Enolase 1 (ENO1) compositions and uses thereof

Abstract

The present invention provides a composition comprising Eno1 for delivery to muscle. In addition, the present invention provides a method for normalizing blood sugar in a subject with elevated blood sugar, comprising administering enolase 1 (Eno1) to the subject, thereby normalizing the subject's blood sugar. The present invention also provides a method for treating one or more conditions of a subject, the condition including impaired glucose tolerance, insulin resistance, prediabetes and diabetes, especially type 2 diabetes, the method comprising administering enolase 1 (Eno1) to the subject, thereby treating the subject's condition. In a specific method of the present invention, Eno1 is delivered to muscle.

Description

[0001] Related Applications

[0002] This application claims priority to U.S. Provisional Patent Application No. 61 / 926,913 filed on January 13, 2014, U.S. Provisional Patent Application No. 62 / 009,783 filed on June 9, 2014, and U.S. Provisional Patent Application No. 62 / 100,881 filed on January 7, 2015, the contents of each of which are incorporated herein by reference in their entirety.

[0003] Submission of sequence listing

[0004] The sequence listing associated with this application was submitted electronically via EFS-Web and is hereby incorporated by reference in its entirety into the specification. The text file containing the sequence listing is named 119992_10619_Sequence_Listing. The size of the text file is 33KB, and the text file was generated on January 13, 2015. Background Art

[0005] As blood sugar levels rise after meals, insulin secretion and stimulate cells of peripheral tissues (skeletal muscle and fat) to actively absorb glucose from the blood as an energy source. Loss of glucose homeostasis as a result of insulin secretion or dysfunction usually leads to metabolic disorders, such as diabetes, which may be jointly caused by obesity or further aggravated by obesity. Because these conditions may reduce the quality of life or even be fatal, it is necessary to restore strategies for properly removing glucose from the bloodstream.

[0006] Although diabetes may develop secondary to any condition that causes extensive damage to the pancreas (e.g., pancreatitis, tumors, administration of specific drugs such as corticosteroids or pentamidine, iron overload (i.e., hemochromatosis), acquired or inherited endocrinopathies, and surgical resection), the most common forms of diabetes typically result from a primary disorder of the insulin signaling system. There are two major types of diabetes, type 1 diabetes (also known as insulin-dependent diabetes mellitus (IDDM)) and type 2 diabetes (also known as insulin-independent or non-insulin-dependent diabetes mellitus (NIDDM)), which, despite their different pathogenesis, share common long-term complications.

[0007] Type 1 diabetes, which accounts for approximately 10% of all primary diabetes cases, is an organ-specific autoimmune disease characterized by extensive destruction of the insulin-producing β cells of the pancreas. The resulting reduction in insulin production inevitably leads to an imbalance in glucose metabolism. Although the administration of insulin provides considerable benefits to patients with this condition, the short serum half-life of insulin is a major obstacle to maintaining normal blood sugar. An alternative treatment is pancreatic islet transplantation, but this strategy has only achieved limited success.

[0008] Type 2 diabetes, which affects a large proportion of the population, is characterized by the disorder of insulin secretion and / or the reduction of the response of peripheral tissues to insulin, that is, insulin resistance. Although the pathogenesis of type 2 diabetes is still unclear, epidemiological studies have shown that this form of diabetes is produced by the collection or polymorphism of multiple genetic defects, which causes its own susceptibility risk and is changed by environmental factors, including overweight, diet, inactivity, medicine and excessive drinking. Although various therapeutic treatments can be used to control type 2 diabetes, they are relevant to various debilitating side effects. Therefore, it is often recommended to be diagnosed as suffering from type 2 diabetes or to adopt a healthier lifestyle in the patient who is in the risk of suffering from type 2 diabetes, including weight loss, diet change, exercise and appropriate drinking. Then, such lifestyle changes are not enough to reverse the blood vessels and organ damage caused by diabetes. Summary of the invention

[0009] In one aspect, the present invention provides a composition comprising enolase 1 (Eno1) or a fragment thereof for delivery to muscle. In one aspect, the present invention provides a method for normalizing blood sugar in a subject with elevated blood sugar, the method comprising administering a composition comprising Eno1 or a fragment thereof to the subject, thereby normalizing blood sugar in the subject. In one aspect, the present invention provides a method for treating one or more conditions of a subject, the conditions including impaired glucose tolerance, insulin resistance, prediabetes and diabetes, especially type 2 diabetes, the method comprising administering a composition comprising Eno1 or a fragment thereof to the subject, thereby treating the condition of the subject. In a specific method of the present invention, Eno1 is delivered to muscle.

[0010] The present invention provides a pharmaceutical composition comprising Eno1 or a fragment thereof for delivery to muscle cells.

[0011] In certain embodiments, Enol comprises an Enol polypeptide or a fragment thereof. In certain embodiments, Enol comprises an Enol nucleic acid or a fragment thereof. In certain embodiments, Enol comprises human Enol, eg, a human Eno 1 polypeptide or a human Eno 1 nucleic acid, or a fragment thereof.

[0012] In certain embodiments, the composition further comprises microparticles. In certain embodiments, the composition further comprises nanoparticles. In certain embodiments, Eno 1 or a fragment thereof is biologically active. In certain embodiments, Eno 1 or a fragment thereof has at least 90% of the activity of purified endogenous human Eno 1 polypeptide.

[0013] In certain embodiments, the composition further comprises an in situ formed composition. In certain embodiments, the composition further comprises a liposome. In certain embodiments, the composition comprises a dendrimer. In certain embodiments, the composition further comprises an expression vector, e.g., encoding Eno1 or a fragment thereof. In certain embodiments, the expression vector comprises a viral vector.

[0014] In certain embodiments, the composition comprises a complex comprising Eno1 or a fragment thereof (e.g., an Eno1 polypeptide, e.g., a human Eno1 polypeptide) and a muscle targeting moiety. In certain embodiments, the muscle targeting moiety comprises a skeletal and / or smooth muscle targeting peptide (MTP). In certain embodiments, the MTP comprises an amino acid sequence selected from ASSLNIA; WDANGKT; GETRAPL; CGHHPVYAC and HAIYPRH. In certain embodiments, the complex comprises a linker, e.g., linking Eno1 and SMTP. In certain embodiments, the linker is selected from a covalent linker, a non-covalent linker, and a reversible linker. In certain embodiments, the complex comprises a pharmaceutically acceptable dendrimer. In certain embodiments, the dendrimer is a PAMAN dendrimer. In certain embodiments, the dendrimer is a G5 dendrimer. In certain embodiments, the dendrimer is an uncharged dendrimer. In certain embodiments, the dendrimer is an acylated dendrimer. In certain embodiments, the dendrimer is a PEGylated dendrimer or an acetylated dendrimer. In certain embodiments, the complex comprises a liposome. In certain embodiments, the complex comprises a microparticle or a nanoparticle. In certain embodiments, the composition comprises an in situ formed composition.

[0015] In certain embodiments, Eno1 is released from the complex upon delivery to muscle cells.

[0016] In certain embodiments, Eno1 or a fragment thereof and the targeting moiety are present in the complex in a ratio of about 1:1 to about 1:30.

[0017] In certain embodiments, the composition is formulated for administration by injection or infusion. In certain embodiments, the composition is formulated for oral administration. In certain embodiments, the composition is formulated for parenteral administration. In certain embodiments, the composition is formulated for intramuscular administration, intravenous administration, or subcutaneous administration.

[0018] The present invention provides a method for lowering blood sugar in a subject with elevated blood sugar, the method comprising administering a pharmaceutical composition comprising Eno1 or a fragment thereof to the subject. In certain embodiments, the pharmaceutical composition comprises any pharmaceutical composition provided herein.

[0019] The present invention provides a method of improving glucose tolerance in a subject with impaired glucose tolerance, the method comprising administering to the subject a pharmaceutical composition comprising Eno 1. In certain embodiments, the pharmaceutical composition comprises any of the pharmaceutical compositions provided herein.

[0020] The present invention provides a method for improving the insulin response of a subject with reduced insulin sensitivity and / or insulin resistance, the method comprising administering a pharmaceutical composition comprising Eno1 or a fragment thereof to the subject. In certain embodiments, the pharmaceutical composition comprises any pharmaceutical composition provided herein.

[0021] The present invention provides a method for treating diabetes in a subject, the method comprising administering a pharmaceutical composition comprising Eno1 or a fragment thereof to the subject. In certain embodiments, the diabetes is type 2 diabetes. In certain embodiments, the diabetes is prediabetes. In certain embodiments, the diabetes is type 1 diabetes. In certain embodiments, the diabetes is gestational diabetes. In certain embodiments, the pharmaceutical composition comprises any pharmaceutical composition provided herein.

[0022] The present invention provides a method of reducing Hb1Ac levels in a subject having elevated Hb1Ac levels, the method comprising administering to the subject a pharmaceutical composition comprising Eno1 or a fragment thereof. In certain embodiments, the pharmaceutical composition comprises any pharmaceutical composition provided herein.

[0023] The present invention provides a method for improving blood sugar control in a subject with abnormal blood sugar control, the method comprising administering a pharmaceutical composition comprising Eno1 or a fragment thereof to the subject. In certain embodiments, the pharmaceutical composition comprises any pharmaceutical composition provided herein.

[0024] In certain embodiments, Eno 1 or a fragment thereof is administered by injection or infusion. In certain embodiments, Eno 1 or a fragment thereof is administered parenterally. In certain embodiments, Eno 1 or a fragment thereof is administered orally. In certain embodiments, Eno 1 or a fragment thereof is administered by a route selected from intramuscular, intravenous, and subcutaneous.

[0025] The present invention provides a method for diagnosing elevated blood glucose levels in a subject, comprising: (a) detecting the level of Eno1 in a biological sample of the subject, and (b) comparing the level of Eno1 in the biological sample with a predetermined threshold, wherein the level of Eno1 below the predetermined threshold indicates the presence of elevated blood glucose in the subject. In a specific embodiment, the method further comprises detecting the level of one or more diagnostic indicators of elevated blood glucose. In a specific embodiment, one or more other diagnostic indicators of elevated blood glucose are selected from HbA1c, fasting blood glucose, postprandial blood glucose, and glucose tolerance. In a specific embodiment, the biological sample is blood or serum. In a specific embodiment, the level of Eno1 is determined by immunoassay or ELISA. In a specific embodiment, step (a) comprises (i) contacting the biological sample with a reagent that selectively binds to Eno1 to form a biomarker complex, and (ii) detecting the biomarker complex. In a specific embodiment, the reagent is an anti-Eno1 antibody that selectively binds to at least one epitope of Eno1.

[0026] In certain embodiments, step (a) comprises determining the amount of Eno1 mRNA in the biological sample. In certain embodiments, an amplification reaction is used to determine the amount of Eno1 mRNA in the biological sample. In certain embodiments, the amplification reaction is (a) polymerase chain reaction (PCR); (b) nucleic acid sequence-based amplification assay (NASBA); (c) transcription-mediated amplification (TMA); (d) ligase chain reaction (LCR); or (e) strand displacement amplification (SDA). In certain embodiments, a hybridization assay is used to determine the amount of Eno1 mRNA in the biological sample. In certain embodiments, an oligonucleotide complementary to a portion of Eno1 mRNA is used in a hybridization assay to detect Eno1 mRNA.

[0027] In a particular embodiment of the invention, the diagnosis of elevated blood glucose is a diagnosis of a disease or condition selected from type 2 diabetes, prediabetes, gestational diabetes and type 1 diabetes.

[0028] The present invention provides a method for diagnosing the presence of elevated blood sugar in a subject, comprising:

[0029] (a) contacting a biological sample with a reagent that selectively binds to Eno1;

[0030] (b) allowing a complex to form between the reagent and Eno1;

[0031] (c) detecting the level of the complex, and

[0032] (d) comparing the level of the complex to a predetermined threshold, wherein a level of the complex above the predetermined threshold indicates that the patient is suffering from elevated blood glucose. In a specific embodiment, the reagent is an anti-Eno1 antibody. In a specific embodiment, the antibody comprises a detectable label. In a specific embodiment, the step of detecting the level of the complex further comprises contacting the complex with a detectable secondary antibody and measuring the level of the secondary antibody. In a specific embodiment, the method further comprises detecting the level of one or more other indicators of elevated blood glucose. In a specific embodiment, one or more other blood glucose indicators are selected from HbA1c levels, fasting blood glucose, postprandial blood glucose and glucose tolerance. In a specific embodiment, the biological sample is blood or serum.

[0033] In a specific embodiment of the invention, the level of the complex is determined by immunoassay or ELISA. In a specific embodiment, elevated blood glucose indicates prediabetes, type 2 diabetes, type 1 diabetes or gestational diabetes. In a specific embodiment, the method further comprises administering a treatment regimen in which the diagnosis indicates that the subject has elevated blood glucose, wherein the treatment regimen is selected from medication and behavioral therapy, or a combination thereof. In a specific embodiment, medication therapy includes treatment with an agent selected from (a) meglitinide, (b) sulfonylurea, (c) dipeptidyl peptidase-4 (DPP-4) inhibitor, (d) biguanide, (e) thiazolidinedione, (f) alpha-glucosidase inhibitor, (g) amylin mimetic; (h) incretin mimetic; (i) insulin; and (j) any combination thereof.

[0034] In certain embodiments, any of the foregoing methods further comprises selecting a subject suspected of having or at risk of having elevated blood glucose.

[0035] In certain embodiments, any of the foregoing methods further comprises obtaining a biological sample from a subject suspected of having or at risk of having elevated blood glucose.

[0036] In a specific embodiment, any of the previous methods further comprises comparing the level of one or more indicators associated with increased blood glucose in the biological sample with the level of one or more indicators associated with increased blood glucose in a control sample, wherein the control sample is selected from: a sample obtained from the same subject at an earlier time point than the biological sample, a sample from a subject with normal blood glucose, a sample from a prediabetic subject, a sample from a type 2 diabetes subject, a sample from a gestational diabetes subject, and a sample from a type 1 diabetes subject.

[0037] The present invention provides a method for monitoring elevated blood glucose in a subject, the method comprising:

[0038] (1) determining the level of Eno1 in a first biological sample obtained from a subject with elevated blood glucose at a first time;

[0039] (2) determining the level of Eno1 in a second biological sample obtained from the subject at a second time, wherein the second time is later than the first time; and

[0040] (3) comparing the level of Enol in the second sample to the level of Enol in the first sample, wherein a change in the level of Enol indicates a change in the elevated blood glucose state in the subject.

[0041] In certain embodiments, the determining steps (1) and (2) further comprise determining the level of one or more other blood glucose indicators selected from the group consisting of HbA1c level, fasting blood glucose, postprandial blood glucose and glucose tolerance.

[0042] In a specific embodiment, before obtaining the second sample, the subject is treated with a drug for elevated blood sugar. In a specific embodiment, the reduced level of Eno1 in the second biological sample compared to the first biological sample indicates an increase in blood sugar in the subject. In a specific embodiment, the increased or equal level of Eno1 in the second biological sample compared to the first biological sample indicates normalization of blood sugar in the subject. In a specific embodiment, the method further includes selecting and / or administering different treatment regimens for the subject based on the subject's blood sugar level. In a specific embodiment, the treatment regimen is selected from drug therapy and behavioral correction therapy. In a specific embodiment, drug therapy includes treatment with an agent selected from (a) meglitinide, (b) sulfonylurea, (c) dipeptidyl peptidase-4 (DPP-4) inhibitor, (d) biguanide, (e) thiazolidinedione, (f) alpha-glucosidase inhibitor, (g) amylin mimetic; (h) incretin mimetic; (i) insulin; and (j) any combination thereof.

[0043] The present invention provides a method for treating elevated blood glucose in a subject, comprising: (a) obtaining a biological sample from a subject suspected of having elevated blood glucose, (b) submitting the biological sample to obtain diagnostic information regarding Eno1 levels, and (c) administering a therapeutically effective amount of an anti-diabetic treatment if the Eno1 level is above a threshold level.

[0044] The present invention provides a method of treating elevated blood glucose in a subject, comprising: (a) obtaining diagnostic information regarding the level of Eno1 in a biological sample, and (b) administering a therapeutically effective amount of an anti-diabetic treatment if the level of Eno1 is above a threshold level.

[0045] The present invention provides a method of treating elevated blood sugar in a subject, comprising:

[0046] (a) obtaining a biological sample from a subject suspected of having elevated blood glucose for identifying diagnostic information regarding Eno1 levels,

[0047] (b) measuring the level of Eno1 in a biological sample,

[0048] (c) if the Eno1 level is below the threshold level, recommending to a health care provider that blood glucose lowering therapy be administered.

[0049] In certain embodiments, the method further comprises obtaining diagnostic information regarding the level of one or more other indicators of elevated blood glucose.

[0050] In certain embodiments, the method further comprises measuring the level of one or more other indicators of elevated blood glucose.

[0051] In certain embodiments, the one or more other indicators of elevated blood glucose are selected from HbA1c levels, fasting blood glucose levels, postprandial blood glucose levels, and glucose tolerance.

[0052] In certain embodiments, if the level of Eno1 is low and at least one other indicator of elevated blood glucose is detected, step (c) further comprises administering a therapeutically effective amount of a glucose-lowering therapy. In certain embodiments, if the level of Eno1 is below a threshold level and at least one other indicator of elevated blood glucose is present, step (c) further comprises recommending administration of a glucose-lowering therapy to a health care provider.

[0053] In certain embodiments, the biological sample is blood or serum. In certain embodiments, the level of Eno1 is determined by immunoassay or ELISA. In certain embodiments, the level of Eno1 is determined by (i) contacting the biological sample with an agent that selectively binds to Eno1 to form a biomarker complex, and (ii) detecting the biomarker complex. In certain embodiments, the agent is an anti-Eno1 antibody that selectively binds to at least one epitope of Eno1.

[0054] In certain embodiments, the level of Enol is determined by measuring the amount of Enol mRNA in a biological sample. In certain embodiments, an amplification reaction is used to measure the amount of Enol mRNA in a biological sample. In certain embodiments, the amplification reaction is (a) polymerase chain reaction (PCR); (b) nucleic acid sequence-based amplification assay (NASBA); (c) transcription-mediated amplification (TMA); (d) ligase chain reaction (LCR); or (e) strand displacement amplification (SDA). In certain embodiments, a hybridization assay is used to measure the amount of Enol mRNA in a biological sample. In certain embodiments, an oligonucleotide complementary to a portion of Enol mRNA is used in a hybridization assay to detect Enol mRNA.

[0055] The present invention provides a kit for detecting Eno1 in a biological sample, comprising at least one reagent for measuring the level of Eno1 in the biological sample, and a set of instructions for measuring the level of Eno1. In a specific embodiment, the reagent is an anti-Eno1 antibody. In a specific embodiment, the kit further comprises a tool for detecting the anti-Eno1 antibody. In a specific embodiment, the tool for detecting the anti-Eno1 antibody is a detectable secondary antibody. In a specific embodiment, the reagent is an oligonucleotide complementary to Eno1 mRNA. In a specific embodiment, the instructions list an immunoassay or ELISA for detecting the level of Eno1 in the biological sample. In a specific embodiment, the instructions list an amplification reaction for analyzing the level of Eno1 mRNA in the biological sample. In a specific embodiment, the amplification reaction is used to measure the content of Enol mRNA in the biological sample. In a specific embodiment, the amplification reaction is (a) polymerase chain reaction (PCR); (b) nucleic acid sequence-based amplification assay (NASBA); (c) transcription-mediated amplification (TMA); (d) ligase chain reaction (LCR); or (e) strand displacement amplification (SDA). In certain embodiments, the instructions list a hybridization assay for determining the amount of Enol mRNA in a biological sample. In certain embodiments, the kit further includes at least one oligonucleotide complementary to a portion of Enol mRNA. In certain embodiments, the kit further includes at least one reagent for measuring HbA1c and / or blood glucose levels in a biological sample. In certain embodiments, the kit further includes instructions for measuring at least one level selected from HbA1c levels, postprandial blood glucose levels, fasting blood glucose levels, and glucose tolerance in a subject from whom the biological sample was obtained.

[0056] The present invention provides a reagent set for use in a method for detecting elevated blood sugar, the reagent set comprising detection reagents for Eno1 and HbA1c.

[0057] The present invention provides a reagent set for use in a method for treating elevated blood sugar, the reagent set comprising detection reagents for Eno1 and HbA1c.

[0058] The present invention provides a reagent set for use in a method for monitoring treatment of elevated blood sugar, the reagent set comprising detection reagents for Eno1 and HbA1c.

[0059] The invention provides a kit comprising a reagent set provided herein and a set of instructions for obtaining diagnostic information regarding the level of one or more elevated blood glucose indicators.

[0060] The present invention provides the use of a reagent set, which comprises a plurality of detection reagents specific for detecting markers of elevated blood sugar in a method for diagnosing and / or treating elevated blood sugar, wherein at least one detection reagent of the reagent set is specific for detecting Eno1, and wherein the remaining one or more detection reagents are specific for detecting marker indicators of elevated blood sugar selected from HbA1c and glucose.

[0061] In certain embodiments of the above methods, glucose flux is increased in skeletal muscle cells of the subject.

[0062] In another aspect, the present invention provides a method for increasing glucose flow in a subject, the method comprising administering a pharmaceutical composition comprising Eno1 or a fragment thereof to the subject. In a specific embodiment, the pharmaceutical composition administered to the subject is any of the above-mentioned pharmaceutical compositions. In another aspect, the present invention provides a method for increasing glycolytic activity or capacity in skeletal muscle cells of a subject, the method comprising administering a pharmaceutical composition comprising Eno1 or a fragment thereof to the subject. In a specific embodiment, the pharmaceutical composition administered to the subject is any of the above-mentioned pharmaceutical compositions.

[0063] In another aspect, the present invention provides a method for increasing mitochondrial free fatty acid oxidation in skeletal muscle cells of a subject, the method comprising administering to the subject a pharmaceutical composition comprising Eno1 or a fragment thereof. In a specific embodiment, the pharmaceutical composition administered to the subject is any of the above pharmaceutical compositions.

[0064] In certain embodiments, the subject has any one or more of elevated blood glucose, impaired glucose tolerance, decreased insulin sensitivity and / or insulin resistance, diabetes, elevated Hb1Ac levels, and abnormal blood glucose level control.

[0065] In certain embodiments of any of the methods described above, the subject is a human.

[0066] In a particular aspect, the present invention relates to a pharmaceutical composition comprising a therapeutically effective amount of Eno1 or a fragment thereof. In a particular embodiment, the pharmaceutical composition is for delivery to a muscle cell. In a particular aspect, the present invention relates to a pharmaceutical composition comprising Eno1 or a fragment thereof and a muscle targeting peptide. In a particular embodiment, the pharmaceutical composition is for delivery to a muscle cell. In a particular embodiment, Eno1 comprises an Eno1 polypeptide or a fragment thereof. In a particular embodiment, Eno1 comprises an Eno1 nucleic acid or a fragment thereof. In a particular embodiment, the composition further comprises an expression vector encoding Eno1 or a fragment thereof. In a particular embodiment, Eno1 or a fragment thereof is biologically active. In a particular embodiment, Eno1 or a fragment thereof has at least 90% of the activity of purifying an endogenous human Eno1 polypeptide. In a particular embodiment, Eno1 is human Eno1. In a particular embodiment, the composition further comprises microparticles. In a particular embodiment, the composition further comprises nanoparticles. In a particular embodiment, the composition further comprises an in situ forming composition. In a particular embodiment, the composition further comprises a liposome. In a particular embodiment, the composition further comprises a dendrimer. In a particular embodiment, the composition comprises a complex comprising an Eno1 polypeptide and a muscle targeting peptide. In certain embodiments, the Eno1 polypeptide is a human Eno1 polypeptide. In certain embodiments, the muscle targeting peptide comprises an amino acid sequence selected from the group consisting of ASSLNIA; WDANGKT; GETRAPL; CGHHPVYAC and HAIYPRH. In certain embodiments, the complex further comprises a linker. In certain embodiments, the linker is selected from the group consisting of a covalent linker, a non-covalent linker and a reversible linker. In certain embodiments, the complex further comprises a pharmaceutically acceptable dendrimer. In certain embodiments, the pharmaceutically acceptable dendrimer is a PAMAN dendrimer. In certain embodiments, the pharmaceutically acceptable dendrimer is a G5 dendrimer. In certain embodiments, the pharmaceutically acceptable dendrimer is an uncharged dendrimer. In certain embodiments, the pharmaceutically acceptable dendrimer is an acylated dendrimer. In certain embodiments, the pharmaceutically acceptable dendrimer is a PEGylated dendrimer or an acetylated dendrimer.

[0067] In a specific embodiment of the aforementioned composition, the complex further comprises a liposome. In a specific embodiment, the complex further comprises a microparticle. In a specific embodiment, the complex further comprises a composition formed in situ. In a specific embodiment, Eno1 is released from the complex when delivered to muscle cells. In a specific embodiment, the dendrimer and ENO1 are present in the complex at a ratio of about 1:1 to about 10:1. In a specific embodiment, the dendrimer and ENO1 are present in the complex at a ratio of about 3:1 to about 5:1. In a specific embodiment, the muscle targeting moiety and the dendrimer are present in the complex at a ratio of about 0.1:1 to about 10:1. In a specific embodiment, the muscle targeting moiety and the dendrimer are present in the complex at a ratio of about 1:1 to about 3:1. In a specific embodiment, Eno1 and the muscle targeting moiety are present in the complex at a ratio of about 1:1 to about 1:30.

[0068] In specific embodiments of the aforementioned compositions, the compositions are formulated for parenteral administration. In specific embodiments, the compositions are formulated for oral administration. In specific embodiments, the compositions are formulated for intramuscular administration, intravenous administration, or subcutaneous administration.

[0069] In a specific aspect, the invention relates to a method of lowering blood glucose in a subject with elevated blood glucose, the method comprising administering any of the compositions described above to the subject, thereby lowering the subject's blood glucose.

[0070] In a specific aspect, the invention relates to a method of improving glucose tolerance in a subject with impaired glucose tolerance, the method comprising administering any of the compositions described above to the subject, thereby improving glucose tolerance in the subject.

[0071] In a particular aspect, the invention relates to a method of increasing the insulin response in a subject having reduced insulin sensitivity and / or insulin resistance, the method comprising administering any of the compositions described above to the subject, thereby increasing the insulin response in the subject.

[0072] In a particular aspect, the present invention relates to a method for treating diabetes in a subject, the method comprising administering any of the above compositions to a subject, thereby treating diabetes in the subject. In a particular embodiment, the diabetes is type 2 diabetes or type 1 diabetes. In a particular embodiment, the diabetes is prediabetes.

[0073] In a specific aspect, the invention relates to a method of reducing Hb1Ac levels in a subject having elevated Hb1Ac levels, the method comprising administering any of the compositions described above to the subject, thereby reducing the Hb1Ac levels in the subject.

[0074] In a specific aspect, the present invention relates to a method for improving blood glucose control in a subject with abnormal blood glucose control, the method comprising administering any of the above compositions to the subject, thereby improving the subject's blood glucose control. In a specific embodiment of the above method, glucose flux in skeletal muscle cells of the subject is increased.

[0075] In a particular aspect, the invention relates to a method of increasing glucose flux in a subject, the method comprising administering any of the compositions described above to the subject, thereby increasing glucose flux in the subject.

[0076] In a specific aspect, the present invention relates to a method for increasing the glycolytic activity or capacity in skeletal muscle cells of a subject, the method comprising administering any of the above compositions to the subject, thereby increasing the glycolytic activity or capacity in skeletal muscle cells of the subject.

[0077] In a specific method, the invention relates to a method of increasing mitochondrial free fatty acid oxidation in skeletal muscle cells of a subject, the method comprising administering any of the compositions described above to the subject, thereby increasing mitochondrial free fatty acid oxidation in skeletal muscle cells of the subject.

[0078] In a specific embodiment of the above method, Enol is administered parenterally. In a specific embodiment of the above method, Enol is administered orally. In a specific embodiment, Enol is administered by a route selected from intramuscular, intravenous, and subcutaneous. In a specific embodiment of the above method, the subject has any one or more of elevated blood glucose, decreased glucose tolerance, decreased insulin sensitivity and / or insulin resistance, diabetes, elevated Hb1Ac levels, and abnormal blood glucose level control.

[0079] In certain embodiments, the above methods further comprise selecting a subject having any one or more of elevated blood glucose, impaired glucose tolerance, decreased insulin sensitivity and / or insulin resistance, diabetes, elevated Hb1Ac levels, and abnormal blood glucose level control.

[0080] In certain embodiments of the foregoing methods, the subject is a human.

[0081] Other embodiments are provided below. BRIEF DESCRIPTION OF THE DRAWINGS

[0082] Figure 1A is a graph showing glucose uptake in smooth muscle myoblasts treated or not with Eno1 and insulin. Figure 1B Shown is glucose uptake in smooth muscle myoblasts treated with 0, 500 or 1000 ug / ml Eno1 but not insulin.

[0083] Figure 2A and2B Shown are Eno1 protein levels in human skeletal muscle myotubes treated with 0, 500 or 1000 μg / ml Eno1. Figure 2C Shown is Eno1 activity in human skeletal muscle myotubes treated with 0, 500 or 1000 μg / ml Eno1.

[0084] Figure 3A The Eno1 activity of native and heat-inactivated Eno1 is shown. Figure 3B Induction of glucose uptake by active and heat-inactivated Eno1 is shown.

[0085] Figure 4A and 4B Shown are the (A) time course and (B) area under the curve (AUC) of glucose clearance in a glucose tolerance test in a diet-induced obesity (DIO) mouse model with or without treatment with Eno1 protein.

[0086] Figure 5 Coomassie blue staining of polyacrylamide gels containing various concentrations of Eno 1 analyzed by SDS-PAGE is shown. L1: Precision Plus Protein Standard Dual Color, L2: Eno 1 (10.0 μg), L3: Eno 1 (1.0 μg), L4: Eno 1 (0.1 μg).

[0087] Figure 6 Silver staining of polyacrylamide gels containing various concentrations of Eno 1 analyzed by SDS-PAGE is shown. L1: Precision Plus Protein Standard Dual Color, L2: Eno 1 (10.0 μg), L3: Eno 1 (1.0 μg), L4: Eno 1 (0.1 μg).

[0088] Figure 7 Western blot analysis of Eno 1 is shown. L1: Precision Plus Protein Standard Dual Color, L2: Eno 1 (10.0 μg), L3: Eno 1 (1.0 μg), L4: Eno 1 (0.1 μg).

[0089] Figure 8 Zeta (ζ) potential measurements of Eno1 / G5-dendrimer / SMTP prepared with a 2:1 ratio of Eno1 to dendrimer SMTP are shown.

[0090] Fig. 9Shown are the normalized activities of Eno1 alone (enolase alone) and Eno1 / G5-dendrimer / SMTP (enolase / G5-SMC) solutions after storage at various temperatures.

[0091] Fig. 10A and 10B Representative fluorescence images of the tissue distribution of (A) fluorescently labeled Eno1-G5-PAMAN dendrimer complexes and (B) fluorescently labeled muscle-targeted Eno-1-G5-PAMAM dendrimer complexes in mice.

[0092] Fig.11 is a graph of body weight of lean mice or DIO mice treated with one of vehicle, G5-PAMAM dendrimer, G5-PAMAM dendrimer+SMTP, G5-PAMAM dendrimer+Enol, G5-PAMAM dendrimer+Eno1+SMTP.

[0093] Fig.12 is a graph showing blood glucose levels in mice with diet-induced obesity after injection of saline or G5-PAMAM dendrimer+Eno1+SMTP (50 ug / kg) at 1, 4 and 24 hours after injection.

