RU2773220C2 - Arginine deiminase encapsulated in erythrocytes, and their use in treatment of cancer and arginase-1 deficiency - Google Patents

Abstract

FIELD: medicine; pharmaceutics.

SUBSTANCE: first invention is the use of a pharmaceutical composition including arginine deiminase encapsulated in erythrocytes, independent of a co-factor, and a pharmaceutically acceptable carrier for the treatment of arginase-1 deficiency. The second invention is the use of a suspension of erythrocytes, in which co-factor-independent arginine deiminase is encapsulated, in the treatment of arginase-1 deficiency for reduction in arginine levels in plasma or blood of a patient suffering from arginase-1 deficiency for at least 50, 60, 70 or 80% during at least 6, 7, 8, 9, 10 or 11 days after the administration. The third invention is a method for the treatment of arginase-1 deficiency, including the administration of a pharmaceutical composition including arginine deiminase encapsulated in erythrocytes and a pharmaceutically acceptable carrier. The fourth invention is the use of erythrocytes, in which arginine deiminase is encapsulated, for the production of a drug for the treatment of arginase-1 deficiency.

EFFECT: group of inventions provides the possibility of treatment of arginase-1 deficiency, leading to a reduction in toxic accumulation of arginine and side products of its metabolism.

34 cl, 7 dwg, 7 tbl, 10 ex

Description

This patent application claims the priority of the previously filed European Patent Application No. 17306122.7, filed August 31, 2017, and Japanese Patent Application No. 2017-254383, filed December 28, 2017.

The present invention relates to an arginine deiminase encapsulated in erythrocytes for use in therapy. In particular, it relates to their use in the treatment of arginase-1 deficiency. It also relates to novel pharmaceutical compositions comprising arginine deiminase from M. arginini encapsulated in erythrocytes and their use in the treatment of diseases in which arginine reduction may be beneficial, such as arginine dependent cancers, in particular arginine auxotrophic cancers, and arginase-1 deficiency.

Prerequisites for the creation of the invention

Arginine is a semi-essential amino acid. It is synthesized during the two-step urea cycle from citrulline through the action of arginosuccinate synthetase and arginosuccinate lyase. Arginine is metabolized to ornithine by arginase, and ornithine, in turn, can be converted to citrulline by a reaction catalyzed by ornithine transcarbamylase.

Some types of tumor cells have been shown to require a supply of arginine, leading to the suggestion that arginine reduction is a possible treatment for these forms of cancer, called arginine auxotrophic cancers. Arginine deiminase, an arginine-degrading enzyme, catalyzes the hydrolysis of arginine to citrulline and ammonia by deamination of the guanidine group. The antitumor activity of arginine deiminase has been the subject of numerous publications. Thus, the activity of arginine deiminase in vivo has been shown in relation to malignant melanoma and hepatocarcinoma. However, the enzyme arginine deiminase has some important drawbacks.

Arginine deiminase is not produced in mammals and is derived from microorganisms, making it a highly antigenic compound in mammals.

In addition, this enzyme has a very short mammalian half-life of about 5 hours and must be administered daily at a high dose in order to be effective.

In order to overcome such drawbacks for cancer treatment, European patent EP1874341 proposes an ingenious approach which consists in encapsulating arginine deiminase into erythrocytes. In EP1874341, especially in example 3, page 10, capture of arginine deiminase from Pseudomonas aeruginosa is disclosed.

Arginase-1 deficiency is a rare genetic disorder that affects the end stage of the liver urea cycle, which converts excreted nitrogen in the form of ammonia into urea for urinary excretion. It is caused by mutations in the ARG1 gene, leading to a partial or complete loss of the enzyme arginase 1, which catalyzes the hydrolysis of arginine to ornithine and urea (see figure 1).

Patients with arginase-1 deficiency show hyperargininemia, progressive neurological and mental impairment, persistent growth retardation, and rare episodes of hyperammonemia.

There are currently no treatments available for arginase-1 deficiency, and treatment outcomes are generally unsatisfactory with a protein-free diet and/or nitrogen scavenger drugs. The main biochemical feature is the accumulation of arginine leading to toxic levels of guanidine compounds and nitric oxide (Sin et al., PLoS ONE, 8(11)). Because arginine and its metabolites are expected to cause neurological-type arginase-1 deficiency, a common treatment goal has been to reduce plasma arginine with a protein-free diet, amino acid supplementation, and administration of nitrogen scavengers. However, with this treatment strategy, normalization or near-normalization of plasma arginine in arginase-1 deficiency is questionable.

Normalization of plasma arginine levels in arginase-1 deficiency is therefore questionable, and arginase enzyme therapy has previously been investigated. For example, Burrage et al., investigated a modified pegylated recombinant human enzyme arginase in mouse models of arginase deficiency (Burrage et al., Human Molecular Genetics, 2015, 24(22)). Although this enzyme resulted in a decrease in plasma arginine levels, it did not improve survival in arginase-deficient mice, probably due to persistence of hyperammonemia. Hyperammonemia is a major challenge in the treatment of arginase deficiency (Burrage et al., Human Molecular Genetics, 24(22)).

Therefore, finding alternative pathways to reduce plasma arginine levels in arginase deficiency has been a major goal for many years.

The reaction catalyzed by arginine deiminase is

This is an equilibrium reaction and there are scientific arguments in the scientific community suggesting that the use of arginine deiminase to treat arginase-1 deficiency is not possible because

- this enzyme is of bacterial origin and can cause allergic reactions;

- the reaction products catalyzed by arginine deiminase are citrulline and ammonia;

- citrulline can be converted back to arginine (see urea cycle in figure 1), and

- Ammonia can be a problem in the context of arginase-1 deficiency when patients have episodes of hyperammonemia.

Against these technical prejudices, Applicant of the present invention surprisingly discovered and showed that arginine deiminase, encapsulated in erythrocytes, may be a novel approach to reduce the toxic accumulation of arginine and its metabolic by-products. The Applicant has found in particular that the reduction in serum arginine levels in mice achieved by administering the composition of the present invention is significant and therefore of therapeutic interest. The inventor has further shown that this reduction in serum arginine levels in arginase-1 deficient mice is not accompanied by significant reductions in ammonia levels. These results are particularly surprising due to the existing technical prejudice that arginine deiminase cannot contribute to the treatment of arginase-1 deficiency due to the production of NH ₃ and possible reverse conversion to citrulline.

Among the various arginine deiminases that can be encapsulated in erythrocytes, Applicant has identified a particular arginine deiminase that has properties that lead to an unexpected improvement in PK/PD parameters.

In particular, arginine deiminase, derived from the bacteria Mycoplasma arginini, improves PK/PD parameters. In addition, said arginine deiminase from Mycoplasma arginini does not require any cofactor, which is a desirable property for its activity in the erythrocyte. Therefore, arginine deiminase from Mycoplasma arginini has technical advantages over other known arginine deiminase and is therefore highly suitable for use in the context of the present invention. In addition, arginine deiminase from Mycoplasma arginini is a homodimer with a molecular weight of 92 kD and is thus suitable for incorporation into erythrocytes.

Thus, the use of said arginine deiminase will be an advance in therapies for reducing arginine, such as anticancer therapy, in particular against arginine auxotrophic cancers, and therapy against arginase-1 deficiency.

The essence of the invention

Thus, it is an object of the invention to provide a pharmaceutical composition comprising arginine deiminase encapsulated in erythrocytes for use in the treatment of arginase-1 deficiency.

The invention relates in particular to a pharmaceutical composition comprising arginine deiminase encapsulated in erythrocytes and a pharmaceutically acceptable carrier for use in the treatment of arginase-1 deficiency.

In one preferred embodiment, the arginine deiminase encapsulated in erythrocytes is M. arginini arginine deiminase.

Another object of the invention is a suspension of erythrocytes encapsulating arginine deiminase from M. arginini. Said suspension is in particular a suspension of erythrocytes encapsulating arginine deiminase from M. arginini in a pharmaceutically acceptable carrier.

Another object of the invention is a pharmaceutical composition comprising a suspension according to the invention, for its use in the treatment of arginase-1 deficiency or arginine-dependent cancers, the treatment or prevention of septic shock, the inhibition of angiogenesis and the treatment of angiogenesis-associated diseases, in particular for its use in the treatment of arginase-1 deficiency arginase-1 or arginine-dependent cancers.

Another object of the invention is a method of treating arginase-1 deficiency, comprising administering to a patient in need thereof an effective amount of a pharmaceutical composition of the invention or administering to a patient in need thereof an effective amount of arginine deiminase encapsulated in erythrocytes.

Another object of the invention is a method of treating arginase-1 deficiency or arginine-dependent cancers, treating or preventing septic shock, inhibiting angiogenesis, or treating angiogenesis-associated diseases, comprising administering to a patient in need thereof an effective amount of a pharmaceutical composition comprising arginine deiminase from M. arginini , or an effective amount of arginine deiminase from M. arginini encapsulated in erythrocytes.

Detailed description

Arginine deiminase

L-цитруллин + NH₃. Соответственно, в контексте настоящего изобретения аргининдеиминаза, используемая в контексте изобретения, также может упоминаться как «фермент» или «ADI»."Arginine deiminase", also called "ADI", is an enzyme that catalyzes the chemical reaction L-arginine + H ₂ O

L-citrulline + NH ₃ . Accordingly, in the context of the present invention, the arginine deiminase used in the context of the invention may also be referred to as "enzyme" or "ADI".

The two substrates of this enzyme are L-arginine and H ₂ O, while its two products are L-citrulline and NH ₃ . Arginine deiminase belongs to the hydrolase family and is listed under EC 3.5.3.6 in the IUBMB enzyme nomenclature. ADI can be derived from various microorganisms such as Bacillus pyocyaneus, Pseudomonas putida, Halobacterium salinarium, Mycoplasma arginini, Mycoplasma hominis, Pseudomonas aeruginosa, Lactobacillus lactis ssp. Lactis and Pseudomonas plecoglossicida (see Rui-Zhi Han et al., Appl. Microbiol. Biotechnol., 2016, 100: 4747-4760).

In one embodiment, the arginine deiminase is a protein.

As used herein, the term "protein" includes single chain peptide molecules as well as polypeptide complexes in which individual constituent polypeptides are linked in covalent and non-covalent ways.

As used herein, the terms "polypeptide" and "peptide" refer to a polymer in which the monomers are amino acids linked together via peptide or disulfide bonds. The terms "unit" and "domain" may also refer to polypeptides and peptides having a biological function.

The arginine deiminase used in the context of the present invention may be of natural, synthetic or artificial origin, or obtained by genetic engineering (for example, by a method for obtaining an enzyme in a host cell, for example, E. coli, after integration of a vector expressing a gene encoding an enzyme), and a person skilled in the art those skilled in the art may refer to, for example, S. Misawa et al., J. of Biotechno., 36, 1994, 145-155, for a description of the production of ADI from M. arginini in E. coli. Arginine deiminases that can be used are described, for example, in EP-A-1 011 717, EP-A-0 414 007, US-A-5 372 942, JP-A-6062867, JP-A-2053490, JP-A -2035081. Equally, the invention includes the use of analogues of this enzyme, such as variants and fragments, which can be, to a significant extent, enzymes that are modified in order to enhance their enzymatic activity, as described, for example, in EP-A-0 981 607.

The present invention preferably uses the improved ADI for use in any disease that would benefit from a reduction in plasma arginine, including, for example, arginine-dependent (autotoxotrophic) cancers and arginase deficiency.

Accordingly, in one preferred embodiment, the arginine deiminase used in the context of the present invention is arginine deiminase from Mycoplasma arginini. Arginine deiminase from Mycoplasma arginini is a homodimer with a molecular weight of 92 kD. The relatively small size of said arginine deiminase, compared to other arginine deiminases, is an advantage for encapsulation in erythrocytes.

In one embodiment, the arginine deiminase from Mycoplasma arginini comprises the amino acid sequence of SEQ ID NO: 1, or variants or fragments thereof. The amino acid sequence of SEQ ID NO: 1 corresponds to the amino acid sequence of arginine deiminase from Mycoplasma arginini, which is available from the GenBank database under NCBI identification number WP_004416214.1, and has been available since August 22, 2017.

As used herein, references to amino acid sequences (also referred to as polypeptides) include both specific amino acid sequences and variants of said sequences.

