Arginine depletion therapy for treating GAMT deficiency

Abstract

To provide a therapy of guanidinoacetate methyltransferase (GAMT) deficiency.SOLUTION: A method and a composition for treating GAMT deficiency or guanidinoacetic acid (GAA) toxicity in a subject include administration of arginine depleted enzyme. A therapeutic preparation can contain arginase, arginine deiminase or a combination thereof, and other compounds if needed, and can be adapted for intravenous or subcutaneous administration to a subject.SELECTED DRAWING: None

Description

[Technical field]

[0001] Sequence Listing A sequence listing entitled 218107_0013_00_WO_594073_ST25.txt, dated October 18, 2019 and having 9,110 bytes is submitted herewith and incorporated into the specification. [Background technology]

[0002] Guanidinoacetate methyltransferase (GAMT) deficiency (MIM 601240) is an autosomal recessive inborn error of creatine synthesis, biochemically reflected by a deficiency in creatine and a marked accumulation of guanidinoacetate (GAA) in the brain and body fluids, resulting in several physical or mental disabilities such as generalized developmental delay / intellectual disability (DD / ID) epilepsy, movement disorders, speech or language delay, and behavioral problems. Affected individuals show a variety of neurological signs, including marked impairments in expressive protolanguage, autistic features, and epilepsy and movement disorders (Stockler-Ipsiroglu, S. et al Molecular Genetics and Metabolism 111(2014)16-25; Dhar SU et al., Mol. Genet. Metab., 2009). Current recommended treatment is based on: (a) Oral supplementation of creatine (administered as creatine monohydrate) is used to increase brain creatine levels. In addition to increasing creatine / phosphocreatine (Cr / PCr) in the brain, oral creatine supplementation can reduce GAA in body fluids and brain, but GAA levels remain significantly elevated in most patients. Even after several months of treatment and despite pharmacological doses of creatine supplementation (0.35-2.0 g / kg / day), Cr / PCr concentrations in the brain of patients remain significantly below the normal range. In addition to Cr supplementation, Cr treatment leads to a decrease in GAA formation by the inhibition of the AGAT enzyme by Cr. By supplementing Cr alone, an approximately 50% decrease in GAA in body fluids can be achieved. Based on published data on Cr / PCr supplementation in the brain after Cr treatment, a preliminary comparison of the slope of brain Cr supplementation with the same dose of Cr between GAMT-D and AGAT-D patients revealed a faster rise in Cr / PCr and a more complete supplementation in the latter group. In contrast to GAMTD, GAA is not increased in AGAT-D (Schultze, Research Gate, March 2005). (b) Strategies to reduce guanidinoacetic acid (GAA) levels include substrate deprivation through an arginine-restricted diet, as well as competitive inhibition of arginine glycine amidinotransferase (AGAT) activity by high-dose L-ornithine supplementation. AGAT is the enzyme responsible for GAA synthesis. (c) Competitive inhibition and substrate deprivation approaches to lower GAA levels in affected patients, which are believed to be neurotoxic, include the use of orally administered L-ornithine (L-ornithine aspartate or L-ornithine hydrochloride) and dietary reduction aimed at reducing arginine intake.

[0003] Diets that aim to reduce arginine intake and decrease total body arginine availability have been shown to be effective in lowering GAA levels and improving disease symptoms associated with high GAA levels, but the required diets are problematic for several reasons. Such diets are difficult to accept because they require protein restriction, and goals often need to be modified over time based on each person's nutritional needs and age requirements. Calculating and tracking protein intake can be a difficult and tedious task for individuals on protein and arginine dietary restrictions. Diets often have palatability and taste issues that affect patients' quality of life and compliance. Dietary restrictions have limited impact because the high proportion of total body arginine is the result of tissue turnover and therefore is not affected by dietary restrictions. (Crombez,EA,and Cederbaum,SD,Mol.Genet.Metab.2005;Burrage,LC,et al.,Hum.Mol.Genet.2015;Carvalho,DR,et al.,Ped.Neurol.2012;Stockler-Ipsiroglu,S.et al.,2014;Viau,KSet al.,Mol.Genet.Metab.2013;Mercimek-Mahmutoglu,S.et al.,Gene,2015) Summary of the Invention

[0004] Provided herein are compounds, compositions and methods for administering arginine depletion enzymes to treat deficiency of guanidinoacetate methyltransferase (GAMT) activity in subjects or treat guanidinoacetate (GAA) toxicity.The compounds and compositions can reduce the arginine level of subjects, and at the same time provide ornithine from arginase-mediated metabolism of arginine.

[0005] Thus, a method for treating a deficiency in guanidinoacetate methyltransferase (GAMT) activity in a subject comprises administering to said subject a pharmaceutical composition comprising a therapeutic amount of an arginine-depleting enzyme. The arginine-depleting enzyme can be a mammalian or bacterial arginase enzyme. The arginine-depleting enzyme can be a human arginase enzyme, a human arginine deiminase enzyme, or a combination of these enzymes. Contemplated enzymes can also include enzyme variants of human arginase enzyme or human arginine deiminase enzyme, which are modified by substitutions, deletions, insertions, or truncations in the amino acid sequence of the enzyme, or a combination thereof. The human arginase enzyme can be a human arginase I enzyme or a human arginase II enzyme. The human arginase enzyme can be a CoA arginine depleting enzyme that is PEGylated and lacks an amino-terminal methionine. +2 It may be Arginase 1 having one or more substitutions or mutations in the polypeptide sequence.

[0006] The treatment / methods of use described herein also contemplate human, bacterial or mammalian Arginase I enzymes that have been engineered with substituted non-native metal cofactors, including cobalt.

[0007] The methods described can use autologous red blood cell ghosts to administer the arginine-depleting enzyme.

[0008] The method of treating a deficiency in GAMT activity is associated with a genetic deficiency in the gene encoding the guanidinoacetate methyltransferase enzyme in a subject.

[0009] The enzymes used in these methods, such as human arginase enzyme or human arginine deiminase enzyme, can be stabilized by association with a stabilizing agent. The stabilizing agent is selected from the group consisting of polyethylene glycol (PEG), synthetic protein polymers, polysialic acid, Fc fusions and albumin. The human arginase enzyme or human arginine deiminase enzyme can be further PEGylated. The size of the PEG can be 1 kD to 10 kD, any integer in between. The human arginase enzyme for use in the methods and compositions can be a PEGylated human arginase I enzyme.

[0010] The method of treatment or pharmaceutical use may be in a mammal, such as a human.

[0011] In another embodiment, 400 mM at pH 7.4 and 37° C. as measured in vitro. -1 s -1 ~4,000mM -1 s -1 k for the hydrolysis of arginine cat / K M Contemplated are methods of treating a deficiency in guanidinoacetate methyltransferase (GAMT) activity or treating guanidinoacetate (GAA) toxicity in a subject using a compound or composition having a human arginase I enzyme having the formula: ##STR00002## It is also contemplated that the human arginase I enzyme and arginase 1 enzyme may include cobalt in a ratio of 2μg Co / mg arginase to 3μg Co / mg arginase for the arginase I enzyme. The arginase I enzyme and arginase 1 enzyme may be generated by contacting the apoenzyme form with cobalt or cobalt ions at a temperature of 30°C to 55°C for 15 minutes to 60 minutes.

[0012] Also contemplated is a method of treating the effects of guanidinoacetic acid (GAA) toxicity in a subject deficient in guanidinoacetate methyltransferase (GAMT) activity, comprising administering to said subject a therapeutic amount of a pharmaceutical composition comprising a PEGylated human arginase I enzyme with a cobalt cofactor. The therapeutic amount of the pharmaceutical composition can be continued for a period of time until the subject shows improvement in the physical or neurological condition. The types of physical or neurological conditions contemplated can include conditions selected from the group consisting of global developmental delay / intellectual disability (DD / ID), epilepsy, movement disorders, speech or language delays, and behavioral disorders. The method contemplates that the therapeutic amount of PEGylated human arginase I or II enzyme (e.g., PEGylated arginase 1 enzyme such as AEB1102), which may include a cobalt cofactor, is about 0.01 mg / kg to about 7.5 mg / kg, about 0.05 mg / kg to about 5 mg / kg, or about 0.1 mg / kg to about 5 mg / kg. The therapeutic amount of the composition can be administered topically, intravenously, intradermally, intraarterially, intraperitoneally, intralesionally, intracranially, intraarticularly, intraprostatically, intrapleurally, intratracheally, intraocularly, intranasally, intravitreally, intravaginally, intrarectally, intramuscularly, subcutaneously, subconjunctivally, intravesicularly, mucosally, intrapericardially, subumbilically, orally, by inhalation, injection, infusion, continuous infusion, by direct local perfusion bath of the target cells, via a catheter, or via a lavage solution. In one embodiment, the method contemplates administration of a pharmaceutical composition adapted for subcutaneous administration to a subject. For subcutaneous formulations, an exemplary pharmaceutical composition can include a therapeutic dose of arginase in potassium phosphate, NaCl, sucrose, and PS80 at pH 6.7. For intravenous administration, an exemplary pharmaceutical composition can include PEGylated human arginase I or II enzyme, saline, and glycerol at pH 7.4. The method contemplates administering a pharmaceutical composition sufficient to reduce serum arginine content in the subject by at least 50%-99%, or 90%-99%, or 90%-99%. The method contemplates administering a pharmaceutical composition sufficient to reduce serum arginine content in the subject by at least 50%-99%, or 90%-99%, or 90%-99%. The method contemplates administering a pharmaceutical composition sufficient to reduce serum arginine content in the subject by at least 50%-99%, or 90%-99%, or 90%-99%.The method also contemplates administering the pharmaceutical composition in an amount sufficient to reduce serum GAA levels in the patient by at least 25%, at least 30%, at least 35%, at least 40%, at least 50%, or more, to an extent that GAA levels can be depleted. The method can use a pharmaceutical composition having a concentration such that the concentration in plasma reaches a maximum level 20-28 hours after a single dose, or reaches a maximum level about 24 hours after a single dose.

[0013] Another contemplated method includes a method for treating the effects of guanidinoacetic acid (GAA) toxicity, comprising administering a therapeutic amount of a pharmaceutical composition comprising an arginine depleting enzyme to a subject in need thereof. The arginine depleting enzyme can be an arginase or arginine deiminase enzyme. A method for treating a deficiency in guanidinoacetic acid methyltransferase (GAMT) activity in a subject is also contemplated, comprising administering a pharmaceutical composition comprising a therapeutic amount of an arginine depleting enzyme to the subject in combination with ornithine supplementation. This method contemplates the use of an arginine depleting enzyme, which can be an arginase or arginine deiminase enzyme. This method also contemplates a pharmaceutical composition that further supplements a high dose of L-ornithine. The L-ornithine in the pharmaceutical composition of this method can be orally administered as L-ornithine aspartate or L-ornithine hydrochloride.

[0014] Also contemplated is a pharmaceutical composition comprising a therapeutic amount of an arginine-depleting enzyme for use as a medicament for use in treating a deficiency in guanidinoacetate methyltransferase (GAMT) activity in a subject, or for treating the effects of guanidinoacetate (GAA) toxicity in a subject.

[0015] Also contemplated are pharmaceutical compositions comprising a therapeutic amount of an arginine-depleting enzyme for use in treating a deficiency in guanidinoacetate methyltransferase (GAMT) activity in a subject, or treating the effects of guanidinoacetate (GAA) toxicity in a subject.

[0016] The following drawings form part of the present specification and are included to further demonstrate certain aspects of the compositions and methods of using the compositions. [Brief description of the drawings]

[0017] [Figure 1] FIG. 1 is a graph showing serum L-arginine depletion in a mouse model. Serum L-Arg concentrations in Balb / c mice treated with a single IP dose of Co-hArgI are maintained below 3-4 μM over a period of 3 days. [Diagram 2] 1 is a graph showing the effect of cobalt loading on the catalytic activity of human Arginase I. [Diagram 3] 1 is a graph of the mean (±SD) concentration time profile of AEB1102 in male monkeys following a single 0.5 mg / kg IV dose. [Figure 4] 1 is a graph of the mean (±SD) concentration time profile of AEB1102 in male monkeys following a single 0.5 mg / kg SC dose. AEB1102 is a PEGylated human arginase I having the sequence of SEQ ID NO:3 lacking the N-terminal methionine and having Co+2 as the metal cofactor. [Figure 5A] 1 is a graph of the mean (±SD) pharmacodynamic profile in male monkeys following a single 0.5 mg / kg IV dose of AEB1102. [Figure 5B] 1 is a graph of the mean (±SD) pharmacodynamic profile in male monkeys following a single 0.5 mg / kg SC dose of AEB1102. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0018] The arginase therapy for arginine depletion and GAA reduction or depletion is described herein.The disclosed method provides certain advantages over the current state of the art, including at least improved substrate depletion effectiveness, substantially greater impact on total body arginine levels, and better reduction or depletion of GAA levels.In addition, this approach provides many benefits to patients, considering that dietary restriction of arginine has a negative impact on patients' quality of life and health.

[0019] The disclosed method represents a paradigm shift in how to approach substrate reduction in GAMT patients. It has the potential to suppress GAA production in GAMT-deficient patients, while being more effective in lowering the availability of arginine as a substrate, and alleviating the requirement of dietary arginine restriction.The disclosed method thus improves quality of life by eliminating the difficulties in performing activities that are adversely affected by the complexity and discomfort of dietary dieting.

