Treatment of Arginase 1 Deficiency

Abstract

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

Description

[Technical field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims priority to U.S. Provisional Patent Application No. 63 / 329,867, filed April 11, 2022, which is incorporated by reference in its entirety into this specification.

[0002] Technical Field

[0002] The present disclosure relates generally to the field of compositions comprising human Arginase 1. Methods of using the compositions are also provided herein.

[0003] 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 by reference in their entirety into this specification. [Background technology]

[0004] background

[0004] ARG1-D is a rare progressive multisystem autosomal recessive disorder (Summar 2013, Diez-Fernandez 2018, Schlune 2015, Waisbren 2018). The disease typically manifests in early childhood and is caused by a deficiency of the enzyme arginase 1 (ARG1 [EC 3.5.3.1]), which leads to two important adverse metabolic effects: accumulation of high levels of arginine and arginine-derived metabolites, leading to episodic elevations of ammonia levels, and dysfunction of the urea cycle.

[0005]

[0005] High plasma arginine levels are believed to be the primary driver of spasticity, developmental delay, and seizures that begin in early childhood and progress throughout life (De Deyn 1997, Waisbren 2018). Lower limb spasticity in early childhood impairs mobility and balance, leading to difficulties walking and climbing stairs and lack of independence. Academic performance and educational attainment are significantly affected by developmental delay and cognitive decline. Neuromuscular and neurocognitive effects result from persistently elevated arginine levels, profoundly affecting daily function and quality of life at a young age and progressively worsening over time, resulting in severe disability and early death (De Deyn 1997, Oeffinger 2008, Prasad 1997, Carvalho 2012).

[0006] In addition to the severe neuromuscular and neurocognitive symptoms that dominate the clinical picture, subjects with ARG1-D exhibit other medically significant disease-related abnormalities, including complications resulting from hyperammonemia, hepatocellular injury, malnutrition, and impaired growth function, which are the result of or accompany the complications of the disease or the requirement for treatment with severe protein restriction.

[0007]

[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 reduce arginine, with or without ammonia scavenger to prevent or treat hyperammonemia.Strict dietary protein restriction can moderately reduce plasma arginine levels to the extent that disease-related abnormalities are improved, thus supporting the usefulness of arginine reduction.

[0008]

[0008] However, this approach is inadequate as most patients continue to have significant hyperargininemia. Furthermore, feeding is difficult, especially in young children, and requires unpleasant supplementation with EAA formulas to maintain adequate essential amino acid intake (Haeberle 2012, Huemer 2016, Lambert 1991, Burrage 2015). The progressive nature of the disease highlights the significant unmet need for therapies that, regardless of current approaches, would lower arginine levels to current guidelines or even to normal levels beyond what is achievable with current standard disease management, potentially slowing or halting the progression of neuromuscular and / or neurocognitive deterioration in patients with ARG1-D. Summary of the Invention [Means for solving the problem]

[0009] overview

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

[0010]

[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, where the pegzilarginase is pegylated human arginase 1 containing a cobalt metal cofactor, and the pegzilarginase is administered weekly at a dose of about 0.05 mg / kg to about 0.2 mg / kg.

[0011]

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

[0012]

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

[0013]

[0013] In one aspect, provided herein is a treatment for Arginase 1 deficiency using cobalt-substituted Arginase 1, with or without combination with another agent.

[0014] The present disclosure also relates to a subset of Arginase 1 deficiency patients who exhibit particular signs or particular symptoms of the disease.

[0015]

[0015] The present disclosure also relates to the prevention of arginine metabolite accumulation.

[0016]

[0016] The present disclosure also relates to dietary modifications (including, but not limited to, elimination or reduction of dietary restrictions) in patients with arginine-1 deficiency treated with cobalt-substituted arginase-1. [Brief description of the drawings]

[0017] BRIEF DESCRIPTION OF THE DRAWINGS [Figure 1A] FIG. 1A shows the clinical trial design scheme. [Figure 1B] FIG. 1B shows main demographic and baseline clinical characteristics. [Diagram 2]

[0018] FIG. 2 shows a waterfall plot of arginine (μM) change from baseline at week 24 during the double-blind period. [Figure 3A]

[0019] FIG. 3A shows a box plot of arginine levels (μM) over time during the double-blind study. [Figure 3B] FIG. 3B shows the percentage of patients who achieved normalization of plasma arginine at different time points. [Figure 4A]

[0020] FIG. 4A shows the change in 2-minute walk test (meters) from baseline at week 24 during the double-blind period for each group. [Figure 4B] FIG. 4B shows a waterfall plot of the change in 2-minute walk test (meters) from baseline at week 24 during the double-blind period for each individual. [Figure 5A]

[0021] FIG. 5A shows the change in GMFM-E from baseline at week 24 during the double-blind period for each group. [Figure 5B] FIG. 5B shows a waterfall plot of the change in GMFM-E from baseline at week 24 during the double-blind period for each individual. [Figure 6]

[0022] FIG. 6 shows box plots of ornithine (μM) over time during the double-blind study period. [Figure 7]

[0023] FIG. 7 shows box plots of alpha-N-acetylarginine (μM) over time during the double-blind study period. [Figure 8]

[0024] FIG. 8 shows box plots of alpha-keto-d-guanidinovaleric acid (μM) over time during the double-blind study period. [Figure 9]

[0025] FIG. 9 shows box plots of arginine acid (μM) over time during the double-blind study period. [Figure 10]

[0026] FIG. 10 shows box plots of guanidinoacetic acid (μM) over time during the double-blind study period. [Figure 11]

[0027] FIG. 11 shows a waterfall plot of the change in GMFM-D from baseline at week 24 during the double-blind period. [Figure 12]

[0028] FIG. 12 shows the responder heatmap for evaluable subjects. [Figure 13]

[0029] FIG. 13 shows selected values ​​from the FAO / WHO / UNU safe levels of protein intake and energy requirements for infants and adults, and during pregnancy and lactation, for healthy populations. [Figure 14]

[0030] Figure 14 shows a summary of the changes from baseline at week 24 for other secondary efficacy endpoints. [Figure 15]

[0031] FIG. 15 shows the dosing strategies tested for IV and SC administration during the adaptive dosing simulation. [Figure 16]

[0032] FIG. 16 shows the distribution of the final dose via the intravenous route. [Figure 17]

[0033] FIG. 17 shows the distribution of the final dose via the subcutaneous route. [Figure 18]

[0034] FIG. 18 shows the relationship between baseline L-arginine and final dose. [Figure 19]

[0035] FIG. 19 shows the number of dose escalations for the intravenous route. [Figure 20]

[0036] FIG. 20 shows the difference between IV and SC dosing. [Figure 21]

[0037] FIG. 21 shows L-arginine times within the recommended range. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0018] Other materials

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

[0019] Detailed Description

[0039] Disclosed herein is a method of treating arginase 1 (ARG1) deficiency (ARG1-D) in a subject, comprising administering PEGylated arginase or human arginase 1 to the subject, where the PEGylated arginase or human arginase 1 is PEGylated human arginase 1 with a cobalt metal cofactor, and the PEGylated arginase or human arginase 1 is administered weekly at a dose of about 0.05 mg / kg to about 0.2 mg / kg. In an embodiment, the subject's plasma arginine level is about 40 μM to about 115 μM after treatment. In an embodiment, patients with a GMFCS classification of ≧II demonstrate greater improvement in mobility after treatment. In an embodiment, the initial dose of PEGylated arginase or human arginase 1 is about 0.1 mg / kg. In an embodiment, subsequent doses of PEGylated arginase or human arginase 1 are adjusted based on the subject's plasma arginine level prior to administration of the subsequent dose.

[0020] definition

[0040] Unless otherwise defined herein, technical and scientific terms used in this detailed description shall have the meanings commonly understood by those skilled in the art. For the purposes of interpreting this specification, the following explanations of terms shall apply where appropriate, and terms used in the singular form shall include the plural form and vice versa, unless the content clearly indicates otherwise. In the event that any explanation of a term provided contradicts any document incorporated herein by reference, the explanation of the term provided below shall prevail.

[0021]

[0041] As used herein, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise.

[0022]

[0042] As used herein, the term "and / or" can be used in this disclosure for either "and" or "or" unless otherwise indicated.

[0023]

[0043] As used herein, "about," "approximately," "substantially," and "significantly" will be understood by those of ordinary skill in the art and will vary to some extent depending on the context in which they are used. If terms are used that are not clear to persons of ordinary skill in the art given the context in which they are 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 plus or minus more than 10% of the particular term.

[0024]

[0044] The term "administering" refers to 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 intravenous (iv) or subcutaneous (sc). The compositions of the present invention can also be administered intramuscularly (im). The composition can be a peggylarginase or human arginase 1 disclosed herein. The composition can be a composition comprising a peggylarginase or human arginase 1 disclosed herein.

[0025]

[0045] The term "dietary liberalization" refers to modification of dietary intake for Arg-1 D patients undergoing any type of dietary restriction as part of disease management, including the addition of any food that is not an arginine-free (or low-arginine) essential amino acid mix.

[0026]

[0046] The term "effective amount" refers to the amount of wild-type arginase, e.g., human arginase 1, or pegylated arginase, e.g., pegylarginase, administered that has a desired effect, e.g., one or more of the following: reduction in plasma levels of arginine, arginic acid (ArgA), GVA, Na-acetylarginine (NAArg), GAA, and homoarginine (HArg). One example is administering the compound to achieve a range of each compound equivalent to the normal levels of arginine, ArgA, GVA, GAA, and / or NAArg in a normal human not suffering from ARG1 deficiency. The effective amount may also improve the patient's muscle strength, ambulatory ability (i.e., ability to run, walk, bike, and climb stairs unsupported), 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)). Normal levels of arginine are described by Luneburg, N. et al., (2011). The effective amount may vary according to factors such as the patient's weight.

[0027]

[0047] The term "Gross Motor Function Classification System" or "GMFCS" scale refers to the criteria for GMFCS Levels I-V provided below. GMFCS Level I Able to walk indoors and outdoors and climb stairs without using hands for support Able to carry out normal activities, e.g. running and jumping o Speed, balance and coordination are decreased. GMFCS Level II Ability to walk indoors and outdoors and climb stairs with handrails 〇 Non-horizontal surfaces, slopes, and crowds are inconvenient o Minimal ability to run or jump. GMFCS Level III Walking on horizontal surfaces using assistive mobility devices indoors and outdoors Able to use handrails to climb stairs Able to propel a manual wheelchair (may require assistance for long distances or on non-horizontal surfaces). GMFCS Level IV o Severely limited walking ability even with assistive devices Uses a wheelchair most of the time and is able to propel the wheelchair independently Able to participate in standing movements. GMFCS Level V Have a physical impairment that limits voluntary control of movement and the ability to maintain head and neck position against gravity Motor function of all parts of the body is impaired Unable to sit or stand on one's own, even with the aid of prosthetic devices 〇 I cannot walk on my own, but I can use an electric mobility device.

[0028]

[0048] The term "Gross Motor Function Scale" or "GMFM" scale refers to a clinical scale designed to assess gross motor function by observing a subject's ability to initiate and complete a movement. Two versions of the GMFM exist. The GMFM-88 is the original 88-item scale. Items span five domains of gross motor activity: A: lying and turning, B: sitting, C: crawling and kneeling, D: standing, and E: walking, running and jumping.

[0029]

[0049] The GMFM-66 is a 66-item subset of the original 88 items identified via Rasch analysis to best describe gross motor function in young children with cerebral palsy of various abilities. It is a unidimensional scale that provides interval scaling rather than the ordinal scaling of the GMFM-88. Items are ordered in terms of impairment, and units of change have the same meaning across the scale ranging from 0 to 100. The GMFM-66 provides information on the level of impairment for each item, thereby providing information to aid in realistic goal setting.

[0030]

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

[0031]

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

[0032]

[0052] The term "PEGylation" refers to conjugation with polyethylene glycol (PEG), which is widely used as a drug carrier given its high degree of biocompatibility and ease of modification (see, e.g., Harris et al, Clin. Pharmacokinet. 40(7): 539-51, 2001). PEG can be coupled (e.g., covalently bonded) to active agents via hydroxyl groups at the ends of the chains and through 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 novel biomaterials, which retain the biocompatible properties of PEG but have the added advantage of having multiple attachment points per molecule (providing greater drug loading). PEGylated arginase variants can be formulated according to known methods to prepare pharma- ceutically useful compositions. ARG1-D patients can be administered wild-type arginase protein (either arginase I or arginase II) that naturally contains manganese metal cofactor, or wild-type arginase protein that is pegylated and contains manganese metal cofactor. In another example, ARG1-D patients can be administered arginase with cobalt metal cofactor instead of natural manganese metal cofactor. Cobalt metal cofactor-containing arginase can be further pegylated, an exemplary form is Co-Argl-PEG (also referred to herein as AEB1 102, PEGylarginase, or Co-hArgI), for example, as described in U.S. Patent No. 8,440,184, which is incorporated herein by reference in its entirety. An exemplary form, for example, PEGylarginase, has approximately 12 5K (5000 dalton) PEG units per monomer attached to one or more lysines present in the protein sequence of PEGylarginase.

[0033]

[0053] The term "pharmaceutical or pharmacologically acceptable" refers to molecular entities and compositions that do not produce adverse, allergic or other undesirable reactions when appropriately administered to a subject, e.g., a human. The preparation of pharmaceutical compositions containing at least one arginase variant, e.g., a stabilized multimeric arginase or a pegylated arginase isolated by the methods disclosed herein, or additional active ingredients, is known to those of skill in the art in light of the teachings of the present disclosure, as exemplified by REMINGTON'S PHARMACEUTICAL SCIENCES, 18th Ed., 1990. Additionally, for animal (e.g., human) administration, it is understood that preparations should meet sterility, pyrogenicity, general safety and purity standards as required by the FDA Office of Biological Standards.

[0034]

[0054] The term "pharmaceutically acceptable carrier" includes any solvent, dispersion medium, coating, surfactant, antioxidant, preservative (e.g., antibacterial, antifungal), isotonicity agent, absorption delaying agent, salt, preservative, drug, drug stabilizer, gel, binder, excipient, disintegrant, lubricant, dye, and the like material, and combinations thereof, known to those skilled in the art (see, e.g., REMINGTON'S PHARMACEUTICAL SCIENCES, 18th Ed., 1990). Except insofar as any conventional carrier is incompatible with the active ingredient, its use in the pharmaceutical compositions is contemplated.

[0035]

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

[0036]

[0056] The terms "protein" and "polypeptide" refer to compounds that contain amino acids joined through peptide bonds and are used interchangeably.

[0037]

[0057] The term "subject" refers to animals, for example, mammals, including humans.

[0038]

[0058] The terms "treat", "treating" or "treatment" include inhibiting, slowing, halting, alleviating, ameliorating, or reversing the progression or severity of an existing symptom, disorder, condition, or disease associated with ARG1-D. Treatment can be applied prophylactically or therapeutically.

[0039] [Table 1]

[0040] [Table 2]

[0041] [Table 3]

[0042] Arginase

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

[0043]

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

[0044]

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

[0045]

[0062] Mammals have two arginase isozymes (EC 3.5.3.1) that catalyze the hydrolysis of L-arginine to urea and L-ornithine. The arginase I gene is located on chromosome 6 (6q23), is highly expressed in the cytosol of hepatocytes, and functions in nitrogen removal as the final step of the urea cycle. The arginase II gene is found on chromosome 14 (14q24.1). Arginase II is localized in mitochondria in tissues, such as kidney, brain, and skeletal muscle, where it is believed to provide a supply of L-ornithine for proline and polyamine biosynthesis (Lopez et al, FEBSJ. 272: 4540-48, 2005).

[0046]

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

[0047]

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

[0048]

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

[0049]

[0066] Arginase has been investigated for nearly 50 years as a way to degrade extracellular L-arginine (Dillon et al, “Biochemical characterization of the argininedegrading enzymes arginaseand arginine deiminase and their effect on nitric oxide production”, Med. Sci. Monit., 8(7): BR248-253(2002)). Native arginase is cleared from the circulation within minutes (Savoca et al., Cancer Biochem. Biophys. 7: 261-268, 1984), while a single injection of PEG-arginase MW5,000 in rats was sufficient to achieve near-complete arginine depletion for approximately 3 days (Cheng et al., Cancer Res. 67: 309-17, 2007).

[0050]

[0067] Bacterial arginine hydrolase, ADI, has shown good kinetics and stability and has been tested in vitro. Unfortunately, ADI is a bacterial enzyme, and therefore it induces strong immune responses and adverse effects in most patients, making it unsuitable for long-term administration in ARG1-D patients, which requires regular administration.

[0051]

[0068] For clinical use in patients with ARG1-D, it is essential to engineer arginase to allow its persistence in the circulation for an extended period of time (e.g., several days). In the absence of any modifications, human arginase has a half-life of only a few minutes in the circulation, mainly because its size is not large enough to avoid filtration through the kidney. Unmodified human arginase is highly susceptible to deactivation in serum, and it is degraded with a half-life of only 4 hours. In an embodiment, the human arginase is human arginase 1. In an embodiment, the human arginase 1 is pegylated. In an embodiment, the human arginase 1 has a cobalt metal cofactor. In an embodiment, the human arginase 1 is pegylated and has a cobalt metal cofactor. In an embodiment, the human arginase 1 comprises the amino acid sequence of SEQ ID NO:2. In an embodiment, the human arginase 1 comprises the amino acid sequence of SEQ ID NO:3. In embodiments, human Arginase 1 comprises at least one amino acid substitution in the metal binding site with reference to SEQ ID NO: 2 or 3, and / or is truncated by one or more amino acids. In embodiments, human Arginase 1 is pegylated, has a cobalt metal cofactor, and comprises the amino acid sequence of SEQ ID NO: 2. In embodiments, human Arginase 1 is pegylated, has a cobalt metal cofactor, and comprises the amino acid sequence of SEQ ID NO: 3. In embodiments, human Arginase 1 is pegylated, has a cobalt metal cofactor, comprises at least one amino acid substitution in the metal binding site with reference to SEQ ID NO: 2 or 3, and / or is truncated by one or more amino acids.