"Protein variants" may be naturally occurring variants such as splice variants, alleles and isoforms, or they may be produced by recombinant methods. Changes in the amino acid sequence can be made by substitution, deletion or insertion of one or more codons in the nucleotide sequence encoding the protein, resulting in a change in the amino acid sequence of the protein. Optionally, the change is by replacing 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or more amino acids with any other amino acid in protein. In addition, or on the other hand, the change may be by adding or deleting 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18 , 19, 20 or more amino acids in a protein.

"Fragments" of proteins are also covered by the invention. Such fragments may be truncated at the N-terminus or C-terminus, or may be devoid of internal residues, for example when compared to a full-length protein. Some fragments lack internal residues that are not essential for enzymatic activity. Preferably, said fragments are at least about 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 150, 250, 300, 350, 380, 400 or more amino acids in length.

Protein variants may include proteins that have at least about 80% amino acid sequence identity with the polypeptide sequence disclosed herein. Preferably the protein variant will be at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93% , 94%, 95%, 96%, 97%, 98%, 99% amino acid sequence identity with the polypeptide sequence disclosed in the present description. Amino acid sequence identity is defined as the percentage of amino acid residues in a variant sequence that are identical to amino acid residues in a reference sequence, after sequence alignment and gapping as necessary to achieve maximum percent sequence identity, and without considering any conservative substitutions as part of sequence identity. Sequence identity can be determined over the entire length of both the variant and the reference amino acid sequences.

The person skilled in the art should be aware that several nucleic acids can encode the same polypeptide as a result of the degeneracy of the genetic code. In addition, it should be appreciated that those skilled in the art can, using routine techniques, make nucleotide substitutions that do not affect the sequence of the polypeptide encoded by the nucleic acids of the invention to reflect the codon usage of any particular host organism that should polypeptides are expressed. Nucleic acids encoding ADI used in the context of the invention can be modified in any way available in the art. Nucleic acids encoding the ADI used in the context of the invention can be obtained recombinantly, synthetically, or by any other means available to those skilled in the art. They can also be cloned by standard methods such as PCR (polymerase chain reaction).

With a polypeptide having an amino acid sequence of at least, for example, 95% "identical" to a certain amino acid sequence of the present invention, the amino acid sequence of the subject polypeptide is assumed to be identical to the elucidated sequence, except that the sequence of the subject polypeptide may include up to five amino acid changes for every 100 amino acids of the elucidated amino acid sequence. In other words, in order to produce a polypeptide having an amino acid sequence that is at least 95% identical to the elucidated amino acid sequence, up to 5% (5 out of 100) of the amino acid residues in the sequence in question may be inserted, deleted, or replaced with a different amino acid.

In the context of the present application, percent identity is calculated using a global alignment (i.e., two sequences are compared completely over their entire length). Methods for comparing the identity of two or more sequences are known in the art. For example, you can use the "needle" program, which uses the Nudelman-Wunsch global alignment algorithm (Needleman and Wunsch, 1970, J. Mol. Biol., 48: 443-453) to find the optimal alignment (including gaps) of two sequences when considering their full length. The needle program is available for example on the ebi.ac.uk world wide website. The percent identity according to the invention is preferably calculated using the (global) program EMBOSS::needle with a "Gap Open" parameter of 10.0, a "Gap Extend" parameter of 0.5 and a Blosum62 matrix.

Proteins consisting of an amino acid sequence "at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical" to the reference sequence may include mutations such as deletions, insertions and/ or substitutions compared to the reference sequence. In the case of deletions, insertions and/or substitutions, a protein consisting of an amino acid sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to the reference sequence may correspond to the homologous sequence derived from a species other than the reference sequence.

Amino acid substitutions can be conservative or non-conservative. Preferably, the substitutions are conservative substitutions in which one amino acid is substituted for another amino acid with similar structural and/or chemical properties. The substitution preferably corresponds to a conservative substitution as specified herein below in Table 1.

Table 1. Conservative amino acid substitution

Conservative substitutions Amino acid type Ala, Val, Leu, lle, Met, Pro, Phe, Trp Amino acids with aliphatic hydrophobic side chains Ser, Tyr, Asn, Gln, Cys Amino acids with uncharged but polar side chains Asp, Glu Amino acids with acidic side chains Lys, Arg, His Amino acids with basic side chains gly Neutral side chain

Preferably, said variants or fragments retain the biological activity of a protein having the full length amino acid sequence of SEQ ID NO: 1.

In some embodiments, the arginideiminase used in the context of the invention may be a variant or fragment having increased biological activity or enzymatic activity compared to a protein having the full amino acid sequence of SEQ ID NO: 1. Accordingly, said variant or fragment may include amino acid substitutions, which improve its enzymatic activity. "Improved enzymatic activity" as used herein refers to, for example, improved optimum pH, improved Km and kcat values, and improved heat stability. Such mutations are either known to those skilled in the art or can be easily obtained on the basis of general knowledge, as disclosed, for example, in Rui-Zhi Han et al. (Appl. Microbiol. Biotechnol., 2016, 100: 4747-4760).

In one embodiment, the arginideiminase variant or fragment may be modified to increase the in vivo half-life.

Pharmaceutical compositions or suspensions

The terms "pharmaceutical composition" or "therapeutic composition" as used herein refer to a compound or composition capable of producing a desired therapeutic effect when properly administered to a patient.

The invention relates to a pharmaceutical composition and suspension, both of which include arginideiminase encapsulated in erythrocytes. The definitions given in the present description refer to compositions and suspensions according to the invention. The ADI encapsulating erythrocytes are preferably in suspension in a pharmaceutically acceptable carrier.

The terms "pharmaceutically" or "pharmaceutically acceptable" refer to molecules and compositions that do not produce an adverse, allergic or other adverse reaction when administered to a mammal, especially a human, as appropriate.

According to a separate embodiment, the "pharmaceutically acceptable carrier" is a "preserving solution" or "preserving solution for erythrocytes", i. a solution in which the erythrocytes encapsulating the enzyme are suspended in their suitable storage form pending their use for injection. The preservative solution preferably comprises at least one erythrocyte preservation promoting agent, in particular selected from glucose, dextrose, adenine and mannitol.

In one embodiment, the preservative solution is an aqueous solution comprising NaCl and/or adenine. In another embodiment, the preservative solution further comprises at least one compound selected from the group consisting of glucose, dextrose and mannitol. As an example, it may include NaCl, adenine and dextrose. As an example, the preservation solution is AS3 medium. As another example, it may include NaCl, adenine, glucose and mannitol. In another example, the preservation solution is a SAGM medium, such as SAG-mannitol, or an ADsol medium.

The precise form of the pharmaceutical compositions, route of administration, dosage and schedule of administration will naturally depend on the condition being treated, the severity of the disease, the age, weight and sex of the patient, and so on. It should also be borne in mind that the composition and suspensions according to the invention are intended for intravenous or intra-arterial administration, preferably for injection, infusion or perfusion. Accordingly, the pharmaceutical composition of the invention is formulated for intravenous or intra-arterial administration.

The compositions of the invention may or may not be ready-to-use. The ready-to-use pharmaceutical composition of the invention has a hematocrit suitable for administration without dilution. When the composition is not ready for use, it can also be packaged at a higher hematocrit so that it must be diluted prior to administration.

"Hematocrit (Ht or HCT)" is generally known as the volume percentage (vol.%) of red blood cells in the blood. In the context of the present invention, hematocrit can also refer to the volume percentage (vol.%) of red blood cells in a composition or suspension, also referred to as a pharmaceutical composition or red blood cell suspension.

According to the invention, the hematocrit of the ready-to-use pharmaceutical composition is preferably between 40 and 70%, preferably between 45 and 65%, more preferably between 45 and 55%, for example 46 and 54%, 47 and 53%, 48 and 52%, for example, is approximately 50%. When the composition is not ready for use, the hematocrit of the pharmaceutical composition for dilution may be higher, in particular between 50 and 90%, preferably 60 and 90%. In a related embodiment, the dilution of the pharmaceutical composition to obtain hematocrit values of the ready-to-use composition as defined herein above may be carried out with a pharmaceutically acceptable carrier.

"RBCs" are also called red blood cells. Red blood cells are produced in the bone marrow and circulate in the body for about 100-120 days before their components are recycled by macrophages. Almost half of the blood volume (40% - 45%) are red blood cells. Methods for isolating red blood cells are known to those skilled in the art.

In one embodiment, the red blood cells are isolated from the blood of a mammal of the same species as the subject or patient being treated. When the mammal is a human, the erythrocytes are preferably human-derived. In one embodiment, the erythrocytes are taken from the patient himself.

In another embodiment, stem cells are used to generate red blood cells. The skilled person may refer to Russeau et al. (ISBT Science Series (2016), 11 (Suppl. 1), 111–117). Stem cells can be further transformed to express the enzyme in erythrocytes. Those skilled in the art may also refer to US2015/0182588. These documents are incorporated herein by reference.

The arginine deiminase used in the context of the invention is highly active. Activity can be expressed, for example, in E. "Unit (E)" refers to the amount of enzyme that converts 1 µmol of substrate per minute. Accordingly, in the context of the present invention, 1 E refers to the amount of enzyme that converts 1 μmol of arginine per minute, preferably at 37°C. More preferably, 1 E refers to arginine deiminase activity when it is present in buffers commonly used to measure arginine deiminase activity, such as those described in said document (Boyde and Rahmatullah, 1980, Analytical Biochemistry, vol. 107, p. 424-431). In some embodiments, the "unit" as defined herein above may also be referred to as "international unit (IU)((IU))". Accordingly, in some embodiments, 1 E and 1 IU are interchangeable.

Methods for quantitative and/or qualitative assessment of arginine deiminase activity are known in the art. Typically, arginine deiminase activity is measured as detailed herein below in the experimental section, in particular in Example 1 and 2 in the experimental section. The exact experimental conditions used in measuring arginine deiminase activity are known to those skilled in the art and are described, for example, in (Boyde and Rahmatullah, 1980, Analytical Biochemistry, vol. 107, p. 424-431). In one embodiment, the arginine deiminase used in the context of the invention has, prior to encapsulation, a specific activity of 10-100 U/mg, such as 20-80, 30-70, 40-60 U/mg, where mg refers to the amount of purified enzyme.

The term "purified enzyme" in the present description refers to an enzyme having a purity of 90-100%, such as 92-100%, 94-100%, more than 96%, more than 97%, more than 98%, more than 99%, for example, 97 %. Methods for determining the purity of an enzyme are known to those skilled in the art and include, for example, SDS gel analysis or mass spectrometric analysis, preferably SDS gel analysis.

"Arginine deiminase is encapsulated in erythrocytes" means that arginine deiminase is contained within erythrocytes.

In one embodiment, the concentration of encapsulated arginine deiminase in the context of the present invention, also referred to as the concentration of arginine deiminase in erythrocytes, is from 0.1 to 7 mg/ml, preferably 0.5-6.5 mg/ml, for example, 1-6 mg/ml, 1 -5 mg/ml, 1-4 mg/ml, 1-3 mg/ml, preferably 1.2-2.8 mg/ml, 1.4-2.6 mg/ml, 1.6-2.4 mg/ml.

In some embodiments, the concentration of encapsulated arginine deiminase is at least 0.1, 0.2, 0.4, 0.6, 0.8, 1, 1.2, 1.4, 1.6, 1.8, 2, 2.2, 2.4, 2.6, 2.8, 3, 3.5, 4, 4.5, 6 mg/ml. In some embodiments, the concentration of encapsulated arginine deiminase is at most 7, 6.5, 5, 5.5, 5, 4.5, 4, 3.5, 3, 2.5, 2 mg/mL.

In another embodiment, the concentration of encapsulated arginine deiminase is from 1 to 1000 U/ml, preferably 1-500 U/ml, eg 1-400 U/ml, 5-400 U/ml, 5-350 U/ml.

In some embodiments, the concentration of encapsulated arginine deiminase is at least 0.5, 1, 2, 3, 4, 5, 10, 20, 30, 40, 50, 100, 200 to 400 U/mL. In some embodiments, the concentration of encapsulated arginine deiminase is at most 1000, 800, 600, 500, 400, 350, 300, 200, 100 U/mL.

Preferably, the concentration of encapsulated arginine deiminase, in mg/ml or in U/ml, refers to the concentration of arginine deiminase in erythrocytes per ml of the pharmaceutical composition according to the invention or suspension according to the invention, more preferably refers to the concentration of arginine deiminase in erythrocytes per ml of erythrocyte solution of the composition or suspension, and the specified the solution typically has a hematocrit of 50%.