[0020] Compositions and methods are provided for the treatment of GAMT deficiency and / or GAA toxicity with enzymes that deplete L-arginine in serum, i.e., arginine-depleting enzymes. 2+ The use of arginase enzymes and variants thereof in which "X" is replaced with another metal is also contemplated. Exemplary arginase enzymes include the amino acid sequence of human arginase I or the amino acid sequence of human arginase II and a non-native metal cofactor. The nucleotide sequences of human arginase I and human arginase II (SEQ ID NOs: 1 and 2, respectively) and the amino acid sequences of human arginase I and human arginase II (SEQ ID NOs: 3 and 4, respectively) are provided below (where "X" can be methionine or nothing). GATATACCATGGGTTCTTCTCACCATCATCACCACCACAGCTCTGGCGAGA ACCTGTACTTCCAGTCTGCGAAGAGCCGTACGATCGGCATTATTGGTGCGCCGTT CTCTAAAGGTCAGCCACGCGGTGGTGTGGAAGAGGGTCCGACGGTTCTGCGTAA GGCCGGTTTATTAGAAAAGCTGAAAGAGCAGGAGTGCGACGTTAAGGACTACGG TGACTTACCATTCGCGGACATCCCGAATGATAGCCCGTTCCAAATCGTTAAGAAT CCGCGCTCTGTGGGTAAAGCAAGCGAGCAGTTAGCAGGTAAGGTGGCCGAGGTC AAGAAAAACGGTCGTATTAGCCTGGTTTTAGGCGGTGATCATAGCTTAGCAATTG GCTCTATCTCTGGTCATGCCCGTGTGCACCCAGATTTAGGTGTCATTTGGGTTGAC GCCCATACGGATATCAATACGCCATTAACGACCACCAGCGGCAATCTGCATGGC CAGCCGGTTAGCTTCTTACTGAAGGAGCTGAAGGGTAAAATTCCAGATGTTCCGG GCTTTAGCTGGGTCACGCCATGTATTTCTGCCAAGGATATCGTGTACATTGGCTT ACGTGACGTCGACCCAGGTGAGCACTACATCTTAAAGACCCTGGGTATCAAGTA TTTCAGCATGACGGAAGTGGACCGCTTAGGCATCGGCAAGGTGATGGAGGAGAC GCTGAGCTATCTGCTGGGCCGTAAGAAACGTCCAATCCATCTGAGCTTCGATGTT GACGGCTTAGACCCGAGCTTTACGCCAGCCACCGGCACGCCGGTCGTTGGTGGTT TAACGTATCGCGAAGGCCTGTATATCACGGAGGAAATCTATAAGACGGGTTTAC TGAGCGGTCTGGACATTATGGAGGTTAATCCAAGCTTAGGTAAGACGCCGGAAG AAGTTACCCGTACCGTTAACACGGCGGTCGCGATCACGTTAGCATGTTTCGGTTT AGCCCGCGAGGGCAACCATAAACCAATTGATTATCTGAATCCACCGAAGTGAGG ATCCGAATTCG (SEQ ID NO: 1) GATATACCATGGGCAGCAGCCATCATCACCACCATCACAGCTCTGGTGAAAACTT ATACTTCCAAAGCGTCCATAGCGTCGCAGTGATTGGTGCCCCGTTTAGCCAAGGT CAAAAACGCAAGGGTGTTGAACATGGTCCGGCAGCGATCCGCGAAGCAGGTTTA ATGAAGCGTTTAAGCAGCTTAGGCTGTCACTTAAAGGATTTCGGTGATTTAAGCT TT ACGCCGGTCCCAAAGGATGATTT ATAC AAT AATCTGATCGTT AACCC ACGCTC TGTGGGTCTGGCGAACCAGGAGCTGGCGGAGGTCGTGTCTCGTGCAGTCAGCGA CGGTTATAGCTGCGTTACGCTGGGCGGTGATCATAGCTTAGCCATTGGTACGATT TCTGGTCATGCCCGCCATTGCCCGGATCTGTGTGTTGTGTGGGTTGATGCGCACG CGGATATCAATACGCCACTGACCACGTCTAGCGGTAATTTACACGGCCAGCCGGT TAGCTTCTTATTACGTGAGCTGCAAGACAAGGTCCCGCAGTTACCAGGCTTCTCT TGGATCAAACCATGTATCAGCAGCGCATCTATTGTCTACATTGGCCTGCGTGATG TCGACCCACCGGAGCACTTCATCCTGAAGAATTATGACATCCAGTATTTCAGCAT GCGTGACATCGACCGTCTGGGTATCCAAAAAGTTATGGAGCGCACGTTCGATCTG TTAATCGGCAAGCGCCAGCGTCCGATTCACCTGAGCTTTGACATTGACGCCTTTG ACCCGACCCTGGCCCCAGCAACGGGCACGCCAGTGGTTGGTGGTTTAACCTACC GTGAGGGT ATGT AT ATTGCAGAAG AGATCCAT AATACCGGCCTGTT ATCTGCCCT GGATCTGGTTGAAGTCAATCCGCAGCTGGCAACCTCTGAGGAGGAAGCGAAGAC GACCGCCAACCTGGCGGTGGACGTCATCGCCTCTTCTTTCGGCCAGACGCGTGAA GGTGGCCATATCGTGTATGACCAATTACCAACGCCATCTAGCCCGGACGAATCTG AGAACCAAGCACGTGTCCGTATTTGAGGATCCGAATTCG (SEQ ID NO: 2) XSAKSRTIGIIGAPFSKGQPRGGVEEGPTVLRKAGLLEKLKEQECDVKDYGDLPFADI PNDSPFQIVKNPRSVGKASEQLAGKVAEVKKNGRISLVLGGDHSLAIGSISGHARVHP DLGVIWVDAHTDINTPLTTTSGNLHGQPVSFLLKELKGKIPDVPGFSWVTPCISAKDI VYIGLRDVDPGEHYILKTLGIKYFSMTEVDRLGIGKVMEETLSYLLGRKKRPIHLSFD VDGLDPSFTPATGTPVVGGLTYREGLYITEEIYKTGLLSGLDIMEVNPSLGKTPEEVT RTVNTAVAITLACFGLAREGNHKPIDYLNPPK (SEQ ID NO: 3) (Human Arginase I) SVHSVAVIGAPFSQGQKRKGVEHGPAAIREAGLMKRLSSLGCHLKDFGDLSFTPVPK DDLYNNLIVNPRSVGLANQELAEVVSRAVSDGYSCVTLGGDHSLAIGTISGHARHCP DLCVVWVDAHADINTPLTTSSSGNLHGQPVSFLLRELQDKVPQLPGFSWIKPCISSASI VYIGLRDVDPPEHFILKNYDIQYFSMRDIDRLGIQKVMERTFDLLIGKRQRPIHLSFDI DAFDPTLAPATGTPVVGGLTYREGMYIAEEIHNTGLLSALDLVEVNPQLATSEEEAK TTANLAVDVIASSFGQTREGGHIVYDQLPTPSSPDESENQARVRI (SEQ ID NO: 4) (human arginase II)

[0021] In some embodiments, the non-native metal is cobalt (Co 2+ ). Human Arginase I and II proteins have two Mn(II) sites as their native metal cofactor. Either or both of the Mn sites can be replaced with a non-native metal cofactor to generate modified Arginase I or II proteins with a non-native metal cofactor. Thus, as used herein, a "non-native metal cofactor" is a metal other than manganese. The proteins are concentrated at 400 mM Mn(II) at pH 7.4. -1 s -1 Exceeds k cat / K M An exemplary arginase enzyme can be shown to have a pH of 400 mM -1 s -1 ~4,000mM -1 s -1 k cat / K M Another exemplary arginase enzyme can exhibit a 400 mM -1 s -1 ~2,500mM -1 s -1 k cat / K M Also contemplated is an arginase enzyme comprising the amino acid sequence of human arginase I or II and a non-native metal cofactor, the protein exhibiting a 400 mM -1 s -1 Exceeds k cat / K MShows.

[0022] In certain embodiments, the human arginase I or II enzyme or other arginine-depleting enzyme is stabilized by association with a stabilizing agent to extend the half-life of the enzyme in the serum of a patient. As used herein, "association" can include any of a number of types of association, including, but not limited to, covalent or non-covalent bonds, and can also include protein fusions expressed from nucleic acid constructs engineered from hydrogen bonds or hydrophobic interactions and others known to those of skill in the art.

[0023] Stabilizing agents for use in the disclosed methods include, but are not limited to, polyethylene glycol (PEG), often referred to as PEGylation, including various PEGylated polymers and linkers, including, but not limited to, TransCon linker technology available from Ascendis Pharma; conjugates to one or more homogenous synthetic protein polymers, known as extenylation and available under the trade name Xten®; polysialylation, available as PSAylation or Polyxen by Xenetic Biosciences; conjugates to one or more Fc fragments or serum proteins, such as albumin, available as PASylation® by XL-Protein GmbH. See, for example, U.S. Patent Nos. 8,679,479 and 9,050,340, which are incorporated herein by reference. A preferred range for arginase PEGylation is 1,000-10,000 daltons and any range therebetween, such as 5,000 daltons.

[0024] The arginine depletion enzyme useful for carrying out this method can include arginase enzyme, arginine deiminase enzyme, or a combination thereof. The enzyme can be a mammalian enzyme, such as a human or primate enzyme, a recombinant human enzyme, an engineered human enzyme, or an enzyme of other species, either mammalian or bacterial, including, but not limited to, mycoplasma. The non-human arginine depletion enzyme can be delivered, for example, by autologous red blood cell ghost. Red blood cell ghost is also called engineered red blood cell or red blood cell microparticle (RBC MP). See, for example, Villa, CH, et al., (2016), and U.S. Patent No. 10,004,764 from the University of Pittsburgh, regarding the use of such particles as a delivery agent for the enzymes discussed herein.

[0025] Native (wild type) arginine depletion enzymes such as arginase can be modified by substitution of metal cofactors. Arginase enzymes can be modified by substitution of metal cofactors in addition to other modifications such as substitution, deletion, insertion, truncation, or stabilization by conjugation to stabilizing proteins or polymers, such as PEGylation. Exemplary arginine depletion enzymes can include the native (wild type) amino acid sequence of human (or primate) arginase I or II and a non-native metal cofactor. The amino acid sequence can also lack a portion of the native sequence. The non-native metal cofactor for arginase can be cobalt.

[0026] The arginase for use as an active agent in the contemplated composition can lack a portion of the wild-type sequence.For example, the amino acid sequence can be a truncated arginase I or arginase II sequence.In another example, the arginase can be arginase II that lacks the first 21 amino acids of the wild-type sequence.Another arginase (wild-type or engineered mutant) for use in the disclosed composition can lack the N-terminal methionine (e.g., SEQ ID NO:3).

[0027] In another aspect, the arginase enzyme or arginine depletion enzyme, such as the arginase enzyme, can include at least one amino acid substitution. For the exemplary arginase enzyme, the enzyme exhibits increased catalytic activity under physiological conditions, particularly at the pH of human serum (pH 7.4), when compared to native (wild-type) human arginase I or II protein. In some embodiments, the arginase enzyme or variant is a human arginase I protein or a human arginase II protein, such as those described in U.S. Pat. No. 9,050,340, which is incorporated herein by reference. The arginase enzyme can include insertions, deletions, or substitutions of 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acids in the arginase polypeptide sequence. The contemplated arginase enzyme can also be amino-terminally truncated or have the amino-terminal methionine removed (e.g., SEQ ID NO: 3), or have a combination of these mutations. Another exemplary arginase further includes a non-native metal cofactor. In certain embodiments, the non-native metal cofactor is Co. +2 Mn +2 Cofactor Co +2 Substitution with significantly increases catalytic activity and dramatically decreases Km at physiological pH.

[0028] Fusion proteins are also contemplated for use with arginine-depleting enzymes. Fusion proteins can include arginine-depleting enzymes and non-arginine-depleting enzymes. Non-arginine-depleting enzyme sequences can include, for example, at least a portion of an Fc region of an immunoglobulin, for example, to extend the half-life of arginase in serum when administered to a patient. The Fc region or portion thereof can be any suitable Fc region. In certain embodiments, the Fc region or portion thereof is an IgG Fc region. The amino acid sequence having arginase depleting activity is selected from the group consisting of a native or mutated arginine deiminase enzyme, a native or mutated amino acid sequence of human arginase I (e.g., arginine 1), and a native or mutated amino acid sequence of human arginase II enzyme or other arginine-depleting enzymes known in the art. In certain embodiments, dimeric Fc-arginase fusion proteins, albumin, or synthetic protein conjugates are contemplated.

[0029] The arginine depletion enzyme in the fusion protein may be native, mutated, and / or otherwise modified, for example, a metal cofactor modified arginase enzyme. The arginine depletion enzyme may include deletions, substitutions, truncations, or combinations thereof. In one example, an Fc-arginase containing fusion protein is contemplated, in which the arginase is arginase I. In further embodiments, the arginine depletion enzyme lacks a portion of the wild-type sequence. The arginine depletion enzyme may be arginase I or arginase II lacking an N-terminal methionine, and the arginase II lacks the first 21 amino acids of the wild-type arginase II sequence. Alternatively, the arginase enzyme may include a non-native metal cofactor. In these embodiments, one or both sites may be substituted to generate a fusion protein that includes the amino acid sequence of human arginase I or II and a non-native metal cofactor. In some embodiments, the non-native metal cofactor is cobalt. In some embodiments, the arginase includes a mutation in the polypeptide sequence in the form of an amino acid substitution, deletion, insertion, or truncation, or combinations thereof. Exemplary arginase enzymes for use in the present disclosure are more fully described in U.S. Pat. No. 8,440,184, which is incorporated herein by reference in its entirety.

[0030] The present disclosure includes a method of treating a human GAMT patient, comprising administering to the patient a formulation comprising a fusion protein, wherein the fusion protein comprises an amino acid sequence having arginine depletion enzyme activity and at least a portion of the Fc region of a human immunoglobulin. The described formulation can comprise an amino acid sequence having a human arginase activity higher than that exhibited by native human arginase enzyme or human arginine deiminase enzyme under physiological conditions, and further comprising one or more linked polyethylene glycol chains. The formulation can be a pharmaceutical formulation comprising any of the above arginine depletion enzymes, such as an arginase enzyme, and a pharmaceutical acceptable excipient. Such pharmaceutical acceptable excipients are well known to those skilled in the art. It is contemplated that all of the described arginine depletion enzymes are useful for human treatment.

[0031] In some circumstances, the formulations can be administered topically, intravenously, intradermally, intra-arterially, intraperitoneally, intralesionally, intracranially, intra-articularly, intraprostatically, intrapleurally, intratracheally, intraocularly, intranasally, intravitreally, intravaginally, intrarectally, intramuscularly, subcutaneously, subconjunctivally, intravesicularly, mucosally, intrapericardially, subumbilically, orally, by inhalation, injection, infusion, continuous infusion, by direct local perfusion of target cells, via a catheter, or via lavage solution.

[0032] All of the above arginine-depleting enzymes (eg, arginase and arginine deiminase enzymes and variant enzymes thereof) are contemplated in preferred embodiments as purified or isolated proteins, preferably monomeric proteins.

[0033] The term "therapeutically effective" as used herein refers to the amount of active agent and / or therapeutic or pharmaceutical composition (such as therapeutic polynucleotide and / or therapeutic polypeptide) used in a method of treating a subject to achieve a therapeutic effect, e.g., at least one symptom of the condition being treated in the subject is at least improved. The therapeutic effect can also include a reduction or depletion of the level of arginine and / or GAA in the subject.

[0034] The term "treating" as used herein refers to the administration of medical or other therapy to a subject for the purpose of ameliorating, inhibiting, preventing, ameliorating, reversing or other beneficial effect of a particular condition or disease. The term does not require any particular result per se.

[0035] Other objects, features and advantages of the described methods and compositions will become apparent from the following detailed description. It should be understood, however, that the detailed description and specific examples, while indicating exemplary embodiments, are given by way of illustration only, since various changes and modifications within the spirit and scope of the described compositions and methods will become apparent to those skilled in the art from this detailed description.

[0036] Described herein are compositions and methods using enzymes that deplete L-arginine for treating deficiency of guanidinoacetate methyltransferase (GAMT) activity or treating GAA toxicity, which are associated with genetic deficiency of the gene that codes for or affects the expression of guanidinoacetate methyltransferase enzyme.Both natural and mutant or engineered enzymes are contemplated, as well as enzymes with modified metal cofactors, enzymes fused to other polypeptides, and enzymes conjugated to polymers that increase serum persistence, such as high molecular weight polyethylene glycol or polysialic acid.

[0037] I. Arginine-depleting enzymes Arginine-depleting enzymes contemplated herein include arginase enzymes, arginase I and II, and arginine deiminase enzymes, and variants thereof.

[0038] Arginine deiminase is an enzyme class L-arginine iminohydrolase. Other synonyms include arginine dihydrolase, citrulline iminase and L-arginine deiminase. Mycobacterium arginine deiminase catalyzes the formation of L-citrulline from L-arginine, as disclosed in NCBI Reference Sequence WP_003405169.1. Homosapien arginine deiminase enzymes include peptidyl arginine deiminase 3 (also known as PAD3; PDI3, UHS1) and peptidyl arginine deiminase 1 (also called HPAD10, PAD1, PDI, and PDI1).

[0039] Arginase is a manganese-containing enzyme. It is the last enzyme in the urea cycle. Arginase is the fifth and final step in the urea cycle, a series of biophysical reactions in mammals by which the body processes harmful ammonia. Specifically, arginase converts L-arginine into L-ornithine and urea.

