to prepare sterile injectable solutions, preparation methods include vacuum drying and freeze-drying, resulting in powders of the composition described herein plus any additional desired components from their previously sterile filtered solution (see below). Appropriate flowability of the solution can be maintained, for example, by using a coating (such as lecithin), in the case of a dispersion by maintaining a desired particle size, and by using a surfactant. Extended absorption of injectable compositions can be achieved by including agents that delay absorption (e.g., monostearate and gelatin) in the composition.

[0187] The compositions described herein can also be formulated in immunoliposome compositions. Such formulations can be prepared by methods known in the art (e.g., as 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 increased cycle time are disclosed in U.S. Patent No. 5,013,556.

[0188] Compositions comprising sALP and sALP fusion peptides (such as TNALP, e.g., the sALP peptide of SEQ ID NO: 1 or a peptide variant having at least 95% sequence identity with the sequence of SEQ ID NO: 1, e.g., Asphertex Alpha) can also be formulated with a carrier (such as a controlled-release formulation, including implants and microencapsulated delivery systems) that will protect the composition (e.g., the sALP peptide or the sALP fusion peptide) from rapid release. Biodegradable, biocompatible polymers such as ethylene vinyl acetate, polyanhydride, polyglycolic acid, collagen, polyorthoesters, and polylactic acid can be used. Many methods for preparing such formulations are known in the art. See, for example, JR Robinson (1978) “Sustained and Controlled Release Drug Delivery Systems,” Marcel Dekker, Inc., New York.

[0189] When the composition is used in combination with a second active agent, the composition may be formulated together with the second pharmaceutical agent, or the composition may be formulated separately from the second pharmaceutical agent formulation. For example, the pharmaceutical compositions may be mixed and administered together before application, or they may be administered separately at the same or different times.

[0190] Compositions comprising sALP and sALP fusion peptides (such as TNALP, e.g., the sALP peptide of SEQ ID NO: 1 or a peptide variant having at least 95% sequence identity with the sequence of SEQ ID NO: 1, e.g., Asphertex Alpha) may be formulated for administration to a patient in conjunction with intravenous immunoglobulin therapy (IVIG), plasma removal, plasma exchange, or plasma exchange, or, if administered to a fetus, to a woman carrying such a fetus.

[0191] carrier / medium Formulations containing sALP or sALP fusion peptides (such as TNALP, e.g., the sALP peptide of SEQ ID NO: 1 or a peptide variant having at least 95% sequence identity with the sequence of SEQ ID NO: 1, e.g., Aspherex Alpha) are provided to subjects with or susceptible to myasthenia gravis 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 oils, fish oils, and injectable organic esters. Aqueous carriers include water, hydroalcoholic solutions, emulsions, or suspensions, including saline and buffered parenteral mediators, including sodium chloride solutions, Ringer's glucose solutions, glucose plus sodium chloride solutions, lactose-containing Ringer's solutions, or non-volatile oils. For example, pharmaceutically acceptable carriers may include sodium chloride and / or sodium phosphate, wherein the composition comprises, for example, about 150 mM sodium chloride and / or about 25 mM sodium phosphate at a pH of 7.4.

[0192] Intravenous mediators may include fluid and nutritional supplements, electrolyte supplements, such as Ringer's glucose-based supplements. These may include pharmaceutically acceptable salts, such as inorganic acid salts, such as hydrochloride, hydrobromide, phosphate, and sulfate; and organic acid salts, such as acetate, propionate, malonate, and benzoate. Additionally, such mediators may contain excipients, such as wetting agents or emulsifiers, and pH buffers. A detailed discussion of pharmaceutically acceptable mediators is available in Remington's Pharmaceutical Sciences (Mack Pub. Co., NJ 1991).

[0193] The following examples are intended to illustrate, and not to limit, this disclosure. Example

[0194] Example 1. AKP2 - / - Mouse studies AKP2 - / - Knockout mice inactivate genes AKP2 produce, AKP2 The mouse counterpart of the gene encoding human tissue nonspecific alkaline phosphatase (TNSALP). AKP2 - / - Knockout mice have been used as a model of human HPP, reproducing HPP that occurs in infancy (Narisawa et al.). Dev Dyn. 1997; 208 (432-446). This example summarizes the detection compared to wild-type (WT) mice. AKP2 - / - The effects of Asphertex Alpha on muscle fiber composition and strength in mice were investigated, and its efficacy in correcting muscle fiber composition and strength was determined. AKP2 - / - Effects on the muscle weakness phenotype in mice.