In one embodiment, the composition or suspension of the invention in storage solution has an extracellular hemoglobin level maintained at or below 0.5, in particular 0.3, especially 0.2, preferably 0.15, even better 0.1 g /dl for 72 hours and storage at a temperature of 2 to 8°C.

In particular, the composition or suspension of the invention in storage solution is characterized by an extracellular hemoglobin level maintained at or below 0.5, in particular 0.3, especially 0.2, preferably 0.15, even better 0.1 g /dl during the period concluded between 24 hours and 20 days, in particular between 24 and 72 hours, and storage at a temperature of from 2 to 8°C.

"Extracellular hemoglobin level" is preferably measured by the manual method described by G. B. Blakney and A. J. Dinwoodie, Clin. Biochem., 8, 96-102, 1975. There are also automatic devices that allow such a measurement to be made with sensitivity specific to them.

In particular, the composition or suspension of the invention in a preservative solution is characterized by a hemolysis rate maintained equal to or less than 2, especially 1.5, preferably 1% per 72 hours, and maintained at a temperature of 2 to 8°C.

In particular, the composition or suspension according to the invention in a preservative solution is characterized by a hemolysis rate maintained equal to or less than 2, especially 1.5, preferably 1% over a period of 24 hours to 20 days, especially between 24 and 72 hours, and at a temperature of 2 to 8°C.

In one embodiment, the composition of the invention is a suspension. In one embodiment, the suspension according to the invention and the suspension in the context of the invention have an osmolarity of 270 to 350 mosm/l. Methods for measuring osmolarity are known to those skilled in the art. In one example, osmolarity is measured with an osmometer (Loser Micro Osmometer Type 15). Osmolarity is preferably measured under standard conditions (ie at 25° C. and 1 atm).

Therapeutic Methods and Applications

The present inventors have shown that a pharmaceutical composition comprising arginine deiminase encapsulated in erythrocytes reduces blood levels of L-arginine in arginase deficient mice by 73% three days after administration, for example when administered at a dose of 8 ml/kg. Those skilled in the art should be aware that these results indicate that the composition of the invention, as well as the suspension of the invention, can be used to treat arginase deficiency, since elevated serum arginine levels are directly related to the clinical condition of the subject or patient being treated.

The inventors have also shown in mice that, surprisingly, administration of a composition or suspension of the invention does not cause marked changes in ammonia levels in treated mice, and thus potentially does not cause possible episodes of hyperammonemia in the subject or patient being treated.

Accordingly, the present invention relates to a pharmaceutical composition comprising an erythrocyte-encapsulated arginine deiminase and a pharmaceutically acceptable carrier for use in the treatment of arginase-1 deficiency. The invention also relates to a suspension of erythrocytes encapsulating arginine deiminase, in particular arginine deiminase from M. arginini, for use in the treatment of arginase-1 deficiency.

"Arginase-1 deficiency" is an inherited metabolic disease in which the body is unable to process arginine (a building block of protein). It belongs to a group of disorders known as urea cycle disorders. They occur when the body's removal of ammonia is disrupted, which can cause elevated levels of ammonia in the blood (hyperammonemia). In most cases, symptoms appear between one and three years of age. Symptoms may include eating problems, vomiting, poor growth, epileptic seizures, and muscle stiffness with involuntary movements (spasticity). Individuals with arginase-1 deficiency may also have growth retardation, developmental loss, and mental retardation. Arginase-1 deficiency is caused by mutations in the ARG1 gene and is inherited as an autosomal recessive disorder. Most people with arignase-1 deficiency appear healthy at birth and develop normally during infancy. The first signs of arginase-1 deficiency usually appear between one and three years of age. In some cases, symptoms appear earlier or later.

Arginase-1 deficiency may be characterized by one or more signs and symptoms selected from a list including poor growth (present in all people who have arginase deficiency), stiff muscles and increased involuntary movements (spasticity), developmental delay, loss of previously acquired milestones mental retardation, seizures, small head size (microcephaly), balance and coordination problems, behavioral abnormality, diaminoaciduria, mental retardation (severe), neurological speech disorder, abnormality in the EEQ (electroencephalogram), hemiplegia/hemiparesis, hyperammonemia, progressive spastic quadriplegia and seizures.

"Methods for diagnosing arginase-1 deficiency" are known to those skilled in the art and typically include full expanded neonatal screening testing. Typically, a three to four-fold increase in plasma arginine concentrations above the upper limit of normal is highly suggestive of this diagnosis. Diagnosis is further typically confirmed by identification of pathogenic variants of the biallelic ARG1 gene on molecular genetic testing or in the absence of detection of arginase enzyme activity (usually <1% of normal) in red blood cell extracts.

The invention also relates to a method for treating arginase-1 deficiency comprising administering to a patient in need thereof an effective amount of arginine deiminase encapsulated in erythrocytes or an effective amount of a composition of the invention.

In the context of the invention, the term "treatment" as used herein means reversing, alleviating, inhibiting the development or prevention of a disorder or condition to which such a term is applied, or one or more symptoms of such a disorder or condition.

Accordingly, the term "treatment" includes not only treatment leading to a complete cure of a disease, but also treatment showing a reduction in the development of the disease and/or prolongation of the life of the patient.

"Effective amount" means an amount effective, in dosages and for the necessary periods of time, to achieve the desired therapeutic or prophylactic result.

A therapeutically effective amount of a composition or suspension of the invention to produce the desired therapeutic result may vary according to such factors as disease state, age, sex and weight of the individual, and protein capacity. A therapeutically effective amount encompasses an amount in which any toxic or deleterious effects of the inhibitor are outweighed by the therapeutically beneficial effects. A therapeutically effective amount also encompasses an amount sufficient to provide a beneficial effect, such as a clinical benefit.

In some embodiments, the methods of the invention comprise administering a composition of the invention described above, either sequentially or simultaneously in combination with at least one additional treatment that is directed against arginase-1 deficiency.

As used herein, one such additional treatment against arginase-1 deficiency refers to a protein-free diet, administration of nitrogen scavengers, injections of botulinum toxin, or administration of amino acids such as ornithine or lysine.

"Nitrogen scavengers" include, but are not limited to, benzoate, phenylbutyrate, and phenylacetate.

As shown in the present description above, the present inventors have shown that M. arginini arginine deiminase, encapsulated in erythrocytes, reduces blood levels of L-arginine in arginase-1 deficient mice by up to 73% three days after administration, for example, when administered in dose of 8 ml/kg.

Accordingly, those skilled in the art should be aware that a pharmaceutical composition comprising a suspension of erythrocytes encapsulating M. arginini arginine deiminase is effective not only in the treatment of arginase-1 deficiency, but also in the treatment of other diseases in which a reduction in concentration of arginine in the blood.

Accordingly, the invention finds particularly interesting application in argigin dependent cancers, in which the beneficial effect of reducing plasma arginine has been shown (see, for example, F. Izzo et al., 2004, C. M. Ensor et al., 2002, F. W. Holtsberg et al. , 2002, J.S. Bomalaski et al., 2003, and Curley S.A. et al., 2003, cited previously).

However, a decrease in plasma arginine may also be beneficial in the treatment or prevention of septic shock, inhibition of angiogenesis, and associated diseases.

Accordingly, in one embodiment, the present invention relates to a pharmaceutical composition comprising a suspension of erythrocytes encapsulating arginine deiminase from M. arginini, for use in reducing the concentration of arginine in the blood.

Accordingly, in a related embodiment, the present invention relates to a pharmaceutical composition comprising erythrocyte-encapsulated M. arginini arginine deiminase and a pharmaceutically acceptable carrier, for use in reducing blood arginine concentrations.

Definitions relating to "pharmaceutical compositions", "arginideiminase", "suspension", "pharmaceutically acceptable carrier" are given in the present description above and apply mutatis mutandis.

In particular, the reduction of blood arginine concentration is useful in the treatment of arginase-1 deficiency, the treatment of arginine-dependent cancers, the treatment or prevention of septic shock, the inhibition of angiogenesis, and the treatment of associated diseases.

Accordingly, in one embodiment, the invention relates to a pharmaceutical composition comprising a suspension of erythrocytes encapsulating M. arginini arginine deiminase for use in the treatment of arginase-1 deficiency, the treatment of arginine-dependent cancers, the treatment or prevention of septic shock, the inhibition of angiogenesis, and the treatment of associated diseases. .

Accordingly, in a related embodiment, the present invention relates to a pharmaceutical composition comprising an erythrocyte-encapsulated arginine deiminase from M. arginini and a pharmaceutically acceptable carrier, for its use in the treatment of arginase-1 deficiency, the treatment of arginine-dependent cancers, the treatment or prevention of septic shock, for the inhibition of angiogenesis and treatment of associated diseases.

The definition of "deficiency of arginase-1" is given in the present description above.

The term "arginine-dependent cancers" refers to cancers involving cancer cells that require arginine for replication, such tumors are unable to synthesize some or all of the arginine they require, mainly due to the loss of the biosynthetic enzyme asparagine synthase, and therefore require a supply of arginine , therefore, such cancers are also referred to as "arginine auxotrophic cancers". A decrease in plasma arginine will deprive these cells of arginine, which is essential for their development, leading to targeted death of these cells, inhibition of tumor growth, or regression of tumor mass.

Arginine-dependent cancers may be selected from the group including hepatocarcinoma, primary liver cancer, melanoma, breast cancer, neuroblastoma, leukemia, mesothelial cancer, urological cancer, sarcoma, gastric cancer, and brain cancer.

"Mesothelial cancer" refers to, for example, malignant multiple mesothelioma (MRM).

"Urological cancer" includes, for example, renal cell carcinoma, prostate cancer, colon cancer, and bladder cancer, in particular transitional cell carcinoma.

"Sarcoma" means, for example, osteosarcoma.

"Cancer of the brain" means, for example, astrocytoma, oligodendroglioma.

In one aspect, the arginine dependent cancer is a hepatocarcinoma or primary liver cancer. In another aspect, the arginine dependent cancer is melanoma, in particular malignant melanoma, in its various forms such as superficially spreading melanoma and nodular melanoma. According to another aspect, arginine dependent cancer is one of the following forms of cancer:

- mammary cancer,

- neuroblastoma (Gong H. et al., Int. J. Cancer, 2003, 106: 723-8),

- leukemia (Gong H. et al., Leukemia, 2000, Vol. 14, 826-9; Noh E. J. et al., Int. J. Cancer, 2004, 112: 502-8).

"Treatment" is defined herein above.

"Prevention" means prophylactic use (ie, for an individual susceptible to the development of a given disease).

The expression "inhibition of angiogenesis and treatment of associated diseases" refers to the treatment of diseases such as angioma, angiofibroma, arthritis, diabetic retinopathy, retinopathy of prematurity, neovascular glaucoma, corneal disease, involutional and other forms of macular degeneration, pterygium, retinal degeneration, retrolental fibroplasia, psoriasis , telangiectasia, pyogenic granuloma, seborrheic dermatitis, acne, cancer and angiogenesis-related metastases (WO 0209741; Park I.S. et al., Br. J. Cancer, 2003, 89: 907-14).

Another object of the invention is the use of ADI-encapsulating erythrocytes or a suspension of such erythrocytes for the preparation of a medicament for the treatment of the diseases described herein.

The application takes into account the characteristics presented for suspensions and pharmaceutical compositions according to the invention or medicinal product.

Another object of the invention is a method of treating arginase-1 deficiency, comprising administering to a patient in need thereof an effective amount of a pharmaceutical composition of the invention, or administering to a patient in need thereof an effective amount of arginine deiminase encapsulated in erythrocytes, or administering to a patient in need thereof, an effective amount of the drug according to the invention.

Another object of the invention is a method of treating arginase-1 deficiency or arginine-dependent cancers, treating or preventing septic shock, inhibiting angiogenesis, or treating angiogenesis-associated diseases, comprising administering to a patient in need thereof an effective amount of a pharmaceutical composition comprising arginine deiminase from M. arginini , or an effective amount of M. arginini arginine deiminase encapsulated in erythrocytes, or administering to a patient in need thereof an effective amount of a drug according to the invention.

The method of treatment includes administering to a patient in need thereof a "therapeutically effective dose", "therapeutically effective amount", or "effective amount", the effective amount being defined herein above.

In particular, a "therapeutically effective dose", "therapeutically effective amount" or "effective amount" can be determined by one of ordinary skill in the art quite simply. The amount of enzyme actually administered may be determined by the attending physician, taking into account relevant circumstances, including the condition being treated, the chosen route of administration, the compound actually administered, the age, weight and response of the individual patient, the severity of the patient's symptoms, and so on. Effective amounts can typically be consistent with maintaining arginine below a threshold level for a sufficient amount of time.