[0094] Fig.13A Shown are the results of glucose tolerance tests in lean mice and in a diabetic mouse model of diet-induced obesity (DIO) after 1 week of treatment with G5-PAMAM dendrimer+SMTP (DIO NP+SMTP) or G5-PAMAM dendrimer+Enol+SMTP (DIO Enolase-1+NP+SMTP). Fig. 13B Shows Fig.13A The area under the curve (AUC) for each treatment group. Fig. 13C and 13D Shown are the (C) time course and (D) area under the curve (AUC) of glucose clearance in a glucose tolerance test in lean or DIO mice after two weeks of treatment with one of vehicle, G5-PAMAM dendrimer (G5), G5-PAMAM dendrimer + SMTP, G5-PAMAM dendrimer + Enol, G5-PAMAM dendrimer + Eno1 + SMTP.

[0095] Fig.14A and 14BShown are the (A) time course and (B) area under the curve (AUC) of glucose clearance in a glucose tolerance test in lean or DIO mice after four weeks of treatment with one of vehicle, G5-PAMAM dendrimer, G5-PAMAM dendrimer+SMTP, G5-PAMAM dendrimer+Enol, G5-PAMAM dendrimer+Eno1+SMTP.

[0096] Fig.15 Shown are serum lactate levels in lean mice, diet-induced obese (DIO) mice, DIO mice treated with G5-dendrimer (DIO+NP), and DIO mice treated with Eno1 / G5-dendrimer / SMTP complex (DIO+Eno-1+NP+SMTP) after 8 weeks of treatment.

[0097] Fig.16A and 16B Shown are the (A) time course and (B) area under the curve (AUC) of glucose clearance in an intraperitoneal glucose tolerance test in db / db mice (BKS.Cg-m+ / +Leprdb / J) after one week of treatment with either vehicle, G5-PAMAM dendrimer (G5)+SMTP, and G5-PAMAM dendrimer+Eno1+SMTP (25ug / kg or 50ug / kg).

[0098] Fig.17A and 17B Shown is the time course of glucose levels in db / db mice (BKS.Cg-m+ / +Leprdb / J) after two weeks of treatment with one of vehicle, G5-PAMAM dendrimer (G5)+SMTP, and G5-PAMAM dendrimer+Eno1+SMTP (25ug / kg or 50ug / kg), obtained during the time course starting immediately after injection of vehicle, the indicated doses of G5-PAMAM dendrimer+SMTP, and G5-PAMAM dendrimer+Eno1+SMTP. Fig.17A Results from all four dosing regimens are shown. Fig. 17B Results from G5-PAMAM dendrimer + SMTP (G5+SMTP) and G5-PAMAM dendrimer + Eno1 + SMTP (Eno1 + G5 + SMTP) 50 ug / kg are shown to show the significant difference in glucose levels at the 30 minute time point.

[0099] Fig.18Shown is the effect of once daily subcutaneous injection of 25 μg / kg body weight or 50 μg / kg body weight of Eno1 / G5-dendrimer / SMTP complex on postprandial blood glucose levels in the db / db diabetic mouse model after two weeks of treatment. Postprandial glucose was measured without fasting 24 hours after Eno1 injection. "NP" is G5-dendrimer.

[0100] Fig.19 The effect of twice daily (morning and evening) subcutaneous injections of 100 μg / kg body weight or 200 μg / kg body weight of Eno1 / G5-dendrimer / SMTP complex on postprandial blood glucose levels in the db / db diabetic mouse model is shown.

[0101] Fig. 20 Shown are the creatine kinase and caspase 3 activities detected after treatment with G5-PAMAM dendrimer (G5), G5-PAMAM dendrimer + SMTP (G5-SMC), and acylated G5-PAMAM dendrimer + SMTP (G5-SMC-Ac).

[0102] Fig.21 Shown are p-Akt protein levels in human skeletal muscle myotubes treated or not with Eno1 and insulin.

[0103] Fig.22A Shown are Glut1, Glut4, HK2, and myogenin mRNA levels in human skeletal muscle myotubes treated with or without purified Eno1. Fig. 22B Shown are Glut1 protein levels in human skeletal muscle myotubes treated or not with purified Eno 1. Glut1 protein levels are relative units normalized by the median value of ribosomal proteins.

[0104] Fig.23 Shown are glucose-6-phosphate (G6P) levels in glucose-starved (upper panel) and glucose-stimulated (lower panel) human skeletal muscle myotubes treated or not with purified Eno1.

[0105] Fig.24 Shown are phosphoenolpyruvate (PEP) levels in glucose-starved (upper panel) and glucose-stimulated (lower panel) human skeletal muscle myotubes treated or not with purified Eno1.

[0106] Fig.25 Shown are oxygen consumption rates (OCRs) in human skeletal muscle myotube (HSMM) cultures treated sequentially with palmitate, CCCP, and etomoxir, with or without purified Eno1.

[0107] Fig.26Shown are the extracellular acidification rates (ECAR) in human skeletal muscle myotube (HSMM) cultures treated sequentially with glucose, oligomycin, and 2-DG, with or without purified Eno1.

[0108] Fig.27A Shown are mitochondrial content in human skeletal muscle myotubes treated with 500 μg / ml or 1000 μg / ml Eno1 relative to untreated control human skeletal muscle myotubes. Mitochondrial content was determined by adding MitotrackerGreen, a green fluorescent mitochondrial dye, to cells 48 hours after Eno1 treatment.

[0109] Fig.27B Mitochondrial reactive oxygen species (Mito-ROS) production in human skeletal muscle myotubes treated with 500 μg / ml or 1000 μg / ml Enol relative to untreated control human skeletal muscle myotubes (Enol 0 ug / ml) is shown. Mito-ROS was measured by treating cells with dihydrorhodamine 123, an uncharged and non-fluorescent reactive oxygen species (ROS) indicator that can passively diffuse across the membrane, where it is oxidized to cationic rhodamine 123 (which is localized in the mitochondria and exhibits green fluorescence). After dihydrorhodamine 123 treatment, myotubes were trypsinized, washed, and subjected to flow cytometric analysis to measure Mito-ROS levels.

[0110] Fig.28A Shown are 5' AMP-activated protein kinase (AMPK) and phosphorylated AMPK (pAMPK) levels in skeletal muscle myotubes treated with 0, 500 or 1000 μg / ml Eno 1. Lamin A / C was used as a loading control.

[0111] Fig.28B Shown are the ratios of pAMPK (p-AMPK) to AMPK in basal or serum-starved skeletal muscle myotubes treated with 0, 500 or 1000 μg / ml Eno1.

[0112] Fig.29 A schematic diagram of the working model describing the potential role of Nampt in the mode of action of Eno1 is shown.

[0113] Fig.30 Shown is Nampt activity in human skeletal muscle myotubes treated with 500 ug / ml or 1000 μg / ml Eno1 in differentiation medium for 48 hours after 4 days of differentiation relative to untreated control human skeletal muscle myotubes (Eno1 0 ug / ml).

[0114] Fig.31Shown is 2-DG uptake in serum-starved human skeletal muscle myotubes treated with recombinant extracellular Nampt (eNampt).

[0115] Fig.32 Glucose uptake in human skeletal muscle myotubes treated with 0, 500 or 1000 μg / ml Eno1 for 48 hours in differentiation medium after 4 days of differentiation is shown in the presence or absence of the Nampt inhibitor FK866. FK866 was added 24 hours after the start of Eno1 treatment, and myotubes were treated with FK866 for 24 hours. 2-DG uptake was measured after 3 hours of serum starvation. Nampt inhibition by FK866 abolished Eno1-induced glucose uptake.

[0116] Fig.33 A schematic diagram of the glycolytic pathway is shown.

[0117] Fig.34A and 34B Shown are the (A) amino acid sequence (SEQ ID NO: 2) and (B) nucleic acid coding sequence (SEQ ID NO: 1) of human Eno1, variant 1 (NCBI accession number NM_001428.3).

[0118] Fig.35A and 35B The (A) amino acid sequence (SEQ ID NO: 4) and (B) nucleic acid coding sequence (SEQ ID NO: 3) of human Eno1, variant 2 (NCBI accession number NM_001201483.1) are shown. Human Eno1, variant 2 protein is also known as MBP-1.

[0119] Fig.36A The nucleic acid sequence of ENO2 mRNA is shown (SEQ ID NO: 5). Fig.36B The amino acid sequence of Eno2 is shown (SEQ ID NO: 6).

[0120] Fig.37A and 37B The nucleic acid sequences of variant 1 (SEQ ID NO: 7) and variant 2 (SEQ ID NO: 8) of ENO3 mRNA are shown, respectively. Fig.37C Isoform 1 of the Eno3 protein (SEQ ID NO: 9), encoded by both variant 1 and variant 2, is shown. Fig.37D The nucleic acid sequence of variant 3 of ENO3 mRNA is shown (SEQ ID NO: 10). Fig.37EThe amino acid sequence of isoform 2 of Eno3 (SEQ ID NO: 11) is shown, which is encoded by variant 3. Variant 3 of Eno3 mRNA differs in the 5'UTR and lacks two exons in the 5' coding region compared to variant 1. Isoform 2 of Eno3 protein is shorter than isoform 1, but has the same N- and C-termini. DETAILED DESCRIPTION

[0121] Discovery platform technology is used to depict the significantly different molecular imprints that drive the pathophysiology of diabetes. Eno1 is identified as a key node that is significantly regulated in a primary in vitro diabetes model of people by this discovery platform technology. The in vitro and in vivo studies discussed later herein confirm the role of Eno1 in insulin-dependent and non-insulin-dependent glucose uptake, glucose tolerance, insulin sensitivity and / or diabetes, such as type 1 diabetes, type 2 diabetes, prediabetes and gestational diabetes. More specifically, the treatment of human myotubes with Eno1 protein has demonstrated the improvement of both non-insulin-dependent and insulin-dependent glucose uptake in myotubes, indicating the role of Eno1 in the treatment of both type 1 and type 2 diabetes and in the presence and absence of insulin and / or insulin response in glucose uptake in two situations. In addition, the administration of Eno1 protein (alone or in the environment of skeletal muscle targeting dendrimers) improves glucose tolerance in a mouse diet-induced obesity model, and is expected to have similar results in genetic models of type 1 and type 2 diabetes. These results demonstrate that Eno1 is effective in normalizing glucose and insulin responses and therefore suggest that Eno1 may be used to improve glucose tolerance and increase insulin sensitivity / reduce insulin resistance and therefore be useful in treating diabetes.

[0122] I. Definitions

[0123] Enolase 1, (α), also known as ENO1L, α-enolase, enolase-α, tau-crystallin, non-neural enolase (NNE), α-enolase-like 1, phosphopyruvate hydratase (PPH), plasminogen binding protein, MYC promoter binding protein 1 (MPB1) and 2-phospho-D-glycerate hydrolase, is one of the three enolase isozymes found in mammals. The protein and nucleic acid sequences of human Eno1 isoforms are provided herein in Figures 34 and 35. The present application provides human amino acid and nucleic acid sequences for the treatment of human diseases. However, it will be understood that the compositions and methods of the present invention can be easily adapted to the treatment of non-human animals by selecting Eno1 of the species to be treated. The amino acid and nucleic acid sequences of Eno1 of non-human species are known in the art and can be found, for example, at ncbi.nlm.nih.gov / genbank / . In some embodiments, the Eno1 used in the compositions and methods of the present invention is mammalian Eno1. In a preferred embodiment, Eno1 is human Eno1.

[0124] As used herein, "administration of Enol", unless otherwise indicated, is understood to be the administration of Enol protein or a nucleic acid construct for expression of Enol protein. In specific embodiments, Enol protein may include an Enol protein fragment or a nucleic acid encoding an Enol protein fragment. In specific embodiments, the administration of Enol is the administration of Enol protein. In specific embodiments, the administration of Enol is the administration of an Enol polynucleotide. Provided herein are protein and nucleic acid sequences of human Enol. In specific embodiments, the administration of Enol includes the administration of a first variant or a second variant of human Enol. In specific embodiments, the administration of Enol includes the administration of a first variant and a second variant of human Enol. In specific embodiments, the administration of Enol includes the administration of a first variant of human Enol. In specific embodiments, the administration of Enol includes the administration of a second variant of human Enol. In specific embodiments, the administration of Enol includes the administration of only the first variant of human Enol. In specific embodiments, the administration of Enol includes the administration of only the second variant of human Enol.

[0125] As used herein, "biologically active" refers to an Eno1 molecule or fragment thereof having at least one activity of an endogenous Eno1 protein. For example, in some embodiments, a biologically active Eno1 molecule or fragment thereof catalyzes the dehydration of 2-phospho-D-glyceric acid (PGA) to phosphoenolpyruvate (PEP). In some embodiments, a biologically active Eno1 molecule or fragment thereof catalyzes the hydration of PEP to PGA. In some embodiments, a biologically active Eno1 molecule or fragment thereof increases glucose uptake in cells, such as muscle cells, preferably skeletal muscle cells. In some embodiments, a biologically active Eno1 molecule or fragment thereof reduces blood glucose levels, such as postprandial blood glucose levels or blood glucose levels in a glucose tolerance test. In some embodiments, a biologically active Eno1 molecule or fragment thereof binds to Nampt, such as extracellular Nampt (eNampt).

[0126] As used herein, "administered to muscle", "delivered to muscle" or "delivered to muscle cells" including skeletal muscle cells, smooth muscle cells, etc. are understood as providing an effective dose of Enol to muscle, e.g., a preparation, method or combination thereof of muscle cells to provide the desired systemic effect, e.g., normalization of blood glucose in subjects with dysglycemia, e.g., by improving glucose tolerance and / or insulin sensitivity, or treating diabetes. In a specific embodiment, Enol is formulated for direct administration to muscle, and preferably stays in the muscle. In a specific embodiment, the Enol formulation for direct administration to muscle (i.e., intramuscular administration) is preferably a sustained release formulation allowing a relatively low frequency of administration (e.g., once a week or less, every other week or less, once a month or less, every other month or less, once every three months or less, once every four months or less, once every five months or less, once every six months or less). In a specific embodiment, Enol is linked to a targeting moiety to increase the delivery of Enol to muscle, so that it is not necessary to deliver Enol directly to muscle (e.g., subcutaneously or intravenously). It will be understood that administration to muscle does not require the entire dose of Enol to be delivered to muscle or myocytes. In specific embodiments, at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35% of Enol is delivered to muscle, preferably skeletal muscle and / or smooth muscle. In specific embodiments, the amount of non-intramuscularly administered muscle-targeted Enol delivered to myocytes is about 1.5 times or more, 2 times or more, 3 times or more, 4 times or more, 5 times or more, or 6 times or more than the amount of non-targeted Enol delivered to muscle. In specific embodiments, Enol is delivered to skeletal muscle. In specific embodiments, Enol is delivered to smooth muscle. In specific embodiments, Enol is delivered to skeletal and smooth muscle. In specific embodiments, Enol is preferentially or in higher amounts delivered to skeletal muscle compared to smooth muscle. In specific embodiments, at least 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95% or more of Enol delivered to muscle is delivered to skeletal muscle. In certain embodiments, Eno1 is not delivered to smooth muscle.Assays to determine relative targeting of a payload by a targeting moiety are known in the art and are provided, for example, in Samoylova et al., 1999, Muscle Nerve, 22:460-466, which is incorporated herein by reference.

[0127] As used herein, "muscle targeting moiety" includes at least a muscle targeting peptide (MTP), for example, a skeletal muscle and / or smooth muscle targeting peptide (SMTP). In a specific embodiment, the targeting moiety includes a ligand that binds to integrin αvβ5 or αvβ3 integrin. In a specific embodiment, the targeting moiety includes a CD-46 ligand. In a specific embodiment, the targeting moiety includes an adenovirus peton protein, optionally in combination with adenovirus 35 fiber protein. In a specific embodiment, at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35% of muscle-targeted Eno1 is delivered to muscle by the muscle targeting moiety, in some embodiments, preferably skeletal muscle and / or smooth muscle. In a specific embodiment, the amount of non-intramuscularly administered muscle-targeted Eno1 delivered to muscle cells is about 1.5 times or more, 2 times or more, 3 times or more, 4 times or more, 5 times or more, or 6 times or more than the amount of non-targeted Eno1 delivered to muscle.

[0128] As used herein, "muscle targeting peptide" or "MTP" is understood to be a peptide sequence that improves the delivery of its payload (e.g., Eno1) to muscle cells (preferably skeletal muscle and / or smooth muscle cells). MTP is known in the art and is provided, for example, in U.S. Patent No. 6329501; U.S. Patent Publication No. 20110130346 and Samoylova et al., 1999, Muscle and Nerve 22: 460-466, each of which is incorporated herein by reference in its entirety. In a specific embodiment, MTP is a skeletal muscle targeting peptide. "Skeletal muscle targeting peptide" is a peptide sequence that improves the delivery of its payload (e.g., Eno1) to skeletal muscle cells. In a specific embodiment, MTP is a smooth muscle targeting peptide. "Smooth muscle targeting peptide" is a peptide sequence that improves the delivery of its payload (e.g., Eno1) to smooth muscle cells. In a specific embodiment, MTP improves the delivery of its payload (e.g., Eno1) to skeletal cells and smooth muscle cells. In certain embodiments, the MTP, e.g., skeletal muscle targeting peptide and / or smooth muscle targeting peptide, does not enhance the delivery of its payload to cardiomyocytes. MTP, e.g., skeletal muscle targeting peptide includes, but is not limited to, peptides comprising the following sequences: ASSLNIA; WDANGKT; GETRAPL; CGHHPVYAC and HAIYPRH. In preferred embodiments, the MTP comprises the amino acid sequence ASSLNIA.

[0129] As used herein, "payload" is understood to be a moiety for delivery to a target cell via a targeting moiety. In certain embodiments, the payload is a peptide, e.g., an Eno1 peptide. In certain embodiments, the payload is a nucleic acid, e.g., a nucleic acid encoding an Eno1 peptide. In certain embodiments, the payload further includes other components (e.g., dendrimers, liposomes, microparticles) or agents (e.g., therapeutic agents) delivered to a target cell together with the Enol payload.

[0130] As used herein, "connector" is understood to be a part that juxtaposes the targeting moiety and the payload sufficiently close to allow the payload to be delivered to the desired site by the targeting moiety. In specific embodiments, the connector is a covalent connector, for example, a cross-linker, including a reversible cross-linker; a peptide bond, for example, wherein the payload is a protein co-translated with the targeting moiety. In specific embodiments, the connector is covalently linked to one of the payload or the targeting moiety and non-covalently linked to the other. In specific embodiments, the connector includes a dendrimer. In specific embodiments, the dendrimer is covalently linked to the targeting moiety and non-covalently linked to the payload, for example, Eno1. In specific embodiments, the connector is a liposome or microparticle, and the targeting moiety is exposed on the surface of the liposome, and the payload (e.g., Eno1) is encapsulated in the liposome or microparticle. In specific embodiments, the connector and Eno1 are present on the surface of the microparticle connector. In specific embodiments, the targeting moiety is present on the surface of the viral particle and the payload includes a nucleic acid encoding Eno1.

[0131] As used herein, "linked," "operably linked," "joined," and the like refer to a juxtaposition in which the components described are present in a complex that allows them to function in an intended manner. The components can be covalently linked (e.g., peptide bonds, disulfide bonds, non-natural chemical bonds), linked by hydrogen bonds (e.g., knob-into-holes pairing of proteins, see, e.g., U.S. Pat. No. 5,582,996; Watson-Crick nucleotide pairing), or ionically bound (e.g., chelators and metals) directly or through a linker (e.g., a peptide sequence, typically a short peptide sequence; a nucleic acid sequence; or a chemical linker, including the use of linkers for attachment to higher order or larger structures, including microparticles, beads, or dendrimers). As used herein, the components of the complex can be linked to each other by packaging in and / or on liposomes and / or dendrimers, wherein some components of the complex can be covalently linked and some non-covalently linked. Linkers can be used to provide separation between active molecules so that the activity of a molecule is not substantially inhibited (less than 10%, less than 20%, less than 30%, less than 40%, less than 50%) by linking a first molecule to a second molecule. Linkers can be used, for example, to link Eno1 to a targeting moiety. As used herein, under conditions in which the agents of the invention are used (i.e., typically physiological conditions), the linked (but non-covalently bonded) molecules have less than 10 -3 , 10 -4 , 10 -5 , 10 -6 , 10 -7 , 10 -8 , 10 -9 , 10 -10 , 10 -11 or 10 -12 , or any range of binding affinities (Kd) to each other inclusive of those values.

[0132] In certain embodiments, the effective load and the targeting moiety are present in the complex in a molar ratio of about 1: 1. In certain embodiments, the targeting moiety is present in the complex in a molar excess relative to the effective load. In certain embodiments, the ratio of the effective load to the targeting moiety is about 0.1: 1, about 0.2: 1, about 0.3: 1, about 0.4: 1, about 0.5: 1, about 0.6: 1, about 0.7: 1, about 0.8: 1, about 0.9: 1, about 1: 1, about 2: 1, about 3: 1, about 4: 1, about 5: 1, about 6: 1, about 7: 1, about 8: 1, about 9: 1, about 10: 1, about 11: 1, about 12: 1, about 13: 1, about 14: 1, about 15: 1, about 16: 1, about 17: 1, about 18: 1, about 19: 1 or about 20: 1.

[0133] "Dendrimers" are polymeric molecules composed of multiple branched monomers radiating radially from a central core. Due to the structure and synthesis methods used to produce dendrimers, the products from dendrimer synthesis are theoretically monodisperse. When the core of the dendrimer is removed, multiple identical fragments called dendrons are retained, and the number of dendrons depends on the multiplicity of the central core. The number of branching points encountered when moving from the core to the outer edge defines its generation, for example, G-1, G2, G-3, etc., and higher generation dendrimers are larger, more branched, and have more terminal groups than lower generation dendrimers. As used herein, dendrimers are preferably pharmaceutically acceptable dendrimers.

[0134] As used herein, "subject with elevated blood glucose" or "increased blood glucose" is understood to be a subject with elevated blood glucose of sufficient duration and frequency to be considered a pathological state, i.e., a subject who does not produce enough insulin or is not sufficiently sensitive to insulin such that the subject's glucose level remains elevated for an extended period of time after eating, for example, for more than two hours after eating, and / or a subject with elevated fasting blood glucose. In specific embodiments, a subject with elevated blood glucose is understood to be a subject with one or both of a fasting blood glucose of at least 100 mg / dl and a 2-hour blood glucose of at least 140 mg / dl in a 75-g oral glucose tolerance test. In specific embodiments, a subject with elevated blood glucose is understood to be a subject with one or more of a fasting blood glucose of at least 126 mg / dl; a 2-hour blood glucose of at least 200 mg / dl in a 75-g oral glucose tolerance test; or a random blood glucose of at least 200 mg / dl. In certain embodiments, a subject with elevated blood glucose is understood to be a pregnant subject with one or more of a fasting blood glucose of at least 92 mg / dl; a 1-hour blood glucose of at least 180 mg / dl in a 75-g oral glucose tolerance test; and a 2-hour blood glucose of at least 153 mg / dl in a 75-g oral glucose tolerance test. In certain embodiments as used herein, a subject with elevated blood glucose does not include a subject with type 1 diabetes or a pancreatic disease that results in absolute insulin deficiency. In certain embodiments as used herein, a subject with elevated blood glucose includes a subject with type 1 diabetes or a pancreatic disease that results in absolute insulin deficiency.

[0135] As used herein, a "subject with elevated HbA1c" or "subject with elevated A1c" is understood to be a subject having an HbA1c level of at least 5.7%. In a specific embodiment, the subject has an HbA1c level of at least 6.5%.

[0136] As used herein, "diabetes" is intended to refer to type 1 diabetes or type 2 diabetes, or both type 1 and type 2 diabetes, optionally in combination with gestational diabetes. In a specific embodiment, diabetes includes type 2 diabetes. In a specific embodiment, diabetes does not include type 1 diabetes. In a specific embodiment, diabetes includes gestational diabetes. In a specific embodiment, diabetes does not include gestational diabetes. In a specific embodiment, diabetes includes prediabetes. In a specific embodiment, diabetes includes prediabetes, type 1 diabetes, and type 2 diabetes. In a specific embodiment, diabetes includes prediabetes and type 2 diabetes.

[0137] As used herein, "insulin resistance" and "insulin insensitivity" are used interchangeably and refer to a condition, especially a pathological condition, in which the amount of insulin is less effective than in normal subjects in lowering blood glucose, resulting in an increase in blood glucose above the normal range (which is not due to a lack of insulin). Without wishing to be bound by mechanism, the condition is generally associated with reduced signaling through the insulin receptor. Typically, insulin resistance in muscle and adipocytes reduces glucose uptake and storage as glycogen and triglycerides, respectively. Insulin resistance in hepatocytes results in reduced glycogen synthesis and an inability to inhibit glucose production and release into the blood.

[0138] Insulin resistance often exists together with "insulin insufficiency" in the same subject, which also leads to elevated blood sugar levels above the normal range that are not due to a lack of insulin, especially pathological elevations of blood sugar. Insulin insufficiency is a condition associated with a lack of insulin action, in which insulin is present and produced by the body. This is distinct from type 1 diabetes, in which insulin is not produced due to a lack of pancreatic islet cells.

[0139] For the purposes of the methods of the invention, it is not necessary to distinguish whether a subject suffers from insulin resistance / insensitivity, insulin deficiency, or both.

[0140] The term "impaired glucose tolerance" (IGT) or "prediabetes" is used to describe people who have blood glucose levels that fall between normal and hyperglycemia when a glucose tolerance test is given, that is, have abnormal glucose tolerance, for example, pathologically abnormal glucose tolerance. Such people are at a higher risk of developing diabetes, although they are not clinically characterized as having diabetes. For example, impaired glucose tolerance refers to a condition in which the patient has a fasting blood glucose concentration or fasting serum glucose concentration that is higher than 110 mg / dl and lower than 126 mg / dl (7.00 mmol / L), or a 2-hour postprandial blood glucose or serum glucose concentration that is higher than 140 mg / dl (7.78 mmol / L) and lower than 200 mg / dl (11.11 mmol / L). Prediabetes, also known as impaired glucose tolerance or impaired fasting blood glucose, is a major risk factor for developing type 2 diabetes, cardiovascular disease, and death. Much focus has been placed on developing therapeutic interventions that prevent the development of type 2 diabetes by effectively treating prediabetes (Pharmacotherapy, 24:362-71, 2004).

[0141] As used herein, a "pathological" condition reaches a clinically acceptable threshold for a disease or condition. If the pathological condition is not resolved, e.g., blood glucose and / or HbA1c are not normalized, the pathological condition can result in significant adverse effects on the subject, particularly long-term. The pathological condition can be reversed by therapeutic agents, surgery, and / or lifestyle changes. The pathological condition may or may not be chronic. The pathological condition may or may not be reversible. The pathological condition may or may not be terminal.

[0142] "Hyperinsulinemia" is defined as a condition in which a subject has insulin resistance with or without normal blood sugar, in which fasting or postprandial serum or plasma insulin concentrations are elevated above those of a normal lean individual without insulin resistance (i.e., >100 mg / dl in a fasting blood glucose test, or >140 mg / dl in an oral glucose tolerance test).

[0143] The condition of "hyperglycemia" (high blood sugar) is a condition in which blood sugar levels are too high. Typically, hyperglycemia develops when blood sugar levels rise above 180 mg / dl. Symptoms of hyperglycemia include frequent urination, thirst, and weight loss over a long time span.

[0144] The condition of "hypoglycemia" (low blood sugar) is a condition in which the blood sugar level is too low. Typically, hypoglycemia occurs when the blood sugar level drops below 70 mg / dl. Symptoms of hypoglycemia include moodiness, numbness of the limbs (especially in the hands and arms), confusion, tremors, and dizziness. Because this condition occurs when there is excess insulin that exceeds the amount of available glucose, it is sometimes also called an insulin reaction.

[0145] As used herein, "HbA1c level" or "A1c level" is understood to be the hemoglobin A1c (HbA1c) level determined from an HbA1c test, which evaluates the average blood glucose level during the previous two and three months. People without diabetes typically have HbA1c values ​​in the range of 4% to 6%. Prediabetes is characterized by pathological HbA1c levels of 5.7% to 6.5%, wherein HbA1c levels above 6.5% are indicative of diabetes. Each 1% increase in HbA1c reflects an increase in blood glucose levels of approximately 30 mg / dL and an increased risk of complications due to persistent blood glucose elevations. Preferably, the HbA1c value of a patient to be treated according to the present invention is reduced to below 9%, below 7%, below 6%, and most preferably to about 5%. Thus, the excess HbA1c level of the patient to be treated (i.e., an HbA1c level exceeding 5.7%) is preferably reduced by at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or more relative to the level before treatment (i.e., pre-treatment level - post-treatment level / pre-treatment level).

[0146] As used herein, the term "subject" refers to humans and non-human animals, including veterinary subjects. The term "non-human animal" includes all vertebrates, e.g., mammals and non-mammals, such as non-human primates, mice, rabbits, sheep, dogs, cats, horses, cows, chickens, amphibians and reptiles. In a preferred embodiment, the subject is a human and can be referred to as a patient.

[0147] As used herein, the term "treat", "treating" or "treatment" preferably refers to an activity that obtains a beneficial or desired clinical result, including, but not limited to, alleviation or improvement of one or more signs or symptoms of a disease or condition, reduction in the extent of the disease, a stable state of the disease (i.e., no worsening), improvement or alleviation of the disease state. As used herein, treatment may include one or more of reduction in insulin resistance, improvement in insulin sensitivity, reduction in insulin deficiency, improvement or normalization of HbAc1 levels, improvement or normalization of HbAc1 levels, improvement or normalization of blood glucose levels (e.g., postprandial blood glucose levels, fasting blood glucose levels, glucose tolerance), and improvement of at least one sign or symptom of diabetes. Treatment goals in the treatment of diabetes (including type 2 diabetes) include HbAc1 levels of <6.5%; preprandial blood glucose 80-120 mg / dl; and 2-hour postprandial blood glucose <140 mg / dl. Treatment goals for the treatment of prediabetes include reducing HbA1c, blood glucose levels, and glucose responses to normal levels. Treatment does not need to be curative, or to achieve the ideal treatment goal of treatment. The therapeutic outcome does not need to be determined quantitatively. However, in specific embodiments, the therapeutic outcome can be quantified by considering the percentage improvement towards the normal value at the end of the range. For example, metabolic symptoms are characterized by the excess of some measurements (e.g., blood sugar level, HbA1c level) and the deficiency of other measurements (e.g., insulin response). A subject with a fasting blood sugar level of 150 mg / dl will have an excess fasting blood sugar of 50 mg / dl (150 mg / dl-100 mg / dl, the maximum normal blood sugar level). A 20% reduction in excess blood sugar will be a 10 mg / dl reduction in excess blood sugar. Similar calculations can be performed for other values.

[0148] As used herein, "reducing blood sugar levels" means reducing excess blood sugar by at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95% or more to obtain normalized blood sugar levels, i.e., blood sugar levels not higher than 150 mg / dl. Ideally, the blood sugar level before a meal is reduced to a normal blood sugar level, i.e., 150 to 60 mg / dL, 140 to 70 mg / dL, 130 to 70 mg / dL, 125 to 80 mg / dL, and preferably 120 to 80 mg / dL. Such a reduction in blood sugar levels can be obtained by increasing any biological activity associated with glucose clearance of the blood. Therefore, a medicament with the ability to reduce blood sugar levels can increase insulin production, secretion or action. For example, insulin action can be increased by increasing glucose uptake by peripheral tissues and / or by reducing liver glucose production. Alternatively, the agent may reduce intestinal carbohydrate absorption, alter glucose transporter activity (e.g., by increasing GLUT4 expression, intrinsic activity, or translocation), increase the amount of insulin-sensitive tissue (e.g., by increasing myocyte or adipocyte differentiation), or alter adipocyte or myocyte gene transcription (e.g., altering secretion of factors expressed by adipocytes from metabolic pathway genes). Ideally, the agent enhances more than one activity associated with glucose clearance.

