Treatment of arginase 1 deficiency

Abstract

A method of treating an arginase 1 (ARG1) deficiency (ARG1-D) in a subject comprising administering to the subject a polyethylene glycol arginase wherein the polyethylene glycol arginase is a pegylated human arginase 1 comprising a cobalt metal cofactor, and wherein the polyethylene glycol arginase is administered weekly at a dose of from about 0.05 mg / kg to about 0.2 mg / kg.

Description

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims priority to U.S. Provisional No. 63 / 329,867, filed on April 11, 2022, which is incorporated herein by reference in its entirety. Technical Field

[0002] The present disclosure relates generally to the field of compositions comprising human arginase 1. Also provided herein are methods of using the compositions. Sequence Listing

[0003] The contents of the electronic sequence listing (AEGL_011_01WO_SeqList_ST26.xml; size: 4,605 ​​bytes; and creation date: April 7, 2023) are incorporated herein by reference in its entirety. Background Art

[0004] ARG1-D is a rare, progressive, multisystem autosomal recessive disease (Summar 2013, DiezFernandez 2018, Schlune 2015, Waisbren 2018). The disease usually presents in early childhood and is caused by a deficiency of the enzyme arginase 1 (ARG1 [EC 3.5.3.1]), which results in two important deleterious metabolic effects: accumulation of high levels of arginine and arginine-derived metabolites, and impairment of the urea cycle, resulting in episodic increases in ammonia levels.

[0005] High plasma arginine levels are thought to be a key driver of spasticity, developmental delays, and seizures, which develop in early childhood and progress throughout life (De Deyn 1997, Waisbren 2018). Lower-limb spasticity in early childhood impairs mobility and balance, leading to difficulty walking and climbing stairs and lack of independence. Developmental delays and cognitive decline significantly impact school performance and educational achievement. Neuromotor and neurocognitive effects are caused by persistently elevated arginine levels and significantly impact daily function and quality of life in the early years, progressively worsening over time, leading to severe disability and early death (De Deyn 1997, Oeffinger 2008, Prasad 1997, Carvalho 2012).

[0006] In addition to the severe neuromotor and neurocognitive manifestations that dominate the clinical picture, subjects with ARG1-D exhibit other medically important disease-related abnormalities, including complications due to hyperammonemia, hepatocellular injury, malnutrition, and growth impairment. These abnormalities are a consequence of or are associated with disease complications or the severe protein restriction therapy required.

[0007] The goal of long-term management of ARG1-D is to reduce plasma arginine levels without adversely affecting growth and development. Current disease management includes strict protein restriction and EAA supplementation to lower arginine, with or without ammonia scavengers to prevent or treat hyperammonemia. Strict dietary protein restriction can modestly reduce plasma arginine levels, improving disease-related abnormalities to a certain extent, thus supporting the value of arginine reduction.

[0008] However, this approach is insufficient as most patients still have significant hyperargininemia. Furthermore, the diet is challenging, especially in children, and supplementation with unpalatable EAA formulas is required to maintain adequate essential amino acid intake ( 2012, Huemer 2016, Lambert 1991, Burrage 2015). Despite current approaches, the progressive nature of the disease highlights a significant unmet need for treatments that would reduce arginine levels beyond what is achievable with current standard disease management to current guidelines or even to normal levels and potentially slow or halt the progression of neuromotor and / or neurocognitive deterioration in patients with ARG1-D. Summary of the invention

[0009] The delivery of arginase 1 deficiency treatments using cobalt-substituted arginase 1 alone or in combination with one or more other treatments is described. For example, the other treatments delivered in combination with arginase 1 treatment can be dietary restriction and / or ammonia scavengers. Combination treatments including arginase 1 may more completely address complex or multifactorial diseases.

[0010] In one aspect, provided herein is a method for treating Arginase 1 (ARG1) deficiency (ARG1-D) in a subject, comprising administering pegzilarginase to the subject, wherein the pegzilarginase is a pegylated human arginase 1 comprising a cobalt metal cofactor, and wherein the pegzilarginase is administered weekly at a dose of about 0.05 mg / kg to about 0.2 mg / kg.

[0011] In one aspect, provided herein is a method of treating arginase 1 (ARG1) deficiency (ARG1-D) in a subject, comprising administering to the subject human arginase 1, wherein the human arginase 1 is a pegylated human arginase 1 comprising a cobalt metal cofactor, and wherein the human arginase is administered at a dose of about 0.05 mg / kg to about 0.2 mg / kg per week.

[0012] In one aspect, provided herein are combination therapies for treating arginase 1 deficiency, particularly a combination of (i) a cobalt-substituted arginase or a fragment thereof capable of reducing arginine in the blood to within the normal range, and any one or more of (ii) dietary restriction and (iii) an ammonia scavenger.

[0013] In one aspect, provided herein are methods for treating arginase 1 deficiency using cobalt-substituted arginase 1, with or without combination with another agent.

[0014] The present disclosure also relates to subpopulations of Arginase 1 Deficiency patients who exhibit specific disease manifestations or specific symptoms.

[0015] The present disclosure also relates to preventing the accumulation of arginine metabolites.

[0016] The present disclosure also relates to dietary modifications (including but not limited to elimination or reduction of dietary restrictions) in patients suffering from arginase 1 deficiency treated with cobalt-substituted arginase 1. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] Figure 1A The clinical study design is shown. Figure 1B Key demographic and baseline clinical characteristics are shown.

[0018] Figure 2 Shown is a waterfall plot of the change in arginine (μM) from baseline at Week 24 during the double-blind period.

[0019] Figure 3A Shown are box plots of arginine levels (μM) over time during the double-blind period. Figure 3B The percentage of patients achieving normalization of plasma arginine at different time points is shown.

[0020] Figure 4A Shown are the changes from baseline in the 2-minute walk test (meters) for each group at Week 24 during the double-blind period. Figure 4B Shown is a waterfall plot of the change from baseline in the 2-minute walk test (meters) for each subject at Week 24 during the double-blind period.

[0021] Figure 5AShown are the changes from baseline in GMFM-E at Week 24 for each group during the double-blind period. Figure 5B Shown is a waterfall plot of each subject's change from baseline in GMFM-E at Week 24 during the double-blind period.

[0022] Figure 6 Shown are box plots of ornithine (μM) over time during the double-blind period.

[0023] Figure 7 Shown is a box plot of α-N-acetylarginine (μM) over time during the double-blind period.

[0024] Figure 8 Shown is a box plot of α-keto-d-guanidinovalerate (μM) over time during the double-blind period.

[0025] Fig. 9 Shown are box plots of arginine (μM) over time during the double-blind period.

[0026] Fig.10 Shown are box plots of guanidinoacetic acid (μM) over time during the double-blind period.

[0027] Fig.11 Shown is a waterfall plot of the change in GMFM-D from baseline at Week 24 during the double-blind period.

[0028] Fig.12 A responder heat map of evaluable subjects is shown.

[0029] Fig.13 Selected values ​​from FAO / WHO / UNU for safe levels of protein intake and energy requirements for children and adults in the healthy population, and during pregnancy and lactation are shown.

[0030] Fig.14 A summary of the changes from baseline at Week 24 for other secondary efficacy endpoints is shown.

[0031] Fig.15 Shown are the dosing strategies tested for IV and SC administration during the adaptive dosing simulation.

[0032] Fig.16 The final dose distribution via the intravenous route is shown.

[0033] Fig.17 The final dose distribution via subcutaneous route is shown.

[0034] Fig.18 The relationship between baseline L-arginine and final dose is shown.

[0035] Fig.19The number of dose titrations for the intravenous route is shown.

[0036] Fig. 20 The differences between IV and SC administration are shown.

[0037] Fig.21 L-arginine times within the recommended range are shown. Other Materials

[0038] The following patents and applications are incorporated herein by reference in their entirety: U.S. Patent No. 8,440,184; U.S. Reissue Patent No. RE46,423; U.S. Patent Application Publication No. 2019 / 0167770; and U.S. Patent Application Publication No. 2021 / 0189371. DETAILED DESCRIPTION

[0039] Disclosed herein is a method for treating arginase 1 (ARG1) deficiency (ARG1-D) in a subject, comprising administering polyethylene glycol arginase or human arginase 1 to the subject, wherein the polyethylene glycol arginase or human arginase 1 is a pegylated human arginase 1 comprising a cobalt metal cofactor, and wherein the polyethylene glycol arginase or human arginase 1 is administered weekly at a dose of about 0.05 mg / kg to about 0.2 mg / kg. In some embodiments, after treatment, the plasma arginine level of the subject is about 40 μM to about 115 μM. In some embodiments, patients with GMFCS classification ≥ II show greater activity improvement after treatment. In some embodiments, the initial dose of polyethylene glycol arginase or human arginase 1 is about 0.1 mg / kg. In some embodiments, the subsequent dose of polyethylene glycol arginase or human arginase 1 is adjusted based on the plasma arginine level of the subject before the subsequent dose is administered. definition

[0040] Unless otherwise defined herein, the technical and scientific terms used in this specification have the meanings commonly understood by those of ordinary skill in the art. Unless otherwise clearly indicated by the content, for the purpose of interpreting this specification, the following description of the terms will apply, and where appropriate, terms used in the singular will also include the plural form, and vice versa. In the event of any conflict between the description of the terms shown and any document incorporated herein by reference, the description of the terms shown below shall prevail.

[0041] As used herein, nouns modified by unmodified quantifiers include plural referents unless the context clearly dictates otherwise.

[0042] As used herein, in the present disclosure, the term "and / or" is used as either "and" or "or" unless stated otherwise.

[0043] As used herein, "about," "approximately," "substantially," and "significantly" will be understood by one of ordinary skill in the art and will vary to some extent depending on the context in which they are used. If there is a usage of the term that is unclear to one of ordinary skill in the art given the context in which the term is used, "about" and "approximately" will mean up to plus or minus 10%, 5%, or 1% of the particular term and "substantially" and "significantly" will mean more than plus or minus 10% of the particular term.

[0044] The term "administering" refers to the injection of a therapeutically effective amount of a compound and a composition containing the disclosed compound. For example, but not limited to, administration can be intravascular (iv) or subcutaneous (sc). The composition of the present invention can also be administered intramuscularly (im). The compound can be polyethylene glycol arginase or human arginase 1 disclosed herein. The composition can be a composition comprising polyethylene glycol arginase or human arginase 1 disclosed herein.

[0045] The term "dietary liberalization" refers to changes in dietary intake in ARG-1D patients who are undergoing any type of dietary restriction as part of disease management, including the addition of any dietary product that is not an arginine-free (or low-arginine) essential amino acid mixture.

[0046] The term "effective amount" refers to the amount of wild-type arginase, such as human arginase 1, or pegylated arginase, such as pegylated arginase, administered that has a desired effect, such as reducing the plasma level of one or more of the following: arginine, arginine (arginine acid, ArgA), GVA, Na-acetylarginine (NAArg), GAA and homoarginine (HArg). An example is to administer a compound to achieve a range of arginine, ArgA, GVA, GAA and / or NAArg that is equivalent to the normal level of each compound in a normal person who does not suffer from ARG1 deficiency. The effective amount can also improve muscle strength, the patient's ambulation ability (i.e., the ability to run, walk, ride a bicycle, climb stairs without assistance) and improve cognitive ability (e.g., Wechsler Intelligence Scale for Children (WISC) test improvement) and / or adaptive behavior (e.g., Adaptive Behavior Assessment Scale (ABAS) or Vineland Adaptive Behavior Scale (VABS) test improvement) (Lopata et al., "Comparison of Adaptive Behavior Measures for Children with HFASDs", Autism Research and Treatment, Vol. 2013, pp. 1-10, (2013)). Luneburg, N. et al., (2011) describes normal levels of arginine. The effective amount may vary with factors such as patient weight.

[0047] The term "Gross Motor Function Classification System" or "GMFCS" scale refers to the criteria for GMFCS levels I to V provided below. GMFCS Level I οCan walk indoors and outdoors and climb stairs without using hands οCan carry out daily activities, such as running and jumping o Have reduced speed, balance and coordination. GMFCS Level II οAbility to walk indoors and outdoors and climb stairs using handrails οHave trouble walking on uneven surfaces, slopes, or in crowds ο Has only minimal ability to run or jump. GMFCS Level III ο Use assistive mobility devices to walk on level surfaces indoors and outdoors οBe able to climb stairs using handrails o Can push a manual wheelchair (may need assistance for long distances or uneven surfaces). GMFCS Level IV ο Severely limited ability to walk, even with assistive devices οUse a wheelchair most of the time and can propel their own electric wheelchair ο May be involved in standing transfers. GMFCS Level V o Have a physiological disorder that limits voluntary control of movement and the ability to maintain head and neck position against gravity οAll aspects of motor function are impaired οUnable to sit or stand independently even with adaptive devices o Unable to walk independently but may be able to use motorized devices for movement.

[0048] The term "gross motor function measure" or "GMFM" scale refers to a clinical measure designed to assess gross motor function by observing a subject's ability to initiate and complete certain movements. There are two versions of the GMFM. The GMFM-88 is the original 88-item measure. Items cover five dimensions of gross motor range of motion: A: lying and rolling, B: sitting, C: crawling and kneeling, D: standing, and E: walking, running, and jumping.

[0049] The GMFM-66 is a 66-item subset of the original 88 items determined through Rasch analysis to best describe gross motor function in children with CP of varying abilities. It has a unidimensional scale that provides an interval scale rather than the ordinal scale of the GMFM-88. Items are ordered by difficulty, and the units of change have the same meaning on a scale from 0 to 100. The GMFM-66 provides information about the difficulty level of each item, thereby providing information to help set realistic goals.

[0050] The term "personalized disease management" refers to the treatment given to a specific ARG1-D patient based on the treating physician's assessment, which generally includes a prescribed diet with strict protein restriction and essential amino acid (EAA) supplementation and / or the use of ammonia scavengers.

[0051] The term "natural protein" refers to any protein that is not a mixture of only essential amino acids, including beans, legumes, lentils, cereals, breads, vegetables, fish, meat, eggs, milk, whey protein, yogurt, cheese, nuts and other foods consumed as part of a nutritious diet.

[0052] The term "pegylation" refers to conjugation with polyethylene glycol (PEG), which has been widely used as a drug carrier due to its high biocompatibility and ease of modification (see Harris et al, Clin. Pharmacokinet. 40 (7): 539-51, 2001). PEG can be coupled (e.g., covalently linked) to active agents through the hydroxyl groups at the chain ends as well as via other chemical methods; however, PEG itself is limited to at most two active agents per molecule. In a different approach, copolymers of PEG and amino acids have been explored as new biomaterials that would retain the biocompatibility properties of PEG, but would have the additional advantage of having many attachment points per molecule (providing greater drug loading). The pegylated arginase variants can be formulated according to known methods to prepare pharmaceutically useful compositions. Wild-type arginase proteins (arginase I or arginase II) that naturally contain a manganese metal cofactor, or wild-type arginase proteins that have been pegylated and contain a manganese metal cofactor, can be administered to ARG1-D patients. In another example, arginase with a cobalt metal cofactor instead of the native manganese metal cofactor can be administered to ARG1-D patients. Arginase containing a cobalt metal cofactor can be further pegylated, with an exemplary form being Co-Argl-PEG (also referred to herein as AEB1 102, pegylated arginase, or Co-hArgI), such as described in U.S. Pat. No. 8,440,184, which is incorporated herein by reference in its entirety. An exemplary form, such as pegylated arginase, has approximately twelve 5K (5000 Dalton) PEG units per monomer linked to one or more lysines present in the protein sequence of pegylated arginase.

[0053] The term "pharmaceutically acceptable or pharmacologically acceptable" refers to molecular entities and compositions that do not produce adverse reactions, allergic reactions or other untoward reactions when administered to a subject (e.g., a human), as appropriate. In light of the present disclosure, the preparation of pharmaceutical compositions containing at least one arginase variant (e.g., a stable multimeric arginine or PEGylated arginase isolated by the methods disclosed herein) or additional active ingredients will be known to those skilled in the art, as exemplified in REMINGTON'S PHARMACEUTICAL SCIENCES, 18th Edition, 1990. In addition, for animal (e.g., human) administration, it will be understood that the formulations should meet the sterility, pyrogenicity, general safety and purity standards required by the FDA Office of Biological Standards.

[0054] The term "pharmaceutically acceptable carrier" includes any and all solvents, dispersion media, coatings, surfactants, antioxidants, preservatives (e.g., antibacterial agents, antifungal agents), isotonic agents, absorption delaying agents, salts, preservatives, drugs, drug stabilizers, gelling agents, binders, excipients, disintegrants, lubricants, dyes, similar materials and combinations thereof, as would be known to one of ordinary skill in the art (see, e.g., REMINGTON'S PHARMACEUTICAL SCIENCES, 18th Edition, 1990). Unless any conventional carrier is incompatible with the active ingredient, its use in the pharmaceutical composition is contemplated.

[0055] The term "major source" refers to substances that constitute at least 50% of the total source. For example, the major source of a subject's protein intake is natural protein, meaning that natural protein constitutes at least 50% of the subject's protein intake source.

[0056] The terms "protein" and "polypeptide" refer to compounds comprising amino acids linked by peptide bonds and are used interchangeably.

[0057] The term "subject" refers to animals, such as mammals, including humans.

[0058] The terms "treat" and variations thereof include inhibiting, slowing, halting, reducing, ameliorating or reversing the progression or severity of an existing symptom, disorder, condition or disease associated with ARG1-D. Treatment may be applied prophylactically or therapeutically. abbreviation Arginase

[0059] In one aspect, arginase is disclosed herein. Wild-type arginase is an enzyme that contains manganese. It is the final enzyme of the urea cycle. Arginase is the fifth and final step in the urea cycle, a series of biophysical reactions in mammals during which the body processes harmful ammonia. Specifically, arginase converts L-arginine to L-ornithine and urea.

[0060] L-arginine is a nitrogen-supplying substrate for nitric oxide synthase (NOS), which produces L-citrulline and nitric oxide (NO). Although the K of arginase (2 to 5 mM) has been reported, M The K of NOS is much higher than that of L-arginine (2 to 20 mM). M , but arginase may also play a role in regulating NOS activity. Under certain conditions, arginase I undergoes Cys-S-nitrosylation, resulting in a higher affinity for L-arginine and reduced availability of NOS substrate.

[0061] Arginase is a homotrimeric enzyme with an a / b fold of parallel eight-stranded b-sheets surrounded by several helices. The enzyme contains a binuclear metal cluster that is essential for the generation of hydroxide for the nucleophilic attack on the guanidino carbon of L-arginine. The natural metal cofactor of arginase is Mn 2+ These Mn 2+ The ion coordinates to the water, orienting and stabilizing the molecule and allowing the water to act as a nucleophile and attack the L-arginine, hydrolyzing it into ornithine and urea.

[0062] Mammals have two arginase isozymes (EC 3.5.3.1) that catalyze the hydrolysis of L-arginine into urea and L-ornithine. The arginase I gene is located on chromosome 6 (6q23), is highly expressed in the cytoplasm of hepatocytes, and plays a role in the final step of the urea cycle, nitrogen removal. The arginase II gene is present on chromosome 14 (l4q24.1). Arginase II is located in mitochondria in tissues (e.g., kidney, brain, and skeletal muscle), where it is thought to provide L-ornithine for proline and polyamine biosynthesis (Lopez et al, FEBS J. 272: 4540-48, 2005).

[0063] Wild-type human arginase II has the following sequence (Uniprot P78540):

[0064] Wild-type human arginase I has the following sequence (Uniprot / P05089):

[0065] Wild-type human arginase I without the N-terminal methionine has the following sequence:

[0066] Arginase has been studied for nearly 50 years as a method for degrading extracellular L-arginine (Dillon et al, "Biochemical characterization of the arginine degrading enzymes arginase and arginine deiminase and their effect on nitric oxide production", Med. Sci. Monit., 8 (7): BR248-253 (2002)). Although native arginase is cleared from the circulation within minutes (Savoca et al., Cancer Biochem. Biophys. 7: 261-268, 1984), a single injection of PEG-arginase MW 5000 in rats is sufficient to achieve near-complete arginine depletion in about 3 days (Cheng et al., Cancer Res. 67: 309-17, 2007).

[0067] The bacterial arginine hydrolase ADI has been tested in vitro showing good kinetics and stability. Unfortunately, ADI is a bacterial enzyme and therefore it induces strong immune responses and adverse effects in most patients and is not suitable for long-term administration in ARG1-D patients requiring regular administration.

[0068] For clinical use in patients with ARG1-D, arginase must be modified to allow it to persist in the circulation for a long time (e.g., several days). In the absence of any modification, the half-life of human arginase in the circulation is only a few minutes, mainly because its size is not large enough to avoid filtration through the kidneys. Unmodified human arginase is easily inactivated in serum, and its half-life for degradation is only four hours. In some embodiments, human arginase is human arginase 1. In some embodiments, human arginase 1 is pegylated. In some embodiments, human arginase 1 has a cobalt metal cofactor. In some embodiments, human arginase 1 is pegylated and has a cobalt metal cofactor. In some embodiments, human arginase 1 comprises the amino acid sequence of SEQ ID NO: 2. In some embodiments, human arginase 1 comprises the amino acid sequence of SEQ ID NO: 3. In some embodiments, human arginase 1 comprises at least one amino acid substitution and / or is truncated by one or more amino acids in the metal binding site, with reference to SEQ ID NO: 2 or 3. In some embodiments, human arginase 1 is pegylated, has a cobalt metal cofactor, and comprises the amino acid sequence of SEQ ID NO: 2. In some embodiments, human arginase 1 is pegylated, has a cobalt metal cofactor, and comprises the amino acid sequence of SEQ ID NO: 3. In some embodiments, human arginase 1 is pegylated, has a cobalt metal cofactor, and comprises at least one amino acid substitution in the metal binding site and / or is truncated by one or more amino acids, with reference to SEQ ID NO: 2 or 3.

[0069] In some embodiments, the polyethylene glycol arginase disclosed herein has the sequence of arginase I, SEQ ID NO: 2, and has a cobalt metal cofactor instead of a manganese metal cofactor. In some embodiments, the polyethylene glycol arginase disclosed herein has the sequence of arginase I, SEQ ID NO: 3, and has a cobalt metal cofactor instead of a manganese metal cofactor. In some embodiments, the polyethylene glycol arginase is pegylated as described in U.S. Patent No. 8,440,184 and prepared as described in U.S. Patent Application Publication No. 2021 / 0189371.

[0070] In some aspects of the present disclosure, methods and compositions related to PEGylated arginase are disclosed. Specifically, PEGylation of arginase at a modified cysteine ​​residue (e.g., replacing the third residue at the N-terminus) can be used to produce a homogeneous PEGylated arginase composition. Also disclosed is a method for isolating PEGylated arginase based on a temporary interruption of polymerization.

[0071] Pegylation is the process by which a PEG polymer chain is covalently attached to another molecule (usually a drug or therapeutic protein). "Pegylation" can be achieved by incubating a reactive derivative of PEG with a target macromolecule. The increase in the hydrodynamic size (size in solution) of a drug or therapeutic protein prolongs its circulation time by reducing renal clearance. Pegylation can also provide water solubility to hydrophobic drugs and proteins. Human arginase 1 disclosed herein, including PEGylated arginase, can be pegylated as described in U.S. Patent No. 8,440,184.

