Methods and compositions for treating fructose intolerance and fructose malabsorption - Patent Application 20070233633

Abstract

Disclosed are pharmaceutical compositions comprising glucose (xylose) isomerase, glucose oxidase enzymes and peroxide degrading enzymes (eg, catalase enzymes) that may be used, inter alia, to treat fructose intolerance and fructose malabsorption.

Description

[Technical field]

[0001] REFERENCE TO RELATED APPLICATIONS This application claims the benefit of and priority to U.S. Provisional Patent Application No. 63 / 232,922, filed August 13, 2021, the disclosure of which is incorporated by reference in its entirety for all purposes.

[0002] FIELD OF THEINVENTION The present invention relates generally to methods and compositions for treating fructose intolerance and fructose malabsorption, and more specifically, the present invention relates to compositions comprising glucose (xylose) isomerase enzymes, glucose oxidase enzymes and peroxide degrading enzymes (e.g., catalase enzymes) and their use in treating fructose intolerance and fructose malabsorption. [Background technology]

[0003] background Increased sugar consumption is believed to contribute to obesity and diabetes and their associated cardiometabolic risks. As a result of its unique metabolic properties, the fructose component of sugar can be particularly harmful. A high fructose diet can produce all of the key features of metabolic syndrome.

[0004] Fructose is a six-carbon monosaccharide (polyhydroxyketone) that is widely consumed in the Western diet. It is typically ingested in three forms: as a pure monosaccharide; as a disaccharide sucrose, such as glucose bound to fructose, which can be hydrolyzed by the enzyme sucrase; and as polymeric forms, such as oligosaccharides and polysaccharides. The polymeric forms of fructose are variably described as inulin, fructans, and fructo-oligosaccharides. Fructose in its various forms is associated with irritable bowel syndrome (IBS), which contributes to the development of obesity (especially in the United States), and is thought to play a role in the pathogenesis of nonalcoholic fatty liver disease. Lambertz et al. (2017) FRONTIERS IN IMMUNOLOGY 8: 1, DiNicolantonio and Lucan (2015) MED. HYPOTHESES 85(3): 295.

[0005] There has been a significant increase in fructose consumption in the United States. This high level of fructose consumption is believed to result from the use of high fructose corn syrup (HFCS) as a sweetener in food manufacturing. HFCS is a cheaper alternative to sucrose and contains up to 80% fructose. Gibson et al. (2007) ALIMENT. PHARMACOL THER. 25: 349. Sugar-sweetened beverages (SSBs) are the major source of added sugar in the diet worldwide and include soda, fruit-flavored beverages, and sports drinks. On average, SSBs contribute about 7% of daily calories and nearly 50% of added sugars in the diet. Hannou et al. (2018) J. CLIN. INVEST. 128(2): 545, Tappy and Le (2010) PHYSIOL. REV. 90: 23.

[0006] There are two distinct types of fructose intolerance - hereditary fructose intolerance (HFI) and dietary fructose intolerance (DFI). Hou et al. (2019) FRONTIERS IN GENETICS 10: 1. HFI is a rare genetic disorder of fructose metabolism associated with deficiency of the enzyme aldolase B (ALDOB) and intracellular accumulation of toxic fructose 1-phosphate in the liver, intestine and kidneys. Fructose ingestion causes acute symptoms such as vomiting and diarrhea, as well as life-threatening symptoms such as acute hypoglycemia, lethargy, renal tubular acidosis and acute liver failure. Di Dato et al.(2019) NUTRIENTS 11: 2397. Chronic ingestion of fructose results in inadequate food intake, failure or retardation of growth, and liver and kidney damage. If diagnosed and treated early, HFI is preventable, but can be fatal without prompt intervention. DFI involves the situation in which free fructose becomes available for fermentative metabolism by luminal bacteria before it can be absorbed across the small intestinal mucosa.

[0007] The majority of fructose absorption occurs through two major transporters known as GLUT5 and GLUT2. Ferraris et al.(2018) ANNU. REV. NUTR. 38: 41. GLUT5 is a facultative transporter (it depends on a concentration gradient for the movement of substrates across it) and is specific for fructose. It is found in the apical cell membrane. Luminal uptake of fructose is ensured as fructose is rapidly removed from the circulation. Although this uptake mechanism has a low capacity, the transporter is present along the length of the small intestine. GLUT2 is a low affinity facultative transporter that can transport glucose, fructose and galactose. It is constitutively present on the basolateral membrane where it transports hexose down the concentration gradient outside the cell. The capacity of the intestine to absorb fructose is saturable, and the capacity of healthy adults to absorb free fructose typically ranges from less than 5 g to more than 50 g per day. Ferraris et al.(2018) ANNU. REV. NUTR. 38: 41. Unabsorbed fructose can impose an osmotic load on the distal small intestine and colon, contributing to gastrointestinal symptoms. In addition, fructose can serve as a substrate for bacterial fermentation, leading to the formation of gases and other bacterial metabolic products that can affect gut motility and cause various symptoms such as abdominal pain and bloating. Excessive fructose consumption can have profound effects on lipid metabolism, contributing to both adiposity and increased circulating triglyceride levels in the form of very low density lipoproteins, inducing hyperinsulinemia, increasing adiposity and hypertension.

[0008] To date, the only available treatment for fructose intolerance and fructose malabsorption is a diet that does not contain fructose, sorbitol and sucrose (FSS). The elimination of all these sugars is not easily feasible due to the hidden small amount of fructose contained in many foods. Therefore, there is an ongoing need for new and effective pharmaceutical compositions and treatments for controlling fructose absorption and treating fructose intolerance (e.g., hereditary fructose intolerance and dietary fructose intolerance) and fructose malabsorption. Summary of the Invention

[0009] Summary of the Invention The present invention is based, in part, on the discovery of pharmaceutical compositions comprising the enzymes glucose (xylose) isomerase (also known as fructose isomerase), glucose oxidase and peroxide-degrading enzymes (e.g., catalase enzymes) that can be used to treat fructose intolerance (e.g., genetic or dietary fructose intolerance) and / or fructose malabsorption in a subject in need of such treatment.

[0010] Thus, in one aspect, the present invention provides a pharmaceutical composition comprising (or consisting essentially of) a glucose (xylose) isomerase enzyme, a glucose oxidase enzyme, a peroxide degrading enzyme (e.g., a catalase enzyme) and a pharma- ceutically acceptable excipient.

[0011] In some embodiments, the glucose (xylose) isomerase enzyme is spray dried or freeze dried. The glucose (xylose) isomerase enzyme can be a microbial glucose (xylose) isomerase enzyme or a functional fragment or variant thereof. The glucose (xylose) isomerase enzyme can be from Streptomyces murinus. In some embodiments, the glucose (xylose) isomerase enzyme comprises SEQ ID NO:5 or a functional fragment or variant thereof. In some embodiments, the pharmaceutical composition comprises about 1,000 to about 250,000 International Units (IU) of the glucose (xylose) isomerase enzyme.

[0012] In some embodiments, the glucose oxidase enzyme is spray dried or freeze dried. The glucose oxidase enzyme can be a microbial glucose oxidase enzyme or a functional fragment or variant thereof. The glucose oxidase enzyme can be derived from Aspergillus niger, for example, the glucose oxidase enzyme comprises SEQ ID NO:1, SEQ ID NO:2, or a functional fragment or variant thereof. In some embodiments, the medicament comprises about 1,000 to about 250,000 international units (IU) of the glucose oxidase enzyme.

[0013] In some embodiments, the peroxide decomposing enzyme is spray dried or freeze dried. In some embodiments, the peroxide decomposing enzyme can be a catalase enzyme or a peroxidase enzyme. In some embodiments, the peroxide decomposing enzyme is a catalase enzyme, which can be a bacterial catalase enzyme, or a functional fragment or variant thereof. In some embodiments, the catalase enzyme is from Aspergillus niger, e.g., the catalase enzyme comprises SEQ ID NO:3, SEQ ID NO:4, or a functional fragment or variant thereof. In some embodiments, the medicament comprises about 1,000 to about 250,000 international units (IU) of the catalase enzyme.

[0014] Accordingly, the ratio of international units of glucose (xylose) isomerase to international units of glucose oxidase enzyme in the pharmaceutical composition is in the range of 0.1 to 10. Additionally or alternatively, the ratio of international units of glucose oxidase to international units of peroxidase enzyme in the pharmaceutical composition is in the range of 0.1 to 10.