[0040] L-arginine is the nitrogen donor substrate for nitric oxide synthase (NOS), which produces L-citrulline and NO. M Arginase may also play a role in regulating NOS activity, although its affinity (2-5 mM) for L-arginine has been reported to be much higher than that of NOS (2-20 μM). 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.

[0041] Arginase is a homotrimeric enzyme with an α / β fold of a parallel eight-stranded β sheet surrounded by several helices. The enzyme contains a binuclear metal cluster, essential for generating 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 water, orienting and stabilizing the molecule, allowing water to act as a nucleophile to attack L-arginine, hydrolyzing it to ornithine and urea.

[0042] 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 (6q.23), 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 present on chromosome 14 (14q24.1). Arginase II is localized in the mitochondria of tissues such as kidney, brain, and skeletal muscle, and is thought to provide L-ornithine for proline and polyamine biosynthesis (Lopez et al., 2005).

[0043] Arginase has been studied for nearly 50 years as a way to degrade extracellular L-arginine (Dillon et al., 2002). Some promising clinical results have been achieved by introducing arginase via transhepatic arterial embolization, after which several patients experienced partial remission of HCC (Cheng et al., 2005). However, arginase has a high K M (approximately 2-5 mM) and exhibits very low activity at physiological pH values, necessitating high doses for chemotherapy purposes in patients (Dillon et al., 2002). Although native arginase is cleared from the circulation within minutes (Savoca et al., 1984), a single injection of PEG-Arginase MW 5,000 in rats was sufficient to achieve nearly complete arginine depletion for approximately 3 days (Cheng et al., 2007).

[0044] reported the surprising observation that many human HCC cell lines do not express ornithine transcarbamylase (OTC) (in addition to argininosuccinate synthetase, ASS), and thus human HCC cells are sensitive to PEG-arginase (Cheng et al., 2007). In mice implanted with Hep3b hepatocellular carcinoma cells, weekly administration of PEG-arginase delayed tumor growth, which was accentuated by coadministration of 5-fluorouracil (5-FU). However, PEG-arginase was used at very high doses that are impractical for use in human therapy, reflecting the low physiological activity of that PEG-arginase.

[0045] To address these issues, bacterial arginine hydrolase, arginine deiminase or ADI, which shows good kinetics and stability, has been tested in vitro and in the clinic.Unfortunately, ADI is a bacterial enzyme, and therefore ADI induces strong immune responses and adverse effects in most patients.However, for patients who do not develop significant adverse reactions, a surprising proportion show stable disease or remission.

[0046] For clinical use, arginase should be engineered to be able to persist in circulation for long periods (e.g., days). In the absence of any modifications, human arginase has a half-life in circulation of only a few minutes, the main reason for this being that its size is not large enough to avoid filtration by the kidney. Unmodified human arginase is highly susceptible to inactivation in serum and is degraded with a half-life of only 4 hours.

[0047] L-arginine is the only substrate for nitric oxide synthase (NOS), which produces L-citrulline and NO. M Arginase can also play a role in regulating NOS activity, although its affinity (2-5 mM) for L-arginine has been reported to be much higher than that of NOS (2-20 μM) (Durante et al., 2007). 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 (Santhanam et al., 2007). Arginase is a homotrimeric enzyme with an α / β fold of a parallel eight-stranded β sheet surrounded by several helices. The enzyme contains a binuclear metal cluster, essential for generating hydroxide for the nucleophilic attack on the guanidinium carbon of L-arginine (Cama et al., 2003; Dowling et al., 2008). The native metal of arginase is Mn 2+ Natural metal cofactors (Mn 2+ The arginase enzyme, which has a pH optimum of 9. At physiological pH, the enzyme has a 10-fold lower k cat / K M Natural Mn 2+ The low catalytic activity exhibited by native human arginase, including arginase enzyme, poses a problem for human therapy because it means that impractical doses of the enzyme may have to be used to achieve therapeutically appropriate reductions in plasma L-arginine levels.

[0048] In some embodiments, the natural metal cofactor (Mn2+ Natural and mutant arginase in which the natural metal (Mn) has been replaced with another metal are contemplated. The replacement of the metal cofactor in human arginase has been found to have a beneficial effect on the rate and stability of L-arginine hydrolysis under physiological conditions when compared to natural human arginase containing the natural metal cofactor. 2+ Substitution of Mn(II) with other divalent cations can be used to shift the pH optimum of the enzyme to lower values ​​and thus achieve a high rate of L-arginine hydrolysis under physiological conditions. Since human arginase I and II proteins have two Mn(II) sites, either or both sites can be substituted to generate mutant arginase I or II proteins with non-native metal cofactors. A suitable non-native metal is cobalt.

[0049] The non-native metal for the arginase enzyme is cobalt (Co 2+ ). Mn 2+ Instead of Co 2+ Incorporation of human arginase I or human arginase II results in dramatically higher activity at physiological pH. 2+ The human arginase I enzyme ("Co-hArgl"), which contains Mn 2+ In vitro k cat / K M It was found that a pharmacological preparation of Co-hArg was able to eliminate serum L-Arg for 3 days in mice with a single injection. Furthermore, a pharmacological preparation of Co-hArgl was able to shrink HCC tumor xenografts in nude mice, whereas a pharmacological preparation of Mn-hArgl (Mn +2 It was found that the administration of arginase I (containing human arginase I enzyme) only slowed tumor growth (Ensor et al., 2002). See also U.S. Patent Nos. 9,050,340 and 8,679,479, which are incorporated herein by reference.

[0050] Methods and compositions related to PEGylated arginase enzymes and arginine deiminase enzymes, including PEGylated human arginase I and II enzymes, are disclosed. Specifically, PEGylation of arginase at engineered cysteine ​​residues (e.g., replacing the third residue at the N-terminus) can be used to generate homogeneous PEGylated arginase compositions. Methods for isolating PEGylated arginase based on the temporary interruption of polymerization are also disclosed.

[0051] An exemplary human Arginase I enzyme may comprise SEQ ID NO:3, or may further comprise one or more of the amino acid substitutions at His101, Asp124, His126, Asp128, Asp232, Asp234, Trp122, Asp181, Ser230, His120, Asp143, His145, Asp147, Asp251, Asp253, Trp141, Asp200, Ser249, Cys303, or Glu256. Alternatively, it may be a native Arginase I with one or more of the mutations set forth in this paragraph, with a native metal cofactor, or using another metal cofactor such as cobalt. Many mutations increase catalytic activity and improve the K of L-arginine under physiological conditions. mIt has been found to dramatically reduce the activity of the arginase I protein and variants described herein. Optionally, the substitution mutation of the arginase I protein and variants described herein can be selected from the group consisting of one or more of the following: Asp181Ser, Ser230Cys, Ser230Gly, Cys303Phe, Cys303Ile, Glu256Gln, Asp181Glu and Ser230Ala. In some aspects, the present invention provides an embodiment in which two or more mutations are introduced into the human arginase I enzyme. The human arginase I enzyme can include at least two amino acid substitutions. The human arginase I enzyme can have substitutions at Asp181Glu and Ser230Ala. These variants are also considered to have or lack an N-terminal methionine. Arginase 1 is, for example, a human arginase I having the sequence of SEQ ID NO: 3, in which the N-terminal methionine is missing, the cofactor is cobalt, and is PEGylated.

[0052] PEGylation is the process of covalently attaching poly(ethylene glycol) polymer chains to another molecule, usually a drug or therapeutic protein. "PEGylation" is usually accomplished by incubating a reactive derivative of PEG with the target macromolecule. Covalent attachment of PEG to a drug or therapeutic protein can increase the hydrodynamic size (size in solution) of the drug, "masking" the drug from the host's immune system (reducing immunogenicity and antigenicity) and extending its circulation time by decreasing renal clearance. PEGylation can also confer water solubility to hydrophobic drugs and proteins.

[0053] The first step in PEGylation is the appropriate functionalization of the PEG polymer at one or both termini. PEG activated at each end with the same reactive moiety is known as "homobifunctional", whereas when the functional groups present are different, the PEG derivative is termed "heterobifunctional" or "heterofunctional". Chemically active or activated derivatives of the PEG polymer are prepared to allow the PEG to be attached to the desired molecule.

[0054] The selection of suitable functional groups for PEG derivatives is based on the type of available reactive groups on the molecule that will be attached to PEG. For proteins, typical reactive amino acids include lysine, cysteine, histidine, arginine, aspartic acid, glutamic acid, serine, threonine, and tyrosine. N-terminal amino groups and C-terminal carboxylic acids can also be used.

[0055] The technique used to form first generation PEG derivatives is generally to react PEG polymers with groups that react with hydroxyl groups, typically anhydrides, acid chlorides, chloroformates, and carbonates. In second generation PEGylation chemistries, more efficient functional groups such as aldehydes, esters, and amides are available for conjugation.

[0056] As the applications of PEGylation become more developed and complex, the need for heterobifunctional PEGs for conjugation increases. These heterobifunctional PEGs are useful for linking two entities where a hydrophilic, flexible, and biocompatible spacer is required. The preferred terminal groups of heterobifunctional PEGs are maleimide, vinyl sulfone, pyridyl disulfide, amine, carboxylic acid, and NHS (N-hydroxysuccinimide) ester.

[0057] The most common modifiers or linkers are based on methoxy PEG (mPEG) molecules. Their activity relies on the addition of protein-modifying groups to the alcohol termini. In some instances, polyethylene glycol (PEG diol) is used as a precursor molecule. The diol is then modified at both ends to create hetero- or homodimeric PEG-linked molecules (as shown in the examples with PEG bis-vinylsulfone).

[0058] The arginine-depleted enzymes and variants thereof contemplated herein 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 exhibit strong cysteinyl specificity under mild conditions and neutral to slightly alkaline pH, but each has some drawbacks. Amides formed with maleimides may be somewhat unstable under alkaline conditions, so formulation options with this linker may be limited in some ways. Amide bonds formed with iodoPEG are more stable, but free iodine can modify tyrosine residues under some conditions. PEG thiols form 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 iodoPEG. However, the thioether bond formed is quite stable. The slow reaction rate also makes the PEG-vinylsulfone reaction easier to control.

[0059] 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. Alternatively, site-directed mutagenesis can be used to incorporate cysteinyl PEGylation sites in thiol-specific linkers. The cysteine ​​mutation must be designed so that it is accessible to PEGylation reagents and still biologically active after PEGylation.

[0060] Amine-specific modifiers include PEG NHS esters, PEG tresylates, PEG aldehydes, PEG isothiocyanates, and several others. All react under mild conditions and are very specific for amino groups. PEG NHS esters are probably one of the more reactive agents. However, their high reactivity can make large-scale control of the PEGylation reaction difficult. PEG aldehydes form imines with amino groups, which are then reduced to secondary amines with sodium cyanoborohydride. Unlike sodium borohydride, sodium cyanoborohydride does not reduce disulfide bonds. However, this chemical is highly toxic and becomes volatile, especially at reduced pH, and must be handled with care.

[0061] Site-specific PEGylation can be challenging because most proteins have multiple lysine residues. Fortunately, because these reagents react with unprotonated amino groups, it is possible to direct PEGylation to lower pK amino groups by performing the reaction at a lower pH. In general, the pK of α-amino groups is 1-2 pH units lower than the ε-amino groups of lysine residues. High selectivity for the N-terminus can often be achieved by PEGylating molecules at pH 7 or lower. However, this is only possible if the N-terminal portion of the protein is not required for bioactivity. Nevertheless, the pharmacokinetic benefits of PEGylation can outweigh any significant loss of in vitro bioactivity, resulting in products with much greater in vivo bioactivity, regardless of the chemistry of PEGylation.

[0062] When developing a PEGylation procedure, there are several parameters to consider. Fortunately, there are usually no more than four or five parameters. A "design of experiments" approach to optimizing PEGylation conditions can be very useful. For thiol-specific PEGylation reactions, parameters to consider include protein concentration, PEG-to-protein ratio (on a molar basis), temperature, pH, reaction time, and possibly the exclusion of oxygen. Oxygen can cause intermolecular disulfide formation with the protein, which would reduce the yield of the PEGylated product. For amine-specific modifications, the same factors should be considered (except oxygen), except that pH can be even more important, especially when targeting N-terminal amino groups.

[0063] For both amine-specific and thiol-specific modifications, the reaction conditions can affect the stability of the protein. This can be limited by temperature, protein concentration, and pH. In addition, the reactivity of the PEG linker should be known before initiating the PEGylation reaction. For example, if the PEGylation agent is only 70% active, the amount of PEG used should ensure that only active PEG molecules are counted in the stoichiometry of the protein-to-PEG reaction. Methods for determining the reactivity and quality of PEG are discussed below.

[0064] II. Proteins and Peptides Compositions are provided that include at least one protein or peptide, such as a stabilized arginase multimer. These polypeptides may be in a fusion protein or may be conjugated to an agent as described herein.

[0065] As used herein, a protein or peptide generally refers to, but is not limited to, a protein of more than about 200 amino acids up to the full length sequence translated from a gene, a polypeptide of more than about 100 amino acids, and / or a peptide of about 3 to about 100 amino acids. For convenience, the terms "protein", "polypeptide", and "peptide" are used interchangeably herein. The term protein or polypeptide is used interchangeably with enzyme unless otherwise specified.

[0066] In certain embodiments, the size of the at least one protein or peptide is, but is not limited to, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, about 110, about 120, about 130, about 140, about 150, about 160, about 170, about 180, about 190, about 200, about 210, about 220, about 230, about 240, about 250, about 275, about 300, about 325, about 350, about 375, about 400, about 425, about 450, about 475, about 500, about 525, about 550, about 575, It may contain about 600, about 625, about 650, about 675, about 700, about 725, about 750, about 775, about 800, about 825, about 850, about 875, about 900, about 925, about 950, about 975, about 1000, about 1100, about 1200, about 1300, about 1400, about 1500, about 1750, about 2000, about 2250, about 2500 or more amino acid residues.

[0067] As used herein, "amino acid residue" refers to any naturally occurring amino acid, any amino acid derivative, or any amino acid mimic known in the art. In certain embodiments, the residues of a protein or peptide are consecutive and do not include any non-amino acid that interrupts the sequence of amino acid residues. In other embodiments, the sequence may include one or more non-amino acid moieties. The sequence of residues of a protein or peptide may also be interrupted by one or more non-amino acid moieties.

[0068] Thus, the term "protein or peptide" encompasses amino acid sequences that contain at least one of the 20 common amino acids found in naturally occurring proteins, or at least one modified or unusual amino acid, including, but not limited to, those shown in Table 1 below. [Table 1]

[0069] Proteins or peptides can be produced by any technique known to those skilled in the art, such as expressing proteins, polypeptides or peptides by standard molecular biology techniques, isolating proteins or peptides from natural sources, or chemically synthesizing proteins or peptides. Nucleotides and protein, polypeptide and peptide sequences corresponding to various genes can be found in computerized databases that have been previously disclosed and are known to those skilled in the art. Such databases include the Genbank and GenPept databases of the National Center for Biotechnology Information (available on the World Wide Web at ncbi.nlm.nih.gov). The coding regions of known genes can be amplified and / or expressed using the techniques disclosed herein or as known to those skilled in the art. Alternatively, various commercial preparations of proteins, polypeptides and peptides are known to those skilled in the art.