[0149] "Altering the insulin signaling pathway so that blood glucose levels are reduced" means altering (by increasing or decreasing) any activity involved in insulin signaling, such that the overall result is an increase in plasma glucose clearance and normalization of blood glucose. For example, altering the insulin signaling pathway, thereby causing an increase in insulin production, secretion or action, increasing glucose uptake in peripheral tissues, decreasing hepatic glucose production or decreasing intestinal carbohydrate absorption.

[0150] A "therapeutically effective amount" is an amount sufficient to treat a disease in a subject. A therapeutically effective amount can be administered in one or more administrations.

[0151] A variety of treatments for type 2 diabetes are known in the art, including both pharmacological and behavioral interventions. Drugs used for the treatment of type 2 diabetes include, but are not limited to, meglitinides (repaglinide (Prandin) and nateglinide (Starlix)); sulfonylureas (glipizide (Glucotrol), glimepiride (Amaryl) and glibenclamide (DiaBeta, Glynase)); dipeptidyl peptidase-4 (DPP-4) inhibitors (saxagliptin (Onglyza), sitagliptin (Januvia) and linagliptin (Tradjenta)); biguanides (metformin (Fortamet, Glucophage)); thiazolidinediones (rosiglitazone (Avandia) and pioglitazone (Actos)); and alpha-glucosidase inhibitors (acarbose (Precose) and miglitol (Glyset)). Insulin is generally used only in the treatment of advanced type 2 diabetes and includes rapid-acting insulins (insulin aspart (NovoLog), insulin glulisine (Apidra), and insulin lispro (Humalog); short-acting insulins (regular insulin (Humulin R, Novolin R)); intermediate-acting insulins (human insulin NPH (Humulin N, Novolin N)) and long-acting insulins (insulin glargine (Lantus) and insulin detemir (Levemir)). Treatment for diabetes may also include behavioral modification, including exercise and weight loss, which may be promoted by the use of medications or surgery. Treatments for elevated blood sugar and diabetes may be combined. For example, medications may be combined with behavioral modification treatments.

[0152] As used herein, "diagnosis" etc. refers to a clinical or other evaluation of a patient's condition based on observation, testing or circumstances and is used to identify a subject as suffering from a disease, disorder or condition based on the presence of at least one indication, such as signs or symptoms of the disease, disorder or condition. Typically, diagnosis using the methods of the present invention includes observation of a subject for multiple indications of a disease, disorder or condition combined with the methods provided herein. Diagnostic methods provide indications of the presence or absence of a disease. A single diagnostic test typically does not provide a definite conclusion about the disease condition of the tested subject.

[0153] As used herein, "monitoring" is understood to mean evaluating at least one sign or symptom of a disease in a subject at a first time point and at a second time point thereafter, comparing the weight or severity of the disease, and determining whether the disease is getting worse or getting better over time.

[0154] The terms "administer," "administering," or "administration" include any method of delivering a pharmaceutical composition or agent to a subject's system or to a specific area in or on a subject. In specific embodiments, the agent is administered enterally or parenterally. In specific embodiments of the invention, the agent is administered intravenously, intramuscularly, subcutaneously, intradermally, intranasally, orally, transdermally, or transmucosally. In certain preferred embodiments, the agent is administered by injection or infusion, e.g., intravenously, intramuscularly, subcutaneously. In certain embodiments, administration includes the use of a pump. In certain embodiments, the agent is administered locally or systemically. Administration of an agent may be performed by multiple people working in collaboration. Administering an agent includes, for example, prescribing the agent to be administered to a subject and / or providing instructions for taking a particular agent directly or by another means (by self-delivery, e.g., by oral delivery, subcutaneous delivery, intravenous delivery through a central line, etc.; or for delivery by a trained person, e.g., intravenous delivery, intramuscular delivery, etc.).

[0155] As used herein, the term "co-administration" refers to the administration of Eno1 before, simultaneously or substantially simultaneously with, after, or intermittently with the administration of an agent for the treatment of diabetes, prediabetes, glucose intolerance, or insulin resistance. The Eno1 preparation provided herein can be used in a combination therapy with at least one other therapeutic agent for the treatment of diabetes, prediabetes, glucose intolerance, or insulin resistance. Eno1 and / or its pharmaceutical preparations and other therapeutic agents can act additively or more preferably synergistically. In one embodiment, Eno1 and / or its preparations are administered simultaneously with the administration of another therapeutic agent for the treatment of diabetes, prediabetes, glucose intolerance, or insulin resistance. In another embodiment, Eno1 and / or its preparations are administered before or after the administration of another therapeutic agent for the treatment of diabetes, prediabetes, glucose intolerance, or insulin resistance.

[0156] As used herein, the term "sample" refers to a collection of similar fluids, cells or tissues separated from a subject. The term "sample" includes any body fluid (e.g., urine, serum, blood, lymph, gynecological fluid, cystic fluid, ascites fluid, intraocular fluid and fluid collected by bronchial lavage and / or peritoneal lavage), ascites, tissue samples or cells from a subject. Other subject samples include tear drops, serum, cerebrospinal fluid, feces, sputum and cell extracts. In specific embodiments, the sample is urine or serum. In specific embodiments, the sample includes cells. In other embodiments, the sample does not include cells.

[0157] As used herein, the term "control sample" refers to any clinically relevant comparative sample, including, for example, a sample from a healthy subject who does not suffer from any of impaired glucose tolerance, increased blood sugar, insulin resistance, diabetes or prediabetes; or a sample from a subject at an earlier time point in the subject (e.g., before treatment, at an earlier stage of treatment). The control sample can be a purified sample, protein and / or nucleic acid provided with a kit. Such a control sample can be diluted, for example, in a serial dilution to allow quantitative measurement of the analyte in the test sample. The control sample can include a sample derived from one or more subjects. The control sample can also be a sample prepared from a subject to be evaluated at an earlier time point. For example, the control sample can be a sample obtained from a subject to be evaluated before the onset of abnormal blood sugar levels or A1c levels, at an earlier stage of the disease, or before administering a treatment or partial treatment. The control sample can also be a sample from an animal model or from a tissue or cell line derived from an animal model, the animal model being an animal model of impaired glucose tolerance, increased blood sugar, insulin resistance, diabetes or prediabetes. The level of Eno1 activity or expression in a control sample consisting of a set of measurements can be determined, for example, based on any suitable statistical measure, such as, for example, a measure of central tendency including the mean, median or mode value.

[0158] The term "control level" refers to a recognized or predetermined level of signs of impaired glucose tolerance, elevated blood sugar, insulin resistance, diabetes or prediabetes in a subject or subject sample. The following levels are considered normal levels:

[0159] --Fasting blood glucose less than or equal to 100 mg / dl.

[0160] --HbA1c less than or equal to 5.7%.

[0161] --Oral glucose tolerance test less than or equal to 140 mg / dl.

[0162] Levels above these levels are understood to be pathological.

[0163] As used herein, the "predetermined threshold value" of a biomarker refers to a biomarker (e.g., expression level or amount (e.g., ng / ml) in a biological sample) or other indicator level of elevated blood glucose in a corresponding control / normal sample or control / normal sample group obtained from a normal or healthy subject (e.g., a subject without abnormal blood glucose). The predetermined threshold value can be determined before or simultaneously with the measurement of the marker level in the biological sample. The control sample can be from the same subject or from different subjects at a previous time.

[0164] As used herein, the sample obtained at "early time point" is a sample obtained in the past sufficient time, so that clinically relevant information can be obtained in the sample from the earlier time point compared to the later time point. In a specific embodiment, the earlier time point is at least four weeks earlier. In a specific embodiment, the earlier time point is at least six weeks earlier. In a specific embodiment, the earlier time point is at least two months earlier. In a specific embodiment, the earlier time point is at least three months earlier. In a specific embodiment, the earlier time point is at least six months earlier. In a specific embodiment, the earlier time point is at least nine months earlier. In a specific embodiment, the earlier time point is at least one year earlier. Multiple subject samples (e.g., 3, 4, 5, 6, 7 or more) can be obtained with time at regular or irregular intervals, and the trend of marker level changes can be analyzed. Suitable intervals for testing specific subjects can be determined by those skilled in the art based on routine considerations.

[0165] As used herein, the term "acquire" is understood to mean make, buy, or otherwise possess.

[0166] As used herein, "detecting", "detection", etc. are understood to refer to an assay performed to identify a specific analyte in a sample, for example, the level of Eno1 expression or activity in a sample. The amount of analyte or activity detected in a sample may be zero or below the detection level of the assay or method. Detection or detection may also include measurement of glucose and / or HbAc1 levels.

[0167] The term "modulate" or "modulation" refers to upregulation (i.e., activation or stimulation), downregulation (i.e., inhibition or suppression), or both (in combination or alone) of levels. A "modulator" is a compound or molecule that performs regulation and can be, for example, an agonist, antagonist, activator, stimulator, repressor, or inhibitor.

[0168] The term "expression" is used herein to refer to the process by which a polypeptide is produced from DNA. The process includes transcribing a gene into mRNA and translating this mRNA into a polypeptide. Depending on the context in which it is used, "expression" can refer to the production of RNA, protein, or both.

[0169] The term "expression level of a gene" or "gene expression level" refers to the level of mRNA and pre-mRNA nascent transcription products, transcription product processing intermediates, mature mRNA and degradation products, or the level of protein encoded by a gene in a cell.

[0170] As used herein, the term "amplification" refers to any known in vitro process for obtaining multiple copies ("amplicons") of a target nucleic acid sequence or its complementary sequence or fragment. In vitro amplification refers to the production of an amplified nucleic acid that may contain less than the complete target region sequence or its complementary sequence. Known in vitro amplification methods include, for example, transcription-mediated amplification, replicase-mediated amplification, polymerase chain reaction (PCR) amplification, ligase chain reaction (LCR) amplification, and strand displacement amplification (SDA, including multiple strand displacement amplification method (MSDA)). Replicase-mediated amplification uses self-replicating RNA molecules and replicase, such as Q-β-replicase (e.g., Kramer et al., U.S. Patent No. 4,786,600). PCR amplification is well known and uses DNA polymerase, primers, and thermal cycles to synthesize multiple copies of two complementary chains of DNA or cDNA (e.g., Mullis et al., U.S. Patent Nos. 4,683,195, 4,683,202, and 4,800,159). LCR amplification uses at least four separate oligonucleotides to amplify a target and its complementary strand by using multiple cycles of hybridization, ligation, and denaturation (e.g., European Patent Application Publication No. 0 320 308). SDA is a method in which primers contain a recognition site for a restriction endonuclease that allows the endonuclease to cleave one strand of a hemimodified DNA duplex that includes the target sequence, followed by a series of primer extension and strand displacement steps to amplify (e.g., Walker et al., U.S. Pat. No. 5,422,252). Two other known strand displacement amplification methods do not require endonuclease cleavage (Dattagupta et al., U.S. Pat. No. 6,087,133 and U.S. Pat. No. 6,124,120 (MSDA)). Those skilled in the art will appreciate that the oligonucleotide primer sequences of the present invention can be readily used in any in vitro amplification method based on primer extension by a polymerase (see generally Kwoh et al., 1990, Am. Biotechnol. Lab. 8: 14-25 and Kwoh et al., 1989, Proc. Natl. Acad. Sci. USA 86, 1173-1177; Lizardi et al., 1988, BioTechnology 6: 1197-1202; Malek et al., 1994, Methods Mol. Biol., 28: 253-260 and Sambrook et al., 2000, Molecular Cloning--A Laboratory Manual (Molecular Cloning--Laboratory Manual), 3rd Edition, CSH Laboratories). As is well known in the art, oligomers are designed to bind to complementary sequences under selected conditions.

[0171] As used herein, the term "antigen" refers to a molecule, such as a peptide, polypeptide, protein, fragment or other biological moiety, that elicits an antibody response in a subject or is recognized and bound by an antibody.

[0172] As used herein, the term "complementary" refers to the broad concept of sequence complementarity between the regions of two nucleic acid chains or between two regions of the same nucleic acid chain. It is known that the adenine residues of the first nucleic acid region can form specific hydrogen bonds ("base pairing") with the residues of the second nucleic acid region (which is antiparallel to the first region) (if the residue is thymine or uracil). Similarly, it is known that the cytosine residues of the first nucleic acid chain can base pair with the residues of the second nucleic acid chain (which is antiparallel to the first chain) (if the residue is guanine). If at least one nucleotide residue in the first region can base pair with the residues in the second region when the two regions are arranged in an antiparallel manner, the first region of the nucleic acid is complementary to the second region of the same or different nucleic acids. Preferably, the first region includes the first part and the second region includes the second part, whereby when the first and second parts are arranged in an antiparallel manner, at least about 50% of the first part, and preferably at least about 75%, at least about 90%, or at least about 95% of the nucleotide residues can base pair with the nucleotide residues in the second part. More preferably, all nucleotide residues in the first part can base pair with the nucleotide residues in the second part.

[0173] As used herein, the phrase "specific binding" or "specifically binds" when used in conjunction with the interaction of an antibody with a protein or peptide means that the interaction is dependent on the presence of a specific structure (i.e., an antigenic determinant or epitope) on the protein; in other words, the antibody recognizes and binds to a specific protein structure rather than the protein as a whole. For example, if the antibody is specific for epitope "A", the presence of a protein containing epitope A (or free, unlabeled A) in a reaction containing labeled "A" and the antibody will reduce the amount of labeled A bound to the antibody.

[0174] The phrase "specific identification" is understood to mean detection of a target marker with sufficiently low analytical background and cross-reactivity of the reagents used so that the detection method is diagnostically useful. In a specific embodiment, the reagents used for the specific identification of a marker bind only one isoform of the marker. In a specific embodiment, the reagents used for the specific identification of a marker bind more than one isoform of the marker. In a specific embodiment, the reagents used for the specific identification of a marker bind all known isoforms of the marker.

[0175] As used herein, the phrase "subject suspected of elevated blood sugar" refers to a subject who has one or more signs or symptoms indicating elevated blood sugar or associated with elevated blood sugar, or is being screened for elevated blood sugar (e.g., in a routine physical examination). Subjects suspected of elevated blood sugar may also have one or more risk factors. Subjects suspected of elevated blood sugar have generally not been tested for abnormal glucose levels, metabolism, or reactions. However, "subjects suspected of elevated blood sugar" include individuals who have received a preliminary diagnosis (e.g., a single occurrence of elevated blood sugar, but not confirmed) but whose degree of elevated blood sugar is unknown. The term further includes people who have had elevated blood sugar (e.g., individuals who have maintained normal blood sugar and / or HbA1c levels for an extended period of time for treatment of elevated blood sugar, e.g., at least 3 months, at least 6 months, etc.).

[0176] The articles "a", "an", and "the" are used herein to refer to one or more than one (i.e., at least one) of the grammatical object of the article, unless otherwise clearly indicated to the contrary. For example, "an element" means one element or more than one element.

[0177] The term "including" is used herein to mean, and is used interchangeably with, the phrase "including but not limited to".

[0178] The term "or" is used herein to mean, and is used interchangeably with, the term "and / or," unless the context clearly indicates otherwise.

[0179] The term "such as" is used herein to mean, and is used interchangeably with, the phrase "such as, but not limited to".

[0180] Unless expressly stated or apparent from the context, as used herein, the term "about" is understood to be within the normal tolerance range in the art, e.g., within 2 standard deviations of the mean. Approximately can be understood to be within 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, 0.5%, 0.1%, 0.05%, or 0.01% of the stated value. Unless otherwise apparent from the context, all numerical values ​​provided herein are modified by the term about.

[0181] The enumeration of a list of chemical groups in any definition of a variable herein includes definitions of that variable as any single group or combination of the listed groups. The enumeration of embodiments for a variable or aspect herein includes that embodiment as any single embodiment or in combination with any other embodiment or portion thereof.

[0182] Any composition or method provided herein can be combined with one or more of any other compositions and methods provided herein.

[0183] 45,46,47,48,49,50,51,52,53,54,55,56,57,58,59,60,61,62,63,64,65,66,67,68,69,70,71,72,73,74,75,76,77,78,79,80,81,82,83,84,85,86,87,88,89,90,91,92,93,94,100,101,102,103,104,105,106,107,108,109,110,111,112,113,114,115,116,117,118,119,120,

[0184] The preferred embodiments of the present invention will now be described in detail. Although the present invention will be described in conjunction with the preferred embodiments, it will be understood that it is not intended to limit the present invention to those preferred embodiments. On the contrary, it is intended to encompass alternatives, changes and equivalents, as may be included in the spirit and scope of the present invention as defined by the appended claims.

[0185] IIA. Enolase 1

[0186] Enolase 1, (α), also known as ENO1L, α-enolase, enolase-α, tau-crystallin, non-neural enolase (NNE), α-enolase-like 1, phosphopyruvate hydratase (PPH), plasminogen binding protein, MYC promoter binding protein 1 (MPB1) and 2-phospho-D-glycerol hydrolase, is one of three enolase isozymes found in mammals. Each isozyme is a homodimer consisting of 2 α, 2 γ or 2 β subunits and acts as a glycolytic enzyme. In addition, α-enolase acts as a structural crystal-like protein (tau-crystallin) in monomeric form. Variable splicing of this gene obtains shorter isoforms, which have been shown to bind to the c-myc promoter and act as a tumor suppressor. Several pseudogenes have been identified, including one on the long arm of chromosome 1. α-Enolase has also been identified as an autoantigen in Hashimoto's encephalopathy. More information about human Eno1 can be found, for example, in the NCBI gene database under Gene ID No. 2023 (see, www.ncbi.nlm.nih.gov / gene / 2023, incorporated herein by reference in the version available on the filing date of this application).

[0187] Eno1 variants

[0188] Two isoforms of human Eno1 are known. The protein and mRNA sequence of human enolase 1, (α) (ENO1), transcript variant 1, mRNA can be found in GenBank Accession No. NM_001428 (see www.ncbi.nlm.nih.gov / nuccore / NM_001428.3, which is incorporated herein by reference in the version available on the date of filing of this application). This variant encodes a longer isoform, which is localized in the cytosol and has α-enolase activity. It has been reported that the monomer form of this isoform plays a role as a structural crystal protein (tau-crystal protein), while the dimer form plays a role as an enolase. In a preferred embodiment of the present invention, Eno1 is a transcript variant of Eno1.

[0189] The protein and mRNA sequence of human enolase 1, (α) (ENO1), transcript variant 2, mRNA can be found at GenBank Accession No. NM_001201483 (see www.ncbi.nlm.nih.gov / nuccore / NM_00001201483.1, which is incorporated herein by reference in the version available on the filing date of this application). This variant differs from variant 1 at the 5' end and initiates translation from an in-frame downstream start codon, thereby producing a shorter isoform (MBP-1). This isoform is localized to the nucleus and acts as a transcriptional repressor of the c-myc proto-oncogene by binding to its promoter. In a specific embodiment of the invention, Eno1 is transcript variant 2 of Eno1.

[0190] For example, several other variants of the Eno1 protein are described in the UniProtKB / Swiss-Prot database under accession No. P06733. Examples of variants of the Eno1 protein are shown in Table 1 below.

[0191] Table 1. Eno1 variants

[0192]

[0193]

[0194]

[0195] In a specific embodiment of the invention, Eno1 is one of the variants listed in Table 1.

[0196] Eno1 activity

[0197] Eno1 is a key glycolytic enzyme that catalyzes the dehydration of 2-phospho-D-glycerate (PGA) to phosphoenolpyruvate (PEP) in the final step of the glycolytic pathway. Diaz-Ramos et al., 2012, J Biomed Biotechnol. 2012: 156795 and Fig.33 . Enolase catalyzes the dehydration of PGA to PEP in the Emden Mayerhoff-Parnas glycolytic pathway (catabolic direction). In the anabolic pathway (reverse reaction) during gluconeogenesis, Eno1 catalyzes the hydration of PEP to PGA. Therefore, Eno1 is also called phosphopyruvate hydratase. Metal ions are cofactors that impair the increase in enolase activity; therefore, Eno1 is also called metal-activated metalloenzyme. Magnesium is the natural cofactor that causes the highest activity and is required for the enzyme to have catalytic activity. The relative activation intensity spectrum of other metal ions involved in the enzyme activity shows the following order: Mg 2+ >Zn 2+ >Mn 2+ >Fe(II) 2+ >Cd 2+ >Co 2+ 、Ni 2+ 、Sm 3+ , Tb 3+ and most other divalent metal ions. In the reaction catalyzed by enolase, the α-proton from the carbon adjacent to the carboxylic acid group of PGA is extracted and PGA is converted to an enolate anion intermediate. This intermediate is further processed in a variety of chemical reactions, including racemization, cycloisomerization, and β-elimination of water or ammonia. See Atlas of Genetics and Cytogenetics in Oncology and Haematology database, atlasgeneticsoncology.org / Genes / GC_ENO1.Html.

[0198] Enzymatically active enolase exists in a dimeric form (homo- or heterodimer) and consists of two subunits facing each other in an antiparallel manner. The crystal structures of enolase from yeast and humans have been determined and the catalytic mechanism has been proposed. Diaz-Ramos et al., cited above. The five residues involved in the catalytic activity of this enzyme are highly conserved throughout evolution. In vitro studies have revealed that different mutant enolases at positions Glu168, Glu211, Lys345, Lys396 or His159 show significantly reduced activity levels. The components and conserved parts of enolase are two Mg2+ ions that participate in the conformational changes of the enolase active site and enable binding to substrates or their analogs. Atlas of Genetics and Cytogenetics in Oncology database, cited above. In a specific embodiment, the composition of the present invention comprises a metal ion cofactor. The metal ion cofactor can provide Eno1 with improved stability in the composition and / or improved Eno1 activity in vivo. In one embodiment, the metal ion cofactor is divalent. In one embodiment, the divalent metal ion cofactor is Mg 2+ 、Zn 2+ , Mn 2+ 、Fe(II) 2+ 、Cd 2+ 、Co 2+ or Ni 2+ In one embodiment, the metal ion cofactor is trivalent, for example, Sm 3+ or Tb3 + .

[0199] For example, Eno1 activity can be measured using a pyruvate kinase (PK) / lactate dehydrogenase (LDH) assay. The reaction for this enolase assay is shown below.

[0200]

[0201] 2-Phospho-D-glycerate (DPG)

[0202] The conversion of NADH to NAD can be determined, for example, by measuring the decrease in NADH fluorescence using a PTI Quantamaster 40 spectrophotometer from Photon Technology International, Inc. (pti-nj.com). +Reaction rate of conversion. Kits for measuring Eno1 activity by the colorimetric pyruvate kinase / lactate dehydrogenase assay are also commercially available, for example, from ABCAM (Cambridge, MA; Catalog No. ab117994). The ABCAM Eno1 activity assay is further described in Example 5 below.

[0203] As also described in Example 2, Enol activity was determined by measuring the effect of Eno1 on glucose uptake in human skeletal muscle myotubes (HSMM).

[0204] In certain embodiments, Eno1 or a fragment thereof has at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 110%, 120%, 130%, 140%, 150%, 160%, 170%, 180%, 190%, 200%, 300%, 400% or 500% of the activity of a purified endogenous human Eno1 polypeptide. In certain embodiments, the activity of Eno1, its fragments and purified endogenous human Eno1 polypeptide is determined by the pyruvate kinase / lactate dehydrogenase assay or HSMM glucose uptake assay described above.

[0205] In certain embodiments, the Eno1 polypeptide complexed with a dendrimer as described herein has at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 110%, 120%, 130%, 140%, 150%, 160%, 170%, 180%, 190%, 200%, 300%, 400% or 500% of the activity of a purified endogenous Eno1 polypeptide not complexed with a dendrimer. In certain embodiments, the activity of the Eno1 polypeptide complexed with a dendrimer and the activity of the purified endogenous Eno1 polypeptide not complexed with a dendrimer are determined by the pyruvate kinase / lactate dehydrogenase assay or the HSMM glucose uptake assay described above.

[0206] In certain embodiments, Eno1 complexed with a dendrimer and a targeting peptide as described herein has at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 110%, 120%, 130%, 140%, 150%, 160%, 170%, 180%, 190%, 200%, 300%, 400% or 500% of the activity of a purified endogenous human Eno1 polypeptide not complexed with a dendrimer and a targeting peptide. In certain embodiments, the activity of the Eno1 polypeptide complexed with a dendrimer and a targeting peptide and the activity of the purified endogenous human Eno1 polypeptide not complexed with a dendrimer and a targeting peptide are determined by the pyruvate kinase / lactate dehydrogenase assay or the HSMM glucose uptake assay described above.

[0207] In one embodiment, Eno1 or a fragment thereof in the composition of the present invention (wherein the composition comprises a metal ion cofactor (e.g., a divalent metal ion cofactor, such as Mg 2+ 、Zn 2+ , Mn 2+ 、Fe(II) 2+ 、Cd 2+ 、Co 2+ or Ni 2+ , or trivalent metal ion cofactors such as Sm 3+ or Tb3 + )) has at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 110%, 120%, 130%, 140%, 150%, 160%, 170%, 180%, 190%, 200%, 300%, 400% or 500% of the activity of a purified endogenous human Eno1 polypeptide. In a specific embodiment, the activity of Eno1 or a fragment thereof in a composition of a metal ion cofactor as described above and the activity of a purified endogenous human Eno1 polypeptide are determined by the above-mentioned pyruvate kinase / lactate dehydrogenase assay or HSMM glucose uptake assay.

[0208] Glucose flow

[0209] Regulation of muscle glucose uptake involves a three-step process consisting of: (1) glucose delivery to muscle, (2) glucose transport to muscle via glucose transporter GLUT4 and (3) intramuscular glucose phosphorylation by hexokinase (HK). Physiological regulation of muscle glucose uptake requires glucose to move from the blood to the interstitium and to the intracellular space, and then to be phosphorylated to G6P. Blood glucose concentration, muscle blood flow and recruitment of capillaries to muscle determine the movement of glucose from the blood to the interstitium. Plasma membrane GLUT4 content controls glucose transport into cells. Muscle hexokinase (HK) activity, cellular HK compartmentalization and the concentration of HK inhibitor (G6P) determine the ability of glucose phosphorylation. These three steps - glucose delivery, transport and phosphorylation - include glucose flow, and all three steps are important for glucose flow control. However, steps downstream of glucose phosphorylation can also affect glucose uptake. For example, acceleration of glycolysis or glycogen synthesis can reduce G6P, increase HK activity, increase the ability of glucose phosphorylation, and potentially stimulate muscle glucose uptake. Wasserman et al., 2010, J Experimental Biology, Vol. 214, pp. 254-262.

[0210] The present invention is based, at least in part, on the discovery that Eno1 affects several components of the glucose flux pathway, including increasing the expression of glucose transporters GLUT1 and GLUT4 and hexokinase HK2, and increasing the levels of glycolytic pathway intermediates G6P and PEP, thus indicating that Eno1 treatment acts to increase glucose flux.

[0211] The present invention is also based, at least in part, on the discovery that Eno1 is differentially regulated in myocytes from normal subjects and in myocytes from subjects with type 2 diabetes. The present invention is further based on the surprising discovery that treatment of myocytes with Eno1 increases glucose uptake into the cells and that administration of Eno1 to diet-induced obese mice normalizes glucose tolerance and insulin responses.

[0212] Therefore, the present invention provides a method for treating elevated blood sugar commonly associated with diabetes (including at least type 1 diabetes, prediabetes, type 2 diabetes and gestational diabetes) by administering Eno1 to a subject. In addition, the present invention provides a method for diagnosing and / or monitoring (e.g., monitoring disease progression or treatment) and / or prognosing elevated blood sugar states (e.g., diabetes) in mammals. The present invention also provides a method for treating or adjusting a treatment regimen based on diagnostic information about Eno1 levels in the blood or serum of a subject with elevated blood sugar. The present invention further provides a reagent set and a kit for implementing the methods of the present invention.

[0213] The present invention also provides a method for increasing glucose flow in a subject, comprising administering a pharmaceutical composition comprising Eno1 or a fragment thereof to the subject. In a specific embodiment, the pharmaceutical composition administered to the subject is any pharmaceutical composition described herein. The present invention also provides a method for increasing glucose flow in skeletal muscle cells of a subject, comprising administering a pharmaceutical composition comprising Eno1 or a fragment thereof to the subject. In a specific embodiment, the pharmaceutical composition administered to the subject is any pharmaceutical composition described herein.

[0214] The present invention also provides a method for increasing glycolytic activity in skeletal muscle cells of a subject, the method comprising administering a pharmaceutical composition comprising Eno1 or a fragment thereof to the subject. In a specific embodiment, the pharmaceutical composition administered to the subject is any pharmaceutical composition described herein.

[0215] The present invention also provides a method of increasing mitochondrial free fatty acid oxidation in skeletal muscle cells of a subject, the method comprising administering a pharmaceutical composition comprising Eno1 or a fragment thereof to the subject. In certain embodiments, the pharmaceutical composition administered to the subject is any pharmaceutical composition described herein.

[0216] As used herein, "increasing glucose flux" is understood to be increasing at least one or more of (1) glucose delivery to muscle, (2) glucose transport into muscle, and (3) glucose phosphorylation within muscle. In certain embodiments, increasing glucose flux comprises increasing glycolytic activity or mitochondrial free fatty acid oxidation in muscle cells.

[0217] IIA. Enolase 2 and Enolase 3

[0218] Enolase 2 (Eno2) is also known as gamma enolase, neuronal enolase, neuron-specific enolase (NSE) or HEL-S-279 and is encoded by the ENO2 gene. Eno2 is a phosphopyruvate phosphatase, a glycolytic enzyme. Eno2 is a homodimer and is found in mature neurons and cells of neuronal origin in the central nervous system (CNS). Neurons release Eno2 into the systemic circulation under various types of stress. Yee et al., 2012, Invest. Ophthalmol. Vis. Sci. Vol. 53, No. 10, pp. 6389-6392. The nucleic acid sequence of ENO2 mRNA and the amino acid sequence of Eno2 are shown in Fig.36A and 36B middle.

[0219] Enolase 3 (Eno3) is also known as beta enolase, muscle enolase, muscle-specific enolase (MSE) or GSD13 and is encoded by the ENO3 gene. Eno3 catalyzes the interconversion of 2-phosphoglycerate and phosphoenolpyruvate. Eno3 is found in adult skeletal muscle cells, where it plays a role in muscle development and regeneration. In adult muscle, more than 90% of enolase activity is caused by Eno3. Mutations in the gene encoding Eno3 are associated with glycogen storage disease. Comi et al., 2001, Ann Neurol. Vol. 50, No. 2, pp. 202-207. Three variants of ENO3 mRNA have been identified, variants 1, 2 and 3. Variants 1 and 2 encode isoform 1 of Eno3 protein, while variant 3 encodes isoform 2 of Eno3 protein. Compared with variant 1, variant 3 of ENO3 mRNA is different in 5'UTR and lacks two exons in the 5' coding region. Isoform 2 of the Eno3 protein is shorter than isoform 1, but has identical N- and C-termini. The nucleic acid sequences of variants 1, 2, and 3 of Eno3 and the amino acid sequences of isoforms 1 and 2 are shown in Figures 37A-37E middle.