[0015] In some embodiments, the pharmaceutical composition is formulated as an oral dosage form.The pharmaceutical composition can be formulated as a solid oral dosage form, such as powder, sachet, granule, pellet, micropellet, tablet or mini tablet.Alternatively, the pharmaceutical composition can be formulated as a liquid oral dosage form, such as elixir, syrup or drop.The pharmaceutical composition can have a shelf life of at least 3 months, 6 months, 9 months, 12 months, 15 months, 18 months, 21 months, 24 months, 36 months, 48 ​​months, 60 months, 72 months, 84 months, 96 months, 108 months or 120 months at room temperature.

[0016] In another aspect, the present invention provides a method for treating fructose intolerance (e.g., dietary fructose intolerance or hereditary fructose intolerance) in a subject in need of such treatment.The method comprises administering (e.g., orally administering) to the subject an effective amount of any of the enzymes or compositions described herein to treat fructose intolerance in the subject.

[0017] In another aspect, the present invention provides a method for treating irritable bowel syndrome (IBS), FODMAP (fermentable oligosaccharides, disaccharides, monosaccharides and polyols) intolerance or malabsorption, obesity, metabolic syndrome, diabetes, hyperglycemia or hyperinsulinemia in a subject who needs to treat IBS, FODMAP (fermentable oligosaccharides, disaccharides, monosaccharides and polyols) intolerance or malabsorption, obesity, metabolic syndrome, diabetes, hyperglycemia or hyperinsulinemia.The method comprises administering (e.g. orally administering) to the subject an effective amount of any of the enzymes or compositions described herein to treat fructose intolerance in the subject.

[0018] In another aspect, the present invention provides a method for reducing fructose levels in a subject that requires reduction of fructose levels.The method comprises administering (e.g., orally administering) to the subject an effective amount of any of the enzymes or compositions described herein to reduce fructose levels in the subject.In some circumstances, the method reduces the level of fructose in the blood of the subject and / or in the gastrointestinal tract of the subject.Alternatively or additionally, the method reduces the level of fructose in a sample (e.g., a body fluid sample, such as blood, serum or plasma) from the subject.

[0019] In some embodiments, the method reduces the level of fructose in the blood of a subject by at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80% or 90% after a meal or snack, as determined by area under the curve (AUC) analysis. In some embodiments, the method reduces the fructose Cmax in the blood of a subject after a meal or snack by at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80% or 90%. In some embodiments, the method reduces the fructose Tmax in the blood of a subject after a meal or snack by at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80% or 90%. In certain embodiments, when administered with food, the method reduces the caloric intake of the food by at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80% or 90%.

[0020] In certain embodiments of any of the aforementioned methods, the subject has dietary fructose intolerance, hereditary fructose intolerance, irritable bowel syndrome (IBS), FODMAP (fermentable oligosaccharides, disaccharides, monosaccharides and polyols) malabsorption and / or intolerance, obesity, metabolic syndrome, diabetes, hyperglycemia or hyperinsulinemia.

[0021] In certain embodiments of any of the foregoing methods, the enzyme or composition is administered to the subject 1, 2, 3, 4 or more than 4 times per day. In certain embodiments, the enzyme or composition is administered to the subject with a meal or snack (e.g., with each meal or snack).

[0022] These and other aspects and features of the present invention are described in the following detailed description and claims. [Brief description of the drawings]

[0023] Description of the drawings The invention may be more fully understood with reference to the following drawings. [Figure 1]FIG. 1 shows the effect of glucose (xylose) isomerase, glucose oxidase and catalase on the conversion of fructose (1%) at pH 7.4 and 37° C. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0024] Detailed Description The present invention is based, in part, on the discovery of pharmaceutical compositions comprising glucose (xylose) isomerase, glucose oxidase enzymes and peroxidolytic enzymes (e.g., catalase enzymes) that can be used to treat various disorders, such as fructose intolerance (e.g., dietary fructose intolerance or hereditary fructose intolerance), irritable bowel syndrome (IBS), FODMAP (fermentable oligosaccharides, disaccharides, monosaccharides and polyols) malabsorption and / or intolerance, obesity, metabolic syndrome, diabetes, hyperglycemia or hyperinsulinemia, in a subject in need of such treatment.

[0025] Various features and aspects of the invention are discussed in detail below.

[0026] I. Enzymes In particular, the present invention provides pharmaceutical compositions comprising glucose (xylose) isomerase, glucose oxidase enzyme and peroxidase enzyme, useful for treating one or more of the disorders described herein, for example. Given that fructose levels may be directly related to these disorders, the methods and compositions described herein are designed to reduce fructose levels in subjects. The use of the compositions described herein facilitates the conversion of fructose to glucose via glucose (xylose) isomerase enzyme. However, considering the natural equilibrium between fructose and glucose, glucose oxidase and peroxidase enzymes shift the equilibrium to remove at least a portion of the glucose produced by glucose (xylose) isomerase enzyme, preventing glucose from being reconverted to fructose.

[0027] As used herein, the term "glucose (xylose) isomerase" refers to an enzyme that is capable of catalyzing at least the following reactions: [ka] The term "catalyst" refers to any enzyme or functional fragment thereof that can catalyze the reaction.

[0028] Glucose (xylose) isomerases include, for example, D-xylose isomerase, D-xylose ketoisomerase, D-xylose ketol-isomerase, D-xylose ketol isomerase, D-xylose:ketol-isomerase, D-xylulose keto-isomerase, glucose isomerase, glucose / xylose isomerase, EC 5.3.1.5, D-XI, GXI, Maxazyme TM , Optisweet TM , Sweetase TM and Spezyme TM It is also called.

[0029] The term glucose (xylose) isomerase includes variants having one or more amino acid substitutions, deletions or insertions relative to the wild-type glucose (xylose) isomerase sequence and / or fusion proteins or conjugates containing glucose (xylose) isomerase. As used herein, the term "functional fragment" of glucose (xylose) isomerase refers to a fragment of full-length glucose (xylose) isomerase that retains, for example, at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90% or 100% of the enzyme activity of the corresponding full-length naturally occurring glucose (xylose) isomerase. Glucose (xylose) isomerase enzyme activity can be assayed by any method known in the art. Exemplary glucose (xylose) isomerase activity assays are available from Abcam (Cambridge, UK; Glucose Isomerase Activity Assay Kit (Colorimetric); ab273289) and Biovision (Milpitas, Calif., USA).

[0030] Exemplary glucose (xylose) isomerase enzymes include glucose (xylose) isomerase enzymes derived from Streptomyces murinus. The amino acid sequence of an exemplary wild-type glucose oxidase enzyme from Streptomyces murinus is shown in SEQ ID NO: 5 (UniProtKB-P37031).

[0031] Further exemplary glucose (xylose) isomerase enzymes include those from Escherichia coli, Klebsiella pneumoniae, Lactobacillus brevis, Lactobacillus pentosus, Bacillus subtilis, Caldicellulosiruptor bescii, Staphylococcus xylosus, Thermoanaerobacter ethanolicus, Thermoanaerobacter thermosulfurogenes, Arthrobacter sp., Actinoplanes missouriensis, Ampullariella sp., Streptomyces violaceoniger, Streptomyces murinus, Streptomyces rochei, and the like. rochei, Streptomyces rubiginosus, Streptomyces olivochromogenes, Thermus thermophilus or Thermotoga neopolitana. Further exemplary glucose (xylose) isomerase enzymes can be found on the World Wide Web at brenda-enzymes.org / enzyme.php?ecno=5.3.1.5#ORGANISM or brenda-enzymes.org / enzyme.php?ecno=5.3.1.5.

[0032] The glucose (xylose) isomerase enzyme may comprise the consensus sequence VX1WX2GREGX3E (SEQ ID NO:6), where X1 is any amino acid, X2 is G or P, and X3 is Y, S, T, A, or E. Alternatively or additionally, the glucose (xylose) isomerase enzyme may comprise the consensus sequence X1EPKPX2X3P (SEQ ID NO:7), where X1 is L, I, V, or M, X2 is any amino acid, and X3 is E or Q.

[0033] As used herein, the term "glucose oxidase" refers to any enzyme or functional fragment thereof that can catalyze the oxidation of β-D-glucose to D-glucono-ω-lactone and hydrogen peroxide and / or the conversion of D-glucono-ω-lactone to gluconic acid. Glucose oxidases typically catalyze at least the following reactions: β-D-glucose + O2 → D-glucono-ω-lactone + H2O2 catalyzes.