[0195] Specifically, the soleus and extensor digitorum longus (EDL) muscles were examined. To determine the muscle fiber composition, fiber size and type were measured or examined. As previously described (see Barton et al. 2005) J OrthopRes. 23 : 259-265; Barton et al. 2012 Faseb J. 26 : 3691-3702; and Evans et al. 2008 Physiol. Genomics 35 (86-95), muscle was harvested, sectioned, and subjected to immunohistochemical examination using antibodies that recognize laminin or myosin heavy chain I, IIa, or IIB. Images were acquired using an epifluorescence microscope on a Leica DMR using OpenLab software. Fiber size and type were determined using MatLAB, where laminin signals define the boundaries of each muscle fiber, and fiber type was detected using anti-myosin antibodies.

[0196] To determine muscle fiber strength, the contractile properties of the soleus and EDL muscles in mice were monitored approximately 2 weeks after birth. Properties to be measured included: maximum force generation capacity at 120 and 100 Hz under ultra-high stimulation current; specific force (force per cross-sectional area); force frequency via calcium regulation and / or fiber type differences; and fatigue using a stimulation duration of 330 ms per second (33% duty cycle). The effect of PPi levels on the contractile function of the soleus and EDL muscles was measured by exposing dissected muscles to PPi concentration ranges (e.g., 2, 4, 8, and 10 µM). In one exemplary experiment, group 1 muscles were exposed to 1 and 8 µM or 4 and 10 µM, and group 2 muscles were exposed to 4 and 8 µM or 2 and 10 µM.

[0197] As a result, from AKP2 - / - No difference in fiber type ratio was observed between the soleus muscle of mice and the soleus muscle of wild-type (WT) mice. Figure 1 Compared to muscle from wild-type (WT) mice, AKP2 - / - Muscles generally have smaller fibers (Figure 2). For example, AKP2 - / - The muscles have a larger percentage of short dimensions (e.g., less than 260 µm). 2 Type I fibers () Figure 2B Type IIa fiber () Figure 2C ) and type IIb fibers ( Figure 2D Interestingly, AKP2 - / - Muscles have small, large myosin IIb fiber clusters, while wild-type muscles lack these fibers. Figure 2D One factor that could explain this is the small proportion (approximately 2%) of IIb fibers within the soleus muscle. Mature soleus muscles contain very few IIb fibers, but immature muscles exhibit a faster muscle phenotype than mature muscles, and therefore the remaining IIb fibers are still noticeable at this age. In summary, AKP2 - / - Mice have smaller fibers, in which the fiber type is not significantly altered.

[0198] For 2-week-old AKP2 - / - Separate muscle function tests were performed on the EDL and soleus muscles of mice and WT controls. Tests were conducted on both males and females to distinguish any sex-dependent differences in force generation.

[0199] Maximum force generation capacity was tested in the EDL and soleus muscles using 120 and 100 Hz stimulation currents. Specific force (force per cross-sectional area) was determined for all muscles. No statistically significant differences in intensity were found between different strains or sexes. Force-frequency relationships were also determined as an assessment of differences in calcium regulation and / or fiber type. No significant differences were found between groups. Fatigue testing was also performed using a stimulation duration of 330 ms per second (33% duty cycle). No significant differences were found between groups. As shown in Figure 3, from... AKP2 - / - No difference in mass was observed between the soleus muscle of mice and the soleus muscle of wild-type (WT) mice. Figure 3A ),strength( Figure 3B ), force frequency ( Figure 3C ) or fatigue parameters ( Figure 3D The difference. Similarly, in the case of... AKP2 - / - No difference in mass was observed between EDL muscle from mice and EDL muscle from wild-type (WT) mice. Figure 4A ),strength( Figure 4B ), force frequency ( Figure 4C ) or fatigue parameters ( Figure 4D The differences between the groups are noted. Note that error bars are not shown for clarity in the accompanying figures for frequency and fatigue. Although the N values ​​for fatigue and force frequency results are low (N=3), the lack of any significant differences between groups suggests that there are indeed no differences. This result is consistent with previously measured fiber type distributions (Figure 2).

