Preparations of polyalkylene oxide-asparaginase, and methods for manufacturing and using the same.

Abstract

Embodiments of the present invention include polyalkylene oxide-asparaginase compositions. In some instances, the composition is a lyophilized, storage-stable composition. In some instances, the lyophilized composition includes one or more of a buffer, a salt, and a sugar. Aspects of the invention further include methods for making the composition. The composition finds use in a variety of applications, including, for example, treating a neoplastic condition in a subject.

Description

[Technical Field]

[0001] [Cross-referencing and incorporation by reference of related applications] This application claims priority to U.S. Provisional Patent Application No. 62 / 344,249, U.S. Provisional Patent Application No. 62 / 344,252, and U.S. Provisional Patent Application No. 62 / 344,256, filed on June 1, 2016, each of which is incorporated into this application by reference in whole. [Background technology]

[0002] [Introduction] L-asparaginase is an enzyme that hydrolyzes the amino acid L-asparagine via a deamination reaction to produce L-aspartic acid and ammonia. E. coli is 2 It contains L-asparaginase I and L-asparaginase II, which are types of asparaginase isozymes. L-asparaginase I is present in the cytosol, and asparaginase II It has low affinity for lagin. However, L-asparaginase II has a peri It is present in the plasm and has a high affinity for L-asparagine. E. coli L-asparaginase II is a tetramer of the same subunit. E. coli L-asparaginase II It is also known as L-asparagine amide hydrolase, type EC-2, EC3.5.1.1.

[0003] L-asparaginase is known to have therapeutic value against leukemia. L-asparaginase is an aminohydrolase that catalyzes the reaction of L-asparagine to L-aspartic acid and ammonia. This reaction occurs in plants, animals, and microorganisms. It plays a major role in metabolism. Currently, the therapeutic activity of this enzyme is being investigated in relation to L-asparagus. It has been established that this condition is caused by the depletion / removal of circulating L-asparagine, an essential nutrient for the proliferation and survival of tumor (leukemia) cells with impaired lagin synthesis ability. L-asparagine synthesis ability is not impaired in normal cells.

[0004] Administration of L-asparaginase to leukemia patients induces selective death of tumor cells through hydrolysis of L-asparagine, leading to the treatment of malignant tumors. In some cases, L-asparaginase itself suffers from the typical disadvantages of protein therapies, such as high clearance and the potential to trigger an immune response in patients being treated with this enzyme. To address these shortcomings, polyethylene glycol-bound derivatives of L-asparaginase (PEG-A) are being developed. Asparaginase can be used. Using L-asparaginase II extracted from E. coli, P EG-asparaginase can be produced, which is substantially non-antigenic, and in patients This indicates a decrease in the rate of clearance from circulation.

[0005] PEG-asparaginase liquid injection formulation (Oncaspar®) is already available in the United States. The National Food and Drug Administration has approved commercial marketing of Oncaspar®. Oncaspar® is approved as a first-line component in multi-agent chemotherapy regimens for patients with acute lymphoblastic leukemia (ALL). In addition, Oncaspar® is used in the treatment of ALL patients who are hypersensitive to asparaginase (e.g., the native form of L-asparaginase). It is approved for this purpose. [Overview of the project]

[0006] Embodiments of the present invention include a polyalkylene oxide-asparaginase composition. In one embodiment, the composition comprises one or more buffers and salts. In another embodiment, the composition is frozen It is a dried storage-stable composition. In one example, this lyophilized composition contains one or more buffers, salts, and sugars. Embodiments of the present invention further include a method of making this composition. Various uses are found for this composition, such as in the treatment of neoplastic conditions in a subject.

Brief Description of the Drawings

[0007] [Figure 1] Figure 1 shows a flow diagram of a process for manufacturing a lyophilized storage-stable composition according to an embodiment of the present disclosure. [Figure 2] Figure 2 shows a graph of purity (%) versus time (weeks) at 40°C for a lyophilized storage-stable composition according to an embodiment of the present disclosure. [Figure 3] Figure 3 shows a graph of potency (IU / mL) versus time (weeks) at 40°C for a lyophilized storage-stable composition according to an embodiment of the present disclosure. [Figure 4] Figure 4 shows a graph of purity (%) versus time (weeks) at 25°C for a lyophilized storage-stable composition according to an embodiment of the present disclosure. [Figure 5] Figure 5 shows a graph of potency (IU / mL) versus time (weeks) at 25°C for a lyophilized storage-stable composition according to an embodiment of the present disclosure. [Figure 6] Figure 6 shows a flow diagram of a process for creating a lyophilized storage-stable composition according to an embodiment of the present disclosure. The final formulation and filtration steps are shown. [Figure 7] Figure 7 shows a flow diagram of a process for creating a lyophilized storage-stable composition according to an embodiment of the present disclosure. The aseptic filling and lyophilization steps are shown. [Figure 8] Figure 8 shows a graph of purity (%) versus time (months) by GF-HPLC for a lyophilized composition according to an embodiment of the present disclosure stored at 2 - 8°C (e.g., 5°C). [Figure 9]Figure 9 shows a graph of potency (activity) (IU / mL) versus time (months) of lyophilized compositions according to embodiments of this disclosure, stored at 2–8°C (e.g., 5°C). [Figure 10] Figure 10 shows a graph of total aggregates versus time (months) by GF-HPLC for lyophilized compositions according to embodiments of the present disclosure, stored at 2–8°C (e.g., 5°C). [Figure 11] Figure 11 shows a graph of purity (%) versus time (months) by GF-HPLC for lyophilized compositions according to embodiments of this disclosure, stored under accelerated conditions (25±3°C; 60%±5%RH). [Figure 12] Figure 12 shows a graph of potency (activity) (IU / mL) versus time (months) of lyophilized compositions according to embodiments of this disclosure, stored under accelerated conditions (25±3°C; 60%±5%RH). [Figure 13] Figure 13 shows a graph of total aggregates versus time (months) measured by GF-HPLC for lyophilized compositions according to embodiments of this disclosure, stored under accelerated conditions (25±3°C; 60%±5%RH). [Figure 14] Figure 14 shows a graph of purity (%) versus time (months) by GF-HPLC for lyophilized compositions according to embodiments of this disclosure, stored under thermal stress conditions (40±2°C; 75%±5%RH). [Figure 15] Figure 15 shows a graph of potency (activity) (IU / mL) versus time (months) of lyophilized compositions according to embodiments of this disclosure, stored under thermal stress conditions (40±2°C; 75%±5%RH). [Figure 16] Figure 16 shows a graph of total aggregates versus time (months) measured by GF-HPLC for freeze-dried compositions according to embodiments of this disclosure, stored under thermal stress conditions (40±2°C; 75%±5%RH). [Modes for carrying out the invention]

[0008] [Definition] In describing embodiments of this disclosure, the following terms are used and as set forth below. It is intended to be defined as follows.

[0009] "Substantially purified" means that the sample contains a large portion of the substance as it was originally present. For example, a substantially purified sample may contain 50% or more of the target substance, 60% or more of the target substance, 75% or more of the target substance, 90% or more of the target substance, 95% or more of the target substance, or even 99% or more of the target substance. Any convenient procedure may be used to purify the target substance, and is not limited to, filtration (e.g., disfiltration, ultrafiltration, etc.), selective precipitation, crystallization, ion exchange chromatography, affinity chromatography, and density-based precipitation.

[0010] "Isolated" means that the compound of interest is in an environment different from the environment in which it naturally occurs. "Isolated" means that the compound of interest in the sample is substantially enriched, and / or the compound of interest is purified or substantially purified.

[0011] The terms “patient” and “subject” are used interchangeably and, in their conventional sense, refer to living organisms that are suffering from or prone to suffering from a condition that can be prevented or treated by administration of the compositions of this disclosure, and include both humans and non-human animals. Examples of subjects include, but are not limited to, humans, chimpanzees and other primates and primates; domestic animals such as cattle, sheep, pigs, goats and horses; domesticated mammals such as dogs and cats; laboratory animals including rodents such as mice, rats and guinea pigs; and birds including chickens, turkeys and other poultry, domestic birds such as ducks and geese, and wild and game birds. The terms do not indicate a specific age; therefore, grown, young and newborn individuals are included.

[0012] "Pharmacologically effective amount" and "therapeutically effective amount" refer to an amount of compound or composition sufficient to treat a particular disease or disorder or one or more symptoms thereof, and / or to prevent that disease or disorder. With respect to tumor conditions, a pharmacochemically or therapeutically effective amount refers, among other things, to an amount sufficient to cause a reduction in the amount and / or incidence of cancer in a patient, and / or to reduce the rate of cancer growth.

[0013] As used herein, the terms “to treat” or “treatment” mean treating or treating a disease or medical condition in a patient, such as a mammal (e.g., a human), and include: (a) preventing the onset of a disease or medical condition, such as prophylactic treatment of a subject; (b) alleviating a disease or medical condition, such as causing the elimination or reduction of the disease or medical condition in a patient; (c) suppressing a disease or medical condition, for example, by delaying or halting its progression; or (d) alleviating the symptoms of a disease or medical condition in a patient.

[0014] The term "physiological state" encompasses conditions suitable for living cells, such as primarily aqueous conditions of pH and salinity that are suitable for living cells.

[0015] Before the embodiments of this disclosure are described in detail, it should be understood that embodiments can vary and are not limited to any specific embodiments described herein. It should also be understood that the terminology used herein is used only to describe specific embodiments and is not intended to be restrictive. The scope of the embodiments of this disclosure is limited only by the appended claims. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as those commonly understood by those skilled in the art to which the invention pertains. Where a range of values ​​is provided, unless the context explicitly indicates otherwise, the values ​​between the upper and lower limits of the range are not limited to any particular value. It should be understood that all other declared or intervening values ​​within the declared range, down to one-tenth of their lower limits, are all included in the embodiments of this disclosure. These upper and lower limits of smaller ranges are independently included in and also included in the embodiments of this disclosure, and are governed by certain excluded limits from the declared range. If a declared range includes one or both limits, the range excluding either or both of the limits included therein is also included in the invention. Certain ranges expressed herein are represented numerically with the term “about” prefixed. As used herein, the term “about” is a literal aid to providing a number close to or approximate to the preceding exact number. To determine whether a given number is close to or approximate to a given number, the number that is close to or approximate provides a substantial equivalent to the particular listed number in the context in which it is expressed.

[0016] All publications, patents, and patent applications referenced herein are incorporated by reference to the same extent as if each individual publication, patent, and patent application were explicitly and individually indicated by reference, so as to be incorporated by reference. Furthermore, each listed publication, patent, and patent application is incorporated by reference herein to disclose and describe the subject matter from which the publication is cited. The cited publications are provided merely as disclosures prior to the filing date of this application and should not be construed as an acknowledgment that the inventions herein are not prior to such publications on the grounds of prior art. Furthermore, the provided publication dates may differ from the actual publication dates, which must be individually verified.

[0017] It should be noted that the claims are drafted to exclude any discretionary elements. Such statements are intended to serve as a basis for precedent for the use of exclusionary terms such as “solely” and “only” in relation to the elements of the claims or the use of “negative” limitations. As will be apparent to those skilled in the art when reading this disclosure, each of the individual embodiments described herein has separate elements and features that are readily separable from or combined with the elements and features of other embodiments without departing from the scope and spirit of the embodiments of this disclosure. Any enumerated method may be performed in the order of the enumerated events or in any logically possible order. Any method and substance similar to or equivalent to those described herein may also be used in the implementation or testing of the embodiments of this disclosure. Representative explanatory methods and substances are disclosed.

[0018] [Detailed description of the invention] Aspects of the present invention include a polyalkylene oxide-asparaginase composition. In some examples, the composition comprises one or more buffers and salts. Aspects of the present invention further include a method for preparing this composition. This composition has various applications, such as in the treatment of neoplastic conditions in subjects.

[0019] In other embodiments, the composition is a lyophilized, storage-stable composition. Embodiments of the present invention include a lyophilized, storage-stable polyalkylene oxide-asparaginase composition. In one example, the lyophilized composition comprises one or more buffers, salts, and sugars. Embodiments of the present invention further include a method for preparing the composition. The composition has various applications, such as the treatment of tumor conditions in subjects (e.g., acute myeloid leukemia (AML) in subjects).

[0020] Aspects of the present invention include administering an effective dose of polyalkylene oxide-asparaginase. Aspects of the present invention further include compositions and kits comprising polyalkylene oxide-asparaginase that can be used to treat a subject.

[0021] In further describing embodiments of this disclosure, compositions (e.g., liquid and lyophilized) are first described in detail. Then, methods of preparation and use, as well as kits containing the compositions, are described.

[0022] [Composition] Aspects of the present disclosure include a polyalkylene oxide-asparaginase composition comprising a polyalkylene oxide group covalently bonded to asparaginase by a linker. This composition may also include one or more buffers and salts. In certain embodiments, this composition is a lyophilized, storage-stable composition. The lyophilized, storage-stable composition may also include one or more salts and sugars.

[0023] The compositions described herein and disclosed herein may include polyalkylene oxide-asparaginase. Polyalkylene oxide-asparaginase comprises asparaginase covalently bonded to one or more polyalkylene oxide groups by linkers. Asparaginase is an enzyme composed of four identical subunits, with one active site per tetramer. For example, asparaginase enzyme is L-asparaginase (for example) If so, it can be L-asparaginase II, which follows the following reaction, and amino acids L-asparagine ((S)-2,4-diamino-4-oxobutanoic acid, or asparagus From ginine (also known as Asn or N), L-aspartic acid (also known as (S)-2-aminosuccinic acid) and ammonia are produced: [ka]

[0024] In some cases, asparaginase reacts with the amino acid L-glutamin according to the following reaction. From ((S)-2,5-diamino-5-oxopentanoic acid, abbreviated as Gln or Q), L-glutamic acid ((S)-2-aminopentanediocytic acid) (and produces ammonia): [ka]

[0025] The above reaction mediated by L-asparaginase is also called a deamination reaction. In one example, the L-asparaginase in the composition is, but is not limited to, E. coli. It is derived from a source of prokaryotes, such as microorganisms containing (i.). In that case, the asparaginase in the composition of interest is E. coli asparaginase. In some cases, the asparaginase is expressed by E. coli. This asparaginase is recovered and purified from a culture medium containing E. coli expressing the asparaginase. In addition to wild-type asparaginase, the asparaginase can also be a non-natural type of asparaginase and / or a synthetically produced asparaginase and / or an active fragment of a natural type and / or synthetic asparaginase. The asparaginases that can be used in embodiments of the present invention are, but are not limited to, those listed below: 9,322,008; 9,127,266; 9,051,561; 8,617,868; 7,807,436; 6,991,788; 6,537,547; 6,436,396; 6,368,845; 6,274,367; 6,251,388; 6,165,735; 6,140,101; 6,087 ,151;6,042,825;5,854,051;5,310,670;4,729,957 and 4,617,271;These disclosures are incorporated herein by reference.

[0026] As described above, the asparaginase in the polyalkylene oxide-asparaginase composition is an asparaginase covalently bonded to one or more polyalkylene oxide groups. For example, this asparaginase may, in a post-translational process, contain asparaginase covalently bonded to one or more polyalkylene oxide groups. Polyalkylene oxide-asparaginase may contain asparaginase covalently bonded to one or more polyalkylene oxide groups at one or more positions of the asparaginase. For example, the polyalkylene oxide group can be covalently bonded to an amino acid residue of the asparaginase. In some embodiments, this polyalkylene oxide group can be covalently bonded to the amino acid side chain of the N-terminal amino acid of the asparaginase. They are covalently bonded. In some embodiments, the polyalkylene oxide group is covalently bonded to the ε-amino group of lysine (K) in asparaginase. In some embodiments, the polyalkylene oxide group is covalently bonded to the amino acid side chain of the N-terminal amino acid of asparaginase and to the ε-amino group of lysine (K). In some cases, the polyalkylene oxide-asparaginase is substantially non-antigenic. "Non-antigenic" or "substantially non-antigenic" means that the composition does not elicit a significant immune response when administered to a subject. In some examples, the polyalkylene oxide-asparaginase has a reduced clearance rate from the subject's circulation compared to unmodified asparaginase. For example, the efflux half-life of polyalkylene oxide-asparaginase may be 1 day or more, 2 days or more, or 3 days or more, or 4 days or more, or 5 days or more, or 6 days or more, or 7 days or more, or 8 days or more, or 9 days or more, or 10 days or more, or 11 days or more, or 12 days or more, or 13 days or more, or 14 days or more, or 15 days or more, or 16 days or more, or 17 days or more, or 18 days or more, or 19 days or more, or 20 days or more, etc. In some embodiments, the efflux half-life of polyalkylene oxide-asparaginase is 3 days or more. In some embodiments, the efflux half-life of polyalkylene oxide-asparaginase is 5 days or more.

[0027] The polyalkylene oxide group bound to asparaginase may be any physiologically acceptable polyalkylene oxide group. Poly(alkylene oxide)(PAO), also known as polyoxyalkylene (POA), is synthesized by polymerization of alkylene oxides (e.g., ethylene oxide, propylene oxide, butylene oxide). Homopolymers are synthesized from only one type of alkylene oxide, while copolymers are synthesized from two or more different alkylene oxides and are known as alkylene oxide copolymers (AOCs). Examples of the former are poly(ethylene oxide) (PEO), a polymer of ethylene oxide (EO), and poly(propylene oxide) (PPO), a polymer of propylene oxide (PO). Poly(ethylene oxide) is also commonly known as polyethylene glycol (PEG) or polyoxyethylene (POE). The molecular weight of such polymers is generally characterized as the average length (or repeating unit). In addition to the standard linear shape, branched or star-shaped poly(alkylene oxides) exist, each serving as a starting point for the growth of the polymer chain. These polymers can be synthesized by initiating a polymerization reaction with a polyfunctional initiator containing multiple hydroxyl groups, amino groups, or thiol groups. For example, using glycerin (which has three hydroxyl groups) as an initiator yields a three-armed branched polymer, while pentaerythritol yields a four-armed polymer. Traditionally, polymers of this type with 3 to 10 arms are called "branched," while those with 10 or more arms are called "star" polymers. "Comb" copolymers are similar to branched and star copolymers, but the initiators of comb copolymers each function as the starting point for the growth of the polymer chain. A polyfunctional polymer having multiple hydroxyl groups, amino groups, or thiol groups arranged along the initiator's backbone. A "grafted" copolymer is formed by adding pendant polymer chains along a polymer backbone having unsaturated C=C bonds, or by reacting the pendant chains. They can be synthesized by adding a pendant functional group (e.g., a hydroxyl group) to a monofunctional polymer chain. All poly(alkylene oxides) contain a single residue corresponding to the molecule used to initiate the polymer synthesis, in addition to the repeating units derived from the alkylene oxide. For linear polymers, the alkylene corresponds to the alkylene oxide used in the synthesis. Glycols (for example, ethylene glycol and ethylene oxide, respectively) This is possible, and therefore the residues derived from the initiator are indistinguishable from the other repeating units of the polymer chain. Low molecular weight molecules other than alkylene glycols are often used as initiators, examples of which are methanol or N-butanol (for linear polymers) and trimethyl This includes rollpropane, glycerol, and pentaerythritol (for branched polymers) or ethylenediamine. The mass of the initiator compared to the mass of the final polymer chain is generally very small and usually negligible. Therefore, the term poly(alkylene oxide) is used herein in a conventional sense and includes both poly(alkylene oxide) initiated from alkylene glycol molecules and poly(alkylene oxide) initiated from other low molecular weight molecules.

[0028] In certain embodiments, the physiologically acceptable polyalkylene oxide group is substantially stable under conditions compatible with living cells, such as temperatures, pH, and salt concentrations that are mostly aqueous and compatible with living cells. In certain embodiments, the polyalkylene oxide The group is water-soluble. The term "water-soluble polymer" refers to a polyalkylene oxide group that is substantially soluble in water, such as in the aqueous state found in the body of a subject. The polyalkylene oxide group of interest includes, but is not limited to, linear polyalkylene glycols. The linear polyalkylene glycol used in specific embodiments of the present invention has the following chemical formula: [ka]

[0029] In the formula, R is selected from the group consisting of hydrogen, lower alkyl groups, and mixtures thereof, R1 is selected from the group consisting of hydrogen and lower alkyl groups, and n is a positive integer. "Lower alkyl group" means an alkyl group having 1 to 4 carbon atoms, i.e., methyl, ethyl, propyl, butyl, and their isomers. R can be selected from the group consisting of hydrogen, methyl, and mixtures thereof, R1 can be selected from the group consisting of hydrogen and methyl, and n can be a positive integer selected to provide the size of the desired polymer.

[0030] In some examples, the poly(alkylene glycol) used in embodiments of the present invention is poly(ethylene glycol), poly(propylene glycol), mixtures thereof, and copolymers of poly(ethylene glycol) and poly(propylene glycol), where one of the terminal hydroxyl groups of the polymer may be substituted with a lower alkyl group. In some embodiments, the polyalkylene oxide is polyethylene glycol (PEG). In certain embodiments, this polyethylene glycol (PEG) has a molecular weight of 1,000 to 20,000 daltons. In certain embodiments, this PEG has a molecular weight of 1,000 to 20,000 daltons, or 1,000 to 19,000 daltons, or 1,000 to 18,000 daltons, or 1,000 to 17,000 daltons, or 1,000 to 16,000 daltons, or 1,000 to 15,000 daltons, or 1,000 to 14,000 daltons, or 1,000 to 13,000 daltons, or 1,000 to 12,000 daltons. The PEG has a molecular weight of 1,000 to 11,000, 1,000 to 10,000, 1,500 to 10,000 daltons, 2,000 to 10,000 daltons, 2,000 to 9,000 daltons, 2,000 to 8,000 daltons, 2,000 to 7,000 daltons, 2,000 to 6,000 daltons, 3,000 to 6,000 daltons, 4,000 to 6,000 daltons, or 4,500 to 5,500 daltons. In a particular embodiment, the PEG has a molecular weight of 2,000 to 10,000 daltons. In a particular embodiment, the PEG has a molecular weight of 4,000 to 6,000 daltons. In a particular embodiment, the PEG has a molecular weight of 5,000 daltons. In one example, the polyethylene glycol is methoxypolyethylene glycol (e.g., monomethoxypolyethylene glycol, or "mPEG").

[0031] As described above, polyalkylene oxide groups can be covalently bound to asparaginase. In some cases, the polyalkylene oxide group is covalently bound to asparaginase via a linker. In this case, the polyalkylene oxide group can be covalently bound to asparaginase via a linker. This linker may be any convenient linker that enables the binding of the polyalkylene oxide group to asparaginase. For example, it may include a reactive functional group that provides a covalent bond between the polyethylene oxide group and asparaginase. In some cases, this linker includes a reactive functional group that provides a covalent bond between the polyethylene oxide group and the amino acids of the amino acid residues of asparaginase. For example, this linker may include a reactive functional group that provides a covalent bond between the polyethylene oxide group and the amino groups of the amino acid residues of asparaginase. Examples of such reactive functional groups include, but are not limited to, p-nitrophenoxy, thiazolidinylthione, and N-hydride Roxisuccinimidyl, or, but not limited to, N-hydroxybenzotriazolyl It contains other suitable reactive functional groups such as halogens, N-hydroxyphthalimidyl, imidazolyl, O-acylurea, pentafluorophenol, or 2,4,6-trichlorophenol. In some cases, the reactive functional group of the linker is N-hydroxysuccinimidyl. Yes. Therefore, in the covalent bonding of the polyalkylene oxide group to asparaginase, this linker may, but is not limited to, include functional groups such as urethane linkers (also known as carbamate linkers) and succinic acid linkers.