[0034] Glucose oxidase is also referred to as EC 1.1.3.4, glucose oxyhydrolase, corylophyline, penatin, glucose aerodehydrogenase, microcid, β-D-glucose oxidase, D-glucose oxidase, D-glucose-1-oxidase, β-D-glucose:quinone oxidoreductase, glucose oxyhydrolase and deoxin-1, which terms are used interchangeably herein unless otherwise indicated. The term glucose oxidase includes variants having one or more amino acid substitutions, deletions or insertions relative to the wild-type glucose oxidase sequence and / or fusion proteins or conjugates containing glucose oxidase. As used herein, the term "functional fragment" of glucose oxidase refers to a fragment of full-length glucose oxidase that retains, for example, at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90% or 100% of the enzyme activity of the corresponding full-length naturally occurring glucose oxidase. Glucose oxidase enzyme activity can be assayed by any method known in the art. An exemplary glucose oxidase activity assay is available from Sigma-Aldrich (Cat. No. MAK097).

[0035] Exemplary glucose oxidase enzymes include those derived from Aspergillus niger. The amino acid sequence of an exemplary wild-type glucose oxidase enzyme from Aspergillus niger is shown in SEQ ID NO:1 (with signal sequence) and SEQ ID NO:2 (without signal sequence). Further exemplary glucose oxidase enzymes can be found on the World Wide Web at brenda-enzymes.org / enzyme.php?ecno=1.1.3.4#ORGANISM or brenda-enzymes.org / enzyme.php?ecno=1.1.3.4.

[0036] As used herein, the term "peroxide decomposition enzyme" refers to any enzyme or functional fragment thereof capable of decomposing hydrogen peroxide. Exemplary peroxide decomposition enzymes include catalase and peroxidase enzymes.

[0037] As used herein, the term "catalase" refers to any enzyme or functional fragment thereof that can catalyze the decomposition of hydrogen peroxide into water and oxygen. Catalases typically catalyze the following reaction: 2H2O2 → O2 + 2H2O catalyzes.

[0038] Catalase is also referred to as EC 1.11.1.6, equilase, caperase, optidase, catalase-peroxidase and CAT, and the terms are used interchangeably herein unless otherwise indicated. The term catalase includes variants with one or more amino acid substitutions, deletions or insertions relative to wild-type catalase sequence and / or fusion proteins or conjugates containing catalase. As used herein, the term "functional fragment" of catalase refers to a fragment of full-length catalase that retains, for example, at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90% or 100% of the enzyme activity of the corresponding full-length naturally occurring catalase. Exemplary catalase activity assays are available from Sigma-Aldrich (Cat. Nos. CAT100 and 219265).

[0039] Exemplary catalase enzymes include catalase enzymes from Aspergillus niger. The amino acid sequence of an exemplary wild-type catalase enzyme from Aspergillus niger is shown in SEQ ID NO:3 (with signal sequence) and SEQ ID NO:4 (without signal sequence). Further exemplary catalase enzymes can be found on the World Wide Web at brenda-enzymes.org / enzyme.php?ecno=1.11.1.6#ORGANISM or brenda-enzymes.org / enzyme.php?ecno=1.11.1.6.

[0040] As used herein, the term "peroxidase" refers to any enzyme or functional fragment thereof that can catalyze an oxidation-reduction reaction by a free radical mechanism that can convert compounds into oxidized or polymerized products. Peroxidases typically catalyze the following reaction: H2O2+AH2→2H2O+A catalyzes.

[0041] The term peroxidase includes variants having one or more amino acid substitutions, deletions or insertions relative to the wild-type peroxidase sequence and / or fusion proteins or conjugates that contain peroxidase.As used herein, the term "functional fragment" of peroxidase refers to a fragment of full-length peroxidase that retains, for example, at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90% or 100% of the enzymatic activity of the corresponding full-length naturally occurring peroxidase.Exemplary peroxidase activity assays are available from Sigma-Aldrich (Cat. No. MAK092).

[0042] Peroxidases represent a large group of enzymes. Exemplary peroxidase enzymes can be found on the World Wide Web at brenda-enzymes.org / enzyme.php?ecno=1.11.1.7#ORGANISM or brenda-enzymes.org / enzyme.php?ecno=1.11.1.7.

[0043] In some embodiments, the glucose (xylose) isomerase, glucose oxidase and / or peroxide degrading enzyme comprises at least one (e.g., 1, 2, 3, 4, 5, 6, 7 or 8) mutation(s) relative to a wild-type glucose (xylose) isomerase, glucose oxidase and / or peroxide degrading enzyme disclosed herein.

[0044] In some embodiments, the glucose (xylose) isomerase, glucose oxidase and / or peroxide decomposition enzyme comprises one or more conservative substitutions with respect to the glucose (xylose) isomerase, glucose oxidase and / or peroxide decomposition enzyme disclosed herein. In other embodiments, the glucose (xylose) isomerase, glucose oxidase and / or peroxide decomposition enzyme comprises one or more non-conservative substitutions with respect to the glucose (xylose) isomerase, glucose oxidase and / or peroxide decomposition enzyme disclosed herein. As used herein, the term "conservative substitution" refers to a substitution with a structurally similar amino acid. For example, conservative substitutions can include those within the following groups: Ser and Cys; Leu, Ile and Val; Glu and Asp; Lys and Arg; Phe, Tyr and Trp; and Gln, Asn, Glu, Asp and His. Conservative substitutions can also be defined by the BLAST (Basic Local Alignment Search Tool) algorithm, BLOSUM substitution matrices (e.g., BLOSUM 62 matrix) or PAM substitution:p matrices (e.g., PAM 250 matrix). Non-conservative substitutions are amino acid substitutions that are not conservative substitutions.

[0045] In certain embodiments, the disclosed glucose (xylose) isomerase, glucose oxidase and / or peroxide degrading enzyme (or a pharmaceutical composition, e.g., a solid pharmaceutical composition comprising same) has a shelf life (e.g., retains at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% of its biological activity) of at least 3 months, at least 6 months, at least 9 months, at least 12 months, at least 15 months, at least 18 months, at least 21 months, at least 24 months, at least 36 months, at least 48 months, at least 60 months, at least 72 months, at least 84 months, at least 96 months, at least 108 months or at least 120 months at room temperature.In some embodiments, the disclosed glucose (xylose) isomerase, glucose oxidase and / or peroxide decomposition enzyme (or a pharmaceutical composition, e.g., a solid pharmaceutical composition, comprising the same) can be stored at room temperature for about 3 to about 120 months, about 3 to about 96 months, about 3 to about 72 months, about 3 to about 48 months, about 3 to about 24 months, about 3 to about 21 months, about 3 to about 18 months, about 3 to about 15 months, about 3 to about 12 months, about 3 to about 9 months, about 3 to about 6 months, 6 to 120 months, 6 to 96 months, 6 to 72 months, 6 to 48 months, 6 to 24 months, 6 to 21 months, 6 to 18 months, 6 to 15 months, 6 to 12 months, 6 to 9 months, 9 to 120 months, 9 to 96 months, 9 to 72 months, 9 to 48 months, 9 to 24 months, 9 to 21 months, 9 to 18 months, 9 to 15 months, 9 to 12 months, 12 to 120 months months, about 12 to about 96 months, about 12 to about 72 months, about 12 to about 48 months, about 12 to about 24 months, about 12 to about 21 months, about 12 to about 18 months, about 12 to about 15 months, about 15 to about 120 months, about 15 to about 96 months, about 15 to about 72 months, about 15 to about 48 months, about 15 to about 24 months, about 15 to about 21 months, about 15 to about 18 months, about 18 to about 120 months, about 18 to about 96 months, about 18 to about 72 months, about 18 to about 48 months, about The composition may have a shelf life of 18 to about 24 months, about 18 to about 21 months, about 21 to about 120 months, about 21 to about 96 months, about 21 to about 72 months, about 21 to about 48 months, about 21 to about 24 months, about 24 to about 120 months, about 24 to about 96 months, about 24 to about 72 months, about 24 to about 48 months, about 48 to about 120 months, about 48 to about 96 months, about 48 to about 72 months, about 72 to about 120 months, about 72 to about 96 months, or about 96 to about 120 months.

[0046] The stability, shelf-life or half-life of glucose (xylose) isomerase, glucose oxidase and / or peroxidolytic enzymes can be measured by any method known in the art, such as enzyme activity or SDS-PAGE analysis, after incubation of the enzymes for a selected time at selected conditions (e.g., temperature, pH and / or humidity conditions). It is understood that the stability, shelf-life or half-life of glucose (xylose) isomerase, glucose oxidase and / or peroxidolytic enzymes depends on the experimental conditions under which it is measured.