[0200] HPP patients, CPPD patients and AKP2 - / -Mice all had elevated PPi levels in their circulation. Therefore, these elevated levels might be in equilibrium with muscle. The effect of PPi concentration on muscle contractile function was then tested. A preliminary study was conducted using 10 µM PPi, and wild-type (WT) muscle showed reversible loss of force production upon exposure to 10 µM PPi. The effects of high PPi on muscle contractile function from WT and wild-type (WT) muscles were then tested. AKP2 - / - The effect of this concentration on muscle force in mice was investigated by testing a series of concentrations (2, 4, 8, 10 µM) to exclude (bracket) [the effects of this concentration]. AKP2 - / - Physiological levels observed in mice and HPP patients. Initial muscle groups were tested at 2 and 8 µM, or 4 and 10 µM PPi concentrations. Second muscle groups were tested at 4 and 8 µM or 2 and 10 µM. Muscles were first tested in normal Ringer's solution, then for 30 minutes each under two testing conditions, and finally returned to normal Ringer's solution. Data from muscles that did not return to normal Ringer's solution for initial force values ​​were discarded. Figure 5B As shown, from AKP2 - / - EDL muscle from mice is more sensitive to elevated PPi than EDL muscle from wild-type (WT) mice. For example, PPi exceeding 4 µM reduces the levels of PPi from mice with elevated PPi. AKP2 - / - The relative force of EDL muscle in mice was significantly different from that in wild-type (WT) mice unless the PPi concentration was at least 10 µM. Figure 5B Conversely, from AKP2 - / - The soleus muscle of mice and wild-type (WT) mice showed similar sensitivity to elevated PPi. Figure 5A ). Future experiments can be conducted to explore the effects of PPi. For example, at elevated PPi, force frequency or fatigue susceptibility can be altered, thus exacerbating weakness. If this is to be done, using a single PPi concentration (e.g., 8 µM) would simplify the study.

[0201] Then to AKP2 - / - Mice were administered Aspheres alpha to determine the effects of alpha-alpha on mice. AKP2 - / - To investigate whether there is a correlation between decreased muscle strength and increased PPi circulating levels in mice, and to evaluate the efficacy of Aspherex alpha in correcting for related phenotypes. Due to untreated... AKP2 - / -Mice typically die at around 12 days of age (lifespan can be extended to 18-20 days with pyridoxine supplementation, but still insufficient for muscle measurements) and are too small to measure muscle strength in vivo, making it difficult to use untreated mice. AKP2 - / - Mice were used as controls to analyze the therapeutic effects of Aspherates Alpha. Conversely, a withdrawal experiment was used. Specifically, mice were treated with Aspherates Alpha from birth until 35 days of age. AKP2 - / - Mice. At this point, some mice were deprived of treatment, and their PPi concentration and muscle strength were measured and compared with mice receiving continuous treatment. The overall study design is summarized below: Table 2. Open-label treatments designed using parallel and randomized controlled trials.

[0202] WT: indicates AKP2 - / - Wild-type mice born in the same littermate From the day of birth until the 35th day after birth, once a day... AKP2 - / - Mice were administered 8.2 mg / kg of Aspherex alpha subcutaneously (SC). Half of the knockout mice then continued to receive Aspherex alpha subcutaneously at the same dosage regimen, while the other half received the control medium subcutaneously at the same dosage regimen. Testing was conducted on day 42. AKP2 - / - Grasping strength was measured in both the mouse group and the untreated wild-type (WT) C57BL / 6-129J mouse group.

[0203] Five trials were conducted. Average scores from these trials were taken and normalized to body weight. Wild-type mice (WT) and mice receiving continuous treatment were measured and compared. AKP 2- / - Mice (Tx-Tx) and mice that stopped treatment after day 35 AKP2 - / - Grasping strength of the forelimbs and hindlimbs of mice (Tx-V). Compared with mice that stopped treatment after day 35. AKP2 - / - Compared to mice (Tx-V), those receiving continuous treatment AKP2 - / - Mice (Tx-Tx) exhibited stronger grip strength in both forelimbs and hindlimbs, demonstrating the beneficial effects of continuous aspartate alpha therapy on myasthenia gravis. Figure 6 ).

[0204] These mouse studies demonstrate the presence of muscle weakness in mouse models (observed in HPP). More surprisingly, they suggest that muscle weakness in HPP may not be attributable to bone defects (which are considered a characteristic feature of HPP), because in wild-type (WT) mice... AKP2 - / - No differences were observed among mice in the proportion of isolated soleus muscle fiber types or in the contractile properties of soleus or EDL muscles, even AKP2 - / - The mice exhibited some degree of smaller muscle fibers. Conversely, muscle weakness in HPP was more correlated with elevated PPi concentrations, as reducing PPi through administration of Aspherex alpha improved [the condition]. AKP2 - / - Mouse muscle grip strength. Therefore, patients with myasthenia gravis characterized by elevated PPi concentrations, even without other symptoms of HPP or not yet diagnosed with HPP, can be treated with Aspherex alpha. Similarly, patients with or susceptible to other myasthenia gravis disorders, such as CPPD and familial hypophosphatemia, can also be treated by administering Aspherex alpha to lower elevated PPi or other alkaline phosphatase substrates (e.g., PLP, PEA, etc.).

