Methods and compositions for treating hyperglycemia and diabetes - Patents.com

Abstract

Disclosed are pharmaceutical compositions comprising a glucose oxidase enzyme and a peroxide decomposing enzyme (eg, a catalase enzyme) that may be used to treat, inter alia, hyperglycemia, diabetes, obesity, metabolic syndrome and / or eating disorders.

Description

[Technical field]

[0001] REFERENCE TO RELATED APPLICATIONS This application claims the benefit of and priority to U.S. Provisional Patent Application No. 63 / 232,920, 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 hyperglycemia, diabetes, obesity, metabolic syndrome and / or eating disorders, and more specifically, the present invention relates to compositions comprising glucose oxidase enzymes and peroxide degrading enzymes (e.g., catalase enzymes) and their use in the treatment of hyperglycemia, diabetes, obesity, metabolic syndrome and / or eating disorders. [Background technology]

[0003] background According to the Centers for Disease Control (CDC), 34.2 million people in the United States have diabetes, and 88 million adults, more than one in three, have prediabetes. Total medical costs and lost work and wages for people diagnosed with diabetes amount to $327 billion. The diabetes epidemic is not limited to the United States. According to the World Health Organization (WHO), the number of adults surviving with diabetes has nearly quadrupled since 1980 to 422 million adults. Diabetes is the leading cause of blindness, kidney failure, heart attacks, strokes, and lower limb amputations. Nearly half of all deaths due to high blood glucose occur before age 70. The WHO estimates that diabetes was the seventh leading cause of death in 2016. According to the World Federation of Diabetes, 4 million people die from diabetes every year.

[0004] It has been shown that aggressive management of blood glucose levels can dramatically reduce diabetes mortality. However, despite the clear benefits of glycemic control, nearly half of people do not meet their glycemic goals. Carbohydrate-rich foods with high glycemic index and glycemic load in particular contribute to increased postprandial glucose (PPG), and carbohydrate absorption from the small intestinal tract can, in turn, contribute to the development of chronic diseases such as obesity and diabetes (Vlachos et al. (2020) Nutrients 12(6): 1561).

[0005] Thus, there is an ongoing need for new, effective pharmaceutical compositions and treatments for controlling carbohydrate absorption and for treating hyperglycemia, diabetes, obesity and / or metabolic syndrome. Summary of the Invention

[0006] Summary of the Invention The present invention is based, in part, on the discovery of a pharmaceutical composition comprising a glucose oxidase enzyme and a peroxide-degrading enzyme (e.g., a catalase enzyme) that can be used to treat hyperglycemia and / or diabetes in a subject in need of such treatment.

[0007] Thus, in one aspect, the present invention provides a pharmaceutical composition comprising (or consisting essentially of) a glucose oxidase enzyme, a peroxidolytic enzyme and a pharma- ceutically acceptable excipient.

[0008] In some embodiments, the glucose oxidase enzyme is spray dried or freeze dried. In some embodiments, the glucose oxidase enzyme is a microbial glucose oxidase enzyme or a functional fragment or variant thereof. In some embodiments, the glucose oxidase enzyme is from Aspergillus niger, e.g., 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.

[0009] In some embodiments, the peroxide decomposing enzyme is spray dried or freeze dried. In some embodiments, the peroxide decomposing enzyme is a catalase enzyme or a peroxidase enzyme, for example, the peroxide decomposing enzyme is a catalase enzyme, which may be a bacterial catalase enzyme or a functional fragment or variant thereof. In some embodiments, the catalase enzyme is from Aspergillus niger, for example, the catalase enzyme comprises SEQ ID NO:3, SEQ ID NO:4, or a functional fragment or variant thereof. In some embodiments, the pharmaceutical comprises about 1,000 to about 250,000 International Units (IU) of catalase enzyme. Depending on the circumstances, the ratio of International Units of glucose oxidase to International Units of peroxide decomposing enzyme in the composition is in the range of 0.1 to 10.

[0010] In some embodiments, the composition is formulated as an oral dosage form. In some embodiments, the composition is formulated as a solid oral dosage form, such as a powder, sachet, granule, pellet, micropellet, tablet or mini tablet. In some embodiments, the composition is formulated as a liquid oral dosage form, such as an elixir, syrup or drop. In some embodiments, the composition is formulated as nanoparticles or nanopreparations.

[0011] In certain embodiments, the composition has 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.

[0012] In some embodiments, the composition does not include a glucoamylase enzyme, a sucrase enzyme, a lactase enzyme, a transglucosidase enzyme, an α-amylase enzyme, or a combination thereof.

[0013] In another aspect, the present invention provides a method of treating diabetes (e.g., type 2 diabetes) in a subject in need of such treatment, comprising administering (e.g., orally administering) to the subject an effective amount of any of the enzymes or compositions described herein to treat diabetes in the subject.

[0014] In another aspect, the present invention provides a method of treating obesity in a subject in need thereof, comprising administering (e.g., orally) to the subject an effective amount of any of the enzymes or compositions described herein to treat obesity in the subject.

[0015] In another aspect, the present invention provides a method of treating metabolic syndrome in a subject in need thereof, comprising administering (e.g., orally) to the subject an effective amount of any of the enzymes or compositions described herein to treat metabolic syndrome in the subject.

[0016] In another aspect, the present invention provides a method of treating hyperglycemia in a subject in need thereof, comprising administering (e.g., orally) to the subject an effective amount of any of the enzymes or compositions described herein to treat hyperglycemia in the subject.

[0017] In another aspect, the present invention provides a method of treating hyperinsulinemia in a subject in need thereof, comprising administering (e.g., orally administering) to the subject an effective amount of any of the enzymes or compositions described herein to treat hyperinsulinemia in the subject.