In some examples, the arginine level may be maintained below a threshold level for at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 days, preferably for at least 3 days.

Typical effective amounts of the enzyme are described in more detail in the present description below in the "Doses" section.

The pharmaceutical compositions may be administered by intravenous or intra-arterial injection, and preferably by injection, infusion or perfusion from a blood bag or the like. The introduction is typically performed intravenously in the upper arm or through a venous catheter.

In particular, from about 10 to about 250 ml of suspension (single dose), pharmaceutical composition or drug according to the invention is administered. Above 20 ml, infusion or perfusion is preferred.

Treatment includes the administration of a single dose or multiple doses according to an accepted protocol. It may be given at monthly, biweekly or weekly intervals, preferably once, twice or three times a week, for the duration of the recommended treatment.

In one embodiment, the treatment in the context of the invention may be to administer one dose each time (each dose), as defined herein below in the "Doses" section.

In one example, treatment in the context of the invention may be to administer the equivalent of 0.001 mg/kg of enzyme per kg of body weight - 1000 mg/kg mg/kg of enzyme per kg of body weight, preferably 0.01 mg/kg of enzyme per kg of body weight - 500 mg /kg of enzyme per kg of body weight, preferably each time (each dose). Preferably, from 0.01 mg/kg of enzyme per kg of body weight to 200 mg/kg of enzyme per kg of body weight is administered, preferably each time (each dose).

In another example, also described in the present description below in the "Doses" section, treatment in the context of the invention may be the introduction of the equivalent of 10 U/kg of enzyme per kg of body weight and 100,000 U/kg mg/kg of enzyme per kg of body weight each time ( each dose), preferably from 10 U/kg of enzyme per kg of body weight to 15000 U/kg of enzyme per kg of body weight each time (each dose), preferably from 500 U/kg of enzyme per kg of body weight to 3500 U/kg of enzyme per kg of body weight each time (each dose).

Doses

Compositions of the invention are preferably packaged or provided in a volume containing a predetermined amount of active material calculated to produce the desired therapeutic effect. Those skilled in the art will appreciate that the amount or dose of composition administered depends, for example, on the condition of the mammal for whom administration is intended (e.g., weight, age, sex, and health status, concurrent treatment, if any, frequency of treatment) , type of administration and type of preparation.

In the context of the present invention, the volume in which the composition for administration is packaged also refers to the dose. In one embodiment, the specified volume is from 10 to 250 ml.

Accordingly, the composition for administration is preferably packaged or provided in a single dose; preferably said dose has the volume defined in the present description above. The packaging is preferably carried out in a container such as, for example, a blood bag of a type suitable for blood transfusion and the like. Preferably, said container contains the entire amount of the encapsulated enzyme as prescribed by the physician. In other words, one dose of erythrocytes encapsulating a given amount of enzyme may be present in one container or pharmaceutical composition. In one embodiment, this single dose is intended to be administered in its entirety to a patient in need thereof. In another embodiment, administration may be achieved in a single dose or multiple doses.

The amount of encapsulated enzyme in a single dose can range from 0.001 mg/kg of the patient's body weight to 1000 mg/kg of the patient's body weight. More preferably, the amount of encapsulated enzyme in a single dose is from 0.01 mg/kg of the patient's body weight to 1000 mg/kg of the patient's body weight. Most preferably, the amount of encapsulated enzyme in a single dose is from 0.01 mg/kg of the patient's body weight to 500 mg/kg of the patient's body weight. Most preferably, the amount of encapsulated enzyme in a single dose is from 0.01 mg/kg of patient's body weight to 200 mg/kg of patient's body weight, or from 0.01 mg/kg of patient's body weight to 100 mg/kg of patient's body weight.

In some embodiments, the amount of encapsulated enzyme in a single dose is at least 0.001, 0.005, 0.01, 0.05, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0 .7, 0.8, 0.9, 1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5, 6, 7, 8, 9, 10, 15, 20 , 25, 30, 35, 40, 45, 50 mg/kg of the patient's body weight. In some embodiments, the amount of encapsulated enzyme in a single dose is at most 200, 150, 100, 80, 70, 60, 50, 45, 40, 35, 30, 25, 20, 25, 15, 10, 5, 1, 0, 5, 0.1 mg/kg patient body weight.

The amount of encapsulated enzyme in a single dose can also range from 10 U/kg patient body weight to 100,000 U/kg patient body weight. More preferably, the amount of encapsulated enzyme in a single dose is from 10 U/kg patient body weight to 80,000 U/kg patient body weight. Most preferably, the amount of encapsulated enzyme in a single dose is from 10 U/kg patient body weight to 50,000 U/kg patient body weight. Most preferably, the amount of encapsulated enzyme in a single dose is from 10 U/kg of patient's body weight to 5000 U/kg of patient's body weight, or from 50 U/kg of patient's body weight to 3500 U/kg of patient's body weight.

In some embodiments, the amount of encapsulated enzyme in a single dose may also be at least 10, 25, 50, 75, 100, 150, 200, 250, 300, 350, 400, 450, 500, 600, 700, 800, 900, 1000 . In some embodiments, the amount of encapsulated enzyme per dose may also be at most 10000, 9500, 9000, 8500, 8000, 7500, 7000, 6500, 6000, 5500, 5000, 4500, 4000, 3500, 3000, 2500, 2000, 1500. 1000, 900, 800, 600, 500, 450, 400, 350, 300, 250, 200, 150, 100 IU/kg patient body weight.

Those skilled in the art will appreciate that the dosages used for administration may be adapted as a function of various parameters, and in particular as a function of the type of administration used, the relevant pathology, or, alternatively, the desired duration of treatment.

A pharmaceutical composition comprising an erythrocyte-encapsulated arginine deimnase and a pharmaceutically acceptable carrier has been described in the art in the context of cancer treatment. Adapting the dosages described in the above publications to the compositions of the invention for use in the treatment of arginase-1 deficiency is within the ability of those skilled in the art.

Encapsulation methods

Red blood cells can be obtained as described in the present description above in the section "Pharmaceutical compositions".

Encapsulation of enzymes in erythrocytes can be performed using a suspension of erythrocytes, which is brought into contact with a hypotonic liquid medium, which leads to the opening of the pores in the erythrocyte membrane. There are three alternatives in the lysis-repair method, which are hypotonic dialysis, hypotonic pre-swell, and hypotonic dilution, all based on the difference in osmotic pressure between the inside and outside of the red blood cells. Hypotonic dialysis is preferred.

An enzyme-encapsulating erythrocyte suspension can be prepared by the following method, including

1 - suspension of erythrocyte sediment in isotonic solution at a hematocrit level equal to or greater than 65%, cooling between +1 and +8°C;

2 - lysis procedure at a temperature maintained between +1 and +8°C, involving the passage of a suspension of erythrocytes at a hematocrit level equal to or greater than 65%, and a hypotonic lysis solution cooled to between +1 and +8°C in a dialysis device such as a dialysis coil or cartridge (a cartridge is preferred);

3 - encapsulation procedure by adding, preferably gradually, the enzyme, which is encapsulated (in particular in a solution obtained in advance), to the suspension before or during lysis at a temperature maintained between +1 and +8°C; and

4 - recovery procedure carried out in the presence of an isotonic or hypertonic, preferably hypertonic, solution at a higher temperature, namely, a component from +30 to +42°C.

In a preferred alternative, the method described in WO-A-2006/016247 (EP 1 773 452; incorporated herein by reference) can be used, comprising

1 - suspension of erythrocyte sediment in isotonic solution at a hematocrit level equal to or greater than 65%, cooling between +1 and +8°C;

2 – measurement of osmotic fragility in an erythrocyte sample from the same sediment;

3 - lysis procedure at a temperature maintained between +1 and +8°C, including the passage of a suspension of erythrocytes at a hematocrit level equal to or greater than 65%, and a hypotonic lysis solution cooled to between +1 and +8°C in a dialysis device such as a dialysis coil or cartridge (a cartridge is preferred); moreover, the lysis parameters are adjusted according to the osmotic fragility measured previously; in particular, depending on the measured osmotic fragility, the flow of the erythrocyte suspension in the dialysis device is controlled or the osmolarity of the lysis solution is adjusted; and

4 - encapsulation procedure by adding, preferably gradually, the enzyme, which is encapsulated (especially in a solution obtained in advance), to the suspension before and during lysis at a temperature maintained between +1 and +8°C; and

5 is a recovery procedure carried out in the presence of an isotonic or hypertonic, preferably hypertonic, solution at a higher temperature, namely +30 to +42°C.

It is important, in the case of dialysis, that the erythrocyte sediment is suspended in an isotonic solution with a hematocrit level equal to or greater than 65%, and preferably equal to or greater than 70%, and this suspension is cooled to a temperature between +1 and +8°C, preferably between +2 and +6°C, usually up to a temperature of about +4°C. According to a certain method, the hematocrit level is from 65 to 80%, preferably from 70 to 80%.

When osmotic fragility is measured, it is advantageous to carry out the measurement on erythrocytes just before the lysis step, in the presence or absence, preferably in the presence, of the enzyme to be encapsulated. For erythrocytes or a suspension containing them, a temperature approaching or identical to the temperature chosen for lysis is advantageous. According to another preferred feature of the invention, the osmotic fragility measurement taken is used immediately, ie the lysis procedure is carried out shortly after the sampling. Preferably, the time interval between sampling and the start of lysis is less than or equal to 30 minutes, even better less than or equal to 25 or even 20 minutes.

With regard to performing the lysis-repair procedure with measurement and according to osmotic fragility, one skilled in the art may refer to WO-A-2006/016247 for details. This document is incorporated herein by reference in its entirety.

An improvement to this encapsulation technique is described in WO 2014/180897, which may be referred to by one skilled in the art, and which is incorporated herein by reference. Thus, according to one embodiment, enzyme-encapsulating erythrocytes are prepared by a method comprising encapsulating the active ingredient in erythrocytes by lysis-reduction, obtaining a suspension or pellet comprising enzyme-containing erythrocytes, and a solution with an osmolality greater than or equal to 280 mOsmol/kg, in in particular between about 280 and about 380 mOsmol/kg, preferably between about 290 and about 330 mOsmol/kg, incubation of the pellet or suspension as such or after addition of the incubation solution, at an osmolality greater than or equal to 280 mOsmol/kg, in particular between about 280 and about 380 mOsmol/kg, preferably between about 290 and about 330 mOsmol/kg. The incubation is in particular carried out for a period greater than or equal to 30 minutes, in particular greater than or equal to 1 hour. The liquid medium of the incubated solution is then removed and the resulting erythrocytes are suspended in a solution capable of injecting the suspension into a patient, preferably a retaining solution capable of injecting the suspension into a patient. The indicated osmolality is the osmolality of the solution in which the erythrocytes or the pellet are suspended at the appropriate moment.

The term "stabilized suspension of erythrocytes" means in particular a suspension having an extracellular hemoglobin content that remains less than or equal to 0.2 g/dl until the suspension is used by humans, the latter can occur, in particular, from 1 to 72 hours after receiving a batch of erythrocytes containing the active ingredient.

The term "ready-to-use stabilized erythrocyte suspension" means a stabilized suspension in a solution that can be injected into a patient, in particular in a preservative solution. Her hematocrit is usually equal to or greater than 35%, 40%, or 45%.

The term "erythrocyte sediment" refers to the concentrate of erythrocytes collected after the separation of erythrocytes from the liquid medium in which they were previously suspended. Separation can be achieved by filtration or centrifugation. Centrifugation is the method commonly used for such separation. The sediment includes some part of the liquid medium. Typically, the sediment has a hematocrit between 70 and 85%.

The term "incubation solution" means a solution in which erythrocytes encapsulating the active ingredient are present during the incubation step. Incubation can be performed over a wide hematocrit range, namely between 10 and 85% hematocrit.

The term "fragile erythrocytes" refers to erythrocytes resulting from the inclusion procedure, which, once suspended in a preservative solution, can be lysed when the suspension is stored at a temperature of 2 to 8°C, in particular after 1-72 hours.

The term "initial hematocrit" refers to the hematocrit before cell loss due to lysis of fragile erythrocytes during incubation.