[0047] In some embodiments, the glucose (xylose) isomerase, glucose oxidase and / or peroxide degrading enzyme has at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% sequence identity to the glucose (xylose) isomerase, glucose oxidase and / or peroxide degrading enzyme disclosed herein.

[0048] For example, in some embodiments, the glucose (xylose) isomerase enzyme has at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% sequence identity to SEQ ID NO: 5. In some embodiments, the glucose oxidase enzyme has at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% sequence identity to SEQ ID NO: 1 or SEQ ID NO: 2. In some embodiments, the peroxidolytic enzyme has at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% sequence identity to SEQ ID NO: 3 or SEQ ID NO: 4. Sequence identity can be determined in a variety of ways within the skill of the art, for example, using publicly available computer software, such as BLAST, BLAST-2, ALIGN or Megalign (DNASTAR) software. BLAST (Basic Local Alignment Search Tool) analysis using the algorithm used by the programs blastp, blastn, blastx, tblastn and tblastx (Karlin et al., (1990) PROC. NATL. ACAD. SCI. USA 87:2264-2268; Altschul, (1993) J. MOL. EVOL. 36, 290-300; Altschul et al., (1997) NUCLEIC ACIDS RES.25:3389-3402, incorporated by reference) is adjusted for searching sequence similarity. For a discussion of basic issues in searching sequence databases, see Altschul et al., (1994) NATURE GENETICS 6:119-129, fully incorporated by reference. Those skilled in the art can determine appropriate parameters for measuring alignment, including any algorithms needed to achieve maximum alignment over the entire length of the sequences being compared. The search parameters for histogram, description, alignment, expect (ie, the statistical significance threshold for reporting matches against database sequences), cutoff, matrix and filter are at default settings.The default scoring matrix used by blastp, blastx, tblastn and tblastx is the BLOSUM62 matrix (Henikoff et al., (1992) PROC. NATL. ACAD. SCI. USA 89:10915-10919, fully incorporated by reference). The four blastn parameters can be adjusted as follows: Q=10 (gap creation penalty); R=10 (gap extension penalty); wink=1 (generates word hits at every wink.sup.th position along the query); and gapw=16 (sets the window width over which gapped alignments are generated). The equivalent Blastp parameter settings can be Q=9; R=2; wink=1; and gapw=32. Searches may also be performed using NCBI (National Center for Biotechnology Information) BLAST Advanced Option parameters (e.g.: -G, cost for open gap [integer]: default = 5 for nucleotides / 11 for proteins; -E, cost for extension gap [integer]: default = 2 for nucleotides / 1 for proteins; -q, penalty for nucleotide mismatch [integer]: default = -3; -r, reward for nucleotide match [integer]: default = 1; -e, expectation value [real number]: default = 10; -W, word size [integer]: default = 11 for nucleotides / 28 for megablast / 3 for proteins; -y, dropoff for blast extension in bits (X): default = 20 for blastn / 7 for others; -X, X dropoff value for gapped alignments (in bits): default = 15 for all programs but not applicable to blastn; and -Z, final X dropoff value for gapped alignments (in bits): 50 for blastn, 25 for others).ClustalW for pairwise protein alignments can also be used (default parameters can include, for example, Blosum62 matrix and Gap Opening Penalty=10 and Gap Extension Penalty=0.1). The best fit comparison between sequences available in the GCG package version 10.0 uses DNA parameters GAP=50 (gap creation penalty) and LEN=3 (gap extension penalty), with equivalent settings for protein comparisons being GAP=8 and LEN=2.

[0049] It is contemplated that the disclosed glucose (xylose) isomerase, glucose oxidase and / or peroxidase can be modified, genetically engineered or chemically conjugated.For example, it is contemplated that the disclosed glucose (xylose) isomerase, glucose oxidase and / or peroxidase can be conjugated to an effector agent using standard in vitro conjugation chemistry.When the effector agent is a polypeptide, the glucose (xylose) isomerase, glucose oxidase and / or peroxidase can be chemically conjugated to the effector or linked to the effector as a fusion protein.The construction of a fusion protein is within the ordinary skill of the art.

[0050] In some embodiments, depending on the particular mode of administration or site of activity, the disclosed glucose (xylose) isomerase, glucose oxidase and / or peroxidolytic enzyme can be modified with a moiety that improves its stability and / or retention in circulation, for example in blood, serum or other tissues. For example, the disclosed glucose (xylose) isomerase, glucose oxidase and / or peroxidolytic enzyme can be conjugated to a polymer, for example a substantially non-antigenic polymer, for example a polyalkylene oxide or polyethylene oxide. In some embodiments, the disclosed glucose (xylose) isomerase, glucose oxidase and / or peroxidolytic enzyme is conjugated to a water-soluble polymer, for example a hydrophilic polyvinyl polymer, for example polyvinyl alcohol or polyvinylpyrrolidone. Examples of such polymers include polyalkylene oxide homopolymers, for example polyethylene glycol (PEG) or polypropylene glycol, polyoxyethylenated polyols, copolymers thereof and block copolymers thereof. Further useful polymers include polyoxyalkylenes such as polyoxyethylene, polyoxypropylene and block copolymers of polyoxyethylene and polyoxypropylene, polymethacrylates, carbomers and branched or unbranched polysaccharides.

[0051] II. Enzyme Production Methods for producing the glucose (xylose) isomerase, glucose oxidase and / or peroxide decomposition enzyme of the present invention are known in the art. For example, the DNA molecule encoding glucose (xylose) isomerase, glucose oxidase and / or peroxide decomposition enzyme can be chemically synthesized using the sequence information provided herein. The synthetic DNA molecule can be ligated to other appropriate nucleotide sequences, such as expression control sequences, to produce a conventional gene expression construct encoding the desired glucose (xylose) isomerase, glucose oxidase and / or peroxide decomposition enzyme.

[0052] Nucleic acids encoding the desired glucose (xylose) isomerase, glucose oxidase and / or peroxide decomposition enzymes can be incorporated (ligated) into an expression vector, which can be introduced into a host cell by conventional transfection or transformation techniques. The transformed host cell can be grown under conditions that allow the host cell to express the genes encoding glucose (xylose) isomerase, glucose oxidase and / or peroxide decomposition enzymes.

[0053] In certain embodiments, nucleic acids encoding recombinant glucose (xylose) isomerase, glucose oxidase and / or peroxide degrading enzymes of the invention can be codon optimized for expression in a heterologous cell, such as a yeast cell (e.g., a Pichia cell) or an E. coli cell, using methods known in the art.

[0054] Specific expression and purification conditions vary according to the expression system used.For example, if gene is expressed in E. coli, gene can be cloned into expression vector by placing engineered gene downstream of suitable bacterial promoter, such as Trp or Tac and prokaryotic signal sequence.Expressed secretory protein accumulates in refractile or inclusion bodies and can be harvested after cell disruption by French press or sonication.Refractile bodies are then solubilized and protein is refolded and cleaved by methods known in the art.

[0055] Glucose (xylose) isomerase, glucose oxidase and / or peroxide decomposition enzyme can be produced by growing (culturing) a host cell transfected with an expression vector encoding such glucose (xylose) isomerase, glucose oxidase and / or peroxide decomposition enzyme under conditions that allow expression of the glucose (xylose) isomerase, glucose oxidase and / or peroxide decomposition enzyme. After expression, the glucose (xylose) isomerase, glucose oxidase and / or peroxide decomposition enzyme can be harvested and purified or isolated using techniques known in the art, for example affinity tags such as glutathione-S-transferase (GST) and histidine tags.

[0056] In some embodiments, the glucose (xylose) isomerase, glucose oxidase and / or peroxide decomposition enzyme are dried, e.g., spray dried. Pharmaceutical proteins can be dried in many ways, for example, by removing water, organic solvent or liquid polymer by means of drying with N2, air or inert gas, vacuum oven drying, freeze drying, washing with a volatile organic solvent followed by evaporation of the solvent, evaporation in a fume hood, tray drying, fluid bed drying, spray drying, vacuum drying or roller drying.

[0057] By spray drying glucose (xylose) isomerase, glucose oxidase and / or peroxide decomposition enzyme, water is separated from the glucose (xylose) isomerase, glucose oxidase and / or peroxide decomposition enzyme preparation, allowing continuous production of dry solids in powder, granule or agglomerate form from liquid feedstocks such as emulsions and pumpable suspensions. Spray drying involves atomizing a liquid feedstock containing glucose (xylose) isomerase, glucose oxidase and / or peroxide decomposition enzyme into a spray of droplets and contacting the droplets with hot air or gas in a drying chamber. The spraying process can be carried out using a two-fluid atomizer that mixes the liquid feedstock with a drying gas such as compressed air or nitrogen. The operating conditions and dryer design are selected according to the drying characteristics of glucose (xylose) isomerase, glucose oxidase and / or peroxide decomposition enzyme and the desired powder quality. An exemplary method for spray drying enzymes is described in US Patent Application Publication No. 2015 / 0353913. The glucose (xylose) isomerase, glucose oxidase and peroxide decomposition enzyme may be spray dried separately or together, or may be spray dried together.