[0220] Eno2 and / or Eno3 can also replace Eno1 for the methods, pharmaceutical compositions, reagent groups and kits described herein. For example, Eno2 and / or Eno3 can be used for treating the elevated blood sugar levels usually associated with diabetes (including at least type 1 diabetes, prediabetes, type 2 diabetes and gestational diabetes) by applying a pharmaceutical composition comprising Eno2 and / or Eno3 to a subject. In addition, Eno2 and / or Eno3 can be used for diagnosing and / or monitoring (e.g., monitoring disease progression or treatment) and / or prognosis of elevated blood sugar states (e.g., diabetes) in mammals. Eno2 and / or Eno3 can also be used for treating or regulating a treatment regimen based on diagnostic information of Eno2 or Eno3 levels in the blood or serum of a subject with elevated blood sugar, as well as for implementing the reagent group and kit of the present invention. The present invention also relates to a pharmaceutical composition comprising Eno2 and / or Eno3, for example, for delivery to muscle cells.

[0221] III. Diabetes Diagnosis and Classification

[0222] Diabetes mellitus (DM), often referred to simply as diabetes, is a group of metabolic diseases in which an individual has high blood sugar, either because the body does not produce enough insulin or because the cells cannot respond to the insulin that is produced. This high blood sugar produces the classic symptoms of polyuria (frequent urination), polydipsia (increased thirst), and polyphagia (increased hunger).

[0223] Type 2 diabetes is caused by insulin resistance, a condition in which cells cannot properly use insulin, sometimes combined with an absolute insulin deficiency. The defect in the responsiveness of body tissues to insulin is believed to involve, at least in part, the insulin receptors. However, the specific defect is unknown.

[0224] In the early stages of type 2 diabetes, the primary abnormality is decreased insulin sensitivity. At this stage, high blood sugar levels can be reversed by a variety of measures and medications that increase insulin sensitivity or decrease glucose production by the liver. Prediabetes refers to a condition in which an individual's blood sugar levels are higher than normal but not high enough to warrant a diagnosis of type 2 diabetes.

[0225] Type 2 diabetes is due to insufficient insulin production by beta cells in the case of insulin resistance. Insulin resistance (which is the inability of cells to respond appropriately to normal levels of insulin) occurs primarily in muscle, liver, and adipose tissue. In the liver, insulin normally inhibits glucose release. However, in the case of insulin resistance, the liver inappropriately releases glucose into the blood. The ratio of insulin resistance to beta cell dysfunction varies between individuals, with some individuals having primarily insulin resistance and only a minor defect in insulin secretion, while other individuals have mild insulin resistance and primarily a lack of insulin secretion.

[0226] Other potentially important mechanisms associated with type 2 diabetes and insulin resistance include: increased lipid breakdown within adipocytes, resistance to and deficiency of incretins, high glucagon levels in the blood, increased salt and water retention by the kidneys, and inappropriate regulation of metabolism by the central nervous system. However, not all people with insulin resistance develop diabetes, as impairment of insulin secretion by the pancreatic beta cells is also required.

[0227] Type 1 diabetes is caused by the body's inability to produce insulin and currently requires treatment with insulin injections. Type 1 diabetes is characterized by the loss of insulin-producing beta cells in the pancreas, the islets of Langerhans, resulting in insulin deficiency. Most affected people are otherwise healthy and have a healthy weight when the disease begins. Sensitivity and responsiveness to insulin are usually normal, especially in the early stages. However, especially in the later stages, insulin resistance may occur, including insulin resistance caused by clearance of administered insulin by the immune system.

[0228] A. Diagnostic criteria

[0229] The American Diabetes Association discloses the diagnosis and classification criteria for diabetes in Diabetes Care, 36: S67-74, 2013, which is incorporated by reference into the text, which provides more detailed definitions of various types of diabetes. The diagnostic criteria for diabetes are further discussed below. The references are classified as type 1 diabetes or type 2 diabetes as follows:

[0230] I. Type 1 diabetes (β-cell destruction, usually leading to absolute insulin deficiency)

[0231] A. Immune-mediated

[0232] B. Idiopathic

[0233] II. Type 2 diabetes (can range from predominantly insulin resistance with relative insulin deficiency to predominantly secretory defect with insulin resistance)

[0234] III. Other specific types

[0235] IV. Gestational diabetes

[0236] Methods for performing diagnostic or evaluation methods are provided herein. The diagnostic criteria for diabetes provided herein are as follows:

[0237] Criteria used for the diagnosis of diabetes

[0238]

[0239]

[0240] *In the absence of unequivocal hyperglycemia, criteria 1-3 should be confirmed by repeat testing.

[0241] The diagnostic criteria for increased risk of diabetes / prediabetes provided in this article are as follows:

[0242] Criteria for Increased Risk of Diabetes (Prediabetes)*

[0243]

[0244] *For all three tests, the risk is continuous, extending below the lower end of the range and becoming disproportionately high at the high end of the range.

[0245] The diagnostic criteria for gestational diabetes mellitus provided in this article are as follows:

[0246] Screening and diagnosis of gestational diabetes mellitus (GDM)

[0247]

[0248]

[0249] Blood glucose measurement for diagnosis and / or monitoring of elevated blood glucose or diabetes can be cumbersome due to specific time requirements relative to eating (e.g., fasting blood glucose) or the amount of time required to perform the test (e.g., with an oral glucose tolerance test). In addition, the diagnostic criteria clearly require that in the absence of clear hyperglycemia, criteria 1-3 should be confirmed by repeat testing. Using HbA1c levels as a diagnostic indicator can be advantageous because it provides an indication of blood glucose levels over time, i.e., about 1-2 months prior, and does not require a special schedule for testing. Similarly, Eno1 levels can be determined without special schedule requirements or food consumption restrictions or requirements.

[0250] Thus, in some aspects, the present invention relates to a method for diagnosing the presence of elevated blood glucose in a subject, comprising: (a) contacting a biological sample with an agent that selectively binds to Eno1; (b) allowing a complex to form between the agent and Eno1; (c) detecting the level of the complex, and (d) comparing the level of the complex to a predetermined threshold, wherein a level of the complex in the sample below the predetermined threshold indicates that the patient is suffering from elevated blood glucose. In a specific embodiment, the agent that selectively binds to Eno1 is an anti-Eno1 antibody. In a specific embodiment, the antibody comprises a detectable label.

[0251] In some embodiments of the above method, the step of detecting the level of the complex further comprises contacting the complex with a detectable second antibody and measuring the level of the second antibody. The method may further comprise detecting the level of one or more other indicators of elevated blood glucose. The one or more other indicators of blood glucose may be selected from HbA1c levels, fasting glucose levels, postprandial glucose levels, and glucose tolerance.

[0252] In some embodiments of the above methods, the biological sample is blood or serum. In some embodiments, the level of the complex is detected by immunoassay or ELIS. In some embodiments, the presence of elevated blood glucose in the subject indicates a disease or condition selected from prediabetes, type 2 diabetes, type 1 diabetes, and gestational diabetes.

[0253] B. Secondary pathologies of diabetes, insulin resistance, and insulin insufficiency

[0254] Abnormal blood sugar regulation caused by diabetes (both type 1 and type 2), insulin resistance and insufficient insulin is associated with secondary pathology, many of which are caused by poor circulation. Such secondary pathology includes macular degeneration, peripheral neuropathy, ulcers and reduced wound healing and reduced renal function. It has been shown that glucose levels and / or HbAc1 levels are maintained within the normal range to reduce the occurrence of these secondary pathologies. It should be understood that the normalization of blood sugar, insulin and HbAc1 levels will reduce the occurrence of secondary pathology by limiting primary pathology (e.g., impaired glucose tolerance, increased blood sugar). In a specific embodiment, Enol is not used to treat the secondary pathology related to impaired glucose tolerance, increased blood sugar, insulin resistance, insufficient insulin, diabetes or prediabetes. In a specific embodiment, Enol is used to treat the secondary pathology related to impaired glucose tolerance, increased blood sugar, insulin resistance, insufficient insulin, diabetes or prediabetes.

[0255] IV. Dosage and Mode of Administration

[0256] The techniques and dosages of administration vary depending on the type of compound (eg, protein and / or nucleic acid, alone or complexed with microparticles, liposomes or dendrimers) and are well known or readily determined by those skilled in the art.

[0257] The therapeutic compounds of the present invention can be administered in unit dosage form together with a pharmaceutically acceptable diluent, carrier or excipient. Administration can be parenteral, intravenous, subcutaneous, oral, external (topical) or local (local). In a specific embodiment, administration is not oral. In a specific embodiment, administration is not external. In a specific preferred embodiment, administration is systemic. Medicament administration can be performed by multiple personnel working in collaboration. Administration of medicaments includes, for example, prescribing a prescription for a medicament to be administered to a subject and / or providing instructions for taking a specific medicament directly or by another person, either by self-delivery, for example, as oral delivery, subcutaneous delivery, intravenous delivery through a central line, etc., or by trained professionals, for example, intravenous delivery, intramuscular delivery, subcutaneous delivery, etc.

[0258] The composition may be in the form of a pill, tablet, capsule, liquid or sustained release tablet for oral administration; or a liquid for intravenous, subcutaneous or parenteral administration; or a polymer or other sustained release vehicle for systemic administration.

[0259] Methods for preparing formulations known in the art can be found in, for example, "Remington: The Science and Practice of Pharmacy" (20th edition, editor A.R. Gennaro, 2000, Lippincott Williams & Wilkins, Philadelphia, Pa). Formulations for parenteral administration can, for example, contain excipients, sterile water, saline, polyalkylene glycols (such as polyethylene glycol), oils of plant origin, or hydrogenated naphthalene. Biocompatible, biodegradable lactide polymers, lactide / glycolide copolymers, or polyoxyethylene-polyoxypropylene copolymers can be used to control the release of the compound. Nanoparticle formulations (e.g., biodegradable nanoparticles, solid lipid nanoparticles, liposomes) can be used to control the biodistribution of the compound. Other potentially useful parenteral delivery systems include ethylene-vinyl acetate copolymer particles, osmotic pumps, implantable infusion systems, and liposomes. The concentration of the compound in the formulation varies according to a variety of factors, including the dose and route of administration of the drug to be administered.

[0260] The compound can optionally be administered as a pharmaceutically acceptable salt, such as a non-toxic acid addition salt or metal complex commonly used in the pharmaceutical industry. Examples of acid addition salts include organic acids such as acetic acid, lactic acid, pamoic acid, maleic acid, citric acid, malic acid, ascorbic acid, succinic acid, benzoic acid, palmitic acid, suberic acid, salicylic acid, tartaric acid, methanesulfonic acid, toluenesulfonic acid or trifluoroacetic acid, etc.; polymeric acids such as tannic acid, carboxymethyl cellulose, etc.; and inorganic acids such as hydrochloric acid, hydrobromic acid, sulfuric acid, phosphoric acid, etc. Metal complexes include zinc, iron, etc. Metal complexes include zinc, iron, etc.

[0261] Preparations for oral use include tablets containing the active ingredient in a mixture with nontoxic pharmaceutically acceptable excipients. These excipients can be, for example, inert diluents or fillers (e.g., sucrose and sorbitol), lubricants, glidants and anti-adherents (e.g., magnesium stearate, zinc stearate, stearic acid, silica, hydrogenated vegetable oil or talc). Preparations for oral use can also be provided as chewable tablets, or as hard gelatin capsules (wherein the active ingredient is mixed with an inert solid diluent), or as soft gelatin capsules (wherein the active ingredient is mixed with water or an oil medium).

[0262] The dosage and timing of administration of the compound will vary according to a variety of clinical factors, including the overall health of the subject and the severity of the symptoms of the disease (eg, Diabetes, pre-diabetes).

[0263] A. Preparations for long-acting injectable drugs

[0264] Biologics and other agents that are subject to high first-pass clearance may not be suitable for oral administration and may need to be administered by a parenteral route. However, compliance with treatment regimens for injectable drugs may be low because subjects often resist self-administration of agents by injection, e.g., subcutaneous injection, especially when the disease does not make the subject uncomfortable. Other routes of administration by injection, e.g., intravenous, intramuscular, generally require administration by trained professionals, making frequent administration of agents inconvenient and often painful.

[0265] Formulations that provide sustained delivery of injectable medicaments have been prepared, including, but not limited to, oil-based injections, injectable drug suspensions, injectable microspheres, and in situ injection systems. Long-acting injectable formulations offer many advantages over conventional formulations of the same compound. These advantages include at least the following: predictable drug release characteristics over a defined period of time after each injection; better patient compliance; ease of administration; increased systemic availability by avoiding first-pass metabolism; reduced dosing frequency (i.e., fewer injections) without compromising the effectiveness of the treatment; reduced incidence of side effects; and reduced overall cost of medical care.

[0266] 1. Oil-based injections and injectable drug suspensions.

[0267] Conventional long-acting injections are composed of lipophilic drugs in aqueous solvents (as suspensions) or lipophilic drugs dissolved in vegetable oils. Commercially available oil-based injection drugs for intramuscular administration include, but are not limited to, haloperidol decanoate, fluphenazine decanoate, testosterone enanthate and estradiol valerate. The frequency of application of these long-acting preparations is about every few weeks. In suspension preparations, the rate-limiting step of drug absorption is the dissolution of drug particles in the tissue fluid around the preparation or drug preparation. Forming poorly water-soluble salts can be used to control the dissolution rate of drug particles to extend absorption. However, several other factors, such as the diffusion degree of injection site, injection volume, drug depot (depot) at the injection site and the absorption and distribution of the oil solvent itself may affect the overall pharmacokinetic properties of the drug. Adjusting these factors to provide the desired drug release characteristics is within the capabilities of those skilled in the art.

[0268] 2. Polymer-based microspheres and in-situ molding.

[0269] The development of polymer-based long-acting injections is one of the most suitable strategies for macromolecules such as peptides and protein drugs. Commercially available microsphere preparations include, but are not limited to, leuprolide acetate, triptorelin pamoate, octreotide acetate, lanreotide acetate, risperidone and naltrexone. Commercially available in situ forming implants include leuprolide acetate, and in situ forming implants containing paclitaxel and bupivacaine are in clinical trials. These preparations are used for intramuscular administration. The advantages of polymer-based preparations for macromolecules include: in vitro and in vivo stability of macromolecules, improvement of systemic availability, extension of biological half-life, enhancement of patient convenience and compliance, and reduction of dosing frequency.

[0270] The most critical factor in the design of injectable microspheres and in situ formed preparations is the selection of suitable biodegradable polymers. The release of drug molecules from biodegradable microspheres is controlled by diffusion through the polymer matrix and polymer degradation. The properties of polymers, such as copolymer composition ratio, polymer crystallinity, glass transition temperature and hydrophilicity play a key role in the release process. Although structure, inherent polymer properties, core solubility, polymer hydrophilicity and polymer molecular weight affect drug release kinetics, the possible mechanisms of drug release from microspheres are as follows: initial release from the surface, release through pores, diffusion through a complete polymer barrier, diffusion through a water-swellable barrier, polymer erosion and bulk degradation. All these mechanisms work together in the release process. Polymers used for microspheres and in situ forming formulations include, but are not limited to, a variety of biodegradable polymers that have been extensively studied for controlled drug delivery over the past few decades, including polylactide (PLA), polyglycolide (PGA), poly(lactide-co-glycolide) (PLGA), poly(ε-caprolactone) (PCL), polygluconic acid esters, polyanhydrides, polyorthoesters, poly(dioxanones), and polyalkylcyanoacrylates. Thermally induced gelling systems used in in situ forming formulations exhibit thermoreversible sol / gel transitions and are characterized by lower critical solution temperatures. They are liquids at room temperature and produce gels at lower critical solution temperatures and above. In situ curing organogels consist of water-insoluble amphoteric liquids that swell in water and form various types of lyotropic liquid crystals.

[0271] B. Targeted Drug Delivery

[0272] The delivery of drug to its site of action can improve the therapeutic index by reducing the amount of drug required to provide the desired systemic effect. The method or preparation that can limit systemic exposure can be used, for example, intramuscular injection, intrasynovial injection, intrathecal injection, intraocular injection, drug administration to the target tissue and drug delivery to the site of action. The multiple sustained delivery preparations discussed above are for intramuscular administration and are provided to the local delivery of muscle tissue. Alternatively, the targeting moiety can be associated or connected with the therapeutic payload for application to the target site. The targeting moiety can include any one of the multiple parts in conjunction with a specific cell type.

[0273] 1. Targeting part

[0274] Specific embodiments of the present invention include the use of targeting moieties, including relatively small peptides (e.g., 25 amino acids or less, 20 amino acids or less, 15 amino acids or less, 10 amino acids or less) as discussed above, muscle targeting peptides (MTPs) including smooth muscle and / or skeletal muscle targeting peptides, αvβ3 integrin ligands (e.g., RGD peptides and peptide analogs), αvβ5 integrin ligands, or CD46 ligands. It will be understood that such peptides may include one or more chemical modifications to allow for the formation of a complex with Eno1, thereby altering the pharmacokinetic and / or pharmacodynamic properties of the peptide. In specific embodiments, the targeting moiety may be a small molecule, such as an RGD peptide mimetic. In specific embodiments, the targeting moiety may include a protein and, optionally, a fiber protein from adenovirus 35. In specific embodiments, the viral protein is present on the viral particle. In specific embodiments, the viral protein is not present on the viral particle. In specific embodiments, the targeting moiety may be an antibody, an antibody fragment, an antibody mimetic, or a T-cell receptor.

[0275] 2. Targeting complex

[0276] The targeted Eno1 complex can be administered by other routes other than intramuscular injection (e.g., subcutaneous injection, intravenous injection) while providing delivery of Eno1 to muscle. The targeted complex can include one or more targeting moieties directly or indirectly attached to Eno1. The formation of the targeted complex does not substantially or irreversibly inhibit the activity of Eno1 and its effects on normalizing blood glucose levels and insulin response. In certain embodiments, the use of the targeted complex can reduce the total amount of Eno1 required to provide an effective dose. Some exemplary, non-limiting embodiments of the targeted complex are discussed below.

[0277] In certain embodiments, the payload and the targeting moiety are present in the complex in a molar ratio of about 1:1. In certain embodiments, the targeting moiety is present in the complex in a molar excess (e.g., 2:1, 3:1, 4:1, 5:1, 6:1, 7:1, 8:1, 9:1, 10:1, 11:1, 12:1, 13:1, 14:1, 15:1, 16:1, 17:1, 18:1, 19:1, 20:1, 21:1, 22:1, 23:1, 24:1, 25:1, 26:1, 27:1, 28:1, 29:1, 30:1 or higher; or any range included by any two values). In certain embodiments, the payload and the targeting moiety are about 1:5-1:15; about 1:7-1:13, about 1:8-1:12.

[0278] It will be understood that the compositions and methods of the present invention include the administration of more than one targeting moiety-payload complex (i.e., a targeting moiety-payload complex population). Therefore, it will be understood that the number of targeting moieties for each payload can represent the average number of targeting moieties for each payload in the complex population. In a specific embodiment, at least 70% of the complexes have a selected molar ratio of a targeting moiety to a payload. In a specific embodiment, at least 75% of the complexes have a selected molar ratio of a targeting moiety to a payload. In a specific embodiment, at least 80% of the complexes have a selected molar ratio of a targeting moiety to a payload. In a specific embodiment, at least 85% of the complexes have a selected molar ratio of a targeting moiety to a payload. In a specific embodiment, at least 90% of the complexes have a selected molar ratio of a targeting moiety to a payload.

[0279] a. Connector

[0280] A variety of chemical linkers are known in the art and are available from commercial sources (e.g., Pierce ThermoFisher Scientific Inc., see, e.g., www.piercenet.com / cat / crosslinking-reagents). Such reagents can be used to chemically link one or more targeting moieties to Enol reversibly or irreversibly. Linkers can also be used to attach the targeting moiety and Eno1 to a structure, e.g., a microparticle, a dendrimer, rather than directly linking the targeting moiety to Eno1. In a particular embodiment, the linker that links Eno1 to the targeting complex is reversible, so that after administration, Eno1 is released from the complex, preferably substantially at the muscle.

[0281] b. Peptide bond

[0282] As used herein, a targeting complex may include transformation of Eno1 with a peptide targeting moiety. Methods of generating expression constructs including an amino acid sequence for targeting Eno1 are well within the capabilities of those skilled in the art.

[0283] c. Liposomes

[0284] Liposomal delivery systems are known in the art and include formulations that limit systemic exposure, thereby reducing systemic exposure and off-target effects. For example, is a composition for treating certain types of cancer in which doxorubicin is encapsulated in long-circulating PEGylated liposomes, the liposomes further comprising cholesterol. Various liposomal formulations of amphotericin B (including and ) are formulated for intravenous administration in liposomes or lipid complexes containing various phospholipids, cholesterol, and cholesteryl sulfate. is verteporfin formulated as liposomes in egg phosphatidylglycerol and DMPC for intravenous administration. Liposomal preparations are also known for intramuscular injection. is an inactivated hepatitis A virus. is the hemaglutinin of inactivated influenza virus strains A and B. Both viral preparations are formulated in a combination of DOPC and DOPE. Such liposomes, or other physiologically acceptable liposomes, can be used for packaging of Eno1 and subsequently surface modified with targeting moieties to deliver Eno1 to muscle. Other moieties that regulate the intracellular trafficking of the liposomes may also be included. When the liposomes are taken up into cells, the liposomes release Eno1, thereby allowing it to have its therapeutic effect.

[0285] d. Dendrimer

[0286] Dendrimers can be used as scaffolds for attaching multiple targeting moieties to one or more Eno 1 molecules. In certain embodiments, the dendrimer is modified with targeting moieties prior to coupling to Eno 1.

[0287] e. Microparticles

[0288] The microparticles can be used as scaffolds for attaching multiple targeting moieties to one or more Eno1 molecules, which are attached to or encapsulated within the microparticles. In certain embodiments, the microparticles are modified with targeting moieties prior to coupling to Eno1.

[0289] f. Viral vectors

[0290] Viral tropism has long been studied and used to direct viruses to target cell types. Parker et al., 2013 (Gene Therapy, 20: 1158-64) have developed adenovirus serotype 5 capsites with serotype 35 fibers and peton to enhance delivery to skeletal and / or smooth muscle. Such viral vectors and other viral vectors can be used to deliver Eno1 expression constructs to muscle cells.

[0291] C. Dendrimer

[0292] Dendrimers can be used as a backbone for targeting complexes in the context of the present invention for delivering non-intramuscularly administered Eno 1 to muscle. Alternatively, dendrimers can be used to modulate the pharmacokinetic and pharmacodynamic properties of intramuscularly administered Eno 1. In the compositions and methods of the present invention, dendrimers are understood to be pharmaceutically acceptable dendrimers.

[0293] Dendrimer-based platforms have gained attention for use in pharmaceutical applications. Similar to other polymer carriers, dendrimers can be synthesized to avoid structural toxicity and immunogenicity. The ability of dendrimers to mimic the size, solubility, and shape of human proteins makes this technology ideal for many therapeutic and diagnostic applications. The size of 1-10 nanometers enables dendrimers to diffuse effectively across the vascular endothelium, internalize into cells, and be quickly cleared by the kidneys. This helps to avoid long-term toxicity and reduce the need for fast degradation platforms. The availability of multiple reactive surface groups enables dendrimers to carry higher payloads of functional molecules, thereby enhancing targeted delivery to the site of action, thereby improving efficacy.

[0294] Dendrimers have been produced or are in commercial development for several biomedical applications. A topical, polylysine dendrimer-based antiseptic, VivaGel TM , has been developed by Starpharma. Dendrimer-based materials for gene transfection. Dendrimer-based diagnostic tools include Gadomer-17, a magnetic resonance imaging (MRI) contrast agent containing a polylysine dendrimer functionalized with a gadolinium chelate, and CS, a biosensor for cardiac markers to rapidly diagnose heart attacks.

[0295] Dendrimers are defined by their core-shell structure, wherein the size and number of functional surface groups are approximately doubled by each additional shell (or generation) added to the core. The shell is synthesized by alternating monomer reactions in a manner known in the art. Specialized dendrimer backbones can be synthesized by varying the monomer units. The biological properties of dendrimers are largely affected by the chemical backbone and surface termination. For dendrimers to be suitable carriers for in vivo drug delivery, they must be non-toxic, non-immunogenic, and able to target and reach specific locations by passing through suitable barriers, while being sufficiently stable to remain in circulation. The vast majority of dendrimers synthesized and disclosed in the literature are insoluble under physiological conditions or are unable to remain soluble after the addition of functional molecules and are not suitable for biological applications. However, several classes of dendrimers have been shown to be useful scaffolds for biomedical applications; examples include polyesters, polylysine, and polypropyleneimine (PPI or DAB) dendrimers.

[0296] The most widely used dendrimer in biomedical applications is poly(amidoamine) (PAMAM) dendrimer. The polyamide backbone synthesized from repeated reactions of methyl acrylate and ethylene-diamine helps the macromolecule remain water soluble and minimizes immunogenicity. Different generations of PAMAM dendrimers are also able to mimic the size and properties of globular proteins that are readily found in the body. The amine-terminated surfaces of all generations of PAMAM dendrimers allow for easy surface modification, enabling the platform to carry and dissolve hydrophobic therapeutic molecules, such as methotrexate, under physiological conditions. If the surface amines are neutralized or appropriately modified (e.g., acylated), PAMAM dendrimers exhibit little nonspecific toxicity.

[0297] Active targeting uses molecules (such as targeting moieties) to mediate the delivery of their payload (drug or other) to cells by binding to cell-specific molecules. Targeting moieties, such as those provided herein, often bind through receptors that are highly expressed on target cells. The interaction between targeting ligands and cell surface receptors allows therapeutic agents or payloads to selectively reach muscle cells and even be directed into cells through receptor-mediated processes.

[0298] Compared with single ligand, the multivalent effect associated with presenting multiple binding ligands on the dendrimer surface enhances the uptake of dendritic scaffolds. The multivalent interaction caused by the simultaneous combination of multiple ligands allows dendritic polymers to improve the binding affinity of the platform, even when a single ligand has low affinity to the receptor of the target. The PAMAM platform has been successfully used as a scaffold for connecting multivalent targeting molecules (including antibodies, peptides, T-antigens and folic acid). The targeting ligand anchors the dendritic polymer to the position where the specific receptor is expressed on the cell surface. Targeted dendritic polymer-drug conjugates specifically deliver higher doses to the targeted cells while avoiding normal cells, thus avoiding potential systemic toxicity.

[0299] Neutralizing the surface amines of PAMAM dendrimers with acetyl groups minimizes toxicity and nonspecific dendrimer uptake. Acetyl capping of dendrimers also allows for improved clearance from the body, minimizing the effects of long-term treatment. PEGylation of amino-terminated PAMAM dendrimers reduces immunogenicity and improves solubility. PEG-terminated dendrimers have an increased blood flow half-life compared to cationic parent materials. Hydroxyl- and methoxy-terminated polyester dendrimers have been shown to be non-toxic in vivo at concentrations up to 40 mg / kg. Toxicity differences between cationic and anionic dendrimers have been demonstrated in vivo. Using a zebrafish embryo model, carboxyl-terminated dendrimers were significantly less toxic than G4 amine-terminated dendrimers. In the same study, surface modification with RGD also reduced toxicity.

[0300] It will be understood that all of the dendritic polymers described above and herein can be used in the Enol compositions of the present invention and methods of use thereof.

[0301] In certain embodiments, the ratio of the number of dendrimer molecules to the number of Eno1 molecules in the complex comprising a dendrimer and Eno1 is from about 1:1 to about 10:1, for example, about 1:1, about 2:1, about 3:1, about 4:1, about 5:1, about 6:1, about 7:1, about 8:1, about 9:1 or about 10:1. In one embodiment, the ratio of the number of dendrimer molecules to the number of Eno1 molecules in the complex comprising a dendrimer and Eno1 is from about 3:1 to 7:1, for example, 3:1, 4:1, 5:1, 6:1 or 7:1. In one embodiment, the ratio of the number of dendrimer molecules to the number of Eno1 molecules in the complex comprising a dendrimer and Eno1 is from 4:1 to 6:1, for example, 3:1, 4:1 or 5:1. In one embodiment, the ratio of the number of dendrimer molecules to the number of Eno1 molecules in the complex comprising a dendrimer and Eno1 is 3: 1 to 5: 1, for example, 3: 1, 4: 1 or 5: 1. In yet another embodiment, the ratio of the number of dendrimer molecules to the number of Eno1 molecules in the complex comprising a dendrimer and Eno1 is 4: 1 to 5: 1. In another embodiment, the ratio of the number of dendrimer molecules to the number of Eno1 molecules in the complex comprising a dendrimer and Eno1 is 3: 1 to 4: 1. In a further preferred embodiment, the ratio of the number of dendrimer molecules to the number of Eno1 molecules in the complex comprising a dendrimer and Eno1 is about 5: 1.

[0302] The optimal ratio of dendrimer to Eno1 in the complex can be tested and selected by measuring the Eno1 activity of the dendrimer / Eno1 complex (e.g., compared to uncomplexed Eno1) using any conventional method known in the art, such as, for example, a pyruvate kinase (PK) / lactate dehydrogenase (LDH) assay or any other assay described herein. The optimal ratio of dendrimer to Eno1 can also be tested and selected by evaluating the effect of the dendrimer / Eno1 complex on glucose uptake in an in vitro assay, for example, by measuring glucose uptake in human skeletal muscle microtubules (HSMM) as described in Example 2 herein, or any similar assay known in the art. The optimal ratio of dendrimer to Eno1 can also be tested and selected by measuring the effect of the dendrimer / Enol complex on blood glucose levels in vivo, for example, by measuring the effect of the dendrimer / Eno1 complex on blood glucose in a diabetic mouse model as described in Examples 7 and 8 herein, or any similar model or assay known in the art. An optimal ratio of dendrimer to Eno 1 in the complex will preferably preserve the activity of Eno 1 in vitro and / or in vivo, and / or provide for delivery of Eno 1 to cells.

[0303] It will be understood that the compositions and methods of the present invention include the administration of more than one dendrimer-Eno1-targeting peptide complex (i.e., a population of dendrimer-Eno1-targeting peptide complexes). Therefore, it will be understood that the number of dendrimers per Eno1 molecule can represent the average number of dendrimers per Eno1 in the population of complexes. In a specific embodiment, at least 70% of the complexes have a selected molar ratio of dendrimer to Eno1. In a specific embodiment, at least 75% of the complexes have a selected molar ratio of dendrimer to Eno1. In a specific embodiment, at least 80% of the complexes have a selected molar ratio of dendrimer to Eno1. In a specific embodiment, at least 85% of the complexes have a selected molar ratio of dendrimer to Eno1. In a specific embodiment, at least 90% of the complexes have a selected molar ratio of dendrimer to Eno1.

[0304] In a specific embodiment, the ratio of the number of dendrimer molecules to the number of targeting peptides in the dendrimer / Eno1 / targeting peptide complex is 1:0.1 to 1:10, 1:1 to 1:10, 1:1 to 1:5, or 1:1 to 1:3. In a specific embodiment, the ratio of the number of dendrimer molecules to the number of targeting peptides is about 1:1, 1:2, 1:3, 1:4, 1:5, 1:6, 1:7, 1:8, 1:9 or 1:10. In a preferred embodiment, the ratio of the number of dendrimer molecules to the number of targeting peptides in the dendrimer / Eno1 / targeting peptide complex is about 1:1. In a preferred embodiment, the ratio of the number of dendrimer molecules to the number of targeting peptides in the dendrimer / Eno1 / targeting peptide complex is about 1:2. In a preferred embodiment, the ratio of the number of dendrimer molecules to the number of targeting peptides in the dendrimer / Eno1 / targeting peptide complex is about 1:3.

[0305] In certain embodiments, the ratio of the number of targeting peptides to the number of dendrimer molecules in the dendrimer / Eno1 / targeting peptide complex is at least 1: 1, at least 2: 1, at least 3: 1, at least 4: 1, at least 5: 1, at least 6: 1, at least 7: 1, at least 8: 1, at least 9: 1 or at least 10: 1. In one embodiment, the ratio of the number of targeting peptides to the number of dendrimer molecules in the dendrimer / Eno1 / targeting peptide complex is at least 3: 1.