The method may include, in particular, the following steps:

(a) encapsulating the enzyme in erythrocytes, including bringing the erythrocytes into contact with a hypotonic medium (allowing opening of pores in the erythrocyte membrane), contacting the active ingredient (to allow its introduction into erythrocytes), reducing erythrocytes, in particular, using an isotonic or hypertonic medium , predominantly hypertensive

(b) obtaining or preparing a suspension or precipitate comprising erythrocytes comprising an enzyme and a solution with an osmolality greater than or equal to 280 mOsmol/kg, in particular from about 280 to about 380 mOsmol/kg, preferably from about 290 to about 330 mOsmol/ kg,

(c) incubating the pellet or suspension from step (b) as such or after adding the incubation solution at an osmolality greater than or equal to 280 mOsmol/kg, in particular from about 280 to about 380 mOsmol/kg, preferably from about 290 to about 330 mOsmol/kg, for a period greater than or equal to 30 minutes, in particular greater than or equal to 1 hour,

(d) removing the liquid medium of the incubated suspension from step (c),

(e) suspending the erythrocytes obtained in step (d) in a solution capable of injecting the suspension into a patient, preferably a preservative solution capable of injecting the suspension into a patient.

According to the first method, the step following the encapsulation by lysis-recovery, namely step (b), comprises at least 1 washing cycle, preferably 2 or 3 washing cycles by diluting the resulting suspension or precipitate in the lysis-recovery step or step (a) in solution at an osmolality of at least greater than 280 mOsmol/kg, in particular from about 280 to about 380 mOsmol/kg, preferably from about 290 to about 330 mOsmol/kg, and then obtaining a precipitate or suspension of erythrocytes. This pellet or suspension comprises erythrocytes comprising the enzyme and a solution with an osmolality greater than or equal to 280 mOsmol/kg, in particular from about 280 to about 380 mOsmol/kg, preferably from about 290 to about 330 mOsmol/kg. Then apply the following steps, for example, (c), (d) and (e).

According to the second method, in the lysis-recovery step or step (a), the recovery of erythrocytes with an isotonic or hypertonic medium produces a suspension of erythrocytes, which can then be incubated, for example, the suspension from step (b), in a solution with an osmolality greater than or equal to 280 mOsmol /kg, in particular from about 280 to about 380 mOsmol/kg, preferably from about 290 to about 330 mOsmol/kg. In other words, the lysis-recovery step or step (a) comprises a step of restoring erythrocytes, wherein the suspended erythrocytes encapsulating the enzyme are mixed with an isotonic or hypertonic recovery solution, preferably hypertonic, to form a suspension of erythrocytes with an osmolality greater than or equal to 280 mOsmol/kg, in particular from about 280 to about 380 mOsmol/kg, preferably from about 290 to about 330 mOsmol/kg. In this method, the incubation step or step (c) comprises the incubation of the suspension resulting from the reconstitution. The incubation is carried out for a period greater than or equal to 30 minutes, in particular greater than or equal to 1 hour. The following steps are then applied, for example (d) and (e).

The steps following the lysis-recovery step, for example (b)-(e), are carried out under conditions that result in lysis of fragile erythrocytes or most of them, namely, more than 50%, 60%, 78%, 80% or 90% or more. In order to do this, it is possible to influence the incubation period, the incubation temperature and the osmolality of the solution in which the erythrocytes are suspended. The higher the osmolality, the longer the incubation can be. Thus, the lower the osmolality, the shorter the incubation can be in order to obtain the same effect. Also, the higher the temperature, the shorter the incubation time can be and vice versa. Subsequent one or more wash cycles will remove cellular derbis and extracellular hemoglobin as well as extracellular enzyme.

According to the invention, the washing cycle includes diluting the suspension or precipitating the erythrocytes and then separating the erythrocytes from the washing solution. Preferably, the washing step comprises preferably 2 or 3 wash-separation cycles. The separation can be carried out in various ways, such as filtration and centrifugation. Centrifugation is preferred.

Incubation is not limited to the hematocrit of the suspension. In this regard, it is possible to incubate a suspension having an initial hematocrit usually from 10 to 85%, in particular from 40 to 80%. This also applies to the sediment with a hematocrit of 70% and, like the suspension, below the indicated value.

The removal step or step (d) aims to remove the liquid part of the suspension or the incubated precipitate in order to remove, in particular, the cellular derby and extracellular hemoglobin, and hence also the extracellular enzyme.

According to the first method, in the case of a removal step or step (d), separation, in particular centrifugation, is carried out, and this applies specifically to a suspension. This separation may be followed by one or more, for example 2 or 3, washing cycles by dilution in isotonic saline and subsequent separation, in particular by centrifugation.

According to the second method, in the case of a removal step or step (d), a dilution is carried out before separation, in particular centrifugation, this being applicable to the suspension or to the sediment. The dilution can be carried out, in particular, with isotonic rinsing solution or storage solution.

The final step or step (e) is to obtain the final suspension, one that can be administered to the patient without any other treatment.

According to the first method, in the case of this step, the dilution of the erythrocyte sediment from the removal step or step (d) is performed with an injection solution, in particular a storage solution.

According to the second method, in the case of this step, one or more washing cycles of the erythrocyte sediment formed in the removal step or step (d) are carried out with an injection solution, in particular a storage solution, by dilution followed by separation. After washing, the erythrocytes are resuspended in an injection solution, in particular a preservative solution.

The method of the invention may also include one, more or all of the following features:

- the incubation step or step (c) is performed at a temperature of from about 2 to about 39°C for a time sufficient to ensure lysis of fragile red blood cells;

- the incubation step or step (c) is carried out at a low temperature, namely from about 2 to about 10°C, in particular from about 2 to about 8°C, and continues for about 1 hour - about 72 hours, in particular, from about 6 hours to about 48 hours, preferably from about 19 hours to about 30 hours;

- the incubation step or step (c) is carried out at a higher temperature of from about 20 to about 39°C, in particular at room temperature (25°C ± 5°C), and continues for from about 30 minutes to about 10 hours, in particular from about 1 hour to about 8 hours, preferably from about 2 hours to about 4 hours; it is possible to work even at a higher temperature than room temperature, but this may have a negative effect on cell yield, P50 and/or 2.3 DPG content;

- in the incubation stage or stage (c) the suspension is at an initial hematocrit between 10 and 85%, in particular between 40 and 80%; you can incubate the precipitate after separation, having a hematocrit, for example, from 70 to 85%, or a dilute precipitate having a hematocrit between about 40 and 70%;

- the stage of incubation includes stirring the suspension;

- the incubation step does not include any mixing;

- as a solution for washing and/or incubation using a measured amount of an aqueous solution of NaCl to obtain the desired osmolality; so, for example, the solution may include 0.9% NaCl; this solution may also include, for example, besides NaCl, glucose, in particular glucose monohydrate, monosodium phosphate dihydrate, disodium phosphate dodecahydrate; as an example, the composition comprises 0.9% of NaCl, 0.2% glucose monohydrate, 0.034% monosodium phosphate dihydrate, 0.2% disodium phosphate dodecahydrate;

- washing in the final stage or stage (e) is performed with a preservative solution;

- the osmolality of the solution (liquid portion) in a ready-to-use suspension or a suspension that can be injected into a patient is between about 280 and about 380 mOsmol/kg, preferably between about 290 and 330 mOsmol/kg;

- the hematocrit of the ready-to-use suspension or suspension that can be injected into the patient is equal to or greater than 35%, 40% or 45%;

- all stages of washing and incubation are performed with a preservative solution;

- the wash solution in step (b) and/or the wash solution in step (e) and the preservative solution all have the same composition and include the compound(s) promoting erythrocyte conservation;

the preservative solution (and wash solution(s) or incubation solutions, if necessary) is an aqueous solution comprising NaCl, adenine and at least one of glucose, dextrose and mannitol;

- preservative solution (and solution(s) for washing or for incubation, if necessary) includes NaCl, adenine and dextrose, preferably AS3 medium;

- Preservative solution (and solution(s) for washing or for incubation, if necessary) includes NaCl, adenine, glucose and mannitol, preferably SAG-mannitol or ADsol medium.

The methods of the invention specifically include the following step:

(a) encapsulating the enzyme in erythrocytes, including contacting a hypotonic medium to allow opening of pores in the erythrocyte membrane, contacting the enzyme to allow it to enter the erythrocytes, restoring the erythrocytes with an isotonic or hypertonic medium. It should be noted that the enzyme may be present in the erythrocyte suspension prior to lysis of the latter, or may also be added during lysis or after lysis, but always before recovery. In one embodiment of this step (a), the method includes the following substeps:

(a1) the presence of a suspension of red blood cells with a hematocrit equal to or greater than 60% or 65%,

(a2) measuring the osmotic fragility of the erythrocytes in this suspension,

(a3) a procedure for lysis and internalization of the active ingredient(s), comprising passing a suspension of erythrocytes through a dialysis device, in particular a dialysis cartridge, in the direction opposite to the lysis solution, and adjusting the flow of the suspension of erythrocytes or adjusting the flow rate of the solution for lysis or adjust the osmolality of the lysis solution depending on the osmotic fragility measured in (a2),

(a4) erythrocyte recovery procedure.

The definitions given in the present description above in the sections "Argigindeiminase", "Pharmaceutical compositions or suspensions", "Therapeutic use" and "Doses", such as "erythrocytes", "hematocrit level", "arginideiminase", apply mutatis mutandis in the section " encapsulation methods.

Throughout this application, features described in one section are fully applicable in other sections of this description. For example, the description referring to "arginideiminase" given in the "Argigindeiminase" section is fully applicable to the section titled "Pharmaceutical compositions or suspensions", the section titled "Therapeutic methods and uses", the section "Methods of encapsulation" and the section "Doses" .

Throughout this application, the term "and/or" is a grammatical construct that can be interpreted to mean that one or more cases can occur if they are related. For example, the phrase "protein-free diet and/or nitrogen scavenger drugs" in the expression "outputs are generally poor with a protein-free diet and/or nitrogen scavenger drugs" indicates that outcomes are generally poor for an individual treated with either a protein-free diet or nitrogen scavenger drugs. or a combination of both, i.e., a protein-free diet and nitrogen scavenger drugs.

Throughout this application, the term "inclusion" should be interpreted as covering all specifically mentioned features, as well as optional, additional, unspecified. As used herein, the use of the term "inclusion" also discloses an embodiment in which there are no features other than those specifically mentioned (ie, "consists of"). In addition, the indication of the singular does not exclude the plural. The mere fact that certain criteria are listed in relation to each other in various dependent claims does not indicate that a combination of these criteria cannot be used to advantage.

The invention will now be described in more detail with reference to the following figures and examples. All literature and patent documents cited in the present description are incorporated herein by reference. Since the invention is explained and described in detail in the foregoing description, the examples are to be considered as illustrative or illustrative and not as limiting.

Brief description of the sequences

SEQ ID NO: 1 shows the amino acid sequence of the full-length arginine deiminase from Mycoplasma arginini, which is available from the NCBI database under accession number WP_004416214.1.

FIGURES

Figure 1. Schematic representation of the urea cycle showing the role of enzymes (NAGS, CPS1, ASS1, ASL, ARG1, OTC) and transporters (ORNT1 and citrine) in the conversion of ammonia nitrogen to urea.

Figure 2. Graph showing the pharmacokinetics (PK) of CFSE-labeled ADI-encapsulating erythrocytes. The ERY-ADI 6 product is prepared by lysis-recovery of a suspension containing 5 mg/ml ADI. Fluorescent labeling of products (CFSE) allows tracing of erythrocytes in vivo. The product injected intravenously into C57BL/6 mice (8 ml/kg) has excellent stability with an estimated half-life of 18 to 22 days after administration.

Figure 3. Graph showing the pharmacodynamics of ADI-encapsulating erythrocytes over 16 days. The ERY-ADI 6 product is prepared by lysis-recovery of a suspension containing 5 mg/ml ADI. The product is administered intravenously (IV) to C57BL/6 mice (8 ml/kg). The level of L-arginine in plasma is measured by HPLC-MS-MS. The level of L-arginine in untreated C57BL/6 mice was estimated to be between 75 and 125 μM. The ERY-ADI 6 product leads to a rapid decline 15 min after administration, reducing the level of L-arginine to 0 μM within 13 days. On day 16, two of three mice show complete depletion of plasma L-arginine. Plasma L-Arginine in the third mouse is 13 μM. The mean plasma L-arginine level for this group of mice on day 16 is 4.33 ± 7.51 μM.