[0070] III. Nucleic Acids and Vectors Nucleic acid sequences encoding fusion proteins for the arginine-depleting enzymes discussed herein, such as stabilized multimeric arginase, are also contemplated. Depending on the expression system used, the nucleic acid sequence can be selected based on conventional methods. For example, human arginase I and II contain multiple codons that are rarely utilized in E. coli protein synthesis, which can interfere with expression, and therefore the respective genes or variants thereof can be codon-optimized for E. coli expression. Various vectors can also be used to express the protein of interest, such as fusion multimeric arginase or cysteine-substituted arginase. Exemplary vectors include, but are not limited to, plasmid vectors, viral vectors, transposons, or liposome-based vectors.

[0071] IV.Host cells Host cells, preferably eukaryotic cells, useful for producing the arginine depletion enzymes and enzyme variants thereof discussed herein are any that can be transformed to allow expression and secretion of arginase and its fusion multimers. Host cells can be bacteria, mammalian cells, yeast, or filamentous fungi. Various bacteria include the genera Escherichia and Bacillus. Yeasts belonging to the genera Saccharomyces, Kluyveromyces, Hansenula, or Pichia find use as suitable host cells. Various types of filamentous fungi can be used as expression hosts, including the following genera: Aspergillus, Trichoderma, Neurospora, Penicillium, Cephalosporium, Achlya, Podospora, Endothia, Mucor, Cochliobolus, and Pyricularia.

[0072] Examples of host organisms that can be used are bacteria, such as Escherichia coli MC1061, derivatives of Bacillus subtilis BRB1 (Sibakov et al., 1984), Staphylococcus aureus SAI123 (Lordanescu, 1975) or Streptococcus lividans (Hopwood et al., 1985); yeasts, such as Saccharomyces cerevisiae AH 22 (Mellor et al., 1983) and Schizosaccharomyces pombe; filamentous fungi, such as Aspergillus nidulans, Aspergillus awamori, awamori (Ward, 1989), and Trichoderma reesei (Penttila et al., 1987; Harkki et al., 1989).

[0073] Examples of mammalian host cells include Chinese hamster ovary cells (CHO-K1; ATCC CCL61), rat pituitary cells (GH 1 , ATCC CCL82), HeLaS3 cells (ATCC CCL2.2), rat hepatoma cells (H-4-II-E; ATCC CRL 1548), SV40-transformed monkey kidney cells (COS-1, ATCC CRL1650), and mouse embryo cells (NIH-3T3; ATCC CRL 1658). The above examples are illustrative of the many possible host organisms known in the art, but are not limiting. In principle, any host capable of secretion, whether prokaryotic or eukaryotic, can be used.

[0074] Mammalian host cells expressing arginase and / or their fusion multimers are cultured under conditions typically used to culture the parent cell line. Generally, the cells are cultured in standard culture media containing physiological salts and nutrients, such as standard RPMI, MEM, IMEM or DMEM, typically supplemented with 5-10% serum, such as fetal bovine serum. Culture conditions are also standard, e.g., cultures are incubated at 37°C in stationary or roller cultures until the desired level of protein is achieved.

[0075] V. Protein Purification Protein purification techniques are well known to those skilled in the art. These techniques require, at one level, homogenization and crude fractionation of cells, tissues or organs into polypeptide and non-polypeptide fractions. The protein or polypeptide of interest can be further purified using chromatographic and electrophoretic techniques to achieve partial or complete purification (or purification to homogeneity), unless otherwise specified. Analytical methods particularly suited to the preparation of pure peptides are ion exchange chromatography, gel exclusion chromatography, polyacrylamide gel electrophoresis, affinity chromatography, immunoaffinity chromatography, and isoelectric focusing. A particularly efficient method of purifying peptides is fast performance liquid chromatography (FPLC) or even high performance liquid chromatography (HPLC).

[0076] A purified protein or peptide having an arginine depletion enzyme or variant thereof as described herein can also refer to a composition that can be isolated from other components, where the protein or peptide is purified to any degree relative to its naturally occurring state. Thus, an isolated or purified protein or peptide also refers to a protein or peptide that is free from the environment in which it may naturally occur. In general, "purified" refers to a protein or peptide composition that has been subjected to fractionation to remove various other components and that retains its expressed biological activity. When the term "substantially purified" is used, this designation refers to a composition in which the protein or peptide forms a major component of the composition, for example, a composition in which the protein constitutes about 50%, about 60%, about 70%, about 80%, about 90%, about 95% or more of the composition.

[0077] Various techniques suitable for use in protein purification are well known to those skilled in the art.These include, for example, precipitation with ammonium sulfate, PEG, and antibodies, or by heat denaturation, followed by centrifugation; chromatographic steps such as ion exchange, gel filtration, reverse phase, hydroxylapatite and affinity chromatography; isoelectric focusing; gel electrophoresis; and combinations of these techniques with other techniques.As is generally known in the art, the order of carrying out various purification steps may be changed or certain steps may be omitted, and still result in a method suitable for preparing substantially purified protein or peptide.

[0078] Various methods for quantifying the degree of purification of a protein or peptide will be known to those skilled in the art in light of the present disclosure. These include, for example, determining the specific activity of an active fraction or assessing the amount of polypeptide within the fraction by SDS / PAGE analysis. A preferred method for assessing the purity of a fraction is to calculate the specific activity of the fraction and compare it to the specific activity of the initial extract, thus calculating the purity therein, as assessed by a "-fold purification value". The actual units used to express the amount of activity will, of course, depend on the particular assay technique chosen after purification, and whether the expressed protein or peptide exhibits detectable activity.

[0079] There is no general requirement that proteins or peptides always be provided in their most purified state. In fact, it is believed that in certain embodiments, substantially less purified products may be useful. Partial purification may be achieved by using fewer purification steps in combination, or by utilizing different forms of the same general purification scheme. For example, it is understood that cation exchange column chromatography performed using HPLC equipment generally results in a greater "fold" of purification than the same technique using a low pressure chromatography system. Methods that exhibit a lower relative degree of purification may have advantages in total recovery of protein product or in maintaining the activity of the expressed protein.

[0080] In certain embodiments, proteins or peptides may be isolated or purified, for example stabilized arginase multimeric fusion proteins, or arginase before or after PEGylation. For example, such arginase variants can include His tags or affinity epitopes to facilitate purification. Affinity chromatography is a chromatographic procedure that relies on the specific affinity between the substance to be isolated and a molecule to which it can specifically bind. This is a receptor-ligand type interaction. The column material is synthesized by covalently attaching one of the binding partners to an insoluble matrix. The column material can then specifically adsorb the substance from the solution. Elution occurs by changing the conditions to those in which binding does not occur (e.g., changing pH, ionic strength, temperature, etc.). The matrix should be a material that does not itself adsorb molecules to a significant extent and has a wide range of chemical, physical and thermal stability. The ligand should be attached in a way that does not affect its binding properties. The ligand should also provide a relatively tight bond. In addition, it must be possible to elute the substance without destroying the sample or the ligand.

[0081] Size Exclusion Chromatography (SEC) is a chromatographic method in which molecules in solution are separated based on their size, or in more technical terms, their hydrodynamic volume. It is usually applied to large molecules or macromolecular complexes such as proteins and industrial polymers. Typically, when an aqueous solution is used to transport the sample through the column, the technique is known as gel filtration chromatography, as opposed to the name gel permeation chromatography, which is used when organic solvents are used as the mobile phase.

[0082] The basic principle of SEC is that particles of different sizes will elute (filter) through the stationary phase at different rates. This separates a solution of particles based on size. If all particles are loaded at or near the same time, particles of the same size should elute together. Each size exclusion column has a range of molecular weights that it can separate. The exclusion limit defines the molecular weight at the upper end of this range, where molecules are too large to be captured by the stationary phase. The permeation limit defines the molecular weight at the lower end of the separation range, where molecules of a small enough size can completely penetrate the pores of the stationary phase and all molecules below this molecular mass are so small that they elute as a single band.

[0083] High performance liquid chromatography (or high pressure liquid chromatography, HPLC) is a form of column chromatography frequently used in biochemistry and analytical chemistry to separate, identify, and quantify compounds. HPLC utilizes a column that holds the chromatographic packing material (the stationary phase), a pump that moves the mobile phase through the column, and a detector that indicates the retention time of the molecules. Retention time varies depending on the interactions between the stationary phase, the molecules being analyzed, and the solvent used.

[0084] VI. Pharmaceutical Compositions It is contemplated that the arginine depletion enzymes and variants thereof, such as arginine deiminase enzymes and / or arginase enzymes, are in compositions that can be administered systemically, either intravenously (iv), intrathecally, and / or intraperitoneally (ip) or subcutaneously. They can be administered alone or in combination with other treatments associated with GAA toxicity.

[0085] The contemplated compositions can be formulated with physiologically acceptable liquid, gel or solid carriers, diluents and excipients.These therapeutic (pharmaceutical) preparations can be administered for clinical use in humans in the same manner as other therapeutic biological agents.In general, the dosage required for therapeutic effectiveness varies according to the type of use and the mode of administration, and the specific requirements of each individual subject.

[0086] Typically, such compositions are prepared as injection liquid solution or suspension.Suitable diluents and excipients are, for example, water, physiological saline, dextrose, glycerol, and other diluents approved by FDA, and their combinations.In addition, if desired, composition may contain a small amount of auxiliary substances, such as wetting agents or emulsifiers, stabilizers or pH buffers.

[0087] When clinical applications are contemplated, it may be necessary to prepare pharmaceutical compositions, i.e., expression vectors, viral stocks, proteins, antibodies, and drugs, in a form appropriate for the intended application. In general, the "pharmaceutical compositions" and "pharmaceutical formulations" discussed herein contain one or more effective amounts of arginase variants or additional agents dissolved or dispersed in a pharmaceutically acceptable carrier. The phrase "pharmaceutical or pharmacologically acceptable" refers to molecular entities and compositions that do not cause adverse allergic or other untoward reactions when administered to an animal, e.g., a human, as appropriate. The preparation of pharmaceutical compositions containing at least one arginase enzyme or variant thereof, such as a stabilized multimeric arginase or PEGylated arginase isolated by the methods disclosed herein, or an additional active ingredient, may be prepared according to the methods described in Remington's Pharmaceutical Sciences, 18, incorporated herein by reference. th It will be known to those of skill in the art in light of the present disclosure, as exemplified by U.S. Pat. No. 6,393,341, Ed., (1990). Moreover, it will be understood that for animal (e.g., human) administration, preparations should meet sterility, pyrogenicity, general safety, and purity standards as required by the FDA Office of Biological Standards.

[0088] As used herein, a "pharmaceutically acceptable carrier" includes any and all solvents, dispersion media, coatings, surfactants, antioxidants, preservatives (e.g., antibacterial, antifungal), isotonic agents, absorption delaying agents, salts, preservatives, drugs, drug stabilizers, gels, binders, excipients, disintegrants, lubricants, sweeteners, flavorings, dyes, similar materials, and combinations thereof known to those of skill in the art (see, e.g., Remington's Pharmaceutical Sciences, 18 th (See, Ed. (1990)). Except insofar as any conventional carrier is incompatible with the active ingredient, its use in the pharmaceutical compositions is contemplated.

[0089] The compositions of the contemplated arginine deiminase enzymes and arginase enzymes and their variants described herein can be formulated with carriers depending on whether they are administered in solid, liquid or aerosol form and whether they need to be sterile for routes of administration such as injection.The pharmaceutical compositions can be administered intravenously, intradermally, transdermally, intrathecally, intraarterially, intraperitoneally, intranasally, intravaginally, intrarectally, topically, intramuscularly, subcutaneously, mucosally, orally, topically, locally, by inhalation (e.g., aerosol inhalation), by injection, by infusion, by continuous infusion, by direct local perfusion bath of target cells, via catheter, via lavage solution, in lipid compositions (e.g., liposomes), or by other methods or any combination of the above methods as known to those skilled in the art (e.g., Remington's Pharmaceutical Sciences, 18, incorporated herein by reference). th (Ed. (1990)).

[0090] The arginine depletion enzyme can be incorporated into the composition in the form of free base, neutral or salt. Pharmaceutically acceptable salts include acid addition salts, such as those formed with free amino groups of proteinaceous compositions, or with inorganic acids, such as hydrochloric acid or phosphoric acid, or with 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 ferric hydroxide, or organic bases, such as isopropylamine, trimethylamine, histidine or procaine. Once formulated, the solution is administered in a manner compatible with the dosage formulation and in a therapeutically effective amount. The formulation is easily administered in a variety of dosage forms, such as formulations for parenteral administration, such as injections, or formulations for aerosols for delivery to the lungs, or formulations such as drug release capsules for administration in the digestive tract.

[0091] The contemplated compositions suitable for administration are provided in a pharma- ceutically acceptable carrier with or without an inert diluent. The carrier should be assimilable and includes liquid, semi-solid, i.e., paste, or solid carriers. Any conventional vehicle, agent, diluent or carrier is used in the administrable compositions for use in carrying out the described treatment and use methods, except where it is deleterious to the recipient or to the therapeutic effectiveness of the composition contained therein. Examples of carriers or diluents include fats, oils, water, saline, lipids, liposomes, resins, binders, and bulking agents, and the like, or combinations thereof. The compositions can also include various antioxidants to retard oxidation of one or more components. In addition, the activity of microorganisms can be prevented by preservatives, such as various antibacterial and antifungal agents, including, but not limited to, parabens (e.g., methylparaben, propylparaben), chlorobutanol, phenol, sorbic acid, thimerosal, or combinations thereof.

[0092] A contemplated composition can be combined with the carrier in any convenient and practical manner, i.e., by solution, suspension, emulsion, mixing, encapsulation, absorption, etc. Such procedures are routine to those of ordinary skill in the art.

[0093] The composition can be combined with semi-solid or solid carrier or mixed thoroughly.Mixing can be carried out by any convenient method, such as grinding.Stabilizers can also be added during the mixing process to protect the composition from loss of therapeutic activity, i.e., denaturation in the stomach.Examples of stabilizers for use in the composition include buffers, amino acids such as glycine and lysine, carbohydrates such as dextrose, mannose, galactose, fructose, lactose, sucrose, maltose, sorbitol, mannitol, etc.

[0094] A pharmaceutical lipid vehicle can be used in compositions that include an arginase variant, one or more lipids, and an aqueous solvent. As used herein, the term "lipid" is defined to include any of a wide range of substances that are characteristically insoluble in water and extractable with an organic solvent. This broad class of compounds is well known to those skilled in the art, and the term "lipid" as used herein is not limited to any particular structure. Examples include compounds that contain long-chain aliphatic hydrocarbons and their derivatives. Lipids can be natural or synthetic (i.e., engineered or produced by humans). However, lipids are usually biological materials. Biological lipids are well known in the art, and include, for example, neutral lipids, phospholipids, phosphoglycerides, steroids, terpenes, lysolipids, glycosphingolipids, glycolipids, sulfatides, lipids with ether- and ester-linked fatty acids, polymerizable lipids, and combinations thereof. Of course, compounds other than those specifically described herein that are understood by those skilled in the art as lipids are also encompassed by the compositions and methods described herein.

[0095] Those skilled in the art will be familiar with the range of techniques that can be used to disperse compositions in lipid vehicles.For example, the stabilized multimer or PEGylated arginase can be dispersed in a solution containing lipids, dissolved in lipids, emulsified in lipids, mixed with lipids, combined with lipids, covalently bound to lipids, contained in lipids as a suspension, contained or complexed in micelles or liposomes, or associated with lipids or lipid structures by any means known to those skilled in the art.Dispersion may or may not form liposomes.