[0018] In another aspect, the present invention provides a method of treating an eating disorder (e.g., binge eating or carbohydrate hyperphagia) in a subject in need of such treatment, comprising administering (e.g., orally administering) to the subject an effective amount of any of the enzymes or compositions described herein to treat the eating disorder in the subject.

[0019] In another aspect, the present invention provides a method for reducing the level of glucose in a subject that requires such reduction.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 the level of glucose in the subject.The method can reduce the level of glucose in the blood and / or gastrointestinal tract of the subject, or can reduce the level of glucose in a sample (e.g., a body fluid sample, such as blood, serum or plasma sample) from the subject.

[0020] In some embodiments, the method reduces the level of glucose in the blood of the 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 glucose Cmax in the blood of the 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 glucose Tmax in the blood of the subject after a meal or snack by at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80% or 90%. In some embodiments, when administered with food, the method reduces the glycemic index of the food by at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90%. In some 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%.

[0021] In another aspect, the present invention provides a method for reducing C-peptide and / or insulin levels in a subject in need thereof. The method includes administering (e.g., orally) to the subject an effective amount of any of the enzymes or compositions described herein to reduce C-peptide and / or insulin levels in the subject. The method may reduce C-peptide and / or insulin levels in the subject's blood and / or gastrointestinal tract or may reduce C-peptide and / or insulin levels in a sample (e.g., a body fluid sample, such as a blood, serum, or plasma sample) from the subject. In some embodiments, the method reduces C-peptide and / or insulin levels in the subject's blood by at least about 10%, 20%, 30%, 40%, 50%, or 60% as determined by area under the curve (AUC) analysis 0-240 minutes or 0-360 minutes after a meal or snack.

[0022] In another aspect, the present invention provides a method for reducing the level of HA1c in a subject that requires reducing the level of HA1c.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 the level of HA1c in the subject.The method can reduce the level of HA1c in the blood of the subject or reduce the level of HA1c in the sample (e.g., body fluid sample, such as blood, serum or plasma sample) from the subject.

[0023] In another aspect, the present invention provides a method for reducing the level of glycemic load of a consumed carbohydrate-rich meal, comprising administering (e.g., orally) to a subject an effective amount of any of the enzymes or compositions described herein to reduce the level of glucose in the GI tract available for absorption.

[0024] In certain embodiments of any of the foregoing methods, the subject has diabetes, obesity, metabolic syndrome, hyperglycemia, hyperinsulinemia, and / or an eating disorder (eg, bulimia or carbohydrate hypereating disorder).

[0025] Depending on the circumstances, the enzyme or composition may be administered to the subject 1, 2, 3, 4 or more than 4 times per day. Additionally, the enzyme or composition may be administered to the subject with a meal or snack (e.g., with each meal or snack).

[0026] These and other aspects and features of the present invention are described in the following detailed description and claims. [Brief description of the drawings]

[0027] Description of the drawings The invention may be more fully understood with reference to the following drawings. [Figure 1A]1A-1B show glucose absorption curves during Meal Glucose Tolerance Test (MGTT) IV (with enzyme treatment with 450 mg glucose oxidase and 0.8 ml catalase; black line (box)) compared to MGTT III (without enzyme treatment; black line (circle)). Figure 1A shows glucose absorption after a chow-only meal. Figure 1B shows glucose absorption after a chow meal and glucose powder. All data points correspond to the mean ± SD and AUC is expressed as the mean ± SEM. [Figure 1B] 1A-1B show glucose absorption curves during Meal Glucose Tolerance Test (MGTT) IV (with enzyme treatment with 450 mg glucose oxidase and 0.8 ml catalase; black line (box)) compared to MGTT III (without enzyme treatment; black line (circle)). Figure 1A shows glucose absorption after a chow-only meal. Figure 1B shows glucose absorption after a chow meal and glucose powder. All data points correspond to the mean ± SD and AUC is expressed as the mean ± SEM. [Diagram 2] Figure 2 shows the glucose absorption curves during a meal glucose tolerance test (MGTT) V, including a group receiving enzyme treatment with 900 mg glucose oxidase and 1.6 ml catalase ("treated") and a group without enzyme treatment ("control"). All data points correspond to the mean ± SEM, and AUC is expressed as the mean ± SEM. [Diagram 3] Figure 3 shows glucose absorption curves during meal glucose tolerance test (MGTT) VI, including a group receiving a chow-only diet ("Control diet"), a group receiving a chow diet and glucose powder ("Control diet + glucose"), and a group receiving a chow diet and glucose powder and enzyme treatment with 450 mg glucose oxidase and 0.8 ml catalase ("Treatment diet + glucose"). All data points correspond to the mean ± SEM, and AUC is expressed as the mean ± SEM. [Figure 4A]Figures 4A-4B show C-peptide release curves during meal glucose tolerance test (MGTT) IV (including enzyme treatment with 450 mg glucose oxidase and 0.8 ml catalase, black line (box)) compared to MGTT III (without enzyme treatment, black line (circle)). Figure 4A shows C-peptide release after a chow-only meal. Figure 4B shows C-peptide release after a chow meal and glucose powder. All data points correspond to the mean ± SDM. AUC is expressed as the mean ± SEM. [Figure 4B] Figures 4A-4B show C-peptide release curves during meal glucose tolerance test (MGTT) IV (including enzyme treatment with 450 mg glucose oxidase and 0.8 ml catalase, black line (box)) compared to MGTT III (without enzyme treatment, black line (circle)). Figure 4A shows C-peptide release after a chow-only meal. Figure 4B shows C-peptide release after a chow meal and glucose powder. All data points correspond to the mean ± SDM. AUC is expressed as the mean ± SEM. [Diagram 5] FIG. 5A shows the C-peptide release curves during meal glucose tolerance test (MGTT) V, including a group receiving enzyme treatment with 900 mg glucose oxidase and 1.6 ml catalase ("MGTT V treatment" black line (box)) and a group without enzyme treatment ("MGTT V control" black line (circle)). FIG. 5B shows a comparison of C-peptide release curves between MGTT V, MGTT IV (including enzyme treatment with 450 mg glucose oxidase and 0.8 ml catalase, group 1), and MGTT III (without enzyme treatment, group 1). All data points correspond to the mean ± SDM. [Figure 6]Figure 6 shows C-peptide release curves during meal glucose tolerance test (MGTT) VI, including a group receiving a chow only diet ("Control diet"), a group receiving a chow diet and glucose powder ("Control diet + glucose"), and a group receiving a chow diet and glucose powder and enzyme treatment with 450 mg glucose oxidase and 0.8 ml catalase ("Treatment diet + glucose"). All data points represent the mean ± SDM. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0028] Detailed Description The present invention is based, in part, on the discovery of pharmaceutical compositions comprising a glucose oxidase enzyme and a peroxide-degrading enzyme (e.g., a catalase enzyme) that can be used to treat diabetes (e.g., type 2 diabetes), hyperglycemia, hyperinsulinemia, obesity and metabolic syndrome in a subject in need of such treatment.