Figure 4. Graph representing blood levels of L-arginine in arginase deficient mice after a single intravenous injection of ADI-encapsulating erythrocytes. The ERY-ADI 4 product is prepared by lysis-recovery of a suspension containing 4.5 mg/ml ADI. As a control, a lysis-repair method without the ADI enzyme produces a product (unloaded erythrocytes) called pRBC (recycled red blood cells). On day 0, two products are administered intravenously to arginase deficient mice (4 or 8 ml/kg in the case of ERY-ADI 4 and 8 ml/kg in the case of pRBC). Blood samples were taken from all mice on day 0 (prior to administration of ERY-ADI 4 or pRBC), day 1 and day 3. When ERY-ADI 4 was administered at a dose of 4 ml/kg, blood L-arginine was 7% on days 1 and 3 after injection, respectively. When a double dose of ERY-ADI 4 (8 ml/kg) is administered, the efficacy of ADI-encapsulating erythrocytes in relation to abnormal blood levels of L-arginine is impressive; the next day after injection, the L-arginine level is more than 10 times lower than the baseline L-arginine concentration in this mouse model (35 ± 22 μM vs. 452 ± 45 μM, corresponding to a decrease in blood by 92%). Three (3) days after administration of the ERY-ADI 4 product, the level of L-arginine in the blood is still 4 times lower than the biochemical level in this pathology (119 ± 57 μM vs. 452 ± 45 μM, which corresponds to a decrease in the blood by 73% ). However, when unloaded erythrocytes (pRBC) are administered, no decrease in blood L-arginine is observed. On the contrary, the level of L-arginine in the blood still increases, which shows that unloaded red blood cells do not affect the biochemical course of the disease.

Figure 5. Graph representing blood ammonia levels in arginase deficient mice following intravenous administration of a single dose of ADI-encapsulating erythrocytes. The ERY-ADI 4 product is prepared by lysis-reduction of a suspension containing 4.5 mg/ml ADI. As a control, a lysis-repair method without the ADI enzyme produces a product (unloaded erythrocytes) called pRBC (recycled red blood cells). On day 0, two products are administered intravenously to arginase deficient mice (4 or 8 ml/kg in the case of ERY-ADI 4 and 8 ml/kg in the case of pRBC). Blood samples were taken from all mice on day 0 (prior to administration of ERY-ADI 4 or pRBC) and day 3 for analysis of serum ammonia. Serum ammonia is analyzed as L-arginine ADI conversion results in citrulline and ammonia. No noticeable changes in ammonia levels are observed when mice are treated with ADI encapsulating erythrocytes or unloaded erythrocytes.

Figure 6. Graph showing blood levels of L-arginine in arginase deficient mice following administrations of ERY-ADI once (Group 2), twice (Group 3), or the free form of the ADI enzyme (Group 4).

Figure 7. Graph showing blood levels of L-arginine in arginase deficient mice after administration of Mock RBC (Group 1), ERY-ADI once (Group 2) and twice (Group 3).

EXAMPLES

Example 1 Method for the preparation and characterization of arginine deiminase

Preparation of the strain and isolation of the overproducing clone. The native sequence of ADI from Mycoplasma arginini (GenBank: X54141) is optimized by modifying some codons, since the genetic codes of E. coli and M. arginini are different (creating a new plasmid coded for C1124-ADM-02). The purification method is described by Misawa et al. (Misawa S. et al, 1994, J. of Biotechnol., 36, 1994, 145-155) with some modifications, i.e., use of the bacterially derived HMS174 (DE3) T1R strain instead of the JM101 strain. Other modifications are described below.

Fermentation

Preparation is achieved in a fermenter with a batch of FED_Coli_9 medium under stirring, controlled pressure and pH from preculture 2 at an optical density of 0.05. The growth phase (at 37° C.) occurs until an optical density (600 nm) of 100 is obtained and induction of expression is achieved at 32° C. by adding 1 mM IPTG to the culture medium. The cell pellet is collected 26-27 hours after induction in two steps: the cell culture fluid is concentrated 5-10 times after passing through a 500 kDa hollow fiber, then the cell pellet is recovered by centrifugation at 15900 xg and then stored at -20°C.

cleaning

ADI is obtained as inclusion bodies (IB). Cellular pellets for cell disruption are suspended in lysis buffer. The disrupted cells are then washed, IB is harvested, and IB is stored at -20°C.

Purification of ADI is started by thawing the precipitate of IB in a buffer consisting of 50 mM tris base, pH 8.5, 6 M guanidinium hydrochloride, 10 mM dithiothreitol. Solubilization is achieved with an incubation time of 1 hour at 37 ± 2°C. After purification, a refolding step takes place for 40-45 hours at room temperature in a buffer consisting of 3 mM monopotassium phosphate (KH2PO4), 7 mM dipotassium phosphate (K2HPO4), pH 7.35. After the second purification step, the medium is loaded onto a Q-Sepharose column. Elution is performed with 250 and 500 mM NaCl and the eluted fraction is subjected to tangential flow filtration (TFF). Two upgrade steps (using a Sartobind Q column) and two TFF steps complete the ADI purification. Before storage of ADI at -20°C perform the final filtration (0.2 μm).

Defining properties. Determine the specific activity of the enzyme by measuring the resulting citrulline, as described in example 2. Protein content is determined by recording absorbance at 280 nm. Purity is determined by SDS-PAGE. Osmolarity is measured with an osmometer (Loser microosmometer, type 15). The main properties of one batch of ADI received are summarized in the present description below in Table 2.

Table 2. Main properties of one received lot of ADI

ADI from M. arginini Composition Liquid phase frozen at -80°C
Properties
323 mosm / kg - 16.65 mg / ml
50 mM sodium phosphate, pH 6.5, sucrose 40 mg/ml, lysine 40 mM Specific activity ~ 47 U/mg Purity 97.5%

Example 2 ADI Analysis for Specific Activity Using Citrulline Measurement

This assay is based on a 2-step reaction (Boyde and Rahmatullah, 1980, Analytical Biochemistry, vol. 107, p 424-431):

- first - L-arginine is converted by ADI into citrulline and ammonia;

- second - in the presence of diacetyl monooxime, iron (III) chloride, thiosemicarbazide, sulfuric and phosphoric acids, citrulline turns into a colored chromophore, recorded at 530 nm.

Get a standard curve of L-citrulline to determine the enzymatic activity of all analyzed samples, reading the absorbance at 530 nm. Specific activity (U/mg) is calculated using enzymatic activity (U/ml) and protein content (mg).

Example 3 ADI encapsulation in mouse erythrocytes

Whole blood from C57BL/6 mice (Charles River) is centrifuged at 1000×g for 10 min at 4°C to remove plasma and buffy coat. The erythrocytes are washed three times with 0.9% NaCl (v:v). The frozen ADI is thawed and added to the erythrocyte suspension to obtain a final suspension with a hematocrit of 65% containing an initial ADI concentration of 2-7 mg/mL.

The suspension is then loaded into a hemodialyzer at a flow rate of 120 ml/hour and dialysis is performed against a hypotonic solution (40-50 mOsmol/kg) at a flow rate of 15 ml/min as countercurrent. The suspension is then reconstituted with hypertonic saline (1600-2100 mOsmol/kg) and then incubated for 30 min at 37°C. After three washes in 0.9% NaCl, 0.2% glucose, the suspension is placed in AS3 preservative solution supplemented with 20% complement-removed plasma.

The resulting products are characterized at time D0 (within 2 hours after their receipt) and at time D1 (ie, after ~18 hours - 24 hours of storage at 2-8°C). Hematological characteristics are obtained using an automatic veterinary unit (Sysmex, PocH-100iV).

results

The activity of ADI in the final products was analyzed in the manner described in Example 4 against the outer calibration range of ADI in aqueous solution. These results show that the activity of ADI in end products increases with the amount of enzyme introduced into the RBC and that up to 2 mg of ADI per ml of end product can be easily encapsulated while maintaining good stability. The main characteristics of the 6 different batches of mouse ERY-ADI products (ERY-ADI 1 - 6) are summarized in this description below in Table 3.

Table 3. Main characteristics of 6 different batches of mouse ERY-ADI end products measured at D0 (2 hours after receipt)

parties ERY-ADI-1 ERY-ADI-2 ERY-ADI-3 ERY-ADI-4 ERY-ADI-5 ERY-ADI-6 Hematological parameters Hematocrit (%) 51.1 51.1 50.8 51.3 51.1 51.3 Body volume (fl) 39.5 40.1 40.6 41.3 40.0 38.60 Taurus hemoglobin (g/dl) 26.6 26.3 25.3 26.7 28.2 25.7 Total hemoglobin (g/dl) 14.8 14.4 14.0 14.8 15.6 13.8 Extracellular Hb (g/dl) 0.3 0.4 0.3 0.8 0.3 0.3 ADI Options Pre-method ADI concentration (mg/ml) 3 4.5 4.5 4.5 3.5 5 ADI concentration inside erythrocytes (mg/ml RBC - 100% Ht) 1.15 1.56 1.90 1.13 1.37 2.70 Extracellular activity (%) 4.8 5.7 4.8 6 4.0 4.1 Intracellular activity (%) 95.2 94.3 95.2 94 96.0 95.9 ADI Encapsulation Yield (%) 38 35 42 25 39 54

Example 4 Assay of Encapsulated ADI in RBCs

The analysis of ADI activity captured by red blood cells and in supernatants is based on the measurement of NH ₃ produced by ADI from L-arginine. NH ₃ ions are analyzed indirectly by the enzymatic action of glutamate dehydrogenase (GLDH) according to the kit sold by Roche Diagnostics (11877984).

Example 5 Pharmacokinetics of ADI Encapsulating RBCs in C57BL/6 Mice

The ERY-ADI 6 mouse product is labeled with CFSE (fluorescent) and intravenously administered to C57BL/6 mice. At each time point (D0 + 15 min, D0 + 6 h, D1, D2, D5, D9, D13 and D16), 3 mice are sacrificed, blood is collected in a tube with lithium heparinate and kept at +4°C, protected from light, to determine pharmacokinetics. The proportion of CFSE-labeled red blood cells in whole blood is determined by flow cytometry. Five microliters of whole blood is diluted in 1 ml PBS, 0.5% BSA, and each sample is run three times (counting 10,000 cells in FL-1; FC500 cytometer, Beckman Coulter). The survival score of ADI-loaded red blood cells is obtained by adding a fraction of ADI-loaded CFSE-labeled erythrocytes at various time points to a fraction of CFSE-labeled ADI-loaded erythrocytes at T0 + 15 min (control, 100%). The different percentages obtained for each time point are indicated in the graph shown in Figure 2, thus illustrating the proportion of ADI-loaded erythrocytes in circulation over time.

Based on half-life calculation, CFSE-labeled ADI-encapsulating erythrocytes have a half-life of 18 to 22 days.

Example 6 Pharmacodynamics of ADI Encapsulating RBCs in C57BL/6 Mice

The ERY-ADI product 6 erythrocytes encapsulating the ADI enzyme was injected intravenously into C57BL/6 mice at a dose of 8 ml/kg. At each time point (D0 + 15 min, D0 + 6 h, D1, D2, D5, D9, D13 and D16) 3 mice are sacrificed, blood is collected in lithium heparinate tubes and stored at +4°C to determine L- plasma arginine.

As can be seen in figure 3, complete depletion of L-arginine in plasma is observed within 13 days immediately (ie 15 minutes) after administration of erythrocytes encapsulating the ADI enzyme. At the very last point of the study (ie, 16 days later), 2 out of 3 mice display complete depletion of plasma L-arginine. The plasma L-arginine level in the third mouse is 13 µM, significantly lower than the physiological plasma L-arginine concentration in this study (100 ± 25 µM).

Example 7 Administration of ADI Encapsulating RBCs to Arginase Deficient Mice (1st Study)

An in vivo study was performed using a mouse model of arginase deficiency (for a full description, see Sin et al., 2013, PLOS One, vol. 8 (11)). These mice lack arginase 1 activity and exhibit the severe pathobiochemical aspects of hyperargininemia commonly seen in humans. Hyperargininemia (or arginase deficiency) is triggered by 5 injections of tamoxifen. Arginine concentrations in the blood begin to rise a few days after the last injection of tamoxifen. In order to demonstrate the efficacy of the mouse product ERY-ADI 4 in reducing blood levels of L-arginine in this mouse model, ERY-ADI 4 (4 and 8 ml/kg) and unloaded erythrocytes (8 ml/kg) were injected intravenously into 15 mice. with arginase deficiency 7 days after the last injection of tamoxifen. Blood is collected on the day (D1) and two days later (D3) after the administration of the three products. The results of this in vivo study are presented in Figures 4 and 5 and in Table 4 below.