[0205] Example 2. Treatment of human muscle weakness As mentioned above AKP2 - / - Studies in knockout mice have shown a correlation between elevated circulating PPi levels (attributed to decreased alkaline phosphatase activity) and reduced muscle strength. This correlation may also exist in patients with HPP. Asphertex alpha treatment can also effectively correct the muscle weakness phenotype in patients with HPP or other muscle weakness disorders characterized by low alkaline phosphatase activity and / or elevated PPi concentrations (such as CPPD and / or familial hypophosphatemia). This embodiment discloses a method for identifying a subgroup of patients with muscle weakness disorders (e.g., HPP, CPPD, familial hypophosphatemia, etc.) having low alkaline phosphatase activity and / or elevated PPi concentrations, and a method for treating or improving the muscle weakness phenotype in patients in such a subgroup with Asphertex alpha. A patient can be identified as one of such a subgroup if they have an increased PPi concentration (or an increased concentration of at least one alkaline phosphatase substrate, such as PLP and PEA) and a muscle weakness phenotype (e.g., loss of muscle strength). In addition, patients can be identified as having low alkaline phosphatase concentrations (Table 4).

[0206] Table 3. Low and normal alkaline phosphatase concentrations in women and men by age group.

[0207] Identification of muscle weakness disorder or phenotype can be performed using routine techniques known in the art. Measurement of PPi (or PLP, PEA, or other alkaline phosphatase substrate) concentrations in such patients can also be performed using routine techniques known in the art and compared with PPi concentrations in normal subjects or subjects without such muscle weakness disorder or phenotype (Table 3). Elevated PPi concentrations can then be identified through this comparison. Table 4. Normal range of pyrophosphate (PPi) levels in infants and children, adolescents and adults.

[0208] More commonly, the level of alkaline phosphatase activity in serum or plasma is measured and compared with age- and sex-adjusted standard data. This was performed on mouse soleus muscle and mouse EDL muscle. AKP2 - / - Knockout mouse studies, intended to understand the underlying causes of hypotonia, would be considered an overly invasive test if performed in humans. Furthermore, because mouse muscle tissue is physiologically plastic, data from mouse muscle are expected to be less affected than those from corresponding human muscle tissue. Therefore, small changes in mouse responses will correlate with larger responses in human muscle tissue. Additionally, since PPi is not a commercially available assay, alkaline phosphatase activity is an acceptable (and inversely correlated) surrogate marker of PPi levels.

[0209] The same or different muscles can be tested to diagnose muscle weakness disorders or phenotypes in animals or humans. For example, various properties of other skeletal or striated muscles, or cardiac or smooth muscles, can be tested. For example, passive mechanical properties (e.g., calcaneal segmental properties) of muscles (e.g., gastrocnemius and Achilles tendon) can be tested using methods known in the art. Viscoelastic properties of muscle stiffness can also be tested.

[0210] Asfutez alpha has previously been shown to be effective in treating patients with HPP, and subcutaneous administration at doses of 3, 6, or 9 mg / kg / week, three times a week or once daily, is recommended. Following similar studies as shown in Example 1, the same or different dosing regimens may be administered to patients with HPP, CPPD, or hypophosphatemia without HPP to treat the muscle weakness phenotype. Multiple endpoints may be used to test treatment efficacy. Some of these endpoints used in HPP treatment include, for example, the Brunincus-Osiris Motor Ability Test, Version 2 (BOT-2), the Radiographic Global Change Impression (RGI-C) scale (7-point scale, where a rating of -3 indicates severe deterioration and a rating of +3 indicates near or complete healing), the Bayley Infant Development Scale, Version 3 (BSID-III), the Child Health Assessment Questionnaire (CHAQ), the Pediatric Outcomes Data Collection Tool Test (PODCI), the Peabody Developmental Motor Scale, Version 2 (PDMS-2), the Six-Minute Walk Test (6MWT), the 12-Point Performance-Oriented Mobility Assessment (POMA-G), and the Modified Performance-Oriented Mobility Assessment (mPOMA-G, e.g., Phillips et al., 2015 Bone Abstracts). 4 (as shown on page 136), and other methods or tests known in the art. Both treatment-naïve patients and patients who have received other alkaline phosphatase therapies can be treated with Asfortes alpha or other relevant peptides with alkaline phosphatase activity.