[0306] It will be understood that the compositions and methods of the present invention include the administration of more than one targeting peptide-Eno1-dendrimer complex (i.e., a population of targeting peptide-Eno1-dendrimer complexes). Therefore, it will be understood that the number of targeting peptides per dendrimer represents the average number of targeting peptides per dendrimer in the population of complexes. In a specific embodiment, at least 70% of the complexes have a selected molar ratio of targeting peptide to dendrimer. In a specific embodiment, at least 75% of the complexes have a selected molar ratio of targeting peptide to dendrimer. In a specific embodiment, at least 80% of the complexes have a selected molar ratio of targeting peptide to dendrimer. In a specific embodiment, at least 85% of the complexes have a selected molar ratio of targeting peptide to dendrimer. In a specific embodiment, at least 90% of the complexes have a selected molar ratio of targeting peptide to dendrimer.

[0307] The optimal ratio of dendrimer to targeting peptide can be selected by measuring targeting of dendrimer / Eno1 / targeting peptide to specific tissues in vivo, for example, by measuring targeting of detectably labeled dendrimer / Eno1 / targeting peptide complexes in vivo as described in Example 6 herein.

[0308] V. Detection, measurement and control of blood sugar level indicators

[0309] Methods for detecting and measuring indicators of elevated blood sugar and glycemic control can vary depending on the nature of the indicator to be measured. Elevated blood sugar can be measured directly (e.g., by determining the amount of glucose in the blood) or indirectly (e.g., by detecting the amount of glycosylated hemoglobin (HbA1c)), and thereby measuring the loss of control of blood sugar levels and the severity of diabetes, which is a reaction product of hemoglobin and glucose. The present invention further provides methods for detecting glycemic control using Eno1.

[0310] The present invention contemplates any suitable means, techniques and / or procedures for detecting and / or measuring the blood glucose level index of the present invention. Those skilled in the art will appreciate that the method for measuring the index of the present invention will depend at least on the type of index (e.g., glucose, ketones, mRNA or polypeptide, including glycosylated polypeptide) and biological sample (e.g., whole blood, serum) detected or measured. Specific biological samples may also require specific specialized processing before measuring the biomarkers of the present invention, for example, the preparation of mRNA in the case of mRNA biomarkers to be measured (e.g., Eno1 mRNA).

[0311] A. Direct or indirect measurement of blood glucose and glycemic control using established markers

[0312] Blood glucose monitoring is a way to directly test the glucose concentration (blood sugar) in the blood at a single time point. Particularly important in the care of diabetes, blood glucose testing is performed by piercing the skin (usually, on a finger) to draw blood, and then applying the blood to a disposable "test strip" of chemical activity. Different manufacturers use different technologies, but most systems measure electrical characteristics, and use this electrical characteristic to determine the glucose level in the blood. This test is commonly referred to as capillary blood glucose. Commercially available blood glucose monitors for periodic or continuous use are known in the art. Glucose monitors for periodic detection of blood glucose levels include, but are not limited to, TRUEResult blood glucose meter (TRUE), ACCU-CHEK blood glucose meter (ACCU-CHEK), OneTouch blood glucose meter (ONETOUCH) and FreeStyle Lite Blood Glucose (FREESTYLE LITE). It will be understood that the normal blood glucose level measured directly will be different according to the amount of time after the last food is eaten, and the normal fasting blood glucose level is lower than the normal postprandial blood glucose level. Direct blood glucose monitoring is also used in glucose tolerance tests to monitor the reaction to taking high doses of glucose and the rate at which glucose is removed from the blood.

[0313] Glycated hemoglobin (hemoglobin A1c, HbA1c, A1C, Hb1C, HbA1c) is a form of hemoglobin that is primarily measured to identify the average blood glucose concentration over an extended period of time, i.e., an indirect measurement of blood glucose. HbA1c is formed in a non-enzymatic glycation pathway by exposure of hemoglobin to blood glucose. When normal blood glucose levels are present, normal amounts of glycated hemoglobin are produced, which are measured as a percentage of total hemoglobin or a specific blood concentration. When blood glucose levels are high, elevated glycated hemoglobin levels are produced. Glycation is an irreversible reaction. Therefore, the amount of glycated hemoglobin in red blood cells reflects the average level of glucose to which the cells have been exposed. Measuring glycated hemoglobin evaluates the effectiveness of treatment by monitoring long-term serum glucose regulation rather than by the snapshot images provided by glucose monitoring. HbA1c levels are proportional to the average blood glucose concentrations over the previous four to three weeks. For example, HbA1c levels can be measured using high performance liquid chromatography (HPLC) or immunoassays. The following is a detailed discussion of methods for detecting and measuring protein analytes.

[0314] B. Detection of Nucleic Acid Indicators

[0315] In certain embodiments, the invention relates to the detection of nucleic acid biomarkers (e.g., Eno1 mRNA biomarkers), optionally in combination with other indicators of blood glucose, to monitor diabetes and / or glycemic control in a subject, e.g., direct measurement of blood glucose, ketones and / or HbA1c.

[0316] In various embodiments, the diagnostic / prognostic methods of the present invention generally involve the determination of the expression level of Eno1 in a blood sample. The determination of gene expression levels in the implementation of the methods of the present invention can be performed by any suitable method. For example, the determination of gene expression levels can be performed by detecting the expression of mRNA expressed from the target gene and / or detecting the expression of a polypeptide encoded by the gene.

[0317] For detecting nucleic acid encoding Eno1, any suitable method can be used, including, but not limited to, Southern blot analysis, northern blot analysis, polymerase chain reaction (PCR) (see, e.g., U.S. Pat. Nos. 4,683,195; 4,683,202 and 6,040,166; "PCR Protocols: A Guide to Methods and Applications", Innis et al. (eds.); 1990, Academic Press: New York), reverse transcriptase PCR (RT-PCR), anchored PCR, competitive PCR (see, e.g., U.S. Pat. No. 5,747,251), rapid amplification of cDNA ends (RACE) (see, e.g., "Gene Cloning and Analysis: Current Innovations", 1997, pp. 99-115); ligase chain reaction (LCR) (see, e.g., EP 01 320 308); single-sided PCR (Ohara et al., Proc. Natl. Acad. Sci., 1989, 86:5673-5677), in situ hybridization, Taqman-based analysis (Holland et al., Proc. Natl. Acad. Sci., 1991, 88:7276-7280), differential display (see, e.g., Liang et al., Nucl. Acid. Res., 1993, 21:3269-3275) and other RNA fingerprinting techniques, nucleic acid sequence-based amplification (NASBA) and other transcription-based amplification systems (see, e.g., U.S. Pat. Nos. 5,409,818 and 5,554,527), Qbeta replicase, strand displacement amplification (SDA), repair chain reaction (RCR), nuclease protection assay, subtraction-based method, wait.

[0318] In other embodiments, the gene expression level of Eno1 can be determined by amplifying complementary DNA (cDNA) or complementary RNA (cRNA) generated from mRNA and analyzing it using a microarray. A variety of different array configurations and methods for producing them are known to those skilled in the art (see, e.g., U.S. Pat. Nos. 5,445,934; 5,532,128; 5,556,752; 5,242,974; 5,384,261; 5,405,783; 5,412,087; 5,424,186; 5,429,807; 5,436,327; 5,472,672; 5,527,681; 5,529,756; 5,545,531; 5,554,501; 5,561,071; 5,571,639; 5,593,839; 5,599,695; 5,624,711; 5,658,734 and 5,700,637). Microarray technology allows for the simultaneous measurement of large numbers of The steady-state mRNA level of a gene is measured. Currently, widely used microarrays include cDNA arrays and oligonucleotide arrays. Analysis using microarrays is usually based on the measurement of the signal intensity received from a labeled probe, which is used to detect cDNA sequences from a sample that hybridize with a nucleic acid probe fixed to a known position on the microarray (see, for example, U.S. Patent Nos. 6,004,755; 6,218,114; 6,218,122 and 6,271,002). Array-based gene expression methods are known in the art and are described in many scientific publications and patents (see, for example, M. Schena et al., Science, 1995, 270: 467-470; M. Schena et al., Proc. Natl. Acad. Sci. USA 1996, 93: 10614-10619; JJ Chen et al., Genomics, 1998, 51: 313-324; U.S. Patent Nos. 5,143,854; 5,445,934; 5,807,522; 5,837,832; 6,040,138; 6,045,996; 6,284,460 and 6,607,885).

[0319] In a specific embodiment, the invention includes a method for identifying a subject suffering from abnormal blood glucose by amplifying and detecting a nucleic acid corresponding to Eno1, optionally in combination with one or more other indicators of elevated blood glucose.

[0320] The nucleic acid used as amplification template can be separated from cells contained in a biological sample according to standard methods (Sambrook et al., 1989). The nucleic acid can be genomic DNA or fragmented or whole cell RNA. In the case of using RNA, it may be necessary to convert the RNA into complementary cDNA. In one embodiment, the RNA is whole cell RNA and is directly used as an amplification template.

[0321] A primer pair that selectively hybridizes with a nucleic acid corresponding to any of the Eno1 nucleotide sequences identified herein is contacted with the isolated nucleic acid under conditions that allow selective hybridization. Once hybridized, the nucleic acid: primer complex is contacted with one or more enzymes that promote template-dependent nucleic acid synthesis. Multiple rounds of amplification, also referred to as "cycling," are performed until a sufficient amount of amplified product is produced. Then, the amplified product is detected. In a specific application, detection can be performed visually. Alternatively, detection can involve indirect product identification by chemiluminescence, radioactive scintigraphy of incorporated radioactive labels or fluorescent labels, or even by a system (AFFYMAX technology; Bellus, 1994) using electrical or thermal pulse signals. After detection, the results seen in a given patient can be compared with statistically significant reference groups of normal patients and patients with elevated blood sugar (e.g., prediabetes, type 2 diabetes, gestational diabetes, or type 1 diabetes patients). In this way, it is possible to associate the amount of nucleic acid detected with various clinical states.

[0322] As defined herein, the term primer refers to any nucleic acid that can initiate the synthesis of a nascent nucleic acid in a template-dependent method. Typically, a primer is an oligonucleotide of ten to twenty, preferably fifteen to twenty nucleotides in length, but longer sequences may also be used. Primers are provided in double-stranded or single-stranded form, although single-stranded form is preferred.

[0323] A variety of template-dependent methods can be used to amplify nucleic acid sequences present in a given template sample. One of the best known amplification methods is the polymerase chain reaction (referred to as PCR), which is described in detail in U.S. Pat. Nos. 4,683,195, 4,683,202, and 4,800,159 and Innis et al., 1990, each of which is incorporated herein by reference in its entirety.

[0324] In PCR, two primer sequences are prepared, which are complementary to the regions on the relative complementary strands of the target nucleic acid sequence. Excessive deoxynucleotide triphosphates are added to the reaction mixture together with a DNA polymerase (e.g., Taq polymerase). If there is a target nucleic acid sequence in the sample, primers will bind to the target nucleic acid and polymerase will cause the primers to extend along the target nucleic acid sequence by adding nucleotides. By raising and lowering the temperature of the reaction mixture, the extended primers will dissociate with the target nucleic acid to form a reaction product, and excess primers will bind to the target nucleic acid and the reaction product, and the process is repeated.

[0325] Reverse transcriptase PCR amplification procedures can be performed to quantify the amount of amplified mRNA. Methods for reverse transcription of RNA into cDNA are well known and described in Sambrook et al., 1989. Alternative methods for reverse transcription utilize thermostable DNA polymerases. These methods are described in WO 90 / 07641 filed December 21, 1990. Polymerase chain reaction methods are well known in the art.

[0326] Another method for amplification is the ligase chain reaction ("LCR"), disclosed in European Application No. 320 308, which is incorporated herein by reference in its entirety. In LCR, two complementary probe pairs are prepared, and in the presence of a target sequence, each pair binds to the opposite complementary strands of the target so that they are adjacent. In the presence of a ligase, the two probe pairs are ligated to form a single unit. By temperature cycling, as in PCR, the bound ligated unit is dissociated from the target and subsequently used as a "target sequence" for ligation of excess probe pairs. U.S. Patent No. 4,883,750 describes a method similar to LCR for binding probe pairs to a target sequence.

[0327] Qbeta replicase, described in PCT Application No. PCT / US87 / 00880, can also be used as yet another amplification method in the present invention. In this method, a repetitive sequence of RNA having a region complementary to a region of the target is added to the sample in the presence of RNA polymerase. The polymerase will replicate the repetitive sequence, which can then be detected.

[0328] Isothermal amplification methods can be used in the present invention for nucleic acid amplification, wherein restriction endonucleases and ligases are used to obtain amplification of target molecules containing nucleotide 5'-[α-thio]-triphosphates in one strand of the restriction site. Walker et al. (1992), which is incorporated herein by reference in its entirety.

[0329] Strand displacement amplification (SDA) is another method for isothermal amplification of nucleic acids, which involves multiple rounds of strand displacement and synthesis, i.e., gap translation. A similar method, called repair chain reaction (PCR), involves annealing several probes in the entire region targeted for amplification, followed by a repair reaction, in which only two of the four bases are present. For ease of detection, the other two bases can be added as biotinylated derivatives. A similar method is used in SDA. Target-specific sequences can also be detected using a cyclic probe reaction (CPR). In CPR, probes with 3' and 5' sequences of nonspecific DNA and an intermediate sequence of specific RNA are hybridized with the DNA present in the sample. During hybridization, the reaction is treated with RNase H, and the product of the probe is identified as a distinct product, which is released after digestion. The original template is annealed with another cyclic probe and the reaction is repeated.

[0330] According to the present invention, further amplification methods described in GB application No. 2 202 328 and PCT application No. PCT / US89 / 01025 can be used, each of which is incorporated by reference in its entirety. In the former application, "modified" primers are used in PCR-like, template- and enzyme-dependent synthesis. Primers can be modified by labeling with a capture portion (e.g., biotin) and / or a detection portion (e.g., enzyme). In the latter application, an excess of labeled primers is added to the sample. In the presence of the target sequence, the probe binds and is cleaved by catalysis. After cleavage, the target sequence is completely released to be bound by the excess probe. The cleavage of the labeled probe gives a signal of the presence of the target sequence.

[0331] Other nucleic acid amplification procedures contemplated include transcription-based amplification systems (TAS), including nucleic acid sequence-based amplification (NASBA) and 3SR. Kwoh et al. (1989); Gingeras et al., PCT application WO 88 / 10315, all of which are incorporated herein by reference. In NASBA, nucleic acids can be prepared for amplification by standard phenol / chloroform extraction, heat denaturation of clinical samples, treatment with lysis buffer and minispin columns for DNA and RNA isolation, or guanidine hydrochloride extraction of RNA. These amplification techniques involve annealing primers with target-specific sequences. After polymerization, the DNA / RNA hybrids are digested with RNase H, while the double-stranded DNA molecules are heat denatured again. In either case, the single-stranded DNA is made completely double-stranded by adding a second target-specific primer followed by polymerization. The double-stranded DNA molecules are then multiply transcribed by a polymerase such as T7 or SP6. In an isothermal cycling reaction, the RNA is reverse transcribed into double-stranded DNA and transcribed once more with a polymerase such as T7 or SP6. The resulting products (whether truncated or intact) are characterized by target-specific sequences.

[0332] Davey et al., European Application No. 329 822 (incorporated herein by reference in its entirety) discloses a nucleic acid amplification method involving cyclically synthesized single-stranded RNA ("ssRNA"), ssDNA and double-stranded DNA (dsDNA), which can be used in accordance with the present invention. The ssRNA is the first template for a first primer oligonucleotide, which is extended by a reverse transcriptase (an RNA-dependent DNA polymerase). The RNA is then removed from the resulting DNA:RNA duplex by the action of ribonuclease H (RNase H, an RNA enzyme specific for RNA in a DNA or RNA duplex). The resulting ssDNA is the second template for a second primer, which also includes a sequence of an RNA polymerase promoter (exemplified by T7 RNA polymerase) that is homologous to the template on the 5' side. This primer is then extended by a DNA polymerase (exemplified by the large "Klenow" fragment of E. coli DNA polymerase 1), thereby forming a double-stranded DNA ("dsDNA") molecule having the same sequence as the original RNA sequence between the primers and additionally having a promoter sequence at one end. This promoter sequence can be utilized by suitable RNA polymerase to prepare many RNA copies of DNA. These copies can re-enter circulation subsequently, causing very rapid amplification. Through the appropriate selection of enzyme, this amplification can be carried out isothermally without the need to add enzyme in each circulation. Due to the cyclic nature of this method, the starting sequence of DNA or RNA form can be selected.

[0333] Miller et al., PCT Application WO 89 / 06700, which is incorporated herein by reference in its entirety, discloses a nucleic acid sequence amplification scheme based on hybridization of a promoter / primer sequence to a single-stranded target DNA ("ssDNA") followed by transcription of many RNA copies of the sequence. This scheme is not cyclic, i.e., new templates are not generated from the resulting RNA transcripts. Other amplification methods include "race" and "One-Sided PCR.TM." Frohman (1990) and Ohara et al. (1989), each of which is incorporated herein by reference in its entirety.

[0334] Methods based on ligating two (or more) oligonucleotides in the presence of a nucleic acid having the sequence of the resulting "di-oligonucleotide" and thereby amplifying a di-oligonucleotide can also be used in the amplification step of the present invention. Wu et al. (1989), which is incorporated herein by reference in its entirety.

[0335] According to the specific analysis format and the specific needs and the target sequence used, the oligonucleotide probe or primer of the present invention can be of any suitable length. In a preferred embodiment, the oligonucleotide probe or primer is at least 10 nucleotides in length (preferably, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32 ...), preferably at least 15 nucleotides in length (15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32 ...), and they can be adaptively changed to be particularly suitable for the selected nucleic acid amplification system and / or the hybridization system used. Longer probes and primers are also within the scope of the present invention, as known in the art. Primers with more than 30, more than 40, more than 50 nucleotides and probes with more than 100, more than 200, more than 300, more than 500, more than 800 and more than 1000 nucleotides in length are also encompassed by the present invention. Of course, longer primers have the disadvantage of being more expensive, and therefore, primers with a length of 15 to 30 nucleotides are generally designed and used in the art. As is well known in the art, probes with a length range of 10 to more than 2000 nucleotides can be used in the methods of the present invention. For the above-mentioned % identity, the size of probes and primers not specifically described (e.g., 16, 17, 31, 24, 39, 350, 450, 550, 900, 1240 nucleotides ...) are also within the scope of the present invention. In one embodiment, the oligonucleotide probe or primer of the present invention specifically hybridizes with Eno1 RNA (or its complementary sequence) or Eno1 mRNA. More preferably, the Eno1 primers and probes are selected to detect Eno1 RNA associated with elevated blood glucose or abnormal blood glucose regulation, such as associated with prediabetes, type 2 diabetes, type 1 diabetes, or gestational diabetes.

[0336] In other embodiments, the detection means can utilize hybridization techniques, for example, wherein a specific primer or probe is selected to anneal to a target biomarker of interest (e.g., Eno1), and thereafter selective hybridization detection is performed. As is generally known in the art, oligonucleotide probes and primers can be designed by considering their melting points for hybridization and the sequences they target (see below and Sambrook et al., 1989, Molecular Cloning--A Laboratory Manual, 2nd edition, CSH Laboratories; Ausubel et al., 1994, see Current Protocols in Molecular Biology, John Wiley & Sons Inc., NY).

[0337] In order to enable hybridization under the test conditions of the present invention, oligonucleotide primers and probes should contain oligonucleotide sequences that are at least 70% (at least 71%, 72%, 73%, 74% or higher), preferably at least 75% (75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89% or higher) and more preferably at least 90% (90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100%) identical to a portion of an Eno1 polynucleotide. The probes and primers of the present invention are those that hybridize under stringent hybridization conditions and those that hybridize to an Eno1 homolog under at least moderate stringency conditions. In a specific embodiment, the probes and primers of the present invention have complete sequence identity to an Eno1 gene sequence (e.g., cDNA or mRNA). It should be understood that other probes and primers can be easily designed based on the Eno1 sequence disclosed herein and used in the present invention by using computer alignment and sequence analysis methods known in the art (see Molecular Cloning: A Laboratory Manual, 3rd edition, edited by Cold Spring Harbor Laboratory, 2000).

[0338] C. Detection of Glucose Peptide Indicators for Glycemic Control

[0339] The present invention contemplates any suitable method for detecting polypeptide indicators of blood glucose, including Eno1 or HbA1c. In a particular embodiment, the detection method is an immunoassay involving antibodies that specifically bind to one or more of Eno1 and hemoglobin, especially specifically to glycated hemoglobin. The steps of various useful immunoassays have been described in the scientific literature, such as, for example, Nakamura et al. (1987), which is incorporated herein by reference.

[0340] Generally, the immunoassay method comprises obtaining a sample suspected of containing a protein or peptide indicator of elevated blood glucose and contacting the sample with an antibody according to the invention under conditions effective to allow formation of an immune complex, as the case may be.

[0341] Immunobinding methods include methods for detecting or quantifying the amount of reactive components in a sample, which require detecting or quantifying any immune complexes formed during the binding process. Here, a sample suspected of containing a protein or peptide indicator of elevated blood sugar is obtained and the sample is contacted with an antibody, and then the amount of immune complexes formed under specific conditions is detected or quantified.

[0342] For the detection of blood glucose indicators, the biological sample analyzed can be any sample suspected of containing a protein or peptide indicator of blood glucose, such as Eno1 or HbA1c. In the case of HbA1c, the biological sample can be, for example, blood, or in the case of Eno1, blood or serum.

[0343] The selected biological sample is contacted with the antibody (e.g., as a detection agent that binds to Eno1, HbA1c, or hemoglobin in the biological sample) under conditions effective to allow the formation of immune complexes (primary immune complexes) and for a sufficient period of time. Typically, complex formation is a matter of simply adding the composition to the biological sample and incubating the mixture for a sufficient period of time for the antibody to form a complex with any antigen present (i.e., bind to any antigen present). After this time, the sample-antibody composition, such as a tissue section, ELISA plate, dot blot, or western blot, is typically washed to remove any non-specifically bound antibody material, thereby allowing only those antibodies that are specifically bound within the primary immune complex to be detected.

[0344] In general, detection of immune complex formation is well known in the art and can be achieved by applying a variety of approaches. These methods are generally based on the detection of labels or markers, such as any radioactive, fluorescent, biological or enzyme tags or labels of standard use in the art. U.S. patents regarding the use of such labels include U.S. Patent Nos. 3,817,837; 3,850,752; 3,939,350; 3,996,345; 4,277,437; 4,275,149 and 4,366,241, each of which is incorporated herein by reference. Of course, as is known in the art, additional advantages may be found by using a second binding ligand such as a second antibody or a biotin / avidin ligand binding arrangement.

[0345] The antibody used in the assay (eg, anti-Eno1 antibody, anti-hemoglobin or anti-glycated hemoglobin antibody) may itself be linked to a detectable label, wherein such label may then be simply detected, thereby allowing the amount of primary immune complexes in the composition to be determined.

[0346] In some embodiments, the first component added in the primary immune complex can be detected by a second binding ligand with binding affinity for the antibody combined. In these cases, the second binding ligand can be connected to a detectable label. The second binding ligand itself is often an antibody, so it can be referred to as a "second" antibody. The primary immune complex is contacted with a second binding ligand or antibody of a mark under the conditions that effectively allow the secondary immune complex to form for a sufficient time. The secondary immune complex is then usually washed to remove the second antibody or the ligand of any non-specifically bound mark, and the mark retained in the secondary immune complex is detected subsequently.

[0347] More methods include detecting primary immune complexes by a two-step method. As described above, a second binding ligand (such as an antibody) with binding affinity to the encoded protein, peptide or corresponding antibody is used to form a secondary immune complex. After washing, the secondary immune complex is again contacted with a third binding ligand or antibody with binding affinity for the second antibody for a sufficient time under conditions that effectively allow immune complexes (tertiary immune complexes) to form. The third ligand or antibody is connected to a detectable label, thereby allowing detection of the tertiary immune complexes thus formed. This system can provide signal amplification if this is desired.

[0348] The immunoassay method of the present invention has obvious practicality in the diagnosis of illness, and the illness such as elevated blood sugar, loss of blood sugar control and diabetes. Here, biological or clinical samples suspected of containing encoded proteins or glycosylated peptides are used. However, these embodiments also have uses for non-clinical samples, such as in the titration of antigen or antibody samples, in the selection of hybridomas, etc.

[0349] The present invention particularly contemplates the use of ELISA as a type of immunoassay analysis. It is contemplated that the biomarker protein or peptide of the present invention will be found in the ELISA test for diagnosis and prognosis monitoring of abnormal blood sugar and diabetes as an immunogen. Immunoassay, in its simplest and most direct sense, is a binding test. Specific preferred immunoassays are various types of enzyme-linked immunosorbent assays (ELISA) and radioimmunoassays (RIA) known in the art. Immunohistochemical detection using tissue sections may be useful. However, it will be readily appreciated that detection is not limited to such technology, and western blotting, dot blotting, etc. may also be used.

[0350] In an exemplary ELISA, the antibody combined with the protein indicator of the present invention is fixed on a selected surface presenting protein affinity, such as the wells of a polystyrene microtiter plate. Then, a test composition suspected of containing a blood glucose level indicator, such as a blood or serum sample, is added to the wells. After combining and washing to remove the immune complex of non-specific binding, the combined antigen can be detected. Detection is usually achieved by adding a second antibody specific for the indicator protein connected to a detectable label. This type of ELISA is a simple "sandwich ELISA". Detection can also be achieved by adding a second antibody, followed by adding a third antibody with binding affinity to the second antibody, the third antibody being connected to a detectable label.

[0351] In another exemplary ELISA, a sample suspected of containing a blood glucose indicator protein is fixed on the hole surface and subsequently contacted with a specific antibody for binding the indicator. After binding and washing to remove the non-specifically bound immune complex, the bound antigen is detected. In the case where the initial antibody is connected to a detectable label, the immune complex can be directly detected. Again, a second antibody having a binding affinity to the first antibody can be used to detect the immune complex, and the second antibody is connected to a detectable label.

[0352] Regardless of the format used, ELISA has certain common features such as coating, incubation or binding, washing to remove non-specifically bound material, and detection of bound immune complexes. These are described below.

[0353] In coating a plate with an antigen or antibody, the wells of the plate are usually incubated with a solution of the antigen or antibody, either overnight or for a specified period of time. The wells of the plate are then washed to remove incompletely adsorbed material. Any remaining available well surfaces are then "coated" with a nonspecific protein that is neutral on the antigen for the test antiserum. These include bovine serum albumin (BSA), casein, and milk powder solutions. Coating allows for blocking of nonspecific adsorption sites on the fixed surface and thereby reduces the background caused by nonspecific binding of the antiserum on the surface.

[0354] In ELISA, it is customary to use secondary or tertiary detection modes rather than direct procedures. Thus, after the protein or antibody is bound to the wells, coated with a non-reactive substance to reduce background, and washed to remove unbound material, the fixed surface is contacted with a control biological sample, such as blood or serum from a subject with normal blood sugar and / or a subject with adequate glycemic control, to be tested under conditions effective to allow immune complex (antigen / antibody) formation. Detection of the immune complex then requires a labeled second binding ligand or antibody, or a second binding ligand or antibody combined with a labeled third antibody or third binding ligand.

[0355] The phrase "under conditions effective to allow immune complex (antigen / antibody) to form" means that the conditions preferably include diluting the antigen and antibody with solutions such as BSA, bovine gamma globulin (BGG) and phosphate buffered saline (PBS) / Tween. These added agents also tend to contribute to the reduction of nonspecific background.

[0356] "Suitable" conditions also mean that the incubation is at a temperature sufficient to allow effective binding and for a sufficient period of time. The incubation step is generally about 1 to about 4 hours, preferably at a temperature of about 25 to 27°C, or at about 4°C overnight, etc.

[0357] After all incubation steps in ELISA, the contacted surfaces are washed to remove non-complexed material. A preferred washing procedure includes washing with a solution such as PBS / Tween or borate buffer. After the formation of specific immune complexes between the test sample and the initially bound material and subsequent washing, the presence of even very small amounts of immune complexes can be determined.

[0358] In order to provide detection means, the second or third antibody has a connected label to allow detection. Preferably, the label is an enzyme that causes color development when incubated with a suitable chromogenic substrate. Therefore, for example, primary or secondary immune complexes are contacted and incubated for a period of time (for example, incubated at room temperature for 2h in a solution containing PBS such as PBS-Tween) with urease, glucose oxidase, alkaline phosphatase or catalase coupled antibodies under conditions conducive to the formation of more immune complexes.

[0359] After incubation with the labeled antibody and subsequent washing to remove unbound material, the amount of label is quantified, for example, by incubation with a chromogenic substrate (such as urea and bromocresol purple). The degree of color generation is then measured, for example, using a visible spectrum spectrophotometer.

[0360] Protein mass spectrometry methods and instruments can also be used to measure, quantify, detect and otherwise analyze the protein biomarkers / indicators of the present invention (e.g., Eno1, HbA1c). Protein mass spectrometry refers to the application of mass spectrometry to study proteins. Although not intended to be limited, two approaches generally use mass spectrometry to characterize proteins. In the first, the intact protein is ionized and then introduced into a mass spectrometer. This approach is referred to as the "top-down" strategy for protein analysis. The two main methods for intact protein ionization are electrospray ionization (ESI) and matrix-assisted laser desorption / ionization (MALDI). In the second approach, proteins are enzymatically digested into smaller peptides using proteases such as trypsin. These peptides are then introduced into a mass spectrometer and identified by peptide mass fingerprinting or tandem mass spectrometry. Therefore, this latter approach (also referred to as "bottom-up" proteomics) uses peptide-level identification to infer the presence of proteins.

[0361] The full protein mass analysis of the biomarker of the present invention can be carried out using time of flight (TOF) MS or Fourier transform ion cyclotron resonance (FT-ICR). Because of their wide mass range, and in the case of FT-ICR, due to its high mass accuracy, these two types of instruments are useful. The most widely used instrument for peptide mass analysis is the MALDI time of flight instrument, because they allow to obtain peptide mass fingerprints (PMF) (1PMF can be analyzed in about 10 seconds) with high stride. Multi-stage quadrupole time of flight and quadrupole ion trap can also be used in the present application.

[0362] Protein indicators can also be measured in complex mixtures of proteins and molecules co-present in biological media or samples, however, it may be necessary to consider the classification of samples herein. It will be understood that the ionization of complex mixtures of proteins may cause the more abundant proteins therein to have a trend of "flooding" or curbing the signal of less abundant proteins from the same sample. In addition, due to the huge number of mixture components, the mass spectrum from the complex mixture may be difficult to explain. Before mass spectrometry, classification can be used to first separate any complex mixture of proteins. There are two methods widely used for classifying proteins or their peptide products from enzyme digestion. The first method is classified by complete protein and is called two-dimensional gel electrophoresis. The second method, high performance liquid chromatography (LC or HPLC), is used for the classification of peptides after enzyme digestion. In some cases, it may be desirable to combine these two technologies. Any other suitable method known in the art for classifying protein mixtures is also considered herein.

[0363] The gel spots identified on the 2D gel can usually be attributed to a protein. If the identity of the protein is desired, the method of in-gel digestion is usually applied, in which the target protein spot is excised and digested by proteolysis. The masses of the peptides obtained from the digestion can be determined by mass spectrometry using peptide mass fingerprinting. If this information does not allow the unambiguous identification of the protein, its peptides can be subjected to tandem mass spectrometry for de novo sequencing.