[0096] The actual dosage of the compositions, pharmaceutical compositions or active agents described herein that can be administered to animal patients can be determined by physical and physiological factors such as the patient's weight, the severity of the condition, the type of disease being treated, past or present therapeutic interventions, sudden illness, and the route of administration. Depending on the dosage and route of administration, the preferred dosage and / or the number of administrations of an effective amount may vary according to the subject's response. The physician responsible for administration will in any case determine the concentration of active ingredient in the composition and the appropriate dose for each individual subject.

[0097] In certain embodiments, the pharmaceutical composition may contain, for example, at least about 0.1% of the active compound. In other embodiments, the active compound may contain, for example, about 2% to about 75% of the weight of the unit, or about 25% to about 60%, and any range derivable therein. Of course, the amount of active compound in each therapeutically useful composition may be prepared so as to obtain a suitable dosage 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 as such, various dosages and treatment regimens may be desirable.

[0098] In other non-limiting examples, dosages can also include about 1 microgram / kg / body weight, about 5 micrograms / kg / body weight, about 10 micrograms / kg / body weight, about 50 micrograms / kg / body weight, about 100 micrograms / kg / body weight, about 200 micrograms / kg / body weight, about 350 micrograms / kg / body weight, about 500 micrograms / kg / body weight, about 1 milligram / kg / body weight, about 5 milligrams / kg / body weight, about 10 milligrams / kg / body weight, about 50 milligrams / kg / body weight, about 100 milligrams / kg / body weight, about 200 milligrams / kg / body weight, about 350 milligrams / kg / body weight, about 500 milligrams / kg / body weight, to about 1000 mg / kg / body weight or more per administration, and any range derivable therein. Non-limiting examples of ranges that can be derived from the numerical values ​​recited herein include ranges based on the above numerical values, such as about 5 mg / kg / body weight to about 100 mg / kg / body weight, about 5 micrograms / kg / body weight to about 500 milligrams / kg / body weight, etc.

[0099] For a pharmaceutical composition including Arginase 1 (SEQ ID NO:3, but lacking the N-terminal methionine, having cobalt as a cofactor, and being PEGylated), an exemplary amount for administration to a subject can be in the range of 0.01 mg / kg to 7.5 mg / kg of the subject's body weight, or 0.05 mg / kg to 5.0 mg / kg, or 0.1 mg / kg to 5.0 mg / kg (and any 0.1 value between 0.1 mg / kg to 5.0 mg / kg).

[0100] The therapeutic efficacy for improving neuromuscular function for the conditions contemplated herein is determined after the first administration of arginine and / or GAA reducing agent.Neuromuscular function can be, but is not limited to, one or more of the following: improved stepping by the patient, walking by the patient, reduced spasticity of the patient, and / or improved attention.Another method contemplates that the subject shows at least one of the following after the first administration of arginine depleting enzyme (e.g., arginase and arginine deiminase): reduced resting spasticity, reduced leg cramps associated with spasticity, adaptive behavior, and improved PROMIS-T score, compared with at least one of spasticity, behavior, and PROMIS-T score before arginase administration.In response to treatment with arginase, one or more toxic metabolites (e.g., GAA) are reduced to normal levels by at least 30%, 40%, 50%, 60%, 70%, 80% from their initial levels, or are eliminated.

[0101] The method of treating a subject is to administer the arginine depletion enzyme composition in repeated doses.After receiving multiple doses, the patient can show improvement in at least one of (a) motility or (b) adaptive behavior, compared with the baseline of the subject's motility or adaptive behavior before treatment.

[0102] A therapeutically effective amount when administered to a patient can also improve muscle strength, the patient's ambulatory ability (i.e., ability to run, walk, bike, climb stairs without support), and improve cognitive ability (e.g., improvement in the Wechsler Intelligence Scale for Children (WISC) test) and / or adaptive behavior (e.g., improvement in the Adaptive Behavior Assessment Scale (ABAS) or Vineland Adaptive Behavior Scale (VABS) test) (Lopata et al., "Comparison of Adaptive Behavior Measures for Children with HFASDs", Autism Research and Treatment, Vol. 2013, pp. 1-10 (2013)).

[0103] VII.Definition The term "aa" refers to an amino acid. Amino acid substitutions are represented by the amino acid position in the molecule using the letter code, e.g., 303 (the letter before the number indicates the amino acid being substituted and the letter after the number indicates the amino acid being introduced).

[0104] Use of the term "or" in the claims is used to mean "and / or" unless expressly indicated to refer to alternatives only or the alternatives are not mutually exclusive, however, the present disclosure supports a definition that refers to alternatives and "and / or" only.

[0105] Throughout this application, the term "about" is used to indicate that a value includes the standard deviation of error for the composition, device, or method being employed to determine the value.

[0106] In accordance with long-standing patent law, the words "a" and "an," when used in conjunction with the word "comprising" in a claim or specification, mean one or more, unless otherwise specified.

[0107] As used herein, the term "portion" when in reference to a protein (as in "a portion of a given protein") refers to fragments of that protein. Fragments can range in size from four amino acid residues to the entire amino acid sequence minus one amino acid.

[0108] As used herein, the terms "protein" and "polypeptide" refer to compounds that contain amino acids linked via peptide bonds and are used interchangeably. The proteins and polypeptides discussed herein may be arginine-depleting enzymes, such as arginine deiminase enzymes, or arginases, such as arginase I or II enzymes, or fusion proteins fused to arginine-depleting enzymes. The proteins may be bacterial or mammalian. Mammalian enzymes may include rodent, human, primate, and the like. These proteins may be natural proteins or genetically modified recombinant proteins. Exemplary proteins include arginine-depleting enzymes that are variants that are truncated at the amino or carboxy terminus or otherwise genetically modified as described herein.

[0109] As used herein, the term "fusion protein" refers to a chimeric protein that includes a protein of interest (i.e., human arginase or a variant thereof) bound (or operably linked) to an exogenous protein fragment (a fusion partner consisting of a non-arginase enzyme or a non-arginine deiminase). The fusion partner may enhance serum half-life, solubility, or both. It may also provide an affinity tag (e.g., His tag) that allows purification of the recombinant fusion protein from the host cell or culture supernatant, or both.

[0110] The terms "in operable combination," "in operable order," and "operably linked" refer to the linking of nucleic acid sequences in such a manner that a nucleic acid molecule capable of directing the transcription of a given gene and / or the synthesis of a desired protein molecule is produced. The terms also refer to the linking of amino acid sequences in such a manner that a functional protein is produced.

[0111] As used herein, the term "K m " refers to the Michaelis-Menten constant of an enzyme and is defined as the concentration of a specific substrate at which a given enzyme produces half its maximum velocity in an enzyme-catalyzed reaction.

[0112] As used herein, the term "k cat " refers to the number of turnovers or the number of substrate molecules that each enzyme site converts to product per unit time when the enzyme is operating at maximum efficiency.

[0113] As used herein, the term "K cat / K m " is the specificity constant, which is a measure of how efficiently an enzyme converts a substrate into a product.

[0114] The term "Mn-hArgI" refers to human Arginase I with a Mn(II) cofactor. The term "Co-hArgI" refers to human Arginase I (mutated or native) with a Co(II) cofactor.

[0115] The term "IC50" is the half-maximal (50%) inhibitory concentration (IC) and is thus a measure of efficacy.

[0116] The term "PEGylation" refers to conjugates with polyethylene glycol (PEG), which is widely used as a drug carrier given its high degree of biocompatibility and ease of modification (Harris et al., 2001). Attachment to various drugs, proteins and liposomes has been shown to improve residence time and reduce toxicity (Greenwald et al., 2000; Zalipsky et al., 1997). PEG can be attached (e.g., covalently bonded) to active agents via the hydroxyl groups at the ends of the chains and by other chemical methods. However, PEG itself is limited to a maximum of two active agents per molecule. In a different approach, copolymers of PEG and amino acids have been investigated as novel biomaterials that retain the biocompatible properties of PEG but have the added advantage of a large number of attachment points per molecule (leading to higher drug loading) and can be synthetically engineered to suit a variety of applications (Nathan et al., 1992; Nathan et al., 1993). In one embodiment, the arginase is PEGylated with polyethylene glycol of 1,000 to 10,000 daltons, e.g., 5,000 daltons. See, e.g., U.S. Patent No. 8,679,479, incorporated herein by reference.

[0117] The term "gene" refers to a DNA sequence that contains control and coding sequences necessary for the production of a polypeptide or its precursor. The polypeptide can be encoded by a full-length coding sequence or by any portion of the coding sequence, so long as the desired enzymatic activity is retained.

[0118] The term "subject" refers to an animal, including a human. A subject may also be a pediatric human patient.

[0119] The term "wild type" or "natural" refers to a gene or gene product that has the characteristics of that gene or gene product when isolated from a naturally occurring source. A wild type gene is one that is most frequently observed in a population and is therefore arbitrarily referred to as the "normal" or "wild type" form of the gene. In contrast, the term "modified" or "mutant" or "mutation" refers to a gene or gene product that exhibits modifications in sequence and / or functional properties (i.e., altered properties) when compared to the wild type gene or gene product. It should be noted that naturally occurring mutants can be isolated. These are identified by the fact that they have altered properties when compared to the wild type gene or gene product. "Natural" can also refer to the metal cofactor of arginase, which is manganese.

[0120] VIII. Kit Kits, such as therapeutic kits, are also provided.For example, the kits can include one or more pharmaceutical compositions as described herein, and optionally instructions for their use.The kits can also include one or more devices for achieving administration of such compositions.For example, the subject kits can include an ampoule pre-filled with stabilized multimeric arginase or isolated PEGylated arginase, optionally formulated as a pharmaceutical product, or lyophilized for use in, for example, a delivery device, or a preloaded syringe for subcutaneous or intravenous injection of the composition into a subject.

[0121] The kit may include a container with a label. Suitable containers include, for example, bottles, vials, and test tubes. The container may be formed from a variety of materials, such as glass or plastic. The container may hold a composition including an antibody effective for therapeutic or non-therapeutic applications, as described above. The label on the container may indicate that the composition is to be used for a particular therapeutic or non-therapeutic application, and may also indicate instructions for either in vivo or in vitro use, such as those described above. An exemplary kit may include the containers described above and one or more other containers containing materials desirable from a commercial and user standpoint, including buffers, diluents, filters, needles, syringes, and package inserts with instructions for use. EXAMPLES

[0122] The following examples serve to illustrate certain aspects of the compounds, compositions, pharmaceutical compositions and methods of use and should not be construed as limiting the scope thereof. In the following experimental disclosure, the following abbreviations apply: eq (equivalent); M (mole); μM (micromole); μM (millimolar); N (normal); mol (mole); mmol (millimolar); μmol (micromole); nmol (nanomole); g (gram); mg (milligram); μg (microgram); L (liter); ml (milliliter); μl (microliter); cm (centimeter); mm (millimeter); μm (micrometer); nm (nanometer); EC (Celsius); MW (molecular weight); PBS (phosphate buffered saline); min (minute).

[0123] [Example 1] Incorporation and determination of metal content in arginase I Mn 2+ and Co 2+Incorporation of hArgl can be achieved by purification of arginase followed by incubation with 10 mM metal at 50 °C for 10 min. To determine the final metal content and identity of the arginase preparations, protein samples of Mn-hArgl (145 μM), Co-hArgl (182 μM) and associated dialysis buffer (100 mM Hepes, pH 7.4) were diluted with 2% nitric acid and analyzed by inductively coupled plasma mass spectrometry (ICP-MS, Department of Geological Sciences, University of Texas, Austin) to quantify the cobalt, iron, manganese and zinc content of the proteins by subtracting the concentration of the metal found in the dialysis buffer from the metal concentration of the final protein sample and dividing by the protein concentration. To determine protein concentration, the extinction coefficient was calculated for hArgl based on the amino acid sequence (Gill and von Hippel, 1989). The total protein concentration of arginase I was calculated based on the calculated ε in a final buffer concentration of 6 M guanidinium hydrochloride, 20 mM phosphate buffer, pH 6.5. 280 =24,180M -1 cm -1 For comparison, arginase concentrations were also calculated by BCA assay using dilutions of BSA as a standard. 2+ The arginase sample incubated with was found to contain 2.1±0.5 equivalents of Co and 0.4±0.1 equivalents of Fe, with no detectable amounts of Zn or Mn. 2+ Samples incubated with Zn contain 1.5 ± 0.2 equivalents of Mn and 0.4 ± 0.1 equivalents of Fe, with no detectable amounts of Zn or Co. Thus, thermal incubation is an efficient method for incorporating cobalt.

[0124] Further studies of cobalt loading demonstrated that higher percentages of cobalt loading were achievable, resulting in higher specific activities. The results of these studies are shown in the table below and in FIG. 3. [Table 2]

[0125] [Example 2] Engineering Fc-Arginase Fusion Proteins to Extend In Vivo Half-Life Fusion to IgG Fc domains has been widely used to extend the in vivo half-life of therapeutic polypeptides such as the TNF-α inhibitor etanercept (Enbrel®). Fc domains bind to the FcγRn receptor, which is expressed on the vascular endothelium and many other tissues (Roopenian and Akilesh, 2007). The affinity of FcγRn for IgG Fc domains is strongly pH-dependent. Binding occurs at the acidic pH of endosomal compartments, allowing the protein to be recycled to the cell surface and thus escape proteolysis. At the cell surface, the Fc domain is released from FcγRn, as the binding affinity is very low at physiological pH. Endosomal recycling via FcγRn is estimated to extend the serum half-life of immunoglobulins at least 4-7-fold, to approximately 7-14 days in humans. Fc fusions exploit this property to confer long half-lives to short-lived molecules. However, human arginase is a homotrimer and therefore, when fused to IgG Fc, which is itself a dimer, the resulting Fc-arginase polypeptide is likely to form high molecular weight aggregates.

[0126] This problem was circumvented by using a mutant form of arginase that disrupts dimerization and is stable in a monomeric form. The dimerization and subunit interface of Arginase I have been studied in some detail (Lavulo et al., 2001). A single amino acid substitution at Glu256Gln has been shown to disrupt dimerization and result in the formation of a monomeric Arginase I enzyme (Sabio et al., 2001). After expression and purification of this mutant, steady-state kinetic analysis revealed a 1,320 s -1 mm -1 k cat / K M The activity was nearly identical to that of Co-hArgl.

[0127] This construct was then cloned into an Fc expression vector, a construct based on the pTRC99a plasmid (Amersham) that contains the DsbA leader sequence followed by the IgG Fc coding region, an EcoRI restriction site and a stop codon. The monomeric arginase gene was placed in frame behind the Fc coding region by digesting both the vector and the gene with EcoRI, and then ligated and transformed into Escherichia coli (E. coli) (BL21) for sequencing and expression. Since IgG Fc is usually a glycosylated protein, expression of recombinant IgG or Fc fusions has been previously performed in recombinant mammalian cells that, unlike bacteria, are capable of N-linked glycosylation. However, glycosylation at Asn297, although important for binding to the activating and inhibitory Fey receptor (FcγRI-III in humans), does not significantly affect affinity or pH-dependent binding to FcγRn (Tao and Morrison, 1989; Simmons et al., 2002). Thus, aglycosylated IgG antibodies expressed in bacteria show serum persistence in primates that is almost indistinguishable from that of fully glycosylated antibodies expressed in mammalian cells (Simmons et al., 2002). In contrast to the prevailing previous concept, IgG antibodies and Fc proteins can be efficiently expressed in E. coli up to g / L levels in fermenters. E. coli expression is technically much simpler and faster. Moreover, since the resulting proteins are aglycosylated, they do not show glycan heterogeneity, which is a key problem in the expression of therapeutic glycoproteins (Jefferis, 2007). The fusion protein is purified by Protein A chromatography and the yield of correctly folded dimeric Fc-Arginase fusion relative to polypeptides unable to dimerize is quantified by FPLC gel filtration chromatography. This preparation yielded a highly active and highly stable form of human Arginase suitable for in vivo testing.