[0211] Example 3. Treatment of muscle weakness in patient 1 The patient was diagnosed with hypotonia combined with low ALP (associated with high PPi), elevated PLP, and elevated urinary PEA. A 6-year-old patient presented with hypotonia of unknown etiology. The patient's other symptoms included cerebellar atrophy, axonal sensory and motor polyneuropathy, and developmental delay, with clinical and biochemical results supporting a diagnosis of hypophospholipase syndrome (HPP). The patient had received continuous physical therapy since birth and had never been able to walk without support, requiring a wheelchair full-time. The patient was unable to feed independently, was G-tube dependent, and could only self-feed by sipping milk, exhibiting both receptive and expressive language delays.

[0212] The initial endocrine assessment was part of a multidisciplinary clinical trial for muscular dystrophy, in which the patient did not utter a single word throughout the entire presentation and was only communicated via computer-based devices. Aside from a few words the patient was able to repeat during neurological assessment, multiple physicians confirmed virtually no verbal output. The patient was treated with a Pulmicort nebulizer. Initial laboratory results were: ALP 149 (normal 150-420 U / L); PLP 172.4 (normal range 20-125 nmol / L); and urinary PEA 180 (normal 0-106 nmol / mgCr).

[0213] Three months after the patient's initial visit, subcutaneous injections of aspartate alpha at 6 mg / kg / week were initiated. At the three-month follow-up appointment, the parents reported that the patient appeared to have greater strength when standing since the start of treatment and had begun to stand from her wheelchair using a walker. While the patient still required significant support, she showed improvement in weight-bearing capacity. She was able to move her legs and demonstrated some upper limb use with fairly good strength. Overall, improved postural control of the trunk and neck was noted. These improvements were attributed to overall improvements in the patient's total muscle tone and strength, confirming the hypothesis generated from in vitro mouse data.

[0214] Several medical professionals and parents noticed a significant increase in the patient's speech. They also noted improvements in the patient's overall language acquisition, including the use of more words and the combination of words into simple sentences. The enhanced speech ability may also be a result of improved muscle tone and strength in response to treatment. Specifically, the 3-month follow-up appointment noted the following improvements after Aspheres Alpha treatment: increased growth rate (5.8 cm / year, compared to 1.3 cm / year before treatment); increased strength; improved speech, i.e., spontaneous speech and even the formation of simple sentences; and improved bone mineral density in the lumbar spine by 0.5 SD. The patient had lost two teeth within one month prior to starting Aspherex Alpha treatment. Radiographic findings included decreased bone mineralization on wrist X-ray (but normal), and decreased bone mineral density, small bone size, and tibial epiphyseal abnormalities on knee X-ray. The patient presented evidence of low bone mineral density. At baseline, DXA scans showed Z-scores of -4.6 for the lumbar spine and -3.3 for the whole body (excluding the head). A repeat DXA scan 3 months after starting Aspherex Alpha showed an improvement of 0.5 SD in the LBD Z-score, although the Z-score remained low. BMD Z-scores for the lumbar spine and the whole body (excluding the head) were -4.1 and -3.3, respectively. Approximately one month after treatment initiation, the patient developed an idiopathic fracture of the humerus; treatment with Aspherex Alpha continued, and the fracture healed well. Renal ultrasound and ophthalmological examinations were normal at baseline.

[0215] Example 4. Treatment of muscle weakness in patient 2 The second patient was identified as having hypotonia combined with low ALP (associated with high PPi), elevated PLP, and elevated urinary PEA. Also noted was a 12-year-old patient with chromosomal duplication, developmental delay, autism spectrum disorder (Asperger's syndrome), and sensory processing difficulties, who had a low ALP level of 90 U / L (normal range 141-460). The level was repeated to be as low as 91 (tested 4 days later). Patients were easily fatigued, requiring rest during normal daily activities involving minimal walking, and unable to walk long distances. Patients also complained of vague pain in the shoulders, upper back, and other areas. Patients reported sometimes waking up with pain and shoulder pain, and persistent leg pain.