[0032] In certain embodiments, the linker includes a urethane linker (also known as a carbamate linker). For example, the reaction of a polypeptide (e.g., asparaginase) of methoxypolyethylene glycol (mPEG) to the amino group of an amino acid via a urethane (carbamate) is shown below. [ka]

[0033] In the reaction shown above, methoxypolyethylene glycol succinimidoyl carbonate (also known as SC-PEG) reacts with polypeptides (e.g., asparaginase). It reacts with the amino group of the amino acid to produce polyethylene glycol-asparaginase with a urethane (carbamate) linker. SC-PEG-asparaginase is produced by Angioli llo, AL., et al., “Pharmacokinetic (PK) and pharmacodynamics (PD) properties of SC-PEG E. coli 1-asparaginase (EZN-2285) in the O ncology Group (COG) study AALL07P4”, 2012 A American Society of Clinical Oncology (ASCO) ) Annual Meeting, Poster 9543;; and Angiolillo, AL., et al., "Pharmacokinetic and Pharmacodynamic Properties of Pegol E scherichia coli L-Asparaginase in the Trea tment of Patients With Acute Lymphoblastic Leukemia: Results From Children's Oncology Group Study AALL07P4”, J Clin. Oncology, 3 It is also described in 2(34),2014,3874-3882.

[0034] In certain embodiments, the linker is a succinate linker (also called a succinyl linker). For example, the linking of amino acids to the amino groups of a polypeptide of methoxypolyethylene glycol (mPEG) (e.g., asparaginase) via a succinate linker is shown below. [ka]

[0035] In the reaction shown above, methoxypolyethylene glycol succinimidyl succinate (also known as SS-PEG) reacts with polypeptides (e.g., asparaginase). Polyethylene glycol-asparagus reacts with the amino group of the ano acid to form a succinic acid linker. It produces ginase. SS-PEG-asparaginase is also described in U.S. Patents 5,122,614; 5,324,844; and 5,612,460, the respective disclosures of which are incorporated herein by reference.

[0036] In certain embodiments, the composition containing polyalkylene oxide-asparaginase is a dehydrated composition. The dehydrated compositions used herein are defined as having a concentration of 25% or less, or 20% or less, by Karl Fischer (KF) titration. The composition contains a low amount of water, such as 15% or less, or 10% or less, or 9% or less, or 8% or less, or 7% or less, or 6% or less, or 5% or less, or 4% or less, or 3% or less, or 2% or less, or 1% or less. In some cases, the dehydrated composition contains 3% or less water by Karl Fischer titration. In some cases, the dehydrated composition contains 1% or less water by Karl Fischer titration. In some cases, the dehydrated composition contains 0.5% or less water by Karl Fischer titration. Any convenient procedure can be used to produce the dehydrated composition, such as increasing the temperature of the composition (e.g., heating), decreasing the pressure, freeze-drying (also known as lyophilization), and combinations thereof.

[0037] In certain embodiments, freeze-drying is used to produce a dehydrated composition, and thus this composition (e.g., a composition containing polyalkylene oxide-asparaginase) is a freeze-dried composition. In some examples, a freeze-dried composition is a composition from which water has been removed by sublimation, where the water in the composition undergoes a phase transition from solid to gas. For example, a freeze-dried composition may be a composition from which water has been removed by freezing the composition (e.g., freezing the water in the composition) and then reducing the pressure around the composition so that the water in the composition sublimes. As described above, freeze-dried compositions have a water content of 25% or less, or 20% or less, or 15% or less, or 10% or less, or 9% or less, or 8% or less, etc., as measured by Karl Fischer titration. The moisture content may be 7% or less, or 6% or less, or 5% or less, or 4% or less, or 3% or less, or 2% or less, or 1% or less, or 0.5% or less, or 0.25% or less, or 0.1% or less. In certain embodiments, the lyophilized composition may contain a low moisture content, as measured by Karl Fischer titration, such as about 0.1% to about 25%, or about 0.25% to about 20%, or about 0.5% to about 15%, or about 1% to about 10%, or about 2% to about 9%, or about 3% to about 8%, or about 4% to about 7%, or about 5% to about 6%. In embodiments, the lyophilized composition may contain a low moisture content, as measured by Karl Fischer titration, such as about 0.1% to about 5%, or about 0.25% to about 4%, or about 0.5% to about 3%, or about 1% to about 2%. In some cases, the lyophilized composition contains 3% or less moisture, as measured by Karl Fischer titration. In other cases, the lyophilized composition contains 1% or less moisture, as measured by Karl Fischer titration. In other cases, the lyophilized composition contains 0.5% or less moisture, as measured by Karl Fischer titration.

[0038] Due to the low moisture content of lyophilized compositions, as described above, lyophilized compositions can be in solid form. In some cases, solid lyophilized compositions are powders. In some cases, lyophilized compositions facilitate the storage of compositions over long periods (e.g., compared to liquid formulations of the same composition). For example, a lyophilized composition can be a storage-stable composition (e.g., a lyophilized storage-stable composition) that is substantially stable over long periods. "Stable," "substantially stable," or "storage-stable" means a composition that does not significantly decompose and / or does not lose activity over extended periods. For example, a storage-stable composition may not contain significant impurities such as 10% or less of impurities, or 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, or 1% or less of decomposition products resulting from the decomposition of the composition over long periods. In certain embodiments, the storage-stable composition may have degradation products of about 1% to about 10%, or about 2% to about 9%, or about 3% to about 8%, or about 4% to about 7%, or about 6% to about 5% over a long period of time. In certain examples, the storage-stable composition may have impurities of 5% or less over a long period of time. In some cases, the storage-stable composition substantially retains its activity over a long period of time, such as retaining 100% or more of its activity, or 99% or more, or 98% or more, or 97% or more, or 96% or more, or 95% or more, or 94% or more, or 93% or more, or 92% or more, or 91% or more, or 90% or more, or 85% or more, or 80% or more, or 75% or more over a long period of time. In some embodiments, the storage-stable composition substantially retains its activity over a long period of time, such as retaining about 75% to about 100%, or about 80% to about 99%, or about 85% to about 98%, or about 90% to about 97%, or about 91% to about 96%, or about 92% to about 95%, or about 93% to about 94% or more of its activity over a long period of time. For example, the storage-stable composition substantially retains 90% or more of its activity over a long period of time.In some cases, the storage-stable composition substantially retains 95% or more of its activity over a long period of time. A long period of time is defined as a period of time of one week or more, or two weeks or more, or three weeks or more, or one month or more, or two months or more, or three months or more, or four months or more, or six months or more, or nine months or more, or one year or more, or 1.5 years (e.g., 18 months) or more, or two years or more, or 2.5 years (e.g., 30 months) or more, or three years or more, or 3.5 years (e.g., 42 months) or more, or four years or more, or 4.5 years (e.g., 54 months) or more, or five years or more. For example, a long period of time can be six months or more. In some cases, a long period of time can be nine months or more. In some cases, a long period of time can be one year (e.g., 12 months) or more. In some cases, a long period of time can be 1.5 years (e.g., 18 months) or more. In some cases, it can be two years (e.g., 24 months) or more. In some embodiments, a long period of time. The storage period can be approximately 1 to 3 weeks, or approximately 1 to 6 months, or approximately 6 to 9 months, or approximately 1 to 1.5 years, or approximately 1 to 2 years, or approximately 1 to 3 years, or approximately 1 to 4 years, or approximately 1 to 5 years. In some embodiments, the storage-stable composition is substantially stable over long periods at room temperature such as 20 to 40°C, or 25 to 35°C, or 25 to 30°C. In some examples, the storage-stable composition is substantially stable over long periods at temperatures lower than room temperature such as 0 to 20°C, 0 to 15°C, 0 to 10°C, or 2 to 8°C.

[0039] In some cases, the composition contains a therapeutically effective amount of polyalkylene oxide-asparaginase. The enzymatic activity of polyalkylene oxide-asparaginase is measured in international units (IU) and corresponds to the amount of enzyme required to produce 1 μmol of ammonia per minute at a pH of 7.3 and a temperature of 37°C. In some cases, the polyalkylene oxide-asparaginase can be present in the composition in amounts ranging from 100 to 5,000 IU / g (for example, in which this polyalkylene oxide-asparaginase is potent (active)), such as 500 to 4,500 IU / g, or 500 to 4,000 IU / g, or 500 to 3,500 IU / g, or 500 to 3,000 IU / g, or 500 to 2,500 IU / g, or 500 to 2,000 IU / g, or 500 to 1,500 IU / g, or 500 to 1,000 IU / g, or 600 to 900 IU / g, or 700 to 800 IU / g. In specific examples, this polyalkylene oxide-asparaginase can be present in the composition in amounts ranging from 500 to 1,000 IU / g. For example, this polyalkylene oxide-asparaginase is present in the composition with a potency (activity) of 500-1,000 IU / g. In specific examples, this polyalkylene oxide-asparaginase is present in the composition in amounts ranging from 700-800 IU / g. For example, this polyalkylene oxide-asparaginase has a potency (activity) in the range of 700-800 IU / g. It may have potency (activity). In certain cases, this polyalkylene oxide-asparaginase is present in the composition at an amount of 750 IU / g. For example, this polyalkylene oxide-asparaginase may have potency (activity) of 750 IU / g.

[0040] In some cases, this composition contains a therapeutically effective amount of polyalkylene oxide-asparaginase, which is present in amounts of 55 IU / mg protein or more, or 60 IU / mg protein or more, or 65 IU / mg protein or more, or 70 IU / mg protein or more, or 75 IU / mg protein or more, or 80 IU / mg protein or more, or 85 IU / mg protein or more, or 90 IU / mg protein or more, or 95 IU / mg protein or more. Alternatively, it has a specific activity of 50 IU / mg protein or more, such as 100 IU / mg protein or more, or 105 IU / mg protein or more, or 110 IU / mg protein or more, or 115 IU / mg protein or more, or 120 IU / mg protein or more, or 125 IU / mg protein or more, or 130 IU / mg protein or more, or 135 IU / mg protein or more, or 140 IU / mg protein or more, or 145 IU / mg protein or more, or 150 IU / mg protein or more. For example, the polyalkylene oxide-asparaginase in this composition may have a specific activity of 85 IU / mg protein or more. In some embodiments, the polyalkylene oxide-asparaginase in the composition has a specific activity of 50-150 IU / mg protein, or 55-145 IU / mg protein, or 60-140 IU / mg protein, or 65-135 IU / mg protein, or 70-130 IU / mg protein, or 75-125 IU / mg protein, or 80-120 IU / mg protein, or 95-105 IU / mg protein. In some cases, the polyalkylene oxide-asparaginase in the composition has a specific activity of 50-150 IU / mg protein, such as 65-140 IU / mg protein, or 70-135 IU / mg protein, or 75-130 IU / mg protein, or 75-125 IU / mg protein. For example, composition The polyalkylene oxide-asparaginate contained within was 75-125 IU / mg of protein. It may have a specific activity within a range.

[0041] In certain embodiments, a composition comprises a therapeutically effective amount of polyalkylene oxide-asparaginase, and the polyalkylene oxide-asparaginase is present in the composition in an amount ranging from 1.5 mg / mL to 14.5 mg / mL, or from 2 mg / mL to 14 mg / mL, or from 2.5 mg / mL to 13.5 mg / mL, or from 3 mg / mL to 13 mg / mL, or from 3.5 mg / mL to 12.5 mg / mL, or from 4 mg / mL to 12 mg / mL, or from 4.5 mg / mL to 11.5 mg / mL, or from 4.5 mg / mL to 11 mg / mL, or from 4.5 mg / mL to 10.5 mg / mL, or from 4.5 mg / mL to 10 mg / mL, or from 4.5 mg / mL to 9.5 mg / mL, or from 4.5 mg / mL to 9 mg / mL, or from 4.5 mg / mL to 8.5 mg / mL, or from 5 mg / mL to 8 mg / mL, etc., within the range of 1 mg / mL to 15 mg / mL. In some cases, the alkylene oxide-asparaginase is present in the composition in an amount ranging from 4.5 mg / mL to 8.5 mg / mL. is present.

[0042] When administered to a subject, this composition provides from 500 to 5,000 IU / m 2 or from 500 to 4,500 IU / m 2 or from 500 to 4,000 IU / m 2 or from 500 to 3,500 IU / m 2 or from 500 to 3,000 IU / m 2 or from 1,000 to 3,000 IU / m 2 or from 1,500 to 3,000 IU / m 2 or from 1,750 to 3,000 IU / m 2 or from 2,000 to 3,000 IU / m 2 or from 2,000 to 2,750 IU / m 2 or from 2,250 to 2,750 IU / m 2 of polyalkylene oxide-asparaginase, etc., within the range of 100 to 5,000 IU / m 2 of polyalkylene oxide-asparagi The composition may contain a sufficient amount of polyalkylene oxide-asparaginase to deliver the enzyme to the subject. For example, this composition may contain 1,500-3,000 IU / m³ 2 The composition may contain a sufficient amount of polyalkylene oxide-asparaginase to deliver the polyalkylene oxide-asparaginase to a subject. In a specific example, this composition may contain 2,000-2,750 IU / m³ 2 The composition may contain a sufficient amount of polyalkylene oxide-asparaginase to deliver the polyalkylene oxide-asparaginase to the subject. In a specific example, this composition may contain 2,250-2,750 IU / m³ 2 The composition may contain a sufficient amount of polyalkylene oxide-asparaginase to deliver the polyalkylene oxide-asparaginase to a subject. For example, this composition may contain 2,500 IU / m³ 2 It may contain a sufficient amount of polyalkylene oxide-asparaginase to deliver the polyalkylene oxide-asparaginase to the subject.

[0043] In certain embodiments, the dosage form administered to the subject is a liquid dosage form, for example, an aqueous dosage form. In some embodiments, in addition to polyalkylene oxide-asparaginase, this dosage form includes a buffer and a salt.

[0044] The compositions of this disclosure may include additional components in addition to polyalkylene oxide-asparaginase. For example, the composition may include a buffer. A buffer suitable for use in the compositions of this disclosure includes a buffer compatible with polyalkylene oxide-asparaginase and suitable for administration to a subject, for example, by injection or intravenous administration. Examples of suitable buffers, but not limited to, include phosphate buffers (e.g., phosphate-buffered saline (PBS)), Dulbecco's phosphate-buffered saline (DPBS), Hank's balanced saline solution (HBSS), Earle's balanced saline solution (EBSS), Tris buffer, Ringer's lactate buffer, and combinations thereof. The buffer included in this composition may be a buffer that maintains the pH of the composition in the range of 6 to 8, or a physiologically acceptable pH of about 7, for example, 7.2, 7.3, or 7.4. In one example, this is a phosphate buffer. The crevice may contain dibasic sodium phosphate (disodium phosphate or sodium hydrogen phosphate; also known as Na2HPO4) and / or monobasic sodium phosphate (monosodium phosphate; also known as NaH2PO4).

[0045] In some cases, the amount of dibasic sodium phosphate in the composition is in the range of 0.05 to 5 wt.%, such as 0.1 to 4.5 wt.%, or 0.1 to 4 wt.%, or 0.1 to 3.5 wt.%, or 0.1 to 3 wt.%, or 0.1 to 2.5 wt.%, or 0.1 to 2 wt.%, or 0.1 to 1 wt.%, or 0.1 to 0.9 wt.%, or 0.1 to 0.8 wt.%, or 0.1 to 0.7 wt.%, or 0.1 to 0.6 wt.%, or 0.2 to 0.6 wt.%, or 0.3 to 0.6 wt.%, or 0.4 to 0.6 wt.%, or 0.5 to 0.6 wt.%. For example, dibasic sodium phosphate can be present in the composition in the range of 0.1 to 1.0 wt.%. In certain cases, dibasic sodium phosphate can be present in a composition in the range of 0.2–0.8 wt.%. In certain cases, dibasic sodium phosphate can be present in a composition in the range of 0.3–0.6 wt.%. In certain cases, dibasic sodium phosphate can be present in a composition in the range of 0.5–0.6 wt.%. For example, dibasic phosphate can be present in a composition in the range of approximately 0.6 wt.%, such as 0.56 wt.% (or 0.558 wt.%). In certain embodiments, the amount of monobasic sodium phosphate in the composition is 0.01 to 1.8 wt.%, or 0.01 to 1.6 wt.%, or 0.01 to 1.4 wt.%, or 0.01 to 1.2 wt.%, or 0.01 to 1.0 wt.%, or 0.01 to 0.8 wt.%, or 0.01 to 0.6 wt.%, or 0.01 to 0.4 wt.%, or 0.0 The range is 0.005 to 2 wt.%, such as 1-0.2 wt.%, or 0.02-0.18 wt.%, or 0.03-0.16 wt.%, or 0.04-0.16 wt.%, or 0.045-0.15 wt.%, or 0.04-0.14 wt.%, or 0.05-0.14 wt.%, or 0.1-0.2 wt.%, or 0.1-0.15 wt.%. For example, monobasic sodium phosphate can be present in a composition in the range of 0.05-0.2 wt.%. In certain examples, monobasic sodium phosphate can be present in a composition in the range of 0.01-0.2 wt.%. In certain examples, monobasic sodium phosphate can be present in a composition in the range of 0.09-0.15 wt.%.In certain cases, monobasic sodium phosphate can be present in a composition in the range of 0.1–0.2 wt.%. In certain cases, monobasic sodium phosphate can be present in a composition in the range of 0.1–0.15 wt.%. For example, monobasic sodium phosphate can be present in a composition in an amount of 0.12 wt.% (or 0.129 wt.%).

[0046] Another additional component that may be included in the compositions of this disclosure is a salt. Suitable salts for use in the compositions of this disclosure include salts that are compatible with polyalkylene oxide-asparaginase and suitable for administration to a subject, for example, by injection or intravenous administration. Examples of suitable salts include, but are not limited to, sodium chloride, potassium chloride, calcium chloride, magnesium chloride, and combinations thereof. In a particular example, this salt is sodium chloride.

[0047] In some cases, the amount of salt (e.g., sodium chloride) in the composition is in the range of 0.05 to 5 wt.%, such as 0.05 to 4 wt.%, or 0.05 to 3 wt.%, or 0.05 to 2 wt.%, or 0.1 to 5 wt.%, or 0.1 to 4 wt.%, or 0.1 to 3 wt.%, or 0.1 to 2 wt.%, or 0.1 to 1.5 wt.%, or 0.1 to 1 wt.%, or 0.2 to 1 wt.%, or 0.3 to 1 wt.%, or 0.4 to 1 wt.%, or 0.5 to 1 wt.%, or 0.6 to 1 wt.%, or 0.7 to 1 wt.%, or 0.8 to 1 wt.%, or 0.8 to 0.9 wt.%. For example, salt (e.g., table salt) can be present in the composition in the range of 0.5 to 1 wt.%. For example, salt (e.g., table salt) can be present in the composition in the range of 0.2 to 2 wt.%. In a specific example, salt ( For example, a salt (e.g., table salt) can be present in the composition in an amount ranging from 0.7 to 1 wt.%. In certain cases, a salt (e.g., table salt) can be present in the composition in an amount ranging from 0.8 to 0.9 wt.%. For example, a salt (e.g., sodium chloride) can be present in the composition in an amount of 0.85 wt.%.

[0048] In certain embodiments, the composition comprising polyalkylene oxide-asparaginase is a lyophilized composition. The lyophilized composition of the present disclosure may also comprise a buffer, a salt, and a sugar in addition to the polyalkylene oxide-asparaginase. For example, an embodiment of the present disclosure comprises a lyophilized, storage-stable composition of polyalkylene oxide-asparaginase comprising a polyalkylene oxide group covalently bonded to the asparaginase by a linker, a buffer, a salt, and a sugar.

[0049] Suitable buffers for use in the lyophilized compositions of this disclosure include buffers compatible with polyalkylene oxide-asparaginase and suitable for administration to subjects, for example, by injection or intravenous administration. Examples of suitable buffers include those described above. In certain embodiments, the phosphate buffer may include dibasic sodium phosphate and monobasic sodium phosphate. In some cases, the amount of dibasic sodium phosphate in the composition is in the range of 0.05 to 1 wt.%, such as 0.1 to 0.9 wt.%, or 0.1 to 0.8 wt.%, or 0.1 to 0.7 wt.%, or 0.1 to 0.6 wt.%, or 0.1 to 0.5 wt.%, or 0.1 to 0.4 wt.%, or 0.2 to 0.4 wt.%, or 0.2 to 0.3 wt.%, or 0.25 to 0.3 wt.%. For example, dibasic sodium phosphate can be present in a composition in an amount ranging from 0.1 to 0.5 wt.%. In certain cases, dibasic sodium phosphate can be present in a composition in an amount ranging from 0.2 to 0.4 wt.%. In certain cases, dibasic sodium phosphate can be present in a composition in an amount ranging from 0.25 to 0.3 wt.%. For example, dibasic sodium phosphate can be present in a composition in an amount of approximately 0.3 wt.%, such as 0.28 wt.% (or 0.279 wt.%). In certain embodiments, the amount of monobasic sodium phosphate in the composition is in the range of 0.005 to 1 wt.%, such as 0.01 to 0.9 wt.%, or 0.01 to 0.8 wt.%, or 0.01 to 0.7 wt.%, or 0.01 to 0.6 wt.%, or 0.01 to 0.5 wt.%, or 0.01 to 0.4 wt.%, or 0.01 to 0.3 wt.%, or 0.01 to 0.2 wt.%, or 0.01 to 0.1 wt.%, or 0.02 to 0.09 wt.%, or 0.03 to 0.08 wt.%, or 0.04 to 0.08 wt.%, or 0.045 to 0.075 wt.%, or 0.04 to 0.07 wt.%, or 0.05 to 0.07 wt.%. For example, monobasic sodium phosphate can be present in a composition in an amount of 0.01 to 0.1 wt.%. In certain cases, monobasic sodium phosphate can be present in a composition in an amount of 0.05 to 0.07 wt.%. In certain cases, monobasic sodium phosphate can be present in a composition in an amount of 0.045 to 0.075 wt.%.For example, monobasic sodium phosphate can be present in the composition in an amount of 0.06 wt.%.

[0050] In certain embodiments, the freeze-dried compositions of the present disclosure include salts. Suitable salts for use in the compositions of the present disclosure include salts compatible with polyalkylene oxide-asparaginase and suitable for administration to subjects, for example, by injection or intravenous administration. Examples of suitable salts are given above. This includes those found in certain salts. In specific examples, this salt is sodium chloride.

[0051] In some cases, the amount of salt (e.g., sodium chloride) in the composition is in the range of 0.05 to 1 wt.%, such as 0.1 to 0.9 wt.%, or 0.1 to 0.8 wt.%, or 0.1 to 0.7 wt.%, or 0.1 to 0.6 wt.%, or 0.1 to 0.5 wt.%, or 0.2 to 0.5 wt.%, or 0.3 to 0.5 wt.%, or 0.4 to 0.5 wt.%, or 0.4 to 0.45 wt.%. For example, the amount of salt (e.g., sodium chloride) in the composition can be in the range of 0.1 to 1 wt.%. In specific examples, the amount of salt (e.g., sodium chloride) in the composition can be in the range of 0.3 to 0.5 wt.%. In a typical example, a salt (e.g., sodium chloride) may be present in the composition in an amount ranging from 0.4 to 0.45 wt.%. For example, a salt (e.g., sodium chloride) may be present in the composition in an amount of approximately 0.4 wt.%, such as 0.425 wt.%. Another component that may be included in the compositions of this disclosure is sugar. Suitable sugars for use in the compositions of this disclosure include sugars that are compatible with polyalkylene oxide-asparaginase and are suitable for administration to subjects, for example, by injection or intravenous administration. Examples of suitable sugars include, but are not limited to, sucrose, mannitol, maltose, trehalose, and 2-hydroxypropyl-β-cyclodextrin (HPCD). Examples include lactose, glucose, fructose, galactose, glucosamine, and combinations thereof. In certain cases, this sugar is a disaccharide. For example, this disaccharide may be sucrose.