[0128] [Example 3] PEGylation of Arginase Arginase I (SEQ ID NO:3) was purified and then made 10 mM in COCl2 and heated at 50°C for 10 minutes. After centrifugation to remove precipitate, the PEG-5000 Arginase was extensively buffer exchanged (PBS with 10% glycerol) using a 100,000 MWCO filtration device (Amicon) and sterilized with a 0.2 micron syringe filter (VWR). All PEGylated enzymes were analyzed for lipopolysaccharide (LPS) content using the Limulus Amebocyte Lysate (LAL) kit (Cape Cod Incorporated).

[0129] PEGylated Co-hArgI was found to have nearly identical serum stability to the wild-type enzyme, 1690 ± 290 s -1 mm -1 k cat / K M The values ​​were shown.

[0130] PEGylated Arginase I preparations in combination with cobalt are further described in US Pat. No. 8,679,479, which is incorporated herein by reference.

[0131] [Example 4] Serum depletion of L-Arg in a mouse model Balb / c mice were treated with a single IP injection of 500 μg of pharmacologically prepared PEGylated Co-hArgI or an equal volume of PBS. Mice were sacrificed by cardiac venipuncture for blood collection at 0, 48, 72 and 96 hours. Blood samples were immediately mixed 50:50 (v / v) with 400 mM pH 4 sodium citrate buffer, allowed to clot for 30 minutes, and centrifuged for serum separation. The resulting serum was then filtered with a 10,000 MWCO device (Amicon) to remove large proteins and precipitates, and the flow-through was collected for analysis. L-arginine standards, control mouse serum and experimental samples were derivatized with OPA (Agilent) and separated on a C18 reversed-phase HPLC column (Agilent) (5 μm, 4.6 x 150 mm) essentially as described in Agilent Technologies (Publication No.: 5980-3088), except for a slight modification of the separation protocol by reducing the flow rate by 1 / 2 and doubling the capture time to obtain better peak separation. To quantify serum L-Arg levels, an L-arginine standard curve was constructed by plotting the L-Arg peak area versus concentration. A single dose of pharmacologically prepared Co-hArgI was sufficient to maintain L-Arg below the detection limit over a 3-day period (Figure 1).

[0132] [Example 5] Subcutaneous and intravenous administration of arginase A single intravenous (IV) or subcutaneous (SC) dose of AEB1102 (PEGylated Arginase I lacking the N-terminal methionine of SEQ ID NO:3 and with cobalt as a cofactor) was administered to male cynomolgus monkeys. The objective of this study was to characterize the pharmacokinetics (PK) and pharmacodynamics (PD) of modified Arginase I (AEB1102) following IV versus SC administration.

[0133] Exposure to AEB1102 was achieved after IV administration at a dose of 0.5 mg / kg. The observed volume of distribution (Vss) was similar to monkey serum volume (45 mL / kg) and the resulting half-life (T1 / 2) was 37.0 ± 1.79 hours. Intravenous administration of 0.5 mg / kg AEB1102 resulted in maximal arginine suppression 24 hours after dosing. Below the limit of quantification (BQL) levels of arginine were achieved in only two of three animals in this dose group. Recovery of arginine appeared to be consistent with AEB1102 levels falling below a mean (±SD) concentration of 7.25 ± 0.433 μg / mL. Recovery to pre-dose levels of arginine was incomplete 168 hours after dosing.

[0134] Following SC administration of AEB1102 at a 0.5 mg / kg dose in two separate formulations, the mean concentration profiles overlapped for Groups 2 and 3, although there was a trend toward slightly higher 24-hour concentrations in Group 3, and absorption appeared to be slightly faster in this group. Median Tmax was the same at 24 hours for both groups. SC T1 / 2 estimates appeared to be slightly shorter in Group 3 (38.1 ± 1.00 hours) versus Group 2 (46.5 ± 3.05 hours). SC administration of 0.5 mg / kg resulted in significant arginine suppression for both formulations of AEB1102 in Groups 2 and 3. Maximal (incomplete) suppression of arginine was achieved in most SC animals at 24 hours post-dose. Recovery of arginine appeared to be consistent with AEB1102 levels falling to below 3.6 to 4.6 μg / mL. Recovery to pre-dose levels of arginine was incomplete at 168 hours post-dose.

[0135] For Group 1, the test article was preformulated at a nominal concentration of 3 mg / mL in phosphate buffered saline (PBS) with 10% glycerol, pH 7.4 (GMP Lot 02) and used as received. Group 2 test article was received as a preformulated 70 mg / mL stock solution in 20 mM potassium phosphate, 37.5 mM NaCl, 5.6% sucrose, and 0.02% PS80, pH 6.7 (Lot No. 144350-0004). This stock was diluted to 20 mg / mL (in PBS) prior to administration. Group 3 test article was received in prefilled syringes containing a solution containing 562.5 mg / mL triacetin, approximately 387 mg / mL sucrose, and 20.7 mg / mL AEB1102 (active; Lot No. 170315P24). Syringes were used as received. [Table 3] A: 10% glycerol in PBS, pH 7.4; B: 562.5 mg triacetin, 386.89 mg sucrose and 50.61 mg AEB1102 (20.7 mg active substance) per mL of formulation; and C: 20 mM potassium phosphate, 37.5 mM NaCl, 5.6% sucrose, and 0.02% PS80, pH 6.7.

[0136] Animals in Group 1 were administered a single IV dose. These doses were administered as a bolus injection via the saphenous (or other appropriate) vein. If a catheter was used for administration, the catheter was flushed with approximately 1 mL of sterile sodium chloride for injection, USP immediately after administration.

[0137] Animals in groups 2 and 3 received a SC dose of AEB1102 once a week for four consecutive weeks. Additionally, these animals received a control saline SC injection at a separate site with the same frequency and dose as the test article. The SC dose was administered as a bolus injection between the skin and underlying layer of tissue in the scapular region on the back of each animal. The injection site was marked after administration and remarked as necessary.

[0138] PK and PD blood samples were collected pre-dose and at 1, 4, 8, 24, 48, 72, 96, 120, 144 and 168 hours post-dose. Blood for PK (1 mL / time point) was collected from the femoral vein / artery (or other suitable vein) into anticoagulant-free tubes. Samples were allowed to clot at room temperature, and after centrifugation, serum samples were divided into two aliquots and stored frozen (-60 to -90 °C) until analysis. Blood for PD (1 mL / time point) was collected from the femoral vein / artery (or other suitable vein) into pre-chilled EDTA SCAT tubes and immediately placed on ice. After centrifugation under refrigerated conditions, 8 µL of acetic acid was added to the resulting plasma (0.4 mL) and the aliquot was mixed by inverting the tube. If the plasma volume per aliquot was less than 0.4 mL (2 aliquots were targeted), the added volume of acetic acid was adjusted to achieve a final concentration of at least 2% (v / v) acetic acid. Aliquots were stored frozen (-60 to -90°C) until analysis.

[0139] Quantification Assay: Analysis of serum AEB1102 concentrations was performed at Intertek Pharmaceutical Services using an enzyme activity assay (Bioanalytical Procedure No. BPAEB1102D) validated in non-human primate serum. The lower limit of quantification (LLOQ) was 0.125 μg / mL in 100% serum. Analysis of plasma arginine concentrations was also performed at Intertek Pharmaceutical Services using an LC-MS / MS assay (Bioanalytical Procedure No. BPARGN1) validated in non-human primate plasma. The LLOQ was 1 μM in 100% plasma. Pharmacokinetic Analysis

[0140] Serum AEB1102 concentration-time data, obtained from Intertek Pharmaceutical Services in a Microsoft Excel (Excel; Microsoft Corp., Seattle, WA) spreadsheet, were analyzed by noncompartmental analysis (NCA) with Phoenix™ WinNonlin® version 7.0 (WinNonlin; Certara USA, Inc., Mountain View, California) using the IV bolus or extravascular administration model as appropriate. Nominal doses and blood draw times were used in the NCA.

[0141] For the IV group, all BQL concentrations were set to missing. This practice allowed for more accurate back-extrapolation and calculation of serum concentrations immediately after injection (C0). For the SC group, BQL concentrations before Cmax were set to 0 and BQL concentrations after Cmax were excluded from the PK analysis.

[0142] The area under the curve from time zero to the last measurable concentration (AUC0-t) was calculated by the linear up / log down method. The elimination constant (λz) was estimated using log / linear regression through the last three or more time points (excluding Tmax). The terminal half-life (T1 / 2) and the AUC from time zero to infinity (AUC0-∞) were calculated using the following formula: T1 / 2=In(2) / λz AUC0-∞=AUC0-t+Ct,pred / λz where Ct,pred is the final concentration predicted by the slope of the terminal phase selected by WinNonlin. The reported PK parameters are listed in the table below with brief definitions.

[0143] Parameter description: Cmax: The maximum observed concentration of a drug measured after administration. Tmax: The time after administration at which the maximum observed concentration of a drug is observed. Tlast: The time post-dose at which the last measurable concentration of drug was observed. AUC0-t: area under the drug concentration-time curve from time 0 to the time post-dose at which the last measurable concentration of the drug was observed.

[0144] Where data permitted, the slope of the terminal elimination phase of each concentration-time curve was determined by log / linear regression and additional parameters, listed below, were also estimated. The goodness of fit (R2) of the slope of the regression line had to be 0.8 or greater to report the following data that are dependent on the terminal phase.

[0145] Additional parameters: T1 / 2: apparent terminal elimination half-life. AUC 0-inf (or AUC 0-∞): area under the concentration-time curve from time 0 to infinity. CL: Volume of serum cleared of drug per unit time after a single IV dose. CL / F: apparent serum volume per unit time cleared of drug after a single SC dose. Vss: steady-state volume of distribution after IV administration. Vz: Terminal volume of distribution after IV administration Apparent terminal volume of distribution after Vz / F:SC administration F: Bioavailability or fraction of dose absorbed after SC administration

[0146] Bioavailability (F) after SC administration was evaluated using the following formula: F=100%*(AUC0-∞,SC / DoseSC) / (AUC0-∞,IV / DoselV)

[0147] Pharmacodynamic analysis: Arginine concentration-time data was obtained in an Excel spreadsheet from Intertek Pharmaceutical Services and plotted with the corresponding AEB1102 concentrations using GraphPad Prism version 7.0 (GraphPad Software, La Jolla, CA).

[0148] statistical analysis Summary statistics [N, mean, standard deviation (SD), minimum (Min), median (Med), maximum (Max), coefficient of variation (CV)] for PK parameters and concentration data were prepared using WinNonlin. If less than 50% of monkeys had measurable concentrations, Min and Med were presented as BQL, Max was the highest measurable concentration, but Mean, SD, and %CV were not reported. If at least 50% of monkeys had measurable concentrations, summary statistics were based on measurable concentrations only. If all of the subjects had BQL concentrations, Mean, Min, Med, and Max were presented as BQL, and SD and %CV were not reported. Graphs were generated using GraphPad Prism (version 7.00 for Windows, GraphPad Software, San Diego California).

[0149] AEB1102 administered IV at 0.5 mg / kg produced maximal arginine suppression 24 hours after dosing. BQL levels of arginine were achieved in only two of three animals in this dose group. Recovery of arginine appeared to coincide with AEB1102 levels falling below a mean (±SD) concentration of 7.25 ± 0.433 μg / mL. Recovery of arginine to pre-dose levels was incomplete 168 hours after dosing.

[0150] Following SC administration of AEB1102 in two separate formulations, the mean concentration profiles overlapped in Groups 2 and 3, although there was a trend toward slightly higher 24-h concentrations in Group 3, suggesting that absorption appeared to be slightly faster in this group. Median Tmax was the same at 24 hours in both groups. SC T1 / 2 estimates appeared to be slightly shorter in Group 3 (38.1 ± 1.00 hours) versus Group 2 (46.5 ± 3.05 hours).

[0151] SC administration of 0.5 mg / kg resulted in significant arginine suppression for both formulations of AEB1102 in groups 2 and 3. Maximal (incomplete) suppression of arginine was achieved in most SC animals at 24 hours post-dose. Arginine recovery appeared to coincide with AEB1102 levels dropping to below 3.6 to 4.6 μg / mL. Again, recovery of arginine to pre-dose levels was incomplete at 168 hours post-dose.

[0152] Pharmacokinetics: The mean PK parameters in male monkeys following IV administration of AEB1102 are summarized in Table 4, and the mean serum concentration-time profiles are shown in FIG. [Table 4]

[0153] The mean PK parameters in male monkeys following SC administration of AEB1102 are summarized in Table 5, and the mean serum concentration-time profiles are shown in FIG. [Table 5]

[0154] Comparison of Figure 3 and Figure 4 demonstrates the difference in arginase 1 (AEB1102) enzyme levels upon intravenous administration compared to the subcutaneous route. In Figure 3, upon IV administration, the drug is at its highest concentration after only 1 hour, whereas the Tmax for SC administration is 24 hours for the SC group. It is believed that the very rapid increase in drug levels with IV administration may be the cause of adverse events in subjects, especially pediatric subjects.

[0155] Pharmacodynamics The mean pharmacodynamic profile in male monkeys following a single dose of 0.5 mg / kg IV or SC is shown in FIG.

[0156] The left panel of Figure 5 shows a rapid decline in arginine levels to approximately 98% within the first hour, remaining below the lower limit of quantification for up to 24 hours. In contrast, SC administration may result in a less dramatic decline in arginine levels in the first few hours after administration, reaching a higher minimum concentration than IV administration, and also providing a longer therapeutic efficacy window.

[0157] [Example 6] In vivo study of arginase-induced reduction of GAA levels in GAMT- / - mouse model The aim of the experiment was to determine whether AEB1102 could reduce GAA levels in a GAMT− / − mouse model, thereby providing evidence supporting the use of arginine-depleting enzymes for the treatment of GAMT-deficient patients.

[0158] GAMT- / - and wild-type C57BL / 6J mice were maintained on a creatine-deficient diet. Mice were administered either 2 mg / kg PBS or AEB1102 by intraperitoneal route. After 72 hours, mice were sacrificed and their blood was collected in tubes containing Nor-NOHA. GAA, ornithine and arginine levels were measured according to Tran et al. (2014).

[0159] Arginase ip treatment reduces arginine in the GAMT- / - mouse model (e.g., from 75.6 μM to 26.2 μM) and concomitantly increases ornithine in both wild-type and GAMT- / - mice. As expected, GAA levels were highly reduced in wild-type mice (from 1.9 to 0.0 μM) but significantly increased in GAMT- / - mice as shown in the table below. AEB1102 treatment reduced the mean GAA plasma levels by approximately 30 μM relative to negative control animals (from 67.3 to 38.6 on average). Data represent the mean ± SEM. The number of animals in each group is indicated by "n" and SEM is the standard error of the mean. [Table 6]

[0160] All compositions and methods of use disclosed and claimed herein can be made and executed without undue experimentation in light of this disclosure. Although the compositions and methods are described with reference to preferred or exemplary embodiments, it will be apparent to one skilled in the art that modifications may be applied to the compositions and methods described herein, and to the steps or sequence of steps of the methods, without departing from the concept, spirit and scope of the materials and methods described. More specifically, it will be apparent that certain agents that are both chemically and physiologically related may be substituted for the agents described herein, while the same or similar results would be achieved. All such similar substitutes and modifications apparent to one skilled in the art are deemed to be within the spirit, scope and concept of the compositions and methods, as further described in the appended claims.