[0029] Various features and aspects of the invention are discussed in detail below.

[0030] I. Enzymes In particular, the present invention provides pharmaceutical compositions comprising the glucose oxidase enzyme and peroxidase enzymes useful for treating disorders such as diabetes (eg, type 2 diabetes), hyperglycemia, hyperinsulinemia, obesity and metabolic syndrome.

[0031] 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.

[0032] 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).

[0033] 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.

[0034] 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.

[0035] 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.

[0036] 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).

[0037] Exemplary catalase enzymes include those derived 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.

[0038] 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.

[0039] 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 comprising peroxidase. As used herein, the term "functional fragment" of peroxidase refers to a fragment of a 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).

[0040] 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.

[0041] In some embodiments, the 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 oxidase and / or peroxide degrading enzyme disclosed herein.

[0042] In some embodiments, the glucose oxidase and / or peroxide decomposition enzyme comprises one or more conservative substitutions relative to the glucose oxidase and / or peroxide decomposition enzyme disclosed herein. In other embodiments, the glucose oxidase and / or peroxide decomposition enzyme comprises one or more non-conservative substitutions relative to the 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 matrix (e.g., BLOSUM 62 matrix) or PAM substitution:p matrix (e.g., PAM 250 matrix). Non-conservative substitutions are amino acid substitutions that are not conservative substitutions.

[0043] In certain embodiments, the disclosed glucose oxidase and / or peroxide-degrading enzyme (or pharmaceutical composition comprising same, e.g., a solid pharmaceutical composition) 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 certain embodiments, the disclosed glucose oxidase and / or peroxide-decomposing enzyme (or a pharmaceutical composition comprising the same, e.g., a solid pharmaceutical composition) may have a shelf life of 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, about 6 to about 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, 12 to 96 months 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 18 to about 2 4 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.

[0044] The stability, shelf-life or half-life of glucose oxidase and / or peroxide decomposing enzymes may 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 oxidase and / or peroxide decomposing enzymes depends on the experimental conditions under which it is measured.

[0045] In some embodiments, the glucose oxidase and / or peroxide decomposing enzyme has at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% sequence identity to the glucose oxidase and / or peroxide decomposing enzyme disclosed herein. For example, 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 peroxide decomposing 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.Search parameters for histogram, description, alignment, expect (i.e., 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). 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 within which gapped alignments are generated). 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.

[0046] It is contemplated that the disclosed glucose oxidase and / or peroxide decomposition enzyme can be modified, genetically engineered or chemically conjugated.For example, it is contemplated that the disclosed glucose oxidase and / or peroxide decomposition enzyme can be conjugated to an effector agent using standard in vitro conjugation chemistry.When the effector agent is a polypeptide, the glucose oxidase and / or peroxide decomposition enzyme 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.

[0047] In some embodiments, depending on the particular mode of administration or site of activity, the disclosed glucose oxidase and / or peroxide decomposition enzymes can be modified with moieties that improve their stability and / or retention in the circulation, e.g., in blood, serum, or other tissues. For example, the disclosed glucose oxidase and / or peroxide decomposition enzymes can be conjugated to a polymer, e.g., a substantially non-antigenic polymer, e.g., a polyalkylene oxide or polyethylene oxide. In some embodiments, the disclosed glucose oxidase and / or peroxide decomposition enzymes are conjugated to a water-soluble polymer, e.g., a hydrophilic polyvinyl polymer, e.g., polyvinyl alcohol or polyvinylpyrrolidone. Examples of such polymers include polyalkylene oxide homopolymers, e.g., polyethylene glycol (PEG) or polypropylene glycol, polyoxyethylated polyols, copolymers thereof, and block copolymers thereof. Further useful polymers include polyoxyalkylenes, e.g., polyoxyethylene, polyoxypropylene, and block copolymers of polyoxyethylene and polyoxypropylene, polymethacrylates, carbomers, and branched or unbranched polysaccharides.

[0048] II. Enzyme Production Methods for producing the glucose oxidase and / or peroxide decomposition enzyme of the present invention are known in the art.For example, the DNA molecule encoding the 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 oxidase and / or peroxide decomposition enzyme.

[0049] The nucleic acid encoding the desired glucose oxidase and / or peroxide decomposition enzyme 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 gene encoding the glucose oxidase and / or peroxide decomposition enzyme.

[0050] In certain embodiments, the nucleic acids encoding the recombinant glucose oxidase and / or peroxide decomposition enzymes of the present invention can be codon optimized for expression in heterologous cells, such as yeast cells (e.g., Pichia cells) or E. coli cells, using methods known in the art.

[0051] Specific expression and purification conditions vary according to the expression system used.For example, when gene is expressed in E. coli, gene is 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.