[0216] The patient had no history of premature tooth loss or fractures. Initial occupational therapy assessment indicated a fine motor skills score of 13 (1st percentile). The age equivalence for response speed was 6.2 years. Visual motor control showed a multi-year delay, at the 7.9-year level. For upper limb speed and dexterity, the patient was at the 4.7-year level. However, regarding tooth loss, the family history showed that one parent began wearing dentures at age 21, and similarly, the maternal grandparents also experienced premature loss of permanent teeth and wore dentures at a young age. The mother's ALP level was found to be 50 U / L (laboratory reference range 40-150). The patient's initial laboratory results were: ALP 82 (normal 150-420 U / L); PLP 210 (normal range 20-125 nmol / L); and urinary PEA 46 (normal 0-44 nmol / mgCr). Although the patient has no history of premature tooth loss, some family members do show signs of premature tooth loss. Radiographic analysis showed normal wrist and knee X-rays. Bone mineral density analysis by DXA scan showed normal BMD (L1-L4 Z-score 2.1, TBLH Z-score 2.4). The patient has no history of fractures. The patient reports pain present in multiple locations at different times, including frequent leg pain sufficient to disrupt sleep, plantar pain lasting 5-10 minutes while standing / walking, tension / pain in fatigued limbs, bilateral knee pain, and vague shoulder / chest pain. Renal ultrasound did not reveal renal calcification. The patient's consistent measurements were at the 90th percentile for height and the 92nd percentile for body weight.

[0217] The patient showed no apparent impairment of physical activity at full rest; however, after walking for more than two minutes, exhibited quadriplegic fatigue and bilateral foot tapping due to impaired knee control attributable to dorsiflexion weakness. The patient was able to walk on their toes but showed significant compensation when attempting heel walking. The patient's initial six-minute walking test was 320 meters, significantly below the age / sex standard of 672 + 55 meters. Secondary to fatigue, the patient required two standing rests against a wall. The patient exhibited progressively increasing gait disturbances, including quadriplegic fatigue and foot tapping attributable to dorsiflexion weakness, and a need for seated rest after 6 MWT. The patient experienced difficulty during exercise and was prone to shortness of breath.

[0218] Patients began subcutaneous injections of aspartate alpha at 6 mg / kg / week and were reassessed after 4 months of treatment. Overall, patients showed multiple improvements, including strength, agility, and endurance. The patient's 6-minute walk test improved to 597 meters (from an initial value of 320 meters). Overall, patients experienced significantly less pain (2 out of 10, instead of 5 out of 10 reported in previous follow-ups) and improved mobility after treatment. These improvements were attributed to overall improvements in overall muscle tone and strength, confirming the hypothesis generated from in vitro mouse data.

[0219] Other implementation plans All publications, patents, and patent applications mentioned in the foregoing description are incorporated herein by reference in their entirety, as if specifically and individually indicated that each individual publication, patent, or patent application is incorporated by reference. Various modifications and variations to the methods, pharmaceutical compositions, and kits of the present invention will be apparent to those skilled in the art and will not depart from the scope and spirit of the claimed invention. While this disclosure has been described in conjunction with specific embodiments, it should be understood that further modifications are possible, and the claimed invention should not be unduly limited to the specific embodiments described.

Claims

1. Use of a therapeutically effective amount of tissue nonspecific alkaline phosphatase (TNSALP) having at least 95% sequence identity with amino acids 1-485 of SEQ ID NO: 1 in the preparation of a composition for treating or improving muscle weakness in subjects with or susceptible to myasthenia gravis. The human subjects described had elevated serum pyrophosphate (PPi) concentrations and low alkaline phosphatase activity. The subjects in this study had HPP without any associated bone mineralization defects. The muscle weakness was caused by elevated pyrophosphate (PPi) concentrations and / or low alkaline phosphatase activity, with the elevated PPi concentrations exacerbating the subjects' muscle weakness. In terms of at least one characteristic selected from fiber type ratio, muscle mass, strength, force frequency, fatigue parameters, and fiber contraction characteristics, the muscles of the subject were not significantly different from those of a normal subject.

2. The use as claimed in claim 1, wherein the TNSALP reduces the concentration of pyrophosphate (PPi) in the subject.

3. The use as claimed in claim 1, wherein the muscle weakness disease is at least one of hypophospholipase syndrome (HPP), calcium pyrophosphate dihydrate crystal deposition (CPPD), and familial hypophosphatemia.

4. The use as described in claim 3, wherein the muscles of the subject having elevated serum pyrophosphate (PPi) concentrations are at least one type of leg muscle.

5. The use as claimed in claim 4, wherein the leg muscle is at least one type selected from the soleus muscle and the extensor digitorum longus (EDL) muscle.

6. The use as claimed in claim 3, wherein the familial hypophosphatemia includes at least one of autosomal dominant hypophosphatemic rickets (ADHR), autosomal recessive hypophosphatemic rickets, X-linked hypophosphatemic rickets, and X-linked hypophosphatemia (XLH).