[0072] The first step of PEGylation can be to suitably functionalize the PEG polymer at one or both terminal domains of a protein or within an amino acid (e.g., lysine). PEGs activated with the same reactive moiety at each terminus are referred to as "homobifunctional," while PEG derivatives are referred to as "heterobifunctional" or "heterofunctional" if the functional groups present are different. Chemically active or activated derivatives of PEG polymers are prepared to attach PEG to the desired molecule.

[0073] The selection of suitable functional groups for PEG derivatives is based on the type of reactive groups available on the molecule to be coupled with PEG. For proteins, typical reactive amino acids include lysine, cysteine, histidine, arginine, aspartic acid, glutamic acid, serine, threonine and tyrosine. N-terminal amino groups and C-terminal carboxylic acids can also be used to connect PEG to polypeptides.

[0074] The technology for forming PEG derivatives includes reacting PEG polymers with groups reactive to hydroxyl groups, which are typically anhydrides, acyl chlorides, chloroformates and carbonates. Pegylation chemicals can also use functional groups that can be used for conjugation, such as aldehydes, esters, amides, etc. Heterobifunctional PEGs are very useful in connecting two entities, where hydrophilicity, flexibility and biocompatibility of the spacer are required. Some preferred end groups of heterobifunctional PEGs are maleimide, vinyl sulfone, pyridyl disulfide, amine, carboxylic acid and H-hydroxysuccinimide (NHS) esters.

[0075] The most common modifiers or linkers are based on methoxy polyethylene glycol (mPEG) molecules. Their activity depends on the addition of protein modifying groups at the alcohol termini. Polyethylene glycol (PEG diol) can be used as a precursor molecule; the diol is subsequently modified at both ends to prepare heterodimeric or homodimeric PEG-linked molecules.

[0076] Proteins are often PEGylated at nucleophilic sites, such as unprotonated thiols (cysteinyl residues) or amino groups. Some examples of cysteinyl-specific modification agents include PEG maleimide, PEG iodoacetate, PEG thiol, and PEG vinyl sulfone. All four are strongly cysteinyl-specific under mild conditions and at neutral to weakly alkaline pH, but each has some disadvantages. The amide formed with maleimide may be somewhat unstable under alkaline conditions, so there may be some limitations in the formulation selection using this linker. The amide linkage formed with iodine-PEG is more stable, but free iodine can modify tyrosine residues under some conditions. PEG thiol forms a disulfide bond with protein thiol, but the linkage may also be unstable under alkaline conditions. Compared with maleimide and iodine-PEG, the reactivity of PEG-vinyl sulfone is relatively slow; however, the thioether linkage formed is very stable. Its slower reaction rate can also make the PEG-vinyl sulfone reaction easier to control.

[0077] Site-specific PEGylation is rarely performed at native cysteinyl residues because these residues are usually in the form of disulfide bonds or are required for biological activity. On the other hand, site-directed mutagenesis can be used to incorporate cysteinyl PEGylation sites for sulfhydryl-specific linkers. Cysteine ​​mutations must be designed to make them accessible to PEGylation reagents and still have biological activity after PEGylation.

[0078] Amine-specific modifiers include PEG NHS esters, PEG tresylate, PEG aldehydes, PEG isothiocyanates, and several others. These amine-specific reagents typically react under mild conditions and are very specific for amino groups.

[0079] Site-specific PEGylation can be challenging due to the multiple lysine residues on most proteins. Fortunately, since these reagents react with unprotonated amino groups, PEGylation can be directed to amino groups with lower pK by performing the reaction at a lower pH. In general, the pK of the alpha amino group is 1 to 2 pH units lower than the epsilon amino group of a lysine residue. By PEGylating the molecule at a pH of 7 or lower, high selectivity for the N-terminus is generally obtained. However, this is only feasible when the N-terminal portion of the protein is not required for biological activity. Nevertheless, the pharmacokinetic benefits of PEGylation generally outweigh the significant loss of in vitro biological activity, resulting in products with much greater in vivo biological activity, regardless of the PEGylation chemistry.

[0080] When developing a PEGylation process, several parameters are considered. Fortunately, there are usually no more than four or five critical parameters. An "experimental design" approach to optimizing PEGylation conditions can be very useful. For thiol-specific PEGylation reactions, parameters to consider include: protein concentration, PEG to protein ratio (on a molar basis), temperature, pH, reaction time, and in some cases, exclusion of oxygen. (Oxygen can contribute to the formation of intermolecular disulfides by proteins, which will reduce the yield of PEGylated products.) For amine-specific modifications, the same factors (except oxygen) should be considered, with the difference that pH may be more critical, particularly when targeting N-terminal amino groups.

[0081] For both amine-specific and thiol-specific modifications, the reaction conditions can affect the stability of the protein. This can be limited by temperature, protein concentration, and pH. In addition, the reactivity of the PEG linker should be understood before starting the PEGylation reaction. For example, if the PEGylation reagent is only 70 percent active, the amount of PEG used should ensure that only active PEG molecules are calculated in the reaction stoichiometry of the protein with PEG. Pharmaceutical composition

[0082] In one aspect, disclosed herein are pharmaceutical compositions comprising arginase. In some embodiments, the arginase is a pegylated arginase, which is a pegylated human arginase 1 comprising a cobalt metal cofactor. In some embodiments, the arginase is human arginase 1. In some embodiments, human arginase 1 is pegylated and has a cobalt metal cofactor.

[0083] Arginase and compositions comprising the same as described herein can be administered systemically or topically. Arginase and compositions comprising the same can be administered intravenously, intrathecally, subcutaneously, intramuscularly, intratumorally and / or intraperitoneally or a combination thereof. Arginase and compositions comprising the same as described herein can be administered alone or in combination with ammonia scavengers (e.g., sodium phenylbutyrate, sodium benzoate, and glyceryl phenylbutyrate).

[0084] Compositions containing arginase or its part can be provided in the form of formulations together with physiologically tolerable liquid, gel or solid carrier, diluent and excipient. Such compositions are usually prepared as liquid solutions or suspensions as injectables. For example, suitable diluents and excipients are water, saline, dextrose, glycerol, etc., and combinations thereof. In addition, if desired, the composition may contain a small amount of auxiliary substances, such as wetting agents or emulsifiers, stabilizers or pH buffers. In the case of considering clinical applications, it may be necessary to prepare pharmaceutical compositions suitable for the intended application form--expression vectors, viral storage liquids, proteins, antibodies and drugs. In general, the pharmaceutical composition of the present invention comprises an effective amount of one or more arginase variants or other reagents dissolved or dispersed in a pharmaceutically acceptable carrier.

[0085] Pharmaceutical compositions containing arginase may include different types of carriers, depending on whether the pharmaceutical composition is to be administered in solid, liquid or aerosol form, and whether it is required to be sterile for such routes of administration (e.g., injection). As will be known to those of ordinary skill in the art, the present invention may be administered intravenously, intradermally, percutaneously, intrathecally, intraarterially, intraperitoneally, intramuscularly, subcutaneously, intratumorally, topically, by injection, infusion, continuous infusion, by catheter, in lipid compositions (e.g., liposomes), or by other methods, or any combination of the foregoing (see, e.g., REMINGTON'S PHARMACEUTICALSCIENCES).

[0086] Arginase variants can be formulated into compositions in free base, neutral or salt form. Pharmaceutically acceptable salts include acid addition salts, such as those formed with free amino groups of proteinaceous compositions, or those formed with inorganic acids such as hydrochloric acid or phosphoric acid or organic acids such as acetic acid, oxalic acid, tartaric acid or mandelic acid. Salts formed with free carboxyl groups can also be derived from inorganic bases, such as hydroxides of sodium, potassium, ammonium, calcium or iron; or organic bases, such as isopropylamine, trimethylamine, histidine or procaine. After formulation, the solution can be administered in a manner compatible with the dosage formulation and in such an amount (such as a therapeutically effective amount). The preparation is easy to administer in a variety of dosage forms, such as formulated for parenteral administration, such as injectable solutions, or for delivery to the lungs in aerosols, or formulated for digestion (alimentary) administration, such as drug release capsules, etc.

[0087] The composition for administration can be provided in a pharmaceutically acceptable carrier with or without an inert diluent. The carrier should be absorbable and include a liquid, semisolid (i.e., paste), or solid carrier. Unless any conventional medium, reagent, diluent, or carrier is harmful to the recipient or to the therapeutic effect of the composition contained therein, its use in an administrable composition for implementing the method of the present invention is suitable.

[0088] Some examples of carriers or diluents include fats, oils, water, saline solutions, lipids, liposomes, resins, adhesives, fillers, etc., or combinations thereof. The composition may also include a variety of antioxidants to delay oxidation of one or more components. In addition, the use of preservatives (e.g., a variety of antibacterial and antifungal agents), including but not limited to parabens (e.g., methylparaben, propylparaben), chlorobutanol, phenol, sorbic acid, thimerosal, or combinations thereof, may improve the longevity of the pharmaceutical composition.

[0089] The composition can be combined with the carrier in any convenient and practical manner, i.e., by solution, suspension, emulsification, mixing, encapsulation, absorption, etc. Such procedures are routine to those skilled in the art.

[0090] Pharmaceutical lipid carriers can be used for compositions comprising arginase variants. The lipid carrier composition can include one or more lipids and an aqueous solvent. As used herein, the term "lipid" refers to any of a wide range of substances that are characteristically insoluble in water and extractable with an organic solvent. Some examples include compounds containing long-chain aliphatic hydrocarbons and derivatives thereof. Lipids can be naturally occurring or synthetic (i.e., designed or produced by humans). However, lipids are typically biological substances.

[0091] Biological lipids include, for example, neutral fats, phospholipids, phosphoglycerides, steroids, terpenes, lysolipids, glycosphingolipids, glycolipids, sulfolipids, lipids with ether and ester-linked fatty acids, and polymerizable lipids, and combinations thereof. Of course, compounds understood to be lipids by those skilled in the art, except those specifically described herein, are also encompassed by the compositions and methods of the present invention.

[0092] The actual dosage amount of the compositions described herein comprising arginase administered to a patient can be determined by physical and physiological factors such as body weight, severity of the condition, type of disease being treated, previous or concurrent therapeutic interventions, patient specific illnesses, and route of administration.

[0093] Depending on the dose and route of administration, the preferred dose and / or number of administrations of the effective amount may vary from subject to subject. The dose will depend on the amount required for the patient to achieve normal levels of at least one or more of the following: arginine, HArg, ArgA, GVA, GAA, and NAArg. Patient levels of ARG1-D are typically assessed for one or more of these five (5) compounds until a normal range is achieved in the patient's plasma. Tissue levels of the five (5) compounds may also be assessed, but may not be necessary or may be performed less frequently than plasma level testing.

[0094] The pharmaceutical composition may include, for example, at least about 0.1% of the active compound. In some embodiments, the active compound may, for example, include about 2% to about 75% of the unit weight, or about 25% to about 60%, and any range derivable therein. Naturally, the amount of the active compound in each therapeutically useful composition can be prepared in such a way that a suitable dose will be obtained in any given unit dose of the compound. Those skilled in the art will consider factors such as solubility, bioavailability, biological half-life, route of administration, product shelf life and other pharmacological considerations for preparing such pharmaceutical preparations, and therefore can expect multiple doses and treatment regimens.

[0095] In some embodiments, the polyethylene glycol arginase is stored at a concentration of about 1 mg / mL to about 8 mg / mL. In some embodiments, the polyethylene glycol arginase is stored at a concentration of at least about 1 mg / mL. In some embodiments, the polyethylene glycol arginase is stored at a concentration of up to about 8 mg / mL. In some embodiments, the polyethylene glycol arginase is stored at a concentration of about 1 mg / mL to about 2 mg / mL, about 1 mg / mL to about 3 mg / mL, about 1 mg / mL to about 4 mg / mL, about 1 mg / mL to about 5 mg / mL, about 1 mg / mL to about 6 mg / mL, about 1 mg / mL to about 7 mg / mL, about 1 mg / mL to about 8 mg / mL, about 2 mg / mL to about 3 mg / mL, about 2 mg / mL to about 4 mg / mL, about 2 mg / mL to about 5 mg / mL, about 2 mg / mL to about 6 mg / mL, about 2 mg / mL to about 7 mg / mL, about 2 mg / mL to about 8 mg / mL, about 3 mg / mL to about In some embodiments, the polyethylene glycol arginase is stored at a concentration of about 1 mg / mL, about 2 mg / mL, about 3 mg / mL, about 4 mg / mL to about 5 mg / mL, about 3 mg / mL to about 6 mg / mL, about 3 mg / mL to about 7 mg / mL, about 3 mg / mL to about 8 mg / mL, about 4 mg / mL to about 5 mg / mL, about 4 mg / mL to about 6 mg / mL, about 4 mg / mL to about 7 mg / mL, about 4 mg / mL to about 8 mg / mL, about 5 mg / mL to about 6 mg / mL, about 5 mg / mL to about 7 mg / mL, about 5 mg / mL to about 8 mg / mL, about 6 mg / mL to about 7 mg / mL, about 6 mg / mL to about 8 mg / mL, or about 7 mg / mL to about 8 mg / mL. In some embodiments, the polyethylene glycol arginase is stored at a concentration of about 1 mg / mL, about 2 mg / mL, about 3 mg / mL, about 4 mg / mL, about 5 mg / mL, about 6 mg / mL, about 7 mg / mL, or about 8 mg / mL. In some embodiments, the PEGylated arginase is stored at a concentration of about 5 mg / mL.

[0096] In some embodiments, human arginase 1 is stored at a concentration of about 1 mg / mL to about 8 mg / mL. In some embodiments, human arginase 1 is stored at a concentration of at least about 1 mg / mL. In some embodiments, human arginase 1 is stored at a concentration of up to about 8 mg / mL. In some embodiments, human arginase 1 is stored at a concentration of about 1 mg / mL to about 2 mg / mL, about 1 mg / mL to about 3 mg / mL, about 1 mg / mL to about 4 mg / mL, about 1 mg / mL to about 5 mg / mL, about 1 mg / mL to about 6 mg / mL, about 1 mg / mL to about 7 mg / mL, about 1 mg / mL to about 8 mg / mL, about 2 mg / mL to about 3 mg / mL, about 2 mg / mL to about 4 mg / mL, about 2 mg / mL to about 5 mg / mL, about 2 mg / mL to about 6 mg / mL, about 2 mg / mL to about 7 mg / mL, about 2 mg / mL to about 8 mg / mL, about 3 mg / mL to about 4 In some embodiments, human arginase 1 is stored at a concentration of about 1 mg / mL, about 2 mg / mL, about 3 mg / mL, about 4 mg / mL, about 5 mg / mL, about 6 mg / mL, about 7 mg / mL, about 8 mg / mL, about 4 mg / mL to about 5 mg / mL, about 4 mg / mL to about 6 mg / mL, about 4 mg / mL to about 7 mg / mL, about 4 mg / mL to about 8 mg / mL, about 5 mg / mL to about 6 mg / mL, about 5 mg / mL to about 7 mg / mL, about 5 mg / mL to about 8 mg / mL, about 6 mg / mL to about 7 mg / mL, about 6 mg / mL to about 8 mg / mL, or about 7 mg / mL to about 8 mg / mL. In some embodiments, human arginase 1 is stored at a concentration of about 1 mg / mL, about 2 mg / mL, about 3 mg / mL, about 4 mg / mL, about 5 mg / mL, about 6 mg / mL, about 7 mg / mL, or about 8 mg / mL. In some embodiments, human arginase 1 is stored at a concentration of about 5 mg / mL. In some embodiments, the weight of human arginase 1 is based on the combined weight of human arginase 1 and polyethylene glycol conjugated to human arginase 1.

[0097] In some embodiments, polyethylene glycol arginase is formulated in a buffer at pH 7.4, the buffer comprising 50 mM sodium chloride, 5 mM potassium phosphor and 1.5% glycerol (w / v / ). In some embodiments, human arginase 1 is formulated in a buffer at pH 7.4, the buffer comprising 50 mM sodium chloride, 5 mM potassium phosphor and 1.5% glycerol (w / v / ).

[0098] In some embodiments, the polyethylene glycol arginase is a liquid. In some embodiments, the polyethylene glycol arginase is lyophilized. In some embodiments, the polyethylene glycol arginase is in a vial or an ampoule. In some embodiments, the vial or ampoule contains about 2 mg or about 5 mg of the polyethylene glycol arginase. In some embodiments, human arginase 1 is a liquid. In some embodiments, human arginase 1 is lyophilized. In some embodiments, human arginase 1 is in a vial or an ampoule. In some embodiments, the vial or ampoule contains about 2 mg or about 5 mg of human arginase 1. Treatment

[0099] In one aspect, disclosed herein are methods of treating arginase 1 (ARG1) deficiency (ARG1-D) in a subject, wherein the method comprises administering arginase to the subject.

[0100] In some embodiments, the arginase is a pegylated arginase, which is a pegylated human arginase 1 comprising a cobalt metal cofactor. In some embodiments, the arginase is human arginase 1. In some embodiments, human arginase 1 is pegylated and has a cobalt metal cofactor.

[0101] In some embodiments, polyethylene glycol arginase is administered daily, weekly, biweekly, monthly. In some embodiments, polyethylene glycol arginase is administered every 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 days. In some embodiments, polyethylene glycol arginase is administered weekly. In some embodiments, human arginase 1 is administered daily, weekly, biweekly, monthly. In some embodiments, human arginase 1 is administered weekly. In some embodiments, human arginase 1 is administered every 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 days.

[0102] In some embodiments, the polyethylene glycol arginase is administered at a dosage of about 0.01 mg / kg to about 0.5 mg / kg. In some embodiments, the polyethylene glycol arginase is administered at a dosage of about 0.05 mg / kg to about 0.2 mg / kg. In some embodiments, the polyethylene glycol arginase is administered at a dosage of at least about 0.01 mg / kg. In some embodiments, the polyethylene glycol arginase is administered at a dosage of at most about 0.5 mg / kg. In some embodiments, the polyethylene glycol arginase is administered at a dosage of at least about 0.05 mg / kg. In some embodiments, the polyethylene glycol arginase is administered at a dosage of at most about 0.2 mg / kg. In some embodiments, polyethylene glycol arginase is present at about 0.01 mg / kg to about 0.05 mg / kg, about 0.01 mg / kg to about 0.1 mg / kg, about 0.01 mg / kg to about 0.15 mg / kg, about 0.01 mg / kg to about 0.2 mg / kg, about 0.01 mg / kg to about 0.25 mg / kg, about 0.01 mg / kg to about 0.3 mg / kg, about 0.01 mg / kg to about 0.35 mg / kg, about 0.01 mg / kg to about 0.4 mg / kg, about 0.01 mg / kg to about 0.45 mg / kg, about 0.01 mg / kg to about 0.5 mg / kg, about 0.05 mg / kg to about 0.1 mg / kg, about 0.05 mg / kg to about 0.15 mg / kg, about 0.05 mg / kg to about 0.2 mg / kg, about 0.05 mg / kg to about 0.25 mg / kg, about 0.05 mg / kg to about 0.3 mg / kg, about 0.05 mg / kg to about 0.35 mg / kg, about 0.05 mg / kg to about 0.4 mg / kg , about 0.05 mg / kg to about 0.45 mg / kg, about 0.05 mg / kg to about 0.5 mg / kg, about 0.1 mg / kg to about 0.15 mg / kg, about 0.1 mg / kg to about 0.2 mg / kg, about 0.1 mg / kg to about 0.25 mg / kg, about 0.1 mg / kg to about 0.3 mg / kg, about 0.1 mg / kg to about 0.35 mg / kg, about 0.1 mg / kg to about 0.4 mg / kg, about 0.1 mg / kg to about 0.45 mg / kg, about 0. 1mg / kg to about 0.5mg / kg, about 0.15mg / kg to about 0.2mg / kg, about 0.15mg / kg to about 0.25mg / kg, about 0.15mg / kg to about 0.3mg / kg, about 0.15mg / kg to about 0.35mg / kg, about 0.15mg / kg to about 0.4mg / kg, about 0.15mg / kg to about 0.45mg / kg, about 0.15mg / kg to about 0.5mg / kg, about 0.2mg / kg to about 0.25mg / kg, about 0.2mg / kg to about 0.3mg / kg, about 0.2mg / kg to about 0.35mg / kg, about 0.2mg / kg to about 0.4mg / kg, about 0.2mg / kg to about 0.45mg / kg, about 0.2mg / kg to about 0.5mg / kg, about 0.25mg / kg to about 0.3mg / kg, about 0.25mg / kg to about 0.35mg / kg, about 0.25mg / kg to about 0.4mg / kg, about 0.25mg / kg to about 0.45mg / kg, about 0.25mg / kg to about 0.5mg / kg, about 0.3 The invention relates to the administration of a dosage of about 0.35 mg / kg to about 0.35 mg / kg, about 0.3 mg / kg to about 0.4 mg / kg, about 0.3 mg / kg to about 0.45 mg / kg, about 0.3 mg / kg to about 0.5 mg / kg, about 0.35 mg / kg to about 0.4 mg / kg, about 0.35 mg / kg to about 0.45 mg / kg, about 0.35 mg / kg to about 0.5 mg / kg, about 0.4 mg / kg to about 0.45 mg / kg, about 0.4 mg / kg to about 0.5 mg / kg, or about 0.45 mg / kg to about 0.5 mg / kg. In some embodiments, the PEGylated arginase is administered at a dose of about 0.01 mg / kg, about 0.05 mg / kg, about 0.1 mg / kg, about 0.15 mg / kg, about 0.2 mg / kg, about 0.25 mg / kg, about 0.3 mg / kg, about 0.35 mg / kg, about 0.4 mg / kg, about 0.45 mg / kg, or about 0.5 mg / kg.