[0052] Glucose oxidase and / or peroxide decomposition enzymes can be produced by growing (culturing) host cells transfected with an expression vector encoding such glucose oxidase and / or peroxide decomposition enzymes under conditions that allow expression of the glucose oxidase and / or peroxide decomposition enzymes. After expression, the glucose oxidase and / or peroxide decomposition enzymes 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.

[0053] In some embodiments, the glucose oxidase and / or peroxidolytic enzyme is dried, for example, spray dried. Pharmaceutical proteins can be dried in many ways, for example, by removing water, organic solvents or liquid polymers 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.

[0054] By spray drying glucose oxidase and / or peroxide decomposition enzyme, water is separated from the 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 spray of droplets of a liquid feedstock containing glucose oxidase and / or peroxide decomposition enzyme, and contacting the droplets with hot air or gas in a drying chamber. The atomization process can be carried out using a two-fluid atomizer that mixes the liquid feedstock with dry air such as compressed air or nitrogen. The operating conditions and dryer design are selected according to the drying characteristics of glucose oxidase and / or peroxide decomposition enzyme and the desired powder quality. Exemplary methods for spray drying enzymes are described in US Patent Application Publication No. 2015 / 0353913. Glucose oxidase and peroxide decomposition enzyme can be spray dried separately or together, and can be spray dried together.

[0055] III. Pharmaceutical Compositions For therapeutic use, the glucose oxidase and / or peroxide decomposition enzyme described herein is 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.

[0056] 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.

[0057] The pharmaceutical composition comprising the glucose oxidase and / or peroxide decomposition enzyme disclosed herein, naturally occurring or recombinant, 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.

[0058] 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.

[0059] 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 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.

[0060] 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.

[0061] In some embodiments, the disclosed compositions include a polyionic agent that can coat, for example, glucose oxidase and / or peroxide decomposition enzyme (e.g., the composition includes a polyionic coating). 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).

[0062] 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 30,00 0 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 100,0 00, 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 75,0 and / or (ii) about 750 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 oxidase enzyme; 50 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 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 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, or about 200,000 to about 250,000 international units (IU) of peroxide decomposition enzyme.

[0063] Depending on the situation, the ratio of the international units of glucose oxidase to the international units of the peroxidase decomposition enzyme is within 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.

[0064] Depending on the circumstances, the amount of glucose oxidase and / or peroxidase decomposition example administered to a subject in 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 of body weight.

[0065] 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 oxidase and / or peroxidase degrading enzyme may be administered in a single dose.

[0066] In some embodiments, the disclosed glucose oxidase and / or peroxide-degrading enzyme or composition is administered to a subject with a meal or snack. In some embodiments, the disclosed glucose oxidase and / or peroxide-degrading enzyme or composition is administered to a subject with each meal or snack that the subject eats. In some embodiments, the disclosed glucose oxidase and / or peroxide-degrading 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 every day, twice every day, three times every day, four times every day, five times every day, six times every day, or more than six times every day.

[0067] According to the situation, the composition can be formulated as powder, granule, pellet, micropellet, mini tablet, nanoparticle or nano preparation.The composition can be encapsulated in capsule, for example, 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, for example, elixir, syrup or drop.

[0068] In certain embodiments, the disclosed compositions do not include (and / or are not administered in combination with) a glucoamylase enzyme. In certain embodiments, the disclosed compositions do not include (and / or are not administered in combination with) a sucrase enzyme. In certain embodiments, the disclosed compositions do not include (and / or are not administered in combination with) a lactase enzyme. In certain embodiments, the disclosed compositions do not include (and / or are not administered in combination with) a transglucosidase enzyme. In certain embodiments, the disclosed compositions do not include (and / or are not administered in combination with) an alpha-amylase enzyme. In certain embodiments, the disclosed compositions do not include (and / or are not administered in combination with) any of a glucoamylase enzyme, a sucrase enzyme, a lactase enzyme, a transglucosidase enzyme, and / or an alpha-amylase enzyme.

[0069] 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 diabetes, obesity, metabolic syndrome, hyperglycemia, hyperinsulinemia and / or eating disorders (e.g., binge eating or carbohydrate hypereating) in a subject.The method comprises administering to the subject an effective amount of (i) glucose oxidase enzyme and (ii) peroxide decomposition enzyme.For example, the method can comprise administering the disclosed pharmaceutical composition comprising (i) spray-dried or freeze-dried glucose oxidase enzyme and (ii) spray-dried or freeze-dried peroxide decomposition enzyme.

[0070] The term "effective amount" as used herein refers to an amount of an active agent (e.g., the disclosed 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 dosages, and is not intended to be limited to a particular formulation or route of administration.

[0071] 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.

[0072] The present invention also provides a method for reducing the level of glucose in a subject, such as a subject with obesity, diabetes, hyperglycemia and / or an eating disorder (e.g., bulimia or hypercarbia). The method comprises administering to the subject an effective amount of (i) glucose oxidase enzyme and (ii) peroxide decomposition enzyme. For example, the method can comprise administering a disclosed pharmaceutical composition comprising (i) spray-dried or freeze-dried glucose oxidase and (ii) spray-dried or freeze-dried peroxide decomposition enzyme. The level of glucose in a subject can refer to the level of glucose in a body fluid (e.g., blood, plasma, serum or urine), tissue, organ, cell and / or gastrointestinal tract in the subject or any of the aforementioned samples.

[0073] Glucose levels, eg, blood glucose levels, can be measured by any method known in the art, eg, by glucometer and / or test strips as described in Example 1 herein.