[0052]

[0069] In an embodiment, the PEGylarginase disclosed herein has the sequence of Arginase I, SEQ ID NO:2, with a cobalt metal cofactor instead of the manganese metal cofactor. In an embodiment, the PEGylarginase disclosed herein has the sequence of Arginase I, SEQ ID NO:3, with a cobalt metal cofactor instead of the manganese metal cofactor. In an embodiment, the PEGylarginase is pegylated as described in U.S. Patent No. 8,440,184 and produced as described in U.S. Patent Application Publication No. 2021 / 0189371.

[0053]

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

[0054]

[0071] PEGylation is the process of covalent attachment of PEG polymer chains to another molecule, usually a drug or therapeutic protein. "PEGylation" can be achieved by incubation of a reactive derivative of PEG with a target macromolecule. Increasing the hydrodynamic size (size in solution) of a drug or therapeutic protein increases its circulation time by reducing renal clearance. PEGylation can also provide water solubility to hydrophobic drugs and proteins. Human arginase 1, including PEGylated arginase, as disclosed herein, can be PEGylated as described in U.S. Pat. No. 8,440,184.

[0055]

[0072] The first step in PEGylation can be suitable functionalization of the PEG polymer at one or both terminal domains of the protein or internally to amino acids, e.g., lysine. PEG activated with the same reactive moiety at each end is known as "homobifunctional", while when the functional groups present are different, the PEG derivative is termed "heterobifunctional" or "heterofunctional". Chemically active or activated derivatives of PEG polymers are prepared to attach PEG to the desired molecule.

[0056]

[0073] The selection of suitable functional group for PEG derivative is based on the type of available reactive group on the molecule to be coupled to PEG.For protein, typical reactive amino acids include lysine, cysteine, histidine, arginine, aspartic acid, glutamic acid, serine, threonine and tyrosine.PEG can also be attached to polypeptide using N-terminal amino group and C-terminal carboxylic acid.

[0057]

[0074] Techniques used to form PEG derivatives include reacting PEG polymers with groups reactive with hydroxyl groups, typically anhydrides, acid chlorides, chloroformates, and carbonates. PEGylation chemistry can also use functional groups utilized in conjugation, such as aldehydes, esters, amides, etc. Heterobifunctional PEGs are extremely useful in the attachment of two entities where a hydrophilic, flexible, biocompatible spacer is required. Preferred terminal groups for heterobifunctional PEGs are maleimide, vinylsulfone, pyridyl disulfide, amine, carboxylic acid, and H-hydroxysuccinimide (NHS) esters.

[0058]

[0075] The most common modifiers, or linkers, are based on methoxypolyethylene glycol (mPEG) molecules. Their activity relies on the addition of protein-modifying groups to the alcohol termini. Polyethylene glycol (PEG diol) can be used as a precursor molecule; the diol is then modified at both termini to create hetero- or homodimeric PEG-linked molecules.

[0059]

[0076] Proteins are generally PEGylated at nucleophilic sites, such as unprotonated thiols (cysteinyl residues) or amino groups. Examples of cysteinyl-specific modification reagents include PEG maleimide, PEG iodoacetate, PEG thiol, and PEG vinylsulfone. All four are strongly cysteinyl-specific under mild conditions, at neutral to slightly alkaline pH, but each has some drawbacks. The amides formed with maleimides may be somewhat unstable under alkaline conditions, and therefore there may be some limitations to formulation options with this linker. The amide bonds formed with iodo-PEG are more stable, but free iodine may modify tyrosine residues under some conditions. PEG thiol forms disulfide bonds with protein thiols, but this bond may also be unstable under alkaline conditions. PEG-vinylsulfone reactivity is relatively slow compared to maleimide and iodo-PEG; however, the thioether bond formed is very stable. This slower reaction rate may also make the PEG-vinylsulfone reaction easier to control.

[0060]

[0077] Site-specific PEGylation at natural cysteinyl residues is rarely performed 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 thiol-specific linkers. The cysteine ​​mutation must be designed so that it is accessible to the PEGylation reagent and remains biologically active after PEGylation.

[0061]

[0078] Amine-specific modifiers include PEG NHS esters, PEG tresylates, PEG aldehydes, PEG isothiocyanates, and several others. These amine-specific agents generally react under mild conditions and are highly specific for amino groups.

[0062]

[0079] Due to the multiple lysine residues on most proteins, site-specific PEGylation can be difficult. Fortunately, because these reagents react with unprotonated amino groups, it is possible to direct PEGylation to amino groups with lower pK by performing the reaction at a lower pH. In general, the pK of the alpha-amino group of a lysine residue is 1-2 pH units lower than the epsilon-amino group. By PEGylating the molecule at pH 7 or lower, high selectivity for the N-terminus can frequently be retained. However, this is only feasible if the N-terminal portion of the protein is not required for biological activity. Nevertheless, the pharmacokinetic benefits from PEGylation frequently outweigh any significant loss in in vitro bioactivity, resulting in a product with significant in vivo bioactivity regardless of the PEGylation chemistry.

[0063]

[0080] There are several parameters to consider when developing a PEGylation procedure. Fortunately, there are usually no more than four or five major parameters. A "design of experiments" approach for optimizing PEGylation conditions can be extremely 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, the exclusion of oxygen. (Oxygen can contribute to intermolecular disulfide formation with proteins, which reduces the yield of PEGylated products.) The same factors (except oxygen) should be considered for amine-specific modifications, except that pH can be even more important, especially when targeting N-terminal amino groups.

[0064]

[0081] For both amine and thiol specific modifications, reaction conditions can affect protein stability. This can be limited by temperature, protein concentration, and pH. In addition, the reactivity of the PEG linker should be known before the start of the PEGylation reaction. For example, if the PEGylation agent is only 70 percent active, the amount of PEG used should ensure that only active PEG molecules are counted in the stoichiometry of the reaction between protein and PEG.

[0065] Pharmaceutical Compositions

[0082] In one aspect, a pharmaceutical composition comprising arginase is disclosed herein.In an embodiment, the arginase is PEGylated arginase, which is PEGylated human arginase 1 with cobalt metal cofactor.In an embodiment, the arginase is human arginase 1.In an embodiment, the human arginase 1 is PEGylated and has cobalt metal cofactor.

[0066]

[0083] The arginase and compositions comprising the same described herein can be administered systemically or locally.The arginase and compositions comprising the same can be administered intravenously, intrathecally, subcutaneously, intramuscularly, intratumorally, and / or intraperitoneally, or combinations thereof.The arginase and compositions comprising the same described herein can be administered alone or in combination with an ammonia scavenger, such as sodium phenylbutyrate, sodium benzoate, and glycerol phenylbutyrate.

[0067]

[0084] The composition containing arginase or a part thereof can be provided together with physiologically tolerable liquid, gel or solid carriers, diluents and excipients in a formulation. Such compositions are typically prepared as injections, either as liquid solutions or suspensions. Suitable diluents and excipients are, for example, water, saline, dextrose, glycerol, and the like, and combinations thereof. In addition, if desired, the composition may contain small amounts of auxiliary substances, such as wetting or emulsifying agents, stabilizers or pH buffering agents. When clinical applications are contemplated, it may be necessary to prepare pharmaceutical compositions-expression vectors, virus stock solutions, proteins, antibodies and drugs in a form appropriate for the intended application. In general, the pharmaceutical compositions of the present invention comprise an effective amount of one or more arginase variants or additional agents dissolved or dispersed in a pharma-ceutically acceptable carrier.

[0068]

[0085] A pharmaceutical composition containing arginase may contain different types of carriers depending on whether it is to be administered in solid, liquid or aerosol form and whether it needs to be sterile for routes of administration such as injection. The present invention may be administered intravenously, intradermally, transdermally, intrathecally, intraarterially, intraperitoneally, intramuscularly, subcutaneously, intratumorally, locally, by injection, infusion, continuous infusion, via catheter, in lipid compositions (e.g., liposomes), or by other methods known to those skilled in the art or any combination of the above (see, e.g., REMINGTON'S PHARMACEUTICAL SCIENCES).

[0069]

[0086] The arginase variants can be formulated in the composition in free base, neutral or salt form. Pharmaceutically acceptable salts include acid addition salts, such as those formed with free amino groups of the proteinaceous composition, 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 sodium, potassium, ammonium, calcium or iron hydroxide; or organic bases, such as isopropylamine, trimethylamine, histidine or procaine. When formulated, the solution can be administered in a manner compatible with the administration formulation and in an amount that is therapeutically effective. The formulations are easily administered in a variety of dosage forms, such as those formulated for parenteral administration, such as injection solutions or aerosols for delivery to the lungs, or those formulated for nutritional administration, such as drug release capsules.

[0070]

[0087] The composition for administration can be provided in a pharma- ceutically acceptable carrier with or without an inert diluent. The carrier should be absorbable, and includes liquid, semi-solid, i.e., paste, or solid carriers. Except insofar as any conventional medium, agent, diluent, or carrier is deleterious to the recipient or to the therapeutic effectiveness of the composition contained therein, its use in an administrable composition for use in carrying out the method of the present invention is appropriate.

[0071]

[0088] Examples of carriers or diluents include fats, oils, water, saline solutions, lipids, liposomes, resins, binders, bulking agents, etc., or combinations thereof. The composition may also include various antioxidants to retard oxidation of one or more components. In addition, the use of preservatives, such as various antibacterial and antifungal agents, including but not limited to parabens (e.g., methylparaben, propylparaben), chlorobutanol, phenol, sorbic acid, methylosal, or combinations thereof, may improve the shelf life of pharmaceutical compositions.

[0072]

[0089] The composition may be combined with the carrier in any convenient and practical manner, i.e., by dissolving, suspending, emulsifying, mixing, encapsulating, absorbing, etc. Such procedures are routine to those skilled in the art.

[0073]

[0090] Pharmaceutical lipid vehicles can be used in compositions containing arginase variants. Lipid vehicle compositions 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 can be extracted using organic solvents. Examples include compounds that contain long-chain aliphatic hydrocarbons and their derivatives. Lipids can be naturally occurring or synthetic (i.e., artificially designed or produced). However, lipids are usually biological materials.

[0074]

[0091] Biological lipids include, for example, neutral lipids, phospholipids, phosphoglycerides, steroids, terpenes, lysolipids, glycosphingolipids, glycolipids, sulfatides, lipids with ether and ether-linked fatty acids, and polymeric lipids, and combinations thereof. Of course, compounds that would be understood by one of skill in the art as lipids, other than those specifically described herein, are also encompassed by the compositions and methods of the present invention.

[0075]

[0092] The actual amount of the arginase-containing compositions described herein to be administered to a patient can be determined based on the patient's physical and physiological factors, such as body weight, severity of the condition, type of disease being treated, previous or current therapeutic interventions, idiopathic disease, and on the route of administration.

[0076]

[0093] Depending on the dosage and route of administration, the preferred dosage and / or the number of times of administration of the effective amount may vary from subject to subject.The dosage depends on the amount required by the patient to achieve normal levels of at least one or more of the following: arginine, HArg, ArgA, GVA, GAA, and NAArg.The ARG1-D patient level of one or more of these five compounds is generally assessed until a normal range is obtained in the patient's plasma.The tissue level of the five compounds may be assessed, but may not be necessary, or may be performed less frequently than the plasma level test.

[0077]

[0094] A pharmaceutical composition may, for example, contain at least about 0.1% of active compound. In embodiments, the active compound may comprise about 2% to about 75%, or about 25% to about 60%, for example, any range derivable therein, of the weight of the unit. Naturally, the amount of active compound in each therapeutically useful composition may be prepared so that a suitable dosage is obtained in any given unit dose of the compound. Factors such as solubility, bioavailability, biological half-life, route of administration, product shelf life, and other pharmacological considerations are contemplated by those skilled in the art of preparing such pharmaceutical formulations, and therefore different dosages and treatment regimes may be desirable.

[0078]

[0095] In an embodiment, the PEGylarginase is stored at a concentration of about 1 mg / mL to about 8 mg / mL. In an embodiment, the PEGylarginase is stored at a concentration of at least about 1 mg / mL. In an embodiment, the PEGylarginase is stored at a concentration of at most about 8 mg / mL. In an embodiment, PEGylarginase is from about 1 mg / mL to about 2 mg / mL, from about 1 mg / mL to about 3 mg / mL, from about 1 mg / mL to about 4 mg / mL, from about 1 mg / mL to about 5 mg / mL, from about 1 mg / mL to about 6 mg / mL, from about 1 mg / mL to about 7 mg / mL, from about 1 mg / mL to about 8 mg / mL, from about 2 mg / mL to about 3 mg / mL, from about 2 mg / mL to about 4 mg / mL, from about 2 mg / mL to about 5 mg / mL, from about 2 mg / mL to about 6 mg / mL, from about 2 mg / mL to about 7 mg / mL, from about 2 mg / mL to about 8 mg / mL, from about 3 mg / mL to about 4 mg / mL, In an embodiment, the PEGylarginase is stored at a concentration of about 1 mg / mL, about 2 mg / mL, about 3 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 an embodiment, PEGylated arginase is stored at a concentration of about 5 mg / mL.

[0079]

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

[0080]

[0097] In an embodiment, PEGylated arginase is formulated in a buffer containing 50 mM sodium chloride, 5 mM potassium phosphor, and 1.5% glycerol (w / v / ) at a pH of 7.4. In an embodiment, human Arginase 1 is formulated in a buffer containing 50 mM sodium chloride, 5 mM potassium phosphor, and 1.5% glycerol (w / v / ) at a pH of 7.4.

[0081]

[0098] In an embodiment, the peggylarginase is liquid. In an embodiment, the peggylarginase is lyophilized. In an embodiment, the peggylarginase is in a vial or ampoule. In an embodiment, the vial or ampoule contains about 2 mg or about 5 mg of peggylarginase. In an embodiment, the human Arginase 1 is liquid. In an embodiment, the human Arginase 1 is lyophilized. In an embodiment, the human Arginase 1 is in a vial or ampoule. In an embodiment, the vial or ampoule contains about 2 mg or about 5 mg of human Arginase 1.

[0082] Treatment Method

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

[0083]

[0100] In an embodiment, the arginase is a pegylated arginase that is a pegylated human arginase 1 that includes a cobalt metal cofactor. In an embodiment, the arginase is human arginase 1. In an embodiment, the human arginase 1 is pegylated and has a cobalt metal cofactor.

[0084]

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

[0085]

[0102] In an embodiment, peggylarginase is administered at a dose of about 0.01 mg / kg to about 0.5 mg / kg. In an embodiment, peggylarginase is administered at a dose of about 0.05 mg / kg to about 0.2 mg / kg. In an embodiment, peggylarginase is administered at a dose of at least about 0.01 mg / kg. In an embodiment, peggylarginase is administered at a dose of at most about 0.5 mg / kg. In an embodiment, peggylarginase is administered at a dose of at least about 0.05 mg / kg. In an embodiment, peggylarginase is administered at a dose of at most about 0.2 mg / kg. In an embodiment, PEGylated arginase is administered at a concentration of 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 ... g / kg~about 0.4mg / kg, about 0.01mg / kg~about 0.45mg / kg, about 0.01mg / kg~about 0.5mg / kg, about 0.05mg / kg~about 0.1mg / kg, about 0.05mg / kg ~0.15mg / kg, approximately 0.05mg / kg~0.2mg / kg, approximately 0.05mg / kg~0.25mg / kg, approximately 0.05mg / kg~0.3mg / kg, approximately 0.05mg / kg~0. 35mg / kg, about 0.05mg / kg to about 0.4mg / kg, about 0.05mg / kg to about 0.45mg / kg, about 0.05mg / kg to about 0.5mg / kg, about 0.1mg / kg to about 0.15mg / 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. 1mg / kg~about 0.4mg / kg, about 0.1mg / kg~about 0.45mg / kg, about 0.1mg / kg~about 0.5mg / kg, about 0.15mg / kg~about 0.2mg / kg, about 0.15mg / kg ~0.25mg / kg, approximately 0.15mg / kg~0.3mg / kg, approximately 0.15mg / kg~0.35mg / kg, approximately 0.15mg / kg~0.4mg / kg, approximately 0.15mg / kg~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 / k g ~ approx. 0.45 mg / kg, approx. 0.2 mg / kg ~ approx. 0.5 mg / kg, approx. 0.25 mg / kg ~ approx. 0.3 mg / kg, approx. 0.25 mg / kg ~ approx. 0.35 mg / kg, approx. 0.25 mg / kg ~ approx. The dose is administered at a dose of 0.25 mg / kg to about 0.5 mg / kg, about 0.3 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 embodiments, PEGylarginase 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.