Table 4. Summary of the results of the in vivo study of ERY-ADI 4 in a mouse model of arginase deficiency. It is shown, in %, a decrease in the levels of arginase in the blood. Negative numbers indicate an increase in arginine levels

% reduction
(L-arg. in the blood) Falsely loaded erythrocytes (8 ml/kg) ERY-ADI 4
(4 ml/kg) ERY-ADI 4
(8 ml/kg) D1 -ten% 60% 92% D3 -thirty% 7% 73%

As can be seen from this table and Figure 4, ADI-encapsulating erythrocytes, when administered at a dose of 4 ml/kg, reduced blood L-arginine by 60% and 7% on days 1 and 3 post-injection, respectively. When a double dose (8 ml/kg) is administered, the effect of ADI-encapsulating erythrocytes on abnormal blood levels of L-arginine is impressive; the day after injection, the level of L-arginine in this mouse model is more than 10 times lower than the baseline concentration of L-arginine (35 ± 22 μM vs. 452 ± 45 μM, corresponding to a decrease in blood by 92%). Three (3) days after administration of the ERY-ADI 4 product, the blood L-arginine level is still 4 times lower than the pathobiochemical level (119 ± 57 μM vs. 452 ± 45 μM, corresponding to a 73% decrease in blood levels). However, when unloaded erythrocytes are administered, no decrease in blood L-arginine is observed. On the contrary, the level of L-arginine in the blood still increases, indicating that unloaded erythrocytes have no effect on the biochemical course of the disease.

At the same time, serum ammonia is analyzed, since the conversion of L-arginine ADI leads to the formation of citrulline and ammonia. As seen in Figure 5, no noticeable changes in ammonia levels are observed when mice are treated with ADI encapsulating erythrocytes or unloaded erythrocytes.

Example 8 Administration of ADI Encapsulating RBCs to Arginase Deficient Mice (Second Study)

In order to confirm that the ERY-ADI mouse product reduces blood levels of L-arginine in this mouse model, a second study was performed in mice treated with tamoxifen according to Example 7. The ERY-ADI product was injected intravenously 3 days after the last tamoxifen injection. One or two intravenous injections of ERY-ADI (8 ml/kg) are planned (groups 2 and 3, respectively). The control group is administered the free form of the ADI enzyme (group 4). Part of the study is a second control group consisting of mice with arginase activity (Group 1).

Blood is collected on the day (D3) and a week later (Day 10). On the day of sacrifice, mice are also bled (day 13).

The results of the second in vivo study are shown in Figure 6.

First, blood levels of arginine are lower in this second study because the authors changed the schedule regarding tamoxifen injections. In this study, ERY-ADI administration is scheduled 3 days instead of 7 days after the last tamoxifen injection, resulting in lower baseline blood arginine levels.

As seen in Figure 6, ADI-encapsulating RBCs, when administered at a dose of 8 ml/kg, reduced blood levels of L-arginine by 81% and 77% compared to baseline levels for groups 2 and 3, respectively, on day 10 ( i.e., 7 days after administration). A second administration of ERY-ADI was planned after blood collection on day 10. Ten (10) days post-administration, the reduction in blood arginine is still quite significant with percentage reductions of approximately 82% and 68% for group 2 and group 3, respectively, compared to base levels. In contrast, no decrease in blood arginine was observed in mice injected with free form ADI on day 10 (group 4). In contrast, the blood arginine concentration reaches 349 µM, reflecting the increase in blood arginine levels in the mouse model of arginase deficiency. Blood levels of L-arginine could not be measured on day 13 in group 4 as all animals died before the 2nd injection of free form ADI.

The second study confirmed the results of the first study with some additional information on the pharmacokinetics of the ERY-ADI product when administered to arginase deficient mice. One single injection allowed for a decrease in blood arginine for at least 10 days, and a second injection of the ERY-ADI product did not result in any side effects in this mouse model.

Example 9 Administration of ADI Encapsulating RBCs to Arginase Deficient Mice (Third Study)

Since no survival was observed when ERY-ADI administration was performed 3 days after the last tamoxifen injection, a third study is planned with an important change in administration schedule. ERY-ADI is injected intravenously on the day before the first tamoxifen injection (day 0). In this study, the last injection of tamoxifen is given on day 5. One or two intravenous injections of ERY-ADI are scheduled (groups 2 and 3, respectively). Group 1 is a control group consisting of arginase deficient mice administered empty red blood cells (RBCs) (ie, processed RBCs without ADI). Blood is collected on day 0, day 11, day 14 (before the second administration of ERY-ADI), and when mice are sacrificed for loss of excess body weight (identified as "end of study").

As seen in Figure 7, ADI-encapsulating erythrocytes, when administered at a dose of 8 ml/kg, reduced blood L-arginine by 71.4% and 78.7% compared to levels in group 1, groups 2 and 3 , respectively, on day 11 (ie, 11 days after administration). On day 14 before the second administration of ERY-ADI (group 3 only), total blood arginine is still reduced by 27.3% and 29.3% in groups 2 and 3, respectively, compared to group 1. In contrast, no decrease in blood arginine in mice injected with empty RBCs (group 1). In this group, the total blood arginine concentration reaches 844 μM at the end of the study, reflecting the increase in total blood arginine in this mouse model of arginase deficiency.

When ERY-ADI is re-injected on day 14 (group 3), total blood arginine is measured a few days later, at the time of ethical sacrifice (identified as "end of study"). Blood arginine is still reduced after the second administration of ERY-ADI, but arginase deficient mice must be euthanized due to significant body weight loss.

This third study confirms the results of the first two studies with some additional information on the pharmacodynamics of the ERY-ADI product when injected into arginase deficient mice. One injection produces a reduction in blood arginine in this mouse model for at least 11 days, and a second injection of ERY-ADI produces an extended reduction in blood arginine for at least 17 days (lifespan of the first mouse that was ethically euthanized) .

However, due to the severity of this mouse model, survival is not observed beyond 15 days after the last tamoxifen injection, no matter how many ERY-ADI injections are performed.

Example 10 Preparation of Human Red Blood Cells Encapsulating Arginine Deiminase

A pouch of leukocyte-deprived human RBCs (provided by Etablissement français du sang) was subjected to three washes with 0.9% NaCl. The arginine deiminase (ADI) solution is gently thawed and added to the RBC suspension to give a final concentration of 60% hematocrit, containing 3 or 5 mg/ml ADI. The suspension is homogenized, loaded into a hemodialyzer at a flow rate of 90 ml/hour and dialyzed against a hypotonic solution at 30 mOsmol/kg. The suspension is then reconstituted with hypertonic saline and then incubated for 30 minutes at 30°C. After 3 washes in 0.9% NaCl, 0.2% glucose, the suspension is placed in AS3 preservative solution (NaCl, NaH ₂ PO ₄ , citric acid, Na-citrate, adenine and glucose, osmolality 288 mosm/kg and pH 5, 88). The products obtained are characterized on day 0, day 1 and day x. Hematological characteristics are obtained using a veterinary automatic unit (Sysmex, PocH-100iV).

It is important to note that the disclosed compositions and suspensions of RBC-encapsulated arginine deiminidase (ADI) do not (or need not) add magnesium, iron, or other enzyme cofactors. Unlike non-encapsulated ADI formulations, which may use magnesium or other cofactors present in the subject's bloodstream, the ADIs of the present disclosure are generally limited to those found in RBCs. In order to address this issue, Applicant specifically chose a cofactor-independent ADI (eg, M. arginini) to ensure long-term in vivo activity of the ADI, without the need to add magnesium or other cofactor products. Applicant represents that other cofactor-independent ADIs can be effectively used in the practice of the disclosed invention.

As used herein, "cofactor-independent ADI" means an ADI that is independent of enzyme cofactors such as vitamins, provitamins, vitamin precursors, or metal ions (eg, magnesium, iron, manganese, etc.). As used herein, "magnesium independent ADI" means an ADI that is independent of magnesium to maintain its enzymatic activity.

Results. The hematological and biochemical characteristics of the 6 end products on day 0 (day of manufacture) are summarized in Table 5 below: three manufactured at 3 mg/mL ADI concentration and three at 5 mg/mL ADI concentration, expressed in terms of pre-dialysis RBC suspension. All ERY-ADI products come from the same ADI lot. In vitro resistance is assessed on day 0, day 1 (table 6) and day 7 (table 7).

Table 5. Hematological and biochemical characteristics of ERY-ADI for VI (day 0)

Regardless of the pre-capture ADI concentration, the capture yield is well reproducible (27.3 to 33.7%).

Table 6. Hematological and biochemical characteristics of ERY-ADI for VI (Day 1)

Table 7. Hematological and biochemical characteristics of ERY-ADI for VI (Day 7)

The uptake of arginine deiminase by human red blood cells appears to be a highly reproducible process. Key parameters are stable at day 0 and day 1 (eg, extracellular hemoglobin, extracellular and intracellular enzyme activity, hematocrit, body volume). On day 7, the measured parameters show that the ERY-ADI product is very stable in vitro, regardless of the concentration of ADI added before the capture (implementation) process.

Conclusion. Arginine deiminase (ADI; EC number 3.5.3.6) captured by red blood cells (RBC) is produced using Erytech's patented ERYCAPS® technology platform. The uptake of therapeutic enzymes by red blood cells can provide long-term therapeutic activity with reduced toxicity.

The introduction of arginine deiminase into red blood cells (ERY-ADI product) significantly improves the pharmacological properties of the enzyme, the decrease in plasma L-arginine is complete within 15 minutes after administration and lasts for 13 days.

Injected into arginase deficient mice, ERY-ADI shows impressive efficacy against the very high blood levels of L-arginine that these mice show. Indeed, 24 hours after administration, the concentration of L-arginine in the blood is reduced by 52% and 92% when 4 or 8 ml/kg are injected, respectively. Three days after administration, the level of L-arginine in the blood remains reduced by 19 and 73%. In addition, despite the production of ammonia by arginine deiminase, the serum level remains comparable to the control level of unloaded RBCs.

These results are confirmed in a second study in which a single injection resulted in a reduction in blood arginine for at least 10 days, and a second injection of ERY-ADI was well tolerated in arginase deficient mice.

Introduction of arginine deiminase into human red blood cells is successful with good reproducibility and stability in vitro.

Based on these results, ERY-ADI appears to be able to counteract the primary biochemical disorder, the rare genetic arginase deficiency disease.

The invention will now be described in the following numbered paragraphs.

1. A pharmaceutical composition comprising arginine deiminase (ADI) encapsulated in erythrocytes, and optionally a pharmaceutically acceptable carrier, for use in the treatment of arginase-1 deficiency, wherein preferably the composition is capable of reducing abnormal plasma or whole blood levels of arginine in a patient or subject, suffering from arginase-1 (Arg1) deficiency to normal physiological or near physiological levels of arginine in plasma or whole blood, and preferably the composition is capable of reducing pathological levels of arginine by at least about 20, 30, 40, 50, 60, 70, or about 80% over a period of at least about 6, 7, 8, 9, 10, or 11 days after administration of a single dose of the composition;

wherein the ADI is optionally one, any combination, or all of the following ADIs: cofactor independent, magnesium independent, iron independent, vitamin independent, provitamin independent; and

and optionally the composition is characterized by any or all of the following ranges of values of these parameters:

hematocrit (%): about 48 - about 51;

body volume (fl): about 82 - about 88;

calf hemoglobin (g/dl): about 72 - about 30;

number of erythrocytes (10 ⁶ / μl): about 5.5 - about 6.2;

total hemoglobin (g/dl): about 13.0 - about 14.5;

extracellular Hb (g / dl): about 0.1 - about 0.2;

concentration of ADI in erythrocytes (mg / ml RBC): about 0.8 - about 1.6;

ADI activity in erythrocytes (U/ml): about 42 - about 49 or about 65 - about 74;

extracellular activity (%): about 0.0 - about 4.0;

intracellular activity (%): about 96 - about 100; and/or

ADI capture output (%): approx. 27 - approx. 34.

2. Pharmaceutical composition for use according to paragraph 1, said composition being a suspension having an osmolarity of 270 to 350 mOsm/l.

3. A pharmaceutical composition for use according to paragraph 1 or 2, wherein the pharmaceutically acceptable carrier is a preservative solution comprising NaCl and adenine.

4. A pharmaceutical composition for use according to any one of paragraphs 1-3, wherein said ADI is an ADI from M. arginini.