[0074] In some embodiments, the method reduces the glucose level measured in a subject (e.g., in the blood of the subject) 5, 15, 30, 60, 90, 120, 180, 240, 300 or 360 minutes after the subject eats a meal or snack.In some embodiments, the method reduces the glucose level measured and / or plotted over time in a subject (e.g., in the blood of the subject), e.g., the method reduces the glucose level measured over time according to area under the curve (AUC) or Cmax analysis as described in Example 1 herein. For example, in certain embodiments, the method reduces glucose levels in a subject about 0 to about 5 minutes, about 0 to about 15 minutes, about 0 to about 30 minutes, about 0 to about 60 minutes, about 0 to about 90 minutes, about 0 to about 120 minutes, about 0 to about 180 minutes, about 0 to about 240 minutes, about 0 to about 300 minutes, about 0 to about 360 minutes, about 5 to about 15 minutes, about 5 to about 30 minutes, about 5 to about 60 minutes, about 5 to about 90 minutes, about 5 to about 120 minutes, about 5 to about 180 minutes, about 5 to about 240 minutes, about 5 to about 300 minutes, or about 5 to about 360 minutes after the subject has eaten a meal or snack.

[0075] In certain embodiments, the method reduces the level of glucose in a subject (e.g., in the subject's blood, e.g., after a meal or snack) by at least about 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80% or 90% relative to the same subject if the subject was not administered glucose oxidase and peroxide degrading enzyme, or relative to a similar subject who was not administered glucose oxidase and peroxide degrading enzyme. In certain embodiments, the method reduces the level of glucose in a subject (e.g., in the subject's blood, e.g., after a meal or snack) by between 10% and about 90%, between about 10% and about 80%, between about 10% and about 60%, between about 10% and about 40%, between about 10% and about 20%, between about 20% and about 80%, between about 20% and about 60%, between about 20% and about 40%, between about 40% and about 80%, between about 40% and about 60%, or between about 60% and about 80% relative to the same subject if the subject were not administered glucose oxidase and peroxide degrading enzyme, or relative to a similar subject who was not administered glucose oxidase and peroxide degrading enzyme.

[0076] In certain embodiments, the method reduces glucose Cmax in the subject's blood after a meal or snack by at least about 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80% or 90% relative to the same subject when the subject is not administered glucose oxidase and peroxide degrading enzyme, or relative to a similar subject when glucose oxidase and peroxide degrading enzyme are not administered. In certain embodiments, the method reduces glucose Cmax in the subject's blood after a meal or snack by about 10% to about 80%, about 10% to about 60%, about 10% to about 40%, about 10% to about 20%, about 20% to about 80%, about 20% to about 60%, about 20% to about 40%, about 40% to about 80%, about 40% to about 60%, or about 60% to about 80% relative to the same subject if the subject were not administered glucose oxidase and peroxide degrading enzyme or relative to a similar subject if the glucose oxidase and peroxide degrading enzyme were not administered.

[0077] In certain embodiments, the method reduces glucose Tmax in the subject's blood after a meal or snack by at least about 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80% or 90% relative to the same subject when the subject is not administered glucose oxidase and peroxide degrading enzyme or relative to a similar subject when glucose oxidase and peroxide degrading enzyme are not administered. In certain embodiments, the method reduces glucose Tmax in the subject's blood after a meal or snack by about 10% to about 80%, about 10% to about 60%, about 10% to about 40%, about 10% to about 20%, about 20% to about 80%, about 20% to about 60%, about 20% to about 40%, about 40% to about 80%, about 40% to about 60% or about 60% to about 80% relative to the same subject if the subject were not administered glucose oxidase and peroxide decomposing enzyme or relative to a similar subject if the glucose oxidase and peroxide decomposing enzyme were not administered.

[0078] In certain embodiments, when administered with food, the method reduces the glycemic index of the food by at least about 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80% or 90% relative to the same subject when the subject was not administered glucose oxidase and peroxide degrading enzyme or relative to a similar subject when glucose oxidase and peroxide degrading enzyme were not administered. In certain embodiments, when administered with food, the method reduces the glycemic index of the food by at least about 10% to about 80%, about 10% to about 60%, about 10% to about 40%, about 10% to about 20%, about 20% to about 80%, about 20% to about 60%, about 20% to about 40%, about 40% to about 80%, about 40% to about 60%, or about 60% to about 80% relative to the same subject if the subject was not administered glucose oxidase and peroxide degrading enzyme or relative to a similar subject if glucose oxidase and peroxide degrading enzyme were not administered.

[0079] In certain embodiments, when administered with food, the method reduces food caloric intake by at least about 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80% or 90% relative to the same subject when the subject was not administered glucose oxidase and peroxide decomposing enzyme or relative to a similar subject when the glucose oxidase and peroxide decomposing enzyme were not administered. In certain embodiments, when administered with food, the method reduces food caloric intake by at least about 10% to about 80%, about 10% to about 60%, about 10% to about 40%, about 10% to about 20%, about 20% to about 80%, about 20% to about 60%, about 20% to about 40%, about 40% to about 80%, about 40% to about 60%, or about 60% to about 80% relative to the same subject if the subject was not administered glucose oxidase and peroxide decomposing enzymes or relative to a similar subject if the glucose oxidase and peroxide decomposing enzymes were not administered.

[0080] The present invention also provides a method for reducing C-peptide and / or insulin levels in a subject, such as a subject with obesity, diabetes, hyperglycemia and / or eating disorders (e.g., bulimia or carbohydrate hypereating disorder). The method comprises administering to the subject an effective amount of (i) glucose oxidase enzyme and (ii) peroxide decomposition enzyme. For example, the method may comprise administering a disclosed pharmaceutical composition comprising (i) spray-dried or freeze-dried glucose oxidase and (ii) spray-dried or freeze-dried peroxide decomposition enzyme. The C-peptide and / or insulin levels in a subject may refer to the levels of C-peptide and / or insulin in a body fluid (e.g., blood, plasma, serum or urine), tissue, organ, cell and / or gastrointestinal tract or any of the aforementioned samples in the subject. C-peptide and / or insulin levels, such as blood C-peptide levels, may be measured by any method known in the art, such as, for example, ELISA as described in Example 1 herein.