[0058] In some embodiments, the glucose (xylose) isomerase, glucose oxidase and / or peroxide decomposition enzyme is immobilized. For example, the glucose (xylose) isomerase, glucose oxidase and / or peroxide decomposition enzyme can be immobilized on IRA-904 resin, calcium alginate beads, Ludox HS-30 or silica beads.

[0059] III. Pharmaceutical Compositions For therapeutic use, the glucose (xylose) isomerase, glucose oxidase and / or peroxidolytic enzyme described herein are preferably combined with a pharma- ceutically acceptable carrier. The term "pharma-ceutically acceptable" as used herein refers to those compounds, materials, compositions and / or dosage forms that are within the bounds of sound medical judgment and suitable for use in contact with human and animal tissues without excessive toxicity, irritation, allergic response or other problems or complications, commensurate with a reasonable benefit / risk ratio.

[0060] The term "pharmaceutical acceptable carriers" as used herein refers to buffers, carriers and excipients suitable for use in contact with human and animal tissues without undue toxicity, irritation, allergic responses or other problems or complications, commensurate with a reasonable benefit / risk ratio. Pharmaceutically acceptable carriers include any of the standard pharmaceutical carriers, such as phosphate buffered saline solution, water, emulsions (such as oil / water or water / oil emulsions) and various types of wetting agents. The compositions may also include stabilizers and preservatives. For examples of carriers, stabilizers and adjuvants, see, for example, Martin, Remington's Pharmaceutical Sciences, 15th Ed., Mack Publ. Co., Easton, PA

[1975] . Pharmaceutically acceptable carriers include buffers, solvents, dispersion media, coatings, isotonic and absorption delaying agents, etc., that are compatible with pharmaceutical administration. The use of such media and agents for pharmaceutical active substances is well known in the art.

[0061] Pharmaceutical compositions containing the naturally occurring or recombinant glucose (xylose) isomerase, glucose oxidase and / or peroxidase disclosed herein may be in unit dosage form and may be prepared in any suitable manner. The pharmaceutical composition should be formulated to be compatible with its intended route of administration. The pharmaceutical composition may be in various forms. For example, these include liquid, semi-solid and solid dosage forms such as liquid solutions, dispersions or suspensions, tablets, pills, powders, liposomes and suppositories. The preferred form depends on the intended mode of administration and therapeutic application.

[0062] Although the compositions are preferably formulated for enteral (e.g., oral) administration, such compositions may be administered by parenteral modes (e.g., intravenous, subcutaneous, intraperitoneal, or intramuscular injection). The phrases "parenteral administration" and "administered parenterally" as used herein refer to modes of administration other than enteral and topical administration, usually by injection, including, but not limited to, intravenous, intramuscular, intraarterial, intrathecal, intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal, transtracheal, subcutaneous, subcuticular, intraarticular, subcapsular, subarachnoid, intraspinal, epidural, and substernal injection and infusion.

[0063] The composition can be formulated as a solution, microemulsion, dispersion, liposome or other ordered structure suitable for stable storage at high concentration. Sterile injectable solution can be prepared by incorporating the agent described herein in the required amount in a suitable solvent with one or a combination of the ingredients listed above, if necessary, and then sterilizing by filtration. Generally, dispersion is prepared by incorporating the agent described herein into a sterile vehicle containing a basic dispersion medium and other required ingredients from those listed above. In the case of sterile powder for preparing sterile injectable solution, the preferred preparation method is vacuum drying and freeze-drying, which produces a powder of the agent described herein and any additional desired ingredients from its solution that has been previously sterile-filtered. The proper fluidity of the solution can be maintained, for example, by using a coating such as lecithin, by maintaining the required particle size in the case of dispersion, and by using surfactants. Prolonged absorption of injectable composition can be achieved by including an agent that delays absorption in the composition, for example, monostearate salt and gelatin.

[0064] Depending on the mode of administration, for example, parenteral administration, it may be desirable to prepare the pharmaceutical preparation as sterile.Sterilization can be achieved by any suitable method, for example, filtration through a sterile filtration membrane.When the composition is lyophilized, filter sterilization can be performed before or after lyophilization and reconstitution.

[0065] In some embodiments, the disclosed compositions include a polyionic agent (e.g., the composition includes a polyionic coating) that can coat, for example, glucose (xylose) isomerase, glucose oxidase, and / or peroxide decomposition enzyme. Exemplary polyionic agents include PSS (poly(sodium 4-styrenesulfonate), PAA (polyacrylic acid sodium salt), PMG (poly(methylene-co-guanidine) hydrochloride), DS (dextran sulfate), PMA (poly(methyl acrylate)), or PVS (polyvinylsiloxane).