[0161] 1. A method for treating a deficiency in guanidinoacetate methyltransferase (GAMT) activity in a subject, comprising administering to the subject a pharmaceutical composition comprising a therapeutic amount of an arginine-depleting enzyme.

[0162] 2. The method of claim 1, wherein the arginine-depleting enzyme is a mammalian or bacterial arginase enzyme.

[0163] 3. The method of claim 1, wherein the arginine depleting enzyme is a human arginine depleting enzyme.

[0164] 4. The method of claim 1, wherein the arginine-depleting enzyme is a human arginase enzyme, a human arginine deiminase enzyme, or a combination thereof.

[0165] 5. The method of 4, wherein one or more of the human arginase enzymes or human arginine deiminase enzymes is modified by substitution, deletion, insertion, or truncation in the amino acid sequence of the enzyme.

[0166] 6. The method of claim 1, wherein the pharmaceutical composition comprises human Arginase I enzyme or human Arginase II enzyme.

[0167] 7. The method of claim 1, wherein the arginine-depleting enzyme is human arginase I enzyme.

[0168] 8. The method of any of claims 6 or 7, wherein the human Arginase I enzyme is engineered with a substituted metal cofactor comprising cobalt.

[0169] 9. The method of claim 2, wherein the arginine-depleting enzyme is administered in erythrocyte ghosts.

[0170] 10. The method of claim 1, wherein the deficiency in GAMT activity is associated with a genetic deficiency in a gene encoding the enzyme guanidinoacetate methyltransferase in the subject.

[0171] 11. The method of claim 4, wherein the human arginase enzyme or human arginine deiminase enzyme is stabilized by association with a stabilizing agent.

[0172] 12. The method of claim 11, wherein the stabilizing agent is selected from the group consisting of polyethylene glycol (PEG), synthetic protein polymers, polysialic acid, Fc fusions and albumin.

[0173] 13. The method of any one of claims 6 to 8, wherein the human Arginase I enzyme is PEGylated.

[0174] 14. The method of any one of claims 1 to 13, wherein the subject is a human.

[0175] 15. The human Arginase I enzyme has a pH of 400 mM at pH 7.4 and 37° C. as measured in vitro. -1 s -1 ~4,000mM -1 s -1 k for the hydrolysis of arginine cat / KM The method of claim 7, wherein:

[0176] 16. The method of claim 8, wherein the human Arginase I enzyme contains cobalt in a ratio of 2 μg Co / mg arginase to 3 μg Co / mg arginase.

[0177] 17. The method of claim 8, wherein the human Arginase I enzyme is produced by contacting Arginase apoenzyme with cobalt or cobalt ions at a temperature between 30° C. and 55° C. for 15 minutes to 60 minutes.

[0178] 18. A method for treating the effects of guanidinoacetic acid (GAA) toxicity in a subject having deficient guanidinoacetic acid methyltransferase (GAMT) activity, comprising administering to the subject a therapeutic amount of a pharmaceutical composition comprising a PEGylated human Arginase I enzyme containing a cobalt cofactor.

[0179] 19. The method of claim 18, wherein administration is continued until the subject shows improvement in his or her physical or neurological condition.

[0180] 20. The method of claim 19, wherein the physical or neurological condition comprises at least one condition selected from the group consisting of global developmental delay / intellectual disability (DD / ID), epilepsy, movement disorder, speech or language delay, and behavioral disorder.

[0181] 21. The method of claim 18, wherein the therapeutic amount of the PEGylated human Arginase I enzyme with a cobalt cofactor is from about 0.01 mg / kg to about 7.5 mg / kg.

[0182] 22. The method of claim 18, wherein the therapeutic amount of the PEGylated human Arginase I enzyme with a cobalt cofactor is from about 0.05 mg / kg to about 5 mg / kg.

[0183] 23. The method of claim 18, wherein the therapeutic amount of the PEGylated human Arginase I enzyme with a cobalt cofactor is from about 0.1 mg / kg to about 5 mg / kg.

[0184] 24. The method of claim 18, wherein the pharmaceutical composition is administered to the subject parenterally.

[0185] 25. The method of claim 18, wherein the pharmaceutical composition is administered topically, intravenously, intradermally, intra-arterially, intraperitoneally, intralesionally, intracranially, intra-articularly, intraprostatically, intrapleurally, intratracheally, intraocularly, intranasally, intravitreally, intravaginally, intrarectally, intramuscularly, subcutaneously, subconjunctivally, intravesicularly, mucosally, intrapericardially, subumbilically, orally, by inhalation, injection, infusion, continuous infusion, by direct local perfusion bath of target cells, via a catheter, or via lavage solution.

[0186] 26. The method of claim 18, wherein the pharmaceutical composition is adapted for subcutaneous administration to the subject.

[0187] 27. The method of claim 26, wherein the pharmaceutical composition comprises a therapeutic dose of arginase in potassium phosphate, NaCl, sucrose and PS80 at pH 6.7.

[0188] 28. A kit for use in the method of claim 18, comprising a syringe containing a solution comprising triacetin, sucrose, and the PEGylated human Arginase I enzyme adapted for subcutaneous administration.

[0189] 29. The method of claim 18, wherein the pharmaceutical composition is adapted for intravenous administration.

[0190] 30. The method of claim 29, wherein the pharmaceutical composition comprises the PEGylated human Arginase I enzyme, saline, and glycerol at a pH of 7.4.

[0191] 31. The method of claim 26, wherein administration of the pharmaceutical composition reduces serum arginine in the patient by 50% to 99%.

[0192] 32. The method of claim 26, wherein administration of the pharmaceutical composition reduces serum arginine in the patient by 90% to 99%.

[0193] 33. The method of claim 26, wherein administration of the pharmaceutical composition reduces serum GAA in the patient by at least 25% to 50%.

[0194] 34. The method of claim 26, wherein the concentration of the pharmaceutical composition in plasma reaches a maximum level 20 to 28 hours after a single administration.

[0195] 35. The method of claim 26, wherein the concentration of the pharmaceutical composition in plasma reaches a maximum level about 24 hours after a single administration.

[0196] 36. A method for treating the effects of guanidinoacetic acid (GAA) toxicity, comprising administering to a subject in need thereof a therapeutic amount of a pharmaceutical composition comprising an arginine-depleting enzyme.

[0197] 37. The method of claim 36, wherein the arginine-depleting enzyme is an arginase or arginine deiminase enzyme.

[0198] 38. A method for treating guanidinoacetate methyltransferase (GAMT) activity deficiency in a subject, comprising administering to the subject a pharmaceutical composition comprising a therapeutic amount of an arginine-depleting enzyme in combination with ornithine supplementation.

[0199] 39. The method of claim 38, wherein the arginine-depleting enzyme is an arginase or arginine deiminase enzyme.

[0200] 40. The method of claim 38, wherein the pharmaceutical composition comprises high-dose L-ornithine supplementation.

[0201] 41. The method of claim 38, comprising orally administering L-ornithine aspartate or L-ornithine hydrochloride.

[0202] 42. A pharmaceutical composition comprising a therapeutic amount of an arginine-depleting enzyme for use as a medicament for use in treating a deficiency in guanidinoacetate methyltransferase (GAMT) activity in a subject, or in treating the effects of guanidinoacetate (GAA) toxicity in a subject.

[0203] 43. A pharmaceutical composition comprising a therapeutic amount of an arginine-depleting enzyme for use in treating a deficiency in guanidinoacetate methyltransferase (GAMT) activity in a subject or for treating the effects of guanidinoacetate (GAA) toxicity in a subject.

[0204] 44. The pharmaceutical composition of any one of claims 42 and 43, wherein the arginine-depleting enzyme is a mammalian or bacterial arginase enzyme.

[0205] 45. The pharmaceutical composition of any one of claims 42 and 43, wherein the arginine depleting enzyme is a human arginine depleting enzyme.

[0206] 46. ​​The pharmaceutical composition of any of claims 42 and 43, wherein the human arginine-depleting enzyme is a human arginase enzyme, a human arginine deiminase enzyme, or a combination thereof.

[0207] 47. The pharmaceutical composition of claim 46, wherein one or more of the human arginase or arginine deaminase enzymes are modified by substitution, deletion, insertion, or truncation of the amino acid sequence of the human arginase or arginine deaminase enzyme.

[0208] 48. The pharmaceutical composition of any one of claims 42 and 43, comprising human Arginase I enzyme or Arginase II enzyme.

[0209] 49. The pharmaceutical composition of any one of claims 42 and 43, wherein the arginine-depleting enzyme comprises a human arginase I enzyme.

[0210] 50. The pharmaceutical composition of any one of claims 48 or 49, wherein the arginine-depleting enzyme comprises a human Arginase I enzyme engineered with a substituted metal cofactor comprising cobalt.

[0211] 51. The pharmaceutical composition of claim 44, wherein the arginine-depleting enzyme is administered in autologous red blood cell ghosts.

[0212] 52. The pharmaceutical composition of any one of claims 42 and 43, wherein the deficiency in GAMT activity is associated with a genetic deficiency of the gene encoding the guanidinoacetate methyltransferase enzyme.

[0213] 53. The pharmaceutical composition of claim 46, wherein the human arginase enzyme or human arginine deiminase enzyme is stabilized by association with a stabilizing agent.

[0214] 54. The pharmaceutical composition of claim 53, wherein the stabilizer is selected from the group consisting of polyethylene glycol (PEG), synthetic protein polymers, polysialic acid, Fc fusions, and albumin.

[0215] 55. The pharmaceutical composition of any one of claims 58-60, wherein the human Arginase I enzyme is PEGylated.

[0216] 56. The pharmaceutical composition of any one of claims 42 to 55, wherein the subject is a human.

[0217] 57. The human Arginase I enzyme has a pH of 400 mM at pH 7.4 and 37° C. as measured in vitro. -1 s -1 ~4,000mM -1 s -1 k for the hydrolysis of arginine cat / K M The pharmaceutical composition of claim 49,

[0218] 58. The pharmaceutical composition of claim 50, wherein the human Arginase I enzyme contains cobalt in a ratio of 2 μg Co / mg arginase to 3 μg Co / mg arginase.

[0219] 59. The method of claim 50, wherein the human Arginase I enzyme is produced by contacting Arginase apoenzyme with cobalt or cobalt ions at a temperature between 30°C and 55°C for 15 minutes to 60 minutes.