[0081] In some embodiments, the method reduces the level of C-peptide and / or insulin in a subject (e.g., in the blood of a subject) measured 5, 15, 30, 60, 90, 120, 180, 240, 300, or 360 minutes after the subject eats a meal or snack. In some embodiments, the method reduces the level of C-peptide and / or insulin in a subject (e.g., in the blood of a subject) measured and / or plotted over time, e.g., the method reduces the level of glucose over time as determined by area under the curve (AUC) or Cmax analysis, e.g., as described in Example 1 herein. For example, in certain embodiments, the methods reduce levels of C-peptide and / or insulin in a subject measured about 0 to about 5 minutes, about 0 to about 15 minutes, about 0 to about 30 minutes, about 0 to about 60 minutes, about 0 to about 90 minutes, about 0 to about 120 minutes, about 0 to about 180 minutes, about 0 to about 240 minutes, about 0 to about 300 minutes, about 0 to about 360 minutes, about 5 to about 15 minutes, about 5 to about 30 minutes, about 5 to about 60 minutes, about 5 to about 90 minutes, about 5 to about 120 minutes, about 5 to about 180 minutes, about 5 to about 240 minutes, about 5 to about 300 minutes, or about 5 to about 360 minutes after the subject has eaten a meal or snack.

[0082] In certain embodiments, the method reduces the levels of C-peptide and / or insulin in a subject (e.g., in the subject's blood, e.g., after a meal or snack) by at least about 5%, 10%, 20%, 30%, 40%, 50% or 60% relative to the same subject if the subject was not administered glucose oxidase and peroxide degrading enzyme, or relative to a similar subject if glucose oxidase and peroxide degrading enzyme were not administered. In certain embodiments, the method reduces levels of C-peptide and / or insulin in a subject (e.g., in the subject's blood, e.g., after a meal or snack) by about 5% to about 60%, about 5% to about 40%, about 5% to about 20%, about 5% to about 10%, about 10% to about 60%, about 10% to about 40%, about 10% to about 20%, about 20% to about 60%, about 20% to about 40%, or about 40% to about 60% relative to the same subject if the subject were not administered glucose oxidase and peroxide degrading enzymes or relative to a similar subject if the glucose oxidase and peroxide degrading enzymes were not administered.

[0083] The present invention also provides a method for reducing the level of HA1c in a subject, for example, a subject with obesity, diabetes, hyperglycemia and / or eating disorder (e.g., bulimia or carbohydrate hypereating disorder). The method comprises administering to the subject an effective amount of (i) glucose oxidase enzyme and (ii) peroxide decomposition enzyme. For example, the method can comprise administering the disclosed pharmaceutical composition comprising (i) spray-dried or freeze-dried glucose oxidase and (ii) spray-dried or freeze-dried peroxide decomposition enzyme. The level of HA1c in a subject can refer to the level of HA1c in a body fluid (e.g., blood, plasma, serum or urine), tissue, organ, cell and / or gastrointestinal tract in the subject or any of the above samples. The level of HA1c can be measured by any method known in the art.

[0084] 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.

[0085] 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.

[0086] 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.

[0087] 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.

[0088] 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.

[0089] 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.

[0090] 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.

[0091] 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.

[0092] 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

[0093] Working Example The following examples are illustrative only and are not intended to limit the scope or content of the invention in any way.

[0094] Example 1 This example describes a study examining the effect of a combination of glucose oxidase and catalase, homogenously mixed with dry feed, on intestinal glucose absorption and C-peptide release in young healthy pigs.

[0095] Study Design / Method The pig model was used due to the similarities between humans and pigs in the morphology and physiology of the gastrointestinal system, enzymatic and hormonal factors, transit time and efficiency of digestion. Both healthy and diabetic pigs were used to test glucose tolerance and insulin sensitivity (see, for example, Koopmans et al. (2011) BMC BIOCHEM12:25 and Pierzynowska et al. (2020) J. DIABETES RES. 2020: 2148740).

[0096] The study included eight pigs (four females and four castrated males). All pigs were 10-12 weeks old upon arrival at the experimental facility and weighed 9.06 ± 0.77 kg (mean ± SEM) at the start of the study.

[0097] During the 1 week adaptation period, the pigs were fed a standard cereal-based diet (Kcynia, SD, Morawski Plant, Poland). To ensure higher and constant stomach pH levels, the standard diet (0.5% of body weight) was fed to the pigs at 3-h intervals (8 times in 24 h; 0900, 1200, 1500, 1800, 2100, 2400, 0300 and 0600 h) starting in the morning at 0900 h. The composition of the standard diet is provided in Table 1. The standard diet contains approximately 50% starch, 17% protein, 3% fat, 28% dietary fiber and 2% H2O. If the standard diet contains approximately 50% starch, 4 grams of diet contains approximately 2 grams of starch, which corresponds to 2 grams of glucose. [Table 1]

[0098] One week after arrival, at the end of the adaptation period, pigs had jugular catheters implanted to allow frequent blood sampling throughout the study.

[0099] After an initial adaptation period and jugular catheterization, pigs were subjected to a dietary glucose tolerance test (MGTT; see Table 3), in which both the chow or glucose-rich chow was supplemented with or without glucose oxidase and catalase enzymes. The effect of enzyme supplementation on glucose utilization and C-peptide release was monitored.

[0100] Details of the glucose oxidase and catalase enzymes used in the study are provided in Table 2. [Table 2]

[0101] Blood samples were collected via a jugular catheter and transferred to BD Vacutainer® glass K3EDTA tubes (BD Diagnostics, New Jersey, USA). Blood samples were immediately placed on ice, after which they were centrifuged at 3000 x g for 15 min at 4°C, and plasma was separated and stored at -80°C until further analysis.