[0066] In certain embodiments, the disclosed compositions and / or doses include: (i) about 750 to about 250,000, about 750 to about 200,000, about 750 to about 150,000, about 750 to about 100,000, 750 to about 75,000, about 750 to about 60,000, about 750 to about 45,000, about 750 to about 30,000, about 750 to about 15,000, about 750 to about 10,000, about 750 to about 7,500, about 750 to about 5,000, about 750 to about 2,500, about 750 to about 1,000, about 1,000 to about 250,000, about 1,000 to about 200,000, about 00 to about 150,000, about 1,000 to about 100,000, 1,000 to about 75,000, about 1,000 to about 60,000, about 1,000 to about 45,000, about 1,000 to about 30,000, about 1,000 to about 15,000, about 1,000 to about 10,000, about 1,000 to about About 7,500, about 1,000 to about 5,000, about 1,000 to about 2,500, about 2,500 to about 250,000, about 2,500 to about 200,000, about 2,500 to about 150,000, about 2,500 to about 100,000, 2,500 to about 75,000, about 2,500 to about 60,0 00, about 2,500 to about 45,000, about 2,500 to about 30,000, about 2,500 to about 15,000, about 2,500 to about 10,000, about 2,500 to about 7,500, about 2,500 to about 5,000, about 5,000 to about 250,000, about 5,000 to about 200,000, about 5,000 to about 150,000, about 5,000 to about 100,000, about 5,000 to about 75,000, about 5,000 to about 60,000, about 5,000 to about 45,000, about 5,000 to about 30,000, about 5,000 to about 15,000, about 5,000 to about 10,000, about 5,00 0 to about 7,500, about 7,500 to about 250,000, about 7,500 to about 200,000, about 7,500 to about 150,000, about 7,500 to about 100,000, 7,500 to about 75,000, about 7,500 to about 60,000, about 7,500 to about 45,000, about 7,500 to About 30,000, about 7,500 to about 15,000, about 7,500 to about 10,000, about 10,000 to about 250,000, about 10,000 to about 200,000, about 10,000 to about 150,000, about 10,000 to about 100,000, 10,000 to about 75,000, about 10,000 to about 60,000, about 10,000 to about 45,000, about 10,000 to about 30,000, about 10,000 to about 15,000, about 15,000 to about 250,000, about 15,000 to about 200,000, about 15,000 to about 150,000, about 15,000 to about 100, 000, 15,000 to about 75,000, about 15,000 to about 60,000, about 15,000 to about 45,000, about 15,000 to about 30,000, about 30,000 to about 250,000, about 30,000 to about 200,000, about 30,000 to about 150,000, about 30,000 ~100,000, ~30,000~75,000, ~30,000~60,000, ~30,000~45,000, ~45,000~250,000, ~45,000~200,000, ~45,000~150,000, ~45,000~100,000 , about 45,000 to about 75,000, about 45,000 to about 60,000, about 60,000 to about 250,000, about 60,000 to about 200,000, about 60,000 to about 150,000, about 60,000 to about 100,000, about 60,000 to about 75,000, about 75,000 to about 250,000, about 75,000 to about 200,000, about 75,000 to about 150,000, about 75,000 to about 100,000, about 100,000 to about 250,000, about 100,000 to about 200,000, about 100,000 to about 150,000, about 150,000 to about 250,000, about 150,000 to about 200,000, about 200,000 to about 250,000 International Units (IU) of glucose (xylose) isomerase enzyme; and / or (ii) about 750 to about 250,000, about 750 to about 200,000, about 750 to about 150,000, About 750 to about 100,000, 750 to about 75,000, about 750 to about 60,000, about 750 to about 45,000, about 750 to about 30,000, about 750 to about 15,000, about 750 to about 10,000, about 750 to about 7,500, about 750 to about 5,000, about 750 to about 2,50 0, about 750 to about 1,000, about 1,000 to about 250,000, about 1,000 to about 200,000, about 1,000 to about 150,000, about 1,000 to about 100,000, 1,000 to about 75,000, about 1,000 to about 60,000, about 1,000 to about 45,000, about 1,000 to about 30,000, about 1,000 to about 15,000, about 1,000 to about 10,000, about 1,000 to about 7,500, about 1,000 to about 5,000, about 1,000 to about 2,500, about 2,500 to about 250,000, about 2,500 to about 200,000, about 2,500 to about 1 50,000, about 2,500 to about 100,000, 2,500 to about 75,000, about 2,500 to about 60,000, about 2,500 to about 45,000, about 2,500 to about 30,000, about 2,500 to about 15,000, about 2,500 to about 10,000, about 2,500 to about 7,500 , about 2,500 to about 5,000, about 5,000 to about 250,000, about 5,000 to about 200,000, about 5,000 to about 150,000, about 5,000 to about 100,000, 5,000 to about 75,000, about 5,000 to about 60,000, about 5,000 to about 45,000, about ,000 to about 30,000, about 5,000 to about 15,000, about 5,000 to about 10,000, about 5,000 to about 7,500, about 7,500 to about 250,000, about 7,500 to about 200,000, about 7,500 to about 150,000, about 7,500 to about 100,000, 7,50 0 to about 75,000, about 7,500 to about 60,000, about 7,500 to about 45,000, about 7,500 to about 30,000, about 7,500 to about 15,000, about 7,500 to about 10,000, about 10,000 to about 250,000, about 10,000 to about 200,000, about 10,00 0 to about 150,000, about 10,000 to about 100,000, 10,000 to about 75,000, about 10,000 to about 60,000, about 10,000 to about 45,000, about 10,000 to about 30,000, about 10,000 to about 15,000, about 15,000 to about 250,000, about 15,000 to about 200,000, about 15,000 to about 150,000, about 15,000 to about 100,000, 15,000 to about 75,000, about 15,000 to about 60,000, about 15,000 to about 45,000, about 15,000 to about 30,000, about 30,000 to about 250 ,000, about 30,000 to about 200,000, about 30,000 to about 150,000, about 30,000 to about 100,000, about 30,000 to about 75,000, about 30,000 to about 60,000, about 30,000 to about 45,000, about 45,000 to about 250,000, about 45,000 to about 200,000, about 45,000 to about 150,000, about 45,000 to about 100,000, about 45,000 to about 75,000, about 45,000 to about 60,000, about 60,000 to about 250,000, about 60,000 to about 200,000, about 60,000 to about 15 0,000, about 60,000 to about 100,000, about 60,000 to about 75,000, about 75,000 to about 250,000, about 75,000 to about 200,000, about 75,000 to about 150,000, about 75,000 to about 100,000, about 100,000 to about 250,000, and / or (iii) about 750 to about 250,000, about 750 to about 200,000, about 750 to about 150,000, about 750 to about 100,000, 750 to about 75,000, about 750 to about 60,000, about 750 to about 45,000, about 750 to about 30,000, about 750 to about 15,000, about 750 to about 50 ... About 10,000, about 750 to about 7,500, about 750 to about 5,000, about 750 to about 2,500, about 750 to about 1,000, about 1,000 to about 250,000, about 1,000 to about 200,000, about 1,000 to about 150,000, about 1,000 to about 100,000, 1,00 0 to about 75,000, about 1,000 to about 60,000, about 1,000 to about 45,000, about 1,000 to about 30,000, about 1,000 to about 15,000, about 1,000 to about 10,000, about 1,000 to about 7,500, about 1,000 to about 5,000, about 1,000 to about 2,50 0, about 2,500 to about 250,000, about 2,500 to about 200,000, about 2,500 to about 150,000, about 2,500 to about 100,000, 2,500 to about 75,000, about 2,500 to about 60,000, about 2,500 to about 45,000, about 2,500 to about 30,000 , about 2,500 to about 15,000, about 2,500 to about 10,000, about 2,500 to about 7,500, about 2,500 to about 5,000, about 5,000 to about 250,000, about 5,000 to about 200,000, about 5,000 to about 150,000, about 5,000 to about 100,000, 5,000 to about 75,000, about 5,000 to about 60,000, about 5,000 to about 45,000, about 5,000 to about 30,000, about 5,000 to about 15,000, about 5,000 to about 10,000, about 5,000 to about 7,500, about 7,500 to about 250,000, about 7,500 to About 200,000, about 7,500 to about 150,000, about 7,500 to about 100,000, about 7,500 to about 75,000, about 7,500 to about 60,000, about 7,500 to about 45,000, about 7,500 to about 30,000, about 7,500 to about 15,000, about 7,500 to about 1 0,000, about 10,000 to about 250,000, about 10,000 to about 200,000, about 10,000 to about 150,000, about 10,000 to about 100,000, 10,000 to about 75,000, about 10,000 to about 60,000, about 10,000 to about 45,000, about ,000 to about 30,000, about 10,000 to about 15,000, about 15,000 to about 250,000, about 15,000 to about 200,000, about 15,000 to about 150,000, about 15,000 to about 100,000, 15,000 to about 75,000, about 15,000 to about 60,0 00, about 15,000 to about 45,000, about 15,000 to about 30,000, about 30,000 to about 250,000, about 30,000 to about 200,000, about 30,000 to about 150,000, about 30,000 to about 100,000, about 30,000 to about 75,000, about 30,0 00 to about 60,000, about 30,000 to about 45,000, about 45,000 to about 250,000, about 45,000 to about 200,000, about 45,000 to about 150,000, about 45,000 to about 100,000, about 45,000 to about 75,000, about 45,000 to about 60,000 0, about 60,000 to about 250,000, about 60,000 to about 200,000, about 60,000 to about 150,000, about 60,000 to about 100,000, about 60,000 to about 75,000, about 75,000 to about 250,000, about 75,000 to about 200,000, about 75, 000 to about 150,000, about 75,000 to about 100,000, about 100,000 to about 250,000, about 100,000 to about 200,000, about 100,000 to about 150,000, about 150,000 to about 250,000, about 150,000 to about 200,000, about 200,It contains 000 to about 250,000 international units (IU) of peroxide decomposition enzyme.

[0067] Depending on the circumstances, the ratio of the international units of glucose (xylose) isomerase to the international units of glucose oxidase enzyme is in the range of 0.1-10, e.g. 0.1-9, 0.1-8, 0.1-7, 0.1-6, 0.1-5, 0.1-4, 0.1-3, 0.1-1 or 0.1-1, 0.5-10, 0.5-9, 0.5-8, 0.5-7, 0.5-6, 0.5-5, 0.5-4, 0.5-3 or 0.5-2, 0.5-1, 1-10, 1-9, 1-8, 1-7, 1-6, 1-5, 1-4, 1-3 or 1-2.

[0068] Depending on the situation, the ratio of the international units of glucose oxidase to the international units of the peroxidase decomposition enzyme is in the range of 0.1 to 10, for example, 0.1 to 9, 0.1 to 8, 0.1 to 7, 0.1 to 6, 0.1 to 5, 0.1 to 4, 0.1 to 3, 0.1 to 1 or 0.1 to 1, 0.5 to 10, 0.5 to 9, 0.5 to 8, 0.5 to 7, 0.5 to 6, 0.5 to 5, 0.5 to 4, 0.5 to 3 or 0.5 to 2, 0.5 to 1, 1 to 10, 1 to 9, 1 to 8, 1 to 7, 1 to 6, 1 to 5, 1 to 4, 1 to 3 or 1 to 2.

[0069] Depending on the circumstances, the amount of glucose (xylose) isomerase, glucose oxidase and / or peroxidase decomposition enzyme administered to a subject at a particular dose may be in the range of about 50 to about 20,000, about 50 to about 15,000, about 50 to about 10,000, about 50 to about 7,500, about 50 to about 5,000, about 50 to about 4,000, about 50 to about 3000, about 50 to about 2000, about 50 to about 1,000, about 50 to about 750, about 50 to about 500 or about 50 to about 150 international units / kg, for example, about 20,000, about 15,000, about 10,000, about 5,000, about 4,000, about 3,000, 2,000, or 1,000 international units / kg.