[0052] In some cases, the amount of sugar (e.g., sucrose) in the composition is in the range of 0.1 to 25 wt.%, such as 0.5 to 20 wt.%, or 1 to 15 wt.%, or 1 to 10 wt.%, or 1 to 9 wt.%, or 1 to 8 wt.%, or 2 to 7 wt.%, or 2 to 6 wt.%, or 3 to 5 wt.%, or 4 to 5 wt.%. For example, sugar (e.g., sucrose) can be present in the composition in an amount of 1 to 10 wt.%. In certain cases, sugar (e.g., sucrose) can be present in the composition in an amount of 3 to 5 wt.%. In certain cases, sugar (e.g., sucrose) can be present in an amount of 4 to 5 wt.%. For example, sugar (e.g., sucrose) can be present in the composition in an amount of 4.5 wt.%.

[0053] In some embodiments, the composition comprises, essentially consists of, or is composed of, a polyalkylene oxide-asparaginase having a potency (activity) in the range of 500 to 1,000 IU / g, an amount of dibasic sodium phosphate in the range of 0.1 to 1.0 wt.%, an amount of monobasic sodium phosphate in the range of 0.01 to 0.2 wt.%, an amount of salt (e.g., sodium chloride) in the range of 0.2 to 2 wt.%, and water.

[0054] In some embodiments, the composition comprises, essentially comprises, or comprises polyalkylene oxide-asparaginase having a potency (activity) in the range of 700-800 IU / g, dibasic sodium phosphate in an amount in the range of 0.2-0.8 wt.%, monobasic sodium phosphate in an amount in the range of 0.1-0.14 wt.%, a salt (e.g., sodium chloride) in an amount in the range of 0.6-1.0 wt.%, and water. In some embodiments, the composition comprises, essentially comprises, or comprises polyalkylene oxide-asparaginase having a potency (activity) in the range of 700-800 IU / g, dibasic sodium phosphate in an amount in the range of 0.5-0.6 wt.%, monobasic sodium phosphate in an amount in the range of 0.09-0.15 wt.%, a salt (e.g., sodium chloride) in an amount in the range of 0.09-0.15 wt.%, and water.

[0055] In some embodiments, the composition comprises, essentially consists of, or is composed of, a polyalkylene oxide-asparaginase having a potency (activity) of 750 IU / g, about 0.6 wt.% of dibasic sodium phosphate, about 0.1 wt.% of monobasic sodium phosphate, about 0.9 wt.% of a salt (e.g., sodium chloride) and water.

[0056] In some embodiments, the composition comprises, essentially consists of, or is composed of, a polyalkylene oxide-asparaginase having a potency (activity) of 750 IU / g, about 0.56 wt.% of dibasic sodium phosphate, about 0.13 wt.% (or 0.129 wt.%) of monobasic sodium phosphate, about 0.85 wt.% of a salt (e.g., sodium chloride) and water.

[0057] In some embodiments, the composition is polyalkylene oxide-asparaginase It contains, essentially consists of, or is composed of, dibasic sodium phosphate, monobasic sodium phosphate, salts (e.g., sodium chloride), and water.

[0058] In other embodiments, the composition comprises, essentially consists of, or is composed of, polyalkylene oxide-asparaginase, dibasic sodium phosphate, monobasic sodium phosphate, salt (e.g., sodium chloride), sugar (e.g., sucrose), and water.

[0059] In some embodiments, the composition comprises, essentially consists of, or is composed of, a polyalkylene oxide-asparaginase having a potency (activity) in the range of 500 to 1,000 IU / g, an amount of dibasic sodium phosphate in the range of 0.1 to 0.5 wt.%, an amount of monobasic sodium phosphate in the range of 0.01 to 0.1 wt.%, an amount of salt (e.g., sodium chloride) in the range of 0.1 to 1 wt.%, an amount of sugar (e.g., sucrose) in the range of 1 to 10 wt.%, and water.

[0060] In some embodiments, the composition comprises, essentially consists of, or is composed of, a polyalkylene oxide-asparaginase having a potency (activity) in the range of 700-800 IU / g, a dibasic sodium phosphate in an amount in the range of 0.2-0.4 wt.%, a monobasic sodium phosphate in an amount in the range of 0.05-0.07 wt.%, a salt (e.g., sodium chloride) in an amount in the range of 0.3-0.5 wt.%, a sugar (e.g., sucrose) in an amount in the range of 3-5 wt.%, and water.

[0061] In some embodiments, the composition comprises, essentially consists of, or is composed of, a polyalkylene oxide-asparaginase having a potency (activity) in the range of 700-800 IU / g, a dibasic sodium phosphate in an amount in the range of 0.25-0.3 wt.%, a monobasic sodium phosphate in an amount in the range of 0.045-0.075 wt.%, a salt (e.g., sodium chloride) in an amount in the range of 0.4-0.45 wt.%, a sugar (e.g., sucrose) in an amount in the range of 4-5 wt.%, and water.

[0062] In some embodiments, the composition comprises polyalkylene oxide-asparaginase having a potency (activity) of 750 IU / g, about 0.3 wt.% of dibasic sodium phosphate, 0.065 wt.% of monobasic sodium phosphate, about 0.4 wt.% of a salt (e.g., sodium chloride), about 4.5 wt.% of a sugar (e.g., sucrose), and water. It includes, is essentially composed of, or is composed of.

[0063] In some embodiments, the composition comprises, essentially consists of, or is composed of, a polyalkylene oxide-asparaginase having a potency (activity) of 750 IU / g, about 0.28 wt.% (or 0.279 wt.%) of dibasic sodium phosphate, 0.06 wt.% of monobasic sodium phosphate, about 0.43 wt.% (or 0.425 wt.%) of a salt (e.g., sodium chloride), about 4.5 wt.% of a sugar (e.g., sucrose), and water.

[0064] In certain cases, the composition (e.g., a liquid or lyophilized composition) is a sterile composition. “Sterile” means that the composition is substantially free of immunogenic components, such as substantially free of pathogenic organisms (e.g., fungi, bacteria, viruses, spores, etc.). In some cases, the composition is contained within a container. Providing a container for the composition facilitates maintaining the composition as a sterile composition. For example, the container may be configured to maintain the composition sealed within the container in a sterile environment. As such, the container may be a sealed container, for example, having a waterproof and / or airtight seal, etc. It may include a seal. This seal may be removable to allow the user to access the contents of the container. In some cases, this seal may be a fragile seal, or in other cases, this seal may be removable from the container. The seal may be configured to allow insertion of a needle, cannula, or syringe into the container. In some cases, a seal configured to allow access to the inside of the container without removing the seal from the container facilitates maintaining the contents of the container (e.g., the composition in the container) in a sterile environment prior to administration of the composition to a subject. Suitable materials for the seal include, but are not limited to, silicone rubber, natural rubber, styrene-butadiene rubber, ethylene-propylene copolymer, polychloroprene, and polyacrylic. The seals include rubber or polymer seals such as relate, polybutadiene, polyurethane, styrene-butadiene, and combinations thereof. For example, in certain embodiments, the seal is a septum that can be penetrated by a needle, syringe, or cannula. The seal can provide convenient access to the sample in the container and can also be a protective barrier lying over the opening of the container. In some examples, the seal is a removable seal such as a screw-on or snap-on lid or other suitable sealing element that is attached to the opening of the container. For example, a screw-on lid can be screwed onto and removed from the opening before and after a sample is added to the container.

[0065] In some cases, this container is a unit dose container. A unit dose container refers to a container containing one or more unit doses for administration to a subject. In some embodiments, a unit dose container contains a predetermined amount of a calculated sufficient amount of a subject composition to produce a desired effect in a subject. Specific embodiments of a composition may be provided in a suitable unit dose container for the individual administration of precise doses. The amount of active composition administered to a subject may depend on the subject being treated, the severity of the distress, and the mode of administration. For example, the unit dose container may contain an amount of composition sufficient to achieve a desired effect in a subject being treated, as disclosed herein. In certain examples, a unit dose container contains a composition having a therapeutically effective amount of polyalkylene oxide-asparaginase. The therapeutically effective amount of polyalkylene oxide-asparaginase is described above. In certain embodiments, the unit dose container is a vial. In some cases, this vial is a sealed vial (e.g., as described above with respect to sealed containers).

[0066] The container may be made of any convenient component compatible with the polyalkylene oxide-asparaginase and other components of the composition. For example, the container may be a solid-compatible container configured to contain a solid (e.g., a lyophilized composition). In some cases, the container may be a liquid-compatible container configured to contain a liquid. The container may also be compatible with both solids and liquids, and the container may be configured to contain both solids and liquids. In some cases, the liquid in the container may be an aqueous solution, and in these cases, the container may be compatible with aqueous compositions. "Compatible" means substantially inert (e.g., does not react significantly with the container) to the liquid and / or composition or other components that come into contact with the container. Examples of suitable container materials include, but are not limited to, glass and plastic. For example, the container may be silicate glass, borosilicate glass, sodium borosilicate glass (e.g., PYREX), but is not limited to these. TM ), may be composed of glass such as fused silica glass, fused silica glass, etc. Other suitable materials for the container include, but are not limited to, plastics such as polypropylene, polymethylpentene, polytetrafluoroethylene (PTFE), perfluoroether (PFE), fluorinated ethylene propylene (FEP), perfluoroalkoxyalkane (PFA), polyethylene terephthalate (PET), polyethylene (PE), polyetheretherketone (PEEK), and polystyrene. In certain examples, as described above, the container may be a vial, and in such cases it may be a glass vial. As described above, the container is a sealed container. This is possible, and in such cases, it may be a sealed glass vial. As will be described in more detail below, the liquid or reconstituted compositions of the present disclosure may be administered to a subject, for example, by injection or intravenously. In certain embodiments, prior to administration of the reconstituted composition to a subject, the solid composition may be combined with a liquid, for example, as described above, to provide a liquid composition suitable for, for example, injection or intravenous administration. In some cases, prior to administration of the composition to a subject, the solid composition may be combined with water (e.g., water for injection, WFI), for example, as described above, to provide a liquid composition suitable for, for example, injection or intravenous administration.

[0067] For example, a lyophilized composition can be reconstituted with water (e.g., water for injection, WFI) to produce a suitable reconstituted dosage unit for injection or intravenous administration to a subject. As described herein, aspects of the disclosure include compositions comprising: polyalkylene oxide-asparaginase comprising a polyalkylene oxide group covalently bonded to asparaginase by a linker; a buffer and a salt. In certain embodiments, as described above, the polyalkylene oxide is polyethylene glycol. In certain embodiments, as described above, the linker is a urethane (carbamate) linker. In certain embodiments, as described above, the asparaginase is E. coli asparaginase. In certain embodiments, as described above, the buffer is a phosphate buffer. In certain embodiments, as described above, is sodium chloride. Thus, certain embodiments of the compositions include polyethylene glycol-asparaginase comprising a polyethylene glycol group covalently bonded to E. coli asparaginase by a urethane linker; a phosphate buffer and a salt. Each component of these compositions (e.g., molecular weight of polyethylene glycol, amount of polyethylene glycol-asparaginase, type of phosphate buffer and The quantity, type and amount of salt are as detailed above.

[0068] As described herein, aspects of the disclosure include a freeze-dried storage-stable composition comprising: polyalkylene oxide-asparaginase comprising asparaginase covalently bonded to a polyalkylene oxide group by a linker; buffer, salt, and sugar. In certain embodiments, as described above, the polyalkylene oxide is polyethylene glycol. In certain embodiments, as described above, the linker is a urethane (carbamate) linker. In certain embodiments, as described above, the asparaginase is E. coli asparaginase. In certain embodiments, as described above, the buffer is a phosphate buffer. In certain embodiments, as described above, the salt is sodium chloride. In certain embodiments, as described above, the sugar is a disaccharide (e.g., sucrose). Thus, a particular embodiment of the freeze-dried storage-stable composition is polyethylene glycol-asparaginase comprising polyethylene glycol groups covalently bonded to E. coli asparaginase by a urethane linker; phosphate buffer, salt, and disaccharide. This includes each component of these compositions (e.g., molecular weight of polyethylene glycol, amount of polyethylene glycol-asparaginase, type and amount of phosphate buffer, type of salt and The quantity is as detailed above.

[0069] The compositions of this disclosure may include, as part of the composition, other components such as additional pharmaceutically acceptable additives or excipients for dose delivery. Additives may include, but are not limited to, carbohydrates, inorganic salts, organic salts, antimicrobial agents, antioxidants, surfactants, water (e.g., water for injection (WFI)), alcohols, polyols, glycerin, vegetable oils, phospholipids, buffers, acids, bases, and any combination thereof. Carbohydrates such as sugars, sugar derivatives such as alditol, aldonic acids, esterified sugars, and / or sugar polymers may also be used. Some carbohydrate additives of interest include, for example, monosaccharides such as fructose, maltose, galactose, glucose, D-mannose, and sorbose; lactose, sucrose, and trehalose. This may include disaccharides such as cellobiose; polysaccharides such as raffinose, melegitose, maltodextrin, dextran, and starch; and algitols such as mannitol, xylitol, maltitol, lactitol, xylitol, sorbitol (glucitol), pyranosylsorbitol, and myo-inositol. Inorganic and organic salts may include, but are not limited to, citric acid, sodium chloride, potassium chloride, sodium sulfate, potassium nitrate, monobasic sodium phosphate, dibasic sodium phosphate, and any combination thereof.

[0070] In certain embodiments, the compositions of the present disclosure may also include antimicrobial agents for preventing or inhibiting the growth of microorganisms, such as benzalkonium chloride, benzethonium chloride, benzyl alcohol, cetylpyridinium chloride, chlorobutanol, phenol, phenylethyl alcohol, phenylmercury nitrate, thiomersal, and any combination thereof.

[0071] The composition may also contain one or more antioxidants. Antioxidants that reduce or prevent oxidation and thus prevent deterioration of the composition may include, for example, ascorbyl palmitate, butylated hydroxyanisole, butylated hydroxytoluene, hypophosphorous acid, monothioglycerol, propyl gallate, sodium bisulfite, sodium formaldehyde sulfoxylate, sodium metabisulfite, and any combination thereof.

[0072] The compositions of this disclosure may comprise one or more surfactants. Suitable surfactants may include, but are not limited to, polysorbates such as "Tween20" and "Tween80," and plurolic such as F68 and F88 (BASF, Mount Olive, New Jersey); sorbitan esters; phospholipids such as lecithin and other phosphatidylcholine and phosphatidylethanolamine; lipids such as fatty acids and fatty acid esters; steroids such as cholesterol; chelating agents such as EDTA; and zinc and other cations. Acids or bases may also be present in the compositions of this disclosure. For example, acids may include, but are not limited to, hydrochloric acid, acetic acid, phosphoric acid, citric acid, malic acid, lactic acid, formic acid, trichloroacetic acid, nitric acid, perchloric acid, sulfuric acid, fumaric acid, and any combination thereof. Examples of bases may include, but are not limited to, sodium hydroxide, sodium acetate, ammonium hydroxide, potassium hydroxide, ammonium acetate, potassium acetate, sodium phosphate, potassium phosphate, sodium citrate, sodium formate, sodium sulfate, potassium sulfate, potassium fumarate, and any combination thereof.

[0073] The amount of any particular additive in a composition varies depending on the properties and function of the additive, the dose-delivery excipient, and the specific needs of the composition. In some cases, the optimal amount of an individual additive is determined through routine experiments, i.e., testing the stability and other parameters of compositions containing various amounts of the additive (ranging from low to high), and then determining the range in which optimal performance is obtained without significant adverse effects. However, generally, additives are present in a composition in amounts ranging from 1% to 99% by weight, such as 5% to 98% by weight, 15% to 95% by weight, 30% or less by weight, 20% or less by weight, or 10% or less by weight. Pharmaceutical additives that can be used in a composition together with additives are described in "Remington: The Science & Practice" ce of Pharmacy”, 22nd ed., Williams & Willia ms,(2012),the “Physician's Desk Reference ”, 70th ed., PDR Network, Montvale, NJ (2015), and Rowe, RC., Handbook of PharmaceuticalExc ipients,7th ed., Pharmaceutical Press, New This information is described in York, NY, (2012), and each of these disclosures is referenced in This is incorporated herein by law.

[0074] [Usage] Aspects of this disclosure also include methods of use of the compositions described herein (e.g., liquid and lyophilized). In certain embodiments, this method of use is a method of deamination of asparagine in a subject. As described above, the asparaginase enzyme mediates a deamination reaction in which the amino acid asparagine is hydrolyzed to produce aspartic acid and ammonia, for example, according to the following reaction: [ka]

[0075] In some cases, the activity of asparaginase reduces asparagine in a subject, such as by decreasing the plasma concentration of asparagine in the subject. Depletion of asparagine in a subject is detrimental to cells in the subject that depend on the presence of asparagine for protein synthesis. For example, protein synthesis in cells that lack the ability to synthesize asparagine themselves (e.g., cells lacking the enzyme asparagine synthase) is adversely affected by the exogenous lack of asparagine, which can result in apoptosis of the cells. In some cases, cells in a subject that depend on asparagine for protein synthesis may have tumor conditions such as cancer. Therefore, the methods of this disclosure include methods for treating tumor conditions in a subject, such as methods for treating cancer in a subject. Accordingly, compositions of this disclosure comprising polyalkylene oxide-asparaginase may be therapeutically effective for treating tumor conditions such as cancer. In certain embodiments, non-tumor cells in a subject are not significantly affected by the polyalkylene oxide-asparaginase compositions of this disclosure. For example, non-tumor cells in a subject may possess the enzyme asparagine synthase and thus retain the ability to synthesize asparagine.

[0076] In certain embodiments, the tumor condition to be treated in a subject is polyalkylene oxide- Examples of tumor conditions suitable for treatment by administering asparaginase to a subject include those dependent on exogenous asparagine. For example, tumor conditions treatable by administering polyalkylene oxide-asparaginase to a subject include cancers such as solid tumors or liquid tumors.

[0077] In certain cases, the tumor condition is characterized by the presence of a solid tumor. Therefore, in some embodiments, the method of the present disclosure is a method for treating a solid tumor in a subject using polyalkylene oxide-asparaginase (e.g., the liquid or reconstituted lyophilized polyalkylene oxide-asparaginase composition of the present disclosure). The method for treating a subject's tumor condition is useful for treating a wide range of solid tumors, including epithelial and non-epithelial malignancies. The types of solid tumors include, but are not limited to, pancreatic cancer, melanoma, squamous cell carcinoma, non-small cell lung cancer (NSCLC), colorectal cancer, breast cancer, ovarian cancer, cervical cancer, prostate cancer, and the like. For example, epithelial malignancies treatable using the subject method include, but are not limited to, esophageal cancer, hepatocellular carcinoma, basal cell carcinoma (a form of skin cancer), squamous cell carcinoma (of various tissues), bladder cancer including transitional cell carcinoma (malignant tumor of the bladder), bronchial cancer, colorectal cancer, gastric cancer, lung cancer including small cell lung cancer and non-small cell lung cancer, adrenocortical carcinoma, thyroid cancer, pancreatic cancer, breast cancer, ovarian cancer, prostate cancer, adenocarcinoma, sweat gland carcinoma, sebaceous gland carcinoma, papillary carcinoma, papillary adenocarcinoma, cystadenocarcinoma, medullary carcinoma, renal cell carcinoma, ductal carcinoma in situ or cholangiocarcinoma, choriocarcinoma, seminomasm, fetal cancer, Wilms' tumor, cervical cancer, uterine cancer, testicular cancer, osteosarcoma, epithelial carcinoma, and nasopharyngeal cancer.

[0078] Non-epithelial malignant tumors treatable using the subject method include, but are not limited to, fibrosarcomas. Myxosarcoma, liposarcoma, chondrosarcoma, chordoma, osteosarcoma, osteosarcoma, (osteogenic s Arcoma, osteosarcoma) includes angiosarcoma, endosarcoma, lymphangiosarcoma, lymphangiosarcoma, synovial tumor, mesothelioma, Ewing's sarcoma, leiomuscular sarcoma, rhabdomyosarcoma, and other soft tissue sarcomas.

[0079] Other solid tumors treatable using the subject method include, but are not limited to, gliomas, astrocytomas, medulloblastomas, craniopharyngiomas, ependymocytes, pinealomas, hemangioblastomas, acoustic neuromas, oligodendrogliomas, meningiomas, melanomas, neuroblastomas, and retinoblastomas.

[0080] In certain cases, the tumor condition is characterized by the presence of a liquid tumor. For example, a liquid tumor may include metastatic cancer cells (e.g., circulating tumor cells (CTCs)), hematological malignancies, and combinations thereof. Examples of liquid tumors may include, but are not limited to, leukemia, lymphoma, and myeloma. In some examples, the cancer is leukemia. Accordingly, in some embodiments, the method is a treatment for leukemia in a subject using polyalkylene oxide-asparaginase (e.g., the liquid or reconstituted lyophilized polyalkylene oxide-asparaginase of the compositions of this disclosure).

[0081] Various types of leukemia may be suitable for treatment using the method of the subject. For example, this leukemia may be acute leukemia. Acute leukemia may be characterized by a rapid increase in immature blood cells. A rapid increase in immature blood cells leads to a densification, which in turn causes significantly less production of healthy blood cells by the bone marrow. Accordingly, the method of the present disclosure includes, for example, a method for treating acute leukemia in a subject by administering to the subject a sufficient dose to treat the subject's acute leukemia of polyalkylene oxide-asparaginase (e.g., liquid or reconstituted lyophilized polyalkylene oxide-asparaginase composition of the present disclosure).

[0082] In other cases, if the leukemia is chronic leukemia, chronic leukemia is characterized by an increase in relatively mature but abnormal white blood cells. Chronic leukemia takes a longer period of time to progress (e.g., several months or several years), and abnormal white blood cells are produced at a significantly higher rate than normal. Accordingly, the method of the present disclosure includes a method for treating a subject's chronic leukemia by administering to the subject a dose of a polyalkylene oxide-asparaginase effective for treating the subject's chronic leukemia (e.g., a liquid or reconstituted lyophilized polyalkylene oxide-asparaginase of the composition of the present disclosure).

[0083] In certain embodiments, the leukemia is lymphoblastic leukemia (also known as lymphocytic leukemia). Lymphoblastic leukemia is characterized by the type of blood cell affected by the leukemia. In lymphoblastic leukemia, abnormal changes occur in blood cells, specifically in bone marrow cells that normally develop into lymphocytes. For example, lymphoblastic leukemia can be B-cell leukemia. Accordingly, the methods of the present disclosure include, for example, a method for treating lymphoblastic leukemia (lymphocytic leukemia) in a subject by administering to the subject a dose of polyalkylene oxide-asparaginase (e.g., liquid or reconstituted lyophilized polyalkylene oxide-asparaginase of the composition of the present disclosure) that is effective in treating the subject's lymphoblastic leukemia (lymphocytic leukemia). For example, certain types of lymphoblastic leukemia treatable by the methods of the subject include acute lymphoblastic leukemia (ALL). In these embodiments, the methods of the present disclosure include, for example, a method for treating acute lymphoblastic leukemia (ALL) in a subject by administering to the subject a dose of polyalkylene oxide-asparaginase (e.g., liquid or reconstituted lyophilized polyalkylene oxide-asparaginase of the compositions of the present disclosure) that is effective in treating the subject's acute lymphoblastic leukemia (ALL). Other types of lymphoblastic leukemia treatable with the methods of the subject include, but are not limited to, chronic lymphoblastic leukemia. The disease is chronic lymphocytic leukemia (CLL). These embodiments include a method of treating a subject's chronic lymphocytic leukemia (CLL) by administering to the subject a dose of a polyalkylene oxide-asparaginase effective for treating the subject's chronic lymphocytic leukemia (e.g., a liquid or reconstituted lyophilized polyalkylene oxide-asparaginase of the compositions of the Disclosure).