[0086]

[0103] In embodiments, human Arginase 1 is administered at a dose of about 0.01 mg / kg to about 0.5 mg / kg. In embodiments, human Arginase 1 is administered at a dose of about 0.05 mg / kg to about 0.2 mg / kg. In embodiments, human Arginase 1 is administered at a dose of at least about 0.01 mg / kg. In embodiments, human Arginase 1 is administered at a dose of at most about 0.5 mg / kg. In embodiments, human Arginase 1 is administered at a dose of at least about 0.05 mg / kg. In embodiments, human Arginase 1 is administered at a dose of at most about 0.2 mg / kg. In an embodiment, human Arginase 1 is 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 ~0.4mg / kg, 0.01mg / kg~0.45mg / kg, 0.01mg / kg~0.5mg / kg, 0.05mg / kg~0.1mg / kg, 0.05mg / kg~0.1 5mg / 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 / k g, about 0.05mg / kg to about 0.4mg / kg, about 0.05mg / kg to about 0.45mg / kg, about 0.05mg / kg to about 0.5mg / kg, about 0.1mg / kg to about 0.15mg / kg, about 0. 1mg / kg~about 0.2mg / kg, about 0.1mg / kg~about 0.25mg / kg, about 0.1mg / kg~about 0.3mg / kg, about 0.1mg / kg~about 0.35mg / kg, about 0.1mg / kg~about 0 .4mg / kg, about 0.1mg / kg to about 0.45mg / 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 / k g, 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~about 0.5mg / kg, about 0.2mg / kg~about 0.25mg / kg, about 0.2mg / kg~about 0.3mg / kg, about 0.2mg / kg~about 0.35mg / kg, about 0.2mg / kg~about 0.4mg / kg, about 0.2mg / kg~about 0.45m g / 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 0.25m g / kg to about 0.5 mg / kg, about 0.3 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 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 embodiments, the weight of human Arginase 1 is based on the combined weight of human Arginase 1 and the polyethylene glycol conjugated to human Arginase 1.

[0087]

[0104] In an embodiment, the initial dose of peggylarginase is about 0.01 mg / kg to about 0.2 mg / kg. In an embodiment, the initial dose of peggylarginase is at least about 0.01 mg / kg. In an embodiment, the initial dose of peggylarginase is at most about 0.2 mg / kg. In an embodiment, the initial dose of PEGylated arginase is from about 0.01 mg / kg to about 0.03 mg / kg, from about 0.01 mg / kg to about 0.05 mg / kg, from about 0.01 mg / kg to about 0.07 mg / kg, from about 0.01 mg / kg to about 0.09 mg / kg, from about 0.01 mg / kg to about 0.1 mg / kg, from about 0.01 mg / kg to about 0.12 mg / kg, from about 0.01 mg / kg to about 0.14 mg / kg, from about 0.01 mg / kg to about 0.16 mg / kg, from about 0.01 mg / kg to about 0.1 0.03mg / kg to about 0.18mg / kg, about 0.01mg / kg to about 0.2mg / kg, about 0.03mg / kg to about 0.05mg / kg, about 0.03mg / kg to about 0.07mg / kg, about 0.03mg / kg to about 0.09mg / kg, about 0.03mg / kg to about 0.1mg / kg, about 0.03mg / kg to about 0.12mg / kg, about 0.03mg / kg to about 0.14mg / kg, about 0.03mg / kg to about 0.16mg / kg, about 0.03mg / kg to about 0.18 ... g ~ approx. 0.2 mg / kg, approx. 0.05 mg / kg ~ approx. 0.07 mg / kg, approx. 0.05 mg / kg ~ approx. 0.09 mg / kg, approx. 0.05 mg / kg ~ approx. 0.1 mg / kg, approx. 0.05 mg / kg ~ approx. 0.12 mg / kg, approx. 0.05 mg / kg~about 0.14mg / kg, about 0.05mg / kg~about 0.16mg / kg, about 0.05mg / kg~about 0.18mg / kg, about 0.05mg / kg~about 0.2mg / kg, about 0.07mg / kg~about 0.09mg / kg, about 0.07m g / kg to about 0.1 mg / kg, about 0.07 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 0.09mg / kg ~ approx. 0.18mg / kg, approx. 0.09mg / kg ~ approx. 0.2mg / kg, approx. 0.1mg / kg ~ approx. 0.12mg / kg, approx. 0.1mg / kg ~ approx. 0.14mg / kg, approx. 0.1mg / kg ~0.16mg / kg, 0.1mg / kg~0.18mg / kg, 0.1mg / kg~0.2mg / kg, 0.12mg / kg~0.14mg / kg, 0.12mg / kg~0.16 mg / kg, about 0.12 mg / kg to about 0.18 mg / kg, about 0.12 mg / kg to about 0.2 mg / kg, about 0.14 mg / kg to about 0.16 mg / kg, about 0.14 mg / kg to about 0.18 mg / kg, about 0.14 mg / kg to about 0.2 mg / kg, about 0.16 mg / kg to about 0.18 mg / kg, about 0.16 mg / kg to about 0.2 mg / kg, or about 0.18 mg / kg to about 0.2 mg / kg. In embodiments, the initial dose of peggylarginase 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 embodiments, the initial dose of peggylarginase is about 0.1 mg / kg.

[0088]

[0105] In an embodiment, the initial dose of human Arginase 1 is about 0.01 mg / kg to about 0.2 mg / kg. In an embodiment, the initial dose of human Arginase 1 is at least about 0.01 mg / kg. In an embodiment, the initial dose of human Arginase 1 is at most about 0.2 mg / kg. In an embodiment, the initial 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 0.1 ... 0.03mg / kg to about 0.18mg / kg, about 0.01mg / kg to about 0.2mg / kg, about 0.03mg / kg to about 0.05mg / kg, about 0.03mg / kg to about 0.07mg / kg, about 0.03mg / kg to about 0.09mg / kg, about 0.03mg / kg to about 0.1mg / kg, about 0.03mg / kg to about 0.12mg / kg, about 0.03mg / kg to about 0.14mg / kg, about 0.03mg / kg to about 0.16mg / kg, about 0.03mg / kg to about 0.18 ... g ~ approx. 0.2 mg / kg, approx. 0.05 mg / kg ~ approx. 0.07 mg / kg, approx. 0.05 mg / kg ~ approx. 0.09 mg / kg, approx. 0.05 mg / kg ~ approx. 0.1 mg / kg, approx. 0.05 mg / kg ~ approx. 0.12 mg / kg, approx. 0.05 mg / kg~about 0.14mg / kg, about 0.05mg / kg~about 0.16mg / kg, about 0.05mg / kg~about 0.18mg / kg, about 0.05mg / kg~about 0.2mg / kg, about 0.07mg / kg~about 0.09mg / kg, about 0.07m g / kg to about 0.1 mg / kg, about 0.07 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 0.09mg / kg ~ approx. 0.18mg / kg, approx. 0.09mg / kg ~ approx. 0.2mg / kg, approx. 0.1mg / kg ~ approx. 0.12mg / kg, approx. 0.1mg / kg ~ approx. 0.14mg / kg, approx. 0.1mg / kg ~0.16mg / kg, 0.1mg / kg~0.18mg / kg, 0.1mg / kg~0.2mg / kg, 0.12mg / kg~0.14mg / kg, 0.12mg / kg~0.16 mg / kg, about 0.12 mg / kg to about 0.18 mg / kg, about 0.12 mg / kg to about 0.2 mg / kg, about 0.14 mg / kg to about 0.16 mg / kg, about 0.14 mg / kg to about 0.18 mg / kg, about 0.14 mg / kg to about 0.2 mg / kg, about 0.16 mg / kg to about 0.18 mg / kg, about 0.16 mg / kg to about 0.2 mg / kg, or about 0.18 mg / kg to about 0.2 mg / kg. In embodiments, the initial 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 embodiments, the initial dose of human Arginase 1 is about 0.1 mg / kg.

[0089]

[0106] In an embodiment, the subsequent dose of peggylarginase is adjusted based on the subject's plasma arginine level before administration of the subsequent dose. In an embodiment, the subsequent dose of peggylarginase is increased when the subject's plasma arginine level before administration of the subsequent dose is higher than about 100 μM. In an embodiment, the subsequent dose of peggylarginase is increased when the subject's plasma arginine level before administration of the subsequent dose is higher than about 115 μM. In an embodiment, the subsequent dose of peggylarginase is increased when the subject's plasma arginine level before administration of the subsequent dose is higher than about 130 μM. In an embodiment, the subsequent dose of peggylarginase is increased when the subject's plasma arginine level before administration of the subsequent dose is higher than about 135 μM. In an embodiment, the subsequent dose of peggylarginase is increased when the subject's plasma arginine level before administration of the subsequent dose is higher than about 150 μM. In an embodiment, the subsequent dose of peggylarginase is increased if the subject's plasma arginine level before administration of the subsequent dose is greater than about 165 μM. In an embodiment, the subsequent dose of peggylarginase is increased if the subject's plasma arginine level before administration of the subsequent dose is greater than about 180 μM. In an embodiment, the subsequent dose of peggylarginase is increased if the subject's plasma arginine level before administration of the subsequent dose is greater than about 195 μM. In an embodiment, the subsequent dose of peggylarginase is increased if the subject's plasma arginine level before administration of the subsequent dose is greater than about 200 μM. In an embodiment, the subsequent dose of peggylarginase is decreased if the subject's plasma arginine level before administration of the subsequent dose is less than about 50 μM. In an embodiment, the subsequent dose of peggylarginase is decreased if the subject's plasma arginine level before administration of the subsequent dose is less than about 40 μM. In an embodiment, the subsequent dose of PEGylarginase is decreased if the subject's plasma arginine level before administration of the subsequent dose is less than about 30 μM. In an embodiment, the subsequent dose of PEGylarginase is decreased if the subject's plasma arginine level before administration of the subsequent dose is less than about 20 μM.

[0090]

[0107] In an embodiment, the subsequent dose of human Arginase 1 is adjusted based on the subject's plasma arginine level before administration of the subsequent dose. In an embodiment, the subsequent dose of human Arginase 1 is increased if the subject's plasma arginine level before administration of the subsequent dose is greater than about 100 μM. In an embodiment, the subsequent dose of human Arginase 1 is increased if the subject's plasma arginine level before administration of the subsequent dose is greater than about 115 μM. In an embodiment, the subsequent dose of human Arginase 1 is increased if the subject's plasma arginine level before administration of the subsequent dose is greater than about 130 μM. In an embodiment, the subsequent dose of human Arginase 1 is increased if the subject's plasma arginine level before administration of the subsequent dose is greater than about 135 μM. In an embodiment, the subsequent dose of human Arginase 1 is increased if the subject's plasma arginine level before administration of the subsequent dose is greater than about 150 μM. In an embodiment, the subsequent dose of human Arginase 1 is increased if the subject's plasma arginine level before administration of the subsequent dose is greater than about 165 μM. In an embodiment, the subsequent dose of human Arginase 1 is increased if the subject's plasma arginine level before administration of the subsequent dose is greater than about 180 μM. In an embodiment, the subsequent dose of human Arginase 1 is increased if the subject's plasma arginine level before administration of the subsequent dose is greater than about 195 μM. In an embodiment, the subsequent dose of human Arginase 1 is increased if the subject's plasma arginine level before administration of the subsequent dose is greater than about 200 μM. In an embodiment, the subsequent dose of human Arginase 1 is decreased if the subject's plasma arginine level before administration of the subsequent dose is less than about 50 μM. In an embodiment, the subsequent dose of human Arginase 1 is decreased if the subject's plasma arginine level before administration of the subsequent dose is less than about 40 μM. In an embodiment, the subsequent dose of human Arginase 1 is decreased if the subject's plasma arginine level before administration of the subsequent dose is less than about 30 μM. In an embodiment, the subsequent dose of human Arginase 1 is decreased if the subject's plasma arginine level before administration of the subsequent dose is less than about 20 μM.

[0091]

[0108] In an embodiment, the subsequent dose of peggyl arginase is adjusted to 0.01 mg / kg to 0.1 mg / kg each time based on the subject's plasma arginine level resulting from the preceding dose. In an embodiment, the subsequent dose of peggyl arginase is adjusted to at least about 0.01 mg / kg each time based on the subject's plasma arginine level resulting from the preceding dose. In an embodiment, the subsequent dose of peggyl arginase is adjusted to at most about 0.2 mg / kg each time based on the subject's plasma arginine level resulting from the preceding dose. In embodiments, subsequent doses of peggylarginase are administered at a dose of 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 ..., or about 0.03 mg / kg, based on the subject's plasma arginine level resulting from the preceding dose. In an embodiment, the subsequent doses of PEGylarginase are adjusted to 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 0.03 mg / kg, about 0.2 mg / kg, about 0.05 mg / kg, about 0.07 mg / kg, about 0.1 mg / kg, about 0.07 mg / kg, about 0.2 mg / kg, or about 0.1 mg / kg to about 0.2 mg / kg. In an embodiment, the subsequent doses of PEGylarginase are adjusted to 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 preceding dose. In an embodiment, subsequent doses of peggylarginase are adjusted to about 0.05 mg / kg each based on the subject's plasma arginine levels resulting from the preceding dose.

[0092]

[0109] In an embodiment, the subsequent dose of human Arginase 1 is adjusted to 0.01 mg / kg to 0.1 mg / kg each time based on the subject's plasma arginine level resulting from the preceding dose. In an embodiment, the subsequent dose of human Arginase 1 is adjusted to at least about 0.01 mg / kg each time based on the subject's plasma arginine level resulting from the preceding dose. In an embodiment, the subsequent dose of human Arginase 1 is adjusted to at most about 0.2 mg / kg each time based on the subject's plasma arginine level resulting from the preceding dose. In embodiments, subsequent doses of human Arginase 1 are administered at a dose of 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 ... In an embodiment, the subsequent doses of human arginase 1 are adjusted to 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 0.03 mg / kg, about 0.2 mg / kg, about 0.05 mg / kg, about 0.07 mg / kg, about 0.1 mg / kg, about 0.07 mg / kg, about 0.2 mg / kg, or about 0.1 mg / kg to about 0.2 mg / kg. In an embodiment, the subsequent doses of human arginase 1 are adjusted to 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 preceding dose. In embodiments, subsequent doses of human Arginase 1 are adjusted to about 0.05 mg / kg each based on the subject's plasma arginine levels resulting from the preceding dose.

[0093]

[0110] In an embodiment, the subject is not premedicated before the first dose of peggyl arginase. In an embodiment, the subject is premedicated before the first dose of peggyl arginase. In an embodiment, the subject is not premedicated before the first dose of human Arginase 1. In an embodiment, the subject is premedicated before the first dose of human Arginase 1.

[0094]

[0111] In an embodiment, the subject's plasma arginine level is at least 200 μM before the first dose of peggylarginase. In an embodiment, the subject's plasma arginine level is at least 210 μM before the first dose of peggylarginase. In an embodiment, the subject's plasma arginine level is at least 230 μM before the first dose of peggylarginase. In an embodiment, the subject's plasma arginine level is at least 250 μM before the first dose of peggylarginase. In an embodiment, the subject's plasma arginine level is at least 270 μM before the first dose of peggylarginase. In an embodiment, the subject's plasma arginine level is at least 290 μM before the first dose of peggylarginase. In an embodiment, the subject's plasma arginine level is at least 310 μM before the first dose of peggylarginase.

[0095]

[0112] In an embodiment, the subject's plasma arginine level is at least 200 μM before the first dose of human Arginase 1. In an embodiment, the subject's plasma arginine level is at least 210 μM before the first dose of human Arginase 1. In an embodiment, the subject's plasma arginine level is at least 230 μM before the first dose of human Arginase 1. In an embodiment, the subject's plasma arginine level is at least 250 μM before the first dose of human Arginase 1. In an embodiment, the subject's plasma arginine level is at least 270 μM before the first dose of human Arginase 1. In an embodiment, the subject's plasma arginine level is at least 290 μM before the first dose of human Arginase 1. In an embodiment, the subject's plasma arginine level is at least 310 μM before the first dose of human Arginase 1.

[0096]

[0113] In an embodiment, the subject is an adult. In an embodiment, the subject is under 18 years of age. In an embodiment, the subject is at least 2 years of age.

[0097]

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

[0098]

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

[0099]

[0116] In an embodiment, the subject's plasma arginine level is about 40 μM to about 115 μM after treatment. In an embodiment, the subject's plasma arginine level is about 40 μM to about 115 μM after two doses of peggylarginase. In an embodiment, the subject's plasma arginine level is about 40 μM to about 115 μM after four doses of peggylarginase. In an embodiment, the subject's plasma arginine level is about 40 μM to about 115 μM after six doses of peggylarginase. In an embodiment, the subject's plasma arginine level is about 40 μM to about 115 μM after eight doses of peggylarginase. In an embodiment, the subject's plasma arginine level is about 40 μM to about 115 μM after ten doses of peggylarginase. In an embodiment, the subject's plasma arginine level is about 40 μM to about 115 μM after twelve doses of peggylarginase. In an embodiment, the subject's plasma arginine level is about 40 μM to about 115 μM after 14 doses of peggylarginase. In an embodiment, the subject's plasma arginine level is about 40 μM to about 115 μM after 16 doses of peggylarginase. In an embodiment, the subject's plasma arginine level is about 40 μM to about 115 μM after 18 doses of peggylarginase. In an embodiment, the subject's plasma arginine level is about 40 μM to about 115 μM after 20 doses of peggylarginase. In an embodiment, the subject's plasma arginine level is about 40 μM to about 115 μM after 22 doses of peggylarginase. In an embodiment, the subject's plasma arginine level is about 40 μM to about 115 μM after 24 doses of peggylarginase.