[0102] Blood glucose concentrations were measured immediately after blood sampling using a glucometer and test strips (Accu-Chek1 Aviva, Roche Diagnostics, Germany). Plasma C-peptide concentrations were measured using a porcine C-peptide ELISA kit (cat#10-1256-01 Mercodia, Uppsala, Sweden) according to the manufacturer's instructions.

[0103] A summary of the study design and individual meal glucose tolerance test (MGTT) conditions is provided in Table 3. All MGTTs were performed in the morning, starting at 0900 hours. On each study day, blood was sampled before feeding (baseline) and at 5, 15, 30, 60, 90, 120, 180 and 240 minutes after feeding. Optionally, blood was additionally sampled at 300 and 360 minutes after feeding. Pigs were deprived of food at 0600 and 1200 hours during the study days.

[0104] Initially, a set of MGTTs (MGTT I, II and III) was performed to find the optimal feed amount and feed / glucose ratio. MGTT I and II revealed that feeding pigs with 2 grams of glucose per kilogram of body weight did not produce a sufficient peak. In MGTT III, pigs were fed (i) 16 grams of standard feed per kilogram of body weight or (ii) 16 grams of standard feed per kilogram of body weight and 8 grams of glucose (mixed with feed) per kilogram of body weight. In MGTT III, glucose absorption peaks were observed for both the feed and the feed / glucose mixture. Consequently, this amount was used in all further MGTTs.

[0105] Further MGTTs (MGTT IV, V and VI) tested the effect of glucose oxidase and catalase supplementation. Glucose oxidase powder was added and homogenized with the feed or feed / glucose mixture. The catalase suspension in a drop from a pipette was added directly to the feed / glucose oxidase or feed / glucose oxidase / glucose mixture. This process ensured homogenization of the individual components.

[0106] At the end of the experiment, the pigs were euthanized with sodium pentobarbiturate (Morbital, Biowet, Pulawy, Poland; 20 mg / kg) and subjected to gross necropsy examination. The stomach and the proximal and distal jejunum were examined for pathological changes. [Table 3]

[0107] All data, except AUC, were expressed as mean ± standard deviation (SD). Distribution of parameters was checked using Shapiro-Wilk normality test. Total area under the curve (AUC) was calculated for postprandial blood glucose and C-peptide levels. AUC was baseline adjusted and compared using one-way ANOVA. Data for AUC were expressed as mean ± standard error of the mean (SEM). Blood glucose and C-peptide levels at different time points were compared using two-way ANOVA. Data were not corrected for multiple comparisons. In all statistical analyses, p ≤ 0.05 was considered significant. All analyses were performed using Prism, version 9.1.0 (GraphPad Software, Inc, San Diego, CA, USA).

[0108] Results – Glucose Absorption The study evaluated whether glucose oxidase and catalase could reduce the absorption of glucose into the blood during a meal glucose tolerance test (MGTT).

[0109] During the MGTT IV, all pigs first received the glucose oxidase and catalase combination. The results are shown in Figure 1. Enzyme supplementation did not affect the palatability of the feed or the feeding behavior of the pigs. Glucose oxidase and catalase supplementation produced a significant reduction in blood glucose levels in both pigs eating feed only (180 and 240 min after feeding, Figure 1A) and pigs eating the feed / glucose mixture (15 and 30 min after feeding, Figure 1B). A significant (p=0.02) 53% reduction in glucose AUC was observed in pigs eating feed only (Figure 1A).

[0110] During MGTT V, four pigs received an increased dose of glucose oxidase and catalase combination compared to MGTT IV. The results are shown in Figure 2. The results were generally consistent with the lower doses. During MGTT VI, three pigs received a chow-only diet, two pigs received a chow-meal / glucose mixture, and two pigs received a chow-meal / glucose mixture and glucose oxidase and catalase combination (same doses as in MGTT IV). The results are shown in Figure 3. A significant 52.5% reduction in glucose AUC was observed in pigs receiving enzyme treatment (p<0.05). A difference in trend was observed for glucose Cmax (which was 157.5 mg / dl at 120 min for animals receiving chow / glucose mixture without enzyme and 110.5 mg / dl at 15 min for animals receiving chow / glucose mixture with enzyme, p=0.06).

[0111] Results – C-peptide release The study also evaluated whether glucose oxidase and catalase could reduce the release of C-peptide (a clinical marker of insulin) during a meal glucose tolerance test (MGTT).

[0112] During the MGTT IV, all pigs first received a combination of glucose oxidase and catalase. The results are shown in Figure 4. Glucose oxidase and catalase supplementation produced a trend toward a decrease in C-peptide AUC for both pigs that ate chow only (Figure 4A) and pigs that ate the chow / glucose mixture (Figure 4B).

[0113] The results for MGTT V are shown in Figure 5. During MGTT V, there was a downward trend in the C-peptide release curve or AUC between the control group not receiving enzyme treatment and the treatment group receiving enzyme treatment.

[0114] The results for MGTT VI are shown in Figure 6. During MGTT VI, there was a downward trend in the C-peptide release curve or AUC between the control group not receiving enzyme treatment and the treatment group receiving enzyme treatment.

[0115] overview Throughout the study, no mortality, adverse clinical signs and pathological macroscopic findings along the gastrointestinal tract were observed following administration of glucose oxidase and catalase enzymes.

[0116] Depending on the experimental condition, supplementation of the diet with glucose oxidase and catalase enzymes resulted in (i) a significant 52% decrease in blood glucose AUC (p<0.05); (ii) a decrease in blood glucose Cmax (30% decrease, p=0.06); and (iii) a decrease in C-peptide AUC (35% decrease for diet and 18% decrease for diet / glucose mixture, p=0.089 and 0.12, respectively).