[0084] In other embodiments, the leukemia is myeloid leukemia. (Also known as myelogenous leukemia.) Myeloid leukemia can be characterized by the blood cells affected by the leukemia. In myeloid leukemia, abnormal changes occur in blood cells, particularly in bone marrow cells that normally develop into red blood cells and / or platelets. Accordingly, the methods of the present disclosure include, for example, a method for treating myeloid leukemia in a subject by administering to the subject a dose of polyalkylene oxide-asparaginase (e.g., liquid or reconstituted lyophilized polyalkylene oxide-asparaginase of the composition of the present disclosure) that is effective in treating the subject's myeloid leukemia. For example, certain types of lymphoblastic leukemia treatable with the methods of the subject are, but are not limited to, acute myeloid leukemia (AML). In these embodiments, the methods of the present disclosure include, for example, a method of treating a subject's acute myeloid leukemia (AML) by administering to the subject a dose of a polyalkylene oxide-asparaginase (e.g., a liquid or reconstituted lyophilized polyalkylene oxide-asparaginase of the compositions of the present disclosure) that is effective in treating the subject's AML.

[0085] Examples of AML include, but are not limited to, AML with recurrent cytogenetic translocations, AML with polyseries cell dysplasia, and other types of AML. For example, AML with recurrent cytogenetic translocations includes, in particular, AML with t(8;2l)(q22;q22), and AMLI(CBF- α) / ETO, acute promyelocytic leukemia (t(l5;l7)(q22;qll-12) and AML with variants, PML / RAR-α), AML with abnormal bone marrow eosinophils (in This includes AML with v(l6)(pl3q22) or t(l6;16)(p13;ql1, CBFb / MYHlIX), and lq23(MLL) abnormalities. Examples of AML with multiseries cell dysplasia may include those associated with and unassociated with previous myelodysplastic syndromes. Other types of acute myeloid leukemia may include, for example, most undifferentiated AML, undifferentiated AML, differentiated AML, acute myelomonocytic leukemia, acute monocytic leukemia, acute erythroleukemia, acute megakaryocytic leukemia, acute basophilic leukemia, and acute panmyelopathy with acute myelofibrosis.

[0086] Other types of myeloid leukemia treatable using the subject method include, but are not limited to, chronic myelogenous leukemia (CML). In these embodiments, the method of the present disclosure includes, for example, a method for treating a subject's chronic myeloid leukemia (CML) by administering to the subject a dose of a polyalkylene oxide-asparaginase (e.g., a liquid or reconstituted lyophilized polyalkylene oxide-asparaginase composition of the present disclosure) that is effective in treating the subject's CML.

[0087] In certain cases, asparaginase can mediate the deamination of glutamine, for example, by hydrolysis of the amino acid glutamine to produce glutamic acid and ammonia according to the following reaction: [ka]

[0088] In some cases, asparaginase activity reduces the concentration of glutamine in a subject, such as by lowering the plasma glutamine concentration in the subject. As discussed above, glutamine depletion in a subject is detrimental to cells in the subject that depend on the presence of glutamine for protein synthesis. For example, protein synthesis in cells that lack the ability to synthesize glutamine themselves (e.g., cells lacking the enzyme glutamine synthase) is adversely affected by the exogenous glutamine deficiency, which may result in apoptosis of the cells. In some cases, cells in a subject that depend on glutamine for protein synthesis may be associated with tumor conditions such as cancer. Accordingly, the methods of the present disclosure include methods for treating tumor conditions in a subject, such as methods for treating cancer in a subject. Accordingly, the methods of the present disclosure include methods for treating tumor conditions in a patient, such as methods for treating cancer in a patient, when the tumor condition may be dependent on exogenous glutamine. For example, embodiments of the subject method include methods for deaminating glutamine in a subject. In certain embodiments, non-tumor cells in a subject are not significantly affected by the polyalkylene oxide-asparaginase composition administered to the subject. For example, non-tumor cells in a subject may possess the enzyme glutamine synthase and thus retain the ability to synthesize glutamine.

[0089] As described above, the compositions of the present disclosure include lyophilized storage-stable compositions. Before administering the composition to a subject, the lyophilized composition is mixed with a liquid to provide a liquid composition suitable for administration, for example, by injection or intravenous administration. In some cases, prior to administration of the composition to a subject, the lyophilized composition may be combined with water (e.g., water for injection, WFI) to provide a liquid composition suitable for administration, for example, by injection or intravenous administration, as described above. For example, the method of the present disclosure may include reconstituting the lyophilized composition of the present disclosure (e.g., lyophilized storage-stable composition). Reconstituting the lyophilized composition can produce a reconstituted dosage unit. In some cases, this reconstituted dosage unit is suitable for administration to a subject, for example, by injection or intravenous administration. In certain embodiments, the reconstitution of the lyophilized composition includes combining the lyophilized composition (e.g., lyophilized storage-stable composition) with water (e.g., water for injection, WFI).

[0090] The liquid or reconstituted dose unit contains a predetermined amount of the composition of this disclosure in a calculated and sufficient quantity to produce the desired therapeutic effect in a subject. The amount of composition in the dose unit (e.g., liquid or reconstituted) administered to a subject may depend on the subject being treated, the severity of the pain, and the mode of administration. For example, a dose unit may contain a therapeutically effective amount of the composition as disclosed herein.

[0091] In certain embodiments of the dosage unit, the amount of polyalkylene oxide-asparaginase may include amounts in the range of 100 to 5,000 IU / mL, such as 500 to 4,500 IU / mL, or 500 to 4,000 IU / mL, or 500 to 3,500 IU / mL, or 500 to 3,000 IU / mL, or 500 to 2,500 IU / mL, or 500 to 2,000 IU / mL, or 500 to 1,500 IU / mL, or 500 to 1,000 IU / mL, or 600 to 900 IU / mL, or 700 to 800 IU / mL. In certain examples, the dosage unit contains 500 to The dose may include amounts in the range of 1,000 IU / mL. In certain examples, the dose unit may include amounts of polyalkylene oxide-asparaginase in the range of 700-800 IU / mL. For example, the dose unit may contain 750 IU / mL of polyalkylene oxide-asparaginase.

[0092] In certain embodiments, the dosage unit is 50 IU / mg protein or more, 55 IU / mg protein or more, 60 IU / mg protein or more, or 65 IU / mg It contains a therapeutically effective amount (e.g., specific activity) of polyalkylene oxide-asparaginase, such as 1 g or more of protein, or 70 IU / mg or more of protein, or 75 IU / mg or more of protein, or 80 IU / mg or more of protein, or 85 IU / mg or more of protein, or 90 IU / mg or more of protein, or 95 IU / mg or more of protein, or 100 IU / mg or more of protein, or 105 IU / mg or more of protein, or 110 IU / mg or more of protein, or 115 IU / mg or more of protein, or 120 IU / mg or more of protein, or 125 IU / mg or more of protein, or 130 IU / mg or more of protein, or 135 IU / mg or more of protein, or 140 IU / mg or more of protein, or 145 IU / mg or more of protein, or 150 IU / mg or more of protein.

[0093] For example, the dosage unit may have a specific activity of 85 IU / mg protein or more. In some embodiments, the dosage unit has a specific activity in the range of 50-150 IU / mg protein, or 55-145 IU / mg protein, or 60-140 IU / mg protein, or 65-135 IU / mg protein, or 70-130 IU / mg protein, or 75-125 IU / mg protein, or 80-120 IU / mg protein, or 85-115 IU / mg protein, or 90-110 IU / mg protein, or 95-105 IU / mg protein. In some cases, the dosage unit has a specific activity in the range of 65-140 IU / mg protein, or 70-135 IU / mg protein, or 75-130 IU / mg protein, or 75-125 IU / mg protein, etc. For example, the aforementioned dosage unit may have a specific activity in the range of 75 to 125 IU / mg of protein.

[0094] In certain embodiments, the dosage units are 1 mg / mL to 15 mg / mL, 1.5 mg / mL to 14.5 mg / mL, or 2 mg / mL to 14 mg / mL, or 2.5 mg / mL to 13.5 mg / mL, or 3 mg / mL to 13 mg / mL, or 3.5 mg / mL to 12.5 mg / mL, or 4 mg / mL to 12 mg / mL, or 4.5 mg / mL to 11.5 mg / mL, or 4.5 mg / mL to 11 mg / The dose contains a therapeutically effective amount (e.g., protein concentration) of polyalkylene oxide-asparaginase in the range of 1 mg / mL to 15 mg / mL, such as mL, or 4.5 mg / mL to 10.5 mg / mL, or 4.5 mg / mL to 10 mg / mL, or 4.5 mg / mL to 9.5 mg / mL, or 4.5 mg / mL to 9 mg / mL, or 4.5 mg / mL to 8.5 mg / mL, or 5 mg / mL to 8 mg / mL. In some cases, the dose unit contains an amount of polyalkylene oxide-asparaginase in the range of 4.5 mg / mL to 8.5 mg / mL.

[0095] When administered to a subject, the dosage unit is 500-5,000 IU / m³. 2It may contain a therapeutically effective amount of polyalkylene oxide-asparaginase to deliver 100-5,000 IU / m2 of polyalkylene oxide-asparaginase to a subject, such as 500-4,500 IU / m2, or 500-4,000 IU / m2, or 500-3,500 IU / m2, or 500-3,000 IU / m2, or 1,000-3,000 IU / m2, or 1,500-3,000 IU / m2, or 1,750-3,000 IU / m2, or 2,000-3,000 IU / m2, or 2,000-2,750 IU / m2, or 2,250-2,750 IU / m2. For example, the dose unit can deliver 1 to 3,000 IU / m2 of polyalkylene oxide-asparaginase to a subject. In a specific example, the dose unit can deliver 2,000 to 2,750 IU / m2 of polyalkylene oxide-asparaginase to a subject. In a specific example, the dose unit can deliver 2,250 to 2,750 IU / m2 of polyalkylene oxide-asparaginase to a subject. For example, the dose unit can deliver 2,500 IU / m2 of polyalkylene oxide-asparaginase to a subject.

[0096] In certain embodiments, the dosage unit may include a buffer such as the buffer described above. For example, the dosage unit may include a phosphate buffer, and such a buffer may include dibasic sodium phosphate and monobasic sodium phosphate.

[0097] In some cases, the dosage unit contains phosphate buffer and, as such, dibasic sodium phosphate and monobasic sodium phosphate. In certain examples, the dosage unit contains an amount of dibasic sodium phosphate in the range of 0.5 to 10 mg / g, such as 1 to 9 mg / g, or 1 to 8 mg / g, or 1 to 7 mg / g, or 2 to 7 mg / g, or 3 to 6 mg / g, or 4 to 6 mg / g, or 5 to 6 mg / g. For example, the dosage unit may contain an amount of dibasic sodium phosphate in the range of 4 to 6 mg / g. In certain examples, the dosage unit may contain an amount of dibasic sodium phosphate in the range of 5 to 6 mg / g. For example, the dosage unit may contain an amount of dibasic sodium phosphate in the range of 5.5 mg / g, such as 5.6 mg / g (or 5.58 mg / g). In certain embodiments, the dosage unit includes an amount of monobasic sodium phosphate in the range of 0.05 to 5 mg / g, such as 0.1 to 4.5 mg / g, or 0.1 to 4 mg / g, or 0.1 to 3.5 mg / g, or 0.1 to 3 mg / g, or 0.1 to 2.5 mg / g, or 0.1 to 2 mg / g, or 0.5 to 2 mg / g, or 1 to 2 mg / g, or 1 to 1.5 mg / g. For example, the dosage unit may include an amount of monobasic sodium phosphate in the range of 1 to 2 mg / g. In certain examples, the dosage unit includes an amount of monobasic sodium phosphate in the range of 1 to 1.5 mg / g. For example, the dosage unit may include an amount of monobasic sodium phosphate of 1.2 mg / g (or 1.29 mg / g).

[0098] In certain embodiments, the dosage unit includes a salt. Suitable salts for use in the dosage unit include salts compatible with polyalkylene oxide-asparaginase and suitable for administration to a subject, for example, by injection or intravenous administration. Examples of suitable salts include, but are not limited to, sodium chloride, potassium chloride, calcium chloride, magnesium chloride, etc., and combinations thereof. In certain embodiments, the dosage unit includes a salt such as sodium chloride. In some cases, the dose unit contains a salt (e.g., sodium chloride) in an amount ranging from 1 to 20 mg / g, such as 1 to 19 mg / g, or 1 to 18 mg / g, or 1 to 17 mg / g, or 1 to 16 mg / g, or 1 to 15 mg / g, or 2 to 15 mg / g, or 3 to 15 mg / g, or 4 to 15 mg / g, or 5 to 14 mg / g, or 5 to 13 mg / g, or 5 to 12 mg / g, or 5 to 11 mg / g, or 5 to 10 mg / g, or 5 to 9 mg / g, or 6 to 9 mg / g, or 7 to 9 mg / g, or 8 to 9 mg / g.

[0099] For example, the dosage unit may contain an amount of salt (e.g., sodium chloride) in the range of 1 to 10 mg / g. In a specific example, the dosage unit may contain an amount of salt (e.g., sodium chloride) in the range of 5 to 10 mg / g. In a specific example, the dosage unit may contain an amount of salt (e.g., sodium chloride) in the range of 6 to 9 mg / g. In a specific example, the dosage unit may contain an amount of salt (e.g., sodium chloride) in the range of 8 to 9 mg / g. For example, the dosage unit contains an amount of salt (e.g., sodium chloride) in the range of 8.5 mg / g.

[0100] In one example, the formulation administered to the subjects was PEG-asparagus, commercially known as Oncaspar®, which has been approved for commercial marketing by the U.S. Food and Drug Administration. This is a Ginase liquid injection preparation. Oncaspar® (pegaspargase) is L-asparaginase (L-asparagineamide hydrolase) covalently bound to monomethoxypolyethylene glycol (mPEG), and is a clear, colorless, preservative-free, isotonic, sterile phosphate-buffered saline solution at pH 7.3. Each milliliter is 750 ± 150 International Units, dibasic sodium phosphate, USP (5.58 mg), monobasic sodium phosphate, USP (1.20 mg), and sodium chloride, USP (9.5 mg), water for injection, USP.

[0101] In certain embodiments, the dosage unit administered to a subject is a dosage unit prepared from a lyophilized composition, such as a lyophilized storage-stable composition described herein. The dosage unit prepared from the lyophilized composition may be a liquid formulation. In addition to polyalkylene oxide-asparaginase, embodiments of the dosage unit (e.g., a dose reconstituted from a lyophilized composition) may include buffers, salts, and sugars.

[0102] In certain embodiments, the reconstituted dose unit includes a buffer, such as the buffer described in detail above. In some cases, the reconstituted dose unit includes an amount of dibasic sodium phosphate in the range of 0.5 to 10 mg / g, such as 1 to 9 mg / g, or 1 to 8 mg / g, or 1 to 7 mg / g, or 1 to 6 mg / g, or 1 to 5 mg / g, or 1 to 4 mg / g, or 2 to 4 mg / g, or 2 to 3 mg / g, or 2.5 to 3 mg / g. For example, the reconstituted dose unit may include an amount of dibasic sodium phosphate in the range of 1 to 5 mg / g. In certain examples, the reconstituted dose unit may include an amount of dibasic sodium phosphate in the range of 2 to 4 mg / g. In certain examples, the reconstituted dose unit may include an amount of dibasic sodium phosphate in the range of 2.5 to 3 mg / g. For example, the reconstituted dose unit may contain approximately 3 mg / g of dibasic sodium phosphate, such as 2.8 mg / g (or 2.79 mg / g). In certain embodiments, the reconstituted dose unit may contain an amount of monobasic sodium phosphate in the range of 0.05 to 1 mg / g, such as 0.1 to 0.9 mg / g, 0.1 to 0.8 mg / g, 0.2 to 0.8 mg / g, 0.3 to 0.8 mg / g, 0.4 to 0.8 mg / g, 0.45 to 0.75 mg / g, or 0.5 to 0.7 mg / g. For example, the reconstituted dose unit may contain an amount of monobasic sodium phosphate in the range of 0.45 to 0.75 mg / g. In certain examples, the reconstituted dose unit may contain an amount of monobasic sodium phosphate in the range of 0.5 to 0.7 mg / g. For example, the reconstituted dose unit may contain an amount of monobasic sodium phosphate in the range of 0.6 mg / g.

[0103] In certain embodiments, the reconstituted dose unit contains a salt such as the salt described in detail above. In certain examples, the reconstituted dose unit contains a salt such as sodium chloride. In some cases, the reconstituted dose unit contains a salt (e.g., sodium chloride) in an amount ranging from 0.5 to 10 mg / g, such as 1 to 9 mg / g, or 1 to 8 mg / g, or 1 to 7 mg / g, or 1 to 6 mg / g, or 1 to 5 mg / g, or 2 to 5 mg / g, or 3 to 5 mg / g, or 4 to 5 mg / g, or 4 to 4.5 mg / g. For example, the reconstituted dose unit contains an amount of salt (e.g., sodium chloride) in the range of 1 to 10 mg / g. In certain examples, the reconstituted dose unit contains an amount of salt (e.g., sodium chloride) in the range of 3 to 5 mg / g. In certain examples, the reconstituted dose unit contains an amount of salt (e.g., sodium chloride) in the range of 4 to 4.55 mg / g. For example, the reconstituted dose unit contains approximately 4 mg / g of salt (e.g., sodium chloride), such as 4.25 mg / g.

[0104] In certain embodiments, the reconstituted dose unit contains sugars such as those described in detail above. In certain examples, the reconstituted dose unit contains sugars such as disaccharides. In some cases, the reconstituted dose unit contains sugars such as sucrose. In some cases, the reconstituted dose unit contains sugar (e.g., sucrose) in amounts ranging from 1 to 250 mg / g, such as 5 to 200 mg / g, or 10 to 150 mg / g, or 10 to 100 mg / g, or 10 to 90 mg / g, or 10 to 80 mg / g, or 20 to 70 mg / g, or 20 to 60 mg / g, or 30 to 50 mg / g, or 40 to 50 mg / g. For example, the reconstituted dose unit contains sugar (e.g., sucrose) in amounts ranging from 10 to 100 mg / g. In certain examples, the reconstituted dose unit contains sugar (e.g., sucrose) in amounts ranging from 30 to 50 mg / g. The amount may include. In certain examples, the reconstituted dosage unit may contain an amount in the range of 40-50 mg / g of sugar (e.g., sucrose). For example, the reconstituted dosage unit may contain an amount of 45 mg / g of sugar (e.g., sucrose).

[0105] In certain embodiments, the dosage unit (e.g., liquid or reconstituted) is within a range compatible with the physiological state. In some cases, the pH of the dosage unit is in the range of 7 to 8. For example, the pH of the dosage unit may be in the range of 7 to 7.5. In some cases, the pH of the dosage unit is 7.2. In some cases, the pH of the dosage unit is 7.3. In some cases, the pH of the dosage unit is 7.4.

[0106] In certain embodiments, the method may include administering the dosage unit to a subject in order to deaminate asparagine in the subject. The route of administration can be selected, but is not limited, according to various factors such as the condition to be treated, the type of composition and / or device used, and the subject to be treated. Useful routes of administration in the disclosed method are not limited, but include oral and intravenous (iv), intraperitoneal (ip), intramuscular (im), rectal, topical, intraocular, nasal, and parenteral routes such as transdermal. For example, a composition suitable for injection can be administered intravenously, intramuscularly, intradermally, subcutaneously, sublingually, intraosseously, or via other routes of administration. In some cases, administering the dose unit to a subject includes administering the dose unit intravenously to the subject. In other cases, administering the dose unit to a subject includes administering the dose unit intramuscularly to the subject.

[0107] In some examples, administering the reconstituted dosage unit to a subject includes intravenous administration of the reconstituted dosage unit to the subject. In other examples, administering the reconstituted dosage unit to a subject includes intramuscular administration of the reconstituted dosage unit to the subject.

[0108] In certain embodiments, the method includes administering the dose units according to a therapeutic dosing plan. For example, in some cases, a subject to be treated may be prescribed a therapeutic dosing plan by a healthcare provider. In some cases, the therapeutic dosing plan may include, but is not limited to, five doses per day, four doses per day, three doses per day, two doses per day, one dose per day, three doses per week, two doses per week, one dose per week, one dose every two weeks, one dose every three weeks, one dose per month, one dose every five weeks, one dose every six weeks, one dose every seven weeks, one dose every two months, and any combination thereof.

[0109] In some embodiments, the therapeutic drug regimen includes administering one or more doses over a period of time. In certain cases, a single dose (e.g., a single dose unit) may be administered to the subject, and following this initial dose, one or more doses may be administered to the subject over a period of time. In some examples, one or more doses (e.g., one or more dose units) may be administered to the subject, and following this initial dose, one or more doses may be administered to the subject over a period of time. For example, a single dose (e.g., a single dose unit) may be administered to the subject, and following this single dose, one or more doses may be administered to the subject over a period of time. In some cases, one or more doses (e.g., For example, one or more dose units can be administered to a subject, and one or more doses can be administered to the subject at subsequent time points following the initial dose. For example, a single dose (e.g., a single dose unit) can be administered to a subject. The drug can be administered to the test subject, and a single dose can be administered to the subject at a subsequent time point following this single dose. Additional single doses can be administered to the subject at a subsequent time point. In other cases, a single dose (e.g., a single dose unit) can be administered to the subject, and two or more doses can be administered at a subsequent time point following this single dose. Additional single or multiple doses can be administered at a subsequent time point. Additional single doses can be administered to the subject at a subsequent time point.

[0110] In other cases, a single dose (e.g., a single dose unit) can be administered to the subject. And following this single dose, two or more doses may be administered at subsequent times. In certain examples, the therapeutic dosing plan includes multiple phases. The therapeutic dosing plan includes multiple phases, and the dosing schedule differs in each phase of the therapeutic dosing plan. In some cases, a subject is prescribed a therapeutic dosing plan having two phases: an induction phase and a coupling phase. In certain examples, a subject is prescribed a therapeutic dosing plan having two phases, and the dosing schedule for the first phase differs from the dosing schedule for the second phase. For example, a subject is prescribed a therapeutic dosing plan having two phases, an induction phase and a coupling phase, and the dosing schedule for the induction phase differs from the dosing schedule for the coupling phase. In other embodiments, a subject is prescribed a therapeutic dosing plan having three phases: an induction phase, a coupling phase and a maintenance phase. In some examples, a subject is prescribed a therapeutic dosing plan having three phases: an induction phase, a coupling phase and a maintenance phase, and the dosing schedule for each phase differs from that of the other phases. For example, a subject may be prescribed a treatment plan that includes three phases: an induction phase, a coupling phase, and a maintenance phase, in which case the administration schedule for each phase will differ from that of the other phases.

[0111] In certain embodiments, the duration of each phase of the treatment plan may be the same or different. For example, the duration of the time between the induction phases may be 1 week or more, such as 2 weeks or more, or 3 weeks or more, or 4 weeks or more, or 5 weeks or more, or 6 weeks or more, or 7 weeks or more, or 8 weeks or more. In some cases, the duration of the time between the induction phases may be 4 weeks. In some cases, the duration of the time between the induction phases may be The duration is 5 weeks.

[0112] In certain embodiments, the length of time between linked phases is 1 week or more, such as 2 weeks or more, or 3 weeks or more, or 4 weeks or more, or 5 weeks or more, or 6 weeks or more, or 7 weeks or more, or 8 weeks or more, or 9 weeks or more, or 10 weeks or more, or 11 weeks or more, or 12 weeks or more, or 13 weeks or more, or 14 weeks or more, or 15 weeks or more, or 16 weeks or more, or 17 weeks or more, or 18 weeks or more, or 19 weeks or more, or 20 weeks or more, or 21 weeks or more, or 22 weeks or more, or 23 weeks or more, or 24 weeks or more, or 25 weeks or more, or 26 weeks or more, or 27 weeks or more, or 28 weeks or more, or 29 weeks or more, or 30 weeks or more, or 31 weeks or more, or 32 weeks or more. In some cases, the length of time between linked phases is 8 weeks. In some cases, the length of time between linked phases is 27 weeks. In some cases, the length of time between linked phases is 30 weeks.