[0100]

[0117] In an embodiment, the subject's plasma arginine level is about 40 μM to about 115 μM after treatment. In an embodiment, the subject's plasma arginine level is about 40 μM to about 115 μM after two doses of human Arginase 1. In an embodiment, the subject's plasma arginine level is about 40 μM to about 115 μM after four doses of human Arginase 1. In an embodiment, the subject's plasma arginine level is about 40 μM to about 115 μM after six doses of human Arginase 1. In an embodiment, the subject's plasma arginine level is about 40 μM to about 115 μM after eight doses of human Arginase 1. In an embodiment, the subject's plasma arginine level is about 40 μM to about 115 μM after ten doses of human Arginase 1. In an embodiment, the subject's plasma arginine level is about 40 μM to about 115 μM after twelve doses of human Arginase 1. In an embodiment, the subject's plasma arginine level is about 40 μM to about 115 μM after 14 doses of human Arginase 1. In an embodiment, the subject's plasma arginine level is about 40 μM to about 115 μM after 16 doses of human Arginase 1. In an embodiment, the subject's plasma arginine level is about 40 μM to about 115 μM after 18 doses of human Arginase 1. In an embodiment, the subject's plasma arginine level is about 40 μM to about 115 μM after 20 doses of human Arginase 1. In an embodiment, the subject's plasma arginine level is about 40 μM to about 115 μM after 22 doses of human Arginase 1. In an embodiment, the subject's plasma arginine level is about 40 μM to about 115 μM after 24 doses of human Arginase 1.

[0101]

[0118] In an embodiment, the subject's intake of natural protein is increased after one or more doses of peggylarginase. The subject's intake of essential amino acid mixture is reduced after one or more doses of peggylarginase. In an embodiment, the subject's relative intake of essential amino acid mixture compared to natural protein is reduced after one or more doses of peggylarginase. In an embodiment, after one or more doses of peggylarginase, the main source of the subject's protein intake is changed from arginine-free (or low arginine) essential amino acid mixture to the addition of any food containing protein. In an embodiment, after one or more doses of peggylarginase, the main source of the subject's protein intake is changed from arginine-free (or low arginine) essential amino acid mixture to natural protein.

[0102]

[0119] In an embodiment, the subject's total protein intake is greater than 30 g / day after one or more doses of peggylarginase. In an embodiment, the subject's total protein intake is greater than 35 g / day after one or more doses of peggylarginase. In an embodiment, the subject's total protein intake is greater than 40 g / day after one or more doses of peggylarginase. In an embodiment, the subject's total protein intake is greater than 45 g / day after one or more doses of peggylarginase. In an embodiment, the subject's total protein intake is greater than 50 g / day after one or more doses of peggylarginase. In an embodiment, the subject's total protein intake is increased by at least 5% after one or more doses of peggylarginase. In an embodiment, the subject's total protein intake is increased by at least 10% after one or more doses of peggylarginase. In an embodiment, the subject's total protein intake is increased by at least 15% after one or more doses of peggylarginase. In an embodiment, the subject's total protein intake is increased by at least 20% after one or more doses of peggylarginase. In an embodiment, the subject's total protein intake is increased by at least 25% after one or more doses of peggylarginase. In an embodiment, the subject's native protein intake is increased to at least 45% of the total protein intake after one or more doses of peggylarginase. In an embodiment, the subject's native protein intake is increased to at least 50% of the total protein intake after one or more doses of peggylarginase. In an embodiment, the subject's native protein intake is increased to at least 55% of the total protein intake after one or more doses of peggylarginase. In an embodiment, the subject's native protein intake is increased to at least 60% of the total protein intake after one or more doses of peggylarginase. In an embodiment, the subject's native protein intake is increased to at least 65% of the total protein intake after one or more doses of peggylarginase. In an embodiment, the subject's native protein intake is increased to at least 70% of total protein intake after one or more doses of PEGylated arginase.

[0103]

[0120] In an embodiment, the subject's intake of natural protein is increased after one or more doses of human Arginase 1. The subject's intake of essential amino acid mixture is reduced after one or more doses of human Arginase 1. In an embodiment, the subject's relative intake of essential amino acid mixture compared to natural protein is reduced after one or more doses of human Arginase 1. In an embodiment, after one or more doses of human Arginase 1, the subject's main source of protein intake is changed from arginine-free (or low arginine) essential amino acid mixture to the addition of any food containing protein. In an embodiment, after one or more doses of human Arginase 1, the subject's main source of protein intake is changed from arginine-free (or low arginine) essential amino acid mixture to natural protein.

[0104]

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

[0105]

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

[0106]

[0123] In an embodiment, the subject does not have a hyperammonemic episode. In an embodiment, the subject does not have an active infection requiring anti-infective therapy. In an embodiment, the subject does not have an active infection with human immunodeficiency virus, hepatitis B, or hepatitis C. In an embodiment, the subject does not have extreme mobility deficits defined as either inability to be assessed based on the Gillette Functional Assessment Questionnaire (GFAQ) or a score of 1 based on the GFAQ. In an embodiment, the subject does not have a history of hypersensitivity to polyethylene glycol (PEG). In an embodiment, the subject has never been treated with botulinum toxin. In an embodiment, the subject does not have a liver transplant or a hematopoietic cell transplant.

[0107]

[0124] In an embodiment, the mobility of the subject is improved after one or more doses of peggylarginase. In an embodiment, arginine metabolite accumulation is reduced after one or more doses of peggylarginase. In an embodiment, the mobility of the subject is improved after one or more doses of human Arginase 1. In an embodiment, arginine metabolite accumulation is reduced after one or more doses of human Arginase 1. In an embodiment, the accumulated guanidino compounds in the blood and cerebrospinal fluid of the subject can be reduced. In an embodiment, the guanidino compound is selected from the group consisting of alpha-N-acetylarginine, alpha-keto-d-guanidinovaleric acid, arginic acid, and guanidinoacetic acid.

[0108]

[0125] In an embodiment, the peggylarginase is administered at a concentration of about 1 mg / mL to about 8 mg / mL. In an embodiment, the peggylarginase is administered at a concentration of at least about 1 mg / mL. In an embodiment, the peggylarginase is administered at a concentration of at most about 8 mg / mL. In an embodiment, PEGylarginase is from about 1 mg / mL to about 2 mg / mL, from about 1 mg / mL to about 3 mg / mL, from about 1 mg / mL to about 4 mg / mL, from about 1 mg / mL to about 5 mg / mL, from about 1 mg / mL to about 6 mg / mL, from about 1 mg / mL to about 7 mg / mL, from about 1 mg / mL to about 8 mg / mL, from about 2 mg / mL to about 3 mg / mL, from about 2 mg / mL to about 4 mg / mL, from about 2 mg / mL to about 5 mg / mL, from about 2 mg / mL to about 6 mg / mL, from about 2 mg / mL to about 7 mg / mL, from about 2 mg / mL to about 8 mg / mL, from about 3 mg / mL to about 4 mg / mL, In an embodiment, the peggylarginase is administered at a concentration of about 1 mg / mL, about 2 mg / mL, about 3 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 an embodiment, the pegylarginase is administered at a concentration of about 5 mg / mL.

[0109]

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

[0110] Embodiment

[0127] In one set of embodiments (Embodiment Set A), the following is provided: A1. A method of treating arginase 1 (ARG1) deficiency (ARG1-D) in a subject, comprising administering pegylarginase to the subject; 1) PEGylated arginase is pegylated human arginase 1 that contains a cobalt metal cofactor; 2) A method in which pegzyarginase is administered weekly at a dose of about 0.05 mg / kg to about 0.2 mg / kg. A2. The method of embodiment A1, wherein the subject's plasma arginine level is from about 40 μM to about 115 μM after 12 doses of PEGylated arginase. A3. The method of embodiment A1 or A2, wherein the initial dose of peggylarginase is about 0.1 mg / kg. A4. The method of any one of embodiments A1-A3, wherein the subsequent dose of peggylarginase 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 peggylarginase is increased if the subject's plasma arginine level prior to administration of the subsequent dose is greater than about 115 μM, and optionally greater than about 150 μM. A6. The method of embodiment A4, wherein the subsequent dose of peggylarginase is decreased if the subject's plasma arginine level prior to administration of the subsequent dose is below about 50 μM, optionally below about 40 μM. A7. The method of any one of embodiments A4-A6, wherein subsequent doses of peggylarginase are adjusted to 0.01 mg / kg to 0.1 mg / kg each time based on the subject's plasma arginine level resulting from the preceding dose. A8. The method of embodiment A7, wherein subsequent doses of peggylarginase are adjusted to 0.05 mg / kg each time based on the subject's plasma arginine level resulting from the preceding dose. A9. The method of any one of embodiments A1-A8, wherein the subject does not receive premedication prior to the first dose of peggylarginase. A10. The method of any one of embodiments A1-A9, wherein the subject's plasma arginine level is at least 250 μM prior to the first dose of pegzylarginase. A11. The method of any one of embodiments A1-A10, wherein the subject is at least 2 years of age. A12. The method of any one of embodiments A1-A11, wherein the subject is classified as GMFCS level II, III, IV, or V. A13. The method of any one of embodiments A1-A12, wherein the subject is co-administered an ammonia scavenger. A14. The method of any one of embodiments A1-A13, wherein the subject's intake of natural protein is increased following one or more doses of PEGylated arginase. A15. The method of any one of embodiments A1-A14, wherein the subject's relative intake of essential amino acid mixture compared to native protein is reduced following one or more doses of pegylarginase. A16. The method of any one of embodiments A1-A15, wherein after one or more doses of peggylarginase, 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 food containing protein. A17. The method of any one of embodiments A1-A15, wherein after one or more doses of pegzylarginase, the primary source of the subject's protein intake is changed from an arginine-free (or low-arginine) essential amino acid mixture to natural protein. A18. The method of any one of embodiments A1-A17, wherein the subject's total protein intake is greater than 40 g / day after one or more doses of peggylarginase. A19. The method of any one of embodiments A1-A18, wherein the subject's total protein intake is increased by at least 15% after one or more doses of peggylarginase. A20. The method of any one of embodiments A1-A19, wherein the subject's native protein intake is increased to at least 50% of total protein intake after one or more doses of PEGylated arginase. A21. The method of embodiment A20, wherein the subject's native protein intake is increased to at least 65% of total protein intake after one or more doses of PEGylated arginase. A22. The method of any one of embodiments A1-A21, wherein the subject's mobility is improved following one or more doses of peggylarginase. A23. The method of any one of embodiments A1-A22, wherein arginine metabolite accumulation is reduced following one or more doses of peggylarginase. A24. The method of any one of embodiments A1-A23, wherein accumulated guanidino compounds in the subject's blood and cerebrospinal fluid are reduced. A25. The method of embodiment A24, wherein the guanidino compound is selected from the group consisting of alpha-N-acetylarginine, alpha-keto-d-guanidinovaleric acid, arginic acid, and guanidinoacetic acid. A26. The method of any one of embodiments A1-A25, wherein pegylarginase is administered at a concentration of 5 mg / mL. A27. The method of any one of embodiments A1-A26, wherein the pegylarginase is administered intravenously. A28. The method of any one of embodiments A1-A26, wherein the peggylarginase is administered subcutaneously. A29. The method of any one of embodiments A1-A28, wherein the pegylarginase is stored at a concentration of about 5 mg / mL. A30. The method of any one of embodiments A1-A29, wherein the pegylarginase 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-A30, wherein the pegylarginase is liquid. A32. The method of any one of embodiments A1-A30, wherein the pegylarginase is lyophilized. A33. The method of any one of embodiments A1-A32, wherein the pegylarginase 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 pegylarginase.

[0111]

[0128] In another set of embodiments (Embodiment Set B), the following is provided: B1. A method of treating arginase 1 (ARG1) deficiency (ARG1-D) in a subject, comprising administering human arginase 1 to the subject; 1) human arginase 1 is pegylated human arginase 1 containing a cobalt metal cofactor; 2) A method in which human arginase 1 is administered weekly at a dose of about 0.05 mg / kg to about 0.2 mg / kg. B2. The method of embodiment B1, wherein the subject's plasma arginine level is from about 40 μM to about 115 μM after 12 doses of human Arginase 1. 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-B3, wherein the 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 prior to administration of the subsequent dose is greater than about 115 μM, and optionally greater than about 150 μM. B6. The method of embodiment B4, wherein a subsequent dose of human Arginase 1 is decreased if the subject's plasma arginine level prior to administration of the subsequent dose is below about 50 μM, optionally below about 40 μM. B7. The method of any one of embodiments B4-B6, wherein subsequent doses of human Arginase 1 are adjusted to 0.01 mg / kg-0.1 mg / kg each time based on the subject's plasma arginine level resulting from the preceding dose. B8. The method of embodiment B7, in which subsequent doses of human Arginase 1 are adjusted to 0.05 mg / kg each time based on the subject's plasma arginine level resulting from the preceding dose. B9. The method of any one of embodiments B1-B8, wherein the subject does not receive premedication prior to the first dose of human Arginase 1. B10. The method of any one of embodiments B1-B9, wherein the subject's plasma arginine level is at least 250 μM prior to the first dose of human Arginase 1. B11. The method of any one of embodiments B1-B10, wherein the subject is at least 2 years of age. B12. The method of any one of embodiments B1-B11, wherein the subject is classified as GMFCS level II, III, IV, or V. B13. The method of any one of embodiments B1-B12, wherein the subject is co-administered an ammonia scavenger. B14. The method of any one of embodiments B1-B13, wherein the subject's native protein intake is increased following one or more doses of human Arginase 1. B15. The method of any one of embodiments B1-B14, wherein the subject's relative intake of essential amino acid mixture compared to native protein is reduced following one or more doses of human Arginase 1. B16. The method of any one of embodiments B1-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 food containing protein. B17. The method of any one of embodiments B1-B15, wherein after one or more doses of human Arginase 1, the primary source of the subject's protein intake is changed from an arginine-free (or low-arginine) essential amino acid mixture to natural protein. B18. The method of any one of embodiments B1-B17, wherein the subject's total protein intake is greater than 40 g / day after one or more doses of human Arginase 1. B19. The method of any one of embodiments B1-B18, wherein the subject's total protein intake is increased by at least 15% after one or more doses of human Arginase 1. B20. The method of any one of embodiments B1-B19, wherein the subject's native protein intake is increased to at least 50% of total protein intake after one or more doses of human Arginase 1. B21. The method of embodiment B20, wherein the subject's native protein intake is increased to at least 65% of total protein intake after one or more doses of human Arginase 1. B22. The method of any one of embodiments B1-B21, wherein the subject's mobility is improved following one or more doses of human Arginase 1. B23. The method of any one of embodiments B1-B22, wherein arginine metabolite accumulation is reduced following one or more doses of human Arginase 1. B24. The method of any one of embodiments B1-B23, wherein accumulated guanidino compounds in the subject's blood and cerebrospinal fluid are reduced. B25. The method of embodiment B24, wherein the guanidino compound is selected from the group consisting of alpha-N-acetylarginine, alpha-keto-d-guanidinovaleric acid, arginic acid, and guanidinoacetic acid. B26. The method of any one of embodiments B1-B25, wherein human Arginase 1 is administered at a concentration of 5 mg / mL. B27. The method of any one of embodiments B1-B26, wherein the pehtoarginase 1 is administered intravenously. B28. The method of any one of embodiments B1-B26, wherein human Arginase 1 is administered subcutaneously. B29. The method of any one of embodiments B1-B28, wherein human Arginase 1 is stored at a concentration of about 5 mg / mL. B30. The method of any one of embodiments B1-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-B30, wherein the human Arginase 1 is liquid. B32. The method of any one of embodiments B1-B30, wherein the human Arginase 1 is lyophilized. B33. The method of any one of embodiments B1-B32, wherein the human Arginase 1 is in a vial or ampoule. B34. The method of embodiment B33, wherein the vial or ampoule contains about 2 mg or about 5 mg of human Arginase 1.

[0112]

[0129] In yet another set of embodiments (Embodiment Set C), the following is provided: C1. A method of dietary liberalization for patients with Arginase 1 deficiency comprising administering cobalt-substituted recombinant Arginase 1 subcutaneously once a week and increasing natural protein intake. 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 the patient is administered 0.05-0.2 mg / kg of cobalt-substituted recombinant arginase 1. C4. The method of any of embodiments C1-C3, wherein the liberalization is a change from an arginine-free (or low-arginine) essential amino acid mixture as the primary source of protein intake to the addition of any food that contains protein. C5. The method of any of embodiments C1-C3, wherein the liberalization is a change from an arginine-free (or low-arginine) essential amino acid mixture as the primary source of protein intake to natural protein as the primary source of protein intake. C6. The method of any of embodiments C1-C5, wherein the patient is an adult and the protein intake is greater than 40 g / day. C7. The method of any of embodiments C1-C6, wherein the patient is co-administered an ammonia scavenger. C8. The method of any of embodiments C1-C7, wherein the patient's total protein intake is increased by more than 15%. C9. The method of any of embodiments C1-C8, wherein the patient's natural protein intake is greater than 50% of their total protein intake. C10. A method of dietary modification, wherein a patient with ARG1 D is administered cobalt-substituted recombinant Arginase 1 once a week and natural protein intake is increased to at least 65% of total protein intake. C11. The method of embodiment C10, wherein the patient is administered 0.05-0.2 mg / kg of cobalt-substituted recombinant arginase 1. C12. A method for treating protein aversion in a patient having a urea cycle disorder, comprising administering cobalt-substituted recombinant Arginase 1 as described herein. C13. A method of treating Arginase 1 deficiency, wherein a subset of patients classified as GMFCS level II receive cobalt-substituted recombinant Arginase 1 as described herein. C14. A method for preventing arginine metabolite accumulation, comprising administering a cobalt-substituted recombinant arginase 1 as described herein. C15. A method for reducing accumulated guanidino compounds in the blood and cerebrospinal fluid of a patient in need of such reduction, comprising administering cobalt-substituted recombinant Arginase 1 as described herein. C16. The method of embodiment C15, wherein the guanidino compound is selected from the group consisting of alpha-N-acetylarginine, alpha-keto-d-guanidinovaleric acid, arginic acid, and guanidinoacetic acid. C17. The method of embodiment C15 or C16, wherein the patient with accumulated guanidino compounds has a disease selected from the group including GAMT, arginase 1 deficiency, S-adenosylhomocysteine ​​hydrolase deficiency, CNS injury, and renal impairment. EXAMPLES

[0113] Working Example

[0130] Below are descriptions of various methods and materials used in this study. These are presented to provide those of ordinary skill in the art with a complete disclosure and description of how to make and use the invention, and are not intended to limit the scope of what the inventors regard as their invention, nor are they intended to represent all of the experiments that have been performed or can be performed. It should be understood that the exemplary descriptions written in the present tense have not necessarily been performed, and that the descriptions can be performed to generate data, etc. relevant to the teachings of the invention. Efforts have been made to ensure accuracy with respect to numbers used (e.g., amounts, percentages, etc.), but some experimental error and deviation should be accounted for.