5. A pharmaceutical composition for use according to any of paragraphs 1-4, wherein the ADI comprises the amino acid sequence of SEQ ID NO: 1, or a variant or fragment thereof.

6. A pharmaceutical composition for use according to paragraph 5, wherein said variant comprises an amino acid sequence that is at least 80% identical to the amino acid sequence of SEQ ID NO: 1.

7. A pharmaceutical composition for use according to paragraphs 5 or 6, wherein said variant or fragment retains the biological activity of an ADI having the amino acid sequence of SEQ ID NO: 1.

8. Pharmaceutical composition for use according to any one of paragraphs 1-7, wherein the concentration of encapsulated ADI is from 0.1 to 7 mg/ml.

9. A pharmaceutical composition for use according to any of paragraphs 1-8, wherein the pharmaceutical composition is packaged in a dose having a volume of 10 to 250 ml.

10. Pharmaceutical composition for use according to paragraph 9, wherein the amount of encapsulated ADI in a single dose for a patient is from 0.01 mg/kg to 500 mg/kg of encapsulated ADI per kg of body weight of said patient.

11. Suspension of erythrocytes encapsulating ADI from M. arginini.

12. Suspension according to paragraph 11, wherein said ADI comprises the amino acid sequence of SEQ ID NO: 1 or a variant or fragment thereof.

13. The suspension of paragraph 12, wherein said variant includes an amino acid sequence that is at least 80% identical to the amino acid sequence of SEQ ID NO: 1.

14. A pharmaceutical composition comprising a suspension according to any of paragraphs 11-13, for use in the treatment of arginase-1 deficiency or arginine-dependent cancers, the treatment or prevention of septic shock, the inhibition of angiogenesis, and the treatment of angiogenesis-associated diseases.

15. A method of treating arginase-1 deficiency or arginine-dependent cancers, treating or preventing septic shock, inhibiting angiogenesis, and treating angiogenesis-associated diseases, comprising administering to a patient or subject in need thereof a composition or suspension according to any one of paragraphs 1-14.

16. A method of reducing abnormal plasma arginine levels to normal physiological plasma levels in a patient or subject chronically suffering from said abnormal plasma arginine levels, comprising the step of administering more than one dose of a composition or suspension according to any one of paragraphs 1-14.

17. The method of paragraph 16, wherein abnormal plasma levels of arginine are in excess of about 200, 210, 220, 230, 240, 250, 260, 270, 280, 290, 300, 310, 320, 330, 340, 350, 360 , 370, 380, 390, 400, 410, 420, 430, 440, 450, 460, 470, 480, 490, 500, 510, 520, 530, 540, 550, 560, 570, 580, 590 or 600 µM.

18. The method of paragraph 16 or 17, wherein the patient or subject has a known Arg1 mutation or exhibits less than about 5% arginase activity or substantially no arginase activity.

19. The method of paragraph 16 or 17, wherein the abnormal plasma arginine levels are at least about 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or more times higher than in healthy subjects not suffering from abnormal plasma arginine levels.

20. The method of any one of paragraphs 16-19, wherein a single dose of the composition or suspension is sufficient to maintain non-pathologically normal physiological plasma levels of arginine for at least about 1, 2, 3, 4, 5, 6, 7, 8, 9 , 10, 11 or 12 weeks.

21. The method of any one of paragraphs 16-20, wherein the composition or suspension need only be administered once every 2 weeks or once every 4 weeks to maintain physiological plasma levels of arginine.

Further, the invention will be described in the following claims without limitation.

--->

SEQUENCE LIST

<110> ERYTECH PHARMA

<120> Arginine deiminase encapsulated inside erythrocytes and their use

in treating Cancer and Arginase-1 Deficiency

<130> BET 17L3041

<160> 1

<170>PatentIn version 3.5

<210> 1

<211> 410

<212> PRT

<213> Mycoplasma arginini

<400> 1

Met Ser Val Phe Asp Ser Lys Phe Lys Gly Ile His Val Tyr Ser Glu

1 5 10 15

Ile Gly Glu Leu Glu Ser Val Leu Val His Glu Pro Gly Arg Glu Ile

20 25 30

Asp Tyr Ile Thr Pro Ala Arg Leu Asp Glu Leu Leu Phe Ser Ala Ile

35 40 45

Leu Glu Ser His Asp Ala Arg Lys Glu His Lys Gln Phe Val Ala Glu

50 55 60

Leu Lys Ala Asn Asp Ile Asn Val Val Glu Leu Ile Asp Leu Val Ala

65 70 75 80

Glu Thr Tyr Asp Leu Ala Ser Gln Glu Ala Lys Asp Lys Leu Ile Glu

85 90 95

Glu Phe Leu Glu Asp Ser Glu Pro Val Leu Ser Glu Glu His Lys Val

100 105 110

Val Val Arg Asn Phe Leu Lys Ala Lys Lys Thr Ser Arg Glu Leu Val

115 120 125

Glu Ile Met Met Ala Gly Ile Thr Lys Tyr Asp Leu Gly Ile Glu Ala

130 135 140

Asp His Glu Leu Ile Val Asp Pro Met Pro Asn Leu Tyr Phe Thr Arg

145 150 155 160

Asp Pro Phe Ala Ser Val Gly Asn Gly Val Thr Ile His Tyr Met Arg

165 170 175

Tyr Lys Val Arg Gln Arg Glu Thr Leu Phe Ser Arg Phe Val Phe Ser

180 185 190

Asn His Pro Lys Leu Ile Asn Thr Pro Trp Tyr Tyr Asp Pro Ser Leu

195 200 205

Lys Leu Ser Ile Glu Gly Gly Asp Val Phe Ile Tyr Asn Asn Asp Thr

210 215 220

Leu Val Val Gly Val Ser Glu Arg Thr Asp Leu Gln Thr Val Thr Leu

225 230 235 240

Leu Ala Lys Asn Ile Val Ala Asn Lys Glu Cys Glu Phe Lys Arg Ile

245 250 255

Val Ala Ile Asn Val Pro Lys Trp Thr Asn Leu Met His Leu Asp Thr

260 265 270

Trp Leu Thr Met Leu Asp Lys Asp Lys Phe Leu Tyr Ser Pro Ile Ala

275 280 285

Asn Asp Val Phe Lys Phe Trp Asp Tyr Asp Leu Val Asn Gly Gly Ala

290 295 300

Glu Pro Gln Pro Val Glu Asn Gly Leu Pro Leu Glu Gly Leu Leu Gln

305 310 315 320

Ser Ile Ile Asn Lys Lys Pro Val Leu Ile Pro Ile Ala Gly Glu Gly

325 330 335

Ala Ser Gln Met Glu Ile Glu Arg Glu Thr His Phe Asp Gly Thr Asn

340 345 350

Tyr Leu Ala Ile Arg Pro Gly Val Val Ile Gly Tyr Ser Arg Asn Glu

355 360 365

Lys Thr Asn Ala Ala Leu Glu Ala Ala Gly Ile Lys Val Leu Pro Phe

370 375 380

His Gly Asn Gln Leu Ser Leu Gly Met Gly Asn Ala Arg Cys Met Ser

385 390 395 400

Met Pro Leu Ser Arg Lys Asp Val Lys Trp

405 410

<---

Claims (34)

1. Use of a pharmaceutical composition comprising an erythrocyte-encapsulated co-factor-independent arginine deiminase and a pharmaceutically acceptable carrier for the treatment of arginase-1 deficiency. 2. Use according to claim 1, wherein said composition is a suspension having an osmolarity of 270 to 350 mOsm/l. 3. Use according to claim 1 or 2, wherein the pharmaceutically acceptable carrier is a preservative solution comprising NaCl and adenine. 4. Application according to any one of paragraphs. 1-3, wherein said arginine deiminase is arginine deiminase from Mycoplasma arginini . 5. Application according to any one of paragraphs. 1-4, wherein the arginine deiminase comprises the amino acid sequence of SEQ ID NO: 1, or a variant or fragment thereof. 6. Use according to claim 5, wherein said arginine deiminase comprises an amino acid sequence that is at least 80% identical to the amino acid sequence of SEQ ID NO: 1. 7. Use according to claim 5 or 6, wherein said variant or fragment retains the biological activity of an arginine deiminase having the amino acid sequence of SEQ ID NO: 1. 8. Application according to any one of paragraphs. 1-7, wherein the concentration of encapsulated arginine deiminase is from 0.1 to 7 mg/ml. 9. Application according to any one of paragraphs. 1-8, wherein the pharmaceutical composition has a volume of 10 to 250 ml. 10. Use according to claim 9, wherein the amount of encapsulated arginine deiminase in a volume of 10 to 250 ml is from 0.01 mg/kg to 500 mg/kg of encapsulated arginine deiminase per kg of patient body weight. 11. Use of a suspension of erythrocytes encapsulated with cofactor-independent arginine deiminase in the treatment of arginase-1 deficiency to reduce plasma or blood levels of arginine in a patient suffering from arginase-1 deficiency by at least 50%, 60%, 70% or 80% in for at least 6, 7, 8, 9, 10 or 11 days after administration. 12. Use according to claim 11, wherein said arginine deiminase comprises the amino acid sequence of SEQ ID NO: 1, or a variant or fragment thereof. 13. Use according to claim 12, wherein said variant comprises an amino acid sequence that is at least 80% identical to the amino acid sequence of SEQ ID NO: 1. 14. Application according to any one of paragraphs. 11-13 for the treatment of arginase-1 deficiency or arginine-dependent cancers, the treatment or prevention of septic shock, the inhibition of angiogenesis, and the treatment of angiogenesis-associated diseases. 15. A method for treating arginase-1 deficiency, comprising administering a pharmaceutical composition comprising arginine deiminase encapsulated in erythrocytes and a pharmaceutically acceptable carrier. 16. The method of claim 15, wherein said composition is a suspension having an osmolarity of 270 to 350 mOsm/L. 17. The method of claim 15 or 16, wherein the pharmaceutically acceptable carrier is a preservative solution comprising NaCl and adenine. 18. The method according to any one of paragraphs. 15-17, wherein said arginine deiminase is arginine deiminase from Mycoplasma arginini . 19. The method according to any one of paragraphs. 15-18, wherein the arginine deiminase comprises the amino acid sequence of SEQ ID NO: 1, or a variant or fragment thereof. 20. The method of claim 19, wherein said variant comprises an amino acid sequence that is at least 80% identical to the amino acid sequence of SEQ ID NO: 1. 21. The method according to claim 19 or 20, wherein said variant or fragment retains the biological activity of an arginine deiminase having the amino acid sequence of SEQ ID NO: 1. 22. The method according to any one of paragraphs. 15-21, wherein the concentration of encapsulated arginine deiminase is from 0.1 to 7 mg/ml. 23. The method according to any one of paragraphs. 15-22, wherein the pharmaceutical composition has a volume of 10 to 250 ml. 24. The method according to claim 23, wherein the amount of encapsulated arginine deiminase in a single dose for a patient is from 0.01 mg/kg to 500 mg/kg of encapsulated arginine deiminase per kg of body weight of said patient. 25. Use of erythrocytes encapsulated with arginine deiminase for the preparation of a drug for the treatment of arginase-1 deficiency. 26. Use according to claim 25, wherein said erythrocytes are in suspension having an osmolarity of 270 to 350 mOsm/l. 27. Use according to claim 25 or 26, wherein the suspension comprises a preservative solution comprising NaCl and adenine. 28. Application according to any one of paragraphs. 25-27, wherein said arginine deiminase is arginine deiminase from Mycoplasma arginini . 29. Application according to any one of paragraphs. 25-28, wherein the arginine deiminase comprises the amino acid sequence of SEQ ID NO: 1, or a variant or fragment thereof. 30. Use according to claim 29, wherein said arginine deiminase comprises an amino acid sequence that is at least 80% identical to the amino acid sequence of SEQ ID NO: 1. 31. Use according to claim 29 or 30, wherein said variant or fragment retains the biological activity of an arginine deiminase having the amino acid sequence of SEQ ID NO: 1. 32. Application according to any one of paragraphs. 25-31, wherein the concentration of encapsulated arginine deiminase is 0.1 to 7 mg/mL. 33. Application according to any one of paragraphs. 25-32, wherein the erythrocytes are in suspension having a volume of 10 to 250 ml. 34. Use according to claim 33, wherein the amount of encapsulated arginine deiminase is from 0.01 mg/kg to 500 mg/kg of encapsulated arginine deiminase per kg of patient body weight.

Images