[0103] In some embodiments, human arginase 1 is administered at a dose of about 0.01 mg / kg to about 0.5 mg / kg. In some embodiments, human arginase 1 is administered at a dose of about 0.05 mg / kg to about 0.2 mg / kg. In some embodiments, human arginase 1 is administered at a dose of at least about 0.01 mg / kg. In some embodiments, human arginase 1 is administered at a dose of up to about 0.5 mg / kg. In some embodiments, human arginase 1 is administered at a dose of at least about 0.05 mg / kg. In some embodiments, human arginase 1 is administered at a dose of up to about 0.2 mg / kg. In some embodiments, human arginase 1 is present at about 0.01 mg / kg to about 0.05 mg / kg, about 0.01 mg / kg to about 0.1 mg / kg, about 0.01 mg / kg to about 0.15 mg / kg, about 0.01 mg / kg to about 0.2 mg / kg, about 0.01 mg / kg to about 0.25 mg / kg, about 0.01 mg / kg to about 0.3 mg / kg, about 0.01 mg / kg to about 0.35 mg / kg, about 0.01 mg / kg to about 0.4 mg / kg, about 0.01 mg / kg to about 0.5 mg / kg, about 0.01 mg / kg to about 0.6 mg / kg, about 0.01 mg / kg to about 0.7 mg / kg, about 0.01 mg / kg to about 0.8 mg / kg, about 0.01 mg / kg to about 0.9 mg / kg, about 0.01 mg / kg to about 0.10 mg / kg, about 0.01 mg / kg to about 0.15 mg / kg, about 0.01 mg / kg to about 0.25 mg / kg / kg to about 0.45mg / kg, about 0.01mg / kg to about 0.5mg / kg, about 0.05mg / kg to about 0.1mg / kg, about 0.05mg / kg to about 0.15mg / kg, about 0.05mg / kg to about 0.2mg / kg, about 0.05mg / kg to about 0.25mg / kg, about 0.05mg / kg to about 0.3mg / kg, about 0.05mg / kg to about 0.35mg / kg, about 0.05mg / kg to about 0.4mg / kg, about 0.05mg / kg to about 0.45 mg / kg, about 0.05 mg / kg to about 0.5 mg / kg, about 0.1 mg / kg to about 0.15 mg / kg, about 0.1 mg / kg to about 0.2 mg / kg, about 0.1 mg / kg to about 0.25 mg / kg, about 0.1 mg / kg to about 0.3 mg / kg, about 0.1 mg / kg to about 0.35 mg / kg, about 0.1 mg / kg to about 0.4 mg / kg, about 0.1 mg / kg to about 0.45 mg / kg, about 0.1 mg / kg to about 0.5 mg / kg g, about 0.15 mg / kg to about 0.2 mg / kg, about 0.15 mg / kg to about 0.25 mg / kg, about 0.15 mg / kg to about 0.3 mg / kg, about 0.15 mg / kg to about 0.35 mg / kg, about 0.15 mg / kg to about 0.4 mg / kg, about 0.15 mg / kg to about 0.45 mg / kg, about 0.15 mg / kg to about 0.5 mg / kg, about 0.2 mg / kg to about 0.25 mg / kg, about 0.2 mg / kg to about 0.3 mg / kg, about 0.2mg / kg to about 0.35mg / kg, about 0.2mg / kg to about 0.4mg / kg, about 0.2mg / kg to about 0.45mg / kg, about 0.2mg / kg to about 0.5mg / kg, about 0.25mg / kg to about 0.3mg / kg, about 0.25mg / kg to about 0.35mg / kg, about 0.25mg / kg to about 0.4mg / kg, about 0.25mg / kg to about 0.45mg / kg, about 0.25mg / kg to about 0.5mg / kg, about 0.3mg / kg to about 0.3 The invention relates to the present invention to a dosage of about 0.5 mg / kg, about 0.3 mg / kg to about 0.4 mg / kg, about 0.3 mg / kg to about 0.45 mg / kg, about 0.3 mg / kg to about 0.5 mg / kg, about 0.35 mg / kg to about 0.4 mg / kg, about 0.35 mg / kg to about 0.45 mg / kg, about 0.35 mg / kg to about 0.5 mg / kg, about 0.4 mg / kg to about 0.45 mg / kg, about 0.4 mg / kg to about 0.5 mg / kg, or about 0.45 mg / kg to about 0.5 mg / kg. In some embodiments, human arginase 1 is administered at a dose of about 0.01 mg / kg, about 0.05 mg / kg, about 0.1 mg / kg, about 0.15 mg / kg, about 0.2 mg / kg, about 0.25 mg / kg, about 0.3 mg / kg, about 0.35 mg / kg, about 0.4 mg / kg, about 0.45 mg / kg, or about 0.5 mg / kg. In some embodiments, the weight of human arginase 1 is based on the combined weight of human arginase 1 and polyethylene glycol conjugated to human arginase 1. .

[0104] In some embodiments, the starting dose of the polyethylene glycol arginase is about 0.01 mg / kg to about 0.2 mg / kg. In some embodiments, the starting dose of the polyethylene glycol arginase is at least about 0.01 mg / kg. In some embodiments, the starting dose of the polyethylene glycol arginase is at most about 0.2 mg / kg. In some embodiments, the starting dose of polyethylene glycol arginase is about 0.01 mg / kg to about 0.03 mg / kg, about 0.01 mg / kg to about 0.05 mg / kg, about 0.01 mg / kg to about 0.07 mg / kg, about 0.01 mg / kg to about 0.09 mg / kg, about 0.01 mg / kg to about 0.1 mg / kg, about 0.01 mg / kg to about 0.12 mg / kg, about 0.01 mg / kg to about 0.14 mg / kg, about 0.01 mg / kg to about 0.16 mg / kg, about 0.01 mg / kg to about 0.18 mg / kg, about 0.01 mg / kg to about 0.2 mg / kg, about 0.03 mg / kg to about 0.05 mg / kg, about 0.03 mg / kg to about 0.07 mg / kg, about 0.03 mg / kg to about 0.09 mg / kg, about 0.03 mg / kg to about 0.1 mg / kg, about 0.03 mg / kg to about 0.12 mg / kg, about 0.03 mg / kg to about 0.14 mg / kg, about 0.03 mg / kg to about 0.16 mg / kg, about 0.03 mg / kg to about 0.18 mg / kg, about 0.03 mg / kg to about 0.2 mg / kg, about 0.05 mg / kg g / kg to about 0.07 mg / kg, about 0.05 mg / kg to about 0.09 mg / kg, about 0.05 mg / kg to about 0.1 mg / kg, about 0.05 mg / kg to about 0.12 mg / kg, about 0.05 mg / kg to about 0.14 mg / kg, about 0.05 mg / kg to about 0.16 mg / kg, about 0.05 mg / kg to about 0.18 mg / kg, about 0.05 mg / kg to about 0.2 mg / kg, about 0.07 mg / kg to about 0.09 mg / kg, about 0.07 mg / kg to about 0.1 mg / kg, about 0.07 mg / kg to about 0.1 mg / kg, about 0.07 mg / kg to about 0.1 mg / kg. to about 0.12 mg / kg, about 0.07 mg / kg to about 0.14 mg / kg, about 0.07 mg / kg to about 0.16 mg / kg, about 0.07 mg / kg to about 0.18 mg / kg, about 0.07 mg / kg to about 0.2 mg / kg, about 0.09 mg / kg to about 0.1 mg / kg, about 0.09 mg / kg to about 0.12 mg / kg, about 0.09 mg / kg to about 0.14 mg / kg, about 0.09 mg / kg to about 0.16 mg / kg, about 0.09 mg / kg to about 0.18 mg / kg, about 0.09 mg / kg to about 0.2 mg / kg.2mg / kg, about 0.1mg / kg to about 0.12mg / kg, about 0.1mg / kg to about 0.14mg / kg, about 0.1mg / kg to about 0.16mg / kg, about 0.1mg / kg to about 0.18mg / kg, about 0.1mg / kg to about 0.2mg / kg, about 0.12mg / kg to about 0.14mg / kg, about 0.12mg / kg to about 0.16mg / kg, about 0.12mg / kg to about 0.18mg / kg, In some embodiments, the starting dose of polyethylene glycol arginase is about 0.01 mg / kg, about 0.03 mg / kg, about 0.05 mg / kg, about 0.07 mg / kg, about 0.09 mg / kg, about 0.1 mg / kg, about 0.12 mg / kg, about 0.14 mg / kg, about 0.16 mg / kg, about 0.18 mg / kg, or about 0.2 mg / kg. In some embodiments, the starting dose of polyethylene glycol arginase is about 0.01 mg / kg, about 0.03 mg / kg, about 0.05 mg / kg, about 0.07 mg / kg, about 0.09 mg / kg, about 0.1 mg / kg, about 0.12 mg / kg, about 0.14 mg / kg, about 0.16 mg / kg, about 0.18 mg / kg, or about 0.2 mg / kg. In some embodiments, the starting dose of PEGylated arginase is about 0.1 mg / kg.

[0105] In some embodiments, the starting dose of human arginase 1 is about 0.01 mg / kg to about 0.2 mg / kg. In some embodiments, the starting dose of human arginase 1 is at least about 0.01 mg / kg. In some embodiments, the starting dose of human arginase 1 is up to about 0.2 mg / kg. In some embodiments, the starting dose of human arginase 1 is about 0.01 mg / kg to about 0.03 mg / kg, about 0.01 mg / kg to about 0.05 mg / kg, about 0.01 mg / kg to about 0.07 mg / kg, about 0.01 mg / kg to about 0.09 mg / kg, about 0.01 mg / kg to about 0.1 mg / kg, about 0.01 mg / kg to about 0.12 mg / kg, about 0.01 mg / kg to about 0.14 mg / kg, about 0.01 mg / kg to about 0.16 mg / kg, about 0.01 mg / kg to about 0.18 mg / kg, about 0. 01 mg / kg to about 0.2 mg / kg, about 0.03 mg / kg to about 0.05 mg / kg, about 0.03 mg / kg to about 0.07 mg / kg, about 0.03 mg / kg to about 0.09 mg / kg, about 0.03 mg / kg to about 0.1 mg / kg, about 0.03 mg / kg to about 0.12 mg / kg, about 0.03 mg / kg to about 0.14 mg / kg, about 0.03 mg / kg to about 0.16 mg / kg, about 0.03 mg / kg to about 0.18 mg / kg, about 0.03 mg / kg to about 0.2 mg / kg, about 0.05 mg / kg / kg to about 0.07mg / kg, about 0.05mg / kg to about 0.09mg / kg, about 0.05mg / kg to about 0.1mg / kg, about 0.05mg / kg to about 0.12mg / kg, about 0.05mg / kg to about 0.14mg / kg, about 0.05mg / kg to about 0.16mg / kg, about 0.05mg / kg to about 0.18mg / kg, about 0.05mg / kg to about 0.2mg / kg, about 0.07mg / kg to about 0.09mg / kg, about 0.07mg / kg to about 0.1 ...5mg / kg to about 0.12mg about 0.12 mg / kg, about 0.07 mg / kg to about 0.14 mg / kg, about 0.07 mg / kg to about 0.16 mg / kg, about 0.07 mg / kg to about 0.18 mg / kg, about 0.07 mg / kg to about 0.2 mg / kg, about 0.09 mg / kg to about 0.1 mg / kg, about 0.09 mg / kg to about 0.12 mg / kg, about 0.09 mg / kg to about 0.14 mg / kg, about 0.09 mg / kg to about 0.16 mg / kg, about 0.09 mg / kg to about 0.18 mg / kg, about 0.09 mg / kg to about 0.2 mg / kg.2mg / kg, about 0.1mg / kg to about 0.12mg / kg, about 0.1mg / kg to about 0.14mg / kg, about 0.1mg / kg to about 0.16mg / kg, about 0.1mg / kg to about 0.18mg / kg, about 0.1mg / kg to about 0.2mg / kg, about 0.12mg / kg to about 0.14mg / kg, about 0.12mg / kg to about 0.16mg / kg, about 0.12mg / kg to about 0.18mg / kg, In some embodiments, the starting dose of human arginase 1 is about 0.01 mg / kg, about 0.03 mg / kg, about 0.05 mg / kg, about 0.07 mg / kg, about 0.09 mg / kg, about 0.1 mg / kg, about 0.12 mg / kg, about 0.14 mg / kg, about 0.16 mg / kg, about 0.18 mg / kg, or about 0.2 mg / kg. In some embodiments, the starting dose of human arginase 1 is about 0.01 mg / kg, about 0.03 mg / kg, about 0.05 mg / kg, about 0.07 mg / kg, about 0.09 mg / kg, about 0.1 mg / kg, about 0.12 mg / kg, about 0.14 mg / kg, about 0.16 mg / kg, about 0.18 mg / kg, or about 0.2 mg / kg. In some embodiments, the starting dose of human arginase 1 is about 0.1 mg / kg.

[0106] In some embodiments, the subsequent dose of polyethylene glycol arginase is adjusted based on the plasma arginine level of the subject before the subsequent dose is administered. In some embodiments, if the plasma arginine level of the subject is higher than about 100 μM before the subsequent dose is administered, the subsequent dose of polyethylene glycol arginase is increased. In some embodiments, if the plasma arginine level of the subject is higher than about 115 μM before the subsequent dose is administered, the subsequent dose of polyethylene glycol arginase is increased. In some embodiments, if the plasma arginine level of the subject is higher than about 130 μM before the subsequent dose is administered, the subsequent dose of polyethylene glycol arginase is increased. In some embodiments, if the plasma arginine level of the subject is higher than about 135 μM before the subsequent dose is administered, the subsequent dose of polyethylene glycol arginase is increased. In some embodiments, if the plasma arginine level of the subject is higher than about 150 μM before the subsequent dose is administered, the subsequent dose of polyethylene glycol arginase is increased. In some embodiments, if the plasma arginine level of the subject is higher than about 165 μM before the subsequent dose is administered, the subsequent dose of polyethylene glycol arginase is increased. In some embodiments, if the plasma arginine level of the subject is higher than about 180 μM before the subsequent dose is administered, the subsequent dose of polyethylene glycol arginase is increased. In some embodiments, if the plasma arginine level of the subject is higher than about 195 μM before the subsequent dose is administered, the subsequent dose of polyethylene glycol arginase is increased. In some embodiments, if the plasma arginine level of the subject is higher than about 200 μM before the subsequent dose is administered, the subsequent dose of polyethylene glycol arginase is increased. In some embodiments, if the plasma arginine level of the subject is lower than about 50 μM before the subsequent dose is administered, the subsequent dose of polyethylene glycol arginase is reduced. In some embodiments, if the plasma arginine level of the subject is lower than about 40 μM before the subsequent dose is administered, the subsequent dose of polyethylene glycol arginase is reduced. In some embodiments, if the plasma arginine level of the subject is lower than about 30 μM before the subsequent dose is administered, the subsequent dose of polyethylene glycol arginase is reduced. In some embodiments, if the subject's plasma arginine level is below about 20 μM prior to administration of the subsequent dose, the subsequent dose of pegylated arginase is reduced.

[0107] In some embodiments, a subsequent dose of human arginase 1 is adjusted based on the subject's plasma arginine level before the subsequent dose is administered. In some embodiments, if the subject's plasma arginine level is above about 100 μM before the subsequent dose is administered, the subsequent dose of human arginase 1 is increased. In some embodiments, if the subject's plasma arginine level is above about 115 μM before the subsequent dose is administered, the subsequent dose of human arginase 1 is increased. In some embodiments, if the subject's plasma arginine level is above about 130 μM before the subsequent dose is administered, the subsequent dose of human arginase 1 is increased. In some embodiments, if the subject's plasma arginine level is above about 135 μM before the subsequent dose is administered, the subsequent dose of human arginase 1 is increased. In some embodiments, if the subject's plasma arginine level is above about 150 μM before the subsequent dose is administered, the subsequent dose of human arginase 1 is increased. In some embodiments, if the subject's plasma arginine level is above about 165 μM before the subsequent dose is administered, the subsequent dose of human arginase 1 is increased. In some embodiments, if the subject's plasma arginine level is above about 180 μM before the subsequent dose is administered, the subsequent dose of human arginase 1 is increased. In some embodiments, if the subject's plasma arginine level is above about 195 μM before the subsequent dose is administered, the subsequent dose of human arginase 1 is increased. In some embodiments, if the subject's plasma arginine level is above about 200 μM before the subsequent dose is administered, the subsequent dose of human arginase 1 is increased. In some embodiments, if the subject's plasma arginine level is below about 50 μM before the subsequent dose is administered, the subsequent dose of human arginase 1 is reduced. In some embodiments, if the subject's plasma arginine level is below about 40 μM before the subsequent dose is administered, the subsequent dose of human arginase 1 is reduced. In some embodiments, if the subject's plasma arginine level is below about 30 μM before the subsequent dose is administered, the subsequent dose of human arginase 1 is reduced. In some embodiments, if the subject's plasma arginine level is below about 20 μM before the subsequent dose is administered, the subsequent dose of human arginase 1 is reduced.

[0108] In some embodiments, each subsequent dose of PEGylated arginase is adjusted by 0.01 mg / kg to 0.1 mg / kg based on the plasma arginine level of the subject resulting from the previous dose. In some embodiments, each subsequent dose of PEGylated arginase is adjusted by at least about 0.01 mg / kg based on the plasma arginine level of the subject resulting from the previous dose. In some embodiments, each subsequent dose of PEGylated arginase is adjusted by up to about 0.2 mg / kg based on the plasma arginine level of the subject resulting from the previous dose. In some embodiments, each subsequent dose of polyethylene glycol arginase is adjusted by about 0.01 mg / kg to about 0.03 mg / kg, about 0.01 mg / kg to about 0.05 mg / kg, about 0.01 mg / kg to about 0.07 mg / kg, about 0.01 mg / kg to about 0.1 mg / kg, about 0.01 mg / kg to about 0.2 mg / kg, about 0.03 mg / kg to about 0.05 mg / kg, about 0.03 mg / kg to about 0.06 ... In some embodiments, each subsequent dose of polyethylene glycol arginase is adjusted by about 0.01 mg / kg, about 0.03 mg / kg, about 0.05 mg / kg, about 0.07 mg / kg, about 0.1 mg / kg, about 0.2 mg / kg, about 0.05 mg / kg to about 0.07 mg / kg, about 0.05 mg / kg to about 0.1 mg / kg, about 0.05 mg / kg to about 0.2 mg / kg, about 0.07 mg / kg to about 0.1 mg / kg, about 0.07 mg / kg to about 0.2 mg / kg, or about 0.1 mg / kg to about 0.2 mg / kg. In some embodiments, each subsequent dose of polyethylene glycol arginase is adjusted by about 0.01 mg / kg, about 0.03 mg / kg, about 0.05 mg / kg, about 0.07 mg / kg, about 0.1 mg / kg, or about 0.2 mg / kg based on the subject's plasma arginine level resulting from the previous dose. In some embodiments, each subsequent dose of polyethylene glycol arginase is adjusted by about 0.05 mg / kg based on the subject's plasma arginine level resulting from the previous dose.

[0109] In some embodiments, each subsequent dose of human arginase 1 is adjusted by 0.01 mg / kg to 0.1 mg / kg based on the subject's plasma arginine level resulting from the previous dose. In some embodiments, each subsequent dose of human arginase 1 is adjusted by at least about 0.01 mg / kg based on the subject's plasma arginine level resulting from the previous dose. In some embodiments, each subsequent dose of human arginase 1 is adjusted by up to about 0.2 mg / kg based on the subject's plasma arginine level resulting from the previous dose. In some embodiments, each subsequent dose of human arginase 1 is adjusted by about 0.01 mg / kg to about 0.03 mg / kg, about 0.01 mg / kg to about 0.05 mg / kg, about 0.01 mg / kg to about 0.07 mg / kg, about 0.01 mg / kg to about 0.1 mg / kg, about 0.01 mg / kg to about 0.2 mg / kg, about 0.03 mg / kg to about 0.05 mg / kg, about 0.03 mg / kg to about 0.2 mg / kg, about 0.03 mg / kg to about 0.05 mg / kg, about 0.03 mg / kg to about 0.2 mg / kg, about 0.03 mg / kg to about 0.05 mg / kg, about 0.03 mg / kg to about 0.06 ... / kg to about 0.07mg / kg, about 0.03mg / kg to about 0.1mg / kg, about 0.03mg / kg to about 0.2mg / kg, about 0.05mg / kg to about 0.07mg / kg, about 0.05mg / kg to about 0.1mg / kg, about 0.05mg / kg to about 0.2mg / kg, about 0.07mg / kg to about 0.1mg / kg, about 0.07mg / kg to about 0.2mg / kg, or about 0.1mg / kg to about 0.2mg / kg. In some embodiments, each subsequent dose of human arginase 1 is adjusted by about 0.01mg / kg, about 0.03mg / kg, about 0.05mg / kg, about 0.07mg / kg, about 0.1mg / kg, or about 0.2mg / kg based on the subject's plasma arginine level resulting from the previous dose. In some embodiments, each subsequent dose of human arginase 1 is adjusted by about 0.05 mg / kg based on the subject's plasma arginine level resulting from the previous dose.

[0110] In some embodiments, the subject does not receive pre-medication prior to the initial dose of polyethylene glycol arginase. In some embodiments, the subject receives pre-medication prior to the initial dose of polyethylene glycol arginase. In some embodiments, the subject does not receive pre-medication prior to the initial dose of human arginase 1. In some embodiments, the subject receives pre-medication prior to the initial dose of human arginase 1.

[0111] In some embodiments, before the initial dose of polyethylene glycol arginase, the plasma arginine level of the subject is at least 200 μM. In some embodiments, before the initial dose of polyethylene glycol arginase, the plasma arginine level of the subject is at least 210 μM. In some embodiments, before the initial dose of polyethylene glycol arginase, the plasma arginine level of the subject is at least 230 μM. In some embodiments, before the initial dose of polyethylene glycol arginase, the plasma arginine level of the subject is at least 250 μM. In some embodiments, before the initial dose of polyethylene glycol arginase, the plasma arginine level of the subject is at least 270 μM. In some embodiments, before the initial dose of polyethylene glycol arginase, the plasma arginine level of the subject is at least 290 μM. In some embodiments, before the initial dose of polyethylene glycol arginase, the plasma arginine level of the subject is at least 310 μM.

[0112] In some embodiments, prior to the initial dose of human arginase 1, the subject's plasma arginine level is at least 200 μM. In some embodiments, prior to the initial dose of human arginase 1, the subject's plasma arginine level is at least 210 μM. In some embodiments, prior to the initial dose of human arginase 1, the subject's plasma arginine level is at least 230 μM. In some embodiments, prior to the initial dose of human arginase 1, the subject's plasma arginine level is at least 250 μM. In some embodiments, prior to the initial dose of human arginase 1, the subject's plasma arginine level is at least 270 μM. In some embodiments, prior to the initial dose of human arginase 1, the subject's plasma arginine level is at least 290 μM. In some embodiments, prior to the initial dose of human arginase 1, the subject's plasma arginine level is at least 310 μM.

[0113] In some embodiments, the subject is an adult. In some embodiments, the subject is less than 18 years old. In some embodiments, the subject is at least 2 years old.

[0114] In some embodiments, the subject is classified as GMFCS level II. In some embodiments, the subject is classified as GMFCS level III. In some embodiments, the subject is classified as GMFCS level IV. In some embodiments, the subject is classified as GMFCS level V.

[0115] The plasma level of arginine and / or guanidinyl compound can be reduced to a normal range or normal level, which can mean that at some time point after the initial dose and / or repeated doses of arginase are administered, the plasma level of arginine and / or one or more guanidinyl compounds is a value within the range of 40 μM to 115 μM (for arginine), as described in Luneburg, N. et al., (2011). For example, during treatment with arginase as disclosed herein, the patient's plasma level for arginine and / or one or more guanidinyl compounds may fluctuate within and outside the normal range. The patient is considered to have a measured plasma level of arginine and / or guanidinyl compounds that is reduced to a normal level or within the normal range. As another example, after receiving one or more doses of arginase as disclosed herein, the patient may have an average amount of arginine and / or one or more guanidinyl compound plasma levels within the normal range (e.g., 40 μM to 115 μM for arginine, as described in Luneburg, N. et al., (2011)). Thus, after receiving an initial dose and / or repeated doses of arginase, the disclosed compositions and methods herein can reduce the plasma levels of arginine and / or guanidinyl compounds in a subject to normal levels or to a normal range at least once. In some patients, it is observed that the plasma levels of arginine and / or guanidinyl compounds can be maintained at normal levels or within a normal range on average according to the methods disclosed herein.