[0113] In certain embodiments, the length of time between maintenance phases is 2 weeks or more, or 3 weeks or more, or 4 weeks or more, or 5 weeks or more, or 6 weeks or more, or 7 weeks or more, or 8 weeks or more, or 9 weeks or more, or 10 weeks or more, or 12 weeks or more, or 16 weeks or more, or 20 weeks or more, or 24 weeks or more, or 28 weeks or more, or 32 weeks or more, or 36 weeks or more, or 40 weeks or more, or 44 weeks or more, or 48 weeks or more, or 52 weeks or more, or 56 weeks or more, or 60 weeks or more, or 64 weeks or more, or 68 weeks or more, or 72 weeks or more, or 76 weeks or more, or 80 weeks or more. Or 1 week or more, such as 84 weeks or more, or 88 weeks or more, or 92 weeks or more, or 96 weeks or more, or 100 weeks or more, or 104 weeks or more, or 108 weeks or more, or 112 weeks or more, or 116 weeks or more, or 120 weeks or more, or 124 weeks or more, or 128 weeks or more, or 132 weeks or more, or 136 weeks or more, or 140 weeks or more, or 144 weeks or more, or 148 weeks or more, or 152 weeks or more, or 156 weeks or more, or 160 weeks or more, or 164 weeks or more, or 168 weeks or more, or 172 weeks or more, or 176 weeks or more, or 180 weeks or more. In some cases, the length of time between maintenance phases is 8 weeks. In some cases, the length of time between maintenance phases is 88 weeks. In some cases, the length of time between maintenance phases is 104 weeks. In some cases, the length of time between linking phases is 140 weeks. In some cases, the length of time between maintenance phases is 156 weeks. The duration of the maintenance phase is in the range of 88 to 104 weeks. In some cases, the duration of the maintenance phase is in the range of 88 to 140 weeks. In other cases, the duration of the maintenance phase is in the range of 88 to 156 weeks.

[0114] Examples of the therapeutic dosing regimens that can be administered to a subject include, but are not limited to, those described herein. In certain embodiments, the therapeutic dosing regimen includes administering a single dose to a subject in the induction phase and administering multiple doses in the maintenance phase. In certain embodiments, the therapeutic dosing regimen includes administering a single dose to a subject in the induction phase and administering multiple doses in the linkage phase. For example, the multiple doses can be administered by administering a dose to the subject every three weeks (e.g., during the linkage phase). In some cases, a single dose can be administered to the subject once every three weeks. As described above, the linkage phase can be 30 weeks, and therefore a total of 10 doses can be administered to the subject (e.g., a single dose can be administered to the subject once every three weeks for 30 weeks). Additional (or fewer) doses can be administered to the subject between the induction and linkage phases, or after the linkage phase, as described or prescribed by the healthcare provider.

[0115] In other examples, the treatment dosing plan may involve administering a single dose to the subject during the induction phase and multiple doses during the linkage phase, with multiple doses being administered to the subject by administering doses every two weeks. In some cases, a single dose is administered to the subject every two weeks. As described above, the linkage phase may be 30 weeks, and therefore a total of 15 doses may be administered to the subject (for example, a single dose may be administered to the subject once every two weeks for 30 weeks). Additional (or fewer) doses may be administered to the subject between the induction and linkage phases, or after the linkage phase, as described or prescribed by the healthcare provider.

[0116] In other embodiments, the therapeutic dosing plan includes administering a single dose to the subject during the induction phase, multiple doses during the linking phase, and multiple doses during the maintenance phase. For example, multiple doses during the linking phase may be administered to the subject on a specific day following the start of the linking phase. In one example, these multiple doses during the linking phase may be administered to the subject by administering two or more doses simultaneously. For example, these multiple doses during the linking phase may be administered to the subject by administering two or more doses on a specific day following the start of the linking phase, and two or more doses may be administered on subsequent days during the linking phase. An example of this type of therapeutic dosing plan may include administering two doses to the subject on day 15 following the start of the linking phase, and two doses on day 43 following the start of the linking phase. Additional (or fewer) doses may be administered to the subject during the induction and linking phases, or, if desired or prescribed by the healthcare provider, after the linking phase but before the maintenance phase.

[0117] In certain embodiments, multiple doses during the maintenance phase can be administered to a subject on specific days following the start of the maintenance phase. In one example, multiple doses during this maintenance phase can be administered to a subject by simultaneously administering two or more doses to the subject. For example, these multiple doses during the maintenance phase can be administered to a subject by administering two or more doses on specific days following the start of the maintenance phase, and two or more doses on subsequent days during the maintenance phase. An example of this type of therapeutic dosing plan may include administering two doses to a subject on day 2 following the start of the maintenance phase, and two doses on day 22 following the start of the maintenance phase. Another example of a therapeutic dosing plan during the maintenance phase may include administering two doses to a subject on day 4 following the start of the maintenance phase, and two doses on day 43 following the start of the maintenance phase. In some cases, the therapeutic dosing plan may include multiple maintenance phases. In certain examples, the dosing schedule between each of these maintenance phases may be the same, or in other examples, the dosing schedule between each maintenance phase may be the same. Joules may vary. Additional (or fewer) doses may be administered to the subject between the linkage and maintenance phases, or after the maintenance phase, or between different maintenance phases, if desired or prescribed by the healthcare provider.

[0118] In certain embodiments, the dose units of the present disclosure may be administered prior to, simultaneously with, or after other active agents for the treatment of related or unrelated conditions, such as in combination therapy. Examples of such additional treatments include radiotherapy, surgical therapy, and chemotherapy. When provided simultaneously with other active agents, the dose units of the present disclosure may be provided in the same or different formulations. For example, combination therapy can be achieved by administering a pharmaceutical composition having dose units and other active agents, such as at least one chemotherapeutic agent, which, in combination, provide a therapeutically effective dose according to a specific therapeutic dosing plan. The administration of separate pharmaceutical compositions may be simultaneous or at different times (e.g., sequentially, on the same day, or in either order on different days), insofar as the combination of these substances produces a therapeutically effective effect in the subject receiving treatment.

[0119] Accordingly, aspects of the present disclosure further include combination therapy. In certain embodiments, the method of the subject involves administering a therapeutically effective amount of one or more additional active agents. Combination therapy means that a polyalkylene oxide-asparaginase (e.g., as described herein) can be used in combination with other therapeutic agents for treating other single diseases or conditions. In certain embodiments, the compounds of the present disclosure are administered concurrently with the administration of other therapeutic agents as a component of a composition containing the compounds of the present disclosure, or as a component of a different composition. In certain embodiments, the composition containing the compounds of the present disclosure is administered prior to or following the administration of other therapeutic agents.

[0120] The subject compound can be used in combination with any agent useful for treating tumor conditions, such as anticancer agents and antitumor agents. One class of anticancer agents of interest is chemotherapeutic agents. "Chemotherapy" means administering one or more chemotherapeutic agents and / or other agents to a cancer patient by various means, including intravenous, oral, intramuscular, intraperitoneal, intravesical, subcutaneous, percutaneous, oral, or inhalation. Agents of interest that can be used in combination with the subject compound include, but are not limited to, cancer chemotherapeutic agents, agents that act to reduce cell proliferation, antimetabolites, microtubule agonists, hormone modulators, and steroids, natural products, and, for example, bioreaction modifiers, which are described in more detail below.

[0121] Cancer chemotherapeutic agents include non-peptidic (i.e., non-proteinic) compounds that reduce the proliferation of cancer cells, and also include cytotoxic agents and cell proliferation inhibitors. Non-limiting examples of chemotherapeutic agents include alkylating agents, nitrosoureas, antimetabolites, antitumor antibiotics, plant (vinca) alkaloids, and steroid hormones. Peptidic compounds may also be used. Suitable cancer chemotherapeutic agents include drastatin and its active analogs and derivatives; and auristatin and its active analogs and derivatives (e.g., monomethyl auristatin D (MMAD), monomethyl auristatin E (MMAE), monomethyl auristatin F (MMAF), etc.). See, for example, WO96 / 33212, WO96 / 14856, and U.S. Patents 6,323, 315. For example, drastatin 10 or auristatin PE may be included in the antibody-drug conjugates of this disclosure. Important cancer chemotherapy agents include maytansinoids and their active analogs and derivatives (for example) See EP1391213; and Liu et al (1996) Proc. Natl. Acad. Sci. USA 93:8618-8623; Zuokalmai Syn and its active analogs and derivatives (for example, its synthetic analog, KW- Including 2189 and CB1-TMl); and benzodiazepines and their active ana Logs and derivatives (including, for example, pyrrolobenzodiazepines (PBD)).

[0122] Drugs that act to reduce cell proliferation are well known and widely used in the art. Such drugs include, but are not limited to, nitrogen mustard, nitrosourea, ethyleneimine derivatives, alkyl sulfonates, and triazines, as well as mechloretamine and cyclophosphamide (Cytoxan). TM ), melphalan (L-sarcolysin), carmustine (BCNU), lomustine ( CCNU), semustine (methyl-CCNU), streptozocin, chlorozotosin, u It contains alkylating agents including racil mustard, chlormethine, ifosfamide, chlorambucil, pipobromane, triethylenemelamine, triethylenethiophosphoramine, busulfan, dacarbazine, and temozolomide.

[0123] Antimetabolites are not limited to cytarabine (CYTOSAR-U), cytocin. Rabinoside, fluorouracil (5-FU), floxuridine (FudR), 6-thioguanine, 6-mercaptopurine (6-MP), pentostatin, methotrexate, 10- Propargyl-5,8-dideazafolate (PDDF, CB3717), 5,8-dideazateto This product contains folate analogs, pyrimidine analogs, purine analogs, and adenosine deaminase inhibitors, including lahydrofolate (DDATHF), leucovorin, fludarabine phosphate, pentostatin, and gemcitabine.

[0124] Suitable natural products and their derivatives (e.g., vinca alkaloids, antitumor antibiotics, enzymes, lymphokines, and epipodophyllotoxins) are, but are not limited to, ara- C, Paclitaxel (Taxol®), Docetaxel (Taxotere®), Deoxycoformycin, Mitomycin-C, Azathioprine; Brequina brequinar; alkaloids, e.g., vincristine, vinblastine, vinorelbine, vindesine; podophyllotoxins, e.g., etoposide, teniposide; antibiotics, e.g., anthracyclines, daunorubicin hydrochloride (daunomycin, rubidomycin, Cerubidine™, idarubicin, doxorubicin, epirubicin and morpholino derivatives); phenoxizone biscyclopeptide, e.g., dactinomycin; basic compounds Lycopeptides, e.g., bleomycin; anthraquinone glycosides, e.g., plicamycin (mitramycin); anthracendions, e.g., mitoxantrone; azilinopyrroindoldiones, e.g., mitomycin; macrocyclic immunosuppressants, e.g., cyclosporine, FK-506 (including tacrolimus, Prograf, rapamycin, etc.).

[0125] Other antiproliferative cytotoxic drugs include navelbine, CPT-11, and Ana. These include anastrazole, letrozole, capecitabine, reloxafine, cyclophosphamide, ifosfamide, and droloxafine. Microtubule-agonists that possess antiproliferative activity and are suitable for use include, but are not limited to, allocolchicine (NSC406042), halichondrin B (NSC609395), colchicine (NSC757), colchicine derivatives (e.g., NSC33410), dorastatin 10 (NSC376128), meitansine (NSC153858), rhizoxin (NSC332598), paclitaxel (Taxol®), Taxol® derivatives, docetaxel (Taxotere®), thiocolchicine (NSC361792), tritylcysteine, vinblastine sulfate, vincristine sulfate, and, but are not limited to, epotilon A, epotilon B, and discodermolide. Natural and synthetic epothyrons; including estramustine, nocodazole, etc.

[0126] Appropriate hormone modulators and steroids (including synthetic analogs) for use include, but are not limited to, corticosteroids such as prednisone, dexamethoxazole. Metazone, etc.; Estrogens and progestins, e.g., hydroxyprogesterone caprylate, medroxyprogesterone acetate, megestrol acetate, estradiol, clomiphene, tamoxifen, etc.; and adrenal cortical inhibitors, e.g., aminoglutethimide; L7α-ethinylestradiol; diethylstilbestrol, testosterone, flu This includes oxymesterone, dromostanolone propionate, testactone, methylprednisolone, methyltestosterone, prednisolone, triamcinolone, chlorotrianicene, hydroxyprogesterone, aminoglutethimide, estramustine, medroxyprogesterone acetate, leuprolide, flutamide (Drogenil®), toremifene (Fareston®), and Zoladex®. Estrogen stimulates proliferation and differentiation; therefore, compounds that bind to estrogen receptors are used to block this activity. Adrenocortical steroids can inhibit T cell proliferation.

[0127] Other suitable chemotherapeutic agents include metal complexes, such as cisplatin (cis-DDP) and carbo. Platin; urea, e.g., hydroxyurea; and hydrazine, e.g., N-methyl hydrazine This includes radins; epidophyllotoxin; topoisomerase inhibitors; procarbazine; mitoxantrone; leucovorin; tegafur; etc. Other antiproliferative agents of interest include immunosuppressants such as mycophenolic acid, thalidomide, desoxyspergualin, azaserin, leflunomide, mizoribine, azaspiron (SKF105685); Iressa (registered trademark) (ZD1839, 4-(3-chloro-4-fluorophenylamino)-7-methoxy-6-(3-( Includes 4-morpholinyl propoxyquinazoline, etc.

[0128] Taxanes are suitable for use. “Taxane” includes any active taxane derivative or prodrug, as well as paclitaxel. “Paclitaxel” (as used herein, e.g., docetaxel, TAXOL) TM TAXOTERE TM(Docetaxel formulations, to be understood as including analogs, formulations, and derivatives such as the 10-deacetylation (10-desacetyl) analog of paclitaxel and the 3'N-desbenzoyl-3'Nt-butoxycarbonyl analog of paclitaxel) can be readily synthesized using techniques well known to those skilled in the art (WO94 / 07882, WO94 / 07881, WO94 / 07880, See also WO94 / 07876, WO93 / 23555, WO93 / 10076; US 5,294,637; 5,283,253; 5,279,949; 5,274,137; 5,202,448; 5,200,534; 5,229,529; and EP590,267) or, for example, SigmaChemical, St. Louis, Missouri (T7 from Pacific yew (Taxus brevifolia)). It is available from various commercial sources, including 402; or T-1912 from Taxus yannanensis. Paclitaxel should not be understood to refer only to the forms that are normally chemically available from paclitaxel, but also to its analogs and derivatives (e.g., Taxotere as mentioned above). TM This refers to docetaxel and paclitaxel complexes (e.g., paclitaxel-PEG, paclitaxel-dextran, or paclitaxel-xylose). The term "taxane" includes hydrophilic derivatives and hydrophobic derivatives. These are various known derivatives, including both sex derivatives. Taxane derivatives include, but are not limited to, galactose and mannose derivatives described in international patent application WO99 / 18113; piperazino and other derivatives described in WO99 / 14209; taxane derivatives described in WO99 / 09021, WO98 / 22451 and U.S. Patent No. 5,869,680; 6-thio derivatives described in WO98 / 28288; U.S. Patent No. 5,821, This includes sulfonamide derivatives as described in U.S. Patent No. 263; and taxol derivatives as described in U.S. Patent No. 5,415,869. This also includes, but is not limited to, prodrugs of paclitaxel, including those described in WO98 / 58927; WO98 / 13059; and U.S. Patent No. 5,824,701.

[0129] Suitable biological reaction modifiers for use include, but are not limited to, (1) tyrosine kinase (RTK) activity inhibitors; (2) serine / threonine kinase activity inhibitors; (3) tumor-associated antigen antagonists such as antibodies that specifically bind to tumor antigens; (4) apoptosis receptor agonists; (5) interleukin-2; (6) IFN-α; (7) IFN-γ; (8) (9) Includes Ronnie's stimulating factor; and angiogenesis inhibitors.

[0130] Subjects treatable by the methods and compositions of this disclosure may include subjects of any age. In some cases, such subjects may be adults. For example, adult human subjects are 18 years of age or older. Subjects treatable by the methods and compositions of this disclosure may include younger subjects. For example, younger human subjects may be under 18 years of age. In some cases, subjects may be in an age range of 1 month to 18 years, such as 1 year to 18 years, including 2 years to 18 years, including 5 years to 16 years, etc.

[0131] In some cases, the method includes diagnosing whether a subject has AML. A subject can be diagnosed for having AML using any conventional procedure. In some cases, the French, American, and British (FAB) classification systems can be used for the diagnosis and classification of acute myeloid leukemia. Diagnosis of acute myeloid leukemia requires that myeloblasts constitute 30% or more of the bone marrow cells or circulating white blood cells (or 20% based on the latest World Health Organization (WHO) classification system). The hematological nature of the disease defines the various subtypes described below. The FAB nomenclature (M1 to M7) classifies the subtypes of acute myeloid leukemia according to the closest similar normal bone marrow element. In some cases, the method includes a method for determining the suitability of a subject diagnosed with AML for treatment using a polyalkylene oxide asparaginase composition, for example, as described herein. In some cases, the method includes monitoring the effectiveness of the treatment. The effectiveness of the treatment can be monitored using any conventional procedure.

[0132] [Manufacturing method] Aspects of this disclosure include methods for producing the polyalkylene oxide-asparaginase compositions described herein. In particular, the method is a method for producing the liquid polyalkylene oxide-asparaginase compositions described herein. The method may include a method for producing an aqueous composition comprising a polyalkylene oxide-asparaginase having a polyalkylene oxide group covalently bonded to the asparaginase by a linker, a buffer, and a salt.

[0133] Embodiments of a method for producing a polyalkylene oxide-asparaginase composition may include producing an aqueous concentrate. For example, a method for producing this aqueous concentrate may include preparing a solution of asparaginase (e.g., L-asparaginase); polyalkylene The process may include one or more steps of: binding a polyalkylene oxide (e.g., polyethylene glycol) to asparaginase; purifying the polyalkylene oxide-asparaginase; filtering and concentrating the polyalkylene oxide-asparaginase solution; diluting the polyalkylene oxide-asparaginase solution; filtering the polyalkylene oxide-asparaginase solution and filling the polyalkylene oxide-asparaginase solution into sterile containers; and storing the polyalkylene oxide-asparaginase solution.

[0134] The above method for producing an aqueous concentrated composition uses asparaginase (e.g., L-asparaginase). A solution of asparaginase can be prepared. This asparaginase can be mixed with an aqueous solution (e.g., a buffered aqueous solution). Examples of suitable buffers are not limited to phosphate buffer, phosphate-buffered saline (PBS), and Dulbecco's phosphate-buffered saline. Examples include DPBS, Hank's HBSS, Earle's EBSS, Tris buffer, Ringer's lactate buffer, borate buffer, and combinations thereof. In some cases, asparaginase is mixed with phosphate buffer.

[0135] The phosphate buffer may contain dibasic sodium phosphate and monobasic sodium phosphate. In some cases, the amount of dibasic sodium phosphate in the aqueous concentrated composition is in the range of 0.05 to 5 wt.%, such as 0.1 to 4.5 wt.%, or 0.1 to 4 wt.%, or 0.1 to 3.5 wt.%, or 0.1 to 3 wt.%, or 0.1 to 2.5 wt.%, or 0.1 to 2 wt.%, or 0.1 to 1 wt.%, or 0.1 to 0.9 wt.%, or 0.1 to 0.8 wt.%, or 0.1 to 0.7 wt.%, or 0.1 to 0.6 wt.%, or 0.2 to 0.6 wt.%, or 0.3 to 0.6 wt.%, or 0.4 to 0.6 wt.%, or 0.5 to 0.6 wt.%. For example, dibasic sodium phosphate may be present in an aqueous concentrated composition in an amount ranging from 0.1 to 1 wt.%. In certain cases, dibasic sodium phosphate may be present in a composition in an amount ranging from 0.2 to 0.8 wt.%. In certain cases, dibasic sodium phosphate may be present in a composition in an amount ranging from 0.3 to 0.6 wt.%. In certain cases, dibasic sodium phosphate may be present in a composition in an amount ranging from 0.5 to 0.6 wt.%. For example, dibasic sodium phosphate may be present in a composition in an amount of approximately 0.6 wt.%, such as 0.56 wt.% (or 0.558 wt.%). In certain embodiments, the amount of monobasic sodium phosphate in the aqueous concentrated composition is 0.01 to 1.8 wt.%, or 0.01 to 1.6 wt.%, or 0.01 to 1.4 wt.%, or 0.01 to 1.2 wt.%, or 0.01 to 1.0 wt.%, or 0.01 to 0.8 wt.%, or 0.01 to 0.6 wt.%, or 0.01 to 0.4 wt.%, or 0.01 Amounts ranging from 0.005 to 2 wt.% can be present, such as ~0.2 wt.%, or 0.02~0.18 wt.%, or 0.03~0.16 wt.%, or 0.04~0.16 wt.%, or 0.045~0.15 wt.%, or 0.04~0.14 wt.%, or 0.05~0.14 wt.%, or 0.1~0.2 wt.%, or 0.1~0.15 wt.%. For example, monobasic phosphate can be present in an aqueous concentrated composition in an amount ranging from 0.05 to 0.2 wt.%. In a specific example, monobasic sodium phosphate can be present in a composition in an amount of 0.01 to 0.2 wt.%.In certain cases, monobasic sodium phosphate can be present in a composition in an amount of 0.09–0.15 wt.%. In certain cases, monobasic sodium phosphate can be present in a composition in an amount of 0.09–0.2 wt.%. In certain cases, monobasic sodium phosphate can be present in a composition in an amount of 0.1–0.15 wt.%. For example, monobasic sodium phosphate can be present in a composition in an amount of 0.12 wt.% (or 0.129 wt.%).

[0136] Additional components that may be included in the aqueous concentrate composition include salts. Suitable salts include, but are not limited to, sodium chloride, potassium chloride, calcium chloride, magnesium chloride, and combinations thereof. In a specific example, this salt is sodium chloride.

[0137] In some cases, the amount of salt (e.g., sodium chloride) in the composition in the aqueous concentrate is in the range of 0.05 to 5 wt.%, such as 0.05 to 4 wt.%, or 0.05 to 3 wt.%, or 0.05 to 2 wt.%, or 0.1 to 5 wt.%, or 0.1 to 4 wt.%, or 0.1 to 3 wt.%, or 0.1 to 2 wt.%, or 0.1 to 1.5 wt.%, or 0.1 to 1 wt.%, or 0.2 to 1 wt.%, or 0.3 to 1 wt.%, or 0.4 to 1 wt.%, or 0.5 to 1 wt.%, or 0.6 to 1 wt.%, or 0.7 to 1 wt.%, or 0.8 to 1 wt.%, or 0.8 to 0.9 wt.%. For example, a salt (e.g., sodium chloride) can be present in the composition in an amount ranging from 0.5 to 1 wt.%. For example, a salt (e.g., sodium chloride) can be present in the composition in an amount ranging from 0.2 to 2 wt.%. In a specific example, a salt (e.g., sodium chloride) of 0.7 to 1 Amounts in the range of 0 wt.% can be present. In certain examples, the amount of salt (e.g., sodium chloride) in the composition can range from 0.8 to 0.9 wt.%. For example, the amount of salt (e.g., sodium chloride) in the composition can be 0.85 wt.%.

[0138] Other aspects of the present disclosure include methods for producing lyophilized storage-stable compositions as described herein. In particular, the method is a method for producing lyophilized polyalkylene oxide-asparaginase compositions as described herein. The method may include lyophilizing an aqueous composition comprising a polyalkylene oxide-asparaginase having polyalkylene oxide groups covalently bonded to asparaginase by a linker, a buffer, a salt, and a sugar, in a manner sufficient to produce a lyophilized storage-stable polyalkylene oxide-asparaginase composition.

[0139] In certain embodiments, freeze-drying is used to dehydrate an aqueous concentrate. In one example, freeze-drying includes removing water from an aqueous concentrate. This water can be removed by sublimation. For example, the water in the composition may undergo a phase transition from solid to gas. In certain cases, freeze-drying includes freezing the composition (for example, freezing the water in the composition) and then reducing the pressure around the composition so that the water in the composition undergoes sublimation. During freeze-drying, the temperature of the composition may be reduced to a temperature below the freezing point of the water in the composition. For example, the temperature in the composition may be below 0°C, or below -5°C, or below -10°C, or below -15°C. or below -20°C, or below -25°C, or below -30°C, or below -35°C, or below -40°C, or below -45°C, or below -50°C, or below -55°C, or below -60°C, or below -65°C, or below -75°C. It can be done. In some cases, the temperature in this composition is reduced to -45°C. In this case, the temperature in the composition is reduced to -30°C.