[0070] For a 180 kg human, it is contemplated that about 500,000 to about 4,000,000, 500,000 to about 3,000,000, about 500,000 to about 2,000,000, 500,000 to about 1,000,000, 1,000,000 to about 4,000,000 1,000,000 to about 3,000,000, or 1,000,000 to about 2,000,000 International Units of glucose (xylose) isomerase, glucose oxidase and / or peroxidase degrading enzyme may be administered in a single dose.

[0071] In some embodiments, the disclosed glucose (xylose) isomerase, glucose oxidase and / or peroxide decomposition enzyme or composition is administered to a subject with a meal or snack. In some embodiments, the disclosed glucose (xylose) isomerase, glucose oxidase and / or peroxide decomposition enzyme or composition is administered to a subject with each meal or snack that the subject eats. In some embodiments, the disclosed glucose (xylose) isomerase, glucose oxidase and / or peroxide decomposition enzyme or composition is administered to a subject once every 7 days, once every 6 days, once every 5 days, once every 4 days, once every 3 days, once every 2 days, once daily, twice daily, three times daily, four times daily, five times daily, six times daily or more than six times daily.

[0072] Depending on the situation, the composition can be formulated as powder, granule, pellet, micropellet or mini tablet.The composition can be encapsulated in capsule, such as hydroxypropylmethylcellulose (HPMC) capsule, soft gelatin capsule or hard gelatin capsule.Alternatively, the composition can be formulated as tablet dosage form.The composition can also be formulated as liquid oral dosage form, such as elixir, syrup or drop.

[0073] IV. Therapeutic uses The methods and compositions disclosed herein can be used to treat diseases or disorders.For example, the present invention provides a method for treating fructose intolerance (e.g. dietary fructose intolerance or hereditary fructose intolerance), irritable bowel syndrome (IBS), FODMAP (fermentable oligosaccharides, disaccharides, monosaccharides and polyols) malabsorption and / or intolerance, obesity, metabolic syndrome, diabetes, hyperglycemia or hyperinsulinemia.The method comprises administering to a subject an effective amount of (i) glucose (xylose) isomerase enzyme, (ii) glucose oxidase enzyme and / or (iii) peroxide decomposition enzyme.For example, the method can comprise administering the disclosed pharmaceutical composition comprising (i) spray-dried or freeze-dried glucose (xylose) isomerase, (ii) spray-dried or freeze-dried glucose oxidase enzyme and / or (iii) spray-dried or freeze-dried peroxide decomposition enzyme.

[0074] The term "effective amount" as used herein refers to an amount of an active agent (e.g., the disclosed glucose (xylose) isomerase, glucose oxidase and / or peroxide decomposition enzyme) sufficient to produce a beneficial or desired result. An effective amount may be administered in one or more administrations, applications or doses and is not intended to be limited to a particular formulation or route of administration.

[0075] As used herein, "treat," "treating," and "treatment" refer to the treatment of a disease in a subject, e.g., a human. This includes (a) inhibiting the disease, i.e., preventing its onset; and (b) alleviating the disease, i.e., causing regression of the disease state. As used herein, the terms "subject" and "patient" refer to an organism that is treated by the methods and compositions described herein. Such organisms preferably include, but are not limited to, mammals (e.g., mice, monkeys, horses, cows, pigs, dogs, cats, etc.), and more preferably, humans.

[0076] The present invention also provides a method for reducing fructose levels in a subject, such as a subject with fructose intolerance (e.g., dietary fructose intolerance or hereditary fructose intolerance), irritable bowel syndrome (IBS), FODMAP (fermentable oligosaccharides, disaccharides, monosaccharides and polyols) malabsorption and intolerance, obesity, metabolic syndrome, diabetes, hyperglycemia or hyperinsulinemia.The method comprises administering to the subject an effective amount of (i) glucose (xylose) isomerase, (ii) glucose oxidase enzyme and / or (iii) peroxide decomposition enzyme.For example, the method can comprise administering the disclosed pharmaceutical composition comprising (i) spray-dried or freeze-dried glucose (xylose) isomerase, (ii) spray-dried or freeze-dried glucose oxidase and / or (ii) spray-dried or freeze-dried peroxide decomposition enzyme. The level of fructose in a subject can refer to the level of fructose in the subject's bodily fluids (eg, blood, plasma, serum or urine), tissues, organs, cells and / or gastrointestinal tract, or in a sample of any of the foregoing.

[0077] Fructose levels, e.g., blood fructose levels, can be measured by any method known in the art. Exemplary fructose measurement kits are available from Sigma Aldrich (FA20-1KT) and Biovision (K439-100).

[0078] The methods and compositions described herein may be used alone or in combination with other therapeutic agents and / or modalities. The term "administered in combination," as used herein, is understood to mean that two (or more) different therapies are delivered to a subject during the course of the subject's affliction with a disorder, such that the effects of the therapies on the patient overlap at some point. In some embodiments, the delivery of one treatment is still occurring when the delivery of the second begins, so there is an overlap in the administration period. This is sometimes referred to herein as "simultaneous" or "concurrent delivery." In other embodiments, the delivery of one treatment ends before the delivery of the other begins. In some embodiments of either case, the treatments are more effective because of the combined administration. For example, the second treatment is more effective, e.g., a comparable effect is seen with less of the second treatment, or the second treatment reduces symptoms to a greater extent than would be seen if the second treatment were administered in the absence of the first treatment, or a similar situation is seen with the first treatment. In some embodiments, the delivery is such that the reduction in symptoms or other parameters associated with the disorder is greater than that observed with one treatment delivered in the absence of the other. The effect of the two treatments can be partially additive, totally additive, or greater than additive. The delivery can be such that the effect of the first treatment delivered is still detectable when the second is delivered.

[0079] Throughout this description, when compositions are described as having, including, or comprising specific components, or when processes and methods are described as having, including, or comprising specific steps, it is further contemplated that there are compositions of the invention that consist essentially of or consist of the recited components, and that there are processes and methods according to the invention that consist essentially of or consist of the recited process steps.

[0080] In this application, when an element or component is said to be included in and / or selected from a list of described elements or components, it is to be understood that the element or component can be any one of the described elements or components, or the element or component can be selected from a group consisting of two or more of the described elements or components.

[0081] Furthermore, it should be understood that the elements and / or features of the compositions or methods described herein, whether express or implied herein, can be combined in various ways without departing from the spirit and scope of the present invention. For example, when reference is made to a particular compound, the compound can be used in various aspects of the compositions of the present invention and / or in the methods of the present invention, unless otherwise understood from the context. That is, in this application, the embodiments are described and shown in a manner that allows for clear and concise application as described and illustrated, but it is intended and understood that the embodiments can be variously combined or separated without departing from the present teachings and invention(s). For example, it is understood that all features described and shown herein can be applicable to all aspects of the invention(s) described and shown herein.

[0082] The phrase "at least one of" should be understood to include each of the recited items following the phrase and various combinations of two or more of the recited items individually, unless otherwise understood from the context and application. The phrase "and / or" with respect to three or more recited items should be understood to have the same meaning, unless otherwise understood from the context.

[0083] Use of the terms "include," "includes," "including," "have," "has," "having," "contain," "contains," or "containing," including their grammatical equivalents, should generally be understood to be open-ended and open-ended, e.g., not excluding additional, unrecited elements or steps, unless specifically stated or understood from the context to the contrary.

[0084] When the term "about" is used before a quantitative value, the invention also includes the specific quantitative value itself, unless specifically stated otherwise. As used herein, the term "about" refers to a ±10% variation from the nominal value, unless otherwise indicated or inferred.

[0085] It should be understood that the order of steps or order for performing certain actions is immaterial so long as the invention remains operable. Moreover, two or more steps or actions may be performed simultaneously.

[0086] The use of any and all examples or exemplary terms herein, such as "such as" or "including," is intended merely to better describe the invention and does not pose a limitation on the scope of the invention unless and until claimed. No language in the specification should be construed as indicating any non-claimed element as essential to the practice of the invention. EXAMPLES

[0087] Working Example The following examples are illustrative only and are not intended to limit the scope or content of the invention in any way.

[0088] Example 1 This example describes a study testing the effect of glucose (xylose) isomerase on the conversion of fructose in the absence and presence of glucose oxidase.