[0116] In some embodiments, the subject's plasma arginine level after treatment is about 40 μM to about 115 μM. In some embodiments, after 2 doses of polyethylene glycol arginase, the subject's plasma arginine level is about 40 μM to about 115 μM. In some embodiments, after 4 doses of polyethylene glycol arginase, the subject's plasma arginine level is about 40 μM to about 115 μM. In some embodiments, after 6 doses of polyethylene glycol arginase, the subject's plasma arginine level is about 40 μM to about 115 μM. In some embodiments, after 8 doses of polyethylene glycol arginase, the subject's plasma arginine level is about 40 μM to about 115 μM. In some embodiments, after 10 doses of polyethylene glycol arginase, the subject's plasma arginine level is about 40 μM to about 115 μM. In some embodiments, after 12 doses of polyethylene glycol arginase, the subject's plasma arginine level is about 40 μM to about 115 μM. In some embodiments, after 14 doses of polyethylene glycol arginase, the subject's plasma arginine level is about 40 μM to about 115 μM. In some embodiments, after 16 doses of polyethylene glycol arginase, the subject's plasma arginine level is about 40 μM to about 115 μM. In some embodiments, after 18 doses of polyethylene glycol arginase, the subject's plasma arginine level is about 40 μM to about 115 μM. In some embodiments, after 20 doses of polyethylene glycol arginase, the subject's plasma arginine level is about 40 μM to about 115 μM. In some embodiments, after 22 doses of polyethylene glycol arginase, the subject's plasma arginine level is about 40 μM to about 115 μM. In some embodiments, after 24 doses of polyethylene glycol arginase, the subject's plasma arginine level is about 40 μM to about 115 μM.

[0117] In some embodiments, the subject's plasma arginine level after treatment is about 40 μM to about 115 μM. In some embodiments, after 2 doses of human arginase 1, the subject's plasma arginine level is about 40 μM to about 115 μM. In some embodiments, after 4 doses of human arginase 1, the subject's plasma arginine level is about 40 μM to about 115 μM. In some embodiments, after 6 doses of human arginase 1, the subject's plasma arginine level is about 40 μM to about 115 μM. In some embodiments, after 8 doses of human arginase 1, the subject's plasma arginine level is about 40 μM to about 115 μM. In some embodiments, after 10 doses of human arginase 1, the subject's plasma arginine level is about 40 μM to about 115 μM. In some embodiments, after 12 doses of human arginase 1, the subject's plasma arginine level is about 40 μM to about 115 μM. In some embodiments, after 14 doses of human arginase 1, the subject's plasma arginine level is about 40 μM to about 115 μM. In some embodiments, after 16 doses of human arginase 1, the subject's plasma arginine level is about 40 μM to about 115 μM. In some embodiments, after 18 doses of human arginase 1, the subject's plasma arginine level is about 40 μM to about 115 μM. In some embodiments, after 20 doses of human arginase 1, the subject's plasma arginine level is about 40 μM to about 115 μM. In some embodiments, after 22 doses of human arginase 1, the subject's plasma arginine level is about 40 μM to about 115 μM. In some embodiments, after 24 doses of human arginase 1, the subject's plasma arginine level is about 40 μM to about 115 μM.

[0118] In some embodiments, after one or more doses of polyethylene glycol arginase, the intake of natural protein of the object is improved. After one or more doses of polyethylene glycol arginase, the intake of the essential amino acid mixture of the object is reduced. In some embodiments, after one or more doses of polyethylene glycol arginase, the relative intake of the essential amino acid mixture of the object is reduced compared with natural protein. In some embodiments, after one or more doses of polyethylene glycol arginase, the main source of protein intake of the object is changed from an essential amino acid mixture without arginine (or low arginine) to the addition of any dietary product containing protein. In some embodiments, after one or more doses of polyethylene glycol arginase, the main source of protein intake of the object is changed from an essential amino acid mixture without arginine (or low arginine) to natural protein.

[0119] In some embodiments, after one or more doses of polyethylene glycol arginase, the total protein intake of the subject is greater than 30g / day. In some embodiments, after one or more doses of polyethylene glycol arginase, the total protein intake of the subject is greater than 35g / day. In some embodiments, after one or more doses of polyethylene glycol arginase, the total protein intake of the subject is greater than 40g / day. In some embodiments, after one or more doses of polyethylene glycol arginase, the total protein intake of the subject is greater than 45g / day. In some embodiments, after one or more doses of polyethylene glycol arginase, the total protein intake of the subject is greater than 50g / day. In some embodiments, after one or more doses of polyethylene glycol arginase, the total protein intake of the subject is increased by at least 5%. In some embodiments, after one or more doses of polyethylene glycol arginase, the total protein intake of the subject is increased by at least 10%. In some embodiments, after one or more doses of polyethylene glycol arginase, the total protein intake of the subject is increased by at least 15%. In some embodiments, after one or more doses of polyethylene glycol arginase, the total protein intake of the subject is increased by at least 20%. In some embodiments, after one or more doses of polyethylene glycol arginase, the total protein intake of the object is increased by at least 25%. In some embodiments, after one or more doses of polyethylene glycol arginase, the natural protein intake of the object is increased to at least 45% of the total protein intake. In some embodiments, after one or more doses of polyethylene glycol arginase, the natural protein intake of the object is increased to at least 50% of the total protein intake. In some embodiments, after one or more doses of polyethylene glycol arginase, the natural protein intake of the object is increased to at least 55% of the total protein intake. In some embodiments, after one or more doses of polyethylene glycol arginase, the natural protein intake of the object is increased to at least 60% of the total protein intake. In some embodiments, after one or more doses of polyethylene glycol arginase, the natural protein intake of the object is increased to at least 65% of the total protein intake. In some embodiments, after one or more doses of polyethylene glycol arginase, the natural protein intake of the object is increased to at least 70% of the total protein intake.

[0120] In some embodiments, after one or more doses of human arginase 1, the subject's intake of natural protein increases. After one or more doses of human arginase 1, the subject's intake of essential amino acid mixtures decreases. In some embodiments, after one or more doses of human arginase 1, the relative intake of the subject's essential amino acid mixture compared to natural protein decreases. In some embodiments, after one or more doses of human arginase 1, the subject's main source of protein intake is changed from an essential amino acid mixture that does not contain arginine (or low in arginine) to the addition of any dietary product containing protein. In some embodiments, after one or more doses of human arginase 1, the subject's main source of protein intake is changed from an essential amino acid mixture that does not contain arginine (or low in arginine) to natural protein.

[0121] In some embodiments, after one or more doses of human arginase 1, the total protein intake of the subject is greater than 30 g / day. In some embodiments, after one or more doses of human arginase 1, the total protein intake of the subject is greater than 35 g / day. In some embodiments, after one or more doses of human arginase 1, the total protein intake of the subject is greater than 40 g / day. In some embodiments, after one or more doses of human arginase 1, the total protein intake of the subject is greater than 45 g / day. In some embodiments, after one or more doses of human arginase 1, the total protein intake of the subject is greater than 50 g / day. In some embodiments, after one or more doses of human arginase 1, the total protein intake of the subject is increased by at least 5%. In some embodiments, after one or more doses of human arginase 1, the total protein intake of the subject is increased by at least 10%. In some embodiments, after one or more doses of human arginase 1, the total protein intake of the subject is increased by at least 15%. In some embodiments, after one or more doses of human arginase 1, the total protein intake of the subject is increased by at least 20%. In some embodiments, after one or more doses of human arginase 1, the total protein intake of the subject is increased by at least 25%. In some embodiments, after one or more doses of human arginase 1, the natural protein intake of the subject is increased to at least 45% of the total protein intake. In some embodiments, after one or more doses of human arginase 1, the natural protein intake of the subject is increased to at least 50% of the total protein intake. In some embodiments, after one or more doses of human arginase 1, the natural protein intake of the subject is increased to at least 55% of the total protein intake. In some embodiments, after one or more doses of human arginase 1, the natural protein intake of the subject is increased to at least 60% of the total protein intake. In some embodiments, after one or more doses of human arginase 1, the natural protein intake of the subject is increased to at least 65% of the total protein intake. In some embodiments, after one or more doses of human arginase 1, the natural protein intake of the subject is increased to at least 70% of the total protein intake.

[0122] In some embodiments, PEGylated arginase or human arginase 1 or a composition comprising the same is used for dietary liberalization in patients with arginase 1 deficiency. In some embodiments, PEGylated arginase or human arginase 1 or a composition comprising the same is used for treating protein aversion in patients with urea cycle disorders. In some embodiments, PEGylated arginase or human arginase 1 or a composition comprising the same is used for treating diseases or conditions associated with ARG1-D. Related diseases or conditions may be, but are not limited to, GAMT, S-adenosylhomocysteine ​​hydrolase deficiency, CNS damage, and renal damage.

[0123] In some embodiments, the subject does not have an episode of hyperammonemia. In some embodiments, the subject does not have an active infection requiring anti-infective treatment. In some embodiments, the subject does not have an active infection of human immunodeficiency virus, hepatitis B, or hepatitis C. In some embodiments, the subject does not have an extreme active defect, defined as being unable to be assessed on the Gillette Functional Assessment Questionnaire (GFAQ) or scoring 1 on the GFAQ. In some embodiments, the subject does not have a history of hypersensitivity to polyethylene glycol (PEG). In some embodiments, the subject has not been treated with botulinum toxin. In some embodiments, the subject does not have a liver or hematopoietic transplant.

[0124] In some embodiments, after one or more doses of polyethylene glycol arginase, the subject's mobility is improved. In some embodiments, after one or more doses of polyethylene glycol arginase, the accumulation of arginine metabolites is reduced. In some embodiments, after one or more doses of human arginase 1, the subject's mobility is improved. In some embodiments, after one or more doses of human arginase 1, the accumulation of arginine metabolites is reduced. In some embodiments, the accumulation of guanidine compounds in the blood and cerebrospinal fluid of the subject can be reduced. In some embodiments, the guanidine compound is selected from α-N-acetylarginine, α-keto-d-guanidine valeric acid, arginine and guanidinoacetic acid.

[0125] In some embodiments, the polyethylene glycol arginase is administered at a concentration of about 1 mg / mL to about 8 mg / mL. In some embodiments, the polyethylene glycol arginase is administered at a concentration of at least about 1 mg / mL. In some embodiments, the polyethylene glycol arginase is administered at a concentration of up to about 8 mg / mL. In some embodiments, the polyethylene glycol arginase is administered at a concentration of about 1 mg / mL to about 2 mg / mL, about 1 mg / mL to about 3 mg / mL, about 1 mg / mL to about 4 mg / mL, about 1 mg / mL to about 5 mg / mL, about 1 mg / mL to about 6 mg / mL, about 1 mg / mL to about 7 mg / mL, about 1 mg / mL to about 8 mg / mL, about 2 mg / mL to about 3 mg / mL, about 2 mg / mL to about 4 mg / mL, about 2 mg / mL to about 5 mg / mL, about 2 mg / mL to about 6 mg / mL, about 2 mg / mL to about 7 mg / mL, about 2 mg / mL to about 8 mg / mL, about 3 mg / mL to about In some embodiments, the polyethylene glycol arginase is administered at a concentration of about 1 mg / mL, about 2 mg / mL, about 3 mg / mL, about 4 mg / mL to about 5 mg / mL, about 3 mg / mL to about 6 mg / mL, about 3 mg / mL to about 7 mg / mL, about 3 mg / mL to about 8 mg / mL, about 4 mg / mL to about 5 mg / mL, about 4 mg / mL to about 6 mg / mL, about 4 mg / mL to about 7 mg / mL, about 4 mg / mL to about 8 mg / mL, about 5 mg / mL to about 6 mg / mL, about 5 mg / mL to about 7 mg / mL, about 5 mg / mL to about 8 mg / mL, about 6 mg / mL to about 7 mg / mL, about 6 mg / mL to about 8 mg / mL, or about 7 mg / mL to about 8 mg / mL. In some embodiments, the polyethylene glycol arginase is administered at a concentration of about 1 mg / mL, about 2 mg / mL, about 3 mg / mL, about 4 mg / mL, about 5 mg / mL, about 6 mg / mL, about 7 mg / mL, or about 8 mg / mL. In some embodiments, PEGylated arginase is administered at a concentration of about 5 mg / mL.

[0126] In some embodiments, human arginase 1 is administered at a concentration of about 1 mg / mL to about 8 mg / mL. In some embodiments, human arginase 1 is administered at a concentration of at least about 1 mg / mL. In some embodiments, human arginase 1 is administered at a concentration of up to about 8 mg / mL. In some embodiments, human arginase 1 is administered at a concentration of about 1 mg / mL to about 2 mg / mL, about 1 mg / mL to about 3 mg / mL, about 1 mg / mL to about 4 mg / mL, about 1 mg / mL to about 5 mg / mL, about 1 mg / mL to about 6 mg / mL, about 1 mg / mL to about 7 mg / mL, about 1 mg / mL to about 8 mg / mL, about 2 mg / mL to about 3 mg / mL, about 2 mg / mL to about 4 mg / mL, about 2 mg / mL to about 5 mg / mL, about 2 mg / mL to about 6 mg / mL, about 2 mg / mL to about 7 mg / mL, about 2 mg / mL to about 8 mg / mL, about 3 mg / mL to about 4 In some embodiments, human arginase 1 is administered at a concentration of about 1 mg / mL, about 2 mg / mL, about 3 mg / mL, about 4 mg / mL, about 5 mg / mL, about 6 mg / mL, about 7 mg / mL, about 8 mg / mL, about 4 mg / mL to about 5 mg / mL, about 4 mg / mL to about 6 mg / mL, about 4 mg / mL to about 7 mg / mL, about 4 mg / mL to about 8 mg / mL, about 5 mg / mL to about 6 mg / mL, about 5 mg / mL to about 7 mg / mL, about 5 mg / mL to about 8 mg / mL, about 6 mg / mL to about 7 mg / mL, about 6 mg / mL to about 8 mg / mL, or about 7 mg / mL to about 8 mg / mL. In some embodiments, human arginase 1 is administered at a concentration of about 1 mg / mL, about 2 mg / mL, about 3 mg / mL, about 4 mg / mL, about 5 mg / mL, about 6 mg / mL, about 7 mg / mL, or about 8 mg / mL. In some embodiments, human arginase 1 is administered at a concentration of about 5 mg / mL. In some embodiments, the weight of human arginase 1 is based on the combined weight of human arginase 1 and polyethylene glycol conjugated to human arginase 1. Implementation

[0127] In one group of embodiments (Embodiment Group A), there is provided: A1. A method of treating arginase 1 (ARG1) deficiency (ARG1-D) in a subject, the method comprising administering polyethylene glycol arginase to the subject, 1) wherein the polyethylene glycol arginase is a pegylated human arginase 1 comprising a cobalt metal cofactor, and 2) wherein the polyethylene glycol arginase is administered at a dose of about 0.05 mg / kg to about 0.2 mg / kg per week. A2. The method of embodiment A1, wherein the subject's plasma arginine level after 12 doses of the polyethylene glycol arginase is from about 40 μM to about 115 μM. A3. The method of embodiment A1 or A2, wherein the initial dose of polyethylene glycol arginase is about 0.1 mg / kg. A4. The method of any one of embodiments A1 to A3, wherein a subsequent dose of the polyethylene glycol arginase is adjusted based on the subject's plasma arginine level prior to administration of the subsequent dose. A5. The method of embodiment A4, wherein the subsequent dose of the polyethylene glycol arginase is increased if the subject's plasma arginine level is above about 115 μM, optionally above about 150 μM, prior to administration of the subsequent dose. A6. The method of embodiment A4, wherein the subsequent dose of the polyethylene glycol arginase is reduced if the subject's plasma arginine level is below about 50 μM, optionally below about 40 μM, prior to administration of the subsequent dose. A7. The method of any one of embodiments A4 to A6, wherein subsequent doses of the polyethylene glycol arginase are adjusted by 0.01 mg / kg to 0.1 mg / kg each time based on the subject's plasma arginine level resulting from the previous dose. A8. The method of embodiment A7, wherein subsequent doses of the polyethylene glycol arginase are adjusted by 0.05 mg / kg each time based on the subject's plasma arginine level resulting from the previous dose. A9. The method of any one of embodiments A1 to A8, wherein the subject did not receive premedication prior to the initial dose of polyethylene glycol arginase. A10. The method of any one of embodiments A1 to A9, wherein prior to the initial dose of polyethylene glycol arginase, the subject's plasma arginine level is at least 250 μM. A11. The method of any one of embodiments A1 to A10, wherein the subject is at least 2 years old. A12. The method of any one of embodiments A1 to A11, wherein the subject is classified as GMFCS grade II, III, IV, or V. A13. The method of any one of embodiments A1 to A12, wherein an ammonia scavenger is co-administered to the subject. A14. The method of any one of embodiments A1 to A13, wherein following one or more doses of said polyethylene glycol arginase, said subject's intake of native protein is increased. A15. The method of any one of embodiments A1 to A14, wherein following one or more doses of said polyethylene glycol arginase, said subject's relative intake of a mixture of essential amino acids is reduced compared to native protein. A16. The method of any one of embodiments A1 to A15, wherein after one or more doses of the polyethylene glycol arginase, the subject's primary source of protein intake is changed from an arginine-free (or low-arginine) essential amino acid mixture to the addition of any protein-containing dietary product. A17. The method of any one of embodiments A1 to A15, wherein after one or more doses of the polyethylene glycol arginase, the subject's primary source of protein intake is changed from an essential amino acid mixture without arginine (or low in arginine) to natural protein. A18. The method of any one of embodiments A1 to A17, wherein after one or more doses of said polyethylene glycol arginase, said subject's total protein intake is greater than 40 g / day. A19. The method of any one of embodiments A1 to A18, wherein following one or more doses of said polyethylene glycol arginase, said subject's total protein intake is increased by at least 15%. A20. The method of any one of embodiments A1 to A19, wherein following one or more doses of said polyethylene glycol arginase, said subject's natural protein intake is increased to at least 50% of total protein intake. A21. The method of embodiment A20, wherein following one or more doses of the polyethylene glycol arginase, the subject's natural protein intake is increased to at least 65% of total protein intake. A22. The method of any one of embodiments A1 to A21, wherein the subject's mobility is improved after one or more doses of the polyethylene glycol arginase. A23. The method of any one of embodiments A1 to A22, wherein accumulation of arginine metabolites is reduced following one or more doses of said polyethylene glycol arginase. A24. The method of any one of embodiments A1 to A23, wherein the subject has reduced accumulation of guanidine compounds in the blood and cerebrospinal fluid. A25. The method of embodiment A24, wherein the guanidine compound is selected from α-N-acetylarginine, α-keto-d-guanidine valeric acid, arginine and guanidine acetic acid. A26. The method of any one of embodiments A1 to A25, wherein the polyethylene glycol arginase is administered at a concentration of 5 mg / mL. A27. The method of any one of embodiments A1 to A26, wherein the polyethylene glycol arginase is administered intravenously. A28. The method of any one of embodiments A1 to A26, wherein the polyethylene glycol arginase is administered subcutaneously. A29. The method of any one of embodiments A1 to A28, wherein the polyethylene glycol arginase is stored at a concentration of about 5 mg / mL. A30. The method of any one of embodiments A1 to A29, wherein the polyethylene glycol arginase is formulated in a buffer comprising 50 mM sodium chloride, 5 mM potassium phosphate and 1.5% glycerol (w / v) at a pH of 7.4. A31. The method of any one of embodiments A1 to A30, wherein the polyethylene glycol arginase is a liquid. A32. The method of any one of embodiments A1 to A30, wherein the polyethylene glycol arginase is lyophilized. A33. The method of any one of embodiments A1 to A32, wherein the polyethylene glycol arginase is in a vial or ampoule. A34. The method of embodiment A33, wherein the vial or ampoule contains about 2 mg or about 5 mg of the polyethylene glycol arginase.

[0128] In another group of embodiments (Embodiment Group B), there is provided: B1. A method of treating arginase 1 (ARG1) deficiency (ARG1-D) in a subject, the method comprising administering to the subject human arginase 1, 1) wherein the human arginase 1 is PEGylated human arginase 1 containing a cobalt metal cofactor, and 2) wherein the human arginase 1 is administered at a dose of about 0.05 mg / kg to about 0.2 mg / kg per week. B2. The method of embodiment B1, wherein the subject's plasma arginine level after 12 doses of human arginase 1 is from about 40 μM to about 115 μM. B3. The method of embodiment B1 or B2, wherein the initial dose of human arginase 1 is about 0.1 mg / kg. B4. The method of any one of embodiments B1 to B3, wherein a subsequent dose of human arginase 1 is adjusted based on the subject's plasma arginine level prior to administration of the subsequent dose. B5. The method of embodiment B4, wherein the subsequent dose of human arginase 1 is increased if the subject's plasma arginine level is above about 115 μM, optionally above about 150 μM, prior to administration of the subsequent dose. B6. The method of embodiment B4, wherein the subsequent dose of human arginase 1 is reduced if the subject's plasma arginine level is below about 50 μM, optionally below about 40 μM, prior to administration of the subsequent dose. B7. The method of any one of embodiments B4 to B6, wherein subsequent doses of human arginase 1 are adjusted by 0.01 mg / kg to 0.1 mg / kg each time based on the subject's plasma arginine level resulting from the previous dose. B8. The method of embodiment B7, wherein each subsequent dose of human arginase 1 is adjusted by 0.05 mg / kg based on the subject's plasma arginine level resulting from the previous dose. B9. The method of any one of embodiments B1 to B8, wherein the subject did not receive premedication prior to the initial dose of human arginase 1. B10. The method of any one of embodiments B1 to B9, wherein prior to the initial dose of human arginase 1, the subject's plasma arginine level is at least 250 μM. B11. The method of any one of embodiments B1 to B10, wherein the subject is at least 2 years old. B12. The method of any one of embodiments B1 to B11, wherein the subject is classified as GMFCS grade II, III, IV, or V. B13. The method of any one of embodiments B1 to B12, wherein an ammonia scavenger is co-administered to the subject. B14. The method of any one of embodiments B1 to B13, wherein following one or more doses of human arginase 1, the subject's intake of native protein increases. B15. The method of any one of embodiments B1 to B14, wherein after one or more doses of human arginase 1, the subject's relative intake of an essential amino acid mixture compared to natural protein is reduced. B16. The method of any one of embodiments B1 to B15, wherein after one or more doses of human arginase 1, the subject's primary source of protein intake is changed from an arginine-free (or low-arginine) essential amino acid mixture to the addition of any protein-containing dietary product. B17. The method of any one of embodiments B1 to B15, wherein after one or more doses of human arginase 1, the subject's primary source of protein intake is changed from an essential amino acid mixture without arginine (or low in arginine) to natural protein. B18. The method of any one of embodiments B1 to B17, wherein after one or more doses of human arginase 1, the subject's total protein intake is greater than 40 g / day. B19. The method of any one of embodiments B1 to B18, wherein following one or more doses of human arginase 1, the subject's total protein intake is increased by at least 15%. B20. The method of any one of embodiments B1 to B19, wherein following one or more doses of human arginase 1, the subject's natural protein intake is increased to at least 50% of total protein intake. B21. The method of embodiment B20, wherein after one or more doses of human arginase 1, the subject's natural protein intake is increased to at least 65% of total protein intake. B22. The method of any one of embodiments B1 to B21, wherein the subject's mobility is improved after one or more doses of human arginase 1. B23. The method of any one of embodiments B1 to B22, wherein after one or more doses of human arginase 1, accumulation of arginine metabolites is reduced. B24. The method of any one of embodiments B1 to B23, wherein the accumulation of guanidine compounds in the blood and cerebrospinal fluid of the subject is reduced. B25. The method of embodiment B24, wherein the guanidine compound is selected from α-N-acetylarginine, α-keto-d-guanidine valeric acid, arginine and guanidine acetic acid. B26. The method of any one of embodiments B1 to B25, wherein the human arginase 1 is administered at a concentration of 5 mg / mL. B27. The method of any one of embodiments B1 to B26, wherein the human arginase 1 is administered intravenously. B28. The method of any one of embodiments B1 to B26, wherein the human arginase 1 is administered subcutaneously. B29. The method of any one of embodiments B1 to B28, wherein the human arginase 1 is stored at a concentration of about 5 mg / mL. B30. The method of any one of embodiments B1 to B29, wherein the human arginase 1 is formulated in a buffer comprising 50 mM sodium chloride, 5 mM potassium phosphate and 1.5% glycerol (w / v) at a pH of 7.4. B31. The method of any one of embodiments B1 to B30, wherein the human arginase 1 is liquid. B32. The method of any one of embodiments B1 to B30, wherein the human arginase 1 is lyophilized. B33. The method of any one of embodiments B1 to B32, wherein the human arginase 1 is in a vial or an ampoule. B34. The method of embodiment B33, wherein the vial or ampoule contains about 2 mg or about 5 mg of the human arginase 1.