[0140] In certain embodiments, the pressure around the composition is reduced to below standard atmospheric pressure. For example, the pressure around the composition can be reduced to 500T or less, such as 250T or less, or 100T or less, or 50T or less, or 10T or less, or 1T or less, or 500mT or less, or 400mT or less, or 300mT or less, or 200mT or less, or 100mT or less, or 90mT or less, or 80mT or less, or 70mT or less, or 60mT or less, or 50mT or less, or 40mT or less, or 30mT or less, or 20mT or less, or 10mT or less. In some cases, the pressure around the composition is reduced to 60mT or less, such as 50mT or less.

[0141] In some embodiments, freeze-drying may also involve increasing the temperature within the composition while reducing the ambient pressure surrounding it. For example, the temperature within the composition can be increased from the minimum temperature described above to a higher temperature. In some cases, this temperature is increased to promote the sublimation of water within the composition at the reduced ambient pressure.

[0142] Embodiments of a method for producing a lyophilized polyalkylene oxide-asparaginase composition may also include producing an aqueous concentrate that is subsequently lyophilized. A process flow diagram of the method for producing the aqueous concentrate is shown in Figure 1. As shown in Figure 1, the method for producing the aqueous concentrate may include one or more of the following steps: preparing a solution of asparaginase (e.g., L-asparaginase) (10); polyalkylene (20) Binding an oxide (e.g., polyethylene glycol) to asparaginase; (30) Purifying this polyalkylene oxide-asparaginase; (40) Filtering and concentrating the solution of polyalkylene oxide-asparaginase; (50) Diluting the solution of polyalkylene oxide-asparaginase; Filter the solution of polyalkylene oxide-asparaginase and fill the solution of polyalkylene oxide-asparaginase into sterile containers (60); and store the solution of polyalkylene oxide-asparaginase (70).

[0143] The above method for producing an aqueous concentrated composition uses asparaginase (e.g., L-asparaginase). A solution of asparaginase can be prepared. This asparaginase can be mixed with solutions such as aqueous solutions (e.g., buffered aqueous solutions). Suitable buffers include, but are not limited to, phosphate buffer, phosphate-buffered saline (PBS), Dulbecco's phosphate-buffered saline (DPBS), Hank's equilibrium saline solution (HBSS), Earle's equilibrium saline solution (EBSS), Tris buffer, Ringer's lactate buffer, borate buffer, and combinations thereof. In some cases, this asparaginase is mixed with phosphate buffer.

[0144] In some embodiments, this phosphate buffer contains dibasic sodium phosphate and monobasic sodium phosphate. In some cases, the amount of dibasic sodium phosphate in the aqueous concentrate is in the range of 0.05 to 1 wt.%, such as 0.1 to 0.9 wt.%, or 0.1 to 0.8 wt.%, or 0.1 to 0.7 wt.%, or 0.1 to 0.6 wt.%, or 0.1 to 0.5 wt.%, or 0.1 to 0.4 wt.%, or 0.2 to 0.4 wt.%, or 0.2 to 0.3 wt.%, or 0.25 to 0.3 wt.%. For example, dibasic sodium phosphate can be present in the aqueous concentrate in the range of 0.1 to 0.5 wt.%. In certain embodiments, the amount of monobasic sodium phosphate in the aqueous concentrated composition is 0.01-0.9 wt.%, or 0.01-0.8 wt.%, or 0.01-0.7 wt.%, or 0.01-0.6 wt.%, or 0.01-0.5 wt.%, or 0.01-0.4 wt.%, or 0.01-0.3 wt.%, or 0.01 The amount is in the range of 0.005 to 1 wt.%, such as ~0.2 wt.%, or 0.01 to 0.1 wt.%, or 0.02 to 0.09 wt.%, or 0.03 to 0.08 wt.%, or 0.04 to 0.08 wt.%, or 0.045 to 0.075 wt.%, or 0.04 to 0.07 wt.%, or 0.05 to 0.07 wt.%. For example, this monobasic sodium phosphate can be present in an aqueous concentrated composition in an amount ranging from 0.01 to 0.1 wt.

[0145] Additional components that may be included in the aqueous concentrate include salts. Suitable salts include, but are not limited to, sodium chloride, potassium chloride, calcium chloride, magnesium chloride, and combinations thereof. In a specific example, this salt is sodium chloride.

[0146] In some cases, the amount of salt (e.g., sodium chloride) in the aqueous concentrate is in the range of 0.05 to 1 wt.%, such as 0.1 to 0.9 wt.%, 0.1 to 0.8 wt.%, 0.1 to 0.7 wt.%, 0.1 to 0.6 wt.%, 0.1 to 0.5 wt.%, 0.2 to 0.5 wt.%, 0.3 to 0.5 wt.%, 0.4 to 0.5 wt.%, or 0.4 to 0.45 wt.%. For example, the amount of salt (e.g., sodium chloride) in the aqueous concentrate can be in the range of 0.1 to 1 wt.%.

[0147] Other components that may be included in this aqueous concentrate are sugars. Suitable sugars include, but are not limited to, sucrose, mannitol, maltose, trehalose, 2-hydroxypropyl-β-cyclodextrin (HPCD), lactose, glucose, fructose, and galac. This includes toxin, glucosamine, and combinations thereof. In specific examples, this disaccharide is a sugar disaccharide. For example, this disaccharide may be sucrose.

[0148] In some cases, the amount of sugar (e.g., sucrose) in this aqueous concentrate is 0.5-20 wt.%, or 1-15 wt.%, or 1-10 wt.%, or 1-9 wt.%, or 1-8 wt.%, or 2-7 wt.%, or 2-6 wt.%, or 3-5 wt. This is an amount in the range of 0.1 to 25 wt.%, such as .%, or 4-5 wt.%. For example, an amount in the range of 1 to 10 wt.% can be present in this aqueous concentrated sugar composition (e.g., sucrose).

[0149] After the preparation of the asparaginase solution, the asparaginase can be bound to the polyalkylene oxide (e.g., polyethylene glycol) so that the polyalkylene oxide can covalently bind to the asparaginase to form a polyalkylene oxide-asparaginase complex. After the preparation of the polyalkylene oxide-asparaginase, the solution can be purified. In some cases, purification involves passing the solution through a filter to remove certain substances from it. The filtration step can produce substantially purified polyalkylene oxide-asparaginase.

[0150] In one example, the filtered polyalkylene oxide-asparaginase solution is then subjected to a diafiltration and concentration step. This polyalkylene oxide-asparaginase solution can be dialyzed using an ultrafiltration membrane, and a concentrated polyalkylene oxide-asparaginase can be obtained as a result. The concentrate from the diafiltration step can then be diluted so that the solution contains the desired concentration of polyalkylene oxide-asparaginase. Suitable buffers useful for the dilution step include those mentioned above. In specific cases, phosphate buffer is used to dilute the polyalkylene oxide-asparaginase solution, thereby producing the desired aqueous concentrated composition. For example, the concentrate from the diafiltration step can be diluted to contain an amount of polyalkylene oxide-asparaginase such that the resulting aqueous concentrate composition has a potency (activity) in the range of 100 to 5,000 IU / mL, such as 500 to 4,500 IU / mL, or 500 to 4,000 IU / mL, or 500 to 3,500 IU / mL, or 500 to 3,000 IU / mL, or 1,000 to 3,000 IU / mL. In a particular example, this aqueous concentrate composition contains an amount of polyalkylene oxide-asparaginase in the range of 1,500 to 3,000 IU / mL. In some cases, the amount of polyalkylene oxide-asparaginase in this aqueous concentrate composition is greater than the amount of polyalkylene oxide-asparaginase in the reconstituted lyophilized composition described herein. In some cases, the diafiltration produces substantially purified polyalkylene oxide-asparaginase.

[0151] This aqueous concentrate can then be filtered and filled into a sterile container. Suitable container materials for the container include, but are not limited to, polymers such as polypropylene, polymethylpentene, polytetrafluoroethylene (PTFE), perfluoroether (PFE), fluorinated ethylene propylene (FEP), perfluoroalkoxyalkane (PFA), polyethylene terephthalate (PET), polyethylene (PE), polyetheretherketone (PEEK), and polystyrene. For example, this may be a sterile polymer bag. This aqueous concentrate can be stored in the container for a certain period of time and can be processed into a freeze-dried, storage-stable composition of the present disclosure.

[0152] Embodiments of the above method may further include shipping the aqueous concentrate to a remote location. A “remote location” is a location different from the place where the aqueous concentrate is manufactured. For example, a remote location could be another location in the same city (e.g., an office or laboratory), another location in a different city, another location in a different state, another location in a different country, etc., and when one item is indicated as “remote” from another, it means that the two items are in the same room but separated, or in at least different rooms, or in different buildings, and can be at least one mile, ten miles, or more than 100 miles apart.

[0153] In certain embodiments, as described above, the method includes lyophilizing an aqueous concentrate to a degree sufficient to produce a lyophilized, storage-stable polyalkylene oxide-asparaginase composition. In some examples, this lyophilization can be carried out in a unit-dose container. Lyophilizing an aqueous concentrate in a unit-dose container to produce a lyophilized, storage-stable polyalkylene oxide-asparaginase composition facilitates the production of the lyophilized composition in a unit-dose container by eliminating the need, for example, to lyophilize the aqueous concentrate in another container and then transfer the lyophilized composition from the other container to the unit-dose container. As such, in some embodiments, the method includes introducing an aqueous concentrate into a unit-dose container and lyophilizing the aqueous concentrate in the unit-dose container. As described above, the unit-dose container may be a vial, such as a glass vial.

[0154] After freeze-drying, the method may further include sealing the freeze-dried composition in a unit-dose container. For example, a sealed container, which is sealed by attaching a seal or lid to the opening of the unit-dose container, can be stored for a long period of time, such as one week or more, or two weeks or more, or three weeks or more, or one month or more, or two months or more, or three months or more, or four months or more, or six months or more, or nine months or more, or one year or more, or 1.5 years (e.g., 18 months) or more, or two years or more, or 2.5 years (e.g., 30 months) or more, or three years or more, or 3.5 years (e.g., 42 months) or more, or four years or more, or 4.5 years (e.g., 54 months) or more, or five years or more. For example, a long period of time may be six months or less. In some cases, this sealed container can be stored for nine months or more. In some cases, this sealed container can be stored for one year (e.g., 12 months) or more. In some cases, this sealed container can be stored for 1.5 years (e.g., 18 months) or more. In some cases, this sealed container can be stored for more than two years (for example, 24 months).

[0155] [kit] Further provided is a kit comprising one or more of the above-described liquid and / or lyophilized compositions used to carry out the method of the subject. For example, the kit comprises a unit-dose container for containing the liquid composition described herein. Or, for example, the kit comprises a unit-dose container for containing the lyophilized composition described herein. In one example, the kit comprises two or more unit-dose containers, each containing one of the liquid compositions described herein. In another example, the kit comprises two or more unit-dose containers, each containing one of the lyophilized compositions described herein. In some examples, the kit comprises two or more unit-dose containers, one or more of which contain one of the liquid compositions described herein, and one or more of which contain one of the lyophilized compositions described herein. In certain embodiments, the kit comprises packaging material configured to contain the unit-dose containers. This packaging material may be a sealed packaging material, such as a sterile sealed packaging material. Sterile packaging materials can be configured to be sealed from the external environment, so that virtually no pathogens (fungi, bacteria, viruses, spores, etc.) are present inside the packaging material. In some cases, this packaging material may optionally be sealed with water vapor-resistant packaging material under an airtight and / or vacuum seal.

[0156] In certain embodiments, the kit includes a buffer solution. For example, the kit may include a diluent suitable for administration to a subject, such as a diluent buffer solution. The kit further includes other components, such as an administration device or liquid source, which may be used in carrying out the method of the subject. The various components in the kit can be packaged as desired, for example, together or separately. The components of the kit of the subject may be in separate containers, or multiple components may be in a single container, and the container and / or packaging material (or part thereof) of the kit may be sterile, if desired.

[0157] In addition to the components described above, the subject kit may further include instructions for using the components of this kit to carry out the subject method. Instructions for carrying out the subject method are generally recorded on a suitable recording medium. For example, these instructions may be printed on paper or plastic, etc. As such, these instructions may be present in the kit as an accompanying document, on the label of the kit container, or within its components (i.e., attached to the packaging material or part of the packaging material). In other embodiments, these instructions may be stored electronically as a data file, for example, on a portable flash drive, CD-ROM, It resides on appropriate computer-readable storage media such as DVD-ROMs and Blu-rays. In this embodiment, the actual instruction manual is not present in the kit, but instructions for obtaining the instruction manual are provided from a remote source, such as the internet. An example of this embodiment is a kit that includes a URL from which the instruction manual can be viewed and / or downloaded. The instruction manual is downloadable. Along with the instruction manual, an embodiment for obtaining the instruction manual is also recorded on a suitable medium.

[0158] [utility] The subject composition (e.g., liquid or lyophilized storage-stable composition) and method find use when there is a desire to treat a subject's disease or condition by administering polyalkylene oxide-asparaginase. For example, the subject composition (e.g., liquid or lyophilized storage-stable composition) and method find use in the treatment of a subject's tumor condition. In some cases, the subject composition (e.g., liquid or lyophilized storage-stable composition) and method find use in the treatment of a subject's cancer. Examples of cancer types that may be treatable with the subject composition (e.g., liquid or lyophilized storage-stable composition) and method include, but are not limited to, leukemia such as acute lymphoblastic leukemia (ALL) and acute myeloid leukemia (AML). Therefore, the subject's lyophilized storage-stable composition and method find use in providing therapeutically effective treatments for tumor conditions such as cancer, including, but are not limited to, leukemia such as acute lymphoblastic leukemia (ALL) and acute myeloid leukemia (AML).

[0159] The lyophilized storage-stable compositions and the methods of this disclosure find applications in treating subjects of any age. In some cases, the compositions (e.g., liquid or lyophilized storage-stable compositions) and methods of the subject matter find applications in treating adults. For example, human adult subjects may be 18 years of age or older. In other cases, the compositions (e.g., liquid or lyophilized storage-stable compositions) and methods find applications in treating minors. For example, human minor subjects may be under 18 years of age.

[0160] The compositions and methods of the disclosure find applications when a storage-stable composition is desired. For example, the compositions and methods of the disclosure find applications in providing a storage-stable composition that is stable for a long period of time (e.g., substantially does not decompose and / or substantially retains all of its activity). For example, the compositions and methods of the disclosure find applications in providing a storage-stable composition that is stable for a long period of time, such as one week or more, or two weeks or three weeks or more, or one month or more, or two months or more, or three months or more, or four months or more, or six months or more, or nine months or more, or one year or more, or 1.5 years (e.g., 18 months) or more, or two years or more, or 2.5 years (e.g., 30 months) or more, or three years or more, or 3.5 years (e.g., 42 months) or more, or four years or more, or 4.5 years (e.g., 54 months) or more, or five years or more. In some cases, the compositions and methods of the disclosure find applications in providing a storage-stable composition that is stable for nine months or more. In some cases, the compositions and methods of the disclosure find applications in providing storage-stable compositions that are stable for 1 year (e.g., 12 months) or longer. In other cases, the compositions and methods of the disclosure find applications in providing storage-stable compositions that are stable for 1.5 years (e.g., 18 months) or longer. In some cases In particular, the compositions and methods of the disclosure find applications in providing storage-stable compositions that are stable for two years (e.g., 24 months) or longer. In specific embodiments, the compositions and methods of the disclosure find applications in providing storage-stable compositions that increase the shelf life of the composition by up to one week, two weeks, three weeks, one month, two months, three months, four months, six months, nine months, one year, or 1.5 years (e.g., 18 months), or two years, or 2.5 years (e.g., 30 months), or three years, or 3.5 years (e.g., 42 months), or four years or more, or 4.5 years (e.g., 54 months), or five years. In specific embodiments, the compositions and methods of the disclosure find applications in providing storage-stable compositions that increase the shelf life of the composition by one month to five years, or six months to four years, or nine months to three years, or one year to two years.

[0161] In certain embodiments, doses of the Disclosure may be administered prior to, concurrently with, or subsequently to the treatment of one or more other tumor conditions, whether related or unrelated. When administered concurrently with the treatment of other tumor conditions, this is achieved by the administration of a pharmaceutical composition having at least one other active agent, such as a chemotherapy drug, which, in combination, provides a therapeutically effective dose according to a specific therapeutic dosing plan. The administration of separate pharmaceutical compositions or therapies may be carried out simultaneously or at different times (e.g., sequentially, in either order, on the same day or on different days), as long as the combined use of these substances produces a therapeutically effective effect in the treated subject. Thus, the methods and compositions of the Disclosure find applications in the treatment of subjects using combination therapies involving the administration of the polyalkylene oxide-asparaginase of the Disclosure, in combination with one or more additional active agents and / or therapies (e.g., radiotherapy, chemotherapy, immunotherapy, etc.).

[0162] As can be understood from the disclosures provided above, embodiments of this disclosure have a wide range of applications. Accordingly, the examples provided herein are provided for illustrative purposes only and are not intended to be construed as limiting in any way to embodiments of this disclosure. Those skilled in the art will recognize that various non-essential parameters may be changed or modified to produce essentially the same results. Accordingly, the following examples are presented to those skilled in the art to provide a complete disclosure and description of how embodiments of this disclosure may be made and used, and are not intended to limit the scope of the inventors' consideration of their invention, nor are they intended to indicate that all or only the following experiments were performed. While efforts have been made to ensure accuracy with respect to the numbers used (e.g., quantity, temperature, etc.), some degree of experimental error and deviation should be included. Unless otherwise indicated, parts are by weight, molecular weight is weight-average molecular weight, temperature is in degrees Celsius, and pressure is atmospheric pressure or near atmospheric pressure.

[0163] The following embodiments are provided for illustrative purposes only and are not intended to limit the embodiments of this disclosure in any way. While efforts have been made to ensure accuracy regarding the numbers used (e.g., quantities, temperatures, etc.), some degree of experimental error and deviation is, of course, acceptable. [Examples]

[0164] Example 1 Concentrated polyethylene glycol-asparaginase containing SS-PEG linker A concentrated bulk composition was produced according to the following procedure. Following the production of this concentrated bulk composition, a lyophilized composition was produced from the concentrated bulk composition according to the procedure described below. Figure 1 shows a process flow diagram for a method for producing a lyophilized, storage-stable composition according to embodiments of the present disclosure.

[0165] Preparation of L-asparaginase solution The amount of L-asparaginase needed for processing was calculated, and a 6L stainless steel beaker was used. - Weighed into a container and mixed for 5-10 minutes. 5 mg of L-asparaginase to the desired concentration. The amount of phosphate buffer required to dilute to / mL was calculated and weighed. The paraginase was then added to phosphate buffer and mixed in a 7-gallon stainless steel pressure vessel for 10–15 minutes. The samples were removed and subjected to protein and specific activity testing.

[0166] PEGylation The amount of SS-PEG required for this step was calculated and weighed. A 7-gallon stainless steel pressure vessel containing the diluted L-asparaginase solution was heated to 29-31°C under gentle stirring. Once the L-asparaginase solution reached the appropriate temperature range, the mixer speed was increased. The drip pump was started. Once the pH was adjusted to 7.7-7.9 with 0.5NNaOH, the SS-PEG was added to a 7-gallon stainless steel pressure vessel. After 30 minutes The dripping pump was stopped, and the temperature jacket was removed.

[0167] purification The material from this process was then filtered through a 0.45 μm filter (Millipore, Billerica, Massachusetts) using a diafiltration peristaltic pump into another 7-gallon stainless steel pressure vessel. After purification, phosphate buffer was added to the original 7-gallon stainless steel pressure vessel and pumped into the 7-gallon stainless steel pressure vessel as a rinse through the 0.45 μm filter.

[0168] Diafiltration / Concentration The amount of PBS required for the 15X diafiltration was calculated, and the diafiltration system was activated. The 7-gallon stainless steel pressure vessel was placed on a scale. The Millipore Pellicon®-2 diafiltration was set up with a diafiltration peristaltic pump and a membrane with a nominal molecular weight limit of 100,000 Da, pre-treated with 5 L of PBS. The level control system, including the buffer peristaltic pump, was then activated, and the 7-gallon stainless steel pressure vessel was placed on a scale. Yes. For conditioning, the Pellicon(registered trademark)-2 system was used. The container was filled with ethylene glycol-asparaginase and recirculated for 5 minutes. Following conditioning, the permeate waste line was opened and diafiltration was initiated. After 15 and 30 minutes, samples were obtained from the permeate waste line into vials and subjected to activity and protein testing. The material for lyophilization was permeate filtered and then concentrated to ≥18.0 mg / mL and ≥1,850 IU / mL. Upon completion of diafiltration, the volume of the material being processed in the 7-gallon stainless steel pressure vessel was adjusted to reach the desired concentration (≥18.0 mg / mL for processing of the lyophilized formulation) by removing an appropriate amount of permeate. PBS was then pumped through the system and added as a rinse to the 7-gallon stainless steel pressure vessel.

[0169] Quality control testing of the product after diafiltration was performed, including determination of the product's impurity profile. To confirm that the product was retained by the diafiltration membrane, the enzymatic activity (EEA) of the permease was measured at 15 and 30 minutes. Released PEG and N-hydroxysuccinimide (NHS) were measured in the final product. These were components of the impurity profile related to the processing. The official in-process controls for the diafiltration unit operation are shown in Table 1 below. From three formulation raw material compositions for freeze-drying (e.g., concentrated bulk formulation raw material compositions) The generated NHS and free PEG data are shown in Table 2 below. The generated data demonstrate that small changes in the diafiltration / concentration process do not affect the quality of the product.

[0170] [Table 1]

[0171] [Table 2]

[0172] Dilution The substance being processed was mixed in a 7-gallon stainless steel pressure vessel before the sample was removed for activity and protein testing. The volume of the substance being processed was then mixed with PBS to a target protein concentration of 2:18.0 mg / mL (target 20.0 mg / mL), and a target activity of 2:1850 IU / mL for formulation raw materials for lyophilized compositions. The substance was diluted. Before the sample was taken out for quality assurance testing, this diluted processed substance was mixed (see Table 1).

[0173] Sterile filtration The concentrated solution for lyophilization was filtered through a 0.2 μm filter and placed in disposable 20 L sterile bags for storage until lyophilization. Samples were collected and subjected to sterility testing. This lyophilization was performed using polyethylene glycol-asphalt. The bulk formulation raw material composition containing laginase can be stored in a 20L bag at 2-8°C for up to 2 months until freeze-dried.

[0174] Container closure This bulk formulation raw material containing polyethylene glycol-asparaginase for freeze-drying. The formulation is processed into a lyophilized composition in the following 0.2 μm filtration step and delivered into pre-sterilized disposable 20 L bioprocess bags. The bag's constituent materials included a layer of low-density polyethylene (LDPE), linear low-density polyethylene (LLDPE), nylon, and ethylene vinyl alcohol (EVOH). The surface in direct contact with the product (inner layer) was LDPE. Each bag had two openings with tubing fixed to the bag, which received the bulk formulation raw material composition containing polyethylene glycol-asparaginase for lyophilization. The bag is crimped with clamps until ready to be opened. This bag has been irradiated and opened with an exposure of 25-40 kGy, and has undergone horseshoe crab hemocyte lysate (Limulus Amebocyte Lysate: LAL) endotoxin testing, 100% visible seal and air leak testing, and similarly, 100% visible inspection after assembly. The results of the qualification tests indicate that the inner layer of this bag is USP compliant. <88> Class IV plastics testing, USP <87> Cytotoxicity testing, and USP <661> It has been shown to have passed biological reactivity tests, including tests on its physicochemical properties. In addition, this bag has passed USP <788> We complied with the specific requirements for injections, and all extractable material tests complied with the manufacturer's requirements.