[0114] Example 1: Efficacy and safety of peggylarginase in patients with arginase 1 deficiency 1.1 Summary Peglyarginase as a treatment

[0131] Pegylated arginase (Co-ARG1-PEG, AEB1102) is a cobalt-substituted pegylated human recombinant arginase 1 enzyme with improved stability and catalytic activity, as well as extended half-life.

[0115]

[0132] Peggylarginase is being investigated as an enzyme therapy for ARG1-D. The biological activity of peggylarginase has been demonstrated based on non-clinical studies and clinical data from Study CAEB1102-101A (Study 101A) and Study CAEB1102-102A (Study 102A) in patients with ARG1-D. This study (CAEB1102-300A [Study 300A]) seeks to confirm the clinical utility of peggylarginase for the treatment of patients with ARG1-D.

[0116]

[0133] Given the characteristics of PEGylated arginase and the relatively low dose required to reduce arginine levels to within the normal range, both intravenous (IV) and subcutaneous (SC) administration were tested in Study 101A, Study 102A, and Study 300A. A brief description of the results from Study 101A and Study 102A is provided below.

[0117]

[0134] Study 101A, a Phase 1 / 2 open-label study designed to evaluate safety, tolerability, pharmacokinetics, pharmacodynamics (PD), and proof-of-concept in patients with ARG1-D, found that IV pegzylarginase was well tolerated and had a tolerable and manageable safety profile. No subjects discontinued treatment due to adverse events (AEs) and had no detectable anti-drug antibodies (ADAs) at study end. Notably, after eight weekly doses of treatment in the second part of the study, pegzylarginase rapidly and sustainably reduced plasma arginine levels to below the current medical guideline level of 200 μM and within the normal range (40-115 μM) in some subjects. Improved plasma arginine control following pegzylarginase treatment was accompanied by evidence of early clinical benefit in the study, with 9 of 16 subjects showing clinically meaningful improvement in at least one clinical outcome. These results suggest that PEGylarginase has the potential to both ameliorate important disease-related abnormalities and potentially alter disease progression. In addition, in clinical and in vitro and in vivo settings, PEGylarginase has demonstrated approximate dose proportionality for IV and SC administration.

[0118]

[0135] Study 102A, a Phase 2 open-label study designed to evaluate the long-term safety, tolerability, and efficacy of IV and SC peggylarginase in pediatric and adult subjects with ARG1-D, was a LTE study of Study 101A. All 14 subjects who completed Study 101A continued to enroll and were treated with peggylarginase in Study 102A. Results of an ongoing interim analysis of Study 102A, with subjects receiving peggylarginase administered IV or SC weekly at doses of 0.05-0.2 mg / kg for approximately 110-183 weeks, demonstrated consistent and sustained reductions in plasma arginine levels to within the normal range and concomitant reductions in plasma guanidino compound levels. Clinically meaningful improvements in mobility as assessed by neuromuscular function were maintained, and importantly, the duration of long-term follow-up in this study exceeded the timescale over which clinical disease progression is typically observed with the current standard of care.

[0119] Rationale for Phase 3 Trial 300A

[0136] As described above, it has been shown that peggylarginase provides a significant, rapid, and sustained reduction in plasma arginine levels in patients with ARG1-D, allowing substantially improved arginine control over that which can be achieved with an individualized disease management (IDM) approach. The improved control of plasma arginine levels was accompanied by clinical improvement in one or more questionnaires of neuromuscular 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 assess the safety and efficacy of peggylarginase in reducing arginine levels and improving clinical outcomes compared to placebo. The objectives of this study, Study 300A, were to confirm these preliminary findings in a blinded, placebo-controlled design and to gain further understanding of the impact of peggylarginase on a broader range of disease indications. Interim analysis results at the data cutoff date are presented in this report.

[0120] Test Purpose

[0137] The Phase 3 PEACE trial was designed to assess the safety and efficacy of PEGylated arginase's ability to achieve arginine control, as well as to evaluate the relationship between arginine lowering and improved neuromuscular function / mobility.

[0121] 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 peggylarginase. Peggylarginase or placebo was given in a 2:1 ratio of weekly IV infusion to IDM for the first 24 weeks of the double-blind period. After completion of the 24-week DB, subjects entered the LTE period, and all subjects received peggylarginase during the LTE period, although the investigators remained blinded to the randomization treatment for the first 8 weeks. Subjects were transitioned from IV administration to weekly SC dosing at any point during the 8 weeks of the LTE to allow for easy and convenient administration for the remainder of the LTE.

[0122] Associations with major mobility outcomes

[0139] Although not previously tested in patients with ARG1-D prior to the start of the clinical development program for peggylarginase, mobility is important for independence and declines clinically over time as ARG1-D disease progresses. Evidence-based outcome measures are necessary to monitor changes in patient outcomes and can be used to support the need for continued home care or indicate the need for additional support services or devices required for walking. Given the lack of data on the usefulness of neuromuscular assessments in assessing disease burden and treatment effects in ARG1-D, multiple assessments were included to further test the impact of peggylarginase on neuromuscular outcomes, including mobility, in this ultra-rare disease. Patients with ARG1-D may have varying degrees of functional impairment, and preferential assessments, such as 2MWT and GMFM-E, were considered useful as primary secondary endpoints in the assessment of the broader population for clinical trials.

[0123]

[0140] The 2MWT and GMFM-E were utilized in preclinical trials of ARG1-D. Based on insights from the Phase 1 / 2 and open-label extension studies of peggylarginase, two primary mobility outcomes, the 2MWT and the GMFM-E, were identified and selected to assess the clinical benefit of peggylarginase beyond arginine reduction in a Phase 3 study. The 2MWT is a shortened, modified 6-minute version and is a relevant tool for measuring mobility performance outcomes (e.g., the NIH Toolbox Motor Domains-2MWT [Bohannon 2014]). The 2MWT has been validated across a wide range of age groups, including young children (Bohannon 2018) and adults (Bohannon 2014). Furthermore, both the 6-minute walk test and the shortened modification, 2MWT, have been used successfully in young children (Geiger 2007, Maher 2008) and adults (Andersson 2006, Ammann-Reiffer 2018) with cerebral palsy, also characterized by spasticity. The 2MWT is limited as a tool for subjects with walking ability, and cognitive abilities and focus on understanding and engaging with instructions. The second outcome, GMFM-E, has been used to assess functional mobility, which is affected by ARG1-D in similar populations, for example, cerebral palsy. This scale assesses the ability to walk, run, and climb stairs, and may be particularly appropriate for more severely affected individuals. This tool is limited to measuring improvement for those with milder disabilities, as they are often at or near the top scores. This suggests that while the sensitivity of the test is good, there is a greater opportunity to measure increases in scores for subjects with more progressive disease, who can be safely assessed.

[0124] Testing

[0141] The study was conducted during the COVID-19 pandemic, which required modifications to study conduct as a result of site closures and stay-at-home measures to prevent the spread of disease. This required implementation of pauses at the individual subject level based on the duration of the study in which each subject was present when the site closure occurred. These were done systematically and documented in the study documentation. The number of missed visits and assessments due to COVID-19 was small compared to the total number scheduled. Every attempt was made to ensure that data collection was robust and that assessments were performed accurately and collected correctly and on time when permitted. Despite the pandemic, the study was performed satisfactorily and subject safety and data integrity were maintained. Overall, the COVID-19 pandemic did not notably impact the ability to monitor and manage subject safety, data integrity, or efficacy assessments at the time of data cutoff.

[0125] Efficacy Results

[0142] Weekly administration of peggylarginase resulted in an early, consistent, and sustained reduction in plasma arginine levels until week 24 of the double-blind period, compared with no significant change in the placebo group. Conventional guidelines recommended targeting control of plasma arginine levels to <200 μM, as levels below this threshold were always previously unreachable in clinical practice with dietary restriction alone for most patients. Treatment with peggylarginase resulted in normalization of arginine levels in the majority of subjects (90.5%) through 24 weeks of dosing. Subjects in the double-blind period continued on the study in the LTE period, transitioning from IV to SC dosing for ease of administration and improved convenience, with the same IV dose unless arginine levels were affected. Continued administration of peggylarginase via the SC route of administration in those who received peggylarginase during the double-blind period resulted in a similarly consistent and sustained reduction in plasma arginine levels until week 24 of the LTE period. PEGylarginase-naive subjects initially randomized to placebo were initiated on weekly PEGylarginase in the LTE and switched to SC dosing after 8 weeks; 100% (N=11) of those who reached LTE24 were also able to normalize plasma arginine levels.

[0126]

[0143] A primary objective, rarely achievable with current treatment approaches, is to achieve arginine levels that meet guideline recommended levels (<200 μM) and ideally meet normal levels. At week 24 of the double-blind period, over 90% of subjects treated with pegzylarginase achieved arginine values ​​within treatment guidelines (<200 μM) compared to zero in the placebo group. In addition, over 90% of subjects treated with pegzylarginase achieved arginine values ​​within the normal range (40-115 μM) at week 24 of the double-blind period, which is unprecedented in this subject population.

[0127]

[0144] The primary secondary efficacy endpoints focused on neurological / neuromuscular 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 neuromuscular function varied across subjects. A favorable effect on neuromuscular function assessed by GMFM-E and 2MWT was observed in the peggylarginase group. A similar proportion of subjects in the peggylarginase group demonstrated clinically meaningful and / or numerical increases in GMFM-E and 2MWT assessments (approximately 55% [N=11] and 50% [N=10], respectively). Improvements in neuromuscular function were also observed in subjects in the placebo group, but were generally of lower magnitude than those observed in the peggylarginase group. Peggylarginase administration resulted in a longer increase from baseline in mean 2-minute walking distance (2MWT) and a larger improvement from baseline in GMFM-E scores at 24 weeks compared to placebo. Numerical improvements were observed in both GMFM-E and 2MWT, and for GMFM-E there was a favorable trend (p=0.1087) of change from baseline compared to placebo. In subgroup analyses, subjects with more severely restricted mobility (GMFCS≧II) treated with peggylarginase had greater gains than subjects classified as GMFCS I in both 2MWT and GMFM-E, because they were not limited by a maximum improvement threshold or upper limit of the assessment. These differences may reflect greater efficacy in capturing clinical benefit with these outcome tools in patients with more progressive disease. Improvements in patients with more progressive disease point to clinical utility for treatment with peggylarginase in those patients. Given the limitations of these outcome tools, the less pronounced improvements in those with mildly progressive disease do not exclude the potential for prevention of progression, which is also the treatment objective. Some limitations of these specific clinical assessments in some segments of the population include: For the 2MWT, subjects with a GMFCS IV classification were generally wheelchair confined and had severely limited walking ability, if they were able to walk at all (N=4).For the GMFM-E assessment, in those with a GMFCS I classification and not severe disability, the improvement measurements were limited by the upper limit of the test, as some were close to or at the upper or upper boundary of baseline response. In further demonstration of the treatment effect of peggylarginase during the double-blind period, for subjects who completed LTE24, increases from baseline for 2MWT and GMFM-E continued to be observed in subjects treated with peggylarginase, highlighting continued improvement with further therapy, while subjects who transitioned from placebo to peggylarginase showed stable or improved results.

[0128]

[0145] Based on interim analysis results of the ongoing Study 300A after 24 weeks of treatment in the double-blind period, PEGylarginase administered IV weekly at doses ranging from 0.05 to 0.20 mg / kg met its primary endpoint (p<0.0001). PEGylarginase demonstrated consistent and sustained reductions in plasma arginine to both therapeutic and normal levels, while no meaningful changes were observed in the placebo group.

[0129]

[0146] Reductions in plasma arginine were associated with clinically meaningful improvements in mobility. Pegylarginase demonstrated a favorable trend in GMFM-E compared to placebo and numerical improvements in 2MWT compared to placebo.

[0130]

[0147] More peggylarginase-treated subjects met response criteria across multiple domains, with generally greater magnitude of response, compared with placebo-treated subjects.

[0131]

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

[0132]

[0149] During the LTE period, subjects were switched to SC dosing with the same IV dose, unless affected by arginine levels, for up to 104 weeks of exposure, simplifying dosing and improving the ease of treatment. The safety profile was consistent between IV and SC dosing.

[0133]

[0150] During the LTE period, subjects continued to maintain normal arginine levels and maintained or even improved mobility after transition to SC dosing, while those transitioning from placebo to peggylarginase remained stable or demonstrated improvement.

[0134]

[0151] The safety profile was consistent between the DB and LTE periods.

[0135]

[0152] In summary, pegylarginase represents a novel targeted treatment option to address the unmet medical need of ARG1-D, and the overall safety and efficacy data demonstrate a favorable benefit-risk profile for patients suffering from this rare progressive debilitating disease.

[0136] 1.2 Background and Overview of Clinical Efficacy

[0153] PEGylarginase (Co-Arg1-PEG, AEB1102) is a cobalt-substituted PEGylated human recombinant Arginase 1 enzyme with improved stability, stronger catalytic activity, and extended half-life compared to the native enzyme. PEGylarginase cleaves excess plasma arginine into ornithine and urea, the natural degradation products of Arginase 1.

[0137]

[0154] ARG1-D is a rare, debilitating, progressive, inherited neurotoxic metabolic disease associated with increased levels of arginine and its metabolites, associated with a marked reduction in quality of life, increased morbidity, and premature death (Diez-Fernandez 2018; Schlune 2015; Summar 2013; Waisbren 2018).

[0138]

[0155] There are similarities between another inherited metabolic disease, phenylketonuria (PKU) and arginase 1 deficiency (ARG1-D), with regard to inherited liver enzyme deficiency and accumulation of amino acids in the blood. Phenylketonuria is a poorly understood deficiency of phenylalanine hydroxylase (PAH) in the liver that results in high levels of the circulating amino acid phenylalanine (Phe) that, if left untreated or inadequately treated, leads to chronic neurological and psychiatric disorders. Treatment is focused on lowering Phe plasma levels to improve clinical outcomes. PEG-valiase is an approved novel enzyme replacement therapy that helps address a significant unmet need in PKU patients whose blood Phe levels have not been controlled with current treatment options.

[0139]

[0156] Similar to PKU, in ARG1-D, enzyme deficiency in the liver results in high plasma and tissue levels of arginine that, when treated with the current standard of care, results in spasticity, gait disturbance, difficulty walking, and impaired development and cognitive abilities. Similarly, PEGylated arginase, a human arginine metabolic enzyme intended to be used as enzyme replacement therapy in patients with ARG1-D, cleaves excess plasma arginine into ornithine and urea, the natural degradation products of arginase 1. The goal of therapy is to reduce blood arginine concentrations to <200 μM to minimize the toxic effects of excess arginine, excess guanidino compounds (GCs) derived from arginine, and dysregulation of nitric oxide metabolism (Palynziq USPI).

[0140]

[0157] Signs of ARG1-D often appear in early childhood and include neuromuscular and neurocognitive deficits. The clinical picture is ultimately remarkably uniform and characterized by spasticity, gait disturbances, difficulty walking, and impaired development and cognitive abilities; some patients have seizures, but variability in the time frame and progression of symptoms has been observed between patients (De Deyn 1997; Marescau 1990; Waisbren 2018).

[0141]

[0158] Elevated arginine and its metabolites are believed to be a major contributor to disease signs and progression (De Deyn 1997; Marescau 1990; Waisbren 2018). Preclinical models, via gene expression profiling of the motor cortex in ARG1-D mouse knockout models, show that arginase deficiency favors demyelination of the corticospinal tract during postnatal CNS development, altering synaptic density in the motor cortex. Several other diseases show a correlation between spasticity and loss of myelination through multiple etiologies, including multiple sclerosis and hereditary spastic paraplegia. Taken together, literature findings support the importance of both arginine and subsequent metabolite accumulation as the primary driver of neurological signs in ARG1-D, and explain the efficacy of reduction of arginine to reverse neurological impairment via enzyme administration or hepatic expression of ARG1 for functional restoration of oligodendrocytes to ameliorate CNS abnormalities.

[0142]

[0159] Neuromuscular complications of the disease are a hallmark feature, with lower limb spasticity, typically seen in early childhood, impairing mobility and balance and resulting in gait disturbance with difficulty walking and climbing stairs. Patients typically develop some form of lower limb spasticity, with approximately 60%-75% having spasticity at first presentation, which increases with extended follow-up (Carvalho 2012; Huemer 2016). This progressive spastic diplegia results in immobility, increasing inability to perform activities of daily living, and increased burden on caregivers.