[0089] The materials used in this and the following examples were as follows: fructose (Sigma, Cat. No. F2543-100G), glucose (Sigma Cat. No. G8270-100G), phosphate buffered saline (GIBCO Life Technologies Cat. No. 70011-044), sodium acetate (Emplura MERC Cat. No. 1.93644.0521G), glucose assay kit (Lyphochek glucose or Lyphozyme from Agappe Diagnostics Ltd. or Beacon Diagnostics Pvt. Ltd. respectively), fructose assay kit (Sigma Cat. No. FA20-1KT), glucose isomerase (Sigma Cat. No. G4166-50G), glucose oxidase (Sigma Cat. No. G7141-10KU) and catalase (Cat. No. C3515-25MG). Unless otherwise indicated, all reagents and kits were used according to the manufacturer's instructions.

[0090] Fructose (1% solution) was incubated with glucose(xylose) isomerase and / or glucose oxidase, and the conversion of fructose to glucose by glucose(xylose) isomerase and the degradation of glucose to gluconic acid and hydrogen peroxide by glucose oxidase were monitored, as shown in Table 1. To mimic intestinal pH conditions, assays were performed in both acetate buffer, pH 5.0, and phosphate-buffered saline (PBS), pH 7.4. [Table 1]

[0091] Test samples were prepared in 2 mL Eppendorf tubes and incubated at 37° C. with constant agitation. Aliquots were withdrawn at 2 and 4 hours and filtered through centrifuge filters (0.2 μm) to separate the glucose (xylose) isomerase enzyme (if immobilized) and prevent further conversion of fructose to glucose. Samples were then tested for glucose and fructose concentrations using the corresponding assay kits.

[0092] Results are shown in Table 2 for glucose and in Table 3 for fructose. The data show that glucose (xylose) isomerase alone converted fructose to glucose until equilibrium such that there was no further decrease in fructose concentration after 2 hours and no further increase in glucose concentration after 2 hours. However, in the presence of glucose oxidase, there was a further decrease in glucose due to degradation of glucose by glucose oxidase. The results show that the mixture of glucose (xylose) isomerase and glucose oxidase converted or degraded greater than 70 to 80% of the fructose in solution within 3 to 4 hours at pH 5.0 and 7.4. [Table 2] [Table 3]

[0093] Example 2 This example describes a study examining the effect of glucose (xylose) isomerase on the conversion of fructose in the absence and presence of glucose oxidase and catalase.

[0094] Fructose (1% solution) was incubated with glucose(xylose) isomerase, glucose oxidase and / or catalase, and the conversion of fructose to glucose by glucose(xylose) isomerase and the degradation of glucose to gluconic acid and hydrogen peroxide by glucose oxidase were monitored, as shown in Table 4. To mimic intestinal pH conditions, assays were performed in both acetate buffer pH 5.0 and phosphate-buffered saline (PBS) pH 7.4. [Table 4]

[0095] Test samples were prepared in 2 mL Eppendorf tubes and incubated at 37° C. with constant agitation. Aliquots were withdrawn at 1, 2, 3 and 4 hours and filtered through centrifuge filters (0.2 μm) to separate the glucose (xylose) isomerase enzyme (if immobilized) and prevent further conversion of fructose to glucose. Samples were then tested for glucose and fructose concentrations using the corresponding assay kits.

[0096] Results are shown for glucose in Table 5 and for fructose in Table 6. Results at pH 7.4 are also shown in Figure 1. As shown, the presence of catalase improved the degradation of fructose. Incubation for 1 hour with glucose (xylose) isomerase and glucose oxidase resulted in 42% (pH 7.4) or 34% (pH 5.0) of remaining glucose. However, incubation for 1 hour with glucose (xylose) isomerase, glucose oxidase and catalase resulted in 2% (pH 7.4) or 0.2% (pH 5.0) of remaining glucose. Unexpectedly, better results were seen at pH 5.0 compared to pH 7.4. It is hypothesized that this may be due to the pH profile of glucose oxidase and catalase, rather than glucose (xylose) isomerase (which is expected to work better at pH 7.4). These results suggest that in vivo, a combination of glucose (xylose) isomerase, glucose oxidase and catalase may be active in the stomach. [Table 5] [Table 6]

[0097] Sequence Listing [Table 7-1] [Table 7-2]

Claims

1. A pharmaceutical composition comprising a glucose (xylose) isomerase enzyme, a glucose oxidase enzyme, a peroxide degrading enzyme and a pharmaceutically acceptable excipient.

2. Glucose (xylose) isomerase enzyme (a) spray-dried or freeze-dried; (b) a bacterial glucose (xylose) isomerase enzyme or a functional fragment or variant thereof; (c) derived from Streptomyces murinus; and / or (d) the pharmaceutical composition of claim 1, comprising from about 1,000 to about 250,000 international units (IU) of the composition.

3. The glucose oxidase enzyme (a) spray-dried or freeze-dried; (b) a bacterial glucose oxidase enzyme or a functional fragment or variant thereof; (c) derived from Aspergillus niger; and / or 10. The pharmaceutical composition of claim 1, wherein (d) comprises about 1,000 to about 250,000 IU in the composition.

4. Peroxide decomposing enzymes (a) spray-dried or freeze-dried; (b) a catalase enzyme or a peroxidase enzyme; (c) derived from Aspergillus niger; and / or (d) the pharmaceutical composition of claim 1, comprising from about 1,000 to about 2,500,000 IU in the composition. (a) the glucose (xylose) isomerase enzyme comprises SEQ ID NO:5 or a functional fragment or variant thereof; (b) the glucose oxidase enzyme comprises SEQ ID NO:2 or a functional fragment or variant thereof; and / or 3. The pharmaceutical composition of claim 2, wherein (c) the catalase enzyme comprises SEQ ID NO: 4 or a functional fragment or variant thereof.

6. 2. The pharmaceutical composition of claim 1, wherein the ratio of IU of glucose (xylose) isomerase to IU of glucose oxidase enzyme is in the range of 0.1 to 10.

7. 2. The pharmaceutical composition according to claim 1, wherein the ratio of IU of glucose oxidase to IU of peroxidolytic enzyme is in the range of 0.1 to 10.

8. The pharmaceutical composition of claim 1, which is formulated as an oral dosage form.

9. The pharmaceutical composition of claim 8, wherein the oral dosage form is a solid or liquid oral dosage form.

10. 9. The pharmaceutical composition of claim 8, formulated as a powder, satchel, granules, pellets, micropellets, tablets, minitablets, elixir, syrup or drops.

11. 10. The pharmaceutical composition of claim 1, having a shelf life at room temperature of at least 3 months, 6 months, 9 months, 12 months, 15 months, 18 months, 21 months, 24 months, 36 months, 48 months, 60 months, 72 months, 84 months, 96 months, 108 months, or 120 months.

12. 12. The pharmaceutical composition according to any one of claims 1 to 11 for treating fructose intolerance in a subject in need thereof, wherein the pharmaceutical composition is orally administered to the subject.

13. 13. The pharmaceutical composition according to claim 12, wherein the fructose intolerance is hereditary fructose intolerance or dietary fructose intolerance.

14. A pharmaceutical composition according to any one of claims 1 to 11 for reducing fructose levels in a subject, wherein the pharmaceutical composition is orally administered to the subject.

15. 15. The pharmaceutical composition of claim 14, which reduces the level of fructose in the blood of a subject, in the gastrointestinal tract of a subject and / or in a sample derived from a subject.

16. The pharmaceutical composition of claim 15, wherein the sample is a body fluid sample, and the body fluid sample is blood, serum or plasma.

17. The pharmaceutical composition comprising: (a) reducing the level of fructose in the subject's blood by at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90% from 0 to 240 minutes or from 0 to 360 minutes after a meal or snack, as determined by area under the curve (AUC) analysis; (b) reducing fructose Cmax in the subject's blood after a meal or snack by at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90%; (c) reducing the fructose Tmax in the subject's blood after a meal or snack by at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90%; and / or (d) the pharmaceutical composition of claim 14, which, when administered with food, reduces food calorie intake by at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90%.

18. 15. The pharmaceutical composition of claim 14, wherein the subject has dietary fructose intolerance, hereditary fructose intolerance, irritable bowel syndrome (IBS), FODMAP (fermentable oligosaccharides, disaccharides, monosaccharides and polyols) intolerance or malabsorption, obesity, metabolic syndrome, diabetes, hyperglycemia or hyperinsulinemia.

19. The pharmaceutical composition comprises: (a) 1, 2, 3, 4 or more times per day; (b) with a meal or snack, and / or (c) at each meal or snack The pharmaceutical composition of claim 12 , which is administered to a subject.

20. 13. The pharmaceutical composition of claim 12, wherein the subject is a human adult or human pediatric subject.

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