[0364] Characterization of protein mixtures using HPLC / MS may also be referred to in the art as "shotgun proteomics" and MuDPIT (Multidimensional Protein Identification Technology). The peptide mixture resulting from the digestion of the protein mixture is fractionated by one or two liquid chromatography (LC) steps. The eluent from the chromatography stage can be introduced directly into the mass spectrometer by electrospray ionization, or arranged on a series of spots for subsequent mass analysis using MALDI.

[0365] MS can be used to identify protein indicators (e.g., Eno1 or Hb1Ac) using various techniques, all of which are considered herein. Peptide mass fingerprinting uses the mass of proteolytic peptides as input for the predicted mass of a search database, which is produced from the digestion of a series of known proteins. If a protein sequence in a reference list produces a considerable number of predicted masses of matching experimental values, there is some evidence that this protein is present in the original sample. It will be further understood that the development of methods and instruments for automated, data-dependent electrospray ionization (ESI) tandem mass spectrometry (MS / MS) combined with microcapillary liquid chromatography (LC) and database retrieval has significantly improved the sensitivity and speed of gel-separated protein identification. Microcapillary LC-MS / MS has been successfully used for large-scale identification of single proteins directly from a mixture without gel electrophoresis separation (Link et al., 1999; Opitek et al., 1997).

[0366] Several recent methods allow for quantitative protein by mass spectrometry. For example, a stable (e.g., non-radioactive) heavy isotope of carbon (13C) or nitrogen (15N) can be added to one sample, while the other can be labeled with a corresponding light isotope (e.g., 12C and 14N). Before analysis, the two samples are mixed. Due to their mass difference, peptides derived from different samples can be distinguished. The ratio of its peak intensity corresponds to the relative abundance ratio of peptides (and proteins). The most commonly used methods for isotope labeling are SILAC (stable isotope labeling by amino acids in cell culture), trypsin-catalyzed 18O labeling, ICAT (isotope-coded affinity tag), iTRAQ (isobaric isotope labeling for relative and absolute quantification). "Semi-quantitative" mass spectrometry can be performed without the need for sample labeling. Typically, this is performed using MALDI analysis (in linear mode). Peak intensity or peak area from a single molecule (usually a protein) is associated with the amount of protein in the sample. However, a single signal depends on the primary structure of the protein, the complexity of the sample, and the setting of the instrument. Other types of "label-free" quantitative mass spectrometry use spectral counting (or peptide counting) of digested proteins as a means for determining relative protein amounts.

[0367] In one embodiment, any one or more protein indicators (eg, Enol, HbA1c) can be identified and quantified from a complex biological sample using mass spectrometry according to the following exemplary methods, which are not intended to limit the use of the present invention or other mass spectrometry-based methods.

[0368] In the first step of this embodiment, (A) a biological sample containing a complex mixture of proteins (including at least one target indicator), for example, a biological sample suspected of elevated blood sugar, is fractionated and labeled with a stable isotope X. (B) Next, a known amount of an internal standard is added to the biological sample, wherein the internal standard is prepared by fractionating a standard protein identical to the at least one target biomarker of interest and labeling it with a stable isotope Y. (C) This sample obtained is then introduced into an LC-MS / MS device, and a multiple reaction monitoring (MRM) analysis is performed using a multiple reaction monitoring (MRM) transition selected for the internal standard to obtain an MRM chromatogram. (D) The MRM chromatogram is then observed to identify a target peptide biomarker derived from the biological sample that shows the same retention time as a peptide derived from the internal standard (internal standard peptide), and the target protein indicator in the test sample is quantified by comparing the peak area of ​​the internal standard peptide with the peak area of ​​the target peptide indicator.

[0369] Any suitable biological sample can be used as the starting point for LC-MS / MS / MRM analysis, including biological samples derived from blood, urine, saliva, hair, cells, cell tissues, biopsy materials and processed products thereof; and protein-containing samples prepared by genetic recombination technology. Preferred embodiments of the present invention include the use of blood or serum samples.

[0370] Each of the above steps (A) to (D) is further described below.

[0371] Step (A) (grading and labeling). In step (A), the target protein indicator is graded into a set of peptides, which are subsequently labeled with a stable isotope X. In order to grade the target protein, for example, a method for digesting the target protein with a proteolytic enzyme (protease) (such as trypsin) and a chemical cleavage method (such as a method using cyanogen bromide) can be used. Preferably, the digestion by protease. If proteolytic digestion is allowed to be complete, it is known that a given molar amount of protein produces the same molar amount for each trypsin peptide cleavage product. Therefore, measuring the molar amount of the trypsin peptides of a given protein allows the molar amount of the original protein in the sample to be determined. The absolute amount of the peptides (peptide set) derived from the target protein contained in the protease digestion can be completed by measuring the absolute amount of the peptides (peptide set) derived from the target protein contained in the protease digestion. Therefore, in order to allow proteolytic digestion to be complete, it is preferred to carry out reduction and alkylation treatment to reduce and alkylate the disulfide bonds contained in the target protein before protease digestion with trypsin.

[0372] Subsequently, the obtained digestion product (peptide collection, peptides containing target biomarkers in the biological sample) is labeled with a stable isotope X. Examples of stable isotopes X include 1H and 2H of hydrogen atoms, 12C and 13C of carbon atoms, and 14N and 15N of nitrogen atoms. Any isotope can be appropriately selected therefrom. Labeling with stable isotopes X can be performed by reacting the digestion product (peptide collection) with a reagent containing a stable isotope. Preferred examples of such commercially available reagents include (manufactured by Applied Biosystems), which is an amine-specific stable isotope kit. The present invention is composed of 2 or 3 reagents which have a constant mass difference between them as a result of isotopic labeling and bind to the N-terminus of the peptide or the primary amine of the lysine residue ( - Light and - heavy; or -D0, -D4 and -D8) composition.

[0373] Step (B) (internal standard addition). In step (B), a known amount of internal standard is added to the sample obtained in step (A). The internal standard used herein is a digestion product (peptide set) obtained by classifying a protein (standard protein) composed of the same amino acid sequence as the target protein (target biomarker) to be measured and labeling the digestion product (peptide set) obtained with a stable isotope Y. The classification process can be carried out in the same manner as above for the target protein. Labeling with a stable isotope Y can also be carried out in the same manner as above for the target protein. However, the stable isotope Y used herein must have a different mass from the stable isotope X used to label the target protein digestion product. For example, in the case of using the above-mentioned mTRAQ (registered trademark) (produced by Applied Biosystems), when mTRAQ-light is used to label the target protein digestion product, mTRAQ-heavy should be used to label the standard protein digestion product.

[0374] Step (C) (LC-MS / MS and MRM analysis). In step (C), the sample obtained in step (B) is first placed in an LC-MS / MS device, and then multiple reaction monitoring (MRM) analysis is performed using the MRM transition selected for the internal standard. The sample obtained in step (B) (peptide collection labeled with stable isotopes) is first separated by one-dimensional or multidimensional high performance liquid chromatography using the LC (liquid chromatography) of the LC-MS / MS device. Specific examples of such liquid chromatography include cation exchange chromatography, in which separation is performed by utilizing the charge difference between peptides; and reverse phase chromatography, in which separation is performed by utilizing the hydrophobicity difference between peptides. These two methods can be used in combination.

[0375] Subsequently, every kind of peptide separated is subjected to tandem mass spectrometry analysis by using a tandem mass spectrometer (MS / MS mass spectrometer) comprising two mass spectrometers connected in series. Using such a mass spectrometer makes it possible to detect the target protein at a few femtomolar (fmol) levels. In addition, MS / MS analysis makes it possible to analyze the internal sequence information on the peptide, so it is possible to achieve false positive-free identification. Other types of MS analyzers can also be used, including sector magnetic field mass spectrometer (Sector MS), quadrupole mass spectrometer (QMS), time-of-flight mass spectrometer (TOFMS) and Fourier transform ion cyclotron resonance mass spectrometer (FT-ICRMS), and the combination of these analyzers.

[0376] Subsequently, the obtained data is subjected to spectral comparison by a search engine and the peptides detected for each protein experiment are listed. The detected peptides are preferably grouped for each protein, and preferably at least three fragments having an m / z value greater than the precursor ion and at least three fragments having an m / z value of preferably 500 or greater are selected from each MS / MS spectrum in the order of decreasing signal intensity on the spectrum. Two or more fragments are selected from these in descending order of intensity, and the average value of the intensity is defined as the expected sensitivity of the MRR transition. When multiple peptides are detected from a protein, at least two peptides with the highest sensitivity are selected as standard peptides using the expected sensitivity as an index.

[0377] Step (D) (quantification of target protein in test sample). Step (D) includes identifying a peptide derived from the target protein (target biomarker of interest) showing the same retention time as a peptide derived from an internal standard (internal standard peptide) in the MRM chromatogram detected in step (C), and quantifying the target protein in the test sample by comparing the peak area of ​​the internal standard peptide with the peak area of ​​the target peptide. The target protein can be quantified by utilizing a calibration curve of a previously prepared standard protein.

[0378] The calibration curve can be prepared by the following method. First, as described above, a recombinant protein consisting of the same amino acid sequence as the target biomarker protein is digested with a protease (such as trypsin). Subsequently, a known concentration of precursor fragment transition selection standard (PFTS) is individually labeled with two different types of stable isotopes (i.e., one is labeled with a stable isomer for labeling an internal standard peptide (labeled with IS), and the other is labeled with a stable isomer for labeling a target peptide (labeled with T)). Multiple samples are produced by mixing a specific amount of IS-labeled PTFS with various concentrations of T-labeled PTFS. These samples are placed in the above-mentioned LC-MS / MS equipment for MRM analysis. The area ratio of the T-labeled PTFS to the IS-labeled PTFS on the obtained MRM chromatogram (T-labeled PTFS / IS-labeled PTFS) is plotted relative to the amount of T-labeled PTFS to prepare a calibration curve. The absolute amount of the target protein contained in the test sample can be calculated by referring to the calibration curve.

[0379] D. Antibodies and Labels (e.g., Fluorescent Moieties and Dyes)

[0380] In some embodiments, the present invention provides methods and compositions comprising labels for high sensitivity detection and quantification of biomolecules of the present invention (e.g., Eno1 alone or in combination with at least one other indicator of blood glucose and blood glucose control (e.g., HbA1c, ketones)) or for direct measurement of blood glucose. One skilled in the art will recognize that many strategies can be used to label target molecules so that they can be detected or distinguished in a mixture of particles (e.g., labeled anti-Eno1 antibodies or labeled secondary antibodies, or labeled oligonucleotide probes that specifically hybridize to Eno1 mRNA). The labels can be attached by any known means, including methods that utilize nonspecific or specific interactions of the labels and targets. The labels can provide detectable signals or affect the mobility of the particles in an electric field. In addition, labeling can be accomplished directly or by binding to a partner.

[0381] In some embodiments, the label includes a binding partner that binds to a target indicator, wherein the binding partner is linked to a fluorescent moiety. The compositions and methods of the present invention can utilize highly fluorescent moieties, for example, moieties capable of emitting at least about 200 photons when stimulated by laser light emission at the excitation wavelength of the moiety, wherein the laser is focused on a point of no less than about 5 microns in diameter containing the moiety, and wherein the total energy directed by the laser at the point does not exceed about 3 microjoules. The moieties suitable for use in the compositions and methods of the present invention are described in more detail below.

[0382] In some embodiments, the present invention provides a label for detecting a biomolecule, comprising a binding partner of a biomolecule attached to a fluorescent moiety, wherein the fluorescent moiety is capable of emitting at least about 200 photons when stimulated by laser emission at the excitation wavelength of the moiety, wherein the laser is focused on a point of no less than about 5 microns in diameter containing the moiety, and wherein the total energy directed by the laser at the point does not exceed about 3 microjoules. In some embodiments, the moiety comprises a plurality of fluorescent entities, for example, about 2 to 4, 2 to 5, 2 to 6, 2 to 7, 2 to 8, 2 to 9, 2 to 10, or about 3 to 5, 3 to 6, 3 to 7, 3 to 8, 3 to 9, or 3 to 10 fluorescent entities. In some embodiments, the moiety comprises about 2 to 4 fluorescent entities. In some embodiments, the biomolecule is a protein or a small molecule. In some embodiments, the biomolecule is a protein. The fluorescent entity can be a fluorescent dye molecule. In some embodiments, the fluorescent dye molecule comprises at least one substituted indole ring system, wherein the substituent on the 3-carbon of the indole ring contains a chemically reactive group or a coupling substance. In some embodiments, the dye molecule is an Alexa Fluor molecule selected from Alexa Fluor 488, Alexa Fluor 532, Alexa Fluor 647, Alexa Fluor 680, or Alexa Fluor 700. In some embodiments, the dye molecule is an Alexa Fluor molecule selected from Alexa Fluor 488, Alexa Fluor 532, Alexa Fluor 680, or Alexa Fluor 700. In some embodiments, the dye molecule is an Alexa Fluor 647 dye molecule. In some embodiments, the dye molecule includes a first type and a second type of dye molecule, for example, two different Alexa Fluor molecules, for example, in the case where the first type and the second type of dye molecules have different emission spectra. The number ratio of the first type to the second type of dye molecules can be, for example, 4: 1, 3: 1, 2: 1, 1: 1, 1: 2, 1: 3, or 1: 4. The binding partner can be, for example, an antibody.

[0383] In some embodiments, the present invention provides a label for detecting a biological indicator of the present invention, wherein the label comprises a binding partner of the indicator and a fluorescent portion, wherein the fluorescent portion is capable of emitting at least about 200 photons when stimulated by a laser emission light at the excitation wavelength of the portion, wherein the laser is focused on a point of not less than about 5 microns in diameter containing the portion, and wherein the total energy directed by the laser at the point does not exceed about 3 microjoules. In some embodiments, the fluorescent portion comprises a fluorescent molecule. In some embodiments, the fluorescent portion comprises a plurality of fluorescent molecules, for example, about 2 to 10, 2 to 8, 2 to 6, 2 to 4, 3 to 10, 3 to 8 or 3 to 6 fluorescent molecules. In some embodiments, the label comprises about 2 to 4 fluorescent molecules. In some embodiments, the fluorescent dye molecule comprises at least one substituted indole ring system, wherein the substituent on the 3-carbon of the indole ring contains a chemically reactive group or a coupling substance. In some embodiments, the fluorescent molecule is selected from Alexa Fluor 488, Alexa Fluor 532, Alexa Fluor 647, Alexa Fluor 680, or Alexa Fluor 700. In some embodiments, the fluorescent molecule is selected from Alexa Fluor 488, Alexa Fluor 532, Alexa Fluor 680, or Alexa Fluor 700. In some embodiments, the fluorescent molecule is an Alexa Fluor 647 molecule. In some embodiments, the binding partner comprises an antibody. In some embodiments, the antibody is a monoclonal antibody. In other embodiments, the antibody is a polyclonal antibody.

[0384] In various embodiments, the binding partner for detecting the target indicator (e.g., Eno1 or HbA1c) is an antibody or an antigen-binding fragment thereof. The term "antibody" as used herein is a broad term and is used in its ordinary sense, including, without limitation, referring to naturally occurring antibodies and non-naturally occurring antibodies, including, for example, single-chain antibodies, chimeric, bifunctional and humanized antibodies, and antigen-binding fragments thereof. The "antigen-binding fragment" of an antibody refers to the portion of the antibody that participates in antigen binding. The antigen binding site is formed by the amino acid residues of the N-terminal variable ("V") region of the heavy ("H") and light ("L") chains. It will be recognized that the selection of the epitope or molecular region from which the antibody is produced will determine its specificity, for example, specificity for various forms of the molecule (if present), or specificity for the overall (e.g., all, or substantially all molecules).

[0385] The method for producing antibodies is well-defined. Those skilled in the art will recognize that many procedures can be used for the production of antibodies, for example, as described in Antibodies, A Laboratory Manual, edited by Harlow and David Lane, Cold Spring Harbor Laboratory (1988), Cold Spring Harbor, NY. Those skilled in the art will also appreciate that the binding fragments or Fab fragments of simulated antibodies can also be prepared from genetic information by various procedures (Antibody Engineering: A Practical Approach (Antibody Engineering: Practical Methods) (Borrebaeck, C., etc.), 1995, Oxford University Press, Oxford; J. Immunol. 149, 3914-3920 (1992)). Monoclonal and polyclonal antibodies to molecules (e.g., proteins) and markers can also be purchased commercially (R and D Systems, Minneapolis, Minn.; HyTest, HyTest Ltd., Turku Finland; Abcam Inc., Cambridge, Mass., USA, Life Diagnostics, Inc., West Chester, Pa., USA; Fitzgerald Industries International, Inc., Concord, Mass. 01742-3049 USA; BiosPacific, Emeryville, Calif.).

[0386] In some embodiments, the antibody is a polyclonal antibody. In other embodiments, the antibody is a monoclonal antibody.

[0387] Antibodies can be prepared by any of a variety of techniques known to those of ordinary skill in the art (see, e.g., Harlow and Lane, Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory, 1988). Generally, antibodies can be produced by cell culture techniques, including the production of monoclonal antibodies as described herein, or by transfection of antibody genes into suitable bacterial or mammalian cell hosts to allow production of recombinant antibodies.

[0388] Monoclonal antibodies can be prepared using hybridoma methods, such as the techniques of Kohler and Milstein (Eur. J. Immunol. 6: 511-519, 1976), and improvements thereto. These methods involve the preparation of immortalized cell lines capable of producing antibodies with the desired specificity. Monoclonal antibodies can also be prepared by recombinant DNA methods, such as those described in U.S. Patent No. 4,816,567. Conventional procedures can be used to separate and sequence the DNA encoding the antibodies used in the disclosed methods. Recombinant antibodies, antibody fragments and / or their fusions can be expressed in vitro or in prokaryotic cells (e.g., bacteria) or eukaryotic cells (e.g., yeast, insect or mammalian cells), and further purified using known methods as needed.

[0389] More particularly, monoclonal antibodies (MAbs) can be easily prepared by using known techniques, such as those illustrated in U.S. Patent No. 4,196,265 (incorporated herein by reference). Typically, this technique involves immunizing a suitable animal with a selected immunogenic composition (e.g., a purified or partially purified expressed protein, polypeptide or peptide). The immune composition is administered in a manner that effectively stimulates antibody-producing cells. Methods for producing monoclonal antibodies (MAbs) generally begin along the same lines as those used to prepare polyclonal antibodies. Rodents, such as mice and rats, are preferred animals, however, it is also possible to use rabbit, sheep or frog cells. The use of rats can provide specific advantages (Goding, 1986, pp.60-61), but mice are preferred, and BALB / c mice are most preferred because this is the most routinely used and generally gives a higher percentage of stable fusions.

[0390] Animals are injected with antigens as described above. If desired, the antigens can be combined with carrier molecules such as keyhole molecule Hemocyanin coupling. Antigen is usually mixed with adjuvant, such as Freund's complete or incomplete adjuvant. Enhanced injection with the same antigen will be performed at approximately two-week intervals. After immunization, somatic cells with antibody production potential, especially B lymphocytes (B cells), are selected for MAb production schemes. These cells can be obtained from the spleen, tonsils or lymph nodes of biopsy, or from peripheral blood samples. Splenocytes and peripheral blood cells are preferred, the former because they are a rich source of antibody-producing cells in the split plasmablast stage, and the latter because peripheral blood is easily available. Typically, a group of animals are immunized and the spleen of the animal with the highest antibody titer is removed, and spleen lymphocytes are obtained by homogenizing the spleen with a syringe.

[0391] The antibody-producing B lymphocytes from the immunized animal are then fused with cells of immortalized myeloma cells, usually cells of the same species as the immunized animal. Myeloma cell lines suitable for hybridoma production fusion processes are preferably non-antibody-producing, have high fusion efficiency and enzyme deficiencies that make them unable to grow in specific selective culture media that only support the growth of the desired fused cells (hybridomas).

[0392] The selected hybridoma is then serially diluted and cloned into a single antibody-producing cell line, which can then be infinitely propagated to provide MAb. Cell lines can be utilized for MAb production in two basic ways. Samples of hybridomas can be injected (often into the peritoneal cavity) into a histocompatible animal of the type of somatic cells and myeloma cells used to provide the original fusion. The injected animal produces a tumor that secretes the specific monoclonal antibody produced by the fused cell hybrid. The body fluids of the animal, such as serum or ascites, can then be drawn to provide a high concentration of MAb. A separate cell line can also be cultured in vitro, where MAb is naturally secreted into the culture medium, from which a high concentration of MAb can be easily obtained. If desired, the MAb produced by any means can be further purified using filtration, centrifugation and various chromatographic methods, such as HPLC or affinity chromatography.

[0393] A large number of monoclonal antibodies of the present invention can also be obtained by propagating hybridoma cells in vivo. The cell clones are injected into mammals that are tissue compatible with the parental cells, for example, syngeneic mice, to cause the growth of antibody-producing tumors. Optionally, the animals are stimulated with hydrocarbons, especially oils, such as pristane (tetramethylpentadecane), before injection.

[0394] According to the present invention, fragments of the monoclonal antibodies of the present invention can be obtained from the monoclonal antibodies produced according to the above by a method including digestion with an enzyme (such as pepsin or papain) and / or by chemical reduction cleavage of disulfide bonds. Alternatively, an automated peptide synthesizer can be used to synthesize the monoclonal antibody fragments included in the present invention.

[0395] Antibodies can also be derived from recombinant antibody libraries based on amino acid sequences designed in silico and encoded by synthetically generated polynucleotides. Methods for designing and obtaining computer-generated sequences are known in the art (Knappik et al., J. Mol. Biol. 296:254:57-86, 2000; Krebs et al., J. Immunol. Methods 254:67-84, 2001; U.S. Pat. No. 6,300,064).

[0396] Digestion of antibodies can be performed using techniques well known in the art to produce antigen-binding fragments thereof. For example, the proteolytic enzyme papain preferentially cleaves IgG molecules to produce several fragments, two of which (the "F(ab)" fragments) each include a covalent heterodimer (which includes a complete antigen binding site). Pepsin can cleave IgG molecules to provide several fragments, including a "F(ab')2" fragment, which includes two antigen binding sites. "Fv" fragments can be produced by preferential proteolytic cleavage of IgM, IgG, or IgA immunoglobulin molecules, but are more commonly derived using recombinant techniques known in the art. Fv fragments include non-covalent V H ::V L Heterodimers (which include an antigen-binding site) retain much of the antigen recognition and binding capabilities of the native antibody molecule (Inbar et al., Proc. Natl. Acad. Sci. USA 69:2659-2662 (1972); Hochman et al., Biochem. 15:2706-2710 (1976) and Ehrlich et al., Biochem. 19:4091-4096 (1980)).

[0397] Antibody fragments that specifically bind to a polypeptide indicator disclosed herein can also be isolated from scFv libraries using known techniques, such as those described in US Pat. No. 5,885,793.

[0398] Extensive expression systems are available in the art for the production of antibody fragments, including Fab fragments, scFv, VL and VH. For example, expression systems in both prokaryotic and eukaryotic sources can be used for the large-scale production of antibody fragments. Particularly advantageous is the expression system that allows a large amount of antibody fragments to be secreted into the culture medium. The eukaryotic expression system for the large-scale production of antibody fragments and antibody fusion proteins has been described, which is based on mammalian cells, insect cells, plants, transgenic animals and lower eukaryotic cells. For example, economical, large-scale production of antibody fragments can be realized in yeast fermentation systems. The large-scale fermentation of these organisms is well known in the art and is currently used for the large-scale production of several recombinant proteins.

[0399] Antibodies that bind to the polypeptide biomarkers used in the methods of the invention are well known to those of skill in the art and in some cases can be obtained commercially or without undue experimentation.

[0400] In still other embodiments, particularly in cases where oligonucleotides are used as binding partners to detect and hybridize with mRNA biomarkers or other nucleic acid-based biomarkers, the binding partner (e.g., oligonucleotide) may include a label, such as a fluorescent moiety or dye. In addition, any binding partner of the present invention, such as an antibody, may also be labeled with a fluorescent moiety. As the term is used herein, a "fluorescent moiety" includes one or more fluorescent entities whose total fluorescence allows the moiety to be detected in a single molecule detector described herein. Thus, the fluorescent moiety may include a single entity (e.g., a quantum dot or a fluorescent molecule) or multiple entities (e.g., multiple fluorescent molecules). It will be recognized that as a term used herein, when "moiety" refers to a group of fluorescent entities, for example, multiple fluorescent dye molecules, each individual entity may be independently connected to a binding partner or the entities may be connected together, as long as the entity as a group provides sufficient fluorescence to be detected.

[0401] Typically, the fluorescence of the moiety involves a combination of quantum efficiency and lack of photobleaching sufficient to make the moiety detectable above background levels in a single molecule detector with the consistency required for the desired detection limit, accuracy and precision of the assay. For example, in some embodiments, the fluorescence of the fluorescent moiety is such that it allows detection and / or quantification of a molecule, e.g., a marker, in an instrument described herein with a detection limit of less than about 10, 5, 4, 3, 2, 1, 0.1, 0.001, 0.00001, or 0.000001 pg / ml and a coefficient of variation of less than about 20, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, 1% or less, e.g., about 10% or less. In some embodiments, the fluorescence of the fluorescent moiety is such that it allows the detection and / or quantification of molecules, e.g., markers, in an instrument described herein with a detection limit of less than about 5, 1, 0.5, 0.1, 0.05, 0.01, 0.005, 0.001 pg / ml and a coefficient of variation of less than about 10%. "Limit of detection" or LoD, as those terms are used herein, includes the lowest concentration, e.g., the first non-zero value, at which a sample can be identified as containing a target substance. It can be defined by the variability of zeros and the slope of a standard curve. For example, the detection limit of an assay can be determined by running a standard curve, determining the standard curve zero value, and adding 2 standard deviations to that value. The concentration of the target substance that can produce a signal equal to that value is the "lower limit of detection" concentration.

[0402] In addition, the part has properties consistent with its use in the selected test. In some embodiments, the test is an immunoassay in which the fluorescent part is attached to an antibody; the part must have properties that make it not aggregate with other antibodies or proteins or not experience aggregation that exceeds the required test accuracy and precision. In some embodiments, the preferred fluorescent part is a fluorescent part with 1) a high absorption coefficient; 2) a high quantum yield; 3) high photostability (low photobleaching); and 4) a combination of compatibility with the labeling of the target molecule (protein), for example, a dye molecule, so that it can be analyzed using the analyzer and system of the present invention (e.g., without causing precipitation of the target protein, precipitation of the protein to which the part has been attached).

[0403] Any suitable fluorescent moiety can be used. Examples include, but are not limited to, Alexa Fluor dyes (Molecular Probes, Eugene, Oreg.). Alexa Fluor is disclosed in U.S. Patent Nos. 6,977,305; 6,974,874; 6,130,101 and 6,974,305, all of which are incorporated herein by reference. Some embodiments of the present invention utilize dyes selected from Alexa Fluor 647, Alexa Fluor 488, Alexa Fluor 532, Alexa Fluor 555, Alexa Fluor 610, Alexa Fluor 680, Alexa Fluor 700 and Alexa Fluor 750. Some embodiments of the present invention utilize dyes selected from Alexa Fluor 488, Alexa Fluor 532, Alexa Fluor 647, Alexa Fluor 700 and Alexa Fluor 750. Some embodiments of the invention utilize dyes selected from Alexa Fluor 488, Alexa Fluor 532, Alexa Fluor 555, Alexa Fluor 610, Alexa Fluor 680, Alexa Fluor 700, and Alexa Fluor 750. Some embodiments of the invention utilize the Alexa Fluor 647 molecule, which has an absorption maximum between about 650 and 660 nm and an emission maximum between about 660 and 670 nm. The Alexa Fluor 647 dye is used alone or in combination with other Alexa Fluor dyes.

[0404] In some embodiments, the fluorescent labeling part of the indicator used to detect the sample using the analyzer system of the present invention is a quantum dot. Quantum dots (QDs), also known as semiconductor nanocrystals or artificial atoms, are semiconductor crystals containing any number of electrons between 100 and 1,000 and ranging from 2-10nm. Some QDs can be 10-20nm in diameter. QDs have a high quantum yield, which makes them particularly useful for optical applications. QDs fluoresce by forming excitons (which is similar to the excited state of traditional fluorophores), but have a much longer life span of up to 200 nanoseconds. This characteristic provides QDs with low photobleaching. The energy level of QDs can be controlled by changing the size and shape of QDs and the depth of QD potential. An optical feature of small exciton QDs is coloring, which is determined by the size of the dots. The larger the dots, the redder the fluorescence, or more toward the red end of the spectrum. The smaller the dots, the bluer the fluorescence or more toward its blue end. The band gap energy that determines the energy and therefore the fluorescence color is inversely proportional to the square of the size of the QD. Larger QDs have higher energy levels, which are more closely spaced, thus allowing the QDs to absorb photons containing lower energy, i.e., those closer to the red end of the spectrum. Because the emission frequency of the dots depends on the band gap, it is possible to control the output wavelength of the dots with extremely high precision. In some embodiments, proteins detected using a single molecule analyzer system are labeled with QDs. In some embodiments, a single molecule analyzer is used to detect proteins labeled with one QD, and filters are used to allow detection of different proteins at different wavelengths.

[0405] E. Isolated macromolecular blood glucose indicator

[0406] 1. Separated peptide indicator

[0407] One aspect of the present invention relates to isolated indicator proteins and biologically active portions thereof, as well as polypeptide fragments suitable for use as immunogens to produce antibodies against indicator proteins or fragments thereof. In one embodiment, the native indicator protein can be isolated from a cell or tissue source by a suitable purification scheme using standard protein purification techniques. In another embodiment, a protein or peptide comprising a complete indicator protein or a segment thereof is produced by recombinant DNA technology. As an alternative to recombinant expression, such a protein or peptide can be chemically synthesized using standard peptide synthesis techniques. The recombinant protein can be modified, for example, glycosylated, to provide a suitable antigen for HbA1c detection. Similarly, non-glycosylated fragments of hemoglobin can be used to produce antibodies that bind to individual non-glycosylated hemoglobin or total hemoglobin.

[0408] An "isolated" or "purified" protein or biologically active portion thereof is substantially free of cellular material or other contaminating proteins from the cell or tissue source from which the protein is derived, or, when chemically synthesized, substantially free of chemical precursors or other chemicals. The language "substantially free of cellular material" includes protein preparations in which the protein is separated from cellular components from which the protein is isolated or recombinantly produced. Thus, a protein substantially free of cellular material includes protein preparations having less than about 30%, 20%, 10 or 5% (dry weight) of heterologous proteins (also referred to herein as "contaminating proteins"). When the protein or biologically active portion thereof is recombinantly produced, it is also preferably substantially free of culture medium, i.e., culture medium accounts for less than about 20%, 10% or 5% of the volume of the protein preparation. When the protein is produced by chemical synthesis, it is preferably substantially free of chemical precursors or other chemicals, i.e., it is separated from chemical precursors or other chemicals that participate in the synthesis of the protein. Thus, such a protein preparation has less than about 30%, 20%, 10%, 5% (dry weight) of chemical precursors or compounds other than the polypeptide of interest.

[0409] The biologically active portion of the indicator protein includes a polypeptide comprising an amino acid sequence that is sufficiently identical to the amino acid sequence of the indicator protein or is derived from the amino acid sequence of the indicator protein, which includes less than the amino acid of the full-length protein and exhibits at least one activity of the corresponding full-length protein. Typically, the biologically active portion comprises a domain or motif with at least one activity of the corresponding full-length protein. The biologically active portion of the indicator protein can be, for example, a polypeptide of 10, 25, 50, 100 or more amino acids in length. In addition, other biologically active portions in which other regions of the marker protein are deleted can be prepared by recombinant technology, and one or more functional activities of the indicator protein in the native form are evaluated.