[0175] Stability waiting time for pharmaceutical raw materials Concentrated bulk formulation raw materials were monitored for 0, 2, 4, 6, 8, and 12 weeks at 2–8°C. Stability samples were held in sample bags with a 250 mL polyethylene product contact surface. Data from the test design are presented in Table 3 (Lot 1), Table 4 (Lot 2), and Table 5 (Lot 3). Stability data for the concentrated bulk formulation raw material for lyophilized compositions showed that it met the acceptance criteria throughout a 12-week test plan and demonstrated a 2-month waiting period from the manufacture of the concentrated bulk formulation raw material, and the manufacture of the lyophilized composition was permitted.

[0176] [Table 3]

[0177] [Table 4]

[0178] [Table 5]

[0179] Freeze-dried composition A lyophilized composition powder for injection contains 3,750 IU of activated PEGylated L-asparagina. The lyophilized composition was prepared in a single-use vial containing -se (750 IU / mL after reconstitution by WFI of 5.2 mL). The components of the lyophilized composition after reconstitution contained 4.5% sucrose, dibasic sodium phosphate, monobasic sodium phosphate, and sodium chloride. The components of this lyophilized composition are provided in Table 6. In addition, this lyophilized composition has a 20 mm aluminum flip-off seal. It was served in a processed glass vial (Nipro Type 1).

[0180] [Table 6]

[0181] Formulation development of freeze-dried polyethylene glycol-asparaginase The purpose of developing freeze-dried polyethylene glycol-asparaginase formulations is to be less The goal was to achieve a stable, freeze-dried composition suitable for storage for 18 months at 2-8°C or 25°C.

[0182] Evaluating the feasibility of a lyophilized composition of polyethylene glycol-asparaginase. Valuable initial experiments were conducted. Five variations of the lyophilized composition were investigated. Each formulation was in 50 mM phosphate buffer at pH 6.5 with 5 mg / mL polyethylene glycol-asparaginase and 5% w / v of five different antifreeze agents (mannitol, It contained maltose, sucrose, trehalose, and / or one of the following (HPCD). In addition, polyethylene glycol-asparaginase without antifreeze is also used for freeze-drying. were prepared. These six lyophilized compositions were compared to the lots of polyethylene glycol-asparaginase (Oncaspar® (registered trademark)) in the liquid formulations used in the preparation of these different formulations. SEC-purity (GF-HPLC) was used to evaluate the quality of the polyethylene glycol-asparaginase compositions. The results of the study are shown in a table. Table As can be seen from Table 7, sucrose was found to be the most effective in maintaining the purity of polyethylene glycol-asparaginase during lyophilization. The purity (83.0%) of the lyophilized composition containing 5% sucrose was comparable to that (83.8%) of the liquid polyethylene glycol-asparaginase formulation used for the preparation of the six formulations for subsequent lyophilization.

[0183]

Table 7

[0184] Several different additives (e.g., sucrose, trehalose, mannitol, polysorbate 80) were evaluated as potential stabilizing agents to be included in the lyophilized compositions. Four experiments were conducted, each containing four different compositions. Small-scale pilot-scale batches of lyophilized polyethylene glycol-asparaginase were prepared with different additives and their stability was evaluated. The list of formulations tested is shown in Table 8.

[0185]

Table 8

[0186] All lots described in Table 8 were evaluated in stability tests at 5°C, 25°C and 40°C and the test panel (activity, specific activity, protein, pH, purity (GF-HPLC ​​) evaluated by aggregates (GF-HPLC) and microparticles, whereby polyethylene glycol-asparaginase was evaluated for its major quality characteristics at the time of shipment and stability. Based on stability data collected from 16 different formulations described in Table 8, sucrose was identified as a suitable cryoprotectant (e.g., stabilizer). Based on stability data collected from 16 different formulations described in Table 8, sucrose was identified as a suitable cryoprotectant (e.g., stabilizer). Various sucrose concentrations were tested to evaluate the appropriate concentration for a stronger (i.e., less impact on the lyophilization procedure) and more stable product. As summarized in Table 9, batches containing additional different concentrations of pilot-scale sucrose were prepared. The sucrose and PEG-asparaginase concentrations shown in Table 9 indicate the amounts present in the concentrated bulk drug substance. During the lyophilization process, the vials are filled to 2.5 mL prior to the start of lyophilization. These lyophilized vials are reconstituted to 5.0 mL, resulting in final sucrose and PEG-asparaginase concentrations that are approximately half of those shown in the table. This study demonstrated that an increase in sugar content enables higher lyophilization temperatures, less stress on the lyophilized product, and a reduction in the overall lyophilization cycle time, and results in a more dried (more stable) lyophilized product. During the lyophilization process, the vials are filled to 2.5 mL prior to the start of lyophilization. These lyophilized vials are reconstituted to 5.0 mL, resulting in final sucrose and PEG-asparaginase concentrations that are approximately half of those shown in the table. This study demonstrated that an increase in sugar content enables higher lyophilization temperatures, less stress on the lyophilized product, and a reduction in the overall lyophilization cycle time, and results in a more dried (more stable) lyophilized product. This study demonstrated that an increase in sugar content enables higher lyophilization temperatures, less stress on the lyophilized product, and a reduction in the overall lyophilization cycle time, and results in a more dried (more stable) lyophilized product.

[0187]

Table 9

[0188] The five lots described in Table 9 were also evaluated in stability tests at 5°C, 25°C, and 40°C.Quality characteristics such as activity, specific activity, protein, pH, purity (GF-HPLC), aggregates (GF-HPLC), and microparticles were evaluated during this stability study. Quality characteristics such as activity, specific activity, protein, pH, purity (GF-HPLC), aggregates (GF-HPLC), and microparticles were evaluated during this stability study. The purity and potency results from accelerated (25°C) and stressed (40°C) stability studies of these lots are shown in Figures 2–5. These stability data showed that formulations containing 10% sucrose (5% sucrose after reconstitution of the final product with 5 mL / vial of WFI) provided the product with the best stability in terms of purity and potency. Other quality properties were less affected by different formulations and were stable in the lot with 10% sucrose in this case as well. Figure 2 shows the purity of lyophilized PEG-asparaginase compositions of lots SA, SB, SC, SD, SE and IA at 40°C. The graph shows the ratio of degree (%) to time (weeks). Figure 3 shows the potency (IU / mL) versus time of lyophilized PEG-asparaginase compositions of lots SC, SD, SE, and IA at 40°C. The graph shows the purity (per week) versus time (weeks) of the freeze-dried PEG-asparaginase compositions of lots SA, SB, SC, SD, SE, and IA at 25°C. Rough sketch shown. Figure 5 shows lyophilized PEG-ASS of lots SC, SD, SE and IA. This graph shows the efficacy (IU / mL) versus time (weeks) of the paraginase composition at 25°C.

[0189] Freeze drying A buffer solution containing water for injection (WFI), dibasic sodium phosphate, monobasic sodium phosphate, sodium chloride, and sucrose was prepared. This buffer solution was used to dilute concentrated bulk formulation raw materials.

[0190] 90% of the target weight of WFI needed to prepare the buffer solution was added to a beaker. Monobasic sodium phosphate, dibasic sodium phosphate, and sodium chloride were then weighed individually, added to the WFI, and mixed until dissolved. The required amount of sucrose was weighed and added to the mixed buffer solution and mixed until dissolved. The solution was then measured and adjusted to 7.3 ± 0.1 by slowly adding NaOH. WFI was added to the buffer solution as needed, and the density of the concentrated bulk solution was used to weigh the required volume of concentrated bulk solution needed for the batch size. A sample of the buffer was collected for sucrose testing. This concentrated bulk solution was added to the buffer solution, and this final solution was mixed for at least (not less than: NLT) 10 minutes. Once mixing was complete, confirmation pH measurements (7.3 ± 0.1) were performed from the top, middle, and bottom of the container, and samples were collected for testing of protein, density, sucrose, and bioburden during processing before filtration.

[0191] Following the final formulation, this solution underwent sterile filtration. A pre-sterilized filtration tube was used. The tubing assembly was placed in a formulated bulk container, and this bulk was then filtered through two 0.22 μm filters placed on the filtration tubing assembly and filled into a pre-sterilized, ready-to-fill 10 L bioprocess bag. Once all the product had been transferred to this 10 L bioprocess bag, the filtration tubing assembly was removed from the bioprocess bag, and the filters were tested for integrity. One 20 mL sample, before lyophilization, was collected upstream of the filter (unfiltered) and tested according to the specifications of the final pharmaceutical product.

[0192] The diagram shows a flowchart illustrating the final formulation and the sterile filtration process.

[0193] Aseptic filling and freeze-drying Following completion of the sterile filtration, a 10 L biobag containing this bulk drug substance solution was coupled to a filling tubing assembly, and the product vials were then filled to a target fill weight of 2.5 g / vial and partially stoppered using a Flexicon FMB210 filler. During the filling operation, the fill weight was monitored by performing a minimum of one weight check (1 vial) per filled tray (action limit: 2.43 - 2.57 g, warning limit: 2.38 - 2.62 g). When the filling operation was complete, 20 pre-lyophilization vials were tested, and all of the remaining filled vials were transferred to stainless steel lyophilization trays and subsequently filled into a pre-cooled (5°C) 270 ft2 Hull freeze-drying system for lyophilization. The phases included in this lyophilization process are shown in Table 10.

[0194]

Table 10

[0195] ​​​​​​​​​​​​​​​​​​​​​​​​​

[0199] [Table 12]

[0200] stability studies Freeze-dried pharmaceutical product lots 1, 2, and 3 were placed during long-term (2–8°C) and accelerated (25±3°C; 60%±5%RH) stability tests. These lots were also placed during thermal stress stability tests (40±2°C; 75%±5%RH) to evaluate the heat-induced degradation profile of the products.

[0201] Long term stability (2~8°C) Stability data generated from lyophilized drug product lots stored under long-term conditions (2–8°C) are provided in Tables 13–16. Long-term stability data showed that lyophilized drug products stored at 5±3°C remained well within the acceptance criteria at all time points for stability. Moisture content (KF) data were measured for 12 weeks in the range of 0.96%–1.35% (specification = NMT 3.0%) under storage conditions of 2–8°C. Unlike commercially available liquid drug products, which have demonstrated increased activity and decreased purity over time, this trend was not observed in the lyophilized compositions. Stability graphs for purity (Figure 8) and potency (Figure 9), as well as aggregates (Figure 10), at 2–8°C are shown in the accompanying figures.

[0202] [Table 13]

[0203] [Table 14]

[0204] [Table 15]

[0205] [Table 16]

[0206] Accelerated stability (25±3°C; 60%±5%RH) Stability data for lyophilized pharmaceutical product lots stored under accelerated conditions (25±3°C; 60%±5%RH) are provided in Tables 17–20. The stability data showed that the lyophilized pharmaceutical products stored under accelerated conditions remained well within the acceptance criteria at all stability time points. Moisture content (KF) data was measured for 4 weeks in the range of 1.12%–1.23% (specification = NMT 3.0%) under storage conditions of 25°C. The stability chart at 25±3°C is shown in the accompanying figures, which include quality characteristics such as purity (Figure 11), potency (Figure 12), and aggregates (Figure 13).

[0207] [Table 17]

[0208] [Table 18]

[0209] [Table 19]

[0210] [Table 20]

[0211] Thermal stress stability (40±2°C; 75%±5%RH) Stability data for lyophilized drug product lots stored under stress conditions (40±2°C; 75%±5%RH) are provided in Tables 21-24. The stability data showed that the lyophilized drug products stored under stress conditions remained within acceptable limits throughout the study period. Moisture content (KF) data were measured for 4 weeks at 40°C storage conditions, ranging from 1.16% to 1.45% (specification = NMT 3.0%). Stability charts at 40±2°C are shown in accompanying figures, as are potency (Figure 14), purity (Figure 15), and aggregates (Figure 16).

[0212] [Table 21]

[0213] [Table 22]

[0214] [Table 23]

[0215] [Table 24]

[0216] Example 2 SC-PEG linker (i.e., succinimidyl carbonate linker) Composition containing ethylene glycol-asparaginase, PegAsparagase Pegol Succinimidyl carbonate-polyethylene glycol [SC-PEG] (Escherichia coli L-asparaginase) was prepared. The components of this composition are provided in Table 25.

[0217] [Table 25]

[0218] Example 3 Following the preparation of a concentrated bulk composition, a lyophilized composition of peguasparagase pegol can be prepared from the concentrated bulk composition. Figure 1 shows an example of a process flow diagram that can be used to prepare a lyophilized, storage-stable composition according to embodiments of the present disclosure. This lyophilized composition powder for injection can be prepared in a single-use vial containing 3,750 IU of active peguasparagase pegol (750 IU / mL after reconstitution with 5.2 mL of WFI). The components of this lyophilized composition, after reconstitution, include 4.5% sucrose, dibasic sodium phosphate, monobasic sodium phosphate, and sodium chloride. The components of the lyophilized composition are provided in Table 26.

[0219] [Table 26]

[0220] Example 4 The purpose of this study was to provide comparative information on the pharmacokinetics (PK), pharmacodynamics (PD), and immunogenicity of liquid PEG-L-asparaginase (PEG-L-asparaginase; Oncaspar®) and lyophilized PEG-L-asparaginase when administered intravenously to beagle dogs once (on day 1) or once weekly for 4 weeks (days 1, 15, 22, 29, and 36) via slow bolus infusion. Reconstituted lyophilized PEG-L-asparaginase was administered via the same route of administration in this study for intravenous administration in humans. Single-dose and repeated-dose PK / PD studies were necessary to determine and compare the pharmacokinetics and pharmacodynamics of the liquid and reconstituted lyophilized versions at equal doses.

[0221] Beagle dogs (nominal group of 5 dogs / sex / group) were administered 500 IU / kg of liquid peguasparagauze or reconstituted lyophilized peguasparagauze intravenously at a dose of 0.667 mL / kg (see Table 27). These beagles were approximately 6 months old and consisted of males weighing 7.2–10.7 kg and females weighing 5.6 kg–8.2 kg.

[0222] The supplied liquid peg-asparagase (PBS buffer containing 50 mM phosphate and 0.85% physiological saline at pH 7.2–7.45 mL) was used, and no preparation was necessary. To achieve a concentration of 750 IU / mL, the contents of the vial were reconstituted with 5.2 mL of sterile water for injection (WFI) using aseptic techniques to prepare lyophilized peg-asparagase (see Example 1) for administration (using a 21-gauge syringe). Prior to administration, the contents of the vial were gently rotated until completely mixed. This mixture was visually inspected for particulate matter issues such as turbidity and discoloration. A fresh formulation was prepared each day of dose administration, maintained at room temperature, and used within 2 hours of preparation.

[0223] [Table 27]

[0224] Animals in groups 1 and 3 were administered a single dose of liquid pegasparagauze or lyophilized pegasparagauze on day 1, respectively. Animals in groups 2 and 4 were given repeated doses of liquid pegasparagauze or lyophilized pegasparagauze, respectively. A slow bolus (lasting approximately 2 minutes) intravenous injection was administered within 2 hours of preparation of the test item. An indwelling catheter (non-butterfly catheter) was used, and the catheter cap was then cleaned by rinsing with saline to remove any remaining dose. A straight needle was inserted into the catheter cap to ensure that the needle position remained consistent for the entire 2-minute duration.

[0225] For pharmacokinetic and pharmacodynamic analysis, blood samples were taken from all animals on days 1 and 36. Approximately 1.0 mL of whole blood was taken at each time point. Prior to blood collection, the animals were neither anesthetized nor fasted. Blood was collected in a tube containing heparin sodium anticoagulant and placed upright in ice water. To obtain plasma within 5 minutes of the start of blood sample collection, blood samples were centrifuged for 5 minutes (approximately 3000 rpm, approximately 4°C).

[0226] PD: One 125 μL aliquot of plasma was pipetted into a cryotube pre-filled with 125 μL of SeraPrep for asparagine determination. This tube was inverted for 3 hours to mix with SeraPrep and immediately flash-frozen with liquid nitrogen or methanol / dry ice within 15 minutes of collection. All aliquots containing SeraPrep were analyzed by high-performance liquid chromatography (HPLC) (LC-MS / MS) with mass spectrometry detection. It was analyzed for paragine measurement.

[0227] PK: The remaining plasma was divided into two cryotubes for asparaginase activity determination and flash-frozen within 30 minutes of blood sample collection. All aliquots without SeraPrep were analyzed for asparaginase activity by a colorimetric mixed enzyme reaction.

[0228] result Analysis of the freeze-dried pegasparagauze during the aforementioned processing period confirmed that the drug formulation was administered at appropriate concentrations (for batch analysis; expected protein concentrations of 6.6 mg / mL and 741 IU / mL of activity) (Table 28).

[0229] [Table 28]

[0230] The group-mean plasma concentration (Cmax) of pooled asparaginase on day 1, and the area under the pooled group-mean plasma asparaginase concentration-time curve (AUC) estimated up to 552 hours post-administration on day 1. 0.552 The values ​​for each group (males and females combined), as well as those following repeated administration on day 36, are summarized in Table 29.

[0231] [Table 29]

[0232] This study was designed as a parallel-group design, and its data were statistically analyzed using analysis of variance techniques. Cmax and AUC from both days were examined. 0.552 The data were analyzed using an ANOVA model that incorporated formulation, time, sex, and their interactions as factors. These two pegasparagase preparations have Cmax and AUC 0.552 The analysis was conducted, and the two-sided 90% confidence intervals corresponding to the geometric mean ratios are summarized in Table 30.

[0233] [Table 30]

[0234] For Cmax, the confidence interval (0.867~0.970) falls within the rejection region, providing evidence of bioequivalence. AUC 0.552 For this, the confidence interval is (0.841 Since the range (~0.996) falls within the rejection region, there was evidence of biological equivalence.

[0235] The mean maximum plasma concentration (Cmax) of peg asparaginase and the area under the pooled mean plasma asparaginase concentration-time curve (AUC) estimated up to 552 hours after administration on day 1. 0.552 ), as well as the values ​​following repeated administration on day 36, are summarized in Table 31 below for each group (by sex) along with the standard deviation in parentheses.

[0236] [Table 31]

[0237] For lyophilized formulations, the mean maximum plasma concentration (Cmax) of asparaginase and the area under the plasma asparaginase concentration-time curve (AUC) are defined. 0.552 The relationship between the two and the dose levels is expressed as a ratio compared to the liquid formulation and is presented in Table 32.

[0238] [Table 32]

[0239] Cmax and AUC of systemic exposure to asparaginase in dogs 0.552 The values ​​for AUC between the two formulations were similar for administration of reconstituted lyophilized peguasparagauze compared to the liquid formulation, both on day 1 and the subsequent repeated administration on day 36. 0.552 While there was no evidence of a significant difference, there is some evidence that Cmax values ​​differ between different formulations, with Cmax being slightly lower (8%) when administered as the lyophilized product than when administered as the liquid formulation.

[0240] Cmax and AUC of systemic exposure to asparaginase in female dogs 0.552 The values ​​are generally similar to indicators of exposure in males, and Cmax or AUC 0.552 Regarding whole-body exposure, there was no evidence of a statistically significant gender-related difference.

[0241] Other parameters evaluated during the study included viability, clinical observation, body weight, food intake, respiratory rate, body temperature, hematology, coagulation, and blood chemistry; no adverse effects related to the test item were observed for any of these parameters. After repeated intravenous administration (day 36), the Cmax value and range (AUC) of systemic exposure to asparaginase in dogs were measured. 0.552) was higher than the value after a single dose (day 1), and these differences The result was statistically significant (p<0.001). AUC 0.552 The mean cumulative percentage, calculated based on the values ​​(note that different animals provided data on different days), was greater than that indicating asparaginase accumulation occurred after repeated intravenous administration of liquid peg asparagase.

[0242] Overall, these two pegasparagase formulations have Cmax and AUC values ​​such that the corresponding two-sided 90% confidence intervals for the geometric mean rates fall perfectly within the conventional bioequivalence margin of 0.8–1.25. 0.552 They were shown to be biologically equivalent in respect to AUC. 0.552 There was no significant difference between the two formulations in this regard. Systemic exposure to asparaginase was similar for both products, and with repeated dosing, some accumulation occurred in both sexes. Asparagine was completely suppressed for up to 336 hours in all animals, and up to 552 hours in the majority.

[0243] In summary, there were no significant differences between the 500 IU / kg doses of liquid peguasparagase and reconstituted lyophilized peguasparagase, and they had comparable pharmacokinetic, pharmacodynamic, and immunogenicity profiles.

[0244] [Embodiment] In one embodiment, the disclosure provides a lyophilized storage-stable composition comprising a polyalkylene oxide-asparaginase having a polyalkylene oxide group covalently bonded to the asparaginase by a linker. This lyophilized storage-stable composition also comprises a buffer, a salt, and a sugar.

[0245] In some embodiments, the polyalkylene oxide group comprises a polyethylene glycol group. In some embodiments, the polyethylene glycol group has a molecular weight in the range of 2,000 to 10,000 daltons. In some embodiments, the polyethylene glycol group has a molecular weight of 5,000 daltons.

[0246] In some embodiments, this asparaginase is E. coli asparaginase.

[0247] In some embodiments, this linker is a urethane linker. In some embodiments, this linker is a succinic acid linker.

[0248] In some embodiments, this polyalkylene oxide-asparaginase is present in amounts ranging from 500 to 1,000 IU / g.

[0249] In some embodiments, this buffer solution includes a phosphate buffer. In some embodiments, this phosphate buffer solution includes dibasic sodium phosphate and monobasic sodium phosphate. In some embodiments, the dibasic sodium phosphate is present in an amount ranging from 0.1 to 0.5 wt.%. In some embodiments, the monobasic sodium phosphate is present in an amount ranging from 0.01 to 0.1 wt.%.

[0250] In some embodiments, this salt is sodium chloride. In some embodiments, the amount of sodium chloride ranges from 0.1 to 1 wt.%.

[0251] In some embodiments, this sugar is a disaccharide. In some embodiments, this disaccharide contains sucrose. In some embodiments, this sugar contains sucrose in an amount ranging from 1 to 10 wt.%.

[0252] In some embodiments, the composition is present in a unit dose container. This is a vial. In some embodiments, this vial is a sealed glass vial.

[0253] In some embodiments, the polyalkylene oxide-asparaginase is present in an amount of 750 IU / g, the polyalkylene oxide group comprises a polyethylene glycol group having a molecular weight of 5,000 daltons, the asparaginase is E. coli asparaginase, the linker is a urethane linker, the buffer comprises a phosphate buffer containing an amount in the range of 0.25-0.3 wt.% of dibasic sodium phosphate and an amount in the range of 0.05-0.07 wt.% of monobasic sodium phosphate, the salt is sodium chloride in the range of 0.4-0.45 wt.%, and the sugar is sucrose in the range of 4-5 wt.%.

[0254] In some embodiments, the polyalkylene oxide-asparaginase is present in an amount of 750 IU / g, the polyalkylene oxide group comprises a polyethylene glycol group having a molecular weight of 5,000 daltons, the asparaginase is E. coli asparaginase, the linker is a urethane linker, the buffer comprises a phosphate buffer containing 0.279 wt.% of dibasic sodium phosphate and 0.06 wt.% of monobasic sodium phosphate, and the salt is 0.425 wt.% of sodium chloride.

[0255] In some embodiments, the polyalkylene oxide-asparaginase is present in an amount of 750 IU / g, the polyalkylene oxide group comprises a polyethylene glycol group having a molecular weight of 5,000 daltons, the asparaginase is E. coli asparaginase, the linker is succinate linker, the buffer comprises a phosphate buffer containing an amount ranging from 0.25 to 0.3 wt.% of dibasic sodium phosphate and an amount ranging from 0.05 to 0.07 wt.% of monobasic sodium phosphate, the salt is sodium chloride in an amount ranging from 0.4 to 0.45 wt.%, and the sugar is sucrose in an amount ranging from 4 to 5 wt.%.