[0143]

[0160] Currently, there are no approved therapies for the treatment of ARG1-D that significantly reduce plasma arginine, and dietary interventions rarely achieve the goals specified by treatment guidelines, resulting in continued disease progression. Pegzylarginase represents a novel targeted treatment option to address unmet medical needs for patients suffering from ARG1-D. The overall efficacy data for pegzylarginase, spanning durations of 24 to over 120 weeks, demonstrated substantial evidence of efficacy by achieving sustained plasma arginine levels within normal levels (40 to 115 μM), meeting or exceeding treatment guidance (<200 μM) for most patients, and clinically meaningful improvements in mobility at the trial level, and more importantly at the individual subject level, in this ultra-rare disease.

[0144] diagnosis

[0161] Diagnosis can be readily made using routinely available plasma arginine assessments or genetic analysis (Haeberle 2012; Haeberle 2019). Diagnostic delays may occur due to overlapping symptoms with other developmental disorders, such as cerebral palsy or hereditary spastic paraplegia (Carvalho 2012).

[0145] Current standard of care

[0162] There are no pharmacological agents known to effectively reduce arginine levels in patients with ARG1-D, and no agents that specifically target the enzyme deficiency have been approved in any country. Current treatments include a low-protein diet that moderately reduces arginine levels, which in some cases is accompanied by improved neuromuscular function (Haeberle 2012; Huemer 2016; Lambert 1991; Prasad 1997). Pegzylarginase represents the first potential enzyme therapy for ARG1-D. The literature indicates that modest reductions in plasma arginine levels, achieved with strict dietary protein restriction, can result in amelioration of disease symptoms; however, the currently recommended treatment goal of reducing plasma arginine to <200 μM is difficult to achieve, in part because of the difficulty in adhering to strict diets to reduce arginine levels to or near the normal range (Haeberle 2012; Huemer 2016; Lambert 1991; Prasad 1997; Burrage 2015). Thus, substantial reductions in plasma arginine levels using PEGylated arginase have the potential to slow or halt the progression of neuromuscular, neurocognitive, and / or adaptive behavioral deterioration in patients with ARG1-D.

[0146]

[0163] Current treatment of ARG1-D focuses on lowering plasma arginine levels through lifelong dietary protein restriction. Protein intake is limited to the minimum required to maintain protein biosynthesis and growth. Typically, more than half of the dietary protein is provided in the form of an arginine-free essential amino acid (EAA) mixture. Although such dietary modifications can result in a modest reduction in plasma arginine levels, levels remain significantly elevated in most patients, likely due to the fact that arginine flux is largely dependent on whole-body protein turnover and is minimally affected by dietary intake (Wu 1998). Furthermore, the diet is uncomfortable and difficult to maintain and manage, especially in growing infants. Ammonia scavengers can be used to help control ammonia levels and therefore the number and severity of hyperammonemia episodes that occur in some patients with ARG1-D. Liver transplantation has been reported to achieve disease normalization in some patients, and despite limited success with this intervention, transplantation is available to only a minority of patients and carries substantial risks of mortality and morbidity.

[0147] Unmet medical needs

[0164] The lack of treatment options for patients with arginase 1 deficiency that can effectively lower arginine levels or frequently achieve reduction within the normal range and promote lifelong maintenance of normal arginine levels highlights the unmet need for new therapies. Development of such therapeutics could be useful in attempts to minimize exposure to the neurotoxic effects of arginine and its metabolites, providing the potential for normal neurocognitive and neuromuscular development in these patients (Amayreh 2014).

[0148] 1.3 Selection of Study Population Selection Criteria

[0165] For study selection, subjects were required to meet all of the following criteria: 1. Subject and / or parent / guardian provided written informed consent / assent, including adherence to the requirements and limitations listed in the ICF and protocol. 2. One of the following: a current diagnosis of ARG1-D documented in the medical record including elevated plasma arginine levels, mutation analysis resulting in a pathogenic variant, or RBC arginase activity. For entry into this study, subjects also had to meet the following plasma arginine criteria: The mean of all plasma arginine measurements during the screening period prior to the randomization visit (Visit 1, Study Day 1) was ≧250 μM. b. If a subject was rescreened, the only values ​​considered for eligibility assessment were those from the current screening period. 3. Subjects were ≥ 2 years of age on the day of informed consent / assent. 4. Subjects were assessable for clinically meaningful within-subject change (clinical response) in at least one component of one assessment included in the primary secondary / other secondary endpoints. 5. Have received documented confirmation from the investigator and / or dietitian that the subject was able to maintain their diet according to the dietary information provided in the protocol, i.e., was able to maintain their current level of protein consumption, including natural protein and EAA supplements. 6. Subjects receiving ammonia scavenger therapy, antiepileptic drugs, and / or spasticity medications (e.g., baclofen) were receiving a stable dose of that medication for at least 4 weeks prior to randomization and were willing to continue receiving a stable dose during the double-blind and blinded follow-up phases of the study. 7. Female and male subjects. 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 before the first dose on the day of the first dose. If the subject (male or female) engaged in sexual activity that could result in pregnancy, the male or female was surgically sterilized, postmenopausal (amenorrhea for 12 months without another medical cause or high follicle stimulating hormone levels in the postmenopausal range in women not using hormonal contraception or hormone therapy), or agreed to use a highly effective method of birth control during the study and for at least 30 days after the last study medication administration. Highly effective contraceptive methods included combined (estrogen and progesterone-containing) hormonal contraception with inhibition of ovulation; progesterone-only hormonal contraception with inhibition of ovulation; intrauterine device; intrauterine hormone-releasing system; or abstinence (abstaining from heterosexual intercourse during the entire risk period with study treatment).

[0149] Exclusion criteria

[0166] Subjects were excluded from the study if they met any of the following criteria: 1. Had a hyperammonemic episode (defined as an event in which the subject had an ammonia level of ≥ 100 μM and one or more symptoms associated with hyperammonemia requiring hospitalization or emergency room care) within 6 weeks prior to receiving the first dose of study medication. 2. Had an active infection requiring anti-infective therapy within 3 weeks prior to the first dose. 3. Had known active infection with human immunodeficiency virus, hepatitis B, or hepatitis C. 4. Had severe mobility impairment defined as either disability as assessed by the Gillette Functional Assessment Questionnaire (GFAQ) or a score of 1 on the GFAQ. 5. Had other medical conditions or complications that, in the opinion of the investigator, would interfere with study compliance or data interpretation (e.g., severe intellectual disability that would prevent required study assessments). 6. Participated in a prior intervention trial using peggylarginase. 7. Had a history of hypersensitivity to polyethylene glycol (PEG) that, in the investigator's judgment, placed the subject at unacceptable risk for AEs. 8. The subject is being treated with a botulinum toxin-containing regimen or plans to initiate a botulinum toxin-containing regimen during the DB or blinded follow-up phase of the study, or has undergone surgery or botulinum toxin treatment for a spasticity-related complication within 16 weeks prior to the first dose of study treatment in this study. 9. Currently participating in another therapeutic clinical trial or having received any investigational drug within 30 days (or 5 half-lives, whichever is longer) prior to the first dose of study treatment in this study. 10. Prior liver or hematopoietic cell transplant procedure.

[0150] 1.4 Study Objectives and Endpoints

[0167] The overall objective of this study was to determine the safety, tolerability, and efficacy of long-term IV and SC administration of peggylarginase for the treatment of pediatric and adult subjects with ARG1-D and hyperargininemia. The study objectives and endpoints are presented in Table 1 below. This study was designed to assess the effect of combining peggylarginase with a subject's IDM regimen.

[0151] [Table 4]

[0152] 1.5 Overall Study Design and Plan

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

[0153]

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

[0154]

[0170] Upon completion of 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 peggylarginase. Subjects initially randomized to peggylarginase received the optimized dose (adjustments were allowed if clinically indicated) that the subject received during the 24-week double-blind treatment period. Subjects initially randomized to placebo during the 24-week double-blind treatment period began the 8-week DB treatment period at a dose of 0.10 mg / kg, which was allowed to be adjusted during the LTE period based on arginine levels.

[0155]

[0171] After the initial 8 weeks of the blinded LTE period, subjects had the option to receive weekly subcutaneous (SC) peggylarginase if approved by the investigator and sponsor. The first four 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. Subsequent SC doses could be administered outside the study site by appropriately trained home health care personnel if deemed safe and appropriate in the opinion of the investigator in consultation with the sponsor. During the LTE period, all subjects received weekly peggylarginase and IDM.

[0156] Study Design Scheme

[0172] The study design scheme is presented in Figure 1 A. Main demographics and baseline clinical characteristics are presented in Figure 1 B.

[0157]

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

[0158] Personalized disease management

[0174] This study was designed to assess the effect of combining pegylarginase with a subject's IDM regimen, which typically involves a strict protein restriction and EAA supplementation-related diet and / or the use of ammonia scavengers.

[0159]

[0175] Subjects had a stable IDM regimen documented during the screening period prior to study entry, including the use of defined protein amounts and defined EAA amounts and / or defined doses of ammonia scavenger medications, as appropriate. Subjects were willing and able to maintain a consistent dietary protein intake during the DB phase of the study. Subjects and / or their caregivers were advised by the investigator and dietician not to alter their IDM during the DB phase of the study unless there was a clear medical reason to do so. Adherence to IDM was collected on electronic case report forms (eCRFs).

[0160]

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

[0161] Administration of treatment

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

[0162]

[0178] Upon completion of the 24-week DB treatment period of the study, all eligible subjects entered an 8-week blinded follow-up treatment period, during which all subjects received peggylarginase.

[0163]

[0179] Subjects initially randomized to peggylarginase received the optimized dose (adjustments were allowed if clinically indicated) that they received during the 24-week DB treatment period. Subjects initially randomized to placebo during the 24-week DB treatment period began an 8-week blinded treatment period in which they were transitioned to a peggylarginase dose of 0.10 mg / kg, which was adjusted during the LTE period based on arginine levels. All subjects had their final DB assessment (i.e., the week 24 assessment) prior to the start of peggylarginase dosing in the LTE period.

[0164]

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

[0165] Dose Selection

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

[0166]

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

[0167] Assessment of efficacy

[0183] The efficacy of peggylarginase was measured by the magnitude, occurrence, and duration of changes in plasma arginine levels, as well as the following clinical outcome assessments: Neurological and neuromuscular signs Adaptive behavioral changes Caregiver and clinician general perception of change and severity and exit interviews Other clinical outcomes, e.g., frequency of seizures and hyperammonemic episodes Neurocognitive, developmental, and quality of life measures Ornithine and guanidino compounds and other exploratory biomarkers (i.e., guanidinoacetic acid, α-N-acetylarginine, α-keto-δ-guanidinovaleric acid, arginic acid, creatine, etc.)

[0168]

[0184] In addition to the neurological examination performed during the physical examination, several assessments of neurological and neuromuscular function were tested: 2MWT, Functional Mobility Assessment (FMA; Functional Mobility Scale [FMS] and GFAQ), 9-hole pegboard test, GMFM-D and GMFM-E, and the Modified Ashworth Scale (MAS).

[0169]

[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, arginic acid, and creatine.

[0170] Electroencephalogram

[0186] In addition to clinically evident seizures, frequent abnormal electroencephalogram (EEG) activity has been observed in patients with ARG1 D (Carvalho 2012; Huemer 2016; De Deyn 1997). Electroencephalography is a test used to evaluate electrical activity in the brain. EEG is defined as alternating-current type electrical activity recorded after being picked up by metal electrodes and conductive media from the scalp surface (Teplan 2002). EEG results have been shown to be abnormal in a significant proportion of patients with ARG1 D, with EEG recordings showing slow diffuse activity and generalized epileptiform activity (Carvalho 2012; De Deyn 1997).

[0171]

[0187] In this study, all subjects consented to baseline and follow-up routine (i.e., non-sleep, ambulatory, or videotelemetry) ambulatory EEG recordings.

[0172]

[0188] Electroencephalograms (EEGs) were performed on all subjects at screening and at week 24. EEG results were interpreted as normal, non-clinically significant (NCS) abnormal, or clinically significant (CS) abnormal. If the result was reported as a CS abnormality, the abnormality was recorded.

[0173]

[0189] Subjects with EEG abnormalities reported as CS abnormalities were listed for the DB analysis period. No other summary reports or lists were produced for these data.

[0174] Clinical Laboratory Assessment

[0190] A series of clinical markers, shown in Table 2 below, were assessed in all study subjects.

[0175] [Table 5]

[0176] [Table 6]

[0177] Individualized disease management plan (and 3-day food diary)

[0191] Current recommended disease management approaches include 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 increasing or decreasing different components to ensure adequate intake of protein for proper growth and development while reducing plasma arginine levels and avoiding hyperammonemia. General guidelines suggest that diets for patients with ARG1 D include approximately 50% protein from natural food sources and approximately 50% from amino acid supplements. This recommendation could be tailored to achieve appropriate IDM objectives for a particular subject if clinically indicated.

[0178]

[0192] Subjects maintained the IDM regimen prescribed pre-enrollment by the treatment investigator throughout the study. The prescribed IDM regimen was not allowed to be altered during the study unless clinically indicated in the investigator's opinion. The rationale for any change in IDM and the date and nature of any major (>±15% of baseline) prescribed changes were recorded in the eCRF.

[0179]

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

[0180]

[0194] Subjects and / or their caregivers were instructed to record all dietary intakes (natural foods, medical foods / EAAs, and EAA supplements) by the study subjects for 3 consecutive days prior to all clinic visits during screening and baseline periods, and prior to DB6, 12, 18, and 24 visits. The prescribed IDM regimen was also recorded at these visits. The date and time of the last dose of EAA was recorded for each day of the 3-day dietary diary.

[0181] Medical history

[0195] In addition to arginase deficiency, the most frequently reported medical history includes cognitive impairment, hyperammonemia, growth retardation, and increased alanine aminotransferase, which are common clinical findings in ARG1-D.A higher proportion of subjects in the peggylarginase group compared to subjects in the placebo group had a history of hyperammonemia (i.e., before treatment).Growth retardation and cognitive impairment were reported in a lower proportion of subjects in the peggylarginase group than in the placebo group (before treatment).

[0182]

[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 were commonly taken to control ammonia levels, and medications were taken to control other disease symptoms.

[0183]

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

[0184]

[0198] Other frequently used medications in both the peggylarginase and placebo groups included essential amino acid formula, baclofen, ibuprofen, paracetamol, vitamin and mineral food supplements, and cetirizine.

[0185] [Table 7]

[0186] [Table 8]

[0187] [Table 9]

[0188] 1.6 Efficacy Assessment

[0199] The study will consist of two periods, a placebo-controlled, double-blind period and an LTE period.

[0189] Primary Endpoint

[0200] Subjects treated with peggylarginase had a rapid and sustained decrease in mean plasma arginine, with a drop seen at week 1, within normal limits by week 12, and sustained through week 24. Mean plasma arginine in the placebo group remained largely unchanged (Figure 3A). At week 24, mean plasma arginine in the peggylarginase group was 105.49 μM compared to 448.82 μM in the placebo group. Treatment with peggylarginase resulted in a meaningful decrease in arginine (i.e., values ​​<200 μM) compared to placebo starting at week 6, which was maintained through week 24 of treatment.

[0190] Primary endpoint analysis

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

[0191]

[0202] In an individual subject assessment of plasma arginine reduction, nearly all subjects (95%, N=20) in the pegylarginase group had a meaningful change in plasma arginine at week 24 compared to the placebo group (Figure 2).

[0192] Key secondary endpoints a. 2-minute walking test

[0203] The 2-minute walk test (2MWT) is an abbreviated adaptation of the 6-minute version (e.g., National Institutes of Health [NIH] Toolbox Motor Domains-2MWT [Bohannon 2014]) and followed standardized methods defined by the American Thoracic Society (2002). Results of the 2MWT were recorded as distance walked in meters and percent change from baseline.

[0193]

[0204] Subjects treated with peggylarginase demonstrated improved 2MWT compared to the placebo group. At baseline, the mean (SD) walking distance over 2 minutes for subjects receiving peggylarginase was 109.0 meters (SD 55.76 meters), which was greater than 99.9 meters (SD 38.61 meters) for subjects receiving placebo. At week 24, the mean (SD) walking distance over 2 minutes for the peggylarginase group was 115.9 meters (SD 51.81), representing an increase of 7.3 meters from baseline compared to the mean walking distance for the placebo group of 102.3 meters (SD 51.10), which represents a difference of 2.7 meters from baseline. Although the change from baseline to week 24 for the LS mean difference in 2MWT between the peggylarginase and placebo groups (5.5 meters) did not meet statistical significance (p=0.5961) (Figure 4A), this indicated that subjects in the peggylarginase group had greater improvement than subjects in the placebo group (Figures 4A and 4B).

[0194]

[0205] In an individual subject assessment of change from baseline in 2MWT using available baseline and week 24 data (N=29), a greater proportion of subjects in the peggylarginase group (52.6%, N=10) had an improvement in change from baseline in 2MWT distance compared to the placebo group (40%, N=4), and the magnitude of improvement was generally greater in subjects in the peggylarginase group versus the placebo group.

[0195] b. Gross Motor Function Scale-88 Part E

[0206] The Gross Motor Function Scale-88 (GMFM-88, also referred to as GMFM) is a clinical scale designed to evaluate gross motor function by observing a subject's ability to initiate and complete a movement. For this study, only domains D and E were assessed. A scoring system was used by trained observers for each item of the GMFM covering two domains: standing (part D) and walking, running, and jumping (part E).