[0410] Preferred indicator proteins are encoded by the nucleotide sequences provided in the sequence listing. Other useful proteins are substantially identical to one of these sequences (e.g., at least about 40%, preferably 50%, 60%, 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%) and retain the functional activity of the corresponding naturally occurring indicator protein, but differ in amino acid sequence due to natural allelic variation or mutagenesis.

[0411] In order to determine the percent identity of two amino acid sequences or two nucleic acids, the sequences are aligned for optimal comparison purposes (e.g., for optimal alignment with a second amino acid or nucleic acid sequence, a gap can be introduced in the first amino acid or nucleic acid sequence). The amino acid residues or nucleotides at the corresponding amino acid positions or nucleotide positions are then compared. When a position in the first sequence is occupied by the same amino acid residue nucleotide as the corresponding position in the second sequence, the molecules are identical at that position. Preferably, a global alignment is used to calculate the percent identity between the two sequences. Alternatively, a local alignment is used to calculate the percent identity between the two sequences. The percent identity between the two sequences is a function of the number of identical positions shared by the sequences (i.e., % identity = identical position # / total position # (e.g., overlapping positions) × 100). In one embodiment, the two sequences are of the same length. In another embodiment, the two sequences are of different lengths.

[0412] The determination of percent identity between two sequences can be accomplished using a mathematical algorithm. A preferred, non-limiting example of a mathematical algorithm for the comparison of two sequences is the algorithm of Karlin and Altschul (1990) Proc. Natl. Acad. Sci. USA 87:2264-2268, as modified in Karlin and Altschul (1993) Proc. Natl. Acad. Sci. USA 90:5873-5877. Such an algorithm is incorporated into the BLASTN and BLASTX programs of Altschul et al., (1990) J. Mol. Biol. 215:403-410. BLAST nucleotide searches are performed using the BLASTN program with score = 100 and word length = 12 to obtain nucleotide sequences homologous to the nucleic acid molecules of the present invention. BLAST protein searches can be performed using BALSTP with score = 50 and word length = 3 to obtain amino acid sequences homologous to the protein molecules of the present invention. To obtain gapped alignments for comparison purposes, a newer version of the BLAST algorithm, called GAPED BLAST, can be used as described in Altschul et al. (1997) Nucleic Acids Res. 25:3389-3402, which is capable of performing gapped local alignments for use with programs BLASTN, BLASTP, and BLASTX. Alternatively, PSI-Blast can be used for iterative searches, which detect distance relationships between molecules. When using BLAST, GAPED BLAST, and PSI-Blast programs, the default parameters of the corresponding programs (e.g., BLASTX and BLASTN) can be used. See http: / / www.ncbi.nlm.nih.gov. Another preferred non-limiting example of a mathematical algorithm for sequence comparison is the algorithm of Myers and Miller (1988) CABIOS 4:11-17. Such an algorithm is incorporated into the ALIGN program (version 2.0), which is part of the GCG sequence alignment software package. When utilizing the ALIGN program to compare amino acid sequences, a PAM120 weighted residual table can be used, a gap length penalty of 12 and a gap penalty of 4. Yet another useful algorithm for identifying regions of local sequence similarity and alignment is the FASTA algorithm, as described in Pearson and Lipman (1988) Proc. Natl. Acad. Sci. USA 85: 2444-2448. When utilizing the FASTA algorithm to compare nucleotide or amino acid sequences, for example, a PAM120 weighted residual table can be used with a k-tuple value of 2.

[0413] The percent identity between two sequences can be determined using techniques similar to those described above, with or without allowing gaps. When calculating percent identity, only exact matches are counted.

[0414] Another aspect of the present invention relates to antibodies against proteins of the present invention. In a preferred embodiment, the antibody specifically binds to a marker protein or a fragment thereof. The terms "antibody" and "antibodies" are used interchangeably herein to refer to immunoglobulin molecules and fragments and derivatives thereof containing immunologically active portions of immunoglobulin molecules, (i.e., such portions contain antigen binding sites that specifically bind to antigens (such as marker proteins, e.g., epitopes of marker proteins)). Antibodies that specifically bind to proteins of the present invention are antibodies that bind to proteins but do not substantially bind to other molecules in samples (e.g., biological samples) that naturally contain the protein. Examples of immunologically active portions of immunoglobulin molecules include, but are not limited to, single-chain antibodies (scAb), F(ab), and F(ab')2 fragments.

[0415] The isolated protein of the present invention or its fragment can be used as an immunogen to produce antibodies. Full-length protein can be used, or alternatively, the present invention provides antigenic peptide fragments used as immunogens. The antigenic peptide of the protein of the present invention comprises at least 8 (preferably 10, 15, 20 or 30 or more) amino acid residues of the amino acid sequence of one of the proteins of the present invention, and includes at least one epitope of the protein, so that the antibody produced for the peptide forms a specific immune complex with the protein. In a specific embodiment, the protein is post-translationally modified. The preferred epitope included in the antigenic peptide is a region located on the surface of the protein, for example, a hydrophilic region. Hydrophobic sequence analysis, hydrophilic sequence analysis or similar analysis can be used to identify hydrophilic regions. In a preferred embodiment, an isolated marker protein or its fragment is used as an immunogen.

[0416] The present invention provides polyclonal and monoclonal antibodies. As used herein, the term "monoclonal antibody composition" or "polyclonal antibody composition" refers to a population of antibody molecules containing only one type of antigen binding site that can immunoreact with a specific epitope. Preferred polyclonal and monoclonal antibody compositions are those compositions in which antibodies against the protein of the present invention have been selected. Particularly preferred polyclonal and monoclonal antibody preparations are preparations containing only antibodies against marker proteins or fragments thereof. Methods for preparing polyclonal, monoclonal and recombinant antibodies and antibody fragments are well known in the art.

[0417] 2. Separated Nucleic Acid Indicators

[0418] One aspect of the present invention relates to isolated nucleic acid molecules, including nucleic acids encoding Eno1 or a portion thereof. The isolated nucleic acids of the present invention also include nucleic acid molecules sufficient to be used as hybridization probes to identify Eno1 nucleic acid molecules and fragments thereof, for example, those suitable for use as PCR primers for amplification of specific products or mutations of marker nucleic acid molecules. As used herein, the term "nucleic acid molecule" is intended to include DNA molecules (e.g., cDNA or genomic DNA) and RNA molecules (e.g., mRNA) and analogs of DNA or RNA produced using nucleotide analogs. Nucleic acid molecules can be single-stranded or double-stranded, but are preferably double-stranded DNA.

[0419] An "isolated" nucleic acid molecule is a nucleic acid molecule that is separated from other nucleic acid molecules present in the natural source of the nucleic acid molecule. In one embodiment, an "isolated" nucleic acid molecule (preferably a protein coding sequence) is a sequence that does not contain the natural flanks of the nucleic acid in the genomic DNA of the organism from which the nucleic acid is derived (i.e., the sequences located at the 5' and 3' ends of the nucleic acid). For example, in various embodiments, an isolated nucleic acid molecule may contain less than about 5 kb, 4 kb, 3 kb, 2 kb, 1 kb, 0.5 kb, or 0.1 kb of nucleotide sequences that naturally flank the nucleic acid molecule in the genomic DNA of the cell from which the nucleic acid is derived. In another embodiment, an "isolated" nucleic acid molecule (such as a cDNA molecule) may be substantially free of other cellular material or culture medium when produced by recombinant technology, or substantially free of chemical precursors or other chemicals when chemically synthesized. Nucleic acid molecules that are substantially free of cellular material include preparations having less than about 30%, 20%, 10%, or 5% of heterologous nucleic acids (also referred to herein as "contaminating nucleic acids").

[0420] Can use standard molecular biology techniques and the sequence information in the database record described herein to separate nucleic acid molecules of the present invention.Use all or part of such nucleotide sequence, can use standard hybridization and cloning techniques (for example, as edited by Sambrook etc., Molecular Cloning:A Laboratory Manual (molecular cloning: laboratory manual), the 2nd edition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, described in 1989) to separate nucleic acid molecules of the present invention.

[0421] Can, according to standard PCR amplification technique, use cDNA, mRNA or genomic DNA as template and suitable oligonucleotide primer to increase nucleic acid molecules of the present invention.Can the nucleic acid cloned thus amplified be in suitable carrier and characterize by DNA sequence analysis.In addition, can, by standard synthesis technique, for example, use automated DNA synthesizer, prepare all or part of Nucleotide corresponding to nucleic acid molecules of the present invention.

[0422] In another preferred embodiment, the isolated nucleic acid molecule of the present invention includes an Eno1 molecule having a nucleotide sequence complementary to the nucleotide sequence of a marker nucleic acid or to the nucleotide sequence of a nucleic acid encoding Eno1. A nucleic acid molecule complementary to a given nucleotide sequence is a nucleic acid molecule that is sufficiently complementary to a given nucleotide sequence so that it can hybridize with the given nucleotide sequence to form a stable duplex.

[0423] In addition, the nucleic acid molecules of the present invention may comprise only a portion of a nucleic acid sequence, wherein the full-length nucleic acid sequence comprises an Eno1 nucleic acid or an Eno1 protein encoded thereof. Such nucleic acids can be used, for example, as probes or primers. Probes / primers are typically used as one or more substantially purified oligonucleotides. The oligonucleotides typically comprise a nucleotide sequence region that hybridizes under stringent conditions to at least about 15, more preferably at least about 25, 50, 75, 100, 125, 150, 175, 200, 250, 300, 350 or 400 or more consecutive nucleotides of Eno1.

[0424] Probes based on the Eno1 sequence can be used to detect transcripts or genomic sequences corresponding to Eno1. In a specific embodiment, the probe hybridizes to a nucleic acid sequence spanning a splice junction. The probe includes a labeling group such as a radioisotope, a fluorescent compound, an enzyme or an enzyme cofactor attached thereto. Such probes can be used as part of a diagnostic test kit or reagent set for identifying cells, tissues or individuals that express or misexpress Eno1 protein, such as by measuring the level of nucleic acid molecules encoding Eno1 in a sample from a subject, for example, detecting mRNA levels or determining whether a gene encoding Eno1 or its translation control sequence has been mutated or deleted.

[0425] The present invention further includes nucleic acid molecules that differ from the nucleotide sequence of a nucleic acid encoding an Eno1 protein (eg, a protein having a sequence provided in the sequence listing) due to the degeneracy of the genetic code, and thereby encode the same protein.

[0426] Those skilled in the art will recognize that the DNA sequence polymorphism that causes amino acid sequence variation can be present in a colony (for example, a crowd). Due to natural allele variation, such genetic polymorphism can be present between the individuals in a colony. Allele is one of a group of genes that occurs in a given locus. In addition, it will be recognized that the DNA polymorphism that affects RNA expression levels can also exist, which may affect the overall expression level of this gene (for example, by affecting regulation and control or degradation).

[0427] As used herein, the phrase "allelic variant" refers to a nucleotide sequence or a polypeptide encoded by a nucleotide sequence present at a given locus.

[0428] As used herein, the terms "gene" and "recombinant gene" refer to nucleic acid molecules that include an open reading frame encoding a polypeptide corresponding to an indicator of the present invention. Such natural allele variation can generally result in a 1-5% variability in the nucleotide sequence of a given gene. Optional alleles can be identified by sequencing the target gene in many different individuals. This can be easily performed by using hybridization probes to identify the same locus in multiple individuals. Any such nucleotide variations and resulting amino acid polymorphisms or variations that are not intended to change functional activity are within the scope of the present invention.

[0429] In another embodiment, the isolated nucleic acid molecule of the present invention is at least 15, 20, 25, 30, 40, 60, 80, 100, 150, 200, 250, 300, 350, 400, 450, 550, 650, 700, 800 or more nucleotides in length and hybridizes to an Eno1 nucleic acid or a nucleic acid encoding Eno1 under stringent conditions. As used herein, the term "hybridizes under stringent conditions" is intended to describe hybridization and washing conditions under which nucleotide sequences that are at least 60% (65%, 70%, preferably 75%) identical to each other generally remain hybridized to each other. Such stringent conditions are known to those skilled in the art and can be found in sections 6.3.1-6.3.6 of Current Protocols in Molecular Biology, John Wiley & Sons, NY (1989). A preferred, non-limiting example of stringent hybridization conditions is hybridization in 6X sodium chloride / sodium citrate (SSC) at about 45°C, followed by one or more washes in 0.2X SSC, 0.1% SDS at 50-65°C.

[0430] F. Indicator Application

[0431] The present invention provides methods for diagnosing elevated blood sugar in a subject, e.g., prediabetes, type 2 diabetes, type 1 diabetes, gestational diabetes. The present invention further provides methods for prognosticating or monitoring the progress of a subject with elevated blood sugar or monitoring the response of a subject with elevated blood sugar to therapeutic treatment.

[0432] In one aspect, the present invention constitutes the use of diagnostic information obtainable by the methods of the present invention in combination with analyzing, detecting and / or measuring the levels of Eno1 and at least one other indicator of blood glucose (e.g., blood glucose, e.g., postprandial blood glucose, fasting blood glucose, glucose tolerance, ketone levels) and HbA1c levels.

[0433] For example, as described herein, when performing the method for detecting and / or measuring a polypeptide indicator of the present invention, a biological sample is contacted with a detection reagent, e.g., a monoclonal antibody, that selectively binds to the target indicator, thereby forming a protein-protein complex, which is then further detected directly (if the antibody includes a label) or indirectly (if a second detection reagent, e.g., a second antibody, which is then labeled) using a second detection reagent. Thus, the method of the present invention converts the polypeptide indicator of the present invention into a protein-protein complex comprising a detectable primary antibody or a primary antibody and a further secondary antibody. In order to identify the presence of a target biomarker, it is necessary to form such a protein-protein complex, and as a result of performing the method of the present invention, the physical characteristics and properties of the target indicator must be changed.

[0434] The same principles apply when performing the methods of the invention for detecting Eno1 nucleic acid. In particular, when an amplification method is used to detect Eno1 mRNA, the amplification method actually results in the formation of a new population of amplicons - that is, molecules that are newly synthesized and not present in the original biological sample, thereby physically transforming the biological sample. Similarly, when a hybridization probe is used to detect Eno1, a physically new molecular species is actually formed by hybridization of the probe (optionally including a label) with the target biomarker mRNA (or other nucleic acid), which is then detected. As a result of performing the methods of the invention, such a polynucleotide product is effectively newly generated or formed.

[0435] In one embodiment, the invention provides a method for diagnosing elevated blood sugar, for example, prediabetes, diabetes (e.g., type 2 diabetes, type 1 diabetes, gestational diabetes). The method of the present invention can be combined with any other method for the reaction of the therapeutic intervention of the subject to be treated for the occurrence or recurrence of elevated blood sugar prognosis and / or elevated blood sugar to be treated by those skilled in the art. The diagnostic and prognostic methods provided herein can be used to determine whether other and / or more complicated or troublesome tests or monitoring (e.g., glucose tolerance test, continuous glucose monitoring) should be performed on the subject. It will be understood that a single test can rarely be used to diagnose such complex diseases as prediabetes or diabetes. Therefore, it will be understood that the diagnostic, prognostic and monitoring methods provided herein are usually combined with other methods known in the art. For example, the method for detecting Eno1 levels provided by the present invention can be combined with the detection of Hb1Ac levels, the detection of blood sugar levels under fasting or postprandial conditions, or a glucose tolerance test for carrying out.

[0436] Also provided is a method for evaluating the efficacy of a treatment regimen (e.g., drug therapy, behavior modification, surgery, or any other treatment approach for treating elevated blood sugar in a subject). In these methods, the amount of Eno1 in a pair of samples (a first sample obtained from a subject before an earlier time point or treatment regimen and a second sample obtained from a subject at a later time point (e.g., a later time point when the subject has experienced at least a portion of the treatment regimen)) is evaluated. It will be understood that the method of the present invention includes obtaining and analyzing more than two samples (e.g., 3, 4, 5, 6, 7, 8, 9 or more samples) at regular or irregular intervals for evaluating marker levels. Paired comparisons can be performed between continuous or non-continuous subject samples. The trend of marker levels and the rate of change of marker levels can be analyzed for any two or more continuous or non-continuous subject samples. The measurement of Eno1 levels can be performed in combination with other methods for detecting blood sugar monitoring.

[0437] The method of the present invention can also be used to select compounds that can regulate blood sugar by regulating Eno1 expression or activity. In this method, cells, preferably cells with altered insulin sensitivity or altered glucose uptake, are contacted with a test compound, and the ability of the test compound to regulate Eno1 expression and / or activity in the cell is determined, thereby selecting a compound that can regulate Eno1 expression or activity (preferably increase Eno1 expression or activity, thereby increasing glucose uptake in the cell).

[0438] Using the methods described herein, a variety of molecules can be screened to identify molecules that modulate (preferably increase) the expression and / or activity of Eno1. The compounds thus identified can be provided to a subject to normalize blood glucose by increasing blood glucose uptake, increasing insulin sensitivity, and / or reducing insulin resistance, thereby treating elevated blood glucose, e.g., prediabetes or diabetes, e.g., type 2 diabetes, type 1 diabetes, or gestational diabetes.

[0439] The present invention relates to the field of predictive medicine, in which diagnostic assays, prognostic assays, pharmacogenomics and monitoring clinical trials are used for prognostic (predictive) purposes, thereby prophylactically treating individuals. Thus, one aspect of the present invention relates to diagnostic assays for determining the level of Eno1 protein or nucleic acid expression to determine whether an individual is at risk of developing a disease or disorder associated with elevated blood sugar, such as, but not limited to, prediabetes or diabetes, including type 2 diabetes, type 1 diabetes or gestational diabetes. Such an analysis can be used for prognostic or predictive purposes, thereby prophylactically treating an individual before the onset of a disorder.

[0440] Yet another aspect of the invention relates to monitoring the effects of agents (eg, drugs or other therapeutic compounds) or behavioral and / or dietary changes on the expression or activity of Eno 1 in clinical trials. These and other applications are described in further detail in the following sections.

[0441] 1. Diagnostic analysis

[0442] Exemplary methods for detecting the presence or absence or change of indicator proteins or nucleic acids in biological samples include obtaining a biological sample (e.g., blood or serum) from a test subject and contacting the biological sample with a compound or reagent capable of detecting a polypeptide or nucleic acid (e.g., mRNA or cDNA). Therefore, the detection method of the present invention can detect mRNA, cDNA or proteins in biological samples, such as in vitro and in vivo, including proteins modified after translation.

[0443] The method for detecting the presence, absence, level change of indicator protein or nucleic acid in biological sample provided herein includes obtaining a biological sample that may contain or not contain marker protein or nucleic acid to be detected from a subject, contacting the sample with an indicator-specific binding agent (e.g., one or more marker-specific binding agents) that can form a complex with the indicator protein or nucleic acid to be detected, and contacting the sample with a detection reagent for detecting indicator-indicator-specific binding agent complex (if formed). It will be understood that the method for detecting the indicator level in biological sample provided herein includes the step of performing the analysis. In a specific embodiment of the detection method, the level of the indicator protein or nucleic acid in the sample is zero or below the detection threshold.

[0444] The method includes the formation of a transient or stable complex between the indicator and the indicator-specific binding agent. The method requires that the complex (if formed) is formed for a sufficient time to allow the detection reagent to bind to the complex and produce a detectable signal (e.g., a fluorescent signal, a signal from a product of an enzymatic reaction, e.g., a peroxidase reaction, a phosphatase reaction, a β-galactosidase reaction, or a polymerase reaction).

[0445] In certain embodiments, all indicators are detected using the same method. In certain embodiments, all indicators are detected using the same biological sample (e.g., the same body fluid). In certain embodiments, different indicators are detected using different methods. In certain embodiments, indicators are detected in different biological samples (e.g., blood and serum).

[0446] 2. Protein Detection

[0447] In a specific embodiment of the present invention, the indicator to be detected is a protein. In a specific embodiment, the indicator to be detected is a post-translationally modified protein. Multiple analyses are used to detect proteins, wherein the complex between the indicator protein to be detected and the indicator-specific binding agent is not naturally occurring, for example, because one of the components is not a naturally occurring compound or the indicator and the indicator-specific binding agent used for detection are not from the same organism (for example, using an indicator-specific binding antibody from a mouse, rat or goat to detect a human indicator protein). In a preferred embodiment of the present invention, the indicator used for detection is a human indicator protein. In some detection analyses, the human indicator used for detection is bound by a non-human antibody specific for the indicator, and therefore, the complex will not form in nature. The complex of the indicator protein can be directly detected, for example, by using a labeled indicator-specific antibody directly bound to the indicator, or by combining a further component with an indicator-indicator-specific antibody complex to detect. In a specific embodiment, the further component is a second indicator-specific antibody that can simultaneously bind to the indicator with the first indicator-specific antibody. In a specific embodiment, further component is a second antibody in conjunction with the indicator-specific antibody, wherein the second antibody is preferably connected to a detectable label (e.g., fluorescent label, enzyme label, biotin). When the second antibody is connected to an enzymatic detectable label (e.g., peroxidase, phosphatase, beta-galactosidase), the second antibody is detected by contacting the enzymatic detectable label with a suitable substrate to produce a colorimetric, fluorescent or other detectable (preferably quantitatively detectable) product. The antibody used in the method of the present invention can be polyclonal, but in a preferred embodiment, a monoclonal antibody is used. Complete antibodies or fragments or derivatives thereof (e.g., Fab or F (ab') 2) can be used in the method of the present invention. This type of detection strategy of indicator protein can be used in, for example, ELISA, RIA, western blotting and immunofluorescence analysis methods.

[0448] In a specific detection assay, the indicator present in the biological sample for detection is an enzyme, e.g., Eno1, and the detection reagent is an enzyme substrate (e.g., 2-phosphoglycerate (2-PG) or phosphoenolpyruvate (PEP), or an analog of any compound that produces a detectable product). In a preferred embodiment, the substrate that forms a complex with the indicator enzyme to be detected is not a substrate for the enzyme in a human subject.

[0449] In a specific embodiment, the indicator-indicator specific binding agent complex is connected to a solid support for the detection of the indicator. The complex can be formed on a matrix or formed before being captured on a matrix. For example, in ELISA, RIA, immunoprecipitation analysis, western blotting, immunofluorescence analysis, gel enzymology analysis, the indicator used to detect is directly or indirectly connected to a solid support. In ELISA, RIA or immunofluorescence analysis, the indicator is usually indirectly connected to a solid support by an antibody or a binding protein. In western blotting or immunofluorescence analysis, the indicator is usually directly connected to a solid support. For gel enzymology analysis, the indicator is dissolved in a gel, typically an acrylamide gel, wherein the substrate of the enzyme is mixed.

[0450] 3. Nucleic acid testing

[0451] In a specific embodiment of the present invention, the indicator is a nucleic acid, for example, Eno1 nucleic acid. Nucleic acids are detected using a variety of analyses, wherein the complex between the indicator nucleic acid to be detected and the indicator-specific probe is not naturally occurring, for example, because one of the components is not a naturally occurring compound. In a specific embodiment, the analyte includes nucleic acid, and the probe includes one or more synthetic single-stranded nucleic acid molecules, for example, DNA molecules, DNA-RNA hybrids, PNAs, or modified nucleic acid molecules containing one or more artificial bases, sugars, or backbone moieties. In a specific embodiment, the synthetic nucleic acid is single-stranded, including fluorescently labeled DNA molecules. In a specific embodiment, the synthetic nucleic acid is a single-stranded oligonucleotide molecule of about 12 to about 50 nucleotides in length. In a specific embodiment, the nucleic acid to be detected is mRNA, and the complex formed is an mRNA hybridized with a single-stranded DNA molecule, and the single-stranded DNA molecule is complementary to the mRNA. In a specific embodiment, RNA is detected by first generating a DNA molecule (i.e., a cDNA molecule) from an RNA template using a single-stranded DNA hybridized with RNA as a primer, the primer being, for example, a universal poly-T primer to transcribe poly A RNA. The cDNA can then be used as a template for amplification reactions, e.g., PCR, primer extension assays, using marker-specific probes. In certain embodiments, the labeled single-stranded DNA can be hybridized with RNA present in the sample for RNA detection by in situ fluorescence hybridization (FISH) or for RNA detection by northern blotting.

[0452] For example, in vitro techniques for mRNA detection include northern hybridization, in situ hybridization, and rtPCR. In vitro techniques for detecting genomic DNA include Southern hybridization. Techniques for mRNA detection include PCR, northern hybridization, and in situ hybridization. Methods include qualitative and quantitative methods.

[0453] The general principle of such diagnosis, prognosis and monitoring analysis involves preparing a sample or reaction mixture that may contain nucleic acid and probe for detection, under suitable conditions and for a time sufficient to allow the indicator nucleic acid and probe to interact and bind, thereby forming a complex that can be removed and / or detected in the reaction mixture. These analyses can be performed in a variety of ways known in the art, for example, PCR, FISH, northern blotting.

[0454] 4. Detection of expression levels

[0455] The level of Enol can be detected based on absolute expression level or standardized or relative expression level. When monitoring the treatment of a subject or determining whether there is a change in blood sugar level or blood sugar regulation in a subject, the detection of absolute Enol level may be preferred. For example, the expression level of Enol in a subject who is receiving abnormal blood sugar treatment can be monitored, for example, at regular intervals, such as monthly intervals. The regulation of Enol level can be monitored over time to observe the changing trend of Enol level. The expression level of Enol in the subject may be higher than the expression level of Enol in a normal sample, but it may also be higher than the previous expression level, thus indicating the benefit of the treatment regimen to the subject. Similarly, the rate of change of Enol level may be important in subjects treated with behavioral or dietary correction rather than therapeutic intervention. The change or no change of Enol level in an individual subject may be more relevant to the treatment decision for the subject than the level of Enol present in the group. In addition, rapid changes in Enol level in subjects with normal blood sugar may indicate abnormal blood sugar or have a tendency to produce a disorder related to abnormal blood sugar, even if the marker is within the normal range of the group. Eno1 levels may be determined or monitored in conjunction with one or more other indicators of elevated blood glucose, for example, HbA1c, elevated blood glucose (including one or more of elevated postprandial or fasting blood glucose), or decreased glucose clearance in a glucose tolerance test.

[0456] As an alternative to making a decision based on the absolute expression level of Eno1, the expression level can be determined based on the standardized expression level of Eno1. Standardization is performed by comparing the absolute expression level of the indicator with the expression of a gene that is not an indicator (e.g., a housekeeping gene of constitutive expression). Suitable genes for standardization include housekeeping genes such as actin genes, and suitable proteins for standardization in blood or serum include albumin. This standardization allows the comparison of the expression level in a sample (e.g., a sample from a normal blood sugar subject) with another sample (e.g., a sample from a suspected or abnormal blood sugar), or a comparison between samples from different sources.

[0457] Alternatively, expression levels can be provided as relative expression levels compared to appropriate controls (e.g., population controls, earlier time point controls, etc.). Preferably, the samples used in the baseline assay will be samples from subjects with normal blood sugar. The choice of cell source depends on the use of relative expression levels. In addition, as more data accumulate, the average expression value can be corrected, thereby providing improved relative expression values ​​based on the accumulated data.

[0458] 5. Monitoring clinical trials

[0459] Monitoring the effect of an agent (e.g., a drug compound) on the level of a blood glucose indicator can be applied not only to basic drug screening or monitoring the treatment of a single subject, but also to clinical trials. For example, the effectiveness of an agent affecting the expression of Eno1 can be monitored in a clinical trial of a subject receiving treatment for elevated blood sugar. In a preferred embodiment, the present invention provides a method for monitoring the effectiveness of treating a subject with an agent (e.g., an agonist, an antagonist, a peptide mimetic, a protein, a peptide, a nucleic acid, a small molecule, or other drug candidate), comprising the steps of (i) obtaining a pre-administration sample from a subject before administering the agent; (ii) detecting the expression level of the indicator Eno1 in the pre-administration sample and optionally one or more other blood glucose indicators (e.g., blood glucose, ketones, or HbA1c); (iii) obtaining one or more post-administration samples from a subject; (iv) detecting the expression level of the indicator in the post-administration sample; (v) comparing the indicator level in the pre-administration sample with the indicator level in one or more post-administration samples; and (vi) thus changing the administration of the agent to the subject. For example, decreased Eno1 expression and lack of normalization of other indicators during treatment may indicate an ineffective dose and the desire to increase the dose. Conversely, increased Eno1 expression and normalization of other indicators may indicate effective treatment and no need to change the dose.

[0460] VI. Impaired Blood Glucose Levels, Impaired Blood Glucose Level Control, and Treatment of Diabetes

[0461] As demonstrated herein, administration of the Enol protein increases glucose uptake and response, thereby normalizing blood glucose levels and control of blood glucose levels. The present invention provides a method for treating a subject with impaired glucose tolerance, increased blood glucose, insulin resistance, insulin deficiency, and diabetes (e.g., type 2 diabetes, type 1 diabetes, prediabetes, and gestational diabetes) by administering Enol to the subject to improve at least one sign or symptom of the condition. In a specific embodiment, Eno1, preferably transcript variant 1 of Eno1, can be administered to a subject, wherein at least one other agent for treating impaired glucose tolerance, increased blood glucose, insulin resistance, insulin deficiency, or diabetes is administered to the subject. As used herein, the agents may be administered sequentially or simultaneously in any order. The administration of multiple agents to a subject does not require co-formulation of the agents or the same dosing regimen.

[0462] Methods for treating impaired glucose tolerance, increased blood sugar, insulin resistance, insulin deficiency or diabetes (especially type 2 diabetes) using Enol can be combined with known methods and agents for the treatment of diabetes. Many agents and regimens are currently available for the treatment of diabetes. The specific agent selected for treatment depends on the subject, the specific symptoms and the severity of the condition. For example, in certain embodiments, Enol can be administered in combination with diet and / or behavior modification (e.g., caloric restriction) (alone or in combination with weight loss surgery and / or increased physical activity). In certain embodiments, Eno1 can be administered with an agent used to treat type 2 diabetes, e.g., metformin (Glucophage, Glumetza, others), a glitazone, e.g., pioglitazone (Actos), glipizide (Glucotrol), glibenclamide (Diabeta, Glynase), glimepiride (Amaryl), acarbose (Precose), metformin (Glucophage), sitagliptin (Januvia), saxagliptin (Onglyza), repaglinide (Prandin), nateglinide (Starlix), exenatide (Byetta), liraglutide (Victoza), or insulin. Insulin is generally used only in the treatment of advanced type 2 diabetes and includes rapid-acting insulins (insulin aspart (NovoLog), insulin glulisine (Apidra), and insulin lispro (Humalog)); short-acting insulins (regular insulin (Humulin R, Novolin R)); intermediate-acting insulins (insulin NPH human (Humanlin N, Novolin N)) and long-acting insulins (insulin glargine (Lantus) and insulin detemir (Levemir)). Treatments for diabetes may also include behavioral modification, including exercise and weight loss, which may be promoted by the use of medications or surgery. Treatments for elevated blood sugar and diabetes may be combined. For example, medications may be combined with behavioral modification treatments. Insulins used in the treatment of type 1 diabetes include, but are not limited to, insulins that are typically only used in the treatment of later type 2 diabetes and include rapid-acting insulins (insulin aspart (NovoLog), insulin glulisine (Apidra), and insulin lispro (Humalog)); short-acting insulins (regular insulin (Humulin R, Novolin R)); intermediate-acting insulins (insulin NPH human (Humanlin N, Novolin N)) and long-acting insulins (insulin glargine (Lantus) and insulin detemir (Levemir)).