7. The use as claimed in claim 1, wherein the use includes: (i) Identify the subject population that has or is susceptible to myasthenia gravis; (ii) Identify a subgroup of subjects within the population described in step (i), wherein: (a) Subjects in the subgroup have elevated serum pyrophosphate (PPi) concentrations and / or low alkaline phosphatase activity; (b) Elevated pyrophosphate (PPi) concentrations enhanced muscle weakness in subjects in the aforementioned subgroup; or (c) (a) and (b); and (iii) Treat or improve at least one symptom of muscle weakness in the subgroup of subjects described in step (ii).

8. The use as described in claim 7, wherein (a) In terms of at least one property of muscle fiber type ratio and fiber contraction characteristics, the muscles of the subject in step (ii) are not significantly different from the muscles of a normal subject without said type of muscle weakness. (b) The type of muscle weakness described herein is caused by elevated inorganic pyrophosphate (PPi) concentration and / or low alkaline phosphatase activity; (c) The elevated pyrophosphate (PPi) concentration in step (iii) amplifies the muscle weakness in the subject; (d) The TNSALP reduces the pyrophosphate (PPi) concentration in the subject; and / or (e) The subject in step (iii) has or is susceptible to at least one of hypophospholipase syndrome (HPP), calcium pyrophosphate dihydrate crystal deposition (CPPD), and familial hypophosphatemia.

9. The use as claimed in claim 8, wherein the muscle is at least one type of leg muscle.

10. The use as claimed in claim 9, wherein the leg muscle is at least one type selected from the soleus muscle and the extensor digitorum longus (EDL) muscle.

11. The use as claimed in claim 8, wherein the familial hypophosphatemia includes at least one of autosomal dominant hypophosphatemic rickets (ADHR), autosomal recessive hypophosphatemic rickets, X-linked hypophosphatemic rickets, and X-linked hypophosphatemia (XLH).

12. The use as claimed in claim 1, wherein the composition is formulated for: (a) administering the medication to the subject daily for at least one week, one month, three months, six months, or one year; and / or (b) Administered via at least one of the following routes: subcutaneous, intravenous, intramuscular, sublingual, intrathecal, and intradermal.

13. The use as claimed in claim 1, wherein the TNSALP: (a) The amino acid sequence comprising amino acids 1-485 of SEQ ID NO: 1; (b) is a fusion protein; (c) Contains immunoglobulin molecules; (d) Contains a negatively charged peptide; and / or (e) Contains bone-targeting alkaline phosphatase, which comprises a polypeptide having the following structure: Z-sALP-Y-spacer-XW n -V, sALP is the extracellular domain of the alkaline phosphatase. V is absent or has an amino acid sequence of at least one amino acid; X either does not exist or has an amino acid sequence of at least one amino acid; Y either does not exist or has an amino acid sequence of at least one amino acid; Z either does not exist or has at least one amino acid sequence; and W n It is polyaspartic acid or polyglutamic acid, where n = 10 to 16.

14. The use as described in claim 13, wherein (a) The spacer and / or immunoglobulin contains a fragment crystallizable region (Fc); (b) The negatively charged peptide contains D 10 D 16 E 10 and E 16 At least one of them; (c) The TNSALP contains sALP-Fc-D 10 The structure; and / or (d) The TNSALP comprises a dimer comprising a monomer of the amino acid sequence of SEQ ID NO:

1.

15. The method of claim 14, wherein the Fc comprises the amino acid sequence of SEQ ID NO:

20.

16. The use as described in claim 13, wherein the TNSALP: (a) Formulate for administration at a dose of about 0.1 mg / kg / day to about 20 mg / kg / day or an equivalent weekly dose; (b) Formulate for administration at a dose of about 0.5 mg / kg / day to about 20 mg / kg / day or an equivalent weekly dose; (c) Formulate for administration at a dose of about 0.5 mg / kg / day to about 10 mg / kg / day or an equivalent weekly dose; (d) Prepare a dose for administration at a rate of about 1 mg / kg / day to about 10 mg / kg / day or an equivalent weekly dose; or (e) is Aspheres alpha and is administered subcutaneously at a dose of 6 mg / kg once weekly, 3 mg / kg twice weekly, 2 mg / kg three times weekly, or 1 mg / kg six times weekly.