[0129] In yet another group of embodiments (Embodiment Group C), there is provided: C1. A method for dietary liberalization of a patient with arginase 1 deficiency, comprising subcutaneous administration of cobalt-substituted recombinant arginase 1 once a week and increasing the intake of natural protein. C2. The method of embodiment C1, wherein the patient reduces the relative intake of essential amino acids compared to natural proteins. C3. The method of embodiment C1, wherein 0.05 to 0.2 mg / kg of cobalt-substituted recombinant arginase 1 is administered to the patient. C4. The method of any one of embodiments C1 to C3, wherein the liberalization is a change from an arginine-free (or low-arginine) essential amino acid mixture as the main source of protein intake to the addition of any dietary product containing protein. C5. The method of any one of embodiments C1 to C3, wherein the liberalization is a change from an arginine-free (or low-arginine) essential amino acid mixture as the main source of protein intake to natural proteins as the main intake of protein. C6. The method of any one of embodiments C1 to C5, wherein the patient is an adult and the protein intake is greater than 40 g / day. C7. The method of any one of embodiments C1 to C6, wherein an ammonia scavenger is co-administered to the patient. C8. The method of any one of embodiments C1 to C7, wherein the patient's total protein intake is increased by more than 15%. C9. The method of any one of embodiments C1 to C8, wherein the patient's natural protein intake exceeds 50% of total protein intake. C10. A method for dietary modification, wherein cobalt-substituted recombinant arginase 1 is administered once a week to a patient with ARG1 D and natural protein intake is increased to at least 65% of total protein intake. C11. The method of embodiment C10, wherein 0.05 to 0.2 mg / kg of cobalt-substituted recombinant arginase 1 is administered to the patient. C12. A method for treating protein aversion in a patient suffering from a urea cycle disorder comprising administering a cobalt-substituted recombinant arginase 1 as described herein. C13. A method of treating arginase 1 deficiency, wherein a subgroup of patients classified as GMFCS class II receive cobalt-substituted recombinant arginase 1 as described herein. C14. A method for preventing accumulation of arginine metabolites comprising administering a cobalt-substituted recombinant arginase 1 as described herein. C15. A method of reducing accumulation of guanidinyl compounds in the blood and cerebrospinal fluid of a patient in need thereof, comprising administering a cobalt-substituted recombinant arginase 1 as described herein. C16. The method of embodiment C15, wherein the guanidine compound is selected from α-N-acetylarginine, α-keto-d-guanidine valeric acid, arginine and guanidine acetic acid. C17. The method of embodiment C15 or C16, wherein the patient with accumulated guanidinyl compound has a disease selected from the group consisting of GAMT, arginase 1 deficiency, S-adenosylhomocysteine ​​hydrolase deficiency, CNS damage and renal damage. Example

[0130] Below is the description of the various methods and materials used in this study. They are proposed to provide a complete disclosure and description of how to make and use the present invention to those of ordinary skill in the art, and are not intended to limit the scope of the present inventor's invention, nor are they intended to represent that the following experiments have been performed and are all experiments that can be performed. It should be understood that the exemplary descriptions written in the present tense are not necessarily performed, but these descriptions can be performed to generate data related to the teachings of the present invention, etc. Efforts have been made to ensure the accuracy of the numbers used (e.g., amounts, percentages, etc.), but some experimental errors and deviations should be considered. Example 1: Efficacy and safety of PEGylated arginase in patients with arginase 1 deficiency 1.1 Overview Pegylated arginase as a treatment

[0131] Polyethylene glycol arginase (Co-ARG1-PEG, AEB1102) is a cobalt-substituted PEGylated human recombinant arginase 1 enzyme with enhanced stability and catalytic activity and prolonged half-life.

[0132] Pegylated arginase is being studied as an enzyme therapy for ARG1-D. The biological activity of pegylated arginase has been demonstrated based on nonclinical studies in patients with ARG1-D and clinical data from Study CAEB1102-101A (Study 101A) and Study CAEB1102-102A (Study 102A). The current study (CAEB1102-300A [Study 300A]) attempted to determine the clinical utility of pegylated arginase for the treatment of patients with ARG1-D.

[0133] Given the properties of pegylated arginase and the relatively low doses required to reduce arginine levels to within the normal range, both intravenous (IV) and subcutaneous (SC) administration were examined in Study 101A, Study 102A, and Study 300A. A brief summary of the results from Study 101A and Study 102A is shown below.

[0134] Study 101A is a Phase 1 / 2, open-label study designed to evaluate safety, tolerability, pharmacokinetics, pharmacodynamics (PD), and proof of concept in patients with ARG1-D, and found that IV peg-arginase was well tolerated and had an acceptable and manageable safety profile. At the end of the study, no subject discontinued treatment due to adverse events (AEs) or with detectable anti-drug antibodies (ADA). Notably, in Part 2 of the study, after 8 weeks of dosing, peg-arginase rapidly and persistently reduced plasma arginine levels to 200 μM below current medical guideline levels, and in some subjects, within the normal range (40 to 115 μM). Improvements in plasma arginine control following peg-arginase treatment were accompanied by evidence of early clinical benefit in the study, with 9 of 16 subjects showing clinically meaningful improvements in at least 1 clinical outcome. These results suggest that peg-arginase can both improve important disease-related abnormalities and potentially alter disease progression. Additionally, in clinical as well as in vitro and in vivo settings, PEGylated arginase exhibited similar dose proportionality under IV and SC administration.

[0135] Study 102A is a LTE study of Study 101A, a Phase 2, open-label study designed to evaluate the long-term safety, tolerability, and efficacy of IV and SC peg-arginase in children and adult subjects with ARG1-D. In Study 102A, all 14 subjects who completed Study 101A continued to participate and were treated with peg-arginase. Interim results from the ongoing Study 102A (in which subjects received approximately 110 to 183 weeks of peg-arginase, administered weekly IV or SC at a dose of 0.05 to 0.2 mg / kg) showed that plasma arginine levels were continuously and persistently reduced to within the normal range, and plasma guanidine compound levels were associated with a decrease. Clinically meaningful improvements in activity assessed by neuromotor function were maintained, and importantly, the duration of long-term follow-up in this study exceeded the timescale over which clinical disease progression would typically be observed under current standard care. Principles of Phase 3 Study 300A

[0136] As described above, it has been shown that pegylated arginase produces a significant, rapid, and sustained reduction in plasma arginine levels in patients with ARG1-D, allowing for significant improvements in arginine control relative to that achievable with a personalized disease management (IDM) approach. Improved control of plasma arginine levels was accompanied by clinical improvements in one or more instruments of neuromotor function, consistent with the hypothesis that improved plasma arginine control has the potential to slow disease progression in affected patients. A Phase 3 study (Study 300A) was designed to evaluate the safety and efficacy of pegylated arginase in reducing arginine levels and improving clinical outcomes compared to placebo. The goal of the current study (Study 300A) was to confirm these preliminary findings in a blinded, placebo-controlled design and to further gain an understanding of the effects of pegylated arginase on a wider range of disease manifestations. The results of the interim analysis as of the data cutoff date are presented in this report. Purpose

[0137] The Phase 3 PEACE study was designed to evaluate the safety and efficacy of peg-arginase in its ability to achieve arginine control and to assess the relationship between arginine reduction and improvement in neuromotor function / mobility. Study Design

[0138] The study consisted of a 24-week randomized, placebo-controlled, double-blind treatment period, followed by an open-label LTE period of up to 150 weeks, in which all subjects received active peg-arginase. During the first 24 weeks of the double-blind period, peg-arginase or placebo was given weekly IV infusions at a 2:1 ratio, plus IDM. After completing the 24-week DB, subjects transitioned to the LTE period, and the investigators remained blinded to randomized treatment for the first 8 weeks, however, all subjects received peg-arginase during the LTE period. Subjects transitioned from IV administration to weekly SC dosing at any time during the 8 weeks of LTE to enable easy and convenient administration during the remainder of the LTE. Relevance of key active outcomes

[0139] Although not formally studied in patients with ARG1-D prior to initiation of the clinical development program for pegarginase, mobility is important for independence and declines clinically over time with disease progression in ARG1-D. Evidence-based outcome measures are necessary to monitor changes in patient performance, which can be used to support the need for ongoing home health care, or indicate the need for additional support services or devices required for ambulation. Given the lack of data on the utility of neuromotor assessments in assessing disease burden and treatment effects in ARG1-D, a number of assessments were included to further investigate the effects of pegarginase on neuromotor outcomes, including activity in this ultra-rare disease. Patients with ARG1-D may have varying degrees of impairment, and supplemental assessments (e.g., 2MWT and GMFM-E) are believed to be helpful as key secondary endpoints in assessing a broader population in the context of clinical studies.

[0140] The 2MWT and GMFM-E were used in previous clinical studies of ARG1-D. Based on insights from the phase 1 / 2 and open-label extension studies of pegarginase, two key mobility outcomes, the 2MWT and GMFM-E, were identified and selected to assess the clinical benefit of pegarginase in addition to arginine lowering in the phase 3 study. The 2MWT is a relevant tool for measuring mobility performance outcomes and is a shorter modification of the 6-minute version (e.g., NIH Toolbox motor domain-2MWT [Bohannon 2014]). The 2MWT has been validated in a wide range of age groups, including children (Bohannon 2018) and adults (Bohannon 2014). In addition, both the 6-minute walk test and the shorter modification (2MWT) have been used successfully in children (Geiger 2007, Maher 2008) and adults with cerebral palsy (Andersson 2006, Ammann-Reiffer 2018), which is also characterized by spasticity. The 2MWT as a tool is limited to subjects who have the ability to walk and the cognitive ability and attention to understand and follow instructions. The secondary outcome, the GMFM-E, has been used in similar populations, such as cerebral palsy, to assess functional activities affected by ARG1-D. This measure assesses the ability to walk, run, and climb stairs and may be particularly relevant to more severely affected subjects. This tool is limited to measuring improvements in subjects with less disability as they are generally at or near the top score. This suggests that while the sensitivity of the test is good, there is a greater chance of improvement in the measure score in subjects with more advanced disease who can be safely assessed. Study implementation

[0141] The study was conducted during the COVID-19 pandemic, which necessitated changes to the conduct of the study due to site closures and stay-at-home measures to prevent disease spread. This required implementation of pauses at the individual subject level based on the study period each subject was in at the time of site closure. These were systematically conducted and documented in the study files. The number of visits and assessments missed due to COVID-19 was low compared to the total number planned. Every attempt was made to ensure that data collection was reliable, assessments were accurate, and, where possible, were collected correctly and in a timely manner. Despite the pandemic, the study was well conducted, and subject safety and data integrity were maintained. Overall, the COVID-19 pandemic did not significantly impact the ability to monitor and manage subject safety, data integrity, or efficacy assessments as of data cutoff. Effectiveness Results

[0142] Compared with no meaningful changes in the placebo group, once-weekly administration of pegol arginase resulted in an early, consistent and sustained decrease in plasma arginine levels at week 24 of the double-blind phase. Previous guidelines recommend targeting plasma arginine levels to <200 μM because previously in clinical practice, most patients were unable to achieve levels below this threshold with dietary restrictions alone. Treatment with pegol arginase resulted in normalization of arginine levels in the vast majority of subjects (90.5%) by week 24 of dosing. Subjects in the double-blind phase continued the study in the LTE phase and were switched from IV to SC dosing, with the same IV dose for ease of administration and increased convenience unless arginine levels required it. In patients who received pegol arginase in the double-blind phase, continued administration of pegol arginase by the SC route of administration resulted in a similar consistent and sustained decrease in plasma arginine levels at week 24 in the LTE phase. In LTE, subjects not receiving pegylated arginase who were initially randomized to placebo started weekly pegylated arginase, switched to SC administration after 8 weeks, and 100% of subjects who reached LTE24 (N=11) were also able to normalize plasma arginine levels.

[0143] The key goal is to achieve arginine levels that meet guideline recommended levels (<200 μM) and ideally reach normal levels, which are rarely achieved using current treatments. At Week 24 of the double-blind period, more than 90% of subjects treated with pegylated arginase achieved arginine values ​​within the treatment guidelines (<200 μM), compared to none in the placebo group. In addition, at Week 24 of the double-blind period, more than 90% of subjects treated with pegylated arginase achieved arginine values ​​within the normal range (40 to 115 μM), which is unprecedented in this subject population.

[0144] The key secondary efficacy endpoints focus on neurological / neuromotor outcomes. Given the wide age range of subjects recruited to this study and the progressive nature of the disease, the degree of baseline deficit in neuromotor function varies between subjects. Positive effects on neuromotor function were observed in the pegylated arginase group as assessed by GMFM-E and 2MWT. A similar proportion of subjects in the pegylated arginase group showed clinically relevant and / or numerical improvements in GMFM-E and 2MWT assessments (approximately 55% [N=11] and 50% [N=10], respectively). Improvements in neuromotor function were also observed in subjects in the placebo group, but were generally at an order of magnitude lower than the improvements observed in the pegylated arginase group. At week 24, compared with placebo, pegylated arginase administration resulted in a longer improvement in the average distance walked in 2 minutes (2MWT) relative to baseline, and greater improvements in GMFM-E scores relative to baseline. Numerical improvements were observed in both GMFM-E and 2MWT, and for GMFM-E, there was a positive trend relative to baseline compared with placebo (p=0.1087). In subgroup analysis, subjects with more severely limited mobility (GMFCS≥II) treated with peg-arginase had greater gains in both 2MWT and GMFM-E than subjects classified as GMFCS I because they were not limited by the maximum improvement threshold or upper limit of the assessment. These differences may be a reflection of the greater effectiveness of using these outcome tools to obtain clinical benefits in patients with more advanced disease. The improvements in patients with more advanced disease indicate the clinical utility of treatment with peg-arginase in these patients. In view of the limitations of these outcome tools, the less significant improvements in patients with less advanced disease do not hinder the possibility of being used to prevent progression, which is also a treatment goal. Some limitations of these specific clinical assessments in some population sections include the following. For 2MWT, subjects with GMFCS IV classification are usually wheelchair-bound, and even if they can walk, their ability to walk is severely limited (N=4). For the GMFM-E assessment, in patients with GMFCS I classification and less severe disability, the measure of improvement was limited by the upper limit of the test, as some patients were near or at the upper or upper limit of baseline response. In further support of the therapeutic effect of peg-arginase during the double-blind period, for subjects who completed LTE24, improvements from baseline in 2MWT and GMFM-E continued to be observed in subjects treated with peg-arginase, highlighting continued improvement with further treatment, while subjects who switched from placebo to peg-arginase remained stable or improved.

[0145] Based on interim results from the ongoing study 300A after 24 weeks of double-blind treatment, peg-arginase administered weekly IV at a dose of 0.05 to 0.20 mg / kg met the primary endpoint (p<0.0001). Pegylated arginase demonstrated consistent and sustained reductions in plasma arginine to both therapeutic and normal levels, whereas no meaningful changes were observed in the placebo group.

[0146] Reductions in plasma arginine were associated with clinically relevant improvements in activity. Pegylated arginase showed a positive trend in GMFM-E and numerical improvement in 2MWT compared with placebo.

[0147] More subjects treated with pegylated arginase met response criteria in multiple domains, generally with responses orders of magnitude greater than those treated with placebo.

[0148] Pegylated arginase was well tolerated in subjects with ARG1-D. TEAEs were transient, manageable, and either self-limited or resolved with standard medical care.

[0149] During the LTE period, subjects were switched to SC dosing to simplify dosing and improve treatment convenience, with the same IV dose unless arginine levels dictated, with exposure up to 104 weeks. The safety profile was consistent between IV and SC dosing

[0150] During the LTE period, subjects continued to maintain normal arginine levels after switching to SC dosing and maintained or further improved mobility, whereas those switched from placebo to peg-arginase remained stable or showed improvement.

[0151] The security features are consistent between DB and LTE periods.

[0152] In conclusion, PEGylated arginase represents a new targeted therapeutic option addressing unmet medical needs in ARG1-D, with overall safety and efficacy data suggesting a positive benefit-risk profile for patients affected by this rare, progressive and debilitating disease. 1.2 Overview of Background and Clinical Efficacy

[0153] Polyethylene glycol arginase (Co-Arg1-PEG, AEB1102) is a cobalt-substituted PEGylated human recombinant arginase 1 enzyme with enhanced stability, more potent catalytic activity, and extended half-life compared to the native enzyme. Polyethylene glycol arginase cleaves excess plasma arginine into ornithine and urea, which are the natural breakdown products of arginase 1.

[0154] ARG1-D is a rare, debilitating, progressive, inherited, neurotoxic metabolic disease associated with elevated levels of arginine and its metabolites, which significantly reduces quality of life and increases morbidity and premature mortality (Diez-Fernandez 2018; Schlune 2015; Summar 2013; Waisbren 2018).

[0155] There are similarities with another inherited metabolic disorder, phenylketonuria (PKU), and arginase 1 deficiency (ARG1-D), in terms of inherited liver enzyme deficiencies and accumulation of amino acids in the blood. Phenylketonuria is a deficiency of phenylalanine hydroxylase (PAH) in the liver that results in high levels of the circulating amino acid phenylalanine (Phe), which, although poorly understood, leads to chronic neurological and psychiatric disorders if left untreated or undertreated. Treatment focuses on lowering Phe plasma levels to improve clinical outcomes. Pegvaliase is a new approved enzyme replacement therapy that helps address a significant unmet need in PKU patients who are unable to control their blood Phe levels with current treatment options.

[0156] Similar to PKU, in ARG1-D, enzyme deficiency in the liver results in high plasma and tissue levels of arginine, which, when treated with the current standard of care, leads to spasticity, gait disturbance, difficulty walking, and developmental and cognitive disability. Similarly, pegylated arginase, a human arginine metabolizing enzyme intended for use as an enzyme replacement therapy for patients with ARG1-D, cleaves excess plasma arginine into ornithine and urea, which are natural breakdown products of arginase 1. The goal of treatment is to reduce blood arginine concentrations to <200 μM to minimize the toxic effects of excess arginine, excess guanidino compounds (GC) derived from arginine, and nitric oxide metabolism disorders (Palynziq USPI).

[0157] Manifestations of ARG1-D typically present in early childhood and include neuromotor and neurocognitive deficits. The clinical presentation is ultimately strikingly consistent, characterized by spasticity, gait disturbance, difficulty walking, and developmental and cognitive disability, with some patients experiencing seizures, although variation in the timeframe and progression of symptoms is observed between patients (De Deyn 1997; Marescau 1990; Waisbren 2018).

[0158] Elevated arginine and its metabolites are believed to be key contributors to disease manifestation and progression (De Deyn 1997; Marescau 1990; Waisbren 2018). Preclinical models have demonstrated that arginase deficiency promotes demyelination of the corticospinal tract during postnatal CNS development and alters synaptic density in the motor cortex through gene expression profiling of the motor cortex of the ARG1-D mouse knockout model. Several other diseases associate spasticity with loss of myelination through a variety of etiologies, including multiple sclerosis and hereditary spastic paraplegia. In summary, the literature findings both support the importance of arginine and subsequent metabolite accumulation as key drivers of ARG1-D neurological manifestations and exemplify the effectiveness of reducing arginine to reverse neuronal damage through enzyme administration or hepatic expression of ARG1 for functional restoration of oligodendrocytes to improve CNS abnormalities.

[0159] Neuromotor complications of the disease are hallmark features, and lower limb spasticity, often seen in early childhood, impairs mobility and balance, leading to gait disturbances and difficulty walking and climbing stairs. Patients typically present with some form of lower limb spasticity, with approximately 60% to 75% presenting with spasticity at initial presentation, which increases with longer follow-up (Carvalho 2012; Huemer 2016). This progressive spastic diplegia results in increasing immobility, inability to perform activities of daily living, and increased caregiver burden.

[0160] There are currently no approved therapies for the treatment of ARG1-D that significantly lower plasma arginine, and dietary interventions rarely achieve treatment guideline-directed targets, resulting in continued disease progression. Pegylated arginase represents a new targeted therapeutic option that addresses the unmet medical needs of patients affected by ARG1-D. The overall efficacy data for peg-arginase, ranging in duration from 24 weeks to over 120 weeks, demonstrated substantial evidence of effectiveness by achieving sustained plasma arginine levels that met or exceeded therapeutic guidelines (<200 μM) and were within normal levels (40 to 115 μM) in the majority of patients, with clinically meaningful improvements in activity at the study level and, more importantly, at the individual subject level for this ultra-rare disease. diagnosis

[0161] Diagnosis can be readily made using routinely available plasma arginine assessment or genetic analysis ( 2012; 2019). Diagnostic delays may occur due to overlap in symptomatology with other developmental disorders such as cerebral palsy or hereditary spastic paraplegia (Carvalho 2012). Current Standard of Care

[0162] There are no pharmacological agents known to be effective in lowering arginine levels in patients with ARG1-D, and no country has approved agents specifically for the enzyme deficiency. Current treatments include a low-protein diet that modestly lowers arginine levels, and in some cases this is accompanied by improved neuromotor function ( 2012; Huemer 2016; Lambert 1991; Prasad 1997). Pegylated arginase represents the first potential enzyme therapy for ARG1-D. The literature suggests that modest reductions in plasma arginine levels achieved with strict dietary protein restriction can lead to improvements in disease manifestations; however, the currently recommended therapeutic goal of lowering plasma arginine to <200 μM is difficult to achieve, in part because adherence to a strict diet to reduce arginine levels to within or near the normal range is challenging ( 2012; Huemer 2016; Lambert 1991; Prasad 1997; Burrage 2015). Therefore, significantly reducing plasma arginine levels using PEGylated arginase has the potential to slow or halt the progression of neuromotor, neurocognitive, and / or adaptive behavioral deterioration in patients with ARG1-D.