[0256] In some embodiments, the polyalkylene oxide-asparaginase is present in an amount of 750 IU / g, the polyalkylene oxide group comprises a polyethylene glycol group having a molecular weight of 5,000 daltons, the asparaginase is E. coli asparaginase, the linker is succinate linker, the buffer comprises a phosphate buffer containing 0.279 wt.% of dibasic sodium phosphate and 0.06 wt.% of monobasic sodium phosphate, the salt is 0.425 wt.% of sodium chloride, and the sugar is 4.5 wt.% of sucrose.

[0257] In some embodiments, the polyalkylene oxide-asparaginase is present in an amount of 750 IU / g, the polyalkylene oxide group comprises a polyethylene glycol group having a molecular weight of 5,000 daltons, the asparaginase is E. coli asparaginase, the linker is a urethane linker, the buffer comprises a phosphate buffer containing 0.558 wt.% of dibasic sodium phosphate and 0.129 wt.% of monobasic sodium phosphate, and the salt is 0.85 wt.% of sodium chloride.

[0258] In another embodiment, the disclosure relates to polyethylene glycol-asparaginase having polyethylene glycol groups covalently bonded to E. coli asparaginase by succinate linker, The present invention provides a freeze-dried storage-stable composition containing arginase. In another embodiment, the present invention provides a freeze-dried polyethylene glycol-asparaginase having polyethylene glycol groups covalently bonded to E. coli asparaginase by a urethane linker. The present invention provides a freeze-dried storage-stable composition, which may also contain the phosphate buffer, salts, and optionally disaccharides outlined in the embodiments described above.

[0259] In another embodiment, the present disclosure provides a method for deaminating asparagine in a subject by administering a composition disclosed herein.

[0260] In some embodiments, the method comprises reconstituting a lyophilized storage-stable composition according to this disclosure to produce a reconstituted dosage unit, and administering the reconstituted dosage unit to a subject to deaminate asparagine in the subject. In some embodiments, the reconstitution comprises mixing the lyophilized storage-stable composition with water for injection (WFI).

[0261] In some embodiments, the administration unit contains 700-800 IU / mL of polyalkylene oxide-asparaginase.

[0262] In some embodiments, the dosage unit contains 2.5 to 6 mg / g of dibasic sodium phosphate. In some embodiments, the dosage unit contains 2.5 to 3 mg / g of dibasic sodium phosphate. In some embodiments, the dosage unit contains 5 to 6 mg / g of dibasic sodium phosphate. In some embodiments, the dosage unit contains 5.25 to 5.75 mg / g of dibasic sodium phosphate.

[0263] In some embodiments, the dosage unit contains 0.45 to 1.5 mg / g of monobasic sodium phosphate. In some embodiments, the dosage unit contains 0.45 to 0.75 mg / g of monobasic sodium phosphate. In some embodiments, the dosage unit contains 1 to 2 mg / g of monobasic sodium phosphate. In some embodiments, the dosage unit contains 1 to 1.5 mg / g of monobasic sodium phosphate.

[0264] In some embodiments, the dosage unit contains 4 to 9 mg / g of sodium chloride. In some embodiments, the dosage unit contains 4 to 4.5 mg / g of sodium chloride. In some embodiments, the dosage unit contains 8 to 9 mg / g of sodium chloride.

[0265] In some embodiments, the dose unit contains sucrose. In some embodiments, the amount of sucrose is in the range of 40 to 50 mg / g.

[0266] In some embodiments, the reconstituted dose unit delivers 1,500–3,000 IU / m2 of polyalkylene oxide-asparaginase to the subject. In some embodiments, this reconstituted dose unit delivers 2,000–2,750 IU / m2 of polyalkylene oxide-asparaginase to the subject.

[0267] In some embodiments, the method is a treatment for a tumorous condition in a subject. In some embodiments, this tumorous condition is cancer. In some embodiments, this cancer is leukemia. In some embodiments, this leukemia is acute lymphoblastic leukemia (ALL). In some embodiments, this leukemia is acute myeloid leukemia (AML).

[0268] In some embodiments, subjects are prescribed a therapeutic dosing plan including an induction phase, a coupling phase, and a maintenance phase. In some embodiments, the method includes administering a single reconstituted dose unit to the subject during the induction phase and administering multiple reconstituted dose units during the maintenance phase. In some embodiments, these multiple reconstituted dose units are administered to the subject by administering the reconstituted dose unit to the subject every three weeks. In some embodiments, These multiple reconstituted dose units are administered to the reconstituted dose unit subject every two weeks. It is administered to the subject by doing so.

[0269] In some embodiments, the subject is a minor. In some embodiments, the subject is an adult.

[0270] In some embodiments, the polyalkylene oxide-asparaginase is present in an amount of 750 IU / g, the polyalkylene oxide group comprises a polyethylene glycol group having a molecular weight of 5,000 daltons, the asparaginase is E. coli asparaginase, the linker is a urethane linker, the buffer is a phosphate buffer containing an amount of dibasic sodium phosphate in the range of 2.5–3 mg / g and an amount of monobasic sodium phosphate in the range of 0.5–0.7 mg / g, the salt is sodium chloride in the range of 4–4.5 mg / g, and the sugar is sucrose in the range of 40–50 mg / g.

[0271] In some embodiments, the polyalkylene oxide-asparaginase is present in an amount of 750 IU / g, the polyalkylene oxide group comprises a polyethylene glycol group having a molecular weight of 5,000 daltons, the asparaginase is E. coli asparaginase, the linker is a urethane linker, the buffer comprises a phosphate buffer containing 2.79 mg / g of dibasic sodium phosphate and 0.6 mg / g of monobasic sodium phosphate, the salt is 4.25 mg / g of sodium chloride, and the sugar is 45 mg / g of sucrose.

[0272] In some embodiments, the polyalkylene oxide-asparaginase is present in an amount of 750 IU / g, the polyalkylene oxide group comprises a polyethylene glycol group having a molecular weight of 5,000 daltons, the asparaginase is E. coli asparaginase, the linker is succinate linker, the buffer is a phosphate buffer containing an amount of dibasic sodium phosphate in the range of 2.5–3 mg / g and an amount of monobasic sodium phosphate in the range of 0.5–0.7 mg / g, the salt is sodium chloride in the range of 4–4.5 mg / g, and the sugar is sucrose in the range of 40–50 mg / g.

[0273] In some embodiments, the polyalkylene oxide-asparaginase is present in an amount of 750 IU / g, the polyalkylene oxide group comprises a polyethylene glycol group having a molecular weight of 5,000 daltons, the asparaginase is E. coli asparaginase, the linker is succinate linker, the buffer comprises a phosphate buffer containing 2.79 mg / g of dibasic sodium phosphate and 0.6 mg / g of monobasic sodium phosphate, the salt is 4.25 mg / g of sodium chloride, and the sugar is 45 mg / g of sucrose.

[0274] In another embodiment, the disclosure provides a method for producing a lyophilized polyalkylene oxide-asparaginase composition by lyophilizing an aqueous concentrate composition in a manner sufficient to produce a lyophilized, storage-stable polyalkylene oxide-asparaginase composition. The aqueous concentrate composition comprises a polyalkylene oxide-asparaginase having polyalkylene oxide groups covalently bonded to the asparaginase by a linker, a buffer, a salt, and a sugar.

[0275] In some embodiments, this aqueous concentrate composition contains 1,500 to 3,000 IU / mL of polyalkylene oxide-asparaginase.

[0276] In some embodiments, this aqueous concentrate composition contains 0.1-0.5 wt.% dibasic phosphorus. Contains sodium phosphate.

[0277] In some embodiments, this aqueous concentrate composition contains 0.01 to 0.1 wt.% monobasic sodium phosphate.

[0278] In some embodiments, this aqueous concentrate composition contains 0.1 to 1 wt.% sodium chloride. In some embodiments, this aqueous concentrate composition contains sucrose.

[0279] In some embodiments, the amount of sucrose is in the range of 1 to 10 wt.%. In some embodiments, the method also includes a method for producing this aqueous concentrated composition.

[0280] In some embodiments, the method also includes introducing the aqueous concentrated composition into a unit dose container and freeze-drying the aqueous concentrated composition in the unit dose container. In some embodiments, the unit dose container is a vial. In some embodiments, the vial is a glass vial. In some embodiments, the method also includes sealing the freeze-dried composition in the unit dose container.

[0281] In some embodiments, the polyalkylene oxide group comprises a polyethylene glycol group. In some embodiments, the polyethylene glycol group comprises a molecular weight in the range of 2,000 to 10,000 daltons. In some embodiments, the polyethylene glycol group comprises a molecular weight of 5,000 daltons.

[0282] In some embodiments, this asparaginase is E. coli asparaginase.

[0283] In some embodiments, this linker is a urethane linker. In some embodiments, this linker is a succinic acid linker.

[0284] In some embodiments, this polyalkylene oxide-asparaginase is present in an amount of 750 IU / g, the polyalkylene oxide group comprises a polyethylene glycol group having a molecular weight of 5,000 daltons, the asparaginase is E. coli asparaginase, the linker is a urethane linker, the buffer is a phosphate buffer containing an amount ranging from 0.25 to 0.3 wt.% of dibasic sodium phosphate and an amount ranging from 0.05 to 0.07 wt.% of monobasic sodium phosphate, the salt is sodium chloride in an amount ranging from 0.4 to 0.45 wt.%, and the sugar is sucrose in an amount ranging from 4 to 5 wt.%.

[0285] In some embodiments, the polyalkylene oxide-asparaginase is present in an amount of 750 IU / g, the polyalkylene oxide group comprises a polyethylene glycol group having a molecular weight of 5,000 daltons, the asparaginase is E. coli asparaginase, the linker is succinate linker, the buffer is a phosphate buffer containing an amount ranging from 0.25 to 0.3 wt.% of dibasic sodium phosphate and an amount ranging from 0.05 to 0.07 wt.% of monobasic sodium phosphate, the salt is sodium chloride in an amount ranging from 0.4 to 0.45 wt.%, and the sugar is sucrose in an amount ranging from 4 to 5 wt.%.

[0286] In other embodiments, the disclosure provides a kit comprising two or more unit-dose containers, each containing a lyophilized storage-stable composition. The lyophilized storage-stable composition comprises a polyalkylene oxide-asparaginase having a polyalkylene oxide group covalently bonded to the asparaginase by a linker, a buffer, a salt, and a sugar.

[0287] In some embodiments, the polyalkylene oxide group comprises a polyethylene glycol group. In some embodiments, the polyethylene glycol group has a molecular weight in the range of 2,000 to 10,000 daltons. In some embodiments, the polyethylene glycol group has a molecular weight of 5,000 daltons.

[0288] In some embodiments, this asparaginase is E. coli asparaginase.

[0289] In some embodiments, this linker is a urethane linker. In some embodiments, this linker is a succinic acid linker.

[0290] In some embodiments, this polyalkylene oxide-asparaginase is present in amounts ranging from 500 to 1,000 IU / g.

[0291] In some embodiments, this buffer solution includes a phosphate buffer. In some embodiments, this phosphate buffer solution includes dibasic sodium phosphate and monobasic sodium phosphate. In some embodiments, the dibasic sodium phosphate is present in an amount ranging from 0.1 to 0.5 wt.%. In some embodiments, the monobasic sodium phosphate is present in an amount ranging from 0.01 to 0.1 wt.%.

[0292] In some embodiments, this salt is sodium chloride. In some embodiments, the amount of sodium chloride ranges from 0.1 to 1 wt.%.

[0293] In some embodiments, this sugar is a disaccharide. In some embodiments, this disaccharide contains sucrose. In some embodiments, this sugar contains sucrose in an amount ranging from 1 to 10 wt.%.

[0294] In some embodiments, this unit dose container is a vial. In some embodiments, this unit dose container is a glass vial. In some embodiments, this vial is a sealed glass vial. In some embodiments, this unit dose container is sealed.

[0295] In some embodiments, the polyalkylene oxide-asparaginase is present in an amount of 750 IU / g, the polyalkylene oxide group comprises a polyethylene glycol group having a molecular weight of 5,000 daltons, the asparaginase is E. coli asparaginase, the linker is a urethane linker, the buffer comprises a phosphate buffer containing an amount in the range of 0.25-0.3 wt.% of dibasic sodium phosphate and an amount in the range of 0.05-0.07 wt.% of monobasic sodium phosphate, the salt is sodium chloride in the range of 0.4-0.45 wt.%, and the sugar is sucrose in the range of 4-5 wt.%.

[0296] In some embodiments, the polyalkylene oxide-asparaginase is present in an amount of 750 IU / g, the polyalkylene oxide group comprises a polyethylene glycol group having a molecular weight of 5,000 daltons, the asparaginase is E. coli asparaginase, the linker is succinate linker, the buffer comprises a phosphate buffer containing an amount in the range of 0.25-0.3 wt.% of dibasic sodium phosphate and an amount in the range of 0.05-0.07 wt.% of monobasic sodium phosphate, the salt is sodium chloride in the range of 0.4-0.45 wt.%, and the sugar is sucrose in the range of 4-5 wt.%.

[0297] In another embodiment, the Disclosure includes a method for treating acute myeloid leukemia (AML) in a subject. The method of the Disclosure includes, for example, administering to a subject a dose of polyalkylene oxide-asparaginase effective for treating the subject's acute myeloid leukemia (AML), wherein the polyalkylene oxide-asparaginase has a polyalkylene oxide covalently bound to asparaginase by a linker.

[0298] In some embodiments, this polyalkylene oxide group includes a polyethylene glycol group.

[0299] In some embodiments, this polyethylene glycol group has a molecular weight in the range of 2,000 to 10,000 daltons.

[0300] In some embodiments, this polyethylene glycol group has a molecular weight of 5,000 daltons.

[0301] In some embodiments, this asparaginase is E. coli asparaginase. In some embodiments, this linker is a urethane linker. In some embodiments, this linker is a succinic acid linker.

[0302] In some embodiments, this dose includes an amount of polyalkylene oxide-asparaginase in the range of 700-800 IU / g.

[0303] In some embodiments, this dose includes a buffer and a salt. In some embodiments, this dose includes a phosphate buffer. In some embodiments, this phosphate buffer includes dibasic sodium phosphate and monobasic sodium phosphate. In some embodiments, this dose includes 5.25–5.75 mg / g of dibasic sodium phosphate. In some embodiments, this dose includes 1–1.5 mg / g of monobasic sodium phosphate. In some embodiments, this salt is sodium chloride. In some embodiments, this dose includes 8–9 mg / g of sodium chloride.

[0304] In some embodiments, this dose comprises 750 IU / g of polyalkylene oxide-asparaginase, the polyethylene glycol group having a molecular weight of 5,000 daltons, the asparaginase being Escherichia coli asparaginase, the linker being a urethane linker, the buffer comprising a phosphate buffer containing an amount of dibasic sodium phosphate in the range of 5.25–5.75 mg / g and an amount of monobasic sodium phosphate in the range of 1–1.5 mg / g, and the salt comprising 8–9 mg / g of sodium chloride.

[0305] In some embodiments, this dose comprises 750 IU / g of polyalkylene oxide-asparaginase, the polyethylene glycol group having a molecular weight of 5,000 daltons, the asparaginase being Escherichia coli asparaginase, the linker being a urethane linker, the buffer comprising a phosphate buffer containing 5.58 mg / g of dibasic sodium phosphate and 1.29 mg / g of monobasic sodium phosphate, and the salt comprising 8.5 mg / g of sodium chloride.

[0306] In some embodiments, this dose comprises 750 IU / g of polyalkylene oxide-asparaginase, the polyethylene glycol group having a molecular weight of 5,000 daltons, the asparaginase being Escherichia coli asparaginase, the linker being succinic acid linker, the buffer being a phosphate buffer containing 5.25–5.75 mg / g of dibasic sodium phosphate and 1–1.5 mg / g of monobasic sodium phosphate, and the salt containing 8–9 mg / g of sodium chloride.

[0307] In some embodiments, this dose comprises 750 IU / g of polyalkylene oxide-asparaginase, the polyethylene glycol group having a molecular weight of 5,000 daltons, the asparaginase being Escherichia coli asparaginase, the linker being succinic acid linker, the buffer being a phosphate buffer containing 5.58 mg / g of dibasic sodium phosphate and 1.29 mg / g of monobasic sodium phosphate, and the salt comprising 8.5 mg / g of sodium chloride.

[0308] In some embodiments, this phosphate buffer contains dibasic sodium phosphate and monobasic sodium phosphate. In some embodiments, this dose contains 2.5–3 mg / g of dibasic sodium phosphate. In some embodiments, this dose contains 0.45–0.75 mg / g of monobasic sodium phosphate. In some embodiments, this salt is sodium chloride. In some embodiments, this dose contains 4–4.5 mg / g of sodium chloride. In some embodiments, this disaccharide contains sucrose. In some embodiments, the amount of sucrose is in the range of 40–50 mg / g.

[0309] In some embodiments, this dose comprises 750 IU / g of polyalkylene oxide-asparaginase, where the polyethylene glycol group has a molecular weight of 5,000 daltons, the asparaginase is E. coli asparaginase, the linker is a urethane linker, the buffer is a phosphate buffer comprising 2.5–3.5 mg of dibasic sodium phosphate and 0.5–0.7 mg of monobasic sodium phosphate, the salt is 4–4.5 mg / g of sodium chloride, and the sugar is 40–50 mg / g of sucrose.

[0310] In some embodiments, this dose comprises 750 IU / g of polyalkylene oxide-asparaginase, the polyethylene glycol group having a molecular weight of 5,000 daltons, the asparaginase being Escherichia coli asparaginase, the linker being a urethane linker, the buffer being a phosphate buffer containing 2.79 mg / g of dibasic sodium phosphate and 0.6 mg / g of monobasic sodium phosphate, the salt containing 4.25 mg / g of sodium chloride, and the sugar containing 45 mg / g of sucrose.

[0311] In some embodiments, this dose comprises 750 IU / g of polyalkylene oxide-asparaginase, where the polyethylene glycol group has a molecular weight of 5,000 daltons, the asparaginase is E. coli asparaginase, the linker is succinate linker, the buffer is a phosphate buffer comprising 2.5–3.5 mg of dibasic sodium phosphate and 0.5–0.7 mg of monobasic sodium phosphate, the salt is 4–4.5 mg / g of sodium chloride, and the sugar is 40–50 mg / g of sucrose.

[0312] In some embodiments, this dose comprises 750 IU / g of polyalkylene oxide-asparaginase, the polyethylene glycol group having a molecular weight of 5,000 daltons, the asparaginase being Escherichia coli asparaginase, the linker being succinate linker, the buffer being a phosphate buffer containing 2.79 mg / g of dibasic sodium phosphate and 0.6 mg / g of monobasic sodium phosphate, the salt containing 4.25 mg / g of sodium chloride, and the sugar containing 45 mg / g of sucrose.

[0313] In some embodiments, the method also includes creating doses by reconstituting a lyophilized storage-stable composition.

[0314] In some embodiments, this dose is 1,500 to 3,000 IU / m². 2 Polyal Chilenoxide-asparaginase is delivered to the subjects. The dose is 2,000-2,750 IU / m². 2 The polyalkylene oxide-asparaginase is delivered to the subject.

[0315] In some embodiments, subjects are prescribed a therapeutic dosing plan including an induction phase, a coupling phase, and a maintenance phase. In some embodiments, the method includes administering a single reconstituted dose unit to the subject during the induction phase and administering multiple reconstituted dose units during the maintenance phase. In some embodiments, these multiple reconstituted dose units are administered to the subject by administering the reconstituted dose unit to the subject every three weeks. In some embodiments, these multiple reconstituted dose units are administered to the subject by administering the reconstituted dose unit to the subject every two weeks.

[0316] In some embodiments, the subject is a minor. In some embodiments, the subject is an adult.

[0317] While the embodiments described above are provided for illustrative purposes and the examples for clarity of understanding, it will be apparent to those skilled in the art that, considering the teachings of this disclosure, certain changes and modifications can be made thereto without departing from the spirit and scope of the appended claims.

[0318] Accordingly, the foregoing merely describes the principles of the embodiments of this disclosure. It will be understood that, despite not being explicitly stated or indicated herein, a person skilled in the art can devise various arrangements that embody the principles of the embodiments of this disclosure and that fall within the spirit and scope of the invention. Furthermore, all embodiments and conditional statements enumerated herein are primarily intended to help the reader understand the principles of the embodiments of this disclosure and the concepts to which the inventors have contributed to expanding the art, and should be construed as not limiting such specifically enumerated embodiments and conditions. Moreover, all statements herein enumerating the principles, aspects, and embodiments of this disclosure, as well as their specific embodiments, are intended to encompass both their structural and functional equivalents. In addition, such equivalents are intended to include both currently known equivalents and those to be developed in the future, i.e., any elements to be developed that perform the same function, regardless of their structure. The scope of the embodiments of this disclosure is therefore not intended to be limited to the embodiments shown and described herein. Rather, the scope and spirit of the embodiments of this disclosure are embodied by the appended claims.

Claims

1. A liquid composition: Polyalkylene oxide-asparaginase at a concentration of 750 IU per 1 mL of the composition; Dibasic sodium phosphate at a concentration of 0.5–0.6 wt.%; Monobasic sodium phosphate at a concentration of 0.1–0.2 wt.%; Sodium chloride in concentrations of 0.8 to 1.0 wt.%; and water; It consists of, The polyalkylene oxide-asparaginase comprises an asparaginase covalently bonded to a polyalkylene oxide group which is polyethylene glycol. The polyalkylene oxide group is covalently bound to the asparaginase via the carbamate moiety. A liquid composition in which the carbamate portion is derived from a succinimidoyl carbonate (SC) linker.

2. The composition according to claim 1, wherein the asparaginase is L-asparaginase.

3. The composition according to claim 1, wherein the polyethylene glycol is methoxypolyethylene glycol.

4. The composition according to claim 1, wherein the polyethylene glycol has a molecular weight in the range of 2,000 to 10,000 daltons.

5. The composition according to claim 1, wherein the polyethylene glycol has a molecular weight of 5,000 daltons.

6. The composition according to claim 1, further comprising sodium hydroxide, hydrochloric acid, or a combination thereof.

7. The composition according to claim 1, wherein the elimination half-life of the polyalkylene oxide-asparaginase is 10 to 20 days.

8. The composition according to claim 1, wherein the elimination half-life of the polyalkylene oxide-asparaginase is 14 to 18 days.

9. A liquid composition: Polyalkylene oxide-asparaginase at a concentration of 750 IU per 1 mL of the composition; 0.558 wt.% dibasic sodium phosphate; Monobasic sodium phosphate at a concentration of 0.12 wt.%; Sodium chloride at a concentration of 0.85 wt.%; and water; It consists of, The polyalkylene oxide-asparaginase comprises an asparaginase covalently bonded to a polyalkylene oxide group which is polyethylene glycol. The polyalkylene oxide group is covalently bound to the asparaginase via the carbamate moiety. A liquid composition in which the carbamate portion is derived from a succinimidoyl carbonate (SC) linker.

10. A composition according to any one of claims 1 to 9 for use in a method for treating a tumorous condition of a subject.

11. The aforementioned tumor condition is cancer. If the cancer is leukemia, The aforementioned leukemia, (i) Acute lymphoblastic leukemia (ALL), or (ii) Acute myeloid leukemia (AML) The composition for use according to claim 10.

12. The subject is prescribed a treatment plan including an induction phase, a coupling phase, and a maintenance phase. Optionally, the method includes administering a single reconstituted dose unit to the subject during the induction phase and administering a plurality of dose units during the maintenance phase. Furthermore, optionally, the multiple dose units are administered to the subject at intervals of three weeks or longer. A composition for use according to claim 10 or 11.

13. (a) The subject is a minor, or (b) The subject is an adult, A composition for use according to any one of claims 10 to 12.

14. A kit comprising one or more unit-dose containers, each containing the composition described in claim 9.

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