[0220] References The following references, to the extent that they provide exemplary procedural or other details supplementary to those set forth herein, are specifically incorporated herein by reference. Cama et al.,Biochemistry,42:7748-7758,2003. Cheng et al., Cancer Lett., 224:67-80, 2005. Cheng et al., Cancer Res., 67:309, 2007. Cheng et al., Cancer Res., 67:4869-4877, 2007. Dillon et al.,Med.Sci.Monit.,8:BR248-253,2002. Dowling et al.,Cell Mol.Life.Sci.,65(13):2039-55,2008. Durante et al.,Clin.Exp.Pharmacol.Physiol.,34:906-911,2007. Ensor et al., Cancer Res., 62:5443-5450, 2002. Gill and von Hippel, Anal. Biochem., 182:319-326, 1989. Greenwald et al., Crit.Rev.Therap.Drug Carrier Syst., 17:101-161, 2000. Harkki et al.,BioTechnology,7:596-603,1989. Harris et al.,Clin.Pharmacokinet.,40(7):539-51,2001. Hopwood et al.、In:Genetic Manipulation of Streptomyces,A Laboratory Manual,The John Innes Foundation,Norwich,Conn。、1985. Jefferis,R.,Expert Opin.Biol.Ther.,7:1401-1413,2007. Lavulo et al.,J.Biol.Chem.,276:14242-14248,2001. Lopez et al.,FEBSJ.,272:4540-4548,2005. Lordanescu,J.Bacteriol,12:597 601,1975. Marescau,B.,et al.,’’Guanidino compound analysis as a complementary diagnostic parameter for hyperargininemia:follow-up of guanidino compound levels during therapy,’’Pediatr.Res.27:297-303,1990. Mellor et al.,Gene,24:1-14,1983. Nathan et al.,Bioconj.Chem.,4:54-62,1993. Nathan et al.,Macromolecules,25:4476-4484,1992. Penttilaet al.,Gene,61:155-164,1987. Remington’s Pharmaceutical Sciences,18th Ed.Mack Printing Company,1289-1329,1990. RoopenianおよびAkilesh,Nat.Rev.Immunol.,7:715-725,2007. Sabio et al.,FEBS Lett.,501:161-165,2001. Santhanam et al.,Circ.Res.,101:692-702,2007. Savoca et al.,Cancer Biochem.Biophys.,7:261-268,1984. Schulze,A.et al.,「Improving treatment of guanidinoacetate methyltransferase deficiency:reduction of guanidinoacetic acid in body fluids by arginine restriction and ornithine supplementation,」Mol.Genet.Metab.74:413-419,2001. Sibakov et al.,Eur.J.Biochem.,145:567-572,1984. Simmons et al.,J.Immunol.Methods,263:133-147,2002. Tao et al.,J.Immunol.,143:2595-2601,1989. Tran,C.,et al.,「Stable isotope dilution microquantification of creatine metabolites in plasma,whole blood and dried blood spots for pharmacological studies in mouse models of creatine deficiency,」Clin.Chim.Acta 436C:160-168,2014. Villa, C., et al., "Red blood cells: Supercarriers for drugs, biologicals, and nanoparticles and inspiration for advanced delivery systems," Adv. Drug Deliv. Rev. 106(Pt A):88 - 103, 2016. Ward, Embo - Alko Workshop on Molecular Biology of Filamentous Fungi, Helsinki, 119 - 128, 1989. Zalipsky et al., Bioconjug. Chem., 8:111 - 118, 1997. (Sequence Listing) SEQUENCE LISTING <110> AERase, Inc. <120> Arginine Depletion Therapy For Treatment of GAMT Deficiency <130> 218107 - 0013 - 00 - WO - 594073 <150> US 62 / 747,837 <151> 2018 - 10 - 19 <160> 4 <170> PatentIn version 3.5 <210> 1 <211> 1044 <212> DNA <213> Artificial Sequence <220> <223> Synthetic sequence <400> 1 gatataccat gggttcttct caccatcatc accaccacag ctctggcgag aacctgtact 60 tccagtctgc gaagagccgt acgatcggca ttattggtgc gccgttctct aaaggtcagc 120 cacgcggtgg tgtggaagag ggtccgacgg ttctgcgtaa ggccggttta ttagaaaagc 180 tgaaagagca ggagtgcgac gttaaggact acggtgactt accattcgcg gacatcccga 240 atgatagccc gttccaaatc gttaagaatc cgcgctctgt gggtaaagca agcgagcagt 300 tagcaggtaa ggtggccgag gtcaagaaaa acggtcgtat tagcctggtt ttaggcggtg 360 atcatagctt agcaattggc tctatctctg gtcatgcccg tgtgcaccca gatttaggtg 420 tcatttgggt tgacgcccat acggatatca atacgccatt aacgaccacc agcggcaatc 480 tgcatggcca gccggttagc ttcttactga aggagctgaa gggtaaaatt ccagatgttc 540 cgggctttag ctgggtcacg ccatgtattt ctgccaagga tatcgtgtac attggcttac 600 gtgacgtcga cccaggtgag cactacatct taaagaccct gggtatcaag tatttcagca 660 tgacggaagt ggaccgctta ggcatcggca aggtgatgga ggagacgctg agctatctgc 720 tgggccgtaa gaaacgtcca atccatctga gcttcgatgt tgacggctta gacccgagct 780 ttacgccagc caccggcacg ccggtcgttg gtggtttaac gtatcgcgaa ggcctgtata 840 tcacggagga aatctataag acgggtttac tgagcggtct ggacattatg gaggttaatc 900 caagcttagg taagacgccg gaagaagtta cccgtaccgt taacacggcg gtcgcgatca 960 cgttagcatg tttcggttta gcccgcgagg gcaaccataa accaattgat tatctgaatc 1020 caccgaagtg aggatccgaa ttcg 1044 <210> 2 <211> 1080 <212> DNA <213> Artificial Sequence <220> <223> Synthetic sequence <400> 2 gatataccat gggcagcagc catcatcacc accatcacag ctctggtgaa aacttatact 60 tccaaagcgt ccatagcgtc gcagtgattg gtgccccgtt tagccaaggt caaaaacgca 120 agggtgttga acatggtccg gcagcgatcc gcgaagcagg tttaatgaag cgtttaagca 180 gcttaggctg tcacttaaag gatttcggtg atttaagctt tacgccggtc ccaaaggatg 240 atttatacaa taatctgatc gttaacccac gctctgtggg tctggcgaac caggagctgg 300 cggaggtcgt gtctcgtgca gtcagcgacg gttatagctg cgttacgctg ggcggtgatc 360 atagcttagc cattggtacg atttctggtc atgcccgcca ttgcccggat ctgtgtgttg 420 tgtgggttga tgcgcacgcg gatatcaata cgccactgac cacgtctagc ggtaatttac 480 acggccagcc ggttagcttc ttattacgtg agctgcaaga caaggtcccg cagttaccag 540 gcttctcttg gatcaaacca tgtatcagca gcgcatctat tgtctacatt ggcctgcgtg 600 atgtcgaccc accggagcac ttcatcctga agaattatga catccagtat ttcagcatgc 660 gtgacatcga ccgtctgggt atccaaaaag ttatggagcg cacgttcgat ctgttaatcg 720 gcaagcgcca gcgtccgatt cacctgagct ttgacattga cgcctttgac ccgaccctgg 780 ccccagcaac gggcacgcca gtggttggtg gtttaaccta ccgtgagggt atgtatattg 840 cagaagagat ccataatacc ggcctgttat ctgccctgga tctggttgaa gtcaatccgc 900 agctggcaac ctctgaggag gaagcgaaga cgaccgccaa cctggcggtg gacgtcatcg 960 cctcttcttt cggccagacg cgtgaaggtg gccatatcgt gtatgaccaa ttaccaacgc 1020 catctagccc ggacgaatct gagaaccaag cacgtgtccg tatttgagga tccgaattcg 1080 <210> 3 <211> 322 <212> PRT <213> Homo sapiens <220> <221> MISC_FEATURE <222> (1)..(1) <223> Xaa is either methionine or not present <400> 3 Xaa Ser Ala Lys Ser Arg Thr Ile Gly Ile Ile Gly Ala Pro Phe Ser 1 5 10 15 Lys Gly Gln Pro Arg Gly Gly Val Glu Glu Gly Pro Thr Val Leu Arg 20 25 30 Lys Ala Gly Leu Leu Glu Lys Leu Lys Glu Gln Glu Cys Asp Val Lys 35 40 45 Asp Tyr Gly Asp Leu Pro Phe Ala Asp Ile Pro Asn Asp Ser Pro Phe 50 55 60 Gln Ile Val Lys Asn Pro Arg Ser Val Gly Lys Ala Ser Glu Gln Leu 65 70 75 80 Ala Gly Lys Val Ala Glu Val Lys Lys Asn Gly Arg Ile Ser Leu Val 85 90 95 Leu Gly Gly Asp His Ser Leu Ala Ile Gly Ser Ile Ser Gly His Ala 100 105 110 Arg Val His Pro Asp Leu Gly Val Ile Trp Val Asp Ala His Thr Asp 115 120 125 Ile Asn Thr Pro Leu Thr Thr Thr Ser Gly Asn Leu His Gly Gln Pro 130 135 140 Val Ser Phe Leu Leu Lys Glu Leu Lys Gly Lys Ile Pro Asp Val Pro 145 150 155 160 Gly Phe Ser Trp Val Thr Pro Cys Ile Ser Ala Lys Asp Ile Val Tyr 165 170 175 Ile Gly Leu Arg Asp Val Asp Pro Gly Glu His Tyr Ile Leu Lys Thr 180 185 190 Leu Gly Ile Lys Tyr Phe Ser Met Thr Glu Val Asp Arg Leu Gly Ile 195 200 205 Gly Lys Val Met Glu Glu Thr Leu Ser Tyr Leu Leu Gly Arg Lys Lys 210 215 220 Arg Pro Ile His Leu Ser Phe Asp Val Asp Gly Leu Asp Pro Ser Phe 225 230 235 240 Thr Pro Ala Thr Gly Thr Pro Val Val Gly Gly Leu Thr Tyr Arg Glu 245 250 255 Gly Leu Tyr Ile Thr Glu Glu Ile Tyr Lys Thr Gly Leu Leu Ser Gly 260 265 270 Leu Asp Ile Met Glu Val Asn Pro Ser Leu Gly Lys Thr Pro Glu Glu 275 280 285 Val Thr Arg Thr Val Asn Thr Ala Val Ala Ile Thr Leu Ala Cys Phe 290 295 300 Gly Leu Ala Arg Glu Gly Asn His Lys Pro Ile Asp Tyr Leu Asn Pro 305 310 315 320 Pro Lys <210> 4 <211> 333 <212> PRT <213> Homo sapiens <400> 4 Ser Val His Ser Val Ala Val Ile Gly Ala Pro Phe Ser Gln Gly Gln 1 5 10 15 Lys Arg Lys Gly Val Glu His Gly Pro Ala Ala Ile Arg Glu Ala Gly 20 25 30 Leu Met Lys Arg Leu Ser Ser Leu Gly Cys His Leu Lys Asp Phe Gly 35 40 45 Asp Leu Ser Phe Thr Pro Val Pro Lys Asp Asp Leu Tyr Asn Asn Leu 50 55 60 Ile Val Asn Pro Arg Ser Val Gly Leu Ala Asn Gln Glu Leu Ala Glu 65 70 75 80 Val Val Ser Arg Ala Val Ser Asp Gly Tyr Ser Cys Val Thr Leu Gly 85 90 95 Gly Asp His Ser Leu Ala Ile Gly Thr Ile Ser Gly His Ala Arg His 100 105 110 Cys Pro Asp Leu Cys Val Val Trp Val Asp Ala His Ala Asp Ile Asn 115 120 125 Thr Pro Leu Thr Thr Ser Ser Gly Asn Leu His Gly Gln Pro Val Ser 130 135 140 Phe Leu Leu Arg Glu Leu Gln Asp Lys Val Pro Gln Leu Pro Gly Phe 145 150 155 160 Ser Trp Ile Lys Pro Cys Ile Ser Ser Ala Ser Ile Val Tyr Ile Gly 165 170 175 Leu Arg Asp Val Asp Pro Pro Glu His Phe Ile Leu Lys Asn Tyr Asp 180 185 190 Ile Gln Tyr Phe Ser Met Arg Asp Ile Asp Arg Leu Gly Ile Gln Lys 195 200 205 Val Met Glu Arg Thr Phe Asp Leu Leu Ile Gly Lys Arg Gln Arg Pro 210 215 220 Ile His Leu Ser Phe Asp Ile Asp Ala Phe Asp Pro Thr Leu Ala Pro 225 230 235 240 Ala Thr Gly Thr Pro Val Val Gly Gly Leu Thr Tyr Arg Glu Gly Met 245 250 255 Tyr Ile Ala Glu Glu Ile His Asn Thr Gly Leu Leu Ser Ala Leu Asp 260 265 270 Leu Val Glu Val Asn Pro Gln Leu Ala Thr Ser Glu Glu Glu Ala Lys 275 280 285 Thr Thr Ala Asn Leu Ala Val Asp Val Ile Ala Ser Ser Phe Gly Gln 290 295 300 Thr Arg Glu Gly Gly His Ile Val Tyr Asp Gln Leu Pro Thr Pro Ser 305 310 315 320 Ser Pro Asp Glu Ser Glu Asn Gln Ala Arg Val Arg Ile 325 330

Claims

1. 2. A method for treating a deficiency of guanidinoacetate methyltransferase (GAMT) activity in a subject, comprising administering to the subject a pharmaceutical composition comprising a therapeutic amount of an arginine-depleting enzyme.

2. 2. The method of claim 1, wherein the arginine-depleting enzyme is a mammalian or bacterial arginase enzyme.

3. 2. The method of claim 1, wherein the arginine-depleting enzyme is a human arginine-depleting enzyme.

4. 2. The method of claim 1, wherein the arginine-depleting enzyme is a human arginase enzyme, a human arginine deiminase enzyme, or a combination thereof.

5. 5. The method of claim 4, wherein one or more of the human arginase enzymes or human arginine deiminase enzymes are modified by substitution, deletion, insertion, or truncation in the amino acid sequence of the enzyme.

6. 2. The method of claim 1, wherein the pharmaceutical composition comprises human Arginase I enzyme or human Arginase II enzyme.

7. 2. The method of claim 1, wherein the arginine-depleting enzyme is human arginase I enzyme.

8. 8. The method of claim 6 or 7, wherein the human Arginase I enzyme is engineered with a substituted metal cofactor comprising cobalt.

9. The method of claim 2 , wherein the arginine-depleting enzyme is administered in erythrocyte ghosts.

10. 2. The method of claim 1, wherein the deficiency in GAMT activity is associated with a genetic deficiency in a gene encoding the enzyme guanidinoacetate methyltransferase in the subject.

11. 5. The method of claim 4, wherein the human arginase enzyme or human arginine deiminase enzyme is stabilized by association with a stabilizing agent.

12. 12. The method of claim 11, wherein the stabilizing agent is selected from the group consisting of polyethylene glycol (PEG), synthetic protein polymers, polysialic acid, Fc fusions and albumin.

13. The method of any one of claims 6 to 8, wherein the human Arginase I enzyme is PEGylated.

14. The method according to any one of claims 1 to 13, wherein the subject is a human.

15. The human Arginase I enzyme has a pH of 400 mM at pH 7.4 and 37° C. as measured in vitro. －１ s －１ ~4,000mM －１ s －１ k for the hydrolysis of arginine ｃａｔ / K Ｍ The method of claim 7, wherein:

16. 9. The method of claim 8, wherein the human Arginase I enzyme comprises cobalt in a ratio of 2 μg Co / mg Arginase to 3 μg Co / mg Arginase.

17. 9. The method of claim 8, wherein the human Arginase I enzyme is produced by contacting Arginase apoenzyme with cobalt or cobalt ions at a temperature between 30° C. and 55° C. for 15 minutes to 60 minutes.

18. A method for treating the effects of guanidinoacetic acid (GAA) toxicity in a subject having deficient guanidinoacetate methyltransferase (GAMT) activity, comprising administering to the subject a therapeutic amount of a pharmaceutical composition comprising a PEGylated human Arginase I enzyme with a cobalt cofactor.

19. 20. The method of claim 18, wherein administration is continued until the subject shows improvement in his or her physical or neurological condition.

20. 20. The method of claim 19, wherein the physical or neurological condition comprises at least one condition selected from the group consisting of global developmental delay / intellectual disability (DD / ID), epilepsy, movement disorders, speech or language delays, and behavioral disorders.

21. 20. The method of claim 18, wherein the therapeutic amount of said PEGylated human Arginase I enzyme with a cobalt cofactor is from about 0.01 mg / kg to about 7.5 mg / kg.

22. 20. The method of claim 18, wherein the therapeutic amount of said PEGylated human Arginase I enzyme with a cobalt cofactor is from about 0.05 mg / kg to about 5 mg / kg.

23. 20. The method of claim 18, wherein the therapeutic amount of said PEGylated human Arginase I enzyme with a cobalt cofactor is from about 0.1 mg / kg to about 5 mg / kg.

24. 20. The method of claim 18, wherein the pharmaceutical composition is administered to the subject parenterally.

25. 20. The method of claim 18, wherein the pharmaceutical composition is administered topically, intravenously, intradermally, intra-arterially, intraperitoneally, intralesionally, intracranially, intra-articularly, intraprostatically, intrapleurally, intratracheally, intraocularly, intranasally, intravitreally, intravaginally, intrarectally, intramuscularly, subcutaneously, subconjunctivally, intravesicularly, mucosally, intrapericardially, subumbilically, orally, by inhalation, injection, infusion, continuous infusion, by direct local perfusion of target cells, via a catheter, or via lavage solution.

26. 20. The method of claim 18, wherein the pharmaceutical composition is adapted for subcutaneous administration to the subject.

27. 27. The method of claim 26, wherein the pharmaceutical composition comprises a therapeutic dose of arginase in potassium phosphate, NaCl, sucrose and PS80 at pH 6.

7.

28. 20. A kit for use in the method of claim 18, comprising a syringe containing a solution comprising triacetin, sucrose, and the PEGylated human Arginase I enzyme adapted for subcutaneous administration.

29. 20. The method of claim 18, wherein the pharmaceutical composition is adapted for intravenous administration.

30. 30. The method of claim 29, wherein the pharmaceutical composition comprises the PEGylated human Arginase I enzyme, saline, and glycerol at pH 7.

4.

31. 27. The method of claim 26, wherein administration of the pharmaceutical composition reduces serum arginine in the patient by 50% to 99%.

32. 27. The method of claim 26, wherein administration of the pharmaceutical composition reduces serum arginine in the patient by 90% to 99%.

33. 27. The method of claim 26, wherein administration of the pharmaceutical composition reduces serum GAA in the patient by at least 25% to 50%.

34. 27. The method of claim 26, wherein the concentration of the pharmaceutical composition in plasma reaches a maximum level 20 to 28 hours after a single administration.

35. 27. The method of claim 26, wherein the concentration of the pharmaceutical composition in plasma reaches a maximum level about 24 hours after a single administration.

36. 1. A method for treating the effects of guanidinoacetic acid (GAA) toxicity, comprising administering to a subject in need thereof a therapeutic amount of a pharmaceutical composition comprising an arginine-depleting enzyme.

37. 37. The method of claim 36, wherein the arginine-depleting enzyme is an arginase or arginine deiminase enzyme.

38. 1. A method for treating guanidinoacetate methyltransferase (GAMT) activity deficiency in a subject, comprising administering to the subject a pharmaceutical composition comprising a therapeutic amount of an arginine-depleting enzyme in combination with ornithine supplementation.

39. 39. The method of claim 38, wherein the arginine-depleting enzyme is an arginase or arginine deiminase enzyme.

40. 39. The method of claim 38, wherein the pharmaceutical composition comprises high dose L-ornithine supplementation.

41. 39. The method of claim 38, comprising orally administering L-ornithine aspartate or L-ornithine hydrochloride.

42. A pharmaceutical composition comprising a therapeutic amount of an arginine-depleting enzyme for use as a medicament for use in treating a deficiency in guanidinoacetate methyltransferase (GAMT) activity in a subject, or in treating the effects of guanidinoacetate (GAA) toxicity in a subject.

43. A pharmaceutical composition comprising a therapeutic amount of an arginine-depleting enzyme for use in treating a deficiency in guanidinoacetate methyltransferase (GAMT) activity in a subject or for treating the effects of guanidinoacetate (GAA) toxicity in a subject.

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