[0163] Current treatment for ARG1-D focuses on reducing plasma arginine levels through lifelong dietary protein restriction. Protein intake is restricted to the minimum required to maintain protein biosynthesis and growth. Typically, half or more of the dietary protein is given in the form of an essential amino acid (EAA) mixture that does not contain arginine. Such dietary adjustments can produce a modest reduction in plasma arginine levels, but levels remain significantly elevated in most patients, probably due to the fact that arginine flux is largely dependent on systemic protein turnover and is minimally affected by dietary intake (Wu 1998). In addition, the diet is unpalatable and difficult to maintain and manage, especially in growing children. Ammonia scavengers can be used to help control ammonia levels and thereby control the number and severity of hyperammonemic episodes that occur in some patients with ARG1-D. Liver transplantation has been reported to achieve disease normalization in some patients, and despite the limited success of this intervention, transplantation is only applicable to a small number of patients and there is a considerable risk of mortality and morbidity. Unmet medical needs

[0164] The lack of therapeutic options that can effectively reduce arginine levels or routinely achieve a reduction to within the normal range and promote lifelong maintenance of normal arginine levels for patients with arginase 1 deficiency highlights the unmet need for new treatments. The development of such therapeutic agents may be helpful in attempts to minimize exposure to the neurotoxic effects of arginine and its metabolites and provide the potential for normal neurocognitive and neuromotor development in these patients (Amayreh 2014). 1.3 Selection of research population Inclusion criteria

[0165] To be included in this study, subjects needed to meet all of the following criteria: 1. The subject and / or parent / guardian provides written informed consent / permission, which includes compliance with the requirements and restrictions outlined in the ICF and protocol. 2. Current diagnosis of ARG1-D as documented in the medical record, which includes 1 of the following: elevated plasma arginine levels, mutation analysis leading to pathogenic variants, or RBC arginase activity. To enter this study, subjects must also meet the following plasma arginine criteria: a. The mean of all measurements of plasma arginine during the Screening Period prior to the Randomization Visit (Visit 1, Study Day 1) was ≥ 250 μM. b. If a subject is rescreened, the only values ​​considered for eligibility assessment are those for the current screening period. 3. The subject's age on the day of informed consent / permission is ≥ 2 years old. 4. The subject has an evaluable within-subject change (clinical response) in at least 1 component of 1 assessment included in the key secondary / other secondary endpoints. 5. Have received written confirmation from the investigator and / or nutritionist that the subject can maintain their diet according to the dietary information given in the protocol, i.e., can maintain their current level of protein consumption, including natural protein and EAA supplementation. 6. Subjects receiving ammonia scavengers, anti-epileptic drugs, and / or drugs for spasticity (e.g., baclofen) have been taking a stable dose of medication for at least 4 weeks prior to randomization and are willing to remain on a stable dose during the double-blind and blinded follow-up portions of the study. 7. Subjects are female and male. Female subjects of childbearing potential had a negative serum pregnancy test during the screening period before receiving the first dose of study treatment, and a negative urine pregnancy test on the day of the first dose and before the first dose. If the subject (male or female) is engaging in sexual activity that could result in pregnancy, he or she is postoperatively infertile, postmenopausal (12 months of no menstruation without alternative medical reasons, or high follicle-stimulating hormone levels in the postmenopausal range for women who are not using hormonal contraception or hormonal therapy), or agrees to use a highly effective birth control method during the study and for at least 30 days after the last study drug administration. Highly effective contraceptive methods include: combined (estrogen and progestin) hormonal contraception associated with ovulation inhibition; progesterone-only hormonal contraception associated with ovulation inhibition; intrauterine device; intrauterine hormone-releasing system; or abstinence (avoiding heterosexual intercourse during the entire risk period associated with study treatment). Exclusion criteria

[0166] Subjects meeting any of the following criteria were excluded from the study: 1. An episode of hyperammonemia within 6 weeks prior to the first dose of study drug (defined as an event in which the subject had an ammonia level ≥ 100 μM with 1 or more symptoms associated with hyperammonemia requiring hospitalization or emergency room management). 2. Active infection requiring anti-infective treatment within 3 weeks before the first dose. 3. Known active infection with human immunodeficiency virus, hepatitis B or hepatitis C. 4. Have extreme mobility deficits, defined as being unable to assess on the Gillette Functional Assessment Questionnaire (GFAQ) or having a score of 1 on the GFAQ. 5. Suffering from other medical conditions or comorbidities that the investigator believes will interfere with study compliance or data interpretation (e.g., severe intellectual disability that interferes with required study assessments). 6. Participated in previous interventional studies of polyethylene glycol arginase. 7. Have a history of hypersensitivity reaction to polyethylene glycol (PEG), and the investigator judges that the history of hypersensitivity reaction puts the subject at an unacceptable risk of AEs. 8. Subject is currently being treated with a botulinum toxin-containing regimen or plans to start such a regimen during the DB or blinded follow-up portion of the study, or has received surgery or botulinum toxin treatment for spasticity-related complications within 16 weeks prior to the first dose of study treatment in this study. 9. Currently participating in another therapeutic clinical study or having received any investigational agent within 30 days (or 5 half-lives, whichever is longer) before the first dose of study treatment in this study. 10. Previous liver or hematopoietic transplantation. 1.4 Study objectives and endpoints

[0167] The overall objective of this study is to demonstrate the safety, tolerability, and efficacy of long-term IV and SC administration of pegylated arginase for the treatment of pediatric and adult subjects with ARG1-D and hyperargininemia. The study objectives and endpoints are shown in Table 1 below. This study is designed to evaluate the effect of pegylated arginase in combination with an IDM program in subjects. Table 1 1.5 Overall study design and planning

[0168] The study (Study 300A) is a multicenter, randomized, double-blind (DB), placebo-controlled study designed to evaluate the safety and efficacy of peg-arginase and is planned to be conducted in approximately 30 subjects with ARG1-D, with an expected total study duration of approximately 178 weeks per subject. The study consists of: 1. A screening period of 3 to 4 weeks in duration to collect all necessary information to ensure that subjects meet the study eligibility criteria, establish baseline plasma arginine data, collect prescribed dietary data, and determine adherence to the prescribed diet using a food diary. 2. 24-week randomized DB treatment period. 3. An open-label long-term extension (LTE) phase of up to approximately 150 weeks, in which all subjects receive active pegylated arginase. The first 8 weeks of treatment remain blinded to ensure that study data relevant to the randomization phase are collected prior to unblinding.

[0169] After completing all screening assessments and confirming study eligibility, subjects were randomized in a 2:1 ratio to receive weekly IV infusions of pegylated arginase plus IDM or placebo plus individualized disease management (IDM) during a 24-week double-blind treatment period.

[0170] After completing the 24-week double-blind treatment period of the study, all eligible subjects began an 8-week blinded follow-up treatment period during which all subjects received pegylated arginase. Subjects initially randomized to pegylated arginase received the optimized dose they received during the 24-week double-blind treatment period (which allowed adjustment based on clinical indications). Subjects initially randomized to placebo during the 24-week double-blind treatment period began an 8-week DB treatment period at a dose of 0.10 mg / kg, which allowed adjustment based on arginine levels during the LTE period.

[0171] After the first 8 weeks of the blinded LTE period, the subjects chose to receive pegylated arginase by weekly subcutaneous (SC) administration with the approval of the investigator and the sponsor. The first 4 SC doses were given at the study site. The initial mg / kg SC dose was allowed to be the same as the IV dose and could be adjusted based on arginine levels. If the investigator deems it safe and appropriate after consultation with the sponsor, subsequent SC doses may be administered outside the study site by appropriately trained home health care workers. During the LTE period, all subjects received pegylated arginase plus IDM weekly. Study Design

[0172] The research design is Figure 1A Key demographic and baseline clinical characteristics are shown in Figure 1B Shown in.

[0173] A total of 32 patients were enrolled (pegylated arginase, n=21; placebo, n=11). Patients were of varying race and ethnicity; approximately 30% were Hispanic or Latino. The mean of all measurements of plasma arginine during the screening period prior to the randomized visit was ≥250 μM. All patients were managed with dietary protein restriction. Spasticity was evident in 65.6% of patients (n=21 / 32), and functional impairment was evident in >50% of patients with GMFCS levels >2. Subjects receiving ammonia scavenger therapy, antiepileptic drugs, and / or drugs for spasticity (e.g., baclofen) had been taking a stable dose of medication for at least 4 weeks prior to randomization and were willing to remain on a stable dose during the double-blind and blinded follow-up portions of the study. Personalized disease management

[0174] This study was designed to evaluate the effect of peg-arginase in combination with an IDM program in subjects that typically includes a prescribed diet with severe protein restriction and EAA supplementation and / or the use of ammonia scavengers.

[0175] Subjects had a stable IDM plan, as demonstrated during the screening period prior to study participation, including the prescribed amount of protein and the prescribed amount of EAA and / or the use of prescribed doses of ammonia scavenger medications, if applicable. Subjects were willing and able to maintain consistent dietary protein intake during the DB portion of the study. Investigators and nutritionists advised subjects and / or their caregivers not to modify their IDM during the DB portion of the study unless there was a clear medical reason to do so. Compliance with the IDM was recorded in the electronic case report form (eCRF).

[0176] Subjects were required to have a stable, consistent diet throughout the duration of the blinded period, which included the 24-week randomization period and the first 8 weeks of the open-label period of the study. A consistent diet was defined as a diet in which prescribed natural protein medical food EAA supplementation and / or use of ammonia scavengers and caloric intake did not change by more than 15% from baseline. Any prescribed change of more than 15% from baseline was recorded. Treatment administered

[0177] After completing all screening assessments and confirming study eligibility, subjects were randomized in a 2:1 ratio to receive weekly IV infusions of pegylated arginase plus IDM or placebo plus IDM during the 24-week DB treatment period.

[0178] After completing the 24-week DB treatment period of the study, all eligible subjects began an 8-week blinded follow-up treatment period during which all subjects received pegylated arginase.

[0179] Subjects initially randomized to peg-arginase received the optimized dose they received during the 24-week DB treatment period (which was adjusted as clinically indicated). Subjects initially randomized to placebo during the 24-week DB treatment period began transitioning to an 8-week blinded treatment period of peg-arginase at a dose of 0.10 mg / kg, which was adjusted based on arginine levels during the LTE. All subjects underwent a final DB assessment (i.e., Week 24 assessment) prior to initiation of peg-arginase dosing during the LTE period.

[0180] After the first 8 weeks of the blinded LTE period, subjects elected to receive peg-arginase by SC administration with approval of the Investigator and Sponsor. The first 4 SC doses were given at the study site. The initial mg / kg SC dose was the same as the IV dose. Subsequent SC doses were allowed to be administered outside the study site by appropriately trained home health care workers if deemed safe and appropriate by the Investigator in consultation with the Sponsor. During the LTE period, all subjects received peg-arginase plus IDM. The unit dosage strength of the formulation for IV administration is 1 mg / ml. The unit dosage strength of the formulation for SC administration is 5 mg / ml. Pegylated arginase is a cobalt-substituted PEGylated recombinant human arginase 1. Pegylated arginase is formulated in 50 mM NaCl, 1 mM K2HPO4, 4 mM KH2PO4, and 1.5% w / v glycerol. Premedication with a non-sedating antihistamine 30 minutes prior to the start of dosing is recommended. The starting dose is 0.10 mg / kg; dose adjustments are allowed based on pharmacodynamic response. The IV infusion time is approximately 30 minutes. Dose selection

[0181] For subjects randomized to peg-arginase, a starting dose of 0.10 mg / kg IV weekly was selected based on available safety, PK, PD, and clinical results (from Study 101A), which showed that this dose was well tolerated and expected to provide the desired level of plasma arginine control in the majority of the target population.

[0182] For the 14 subjects who completed the study at the time of database lock, the final dose in Study 101A was 0.04 to 0.20 mg / kg. The median final dose was 0.09 mg / kg; therefore, a starting dose of 0.10 mg / kg was selected in this study to represent an effective and well-tolerated dose. Dose adjustments were made based on plasma arginine levels. In general, the dose was adjusted if the subject's plasma arginine level at 168 hours after dosing was outside the range of 50 to 150 μM. Effectiveness evaluation

[0183] The efficacy of PEGylated arginase was measured by the magnitude, onset, and duration of changes in plasma arginine levels and the following clinical outcome assessments: Neurological and neuromotor manifestations Adapting to behavioral changes Nursing staff and clinician global impression of change and severity and exit interview Other clinical outcomes, such as frequency of seizures and hyperammonemic episodes Neurocognitive, developmental, and quality of life measures Ornithine and guanidine compounds and other exploratory biomarkers (i.e., guanidinoacetic acid, α-N-acetylarginine, α-keto-δ-guanidine valeric acid, arginine, creatine, or others)

[0184] In addition to the neurological examination performed along with the physical examination, several assessments of neurological and neuromotor function were tested: 2MWT, Functional Mobility Assessment (FMA; Functional Mobility Scale [FMS] and GFAQ), 9-Hole Pegboard Test, GMFM-D and GMFM-E, and Modified Ashworth Scale (MAS).

[0185] Blood samples were collected for analysis of plasma arginine, ornithine, and guanidino compounds. The guanidino compounds to be measured included guanidinoacetic acid, α-N-acetylarginine, α-keto-δ-guanidinovaleric acid, arginine, and creatine. Electroencephalogram

[0186] In addition to clinically apparent epilepsy, high frequencies of abnormal electroencephalogram (EEG) activity have been observed in patients with ARG1 D (Carvalho 2012; Huemer 2016; De Deyn 1997). The electroencephalogram is a test used to evaluate electrical activity in the brain. The EEG is defined as an alternating type of electrical activity recorded from the surface of the scalp after being picked up by metal electrodes and a conductive medium (Teplan 2002). A significant proportion of patients with ARG1 D have been shown to have abnormal EEG results, with EEG recordings showing slow diffuse activity and generalized epileptic activity (Carvalho 2012; De Deyn 1997).

[0187] In this study, all subjects consented to baseline and follow-up routine (ie, non-sleep, ambulatory, or video telemetry) outpatient EEG recordings.

[0188] Electroencephalograms (EEGs) were performed on all subjects at Screening and Week 24. EEG results were interpreted as normal; abnormal, not clinically significant (NCS); or abnormal, clinically significant (CS). If a result was reported as abnormal CS, it was reported abnormal.

[0189] Subjects with abnormal EEG findings and abnormal, CS-reported findings were included for the DB analysis period. No other summary reports or tabulations were generated for these data. Clinical laboratory evaluation

[0190] A full range of clinical markers as shown in Table 2 below were assessed for all trial subjects. Table 2 Abbreviations: ALT = alanine aminotransferase; ALP = alkaline phosphatase; aPTT = activated partial thromboplastin time; AST = aspartate aminotransferase; BUN = blood urea nitrogen; IGF1 = insulin-like growth factor 1; INR = international normalized ratio; PK = pharmacokinetics; PT = prothrombin time; RBC = red blood cell; sRBP = serum retinol-binding protein; TIBC = total iron-binding capacity; WBC = white blood cell. Personalized disease management plan (and 3-day diary food record)

[0191] The currently recommended approach to disease management involves a framework of strict protein restriction and EAA supplementation and / or the use of ammonia scavengers. These aspects of disease management are individualized for each patient by titrating the different components to lower plasma arginine levels and ensure adequate intake of protein for proper growth and development while avoiding hyperammonemia. General guidelines recommend that the diet of patients with ARG1 D will include approximately 50% protein from natural food sources and approximately 50% from amino acid supplements. This recommendation can be adjusted as clinically indicated to achieve IDM goals appropriate for the specific subject.

[0192] Throughout the study, subjects maintained the IDM regimen prescribed by the treating investigator prior to enrollment. Modifications to the prescribed IDM regimen were not permitted during the study unless deemed clinically indicated by the investigator. The rationale for any change in the IDM and the date and nature of any major (>±15% of baseline) prescribed change were recorded in the eCRF.

[0193] Subjects were required to maintain dietary protein intake levels consistent with their baseline levels throughout the duration of the 8-week blinding period of the randomized DB and LTE phases of the study. A consistent diet was defined as a diet in which natural intact proteins, medical food EAAs, and calories prescribed and consumed were <±15% relative to baseline.

[0194] Subjects and / or their caregivers were instructed to record all dietary intake (whole foods, medical foods / EAAs, and EAA supplements) of study subjects for 3 consecutive days prior to all clinic visits during the screening and baseline periods and prior to DB visits 6, 12, 18, and 24. At these visits, the prescribed IDM regimen was also recorded. The date and time of the last dose of EAA was recorded for each day of the 3-day food diary record. Medical history

[0195] In addition to arginase deficiency, the most frequently reported histories included cognitive impairment, hyperammonemia, developmental delay, and elevated alanine aminotransferase, which are common clinical findings in ARG1-D. A greater proportion of subjects in the pegylated arginase group had a history of hyperammonemia (i.e., prior to treatment) compared with subjects in the placebo group. A lower proportion of subjects in the pegylated arginase group reported developmental delay and cognitive impairment than in the placebo group (prior to treatment).

[0196] In general, the most commonly used medications prior to the study were dietary supplements, essential amino acid formulas, vitamins, and minerals, as the standard of care for ARG1-D is a protein-restricted diet. Ammonia scavengers are often taken to manage ammonia levels and medications are taken to manage other disease symptoms.

[0197] Overall, all 32 subjects (100%) received concomitant medications during the study period (Table 3). The most frequently used concomitant medications were the ammonia scavengers sodium benzoate and glyceryl phenylbutyrate. The proportion of subjects using sodium benzoate or glyceryl phenylbutyrate was similar in the pegarginase and placebo groups. Overall, nearly all subjects in the pegarginase treatment group (95.2%, N=20) and the placebo group (81.8%, N=9) received an ammonia scavenger during the study period.

[0198] Other frequently used medications in both the peg-arginase and placebo groups included essential amino acid formulas, baclofen, ibuprofen, paracetamol, vitamin and mineral dietary supplements, and cetirizine. Table 3 Summary of concomitant medications—preferred names and corresponding ATC categories reported by ≥2 subjects 1.6 Effectiveness Evaluation

[0199] The study consisted of 2 periods, a placebo-controlled double-blind period and a LTE period. Primary End Point

[0200] The mean plasma arginine in subjects treated with PEGylated arginase decreased rapidly and continuously, with a decrease observed at week 1 and within the normal range by week 12, which persisted through week 24. There was little change in mean plasma arginine in the placebo group ( Figure 3A At week 24, mean plasma arginine was 105.49 μM in the peg-arginase group compared with 448.82 μM in the placebo group. Treatment with peg-arginase resulted in a significant decrease in arginine (i.e., values ​​< 200 μM) starting at week 6 and maintained through week 24 of treatment compared with placebo. Primary Endpoint Analysis

[0201] The primary analysis used a mixed effects model with repeated measures (MMRM) approach. Log-transformed plasma arginine data were used in this analysis. One subject withdrew before week 24; the corresponding missing week 24 data were imputed using the last observation carried forward (LOCF) method. At baseline, the geometric mean (SD) plasma arginine level was lower in the peg-arginase group (354.0 μM [SD 1.30 μM]) than in the placebo group (464.7 μM [SD 1.21 μM]). Treatment with peg-arginase resulted in a significant reduction in plasma arginine starting at week 6 compared with placebo (p<0.0001) and maintained through treatment week 24. At week 24, peg-arginase showed a 76.7% reduction in mean plasma arginine compared with placebo.

[0202] When individual subjects were assessed for decreases in plasma arginine, nearly all subjects (95%, N=20) in the peg-arginase group had a meaningful change in plasma arginine at Week 24 compared with the placebo group ( Figure 2 ). Key Secondary End Points a. Two-minute walk test

[0203] The two-minute walk test (2MWT) is a shorter modification of the 6-minute version (e.g., National Institutes of Health [NIH] Toolbox motor domain-2MWT [Bohannon 2014]). A standardized method as defined by the American Thoracic Society (2002) is followed. Results of the 2MWT are recorded as distance completed (in meters) and percent change from baseline.

[0204] Subjects treated with pegarginase demonstrated improvement in the 2MWT compared to the placebo group. At baseline, subjects receiving pegarginase walked a longer mean (SD) distance in 2 minutes of 109.0 meters (SD 55.76 meters) compared to a mean (SD) distance of 99.9 meters (SD 38.61 meters) for subjects receiving placebo. At Week 24, the mean (SD) distance walked in 2 minutes in the pegarginase group was 115.9 meters (SD 51.81) (representing an improvement of 7.3 meters from baseline) compared to a mean distance walked in 2 minutes in the placebo group of 102.3 meters (SD 51.10) (representing a difference of 2.7 meters from baseline). Although the change in the LS mean difference (5.5 meters) in the 2MWT between the pegarginase group and the placebo group from baseline to Week 24 did not reach statistical significance (p=0.5961)( Figure 4A ), but it showed that subjects in the PEG-arginase group had greater improvement than those in the placebo group ( Figure 4A and 4B ).

[0205] When evaluating the change from baseline in the 2MWT for individual subjects with available baseline and Week 24 data (N=29), a greater proportion of subjects in the peg-arginase group (52.6%, N=10) had an improvement in the change from baseline in the 2MWT distance compared to the placebo group (40%, N=4), and the magnitude of the improvement was generally greater in subjects in the peg-arginase group relative to subjects in the placebo group. b. Gross Motor Function Measurement-88 Part E

[0206] The Gross Motor Function Measure-88 (GMFM-88, also referred to as the GMFM) is a clinical measure designed to assess gross motor function by observing the subject's ability to start and complete certain movements. For this study, only dimensions D and E were assessed. A scoring system was used for each item of the GMFM by a trained observer, which covers 2 dimensions: standing (part D) and walking, running, and jumping (part E).

[0207] Subjects treated with pegarginase showed improvement in GMFM-E compared to the placebo group. At baseline, the mean (SD) GMFM-E score for subjects in the pegarginase group was (48.3 [SD 19.93]) and in the placebo group was (46.5 [SD 24.56]). At Week 24, the mean (SD) GMFM-E score in the pegarginase group was 52.0 (SD 21.27), representing an improvement of 4.2 points from baseline - compared to a mean (SD) score of 46.71 (SD 25.71) in the placebo group, representing a decrease of 0.4 (51.10) from baseline. The change in the LS mean difference in GMFM-E score between the pegarginase group and the placebo group from baseline to Week 24 was 4.6 points, representing a trend for improvement (p=0.1087)( Figure 5A ).