[0196]

[0207] Subjects treated with peggylarginase demonstrated improvement in GMFM-E compared to the placebo group. At baseline, the mean (SD) GMFM-E score for subjects in the peggylarginase group was (48.3 [SD 19.93]) and in the placebo group (46.5 [SD 24.56]). At week 24, the mean (SD) GMFM-E score for the peggylarginase group was 52.0 (SD 21.27), representing an increase 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 from baseline to week 24 for the LS mean difference in GMFM-E score between the peggylarginase and placebo groups was 4.6 points, representing a trend toward improvement (p=0.1087) (Figure 5A).

[0197]

[0208] Individual responses are summarized by waterfall plots of GMFM-E and change from baseline at week 24 (Figure 5B). A similar proportion of subjects in the peggylarginase group (55.0%, N=11) had improvement in change from baseline in GMFM-E compared to the placebo group (54.5%, N=6), but the magnitude of improvement was generally greater in peggylarginase subjects compared to placebo subjects (Figure 5B).

[0198]

[0209] In individual subject assessment of change from baseline in GMFM-E, a similar proportion of subjects in the peggylarginase group (55.0%, N=11 / 20) compared with the placebo group (54.5%, N=6 / 10) had improvement in change from baseline in GMFM-E score at week 24, although the magnitude of improvement was generally greater in subjects in the peggylarginase group versus the placebo group.

[0199] c. Responders based on arginine levels less than 200 μM (<200 μM)

[0210] Subjects treated with peggylarginase demonstrated improved plasma arginine levels that met treatment guidelines compared to the placebo group. Current guidelines recommend a treatment goal of plasma arginine of less than 200 μM. The proportion of subjects who could achieve this goal (responders) was assessed by treatment group. Nineteen of 21 subjects in the peggylarginase group (90.5%) achieved arginine values ​​<200 μM and were responders after 24 weeks of treatment compared to zero in the placebo group (Table 4).

[0200] [Table 10]

[0201] d. Responders based on arginine levels within the normal range (40-115 μM)

[0211] The subjects in the peggylarginase group showed an improvement in plasma arginine levels resulting in normal arginine levels compared to the placebo group. While current guidelines recommend plasma arginine of less than 200 μM, achieving values ​​within normal range is an important therapeutic objective. The proportion of subjects who could achieve this treatment objective was assessed for each treatment group. At week 24, 90.5% of the subjects in the peggylarginase group achieved arginine values ​​within normal range and met this response criterion, while no subjects in the placebo group met this response criterion (Table 5 and Figure 3B).

[0202] [Table 11]

[0203] e. Changes in ornithine and guanidino compounds at 24 weeks

[0212] Baseline ornithine values ​​and changes from baseline are presented in FIG. 6, and baseline guanidino compound values ​​and changes from baseline are presented in FIGS. 7-10.

[0204]

[0213] Ornithine and urea are products of the hydrolysis of arginine by arginase I in the final step of the urea cycle. Ornithine levels are generally low in subjects with ARG1-D due to the lack of the enzyme arginase I available to convert it from arginine. A summary of the change in ornithine from baseline is presented in Figure 6.

[0205]

[0214] Subjects treated with peggylarginase had a rapid and sustained increase in mean plasma ornithine, with increases seen at week 1 and continued improvement to week 12 levels that were sustained through week 24. At baseline, the median ornithine level in the peggylarginase group was 32.1 μM and in the placebo group was 29.2 μM. Subjects treated with peggylarginase had an increase in ornithine at week 1 compared to minimal change in the placebo group, which continued to increase with further dosing (FIG. 6). Treatment with peggylarginase resulted in a significant increase in ornithine starting at week 6 compared to placebo (p<0.0001) that was maintained through week 24 of treatment. At week 24, the mean ornithine level in the peggylarginase group was 67.7 μM, demonstrating a 106.9% increase in mean ornithine compared to placebo. Starting at week 6 and maintained through week 24 of treatment, the mean ornithine levels in the peggylarginase group were at least 60 μM.

[0206] f. Guanidino compounds in arginase-1 deficiency

[0215] Guanidino compounds (GCs) are direct and indirect products of arginine metabolism and are commonly elevated in ARG1-D. These compounds may contribute to hyperargininemia as well as pathophysiological changes, such as seizures and liver dysfunction, in ARG1-D, although the exact mechanisms remain incompletely understood (Deshmukh 1991; Franzoi 2018; Yu 2013).

[0207]

[0216] Abnormalities in guanidino compounds, nitric oxide, and homoarginine concentrations have been implicated in playing a pathophysiological role in Arg-1 D. Elevations in guanidino compounds have been observed in animals and humans with hyperargininemia. These arise from elevated arginine levels via the arginine:glycine amidinotransferase reaction. Guanidino compounds such as guanidinoacetate have been shown to be epileptogenic and may contribute to neurological dysfunction and epilepsy in these patients.

[0208]

[0217] At baseline, plasma levels of guanidine compounds were similar between treatment arms, but values ​​of alpha-keto delta-guanidinovaleric acid and alpha N-acetylarginine were progressively higher in the peggylarginase group. Median values ​​of arginic acid, guanidinoacetic acid, alpha-keto delta-guanidinovaleric acid, and alpha N-acetylarginine in the peggylarginase group were 2.5 μM, 3.1 μM, 4.6 μM, and 1.1 μM, respectively, in the peggylarginase group and 2.8 μM, 3.5 μM, 5.4 μM, and 1.7 μM, respectively, in the placebo group.

[0209]

[0218] For the peggylarginase group, a reduction in the levels of all four GCs was observed starting at week 1 and was maintained over time until week 24 of the double-blind period (Figures 7, 8, 9, and 10, respectively). Meanwhile, for the placebo group, the levels of all four GCs fluctuated over time but remained similar to baseline levels until week 24. Treatment with peggylarginase resulted in a significant reduction in GCs compared to placebo starting at week 6 (p-value range: 0.0059 to <0.0001) that was maintained until week 24 of treatment. At week 24, peggylarginase demonstrated a 50% to 70% reduction in GCs (compared to placebo) accompanied by a reduction in plasma arginine levels. Starting at week 6 and maintained through week 24 of treatment, the mean alpha N-acetylarginine level in the PEGylarginase group was reduced to less than 0.5 μM (0.3 μM at week 24); the mean alpha-keto-δ-guanidinovaleric acid level in the PEGylarginase group was reduced to less than 2 μM (1.3 μM at week 24); the mean arginine acid level in the PEGylarginase group was reduced to less than 1 μM (0.7 μM at week 24); and the mean guanidinoacetic acid level in the PEGylarginase group was reduced to less than 2 μM (1.7 μM at week 24).

[0210]

[0219] g. Guanidino compounds in renal disorders

[0220] When disease causes renal impairment, toxic guanidino compounds can accumulate in patients.Arginine produced from citrulline is mostly derived from kidney.Arginine is involved in guanidino compound biosynthesis, which requires cooperation between organs.In renal failure, citrulline can accumulate in plasma in proportion to renal impairment.Therefore, the disorder of arginine and guanidino compound metabolism is predicted in some tissues.

[0211]

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

[0212]

[0222] The metabolism and / or tissue content of several guanidino compounds were perturbed. The experimentally proven toxins α-keto-δ-guanidinovaleric acid and guanidinosuccinic acid (GSA) accumulated in the injured kidney. Nephrectomy also affected guanidino compound levels and metabolism in muscle and liver. Thus, disturbance of renal metabolism and function (i.e., renal injury) may result in the accumulation of toxic guanidino compounds.

[0213]

[0223] There is also evidence that the neurotoxic substance guanidinoacetate can cause seizures when it accumulates above normal levels. Similarly, reduced guanidinoacetate levels in patient plasma have been associated with reduced epileptogenic effects of GAA. There is also evidence that guanidinoacetate plays a pathogenic role in the development of extrapyramidal movement disorders.

[0214]

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

[0215]

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

[0216]

[0226] With regard to the diagnosis and management of CKD, EEG findings can be diagnostically useful as the degree of EEG changes correlates with the severity of encephalopathy. Typical EEG features in uremic neuropathy, such as slowing of alpha rhythm with excess delta and theta waves, are often nonspecific. The presence of triphasic sharp waves on the EEG is considered a specific feature of metabolic encephalopathy. Triphasic waves are typically seen in uraemic g.

[0217] h. Other disabilities

[0227] Secondary increases in GAA are demonstrated in argininemia, S-adenosylhomocysteine ​​hydrolase deficiency (SAHH deficiency), and cobalamin disorders. Arginine supplementation is also known to contribute to mild increases in GAA. Encephalopathy.

[0218] i. Guanidino compounds in guanidinoacetate methyltransferase (GAMT) deficiency

[0228] Guanidinoacetate methyltransferase (GAMT) deficiency (MIM601240) is an autosomal recessive inborn error of creatine synthesis, biochemically reflected by creatine deficiency and significant accumulation of guanidinoacetate (GAA) in the brain and body fluids, which results in physical or mental disabilities, such as global developmental delay / intellectual disability (DD / ID), epilepsy, movement disorders, speech or language development delay, and behavioral problems. Affected individuals exhibit a variety of neurological symptoms, including significant impairments in expressive language, autistic features, and epilepsy and movement disorders. GAA in affected individuals is believed to be neurotoxic.

[0219]

[0229] Strategies for reducing guanidinoacetate (GAA) levels include substrate deprivation via an arginine restricted diet and competitive inhibition of arginine glycine amidinotransferase (AGAT) activity via high-dose L-ornithine supplementation. AGAT is the enzyme responsible for GAA synthesis. Although medical diets aimed at reducing arginine intake and decreasing systemic arginine availability have been shown to be effective in reducing GAA levels and ameliorating disease symptoms associated with high GAA levels, the required diets are problematic for a number of reasons.

[0220] Guanidino compounds in jS-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. Guanidinoacetate is observed to be elevated in S-adenosylhomocysteine ​​hydrolase deficiency.

[0221] k. Change in gross motor function measure part D (GMFM-D)

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

[0222]

[0232] Individual responses are summarized by waterfall plots of GMFM-D and change from baseline at Week 24 (Figure 11). A greater proportion of subjects in the peggylarginase group (65.0%, N=13) compared with the placebo group (54.5%, N=6) had an improvement in the mean change from baseline at Week 24 in GMFM-D scores, with the magnitude of improvement generally being greater in peggylarginase subjects versus placebo subjects.

[0223] l. Arginine and clinical outcomes: individual and combined responses

[0233] Considering the normalization of arginine levels and the composite of functional assessments after 24 weeks of treatment within evaluable subjects, the subject levels and overall response demonstrated a differential response in favor of subjects treated with pegylarginase compared to placebo.

[0224]

[0234] Heat maps were developed to represent subjects who met the threshold for normalization of arginine levels and met the criteria for response in 2MWT, GMFM-E, and GMFM-D to characterize the relationship between reduction in arginine levels and clinical outcomes (Figure 12). In the peggylarginase-treated group, 13 of 17 subjects with normalized arginine levels who were evaluable for response had one or more assessments that met the criteria for meaningful clinical improvement, while no subjects in the placebo group had a clinical improvement corresponding to normalized arginine levels, but 4 of 9 evaluable subjects had clinically meaningful improvement across one assessment domain. There were 8 and 6 subjects in the peggylarginase group who met ≥ 2 criteria for clinical response without exacerbation and without exacerbation, respectively, and 0 subjects in the placebo group.

[0225] m. Improvement in measuring spasticity and other neurological / neuromuscular signs using the modified Ashworth scale

[0235] The Modified Ashworth Scale (MAS) was developed to assess spasticity in subjects with central nervous system lesions and is used to measure the tolerance to passive movement of a joint due to spasticity. The scale utilizes a scoring scale of 0 (no spasticity) to 4 (complete stiffness). Thus, a decrease in MAS score indicates an improvement in spasticity. The mean MAS lower body score was defined as the average of the values ​​for all muscle groups with a score value >0.

[0226]

[0236] At baseline, the mean (SD) MAS lower body score was 0.77 (SD 1.076) in the peggylarginase group and 0.25 (SD 0.433) in the placebo group. By week 24, there was a numerical improvement in the peggylarginase group compared to placebo. The mean change from baseline in MAS lower body score for subjects in the peggylarginase and placebo groups was -0.16 (SD 0.519) and -0.03 (SD 0.056), respectively. These improved scores indicate that four of five subjects with baseline spasticity in the peggylarginase group demonstrated improvement, whereas only one of two subjects in the placebo group showed improvement.

[0227] n. Nutritional management of patients

[0237] Clinical studies of urea cycle disorders (UCDs) have revealed considerable variation in protein tolerance, with age, sex, and genotype all contributing to this variation. Patients with the mildest forms of UCD can tolerate a normal diet, in contrast to those with severe forms who require a very restricted diet. The goal of treatment for patients with UCDs is to maintain normal plasma ammonia and amino acid concentrations with nutritional management and pharmacotherapy. Nutritional therapy for UCDs consists of a protein-restricted diet supplemented with an EAA mixture to ensure adequate intake of essential and conditionally essential amino acids. Sufficient protein-free energy must be administered both to allow normal turnover of protein required at all ages and to prevent net catabolism of body protein.

[0228]

[0238] Long-term management seeks to minimize the nitrogen load on the urea cycle. The amount of natural protein tolerated by each patient must be determined individually, with an increase or decrease to ammonia. The WHO Food and Agriculture Organization of the United Nations has set a "safe level of protein intake", calculated as an age-adjusted average, which can be used as a guideline. Lower protein intakes may still be appropriate, but individualized over-restriction may attenuate growth and cause metabolic instability. If intake is too low, additional supplementation may be applied. An adequate energy supply must also be ensured to prevent catabolism and the resulting hyperammonemia. The FAO / WHO / UNU 2007 Report (summarized in Figure 13, derived from Harberle et al.) can be used as a guideline for energy intake. Patients with reduced mobility have lower energy expenditure and therefore lower energy requirements.

[0229]

[0239] Essential amino acids (also known as indispensable amino acids) are those that humans and other vertebrates cannot synthesize from metabolic intermediates. These amino acids must be supplied from the diet because the human body lacks the metabolic pathways required to synthesize them. There are nine essential amino acids, with possible variations depending on the metabolic status of the individual: 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, can be excluded from the diet. The human body can synthesize them using only the essential amino acids.

[0230]

[0240] Supplementation with EAAs and other essential nutrients is required when natural protein tolerance is too low to achieve normal growth and metabolic stability. Currently, there are no methods to increase natural protein tolerance.

[0231]

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

[0232]

[0242] Deviations from the standard healthy adult metabolic state can expose the body to metabolic conditions that require more than just the standard essential amino acids to be in nitrogen balance. Children and infants may also require more than just the standard essential amino acids. In general, the optimal ratio of essential to non-essential amino acids requires a balance that varies between individuals depending on physiological needs (including different disease states).

[0233]

[0243] Although individual variations exist, the World Health Organization estimates an average total protein requirement of 0.66 g / kg per day, with an intake of approximately 0.18 g / kg per day of essential amino acids and 0.48 g / kg per day of non-essential amino acids.

[0234]

[0244] Protein aversion is a common feature of UCD. Neonatal symptoms of UCD usually appear after the first 24-48 hours after birth upon completion of normal pregnancy and delivery. Common presenting symptoms are anorexia, vomiting, fatigue, hypotension, dyspnea, irritability, and seizures. Patients have been reported in whom a detailed medical history obtained after the onset of severe neurological symptoms revealed severe, previously unrecognized protein aversion. Many infants have a picky eating behavior during the first few years after birth. In contrast to most "normal" picky-eating infants who tend to have more problems with vegetables, infants with UCD develop a protein aversion, particularly with respect to meat, eggs, and dairy products, but prefer fruits and vegetables. Metabolic deterioration in patients may result from non-compliance with the diet. It has been suggested that patients may eat more protein than recommended due to reluctance to give up favorite foods, inadequate compliance by the patient's family, or severe peer pressure.

[0235] o. Food record

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

[0236]

[0246] Median baseline total protein (including native and EAA proteins) was similar between treatment groups, while median total calories was lower in the peggylarginase group versus the placebo group. Median baseline total calories and total protein (including native and EAA proteins) were lower in the peggylarginase group than in the placebo group.

[0237]

[0247] The median changes from baseline for total prescribed EAA protein and total consumed EAA protein from weeks 12 to 24 were 0.0 for both.

[0238]

[0248] The median change from baseline in total natural protein consumed was 0.0 grams for both treatment groups at weeks 12 and 24. Small variations in the change from baseline in median total natural protein consumed from weeks 12 and 24 were observed in the PEGylated arginase group (-0.2 and -0.5, respectively) and the placebo group (0.1 and 0.7, respectively).

[0239] p. Meal management plan (and 3-day food diary)

[0249] Overall, most subjects from the peggylarginase group had an increase of >15% in their total calories consumed, total protein consumed / day, and total natural protein consumed, or remained within 15%. For total EAA protein consumed, the majority of subjects remained within 15% of baseline. However, by week 24, a higher proportion of subjects in the peggylarginase group increased their total protein consumption by more than 15% compared to placebo (42.9% [9 subjects] vs. 18.2% [2 subjects]) (Table 6). Even though a higher proportion of subjects had a higher total protein intake, subjects had normalized arginine, and therefore dietary changes did not negatively affect the ability of peggylarginase to control arginine.