[0117] Together, these results indicate that supplementing the diet with glucose oxidase and catalase enzymes can result in reduced glucose absorption, suggesting that administration of glucose oxidase and catalase enzymes may treat or prevent obesity or metabolic disorders, such as metabolic syndrome and type 2 diabetes mellitus.

[0118] Incorporation by Reference The entire disclosures of each of the patent and scientific literature referenced herein are incorporated by reference for all purposes.

[0119] Equivalent The present invention may be embodied in other specific forms without departing from its spirit or essential characteristics. Therefore, the foregoing embodiments are to be considered in all respects as illustrative and not limiting of the invention described herein. The scope of the invention is therefore indicated by the appended claims rather than the foregoing description, and all changes that come within the meaning and range of equivalence of the claims are intended to be embraced therein.

[0120] Sequence Listing [Table 4-1] [Table 4-2]

Claims

1. A pharmaceutical composition comprising a glucose oxidase enzyme, a peroxide-degrading enzyme and a pharmaceutically acceptable excipient.

2. (I) The glucose oxidase enzyme (a) spray-dried or freeze-dried; (b) a microbial glucose oxidase enzyme or a functional fragment or variant thereof; (c) derived from Aspergillus niger; and / or (d) comprising about 1,000 to about 250,000 international units (IU) in the composition; (II) a peroxide-decomposing enzyme, (a) spray-dried or freeze-dried; (b) is a catalase enzyme or a peroxidase enzyme; and / or (c) comprising about 1,000 to about 250,000 IU in the composition; and / or 2. The pharmaceutical composition according to claim 1, wherein the ratio of IU of glucose oxidase to IU of peroxidolytic enzyme (III) is in the range of 0.1 to 10.

3. The catalase enzyme, (I) a microbial catalase enzyme or a functional fragment or variant thereof; and / or The pharmaceutical composition according to claim 2, wherein (II) is derived from Aspergillus niger. (I) the glucose oxidase enzyme comprises SEQ ID NO:2 or a functional fragment or variant thereof; and / or 4. The pharmaceutical composition of claim 3, wherein (II) the catalase enzyme comprises SEQ ID NO: 4 or a functional fragment or variant thereof.

5. The composition, (a) formulated as an oral dosage form; (b) has 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; and / or 10. The pharmaceutical composition of claim 1, wherein the pharmaceutical composition does not contain (c) a glucoamylase enzyme, a sucrase enzyme, a lactase enzyme, a transglucosidase enzyme, an α-amylase enzyme, or a combination thereof.

6. 6. The pharmaceutical composition of claim 5, formulated as a powder, satchel, granules, pellets, micropellets, tablets, minitablets, elixir, syrup, drops or nanopreparation.

7. 10. The pharmaceutical composition of any one of claims 1 to 6 for use in treating diabetes in a subject in need thereof, wherein the pharmaceutical composition is orally administered to the subject.

8. 8. The pharmaceutical composition according to claim 7, wherein the diabetes is type 2 diabetes.

9. 7. The pharmaceutical composition according to any one of claims 1 to 6, for use in treating obesity, metabolic syndrome, hyperglycemia or hyperinsulinemia in a subject in need thereof, wherein the pharmaceutical composition is orally administered to the subject.

10. A pharmaceutical composition according to any one of claims 1 to 6 for use in a method for reducing glucose levels in a subject, wherein the pharmaceutical composition is orally administered to the subject.

11. The method comprises: (a) reducing the level of glucose in the subject's blood, in the subject's gastrointestinal tract, and / or in a sample derived from the subject; (b) reducing the level of glucose in the subject's blood by at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90% as determined by area under the curve (AUC) analysis 0-240 minutes or 0-360 minutes after a meal or snack; (c) reducing glucose Cmax in the subject's blood by at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90% after a meal or snack; (d) reducing glucose Tmax in the subject's blood by at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90% after a meal or snack; (e) when administered with food, reduces the glycemic load of food by at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90%; and / or (f) the pharmaceutical composition of claim 10, which, when administered with food, reduces food calorie intake by at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90%.

12. A pharmaceutical composition according to any one of claims 1 to 6 for use in a method for reducing C-peptide and / or insulin levels in a subject, wherein the pharmaceutical composition is orally administered to the subject.

13. The method comprises: (a) reducing the level of C-peptide and / or insulin in the blood of a subject and / or in a sample derived from the subject, and / or 13. The pharmaceutical composition of claim 12, wherein (b) the pharmaceutical composition reduces the level of C-peptide and / or insulin in the blood of the subject by at least about 10%, 20%, 30%, 40%, 50%, or 60% as determined by AUC analysis 0-240 minutes or 0-360 minutes after a meal or snack.

14. A pharmaceutical composition according to any one of claims 1 to 6 for use in a method for reducing hemoglobin A1c (HA1c) levels in a subject, wherein the pharmaceutical composition is orally administered to the subject.

15. The pharmaceutical composition of claim 14, wherein the method reduces the level of HA1c in the blood of the subject and / or in a sample derived from the subject.

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

17. A pharmaceutical composition according to any one of claims 1 to 6 for use in a method for reducing the glycemic load of carbohydrates consumed in a carbohydrate-rich diet, wherein the pharmaceutical composition is orally administered to a subject.

18. 20. The pharmaceutical composition of claim 17, wherein administration of the pharmaceutical composition reduces the level of glucose in the gastrointestinal tract of the subject that is available for absorption.

19. The pharmaceutical composition of claim 10, wherein the subject has diabetes, obesity, metabolic syndrome, hyperglycemia, hyperinsulinemia and / or an eating disorder.

20. 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 7 , which is administered to a subject.

21. 8. The pharmaceutical composition of claim 7, wherein the subject is a human adult or human pediatric subject.

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