[0208] Individual responses are summarized in waterfall plots of GMFM-E and change from baseline at Week 24 ( Figure 5B A similar proportion of subjects in the pegarginase group (55.0%, N=11) experienced an improvement in the change from baseline in GMFM-E compared with the placebo group (54.5%, N=6); however, the magnitude of improvement was generally greater in the pegarginase subjects compared with the placebo subjects ( Figure 5B ).

[0209] When evaluating individual subjects for change from baseline in GMFM-E, a similar proportion of subjects in the pegylated arginase group (55.0%, N=11 / 20) had an improvement in the change from baseline in GMFM-E score at Week 24 compared to the placebo group (54.5%, N=6 / 10), however, the magnitude of improvement was generally greater in subjects in the pegylated arginase group relative to the placebo group. c. Responders based on arginine values ​​less than 200 μM (<200 μM)

[0210] Compared to the placebo group, subjects treated with PEGylated arginase showed improvements in plasma arginine levels that were consistent with treatment guidelines. Current guidelines recommend a treatment goal of plasma arginine below 200 μM. The proportion of subjects in the treatment group who were able to achieve this goal (responders) was assessed. Nineteen of the 21 subjects in the PEGylated arginase group (90.5%) became responders after 24 weeks of treatment, reaching arginine values ​​of <200 μM, compared to none in the placebo group (Table 4). Table 4 Proportion of responders with arginine values ​​< 200 μM during the double-blind period d. Responders based on arginine values ​​within the normal range (40 to 115 μM)

[0211] Compared with the placebo group, subjects in the pegylated arginase group showed improvements in plasma arginine levels, resulting in normal arginine levels. Although current guidelines recommend plasma arginine below 200 μM, achieving values ​​within the normal range is an important therapeutic goal. The proportion of subjects who were able to achieve this treatment goal was evaluated by treatment group. At Week 24, 90.5% of the subjects in the pegylated arginase group met the response criteria, with arginine values ​​reaching the normal range, and no subjects in the placebo group met this response criterion (Table 5 and Table 5). Figure 3B ). Table 5. Proportion of responders with arginine values ​​within the normal range (40 to 115 μM) during the double-blind period e. Changes in ornithine and guanidine compounds at week 24

[0212] Baseline ornithine values ​​and changes from baseline Figure 6 The baseline guanidine compound values ​​and the changes from baseline are shown in Figures 7 to 10 Shown in.

[0213] Ornithine and urea are products of the hydrolysis of arginine by arginase I in the final step of the urea cycle. In subjects with ARG1-D, ornithine levels are typically low due to a lack of the enzyme arginase I that is available to convert arginine to ornithine. Changes from baseline in ornithine are summarized in Figure 6 Shown in.

[0214] Subjects treated with pegylated arginase had rapid and sustained increases in mean plasma ornithine, with increases observed at week 1 and continued improvement in levels at week 12 that persisted through week 24. At baseline, median ornithine levels were 32.1 μM in the pegylated arginase group and 29.2 μM in the placebo group. Subjects treated with pegylated arginase had increases in ornithine at week 1, compared with minimal changes in the placebo group, and continued to increase with further dosing ( Figure 6 ). Treatment with PEGylated arginase resulted in a significant increase in ornithine compared to placebo (p<0.0001) starting at week 6 and maintained through treatment week 24. At week 24, the mean ornithine level in the PEGylated arginase group was 67.7 μM, representing a 106.9% increase in mean ornithine compared to placebo. Starting at week 6 and maintained through treatment week 24, the mean ornithine level in the PEGylated arginase group was at least 60 μM. f. Guanidine compounds in arginase-1 deficiency

[0215] Guanidine compounds (GC) are direct and indirect products of arginine metabolism and are often elevated in ARG1-D. In addition to hyperargininemia in ARG1-D, these compounds can also lead to pathophysiological changes such as seizures and liver dysfunction, although the exact mechanism is not fully understood (Deshmukh 1991; Franzoi 2018; Yu 2013).

[0216] Abnormalities in the concentrations of guanidino compounds, nitric oxide, and homoarginine are thought to play a pathophysiological role in Arg-1 D. Increases in guanidino compounds have been observed in animals and humans affected by hyperargininemia. These are caused by elevated arginine levels through the arginine:glycine amidinotransferase reaction. Guanidino compounds (such as guanidinoacetate) have been shown to be epileptogenic and may be the cause of neurological damage and epilepsy in these patients.

[0217] At baseline, plasma levels of guanidine compounds were similar between treatment groups, although values ​​of α-keto-δ-guanidine valerate and αN-acetylarginine were progressively higher in the PEG-arginase group. The median values ​​of arginine, guanidine acetic acid, α-keto-δ-guanidine valerate, and αN-acetylarginine in the PEG-arginase group were 2.5 μM, 3.1 μM, 4.6 μM, and 1.1 μM, respectively, and 2.8 μM, 3.5 μM, 5.4 μM, and 1.7 μM, respectively, in the placebo group.

[0218] For the PEG-arginase group, decreased levels of all four GCs were observed starting from Week 1 and were maintained over time until Week 24 of the double-blind period ( Figure 7 , Figure 8 , Fig. 9 , Fig.10 ). For the placebo group, levels of all four GCs fluctuated over time but remained similar to baseline levels until week 24. Starting from week 6, treatment with pegarginase resulted in a significant reduction in GCs compared with placebo (p value range: 0.0059 to <0.0001), which was maintained until week 24 of treatment. At week 24, pegarginase showed a 50% to 70% reduction in GCs (compared with placebo), which was associated with a reduction in plasma arginine levels. Starting at week 6 and maintained through week 24 of treatment, mean αN-acetylarginine levels in the PEGylated arginase group decreased to less than 0.5 μM (0.3 μM at week 24); mean α-keto-δ-guanidinovaleric acid levels in the PEGylated arginase group decreased to less than 2 μM (1.3 μM at week 24); mean arginine levels in the PEGylated arginase group decreased to less than 1 μM (0.7 μM at week 24); and mean guanidinoacetic acid levels in the PEGylated arginase group decreased to less than 2 μM (1.7 μM at week 24). g. Guanidine compounds in renal injury

[0220] When the disease causes renal damage, toxic guanidine compounds can accumulate in the patient. Arginine produced by citrulline mainly comes from the kidney. Arginine participates in the biosynthesis of guanidine compounds and requires cooperation between organs. In renal insufficiency, citrulline can accumulate in plasma, proportional to renal damage. Therefore, it is expected that arginine and guanidine compound metabolism will be disturbed in several tissues.

[0221] Levillain et al used a nephrectomy model based on ligation of renal artery branches, allowing for simultaneous measurement of arginine and guanidinyl compound metabolism in the injured (left) and healthy (right) kidneys.

[0222] The metabolism and / or tissue content of several guanidine compounds are disturbed. Experimentally proven toxins α-keto-δ-guanidine valeric acid and guanidinosuccinic acid (GSA) accumulate in damaged kidneys. Nephrectomy also affects the levels and metabolism of guanidine compounds in muscle and liver. Therefore, disturbances in renal metabolism and function (i.e., renal damage) can lead to the accumulation of toxic guanidine compounds.

[0223] There is also evidence that the neurotoxic substance guanidinoacetate causes epileptic seizures when it accumulates to higher than normal levels. Similarly, a reduction in the level of guanidinoacetate in a patient's plasma is associated with a reduction in the epileptogenic effects of GAA. There is also evidence that guanidinoacetate plays a pathogenic role in the development of extrapyramidal movement disorders.

[0224] It has long been recognized that uremic toxins may directly or indirectly contribute to CNS damage and neurodegeneration (Arnold et al).

[0225] Patients with chronic kidney disease (CKD) frequently suffer from neurological complications. Uremic encephalopathy is a brain dysfunction caused by the accumulation of toxins produced by acute or chronic renal failure. Guanidine compounds have long been associated with uremic encephalopathy, such as guanidinosuccinic acid (GSA), methylguanidine, guanidine and creatinine, which have been shown to increase in the serum, brain and cerebrospinal fluid of uremic patients. These compounds are known to induce convulsions in experimental settings.

[0226] With regard to the diagnosis and management of CKD, EEG findings can be of diagnostic value, as the extent of EEG changes correlates with the severity of encephalopathy. The typical features of the EEG in uremic neuropathy are often nonspecific, such as slowed alpha rhythm with excess delta and theta waves. The presence of triphasic sharp waves on the EEG is considered a specific feature of metabolic encephalopathy. Triphasic waves are often seen in uremic encephalopathy. h. Other diseases

[0227] Secondary GAA elevation has been demonstrated in argininemia, S-adenosylhomocysteine ​​hydrolase deficiency (SAHH deficiency), and cobalamin disorders. Arginine supplementation is also known to help mild GAA elevations. Encephalopathy. i. Guanidine compounds in guanidinoacetate methyltransferase (GAMT) deficiency

[0228] Guanidinoacetate methyltransferase (GAMT) deficiency (MIM 601240) is an autosomal recessive inborn error of creatine synthesis that biochemically reflects creatine deficiency and significant accumulation of guanidinoacetate (GAA) in the brain and body fluids, resulting in physical or mental disabilities such as global developmental delay / intellectual disability (DD / ID), epilepsy, movement disorders, speech or language delay, and behavioral problems. Affected individuals display significant expressive speech impairment, autistic features, and diverse neurological manifestations, including epilepsy and movement disorders. GAA in affected patients is thought to be neurotoxic.

[0229] Strategies to reduce guanidinoacetate (GAA) levels include substrate deprivation through arginine-restricted diets and competitive inhibition of arginine glycineamidinotransferase (AGAT) activity through high-dose L-ornithine supplementation. AGAT is the enzyme responsible for GAA synthesis. Although medical diets designed to reduce arginine intake and reduce systemic arginine availability have been shown to be effective in reducing GAA levels and improving disease manifestations associated with high GAA levels, the required diets are problematic for many reasons. Guanidine compounds in adenosylhomocysteine ​​hydrolase deficiency

[0230] The AHCY gene encodes S-adenosylhomocysteine ​​hydrolase (EC 3.3.1.1), which catalyzes the hydrolysis of S-adenosylhomocysteine ​​to adenosine and homocysteine. Elevated guanidinoacetate levels have been observed in S-adenosylhomocysteine ​​hydrolase deficiency. k. Changes in the Gross Motor Function Measure Part D (GMFM-D)

[0231] At baseline, the mean (SD) GMFM-D score for subjects in the PEGylated arginase group was 28.0 points (SD 9.61), and for subjects in the placebo group was 29.5 points (SD 12.42). At week 24, the mean (SD) GMFM-D score for subjects in the PEGylated arginase group was 30.5 points (SD 10.09), and for subjects in the placebo group was 28.2 points (SD 13.28). This change demonstrates a numerical improvement in subjects in the PEGylated arginase group.

[0232] Individual responses are summarized in waterfall plots of GMFM-D and change from baseline at Week 24 ( Fig.11A greater proportion of subjects in the pegarginase group (65.0%, N=13) had an improvement in the mean change from baseline in GMFM-E score at Week 24 compared with the placebo group (54.5%, N=6), and the magnitude of improvement was generally greater in the pegarginase subjects relative to the placebo subjects. l. Arginine and clinical outcomes Individual and combined responses

[0233] Considering the normalization of arginine levels and comprehensive functional assessment after 24 weeks of treatment, the responses at the subject level and overall among the evaluable subjects indicated a differential response favoring subjects treated with pegarginase compared with placebo.

[0234] Heat maps were developed to represent subjects who met the threshold for normalization of arginine levels and met the response criteria in the 2MWT, GMFM-E, and GMFM-D to characterize the relationship between arginine level reduction and clinical outcomes ( Fig.12 ). In the pegarginase-treated group, 13 of 17 subjects with normalized arginine levels who were evaluable for response met criteria for meaningful clinical improvement in one or more assessments, while no subject in the placebo group had normalized arginine levels and corresponding clinical improvement, although 4 of 9 evaluable subjects did have clinically meaningful improvement in 1 assessment domain. Regardless of worsening and the absence of worsening, 8 and 6 subjects in the pegarginase group, respectively, met ≥2 clinical response criteria, and 0 subjects in the placebo group. m. Improvement in spasticity and other neurological / neuromotor performance measures using the modified Ashworth Scale

[0235] The modified Ashworth scale (MAS) is developed to assess spasticity in subjects with central nervous system lesions and is used to measure the resistance to passive motion around joints due to spasticity. The scale uses a scoring scale of 0 (no spasticity) to 4 (complete stiffness). Therefore, the reduction in the MAS score indicates an improvement in spasticity. The average MAS lower body score is defined as the mean value of the values ​​of all muscle groups with a score value>0.

[0236] At baseline, the mean (SD) MAS lower body scores were 0.77 (SD 1.076) in the pegarginase group and 0.25 (SD 0.433) in the placebo group. As of Week 24, there was a numerical improvement in the pegarginase group compared to placebo. The mean changes from baseline in MAS lower body scores for subjects in the pegarginase group and the placebo group were -0.16 (SD 0.519) and -0.03 (SD 0.056), respectively. These improved scores indicate that 4 of 5 subjects with baseline spasticity in the pegarginase group showed improvement, while only 1 of 2 subjects in the placebo group showed improvement. n. Nutritional management of patients

[0237] Clinical work on urea cycle disorders (UCD) has revealed that there is considerable variation in protein tolerance, with age, sex, and genotype contributing to this variation. Patients with the mildest forms of UCD can tolerate a normal diet, whereas patients with severe forms of UCD require a very restricted diet. The goal of treatment for patients with UCD is to maintain normal plasma ammonia and amino acid concentrations with nutritional management and drug therapy. Nutritional therapy for UCD consists of a restricted protein diet supplemented with a mixture of EAAs to ensure adequate intake of essential and conditionally essential amino acids. Sufficient protein-free energy must be administered to allow normal turnover of protein required for all ages while preventing net catabolism of body protein.

[0238] Long-term management seeks to minimize the nitrogen load on the urea cycle. The amount of native protein tolerated by each patient must be determined individually and titrated against ammonia. The Food and Agriculture Organization of the WHO has set "safe levels of protein intake", calculated as age-adjusted means, which can be used as a guide. Lower protein intakes may still be adequate, but over-restriction, individualized, may impair growth and lead to metabolic instability. If intake is too low, additional supplementation may be indicated. Adequate energy supply must also be guaranteed to prevent catabolism and subsequent hyperammonemia. The FAO / WHO / UNU 2007 report (in Fig.13 The energy intake of patients with reduced activity will have lower energy expenditure and therefore lower energy requirements.

[0239] Essential amino acids (also known as indispensable amino acids) are amino acids that cannot be synthesized by humans and other vertebrates from metabolic intermediates. These amino acids must be provided from an exogenous diet because the human body lacks the metabolic pathways required to synthesize these amino acids. Although this may vary depending on the metabolic state of the individual, there are nine essential amino acids: phenylalanine, valine, tryptophan, threonine, isoleucine, methionine, histidine, leucine, and lysine. These essential amino acids can be obtained from a single naturally occurring protein source (e.g., an animal-based nutritional source). Non-essential amino acids, also known as dispensable amino acids, may not be included in the diet. The human body can synthesize these amino acids using only the essential amino acids.

[0240] When native protein tolerance is too low to achieve normal growth and metabolic stability, supplementation with EAA and other essential nutrients is required. Currently, there is no method to increase native protein tolerance.

[0241] The typical supplementation approach for UCD is to provide 20% to 30% of total protein intake as EAA supplements. However, protein restriction in patients with ARGD1 may be more severe, and supplementation with EAAs may be as high as 50% of total protein. EAAs may be given as equally divided doses with 3 or 4 main meals to improve utilization and prevent nitrogen overload.

[0242] Deviations from the metabolic state of a standard healthy adult can place the body in a metabolic state where it requires more than the standard essential amino acids to achieve nitrogen balance. Infants and children may also require more than the standard essential amino acids. In general, the optimal ratio of essential to non-essential amino acids needs to be balanced based on the varying physiological requirements between individuals, including different disease states.

[0243] Although there are individual differences, the World Health Organization estimates the average total protein requirement to be 0.66 g / kg / day, the intake of essential amino acids to be approximately 0.18 g / kg / day, and the intake of dispensable amino acids to be 0.48 g / kg / day.

[0244] Protein aversion is a common feature of UCD. Neonatal symptoms of UCD usually appear after the first 24 to 48 hours after birth in infants born at term following a normal pregnancy and delivery. Common presenting symptoms are poor feeding, vomiting, lethargy, hypotonia, respiratory distress, irritability, and seizures. A detailed history obtained after the onset of severe neurological symptoms has been reported in patients revealing severe, previously undetected protein aversion. Many children go through a period of picky eating behavior during the first few years of life. Children with UCD develop protein aversion and are particularly picky about meat, eggs, and dairy products, but do like fruits and vegetables, which is contrary to most "normal" picky children, who tend to have more problems with vegetables. Patients may experience metabolic deterioration due to non-compliance with the prescribed diet. It has been shown that patients may eat more protein than recommended due to reluctance to give up favorite foods, low acceptance by their families, or severe peer pressure. o. Food records

[0245] Prior to enrollment in the study, disease management for all 32 subjects included treatment with dietary protein restriction and amino acid supplementation, consistent with current guidelines for the management of ARG1-D. The study sites were instructed to minimize changes in the subjects' dietary protein intake to within 15% of baseline to keep the diet as stable as possible throughout the study.

[0246] At baseline, median total prescribed protein (including native and EAA proteins) was similar between treatment groups, whereas median total prescribed calories were lower in the peg-arginase group relative to the placebo group. At baseline, median total consumed calories and total consumed protein (including native and EAA proteins) were lower in the peg-arginase group than in the placebo group.

[0247] From week 12 to week 24, the changes from baseline in median total prescribed EAA protein and total consumed EAA protein were both 0.0.

[0248] The median change from baseline in total prescribed native protein was 0.0 g for both treatment groups at Weeks 12 and 24. Small fluctuations in the median change from baseline in total consumed native protein at Weeks 12 and 24 were observed in the peg-arginase group (-0.2 and -0.5, respectively) and the placebo group (0.1 and 0.7, respectively). p.Dietary management plan (and 3-day diary diet record)

[0249] In general, the total calories consumed, total protein consumed / day, and total natural protein consumed by most subjects in the PEG arginase group increased by >15% or remained within 15%. For total consumption of EAA protein, most subjects remained within 15% of baseline. However, by the 24th week, the total protein consumption of a higher proportion of subjects in the PEG arginase group increased by more than 15% (42.9% [9 subjects] relative to 18.2% [2 subjects]) (Table 6) compared with placebo. Although the proportion of subjects with higher total protein intake was higher, the subjects had normalized arginine, and therefore, dietary fluctuations did not negatively affect the ability of PEG arginase to control arginine. Table 6 Overview of dietary management Note: N in the title represents the total number of subjects in each treatment group in the full analysis set. The percentages are based on the total number of subjects in each corresponding treatment group.

[0250] Unlike fats and carbohydrates, proteins are not stored in the body but exist in a state of equilibrium between anabolism (formation) and catabolism (breakdown). Although hyperammonemic crises are less common in patients with arginase deficiency than in other urea cycle defects, they can still occur throughout infancy and adulthood. Current treatment of ARG1-D focuses on attempts to lower plasma arginine levels through lifelong dietary protein restriction. Protein intake is restricted to the minimum required to maintain protein biosynthesis and growth.

[0251] Typically, half or more of the dietary protein is given in the form of an essential amino acid (EAA) mixture that does not contain arginine. Typical physician-recommended protein intake for healthy people is about 10 to 53 grams per day, while the recommended intake for patients with arginase 1 deficiency is about 4 to 40 grams per day (about 25% to 60% less protein). The challenge of adhering to a protein-restricted diet strict enough to lower plasma arginine to below target levels places a significant burden on patients and their families. Adherence to this type of diet can be difficult due to previously established eating habits; may exacerbate eating disorders; and requires regular follow-up to assess disease status. q.EEG

[0252] As a whole, there were no statistically significant differences in EEG results between treatment groups, but individual patients did show improvement. At baseline, except for 1 subject, 28.6% (6 subjects) from the pegylated arginase group and 45.5% (5 subjects) from the placebo group had abnormal clinically insignificant EEGs. At baseline, one subject from the pegylated arginase group had an abnormal clinically significant EEG result. The subject experienced generalized epileptiform discharges. Subjects in the placebo group did not have abnormal clinically significant EEGs.

[0253] At week 24, 23.8% (5 subjects) from the PEG arginase group and 63.6% (7 subjects) from the placebo group had abnormal EEG, but no clinically significant results. Three subjects from the PEG arginase group with (clinically insignificant) abnormal EEG at baseline converted to normal EEG at week 24; all subjects from the placebo group with clinically insignificant abnormal EEG results at baseline had abnormal clinically insignificant EEG results at week 24. At baseline, one subject from the PEG arginase group and two subjects from the placebo group had normal EEG results, and then had abnormal clinically insignificant EEG results at week 24. One subject from the PEG arginase group had an abnormal clinically significant EEG, which was recorded as a pathological wake-sleep EEG. No subject in the placebo group had an abnormal clinically significant EEG. r. EEG and guanidine compounds

[0254] The electroencephalogram (EEG) patients with altered EEG states (description of changes with guanidine compounds) are shown in Table 7 below. Table 7 *Normal range: guanidine acetic acid (0.4 to 3.0 μM), α-keto-d-guanidine valerate (ULN < 0.05 μM), α-N-acetylarginine (0.025 to 0.255 μM), arginine (0.025 to 0.1 μM); Abbreviations: H = above reference range; L = below reference range. 1.7 Patient Summary

[0255] 101-0002: At screening (week 0), the EEG was abnormal and not clinically significant (NCS). After 24 weeks of treatment with pegylated arginase, the EEG was normal. During the screening period, the patient had levels of aN-acetylarginine (NAArg), arginine (AA), and guanidinoacetic acid (GAA) above the normal range. At week 24 of treatment with pegylated arginase, GAA and NAArg had dropped to within the normal range. AA is still considered high, but has decreased from 3.39 to 4.17uM to 0.62 to 0.87uM. From screening (week 0) to week 24 of treatment with pegylated arginase, GVA remained high, but had decreased from 7.21 to 9.1uM to 0.96 to 1.91uM.

[0256] 107-0001: At screening (week 0), EEG was abnormal and not clinically significant (NCS). After 24 weeks of treatment with pegylated arginase, EEG was normal. At screening, this patient's NAArg was 0.12 (normal) to 2.77 (high), and at week 24 it was BLQ to 0.14uM (normal). AA remained above the normal range at screening and treatment week 24, but had decreased from 1.06 to 2.27 uM to 0.11 to 0.36 uM. GAA remained within the normal range (although one measurement at week 24 was BLQ). GVA remained above the normal range, but had decreased from 2.24 to 4.57 at screening to 0.13 to 1.41 uM at week 24.