[0240] [Table 12]

[0241] [Table 13]

[0242]

[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). While hyperammonemic crisis is less common in patients with arginase deficiency than other urea cycle disorders, it can still occur throughout childhood and adulthood. Current treatment of ARG1-D focuses on attempts to lower plasma arginine levels through lifelong dietary protein restriction. Protein intake is limited to the minimum required to maintain protein biosynthesis and growth.

[0243]

[0251] Typically, more than half of the dietary protein is provided in the form of an arginine-free essential amino acid (EAA) mixture. A typical physician-recommended protein intake for a healthy individual is approximately 10-53 grams per day, whereas the recommended intake for an arginase 1 deficiency patient is approximately 4-40 grams per day (about 25-60% less protein). The challenge of adhering to a protein-restricted diet severe enough to reduce plasma arginine below the target level places a significant burden on the patient and their family. Adherence to this type of diet can be difficult due to established eating habits; can exacerbate eating disorders; and requires regular follow-up to assess the disease state.

[0244] Q.Electroencephalogram

[0252] There were no statistically significant differences between the treatment groups overall in EEG results, but individual patients showed improvement. At baseline, 28.6% (6 subjects) from the peggylarginase group and 45.5% (5 subjects) from the placebo group had clinically non-significant abnormal EEGs, with the exception of one subject. One subject from the peggylarginase group had abnormal clinically significant EEG results at baseline. This subject had generalized epileptiform discharges. No subjects in the placebo group had clinically significant abnormal EEGs.

[0245]

[0253] At week 24, there were 23.8% (5 subjects) from the peggylarginase group and 63.6% (7 subjects) from the placebo group with abnormal EEG but not clinically significant results. Three subjects from the peggylarginase group who had baseline abnormal EEG (clinically non-significant) shifted to normal EEG at week 24; all subjects from the placebo group who had baseline abnormal EEG results that were not clinically significant had clinically non-significant abnormal EEG results at week 24. One subject from the peggylarginase group and two subjects from the placebo group had normal EEG results at baseline and then clinically non-significant abnormal EEG results at week 24. One subject from the peggylarginase group had a clinically significant abnormal EEG, which was recorded as a pathological wake-sleep EEG. There were no subjects in the placebo group with clinically significant abnormal EEG.

[0246] r. Electroencephalography and guanidino compounds

[0254] Electroencephalogram (EEG) patients with altered EEG status (and description of the guanidino compound changes) are shown in Table 7 below.

[0247] [Table 14]

[0248] 1.7 Patient Summary

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

[0249]

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

[0250]

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

[0251]

[0258] 129-0001: At screening, EEG was clinically significant (CS) abnormal, whereas after 24 weeks of peggylarginase treatment, EEG was not clinically significant (NCS) abnormal. At screening, GAA was within normal range, which was maintained at 24 weeks of treatment. At screening, NAArg, GVA, and AA levels were above normal range. At 24 weeks of treatment, AA was above normal range but had decreased from 2.11-2.81 uM to 0.38-0.61 uM. At 24 weeks of treatment, NAArg was in normal-high range and had decreased from 1.4-1.92(H) uM to 0.25(N)-0.53(H) uM. At 24 weeks of treatment, GVA was above normal range but had decreased from 3.47-5.57(H) to 0.65-1.05(H).

[0252]

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

[0253]

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

[0254]

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

[0255]

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

[0256] 1.8 Pharmacokinetic Analysis

[0263] Adequate PK exposure was achieved within the dose range of 0.05-0.2 mg / kg IV, consistent with the dose-response relationship observed in previous studies. Maximum observed peggylarginase concentrations (t max The median time to PK exposure (C) generally occurred rapidly (≤4.7 hours after initiation of infusion) and was as expected after a set 0.5-hour QW IV infusion of peggylarginase. max and AUC 0-168 ) increased approximately dose-proportionally over the dose range of 0.05 to 0.2 mg / kg at steady state after repeated QW dosing (weeks 12 and 24). Mean t 1 / 2 was approximately 40 hours (range: 37.3 to 43 hours) and was similar across doses, whether administered as a single dose or at steady state. Steady state was achieved at or before week 12 based on available data and sampling times; however, it is theoretically possible that t 1 / 2 Based on the data, this is expected to be reached after 2 weeks of consistent QW dosing. Exposure was relatively constant for weeks 12 and 24 versus week 1, with negligible accumulation after weekly dosing.

[0257] 1.9 Pharmacodynamic analysis

[0264] PEGylarginase 1 / 2 The rapid onset of action of resulted in an early, consistent and sustained reduction in plasma arginine. At all time points assessed after dosing (weeks 1, 12 and 24), arginine remained below the upper limit of normal (115 μM) for the majority of the QW dosing interval. At weeks 12 and 24, post-dose plasma arginine levels were generally lower than baseline values ​​for all subjects and all doses, and below guideline recommended levels (<200 μM) for all but one subject. Deviations below 40 μM were observed early after IV dosing, with the majority of subjects approaching the normal range by 96 hours, and all subjects having levels above 40 μM at 168 hours.

[0258] 1.10 Efficacy Conclusions Double-blind period

[0265] The objective of this study was to demonstrate efficacy of PEGylarginase versus placebo based on statistically significant reductions in plasma arginine concentrations and neuromuscular assessments. 1 / 2 produced early, consistent and sustained reductions in plasma arginine levels and improved 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 PEGylarginase group and 471.74 [SD 79.928] μM in the placebo group. After 24 weeks of treatment with PEGylarginase, mean plasma arginine values ​​in the PEGylarginase group demonstrated an early, consistent, and sustained reduction.

[0259]

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

[0260]

[0267] At weeks 12 and 24, nearly all subjects receiving PEGylated arginase achieved plasma arginine levels <200 μM (95.2% [20 / 21] and 90.5% [19 / 21], respectively, p<0.0001).

[0261]

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

[0262]

[0269] The mean improvement from baseline in functional mobility as measured by GMFM E was 4.2 (N=20) at week 24 for the peggylarginase group compared to a decrease of 0.4 in the placebo group. The LS mean difference at week 24 was 4.6 (p=0.1087), demonstrating a trend toward clinically meaningful improvement. This improvement exceeds the minimal clinically meaningful difference for patients with similar neuromuscular diseases that is applicable to ARG1-D based on psychometric analysis in patients with this disorder.

[0263]

[0270] An increase from baseline in the mean distance walked over 2 minutes as measured by the 2MWT was demonstrated at week 24 of the double-blind period. In the double-blind period, the mean improvement from baseline at week 24 was 7.3 meters (N=19) in subjects treated with peggylarginase compared to 2.3 meters (N=10) in the placebo group. The mean difference in LS at week 24 between the peggylarginase and placebo groups was 5.5 meters, representing a numerical improvement (p=0.5961).

[0264]

[0271] Subjects with a GMFCS classification of ≧II demonstrated greater improvements in GMFM-E and 2MWT compared to subjects with GMFCS I when treated with pegylarginase compared to placebo.

[0265]

[0272] An increase in ornithine and an approximately 50-70% decrease in GC was accompanied by a reduction in plasma arginine in subjects treated with peggylarginase, with no significant changes noted in the placebo group.

[0266]

[0273] The mean improvement from baseline in GMFM-D was 2.5 (N=20) at week 24 for the peggylarginase group compared with -1.3 for the placebo group, demonstrating numerical improvement and potential clinical relevance.

[0267]

[0274] The mean (SD) improvement from baseline in the Vineland Adaptive Behavior Assessment System (VABS-II) composite score was 1.4 (SD 16.54) in the peggylarginase group and -1.6 (SD 8.78) in the placebo group at week 24. The LS mean difference at week 24 was 4.9, indicating a numerical improvement in favor of peggylarginase. At week 24, the proportion of eligible subjects who met the criteria for clinical response (N=17 for peggylarginase and N=9 for placebo) was higher for each individual component (2MWT, GMFM-D, GMFM-E) in the peggylarginase compared to placebo group. 2MWT, N=7 subjects met criteria: - Peglyarginase group: 29.4% (N=5) - Placebo group: 22% (N=2) GMFM-E, N=11 subjects met criteria: - Peglyarginase group: 52.9% (N=9) - Placebo group: 18.2% (N=2) GMFM-D, N=7 subjects met criteria: - Peglyarginase group: 41.1% (N=7) - Placebo group: 0% (N=0)

[0268]

[0275] When considered as the absence of any worsening of response in any individual component (response criterion definition 1), the composite clinical response was similar in the pegylarginase group (41.2%; N=7) compared with the placebo group (44.4%; N=4) for the response to any single endpoint.

[0269]

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

[0270]

[0277] A higher percentage of subjects met the criteria for a clinically meaningful response in two or more assessments in the peggylarginase group compared to the placebo group, which was zero (47.0% without exacerbation; N=8, 35.3% without exacerbation; N=6). Additionally, the percentage of subjects who achieved a clinical response in all three measures was higher in the peggylarginase group (11.7%; N=2) compared to the placebo group, which was zero. The magnitude of the response was generally higher in subjects treated with peggylarginase compared to placebo.

[0271]

[0278] In the peggylarginase 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 meaningful.

[0272]

[0279] No conclusive differences were observed in other endpoints (i.e., 9-hole pegboard dexterity test, caregiver and clinician global perception of change, VABS-II, FMS / GFAQ, PEDsQL, responder based on neurocognitive assessment).

[0273]

[0280] Following QW IV administration, PK exposure increased approximately dose-proportionally over the dose range evaluated, with negligible accumulation and correlated with arginine depletion.

[0274]

[0281] Anti-drug antibodies were minimal, transient, and resolved without significant clinical impact.

[0275] Hyperammonemic events

[0282] Overall, 21.9% (N = 7) of subjects experienced hyperammonemic episode AEs, with a smaller proportion in the peggylarginase group (14.3% [N = 3]) compared with placebo g. Hyperammonemia was reported in a lower proportion of peggylarginase group subjects (9.5% [N = 2]) compared with placebo g (27.3% [N = 3]). Hyperammonemic encephalopathy was observed in 4.8% (N = 1) of peggylarginase group subjects and 9.1% (N = 1) of placebo group subjects. No hyperammonemic crisis TEAEs were reported.

[0276] Example 2: A brief summary of PEACE

[0283] As described above, PEACE was designed to assess the effect of treatment with pegylarginase (n=21) versus placebo (n=11) from baseline to a pre-specified 24-week treatment period.

[0277] 1.1 Top-line results

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

[0278]

[0285] Normal plasma arginine levels (40-115 μM) were achieved in 90.5% of peggylarginase-treated patients compared with 0 patients in the placebo arm.

[0279]

[0286] Concomitant improvements in primary and secondary mobility assessments in peggylarginase-treated patients compared with patients in the placebo arm. o Gross Motor Function Scale Part E (GMFM-E): Least squares mean scores improved by 4.2 units for peggylarginase-treated patients and worsened by 0.4 units in the placebo arm (p=0.1087), establishing a favorable trend. Two-minute walk test (2MWT): least squares mean distance increased by 7.4 meters in peggylarginase-treated patients and 1.9 meters in the placebo arm (p=0.5961).

[0280]

[0287] Pegzylarginase was well tolerated and safety data were consistent with results from prior clinical trials. Adverse events were generally mild to moderate in severity. There were no study discontinuations due to adverse events.

[0281] 1.2 Patient-level outcome analysis

[0288] In an analysis of individual patients who were Gross Motor Function Classification System (GMFCS) levels I-III using predefined clinical response criteria, there were clinically meaningful differences between peggylarginase-treated patients (n=17) and placebo (n=9). Eleven patients (65%) treated with peggylarginase compared with four patients (44%) who received placebo met or exceeded pre-specified response criteria for at least one mobility assessment. Eight patients (47%) met or exceeded pre-specified clinical response criteria for at least two of the mobility outcomes compared with zero patients who received placebo.

[0282]

[0289] Six of the patients who met or exceeded the clinical response threshold for at least two mobility outcomes also showed no deterioration for any other mobility endpoints. Additional analyses were performed on these six patients to compare improvement matched for age and sex (Table 8 below).

[0283] [Table 15]

[0284] 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 for patients treated with peggylarginase improved by 2.25 units from baseline compared to placebo (p=0.0896). Peggylarginase-treated patients also showed statistically significant biochemical improvements in measures of ornithine and guanidino compounds compared to placebo, consistent with the peggylarginase mechanism of action (Figure 14).

[0285] Example 3: Optimized Dosing Simulation

[0291] Based on cumulative patient data from Phase 3, a dosing simulation was performed to assess different dosing strategies.

[0286]

[0292] The starting dose was administered for 4 weeks after week 5, and the dose could be increased or decreased based on the value of the 168 hour post-dose L-arginine sample using the following algorithm: If L-arginine was >150uM 168 hours after any dose, the dose was increased, up to a maximum of 0.2mg / kg. If L-arginine was <50uM 168 hours after two consecutive doses, the dose was reduced.

[0287]

[0293] The dosing algorithm is optimized to ensure that L-arginine remains within 50-150 uM 168 hours post dose. The dosing strategy shown in Figure 15 was tested for IV and SC administration:

[0288]

[0294] In scenario 1 (0.1 / 0.1 mg / kg) for approximately 75% of subjects, 0.1 mg / kg was sufficient to maintain L-arginine control. A starting dose of 0.05 mg / kg was sufficient to maintain L-arginine control for approximately 44% of subjects. Similar results were seen for the SC dosing route. (Figures 16 and 17)

[0289] 1.1 Relationship between baseline L-arginine and final dose (Figure 18)

[0295] Subjects with higher baseline L-arginine tended to require higher final maintenance doses regardless of dosing strategy. Similar results were seen for the SC dosing route.

[0290] 1.2 Number of dose adjustments required by IV route (Figure 19)

[0296] Scenario 1 (0.1 / 0.1 mg / kg) required the fewest number of dose titrations, while scenario 3 (0.05 / 0.05 mg / kg) required the greatest number of dose titrations (this was a result of the starting dose and the size of the dose increments). Similar results were seen for the SC route of administration.

[0291]

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

[0292]

[0298] SC dosing across all dosing scenarios provided tighter control compared to IV dosing of L-arginine over the dosing interval (shorter time <40uM, longer time 40-115uM). L-arginine values ​​168 hours post-dose correlated with control of L-arginine during the dosing interval and can be used for monitoring and dose titration decisions.

[0293]

[0299] A dose of 100-200uM L-arginine 168 hours later was thought to minimize the time that L-arginine remains <40uM while maximizing the time that L-arginine remains within the normal range but below guideline recommended levels.

[0294]

[0300] Dose escalation of 0.05 mg / kg allows for greater flexibility in dosing but requires more dose titration.

[0295] 1.3 Differences between IV and SC dosing (Figure 20)

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

[0296]

[0302] SC dosing results in lower overall PEGylarginase concentrations compared to IV dosing, and therefore the reduction in L-arginine is not substantial. SC dosing results in less fluctuation in PEGylarginase concentrations compared to IV dosing, and therefore the response is sustained for a longer period of time.

[0297] 1.4 L-Arginine Time Within Recommended Range (Figure 21)

[0303] All dosing strategies and routes of administration resulted in L-arginine remaining <200 uM for nearly the entire dosing interval. The SC route minimized the time L-arginine was <40 uM compared to the IV route. Similarly, SC dosing resulted in L-arginine remaining at 40-200 uM and 40-115 uM (normal range) for extended periods compared to IV dosing.

[0298]

[0304] It will be appreciated by those skilled in the art that modifications may be made to the above-described embodiments without departing from the broad inventive concept thereof. It is understood, therefore, that the invention is not limited to the particular embodiments disclosed, which are intended to cover modifications within the spirit and scope of the invention as defined by this detailed description.

[0299]

[0305] Various publications, articles and patents are cited or described in the Background and throughout this specification; each of these references is incorporated herein by reference in its entirety. The discussion of documents, acts, materials, devices, articles and the like which has been included in the specification is for the purpose of providing a context for the present invention. Such discussion is not an admission that any or all of those matters form part of the prior art with respect to any invention disclosed or claimed.

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Claims

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

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

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

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

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

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

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

8. 8. The method of claim 7, wherein the subsequent doses of PEGylated arginase are adjusted to 0.05 mg / kg each time based on the subject's plasma arginine level resulting from the prior dose.

9. The method of any one of claims 1 to 8, wherein the subject is not premedicated prior to the first dose of the pegzylarginase.

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 first dose of PEGylated 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 level II, III, IV, or V.

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

14. The method of any one of claims 1 to 13, wherein the subject's intake of natural protein is increased after one or more doses of the peggylarginase.

15. 15. The method of any one of claims 1 to 14, wherein the subject's relative intake of essential amino acid mixture compared to natural protein is reduced after one or more doses of the PEGylarginase.

16. 16. The method of any one of claims 1 to 15, wherein after one or more doses of the pegzylarginase, 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 food containing protein.

17. 16. The method of any one of claims 1 to 15, wherein after one or more doses of the pegzylarginase, the primary source of the subject's protein intake is changed from an arginine-free (or low-arginine) essential amino acid mixture to natural protein.

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

19. 19. The method of any one of claims 1-18, wherein the subject's total protein intake is increased by at least 15% after one or more doses of the peggylarginase.

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

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

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 peggylarginase.

23. 23. The method of any one of claims 1 to 22, wherein arginine metabolite accumulation is reduced following one or more doses of the peggylarginase.

24. The method of any one of claims 1 to 23, wherein accumulated guanidino compounds in the blood and cerebrospinal fluid of the subject are reduced.

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

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

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

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

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

30. 30. The method of any one of claims 1 to 29, wherein the pegylarginase is formulated in a buffer containing 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 PEGylated arginase is liquid.

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

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

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

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