17. The use as described in claim 1, comprising at least one of the following: (a) Prior to the administration of the TNSALP, the subject was characterized by having an average walking distance of approximately 350 meters or less within six minutes; (b) The application of the TNSALP resulted in an increase in average walking distance of at least 100 meters or more within six minutes; (c) The subjects, after administration of the TNSALP, exhibited an average walking distance of approximately 500 meters or longer over a six-minute period; (d) The subjects, after administration of the TNSALP, exhibited a reduced dependence on assistive mobility devices, wherein, Optionally, the assistive mobility device is selected from at least one of the following: walker, wheelchair, carrier, cane, and orthosis; (e) Prior to administration of the TNSALP, the subject was characterized by having a plasma PPi concentration of approximately 4.5 μM or greater; (f) Administration of the TNSALP resulted in a median reduction of PPi concentration of at least about 1 μM in plasma samples from the patient; (g) The subjects exhibited plasma PPi concentrations of approximately 2 μM to approximately 5 μM after administration of the TNSALP; (h) Prior to administration of the TNSALP, the subjects were characterized by having a plasma ALP concentration of approximately 90 U / L or less for subjects aged 0 to 14 days; approximately 134 U / L or less for subjects aged 15 days to less than 1 year; approximately 156 U / L or less for subjects aged approximately 1 year to less than 10 years; approximately 141 U / L or less for subjects aged approximately 10 years to less than approximately 13 years; approximately 62 U / L or less for female subjects aged approximately 13 years to less than approximately 15 years; approximately 127 U / L or less for male subjects aged approximately 13 years to less than approximately 15 years; approximately 54 U / L or less for female subjects aged approximately 15 years to less than approximately 17 years; approximately 89 U / L or less for male subjects aged approximately 15 years to less than approximately 17 years; approximately 48 U / L or less for female subjects aged approximately 17 years or older; or approximately 59 U / L or less for male subjects aged approximately 17 years or older. (i) Administration of the TNSALP resulted in a median increase in ALP concentration of at least about 100 U / L or greater in plasma samples from the subject; (j) Following administration of the at least one recombinant polypeptide containing alkaline phosphatase, the subjects exhibited approximately 273 U / L or greater for subjects aged 0 to 14 days; approximately 518 U / L or greater for subjects aged 15 days to less than 1 year; approximately 369 U / L or greater for subjects aged approximately 1 year to less than 10 years; approximately 460 U / L or greater for subjects aged approximately 10 years to less than approximately 13 years; approximately 280 U / L or greater for female subjects aged approximately 13 years to less than approximately 15 years; approximately 517 U / L or greater for male subjects aged approximately 13 years to less than approximately 15 years; approximately 128 U / L or greater for female subjects aged approximately 15 years to less than approximately 17 years; approximately 365 U / L or greater for male subjects aged approximately 15 years to less than approximately 17 years; approximately 95 U / L or greater for female subjects aged approximately 17 years or older; or approximately 164 U / L or greater for male subjects aged approximately 17 years or older. Plasma ALP concentration of U / L or higher; (k) Prior to the administration of the TNSALP, the subject was characterized by having a mean Brunincus-Osiris Motor Ability Test Version 2 (BOT-2) strength score of about 10 or less; (l) Administration of the TNSALP resulted in an average BOT-2 intensity score of approximately 10 or greater for the subjects; (m) Prior to the administration of the TNSALP, the subjects were characterized by having an average BOT-2 running speed and agility score of about 5 or less; (n) Administration of the TNSALP resulted in the subjects having an average BOT-2 running speed and agility score of approximately 5 or greater; (o) Prior to administration of the TNSALP, the subject was characterized by having a mean Child Health Assessment Questionnaire (CHAQ) score of approximately 0.8 or greater; (p) Administration of the TNSALP resulted in the subjects having an average CHAQ score of approximately 0.5 or less; (q) Prior to administration of the TNSALP, the subject was characterized by having a mean Pediatric Outcome Data Collection Tool (PODCI) score of approximately 40 or less; (r) Administration of the TNSALP resulted in a mean PODCI score of approximately 40 or greater for the subjects; (s) Prior to the administration of the TNSALP, the subject was characterized by having an average muscle strength grade of less than about 5; (t) Administration of the TNSALP resulted in an average increase of approximately 1 or greater in the muscle strength grade of the subjects; (u) Administration of the TNSALP resulted in an average increase of approximately 1 or greater in the muscle strength grade of the subjects; (v) Prior to the administration of the TNSALP, the subject was characterized by having a mean handheld force measurement (HHD) value that was approximately 80% less than the predicted HHD value; and / or (w) The administration of the TNSALP resulted in the subject having an average HHD value that was approximately 80% or greater than the predicted HHD value; wherein, optionally, the HHD value represented the subject’s grip strength, knee flexion, knee extension, hip flexion, hip extension, or hip abduction.

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