[0257] 125-0002: At screening (week 0), the EEG was abnormal and not clinically significant (NCS). After 24 weeks of treatment with pegylated arginase, the EEG was normal. At screening, the patient had NAArg, GVA, AA, and GAA levels above the normal range. At week 24 of treatment, NAArg and GAA were within the normal range. AA remained above the normal range but had decreased from 4.69 to 6.80 uM to 0.32 to 0.71 uM. GVA remained above the normal range but had decreased from 3.27 to 5.64 at screening to 0.45 to 1.19 at week 24.

[0258] 129-0001: EEG was abnormal, clinically significant (CS) at screening, and abnormal, clinically non-significant (NCS) after week 24 of pegarginase treatment. GAA was within the normal range at screening, which was maintained at week 24 of treatment. Levels of NAArg, GVA, and AA were above the normal range at screening. At week 24 of treatment, AA was above the normal range but had decreased from 2.11 to 2.81 uM to 0.38 to 0.61 uM. At week 24 of treatment, NAArg was between the normal and high range and had decreased from 1.4 to 1.92 (H) uM to 0.25 (N) to 0.53 (H) uM. At week 24 of treatment, GVA was above the normal range but had decreased from 3.47 to 5.57 (H) to 0.65 to 1.05 (H).

[0259] 147-0002: EEG was normal at screening, but at week 24 of pegarginase treatment, EEG was abnormal and not clinically significant (NCS). NAArg was 0.12 (N) to 0.34 (H) at screening. NAArg was within the normal range at week 24 of treatment. GAA levels were above the normal range at screening, but at week 24 of treatment, GAA was within the normal range. AA was above the normal range at screening and remained so at week 24, although it had decreased from 1.27 to 2.7 uM at screening to 0.2 to 0.46 uM at week 24 of treatment. GVA remained above the normal range, although it had decreased from 1.62 to 3.16 at screening to 0.33 to 0.73 at week 24 of treatment.

[0260] 151-0001: EEG was normal at screening, but at week 24 of treatment with pegylated arginase, EEG was abnormal and clinically significant (CS). At screening, NAArg was 1.00 to 1.70 (above normal range). At week 24 of treatment, NAArg levels were between normal and high (0.13 (N) to 0.35 (H)). AA was above the upper limit of normal at screening and remained above normal after week 24 of treatment, although it had decreased from 2.39 to 3.54 to 0.55 to 1.43. GAA was above the upper limit of normal at screening, 3.37 to 3.87. At week 24 of treatment, GAA decreased to 0.33 (L) to 1.27 (N). GVA was above normal levels, but had decreased from 4.30 to 9.82 at screening to 1.49 to 3.13 at week 24 of treatment.

[0261] 124-0002: At screening, EEG was normal, but at week 24 of receiving placebo, it was abnormal, NCS. At screening, NAArg was above the upper limit of normal and remained above the upper limit of normal throughout the placebo period (0.44 to 2.27 at week 0 and 0.74 to 1.81 at week 24). AA was above normal at screening and remained above normal throughout the placebo period (0.67 to 3.19 at week 0 and 2.36 to 3.48 at week 24). At screening, GVA was 0.81 to 4.04 and 3.80 to 7.60 at week 24, i.e., remained above the normal range. At screening, GAA was 1.06 (N) to 5.80 (H), i.e., between normal and high levels. At week 24, GAA was 4.51 (H) to 6.34 (H), i.e., above the normal range.

[0262] 157-0001: EEG was normal at screening and abnormal at week 24 on placebo, NCS. NAArg, GVA, and AA were above the upper limit of normal at screening and remained above the upper limit of normal at week 24 on placebo. GAA was 2.63 (N) to 3.19 (H) at screening and 2.51 (N) to 3.6 (H) at week 24. 1.8 Pharmacokinetic Analysis

[0263] Adequate PK exposure was achieved with IV administration in the dose range of 0.05 to 0.2 mg / kg, which was consistent with the dose-response relationship observed in previous studies. The median time to maximum observed PEGylated arginase concentration (t max ) generally appeared rapidly (≤4.7 hours after start of infusion), as expected after a nominal 0.5 hour QW IV infusion of pegarginase. max and AUC 0-168 ) was increased in an approximately dose-proportional manner over the dose range of 0.05 to 0.2 mg / kg after repeated QW dosing (Weeks 12 and 24) to achieve steady-state. Mean t 1 / 2 The duration of the study was approximately 40 hours (range: 37.3 to 43 hours), which was similar across doses and whether a single dose was given or the steady state was reached. Based on the available data and the timing of sampling, steady state was reached at or before Week 12; however, it is theoretically expected to be achieved at or before Week 12 based on t 1 / 2 The exposure was achieved after 2 weeks of continuous QW dosing. Exposures at Weeks 12 and 24 were relatively constant relative to Week 1, with negligible accumulation after weekly dosing. 1.9 Pharmacodynamic Analysis

[0264] Rapid onset of action of PEGylated arginase1 / 2 Lead to early, consistent and continuous reduction of plasma arginine.At all time points (week 1, week 12 and week 24) evaluated after administration, arginine remained below the upper limit of normal (115 μM) in most QW dosing intervals.At week 12 and week 24, for all subjects and all dosages, the plasma arginine level after administration was usually lower than the baseline value, and except for 1 subject, it was lower than the guideline recommended level (<200 μM).After IV administration, early observations were made of a shift lower than 40 μM, with most subjects approaching the normal range at 96 hours and all subjects having a level higher than 40 μM at 168 hours. 1.10 Conclusion on effectiveness Double-blind period

[0265] The purpose of this study was to demonstrate the efficacy of peg-arginase relative to placebo based on statistically significant reductions in plasma arginine concentrations and neuromotor assessments. 1 / 2 This resulted in early, consistent, and sustained reductions in plasma arginine levels and improvements in mobility through Week 24 of the placebo-controlled, double-blind period. At baseline, mean plasma arginine was 365.44 μM [SD 93.682] in the PEGylated arginase group and 471.74 [SD 79.928] μM in the placebo group. After 24 weeks of treatment with PEGylated arginase, mean plasma arginine values ​​in the PEGylated arginase group showed an early, consistent, and sustained decrease.

[0266] Plasma arginine levels were reduced by 76.7% compared to placebo (p<0.0001), reaching both within treatment guidelines (<200 μM) and normal levels.

[0267] At weeks 12 and 24, nearly all subjects taking peg-arginase achieved plasma arginine values ​​<200 μM (95.2% [20 / 21] and 90.5% [19 / 21], p<0.0001).

[0268] At weeks 12 and 24, nearly all subjects achieved plasma arginine values ​​within the normal range (40 to 115 μM) (85.7% [18 / 21] and 90.5% [19 / 21], p<0.0001).

[0269] At Week 24, the mean improvement from baseline in functional activity measured by GMF E was 4.2 in the pegarginase group compared to a decrease of 0.4 in the placebo group (N=20). The LS mean difference was 4.6 at Week 24 (p=0.1087), demonstrating a trend toward clinically meaningful improvement. This improvement exceeds the minimal clinically important difference in patients with similar neurological motor disorders, which is applicable to ARG1-D based on a psychometric analysis of patients with this condition.

[0270] Improvements from baseline in mean distance walked in 2 minutes as measured by the 2MWT were demonstrated at Week 24 of the double-blind period. For the double-blind period, the mean improvement from baseline for subjects treated with pegarginase was 7.3 meters (N=19) at Week 24, compared to 2.3 meters in the placebo group (N=10). At Week 24, the LS mean difference between the pegarginase group and the placebo group was 5.5 meters, representing a numerical improvement (p=0.5961).

[0271] When treated with peg-arginase compared with placebo, subjects with GMFCS classification ≥ II showed greater improvements in GMFM-E and 2MWT compared with subjects with GMFCS I.

[0272] In subjects treated with PEGylated arginase, increases in ornithine and decreases in GC of approximately 50% to 70% were accompanied by decreases in plasma arginine, whereas no meaningful changes were found in the placebo group.

[0273] At Week 24, the mean improvement from baseline in GMFM-D was 2.5 in the pegarginase group compared with -1.3 in the placebo group (N=20), indicating a numerical improvement that is potentially clinically relevant.

[0274] At Week 24, the mean (SD) improvement from baseline in the Vineland Adaptive Behavior (VABS-II) composite score was 1.4 (SD 16.54) in the peg-arginase group and -1.6 (SD 8.78) in the placebo group. At Week 24, the LS mean difference was 4.9, indicating that the numerical improvement favored peg-arginase. At Week 24, the proportion of eligible subjects meeting clinical response criteria was greater for each individual component (2MWT, GMFM-D, GMFM-E) in the pegylated arginase group compared with the placebo group (pegylated arginase N=17 and placebo N=9). ο2MWT, N=7 subjects met the criteria: -PEG-arginase group: 29.4% (N=5) - Placebo group: 22% (N=2) οGMFM-E, N=11 subjects met the criteria: -PEG-arginase group: 52.9% (N=9) - Placebo group: 18.2% (N=2) oGMFM-D, N=7 subjects met the criteria: -PEG-arginase group: 41.1% (N=7) - Placebo group: 0% (N=0)

[0275] The composite clinical response was similar in the peg-arginase group (41.2%; N=7) compared with the placebo group (44.4%; N=4) for the response to any individual endpoint, when no worsening of any response in any individual component (response criteria definition 1) was also considered.

[0276] Using the composite clinical response (response criteria definition 2, response with or without worsening), there was a clinically meaningful difference between pegarginase and placebo in favor of pegarginase, with a numerically higher number of responders in the pegarginase group (64.7%; N=11) compared with the placebo group (44.4%; N=4).

[0277] A higher percentage of subjects met the criteria for a clinically meaningful response at 2 or more assessments in the pegylated arginase group (47.0%; N=8 regardless of exacerbations, 35.3% N=6 without exacerbations) compared to the placebo-treated group (none). In addition, a higher percentage of subjects achieved a clinical response in all 3 measures in the pegylated arginase group (11.7%; N=2) compared to the placebo group (none). The magnitude of responses was generally higher in subjects treated with pegylated arginase compared to placebo.

[0278] In the peg-arginase group, the mean improvement from baseline in the MAS lower body score was 0.13 (N=7) at week 24. In the placebo group, the mean improvement from baseline in the MAS lower body score was 0.04 (N=5) at week 24. This difference may be clinically relevant.

[0279] No clear differences were observed in other endpoints (ie, 9-hole pegboard dexterity test, caregiver and clinician global impression of change, responders to VABS-II, FMS / GFAQ, PEDsQL, neurocognitive assessment).

[0280] Following QW IV administration, PK exposure increased in an approximately dose-proportional manner across the dose range evaluated, with negligible accumulation, and was associated with a decrease in arginine.

[0281] Antidrug antibodies were rare, transient, and resolved, with no meaningful clinical impact. Hyperammonemia events

[0282] Overall, 21.9% of subjects (N=7) experienced hyperammonemic episode AEs, with a smaller percentage in the pegarginase group (14.3% [N=3]) compared to placebo. A lower percentage of subjects reported hyperammonemia in the pegarginase group (9.5% [N=2]) compared to the placebo group (27.3% [N=3]). Hyperammonemic encephalopathy was experienced by 4.8% of subjects in the pegarginase group (N=1) and 9.1% of subjects in the placebo group (N=1). No TEAEs of hyperammonemic crisis were reported. Example 2: Brief Overview of PEACE

[0283] As described above, PEACE was designed to evaluate the effect of treatment with pegylated arginase (n=21) versus placebo (n=11) from baseline through a pre-specified 24-week treatment period. 1.1 Topline Results

[0284] The primary endpoint was met, with a highly statistically significant 76.7% reduction in mean plasma arginine in patients treated with peg-arginase compared to placebo (p<0.0001).

[0285] 90.5% of the pegarginase-treated patients achieved normal plasma arginine levels (40 to 115 μM), compared to no patients in the placebo group.

[0286] There were concomitant improvements in key secondary activity assessments in patients treated with peg-arginase compared with patients in the placebo group. o Gross Motor Function Measure Part E (GMFM-E): The least squares mean score improved by 4.2 units in patients treated with pegarginase and worsened by 0.4 units in the placebo group (p=0.1087), establishing a positive trend. o 2-minute walk test (2MWT): The least square mean distance improved by 7.4 m in patients treated with peg-arginase and by 1.9 m in the placebo group (p=0.5961).

[0287] Pegylated arginase was well tolerated and safety data were consistent with those generated in previous clinical trials. Adverse events were generally mild to moderate in severity. No study discontinuations were due to adverse events. 1.2 Patient-level outcome analysis

[0288] In an analysis of individual patients with Gross Motor Function Classification System (GMFCS) grades I to III with predetermined clinical response criteria, clinically important differences were seen between patients treated with pegarginase (n=17) and placebo (n=9). o Eleven patients (65%) treated with pegarginase met or exceeded pre-specified response criteria for at least one activity assessment, compared with four patients (44%) receiving placebo. o Eight patients (47%) met or exceeded pre-specified clinical response criteria for at least two active outcomes, compared with no patients receiving placebo.

[0289] Six of the patients who met or exceeded the clinical response threshold for at least two active outcomes also did not show deterioration in any other active endpoint. Additional analyses were performed on these six patients to compare improvements with age- and sex-matched criteria (Table 8 below). Table 8 Analysis of patient-level outcomes Age is age at recruitment; according to NIH toolbox, standardized definitions for age / sex ±15% of the average distance of the matching *Reflects changes from Week 12; Distance at baseline was not assessed due to age 1.3 Additional Secondary Efficacy Endpoints

[0290] In a post hoc analysis correcting for missing assessments that were inappropriately scored as 0 rather than "not assessed," the least squares mean GMFM-D score improved by 2.25 units from baseline in patients treated with pegarginase compared with placebo (p = 0.0896). Patients treated with pegarginase also showed statistically significant biochemical improvements in measures of ornithine and guanidino compounds compared with placebo, consistent with the mechanism of action of pegarginase ( Fig.14 ). Example 3: Adaptive drug delivery simulation

[0291] Based on accumulating patient data from Phase 3, dosing simulations were run to evaluate various dosing strategies.

[0292] The starting dose is administered for four weeks, and starting in Week 5, the dose can be titrated based on the value of the L-arginine sample 168 hours after dosing using the following algorithm: o If L-arginine > 150 uM 168 hours after any dose, increase the dose with an upper limit of 0.2 mg / kg. o If L-arginine < 50 uM 168 hours after two consecutive doses, reduce the dose.

[0293] The dosing algorithm was optimized to ensure that L-arginine remained within 50 to 150 uM for 168 hours after dosing. Fig.15 Dosing strategy as shown in:

[0294] In Scenario 1 (0.1 / 0.1 mg / kg), 0.1 mg / kg was sufficient to maintain L-arginine control for approximately 75% of subjects. A starting dose of 0.05 mg / kg was sufficient to maintain L-arginine control for approximately 44% of subjects. Similar results were observed for the SC route of administration. Fig.16 and Fig.17 ) 1.1 Relationship between baseline L-arginine and final dose ( Fig.18 )

[0295] Regardless of dosing strategy, subjects with higher baseline L-arginine tended to require a higher final maintenance dose. Similar results were observed for the SC route of administration. 1.2 Number of dose titrations required for IV route ( Fig.19 )

[0296] Regimen 1 (0.1 / 0.1 mg / kg) required the least number of dose titrations, whereas Regimen 3 (0.05 / 0.05 mg / kg) required the most dose titrations (this was a result of the starting dose and the size of the dose escalation). Similar results were observed for the SC route of administration.

[0297] Doses of 0.05 to 0.2 mg / kg (IV and SC) resulted in L-arginine <200 uM (guideline recommended level) for nearly the entire dosing interval (>98%), with similar levels of control regardless of dosing strategy.

[0298] Compared to IV dosing, SC dosing in all dosing regimens resulted in tighter control of L-arginine within the dosing interval (less time < 40 uM and more time between 40 and 115 uM). The L-arginine values ​​at 168 hours after dosing correlate with the control of L-arginine during the dosing interval and can be used for monitoring and dose titration decisions.

[0299] Dosing of 100 to 200 μM L-arginine for 168 hours after administration appears to minimize the time that L-arginine remains <40 uM while maximizing the time that L-arginine remains in the normal range and below guideline recommended levels.

[0300] Dose increments of 0.05 mg / kg allow for greater flexibility in dosing, however more dose titration is required. 1.3 Differences between IV and SC administration ( Fig. 20 )

[0301] Two subjects weighing 31 were mock-treated (in the absence of any anti-drug antibodies). Eight 0.05 mg / kg doses were administered QW. One subject received IV dosing and one subject received SC dosing.

[0302] Compared to IV dosing, SC dosing resulted in an overall lower concentration of PEGylated arginase, so the reduction in L-arginine was less dramatic. Compared to IV dosing, SC dosing had less fluctuation in PEGylated arginase concentrations, so the response lasted for a longer period of time. 1.4 L-arginine time within the recommended range ( Fig.21 )

[0303] For almost the entire dosing interval, all dosing strategies and routes of administration result in L-arginine remaining <200uM. Compared with the IV route, the SC route minimizes the time of L-arginine <40uM. Similarly, compared with IV administration, SC administration results in L-arginine remaining 40 to 200uM and 40 to 115uM (normal range) for a longer period of time. *****

[0304] Those skilled in the art will appreciate that the above embodiments may be modified without departing from the broad inventive concept thereof. Therefore, it should be understood that the present invention is not limited to the disclosed embodiments, but is intended to include modifications within the spirit and scope of the present invention as defined by this specification.

[0305] Various publications, articles, and patents are cited or described throughout the background and specification, each of which is incorporated herein by reference in its entirety. Discussions of documents, acts, materials, devices, articles, and the like that have been included in this specification are for the purpose of providing a background for the present invention. Such discussion does not admit that any or all of these matters constitute part of the prior art with respect to any invention disclosed or claimed. References Amayreh W, Meyer U, Das AM, et al. Treatment of arginase deficiencyrevisited: guanidinoacetate as a therapeutic target and biomarker fortherapeutic monitoring Dev Med Child Neurol.2014;56(1):1021-1024. American Thoracic Society.ATS Statement:Guidelines for the six-minutewalk test.Am J Respir Crit Care Med.2002;166:111-117. 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Claims

1. A method of treating arginase 1 (ARG1) deficiency (ARG1-D) in a subject, the method comprising administering polyethylene glycol arginase to the subject, wherein the PEGylated arginase is PEGylated human arginase 1 comprising a cobalt metal cofactor, and The polyethylene glycol arginase is administered at a dose of about 0.05 mg / kg to about 0.2 mg / kg per week.

2. The method of claim 1, wherein after 12 doses of the polyethylene glycol arginase, the subject's plasma arginine level is about 40 μM to about 115 μM.

3. The method of claim 1 or 2, wherein the initial dose of polyethylene glycol arginase is about 0.1 mg / kg.

4. The method of any one of claims 1 to 3, wherein a subsequent dose of the polyethylene glycol arginase is adjusted based on the subject's plasma arginine level prior to administration of the subsequent dose.

5. The method of claim 4, wherein the subsequent dose of the pegylated arginase is increased if the subject's plasma arginine level prior to administration of the subsequent dose is above about 115 μM, optionally above about 150 μM.

6. The method of claim 4, wherein a subsequent dose of the PEGylated arginase is reduced if the subject's plasma arginine level is below about 50 μM, optionally below about 40 μM, prior to administration of the subsequent dose.

7. The method of any one of claims 4 to 6, wherein subsequent doses of the polyethylene glycol arginase are adjusted by 0.01 mg / kg to 0.1 mg / kg each time based on the subject's plasma arginine level resulting from a previous dose.

8. The method of claim 7, wherein each subsequent dose of the polyethylene glycol arginase is adjusted by 0.05 mg / kg based on the subject's plasma arginine level resulting from a previous dose.

9. The method of any one of claims 1 to 8, wherein the subject has not received premedication prior to the initial dose of polyethylene glycol arginase.

10. The method of any one of claims 1 to 9, wherein the subject's plasma arginine level is at least 250 μM prior to the initial dose of polyethylene glycol arginase.

11. The method of any one of claims 1 to 10, wherein the subject is at least 2 years old.

12. The method of any one of claims 1 to 11, wherein the subject is classified as GMFCS grade II, III, IV, or V.

13. The method of any one of claims 1 to 12, wherein an ammonia scavenger is co-administered to the subject.

14. The method of any one of claims 1 to 13, wherein following one or more doses of the polyethylene glycol arginase, the subject's intake of native protein is increased.

15. The method of any one of claims 1 to 14, wherein following one or more doses of the polyethylene glycol arginase, the subject's relative intake of a mixture of essential amino acids is reduced compared to native protein.

16. The method of any one of claims 1 to 15, wherein after one or more doses of the polyethylene glycol arginase, the subject's primary source of protein intake is changed from an arginine-free (or low-arginine) essential amino acid mixture to the addition of any protein-containing dietary product.

17. The method of any one of claims 1 to 15, wherein following one or more doses of the polyethylene glycol arginase, the subject's primary source of protein intake is changed from an essential amino acid mixture without arginine (or low in arginine) to natural protein.

18. The method of any one of claims 1 to 17, wherein following one or more doses of the polyethylene glycol arginase, the subject's total protein intake is greater than 40 g / day.

19. The method of any one of claims 1 to 18, wherein following one or more doses of the polyethylene glycol arginase, the subject's total protein intake is increased by at least 15%.

20. The method of any one of claims 1 to 19, wherein following one or more doses of the polyethylene glycol arginase, the subject's natural protein intake is increased to at least 50% of total protein intake.

21. The method of claim 20, wherein following one or more doses of the polyethylene glycol arginase, the subject's natural protein intake is increased to at least 65% of total protein intake.

22. The method of any one of claims 1 to 21, wherein the subject's mobility is improved after one or more doses of the polyethylene glycol arginase.

23. The method of any one of claims 1 to 22, wherein accumulation of arginine metabolites is reduced following one or more doses of the polyethylene glycol arginase.

24. The method of any one of claims 1 to 23, wherein the subject has reduced accumulation of guanidinyl compounds in the blood and cerebrospinal fluid.

25. The method of claim 24, wherein the guanidinyl compound is selected from the group consisting of α-N-acetylarginine, α-keto-d-guanidinovaleric acid, arginine, and guanidinoacetic acid.

26. The method of any one of claims 1 to 25, wherein the polyethylene glycol arginase is administered at a concentration of 5 mg / mL.

27. The method of any one of claims 1 to 26, wherein the polyethylene glycol arginase is administered intravenously.

28. The method of any one of claims 1 to 26, wherein the polyethylene glycol arginase is administered subcutaneously.

29. The method of any one of claims 1 to 28, wherein the polyethylene glycol arginase is stored at a concentration of about 5 mg / mL.

30. The method of any one of claims 1 to 29, wherein the polyethylene glycol arginase is formulated in a buffer comprising 50 mM sodium chloride, 5 mM potassium phosphate and 1.5% glycerol (w / v) at a pH of 7.

4.

31. The method of any one of claims 1 to 30, wherein the polyethylene glycol arginase is a liquid.

32. The method of any one of claims 1 to 30, wherein the polyethylene glycol arginase is lyophilized.

33. The method of any one of claims 1 to 32, wherein the polyethylene glycol arginase is in a vial or ampoule.

34. The method of claim 33, wherein the vial or ampoule contains about 2 mg or about 5 mg of the polyethylene glycol arginase.

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