[0463] Thus, in some aspects, the invention relates to a method of treating elevated blood glucose in a subject, comprising: (a) obtaining a biological sample from a subject suspected of having elevated blood glucose, (b) submitting the biological sample to obtain diagnostic information regarding Eno1 levels, and (c) administering a therapeutically effective amount of an anti-diabetic treatment to the subject when the Eno1 level in the sample is above a threshold level.

[0464] In some aspects, the invention relates to a method of treating elevated blood glucose in a subject, comprising: (a) obtaining diagnostic information regarding the level of Eno1 in a biological sample from the subject, and (b) administering to the subject a therapeutically effective amount of an anti-diabetic treatment when the level of Eno1 in the sample is above a threshold level.

[0465] In some aspects, the present invention relates to a method of treating elevated blood glucose in a subject, comprising: (a) obtaining a biological sample from a subject suspected of having elevated blood glucose for identifying diagnostic information regarding Eno1 levels, (b) detecting the level of Eno1 in the biological sample, and (c) recommending to a health care provider a blood glucose-lowering treatment to be administered to the subject when the level of Eno1 in the sample is below a threshold level.

[0466] The above method may further include obtaining diagnostic information about the level of one or more other indicators of elevated blood glucose. In some embodiments, the method further includes measuring the level of one or more other indicators of elevated blood glucose. The one or more other indicators of elevated blood glucose may be selected from HbA1c levels, fasting blood glucose levels, postprandial blood glucose levels, and glucose tolerance.

[0467] In some embodiments of the above methods, if the level of Eno1 in the sample is below the threshold level and at least one other indicator of elevated blood glucose is detected, step (c) further comprises administering a therapeutically effective amount of a glucose-lowering therapy to the subject. In some embodiments, if the level of Eno1 in the sample is below the threshold level and at least one other indicator of elevated blood glucose is detected, step (c) further comprises recommending a glucose-lowering therapy to a health care provider for administration to the subject.

[0468] In some embodiments of the above methods, the biological sample is blood or serum. In some embodiments, the level of Eno1 is determined by immunoassay or ELISA. In some embodiments, the level of Eno1 is determined by (i) contacting the biological sample with an agent that selectively binds to Eno1 to form a biomarker complex, and (ii) detecting the biomarker complex. In some embodiments, the agent that selectively binds to Eno1 to form a biomarker complex is an anti-Eno1 antibody that selectively binds to at least one epitope of Eno1.

[0469] In some embodiments of the above methods, the level of Eno1 is detected by measuring the amount of Enol mRNA in the biological sample. For example, the amount of Enol mRNA can be detected by an amplification reaction. In some embodiments, the amplification reaction is (a) polymerase chain reaction (PCR); (b) nucleic acid sequence-based amplification assay (NASBA); (c) transcription-mediated amplification (TMA); (d) ligase chain reaction (LCR); or (e) strand displacement amplification (SDA).

[0470] In some embodiments, hybridization analysis is used to detect the amount of Enol mRNA in a biological sample. In some embodiments, an oligonucleotide complementary to a portion of Enol mRNA is used in a hybridization assay to detect Enol mRNA.

[0471] VI. Animal Models of Diabetes and Insulin Resistance

[0472] A variety of genetic and induced animal models of metabolic symptoms (such as type 1 diabetes and type 2 diabetes, insulin resistance, hyperlipidemia) are well characterized in the art. Such animals can be used to demonstrate the role of Eno1 in insulin resistance and diabetes treatment. Models of type 1 diabetes include, but are not limited to, NOD mice and streptozotocin-induced diabetes in rats and mice (type 1 diabetes model). Genetic and induced models of type 2 diabetes include, but are not limited to, leptin-deficient ob / ob mice, leptin receptor-deficient db / db mice, and high-fat diet mice or rat models. In each model, the timeline for the development of specific disease characteristics is well known. Eno1 can be administered before or after the onset of symptoms of diabetes or insulin resistance to demonstrate the efficacy of Eno1 in preventing or treating diabetes and / or insulin resistance in these animal models.

[0473] Depending on the particular animal model chosen and the timing of intervention, e.g., before or after the onset of diabetes and / or insulin resistance, the animal models can be used to demonstrate the efficacy of the methods provided herein for preventing, treating, diagnosing and monitoring diabetes and / or insulin resistance.

[0474] VII. Drug Screening

[0475] Administration of Eno1 results in normalization of blood glucose in animals with induced diabetes, making Eno1 an attractive target for identifying new therapeutic agents by screening to detect compounds or entities that enhance Eno1 expression. Thus, the present invention provides methods for identifying compounds potentially useful for regulating blood glucose and diabetes. In particular, the present invention provides methods for identifying compounds potentially useful for regulating Eno1, wherein the compounds regulate blood glucose and diabetes.

[0476] Such an analysis typically includes a reaction between Eno1 and one or more analytical components (e.g., a test compound). The other components may be the test compound itself, or a combination of the test compound and a natural binding partner of Eno1. Compounds identified by those analyses as described herein are useful, for example, for regulating (e.g., inhibiting, alleviating, treating or preventing) a disease. Compounds identified for regulating the expression level of Eno1 are preferably further tested for activities useful in the treatment of abnormal blood sugar and / or diabetes (e.g., normalizing postprandial and / or fasting blood sugar, normalizing glucose clearance and / or insulin levels in a glucose tolerance test, and normalizing HbA1c levels).

[0477] The test compounds used in the screening assays of the present invention can be obtained from any available source, including a system library of natural and / or synthetic compounds. Test compounds can also be obtained by any of a variety of approaches in combinatorial library methods known in the art, including: biological libraries; peptoid libraries (libraries of molecules with new, non-peptide backbones that have the functionality of peptides but resist enzymatic degradation, but the molecules still retain biological activity; see, e.g., Zuchermann et al., 1994, J. Med. Chem. 37: 2678-85); spatially addressed parallel solid or liquid phase libraries; synthetic library methods requiring deconvolution; 'one bead one compound' library methods; and synthetic library methods using affinity chromatography selection. Biological libraries and peptoid library approaches are limited to peptide libraries, while the other four approaches are applicable to small molecule libraries of peptides, non-peptide oligomers or compounds (Lam, 1997, Anticancer Drug Des. 12: 145).

[0478] Examples of methods for molecular library synthesis can be found in the prior art, for example, in: DeWitt et al., (1993) Proc. Natl. Acad. Sci. USA 90:6909; Erb et al., (1994) Proc. Natl. Acad. Sci. USA 91:11422; Zuckermann et al., (1994) J. Med. Chem. 37:2678; Cho et al., (1993) Science 261:1303; Carrell et al., (1994) Angew. Chem. Int. Ed. Engl. 33:2059; Carell et al., (1994) Angew. Chem. Int. Ed. Engl. 33:2061; and Gallop et al., (1994) J. Med. Chem. 37:1233.

[0479] Compound libraries can be present in solution (e.g., Houghten, 1992, Biotechniques 13:412-421), or on beads (Lam, 1991, Nature 354:82-84), chips (Fodor, 1993, Nature 364:555-556), bacteria and / or spores (Ladner, USP 5,223,409), plasmids (Cull et al., 1992, Proc Natl Acad Sci USA 89:1865-1869), or phage (Scott and Smith, 1990, Science 249:386-390; Devlin, 1990, Science 249:386-390). 249:404-406; Cwirla et al., 1990, Proc. Natl. Acad. Sci. 87:6378-6382; Felici, 1991, J. Mol. Biol. 222:301-310; Ladner, supra).

[0480] The screening methods of the present invention include contacting cells, e.g., diseased cells, particularly cells with abnormal insulin response and / or glucose uptake, with a test compound and determining the ability of the test compound to modulate the expression and / or activity of Eno1 in the cells. The expression and / or activity of Eno1 can be determined using any method known in the art, such as those described herein, optionally in combination with a method for detecting blood glucose levels.

[0481] In another embodiment, the present invention provides a test for screening candidate or test compounds that are substrates for Eno1 or its biologically active portion. In yet another embodiment, the present invention provides an assay for screening candidate or test compounds that bind to Eno1 or its biologically active portion. For example, the ability of the test compound to directly bind to Eno1 can be determined by any method known in the art.

[0482] The present invention further relates to new agents identified by the above-mentioned screening assays. Therefore, it is within the scope of the present invention to further use the agents identified as described herein in suitable animal models. For example, agents capable of modulating the expression and / or activity of Eno1 can be used in animal models to determine the efficacy, toxicity or side effects of treatment using such agents. Alternatively, agents identified as described herein can be used in animal models to determine the mechanism of action of such agents. In addition, the present invention relates to the use of new agents identified by the above-mentioned screening assays for treatment as described above.

[0483] In certain embodiments, screening methods are performed using cells contained in multiple wells of a multi-well assay plate. Such assay plates are commercially available, for example, from Stratagene Corp. (La Jolla, Calif.) and Corning Inc. (Acton, Mass.), and include, for example, 48-well, 96-well, 384-well, and 1536-well plates.

[0484] The repeatability of the results is tested by performing more than one analysis using the same candidate compound at the same concentration (e.g., by incubating cells in more than one well of an assay plate). In addition, because candidate compounds may be effective at different concentrations depending on the properties of the compound and the properties of its mechanism of action, different concentrations of candidate compounds can be tested. Typically, candidate compound concentrations of 1 fM to about 10 mM are used for screening. Preferred screening concentrations are generally about 10 pM to about 100 μM.

[0485] The screening methods of the present invention will provide "hits" or "leads," i.e., compounds that have desired but suboptimal biological activity. Lead optimization of these compounds for all physicochemical, pharmacokinetic, and toxicological factors required for clinical usefulness can yield improved drug candidates. The present invention also includes these improved drug candidates and their use as therapeutic agents for regulating blood glucose and insulin response.

[0486] VIII. Kits / reagent sets

[0487] The present invention also provides compositions for diagnosing, prognosing or monitoring the survival of a subject with a disease or disorder, recurrence of a disorder or a disorder to be treated (e.g., abnormal blood sugar and / or diabetes). These kits include one or more of the following: a detectable antibody that specifically binds to Eno1, a detectable antibody that specifically binds to Eno1, reagents for obtaining and / or preparing a subject tissue sample for staining, and instructions for use.

[0488] The present invention also includes a kit for detecting the presence of Eno1 protein or nucleic acid in a biological sample. Such a kit can be used to determine whether a subject suffers from abnormal blood sugar and / or diabetes or is at an increased risk of producing abnormal blood sugar and / or diabetes. For example, the kit can include a marker compound or reagent capable of detecting Eno1 protein or nucleic acid in a biological sample and a tool for determining the amount of protein or mRNA in the sample (e.g., an antibody binding to a protein or fragment thereof, or an oligonucleotide probe binding to DNA or mRNA encoding the protein). The kit can also include instructions for use of the kit for implementing any of the methods provided herein or for interpreting the results obtained using the kit based on the teachings provided herein. The kit can also include reagents for detecting control proteins not associated with abnormal blood sugar in a sample, for example, actin in a tissue sample, albumin in a blood or blood-derived sample for standardizing the amount of Eno1 present in a sample. The kit can also include a purified marker for detection, used as a control or for quantification of an analysis performed using the kit.

[0489] The kit includes a reagent set for use in a method to diagnose abnormal blood sugar in a subject (or identify a subject susceptible to abnormal blood sugar and / or diabetes), the reagent set including at least two detection reagents, including a reagent for detecting Eno1 levels and a reagent for detecting another indicator of blood sugar (e.g., HbA1c).

[0490] For antibody-based kits, the kit can include, for example: (1) a first antibody that binds to Eno1 (e.g., attached to a solid support); and optionally (2) a different second antibody that binds to Eno1 or the first antibody and is coupled to a detectable label. In a specific embodiment, the kit includes (1) a second antibody that binds to a second marker protein (e.g., attached to a solid support); and optionally (2) a different second antibody that binds to HbA1c or hemoglobin (total or unmodified hemoglobin) or binds to the second antibody and is coupled to a detectable label. The first and second marker proteins are different.

[0491] For oligonucleotide-based kits, the kit can include, for example: (1) an oligonucleotide that hybridizes to a nucleic acid sequence encoding Eno1, for example, a detectably labeled oligonucleotide, or (2) a pair of primers that can be used to amplify Eno1 nucleic acid molecules. In certain embodiments, the kit includes a third primer specific for each nucleic acid marker to allow detection using a quantitative PCR method. In certain embodiments, the kit further includes instructions for measuring blood glucose in a subject directly or indirectly (e.g., using HbA1c levels).

[0492] For chromatographic methods, the kit may include markers, including labeled markers, to allow for the detection and identification of one or more blood glucose indicators, e.g., Eno1 and HbA1c, by chromatography. In certain embodiments, the kit for chromatographic methods includes compounds for derivatization of one or more blood glucose indicators. In certain embodiments, the kit for chromatographic methods includes a column for resolving the indicator of the method.

[0493] Reagents specific for the detection of Eno1 allow for the detection and quantification of markers in complex mixtures (e.g., serum, blood). In certain embodiments, the reagent is species specific. In certain embodiments, the Eno1 reagent is not species specific. In certain embodiments, the Eno1 reagent is isoform specific. In certain embodiments, the Eno1 reagent is not isoform specific. In certain embodiments, the reagent detects total Eno1.

[0494] In certain embodiments, the kit for diagnosis, monitoring or characterization of elevated blood glucose and / or diabetes comprises at least one reagent specific for detection of Eno1 expression levels. In certain embodiments, the kit further comprises instructions for detecting (directly or indirectly or both) blood glucose levels in a sample. In certain embodiments, the kit comprises at least one reagent for detecting HbA1c levels.

[0495] In a particular embodiment, the kit may also include, for example, a buffer, a preservative, a protein stabilizer, a reaction buffer. The kit may further include components necessary for detecting a detectable label (e.g., an enzyme or a substrate). The kit may also contain a control sample or a series of control samples that can be measured and compared with the test sample. The control may be a control serum sample or (if appropriate) a control sample of a purified protein or nucleic acid, with an indicator of a known level. Each component of the kit may be packaged in a single container and all of the various containers may be in a single package, together with instructions for explaining the results of the analysis performed using the kit.

[0496] The kits of the invention may optionally include other components useful for performing the methods of the invention.

[0497] For example, in some aspects, the present invention relates to a kit for detecting Eno1 in a biological sample, comprising at least one reagent for measuring the level of Eno1 in the biological sample, and a set of instructions for measuring the level of Eno1. In some embodiments, the reagent is an anti-Eno1 antibody. In some embodiments, the kit further comprises a means for detecting the anti-Eno1 antibody. In some embodiments, the means for detecting the anti-Eno1 antibody is a detectable second antibody. In some embodiments, the reagent for measuring the level of Eno1 is an oligonucleotide complementary to Eno1 mRNA.

[0498] In some embodiments of the above kits, the instructions provide an immunoassay or ELISA for detecting the level of Eno1 in a biological sample. In some embodiments, the instructions provide an amplification reaction for determining the level of Eno1 mRNA in a biological sample. In some embodiments, the amplification reaction is used to detect the amount of Eno1 mRNA in a biological sample. In some embodiments, the amplification reaction is (a) polymerase chain reaction (PCR); (b) nucleic acid sequence-based amplification analysis (NASBA); (c) transcription-mediated amplification (TMA); (d) ligase chain reaction (LCR); or (e) strand displacement amplification (SDA).

[0499] In some embodiments of the above kits, the instructions provide a hybridization assay for detecting the amount of Enol mRNA in a biological sample. In some embodiments, the kit further comprises at least one oligonucleotide complementary to a portion of Enol mRNA.

[0500] The present invention further provides a reagent set and at least one control reagent for detecting one or more blood glucose indicators in a subject's sample. In a specific embodiment, the control reagent is used to detect the indicator in a biological sample, wherein the reagent set provides a control sample containing an indicator used as a positive control and optionally for the indicator dose present in a quantitative biological sample. In a specific embodiment, the reagent set includes a detection reagent for a protein or nucleic acid unrelated to abnormal blood glucose known to exist or not present in a biological sample to provide a positive or negative control respectively. The reagent set can provide a reagent for a control protein unrelated to abnormal blood glucose in a detection sample, for example, the albumin in blood or a blood-derived sample, for the standardization of the indicator dose present in a sample. The reagent set can provide a purified indicator, for example, Eno1, used as a control for detecting or for the quantitative analysis performed using the reagent set.

[0501] In a preferred embodiment, the reagent set includes a reagent for Eno1 detection, preferably in combination with a control reagent. In the reagent set, Eno1 is detected by a reagent specific for the Eno1. In a specific embodiment, the reagent set further includes a reagent for HbA1c detection. In a specific embodiment, the reagent set includes repeated wells, points or sections to allow analysis of biological samples and control samples of various dilutions (e.g., serial dilutions). In a preferred embodiment, the reagent set allows quantitative detection of one or more indicators of blood glucose.

[0502] In a specific embodiment, the reagent set is a protein chip for detecting one or more markers. In a specific embodiment, the reagent set is an ELISA plate for detecting one or more markers. In a specific embodiment, the set is a quantitative PCR plate for detecting one or more markers.

[0503] In a specific embodiment, a detection reagent set is provided on a single device, including a detection reagent and at least one control sample for one or more markers of the present invention. In a specific embodiment, a detection reagent set can be provided on a single device, including a detection reagent and at least one control sample for two or more markers of the present invention. In a specific embodiment, a plurality of reagent sets for detecting different markers of the present invention are provided with at least one unified control sample to facilitate comparison of results between the reagent sets.

[0504] In certain embodiments, the reagent sets and kits further include instructions or suggestions for measuring blood glucose in a subject. In certain embodiments, the kit or reagent set is provided with one or more reagents or devices for blood glucose measurement.

[0505] The present invention also provides a kit for treating at least one diabetes, for example, type 1 diabetes, type 2 diabetes, gestational diabetes, prediabetes, insulin resistance, glucose intolerance, abnormal blood sugar and loss of blood sugar control. The kit includes one or more of Eno1 and instructions for use and (as needed) a device for administration.

[0506] The present invention is further illustrated by the following examples, which should not be construed as limiting.The contents of all references and published patents and patent applications cited throughout this application are hereby incorporated by reference.

[0507] Example

[0508] Example 1 - Use of platform technology to identify enolase 1 (Eno1) as an important active node in diabetes etiology

[0509] In this example, the platform technology described in detail in International Patent Application No. PCT / US2012 / 027615 was used to integrate data obtained from a customized diabetes model and identify new proteins / pathways that drive the pathogenesis of diabetes (particularly type 2 diabetes). The relationship map obtained from this analysis has identified Eno1 as an important active node in the etiology of diabetes. Therefore, Eno1 is an important diabetes treatment target, as well as a diagnostic / prognostic marker associated with diabetes.

[0510] Example 2 - Eno1 regulation of glucose uptake in myotubes

[0511] Using commercially available expression vectors, Eno1 was recombinantly expressed in E. coli as a 6X HIS protein tag. Tagged Eno1 was purified using affinity chromatography methods known in the art. Preferably, the 6X HIS tag was cleaved to produce a protein for use in the methods provided herein.

[0512] Human skeletal muscle myoblasts (HSMM) were obtained from PromoCell and cultured in the growth medium recommended by the retailer. HSMM myoblasts (20,000 cells / well) were differentiated for 7 days in 96-well plates with 2% horse serum prior to the experiment. Cells were treated with human Eno1 (500ug / ml). Cells were washed twice with 200μl of MBSS modified balanced salt solution (MBSS) buffer containing 0.1% BSA, and then serum starved for 4 hours with 100ul MBSS 0.1% BSA. At the beginning of insulin stimulation, 100ul 2× reagent in MBSS 0.1% BSA buffer was added to 100ul starvation medium to make a 1× concentration for the experiment. 2× reagents were: insulin (0, 20nM and 200nM); fluorescent glucose analog 2-NBDG (500uM). Cells were treated with insulin and fluorescent deoxy-glucose analog 2-NBDG for 30 min, then washed twice with MBSS buffer, and then 50 ul MBSS buffer was added to the wells. Glucose uptake was detected with a fluorometer, together with background detection using wells without cells therein. After the fluorometer readout, fixative (formalin, 50 ul) was added to the 50 ul MBSS in the wells, and then 100 ul 1 Um DAPI was added to the 100 ul formalin and MBSS mixture.

[0513] like Figure 1A and 1BAs shown in, treatment of myotubes with Enol significantly increased glucose uptake in the absence and presence of insulin (p=0.025 non-insulin-dependent glucose uptake, untreated vs. Enol treated). These results demonstrate a role for Enol in both insulin-dependent and non-insulin-dependent glucose uptake. The insulin-dependent glucose uptake induced by Enol demonstrates that Enol is intricately associated with the insulin signaling pathway in at least skeletal muscle in subjects that are insulin sensitive. The results also demonstrate a role for Enol in non-insulin-dependent glucose uptake. This observation is important for the treatment of subjects with type 1 and type 2 diabetes who suffer from insulin resistance and also have hyperinsulinemia, such that insulin action is impaired and therefore non-insulin-dependent. These results demonstrate that Enol is useful in stimulating glucose uptake, even in individuals who no longer have normal insulin signaling.

[0514] Cell cultures of human skeletal muscle myotubes were treated with the purified Eno1 protein described above for 48 h to measure Eno1 uptake. Eno1 levels in cells were then determined by Western blotting. Figure 2A and 2B As shown in , the level of Eno1 in cells treated with 500 μg / ml Eno1 or 1000 μg / ml Eno1 had significantly higher levels of Eno1 relative to untreated cells. The level of Eno1 in cells treated with 1000 μg / ml Eno1 was also higher than that in cells treated with 500 μg / ml Eno1. These results indicate that Eno1 is delivered to human skeletal muscle myotubes in a dose-dependent manner.

[0515] To determine the role of Eno1 enzyme activity in glucose uptake, purified Eno1 was heat-inactivated by treatment at 88°C for 90 seconds, and the activity levels of native and heat-inactivated Eno1 were compared. Eno1 activity was determined by colorimetric analysis using the Eno1 human activity assay kit from Abcam (Cambridge, MA; Cat. No. ab117994). Figure 3A As shown in , heat inactivation greatly reduced Eno1 enzyme activity, but some residual activity remained.

[0516] The effects of native and heat-inactivated Eno1 on glucose uptake were compared in human skeletal muscle myotubes as described above. Figure 3BAs shown in , myotubes treated with pristine (active) Eno1 exhibited significantly higher glucose uptake relative to myotubes not treated with Eno1. Myotubes treated with heat-inactivated Eno1 exhibited significantly lower glucose uptake compared to myotubes treated with active Eno1. These results suggest that the effect of Eno1 on glucose uptake is dependent on Eno1 enzyme activity. The increase in glucose uptake observed with heat-inactivated Eno1 relative to the control without Eno1 is likely due to residual Eno1 activity of the heat-inactivated enzyme.

[0517] Example 3 - Mouse Model of Diet-Induced Obese (DIO) Mice

[0518] Two essentially equivalent models of diet-induced obesity are used in the methods provided herein.

[0519] In the first approach, male C57BL / 6J mice were obtained from Jackson Laboratories (Bar Harbor, ME) and initially housed 4-5 / cage at 22°C with a 12:12hr day-night cycle. Starting at 6 weeks of age, mice were fed a high-fat diet (Research Diets Cat#: D12492; 60 kcal% fat, 20 kcal% protein, and 20 kcal% carbohydrates). Lean control mice were also obtained and fed a standard diet. The body weight of DIO mice before the experiment was significantly heavier than that of the lean control group. In one study, DIO mice weighed 38.4 ± 0.6 g, while lean mice weighed 29.9 ± 0.5 g (p < 0.05).

[0520] In the second method, diet-induced obese male C57BL / 6J mice (12 weeks old) and control lean mice (12 weeks old) were obtained from Jackson Laboratories (Bar Harbor, ME) and initially housed 4-5 / cage at 22°C with a 12:12hr day-night cycle. Mice were acclimated to the animal facility for one week prior to treatment and maintained on a high-fat diet (Research Diets Cat#: D12492; 60 kcal% fat, 20 kcal% protein, and 20 kcal% carbohydrates) for the DIO group or a low-fat diet (10% kcal% fat) for the lean group.

[0521] Example 4 - Treatment of glucose intolerance with Eno1 in diet-induced obese (DIO) mice

[0522] The experimental protocol was initiated when mice (n=10 / group) became obese after 7 weeks of high-fat diet maintenance. Mini-osmotic pumps (model 1004, Alzet, Cupertino, CA) were filled with 0.1 ml of Eno1 peptide or vehicle (phosphate buffered saline (PBS), pH 7.0) according to the manufacturer's instructions. The pumps were prepared overnight at 4°C in sterile saline. Mice were anesthetized with isoflurane (1-3% in 100% oxygen) and scrubbed with 70% isopropyl alcohol and povidone-iodine solution before surgery. A small subcutaneous incision was made in the mid-scapular region, the pump was inserted and the wound was sutured. The animals were allowed to recover before returning to their housing cages. Implantation of the subcutaneous mini-osmotic pumps continuously perfused the peptide at a constant rate of 0.11 μl / hr for four weeks. Pump replacement surgery was performed every 4 weeks. The treatment dose of purified Eno1 calculated by the pump perfusion rate was 10 μg / kg body weight.

[0523] After a 6-h fast, a glucose tolerance test (GTT) was performed using conventional methods. Briefly, an initial fasting blood glucose level was determined, followed by an intraperitoneal (ip) injection of 20% dextrose solution at a dose of 1.5 g / kg body weight. 15, 30, 60, 90, and 120 minutes after the glucose injection, the test was performed using Advantage Blood Glucose Meter( Blood glucose levels were measured from the tail vein using Graphpad The software calculated the area under the curve (AUC) during the GTT, and a student t-test was performed for significance between different treatment groups. The results are shown in Figure 4A and 4B middle.

[0524] As can be easily observed, mice treated with Eno1 had a significantly reduced area under the blood glucose curve compared to untreated mice (p=0.017). These data demonstrate that treatment of obese mice with Eno1 protein improves glucose tolerance, as demonstrated by a glucose tolerance test, and indicate that Eno1 is effective in the treatment of insulin resistance, glucose intolerance, and type 2 diabetes.

[0525] Example 5 - Production of PAMAM dendrimers, muscle-targeted Eno1

[0526] Having demonstrated the efficacy of Eno1 in increasing glucose uptake and improving glucose tolerance in myotubes when administered systemically, muscle-targeted Eno1 was generated to analyze its efficacy in improving glucose tolerance. Detectably labeled G5-PAMAM dendrimers containing the muscle targeting peptide (MTP) ASSLNIA and / or Eno1 were generated using the methods described below. Various ratios of MTP to dendrimer were evaluated, including dendrimers containing MTP containing about 10 MTP peptides / dendrimer, about 3 MTP peptides / polymer, or about 1 MTP peptide / dendrimer.

[0527] The process of preparing the Eno1 dendrimer complex included identification of the optimal ratios and concentrations of the reagents. A stock solution of Eno1 was prepared in buffer and the protein solution was mixed with the G5 dendrimer-muscle targeting peptide (MTP) conjugate at different ratios. Various ratios of dendrimer to Eno1 were also evaluated, including dendrimers containing Eno1 that contained about one dendrimer / Eno1 protein molecule or about five dendrimers / Eno1 protein molecules.

[0528] The stability of the Eno1-dendrimer-SMTP complexes was evaluated at different temperatures and the stability over a period of 3-4 months was determined by measuring Eno1 activity using a commercially available Eno1 assay. Selected conjugates were also evaluated using biophysical techniques, including dynamic light scattering (DLS) and UV-Vis spectroscopy, to confirm the complexation between the dendrimer-peptide conjugate and Eno1.

[0529] Determination of Eno1 purity: The purity of a 5.32 mg / L solution of Eno1 protein was checked by Coomassie and silver staining and Western blotting. Several dilutions of Eno1 protein ranging from 10 μg / well to 100 ng / well were prepared and loaded on 12-well, 4-12% TGX gel [BIO-RAD Cat#456-1095 Lot#4000 79200]. Lanes were assigned as follows: Lane 1: Ladder (Precision Plus Protein Standard Dual Color [BIO-RAD Cat#161-0374]; Lane 2: Eno1 (10.0 μg); Lane 3: Eno1 (1.0 μg); Lane 4: Eno1 (0.1 μg); Lane 5: Ladder (Precision Plus Protein Standard Dual Color [BIO-RAD Cat#161-0374]; Lane 6: Eno1 (10.0 μg); Lane 7: Eno1 (1.0 μg); Lane 8: Eno1 (0.1 μg); Lane 9: Ladder (Precision Plus Protein Standard Dual Color [BIO-RAD Cat#161-0374]; Lane 10: Eno1 (10.0 μg); Lane 11: Eno1 (1.0 μg); Lane 12: Eno1 (0.1 μg). SDS-PAGE was run at 200 V for 20-25 min.

[0530] Coomassie staining: After running the gel, the gel was divided into 3 equal parts. One part was stained with Coomassie. In short, the gel was immersed in 100mL Coomassie staining solution (0.025% Coomassie dye in 40% methanol and 7% acetic acid) and heated in a microwave oven for one minute. Then, the gel was stained for 45 minutes with gentle stirring. After the staining was completed, the gel was destained with a destaining solution (40% methanol and 7% acetic acid) until the background staining was acceptable.

[0531] like Figure 5 As shown in , the protein appears as a single band of approximately 47 kDa, which is consistent with the size of Eno1.

[0532] Silver staining: Since Coomassie staining is not a sensitive method for visualization of protein bands, another portion of the gel was stained with silver stain using the BIO-RAD Silver Staining Kit [BIO-RAD Cat#161-0443]. A modified silver staining protocol was followed.

[0533] like Figure 6 As shown in the figure, additional bands are seen in each lane, which correspond to the bands of the ladder. This is due to the leakage of the ladder bands into the adjacent lanes. The three bands marked with arrows are not from the ladder. The most obvious band is about 47 kDa, which is consistent with the size of Eno1. There are two additional bands in the purified protein, but these bands are faint, indicating that the overall purity of Eno1 is relatively high.

[0534] Western blot analysis: The identity of Eno1 was further confirmed by Western blot. For this purpose, the last part of the gel was transferred to 100 mL Tris-glycine buffer...

Claims

1. A pharmaceutical composition comprising a complex of an Eno1 polypeptide and a muscle targeting peptide, wherein the Eno1 polypeptide and the muscle targeting peptide are connected by a linker, and Wherein the muscle targeting peptide is a skeletal muscle targeting peptide.

2. The pharmaceutical composition of claim 1, wherein the Eno1 polypeptide is a human Eno1 polypeptide.

3. The pharmaceutical composition of claim 2, wherein the muscle targeting peptide comprises an amino acid sequence selected from the group consisting of ASSLNIA, WDANGKT, GETRAPL, CGHHPVYAC and HAIYPRH.

4. The pharmaceutical composition of claim 1, wherein the linker is selected from the group consisting of a covalent linker, a non-covalent linker, and a reversible linker.

5. The pharmaceutical composition of any one of claims 1 to 4, wherein the composition further comprises a pharmaceutically acceptable dendrimer.

6. The pharmaceutical composition of claim 5, wherein the pharmaceutically acceptable dendrimer is a PAMAM dendrimer.

7. The pharmaceutical composition of claim 5, wherein the pharmaceutically acceptable dendrimer is a G5 dendrimer.

8. The pharmaceutical composition of claim 5, wherein the pharmaceutically acceptable dendrimer is an uncharged dendrimer.

9. The pharmaceutical composition of claim 5, wherein the pharmaceutically acceptable dendrimer is an acylated dendrimer.

10. The pharmaceutical composition of claim 5, wherein the pharmaceutically acceptable dendrimer is a PEGylated dendrimer or an acetylated dendrimer.

11. The pharmaceutical composition of any one of claims 1-4, wherein the complex further comprises a liposome.

12. The pharmaceutical composition of any one of claims 1-4, wherein the complex further comprises microparticles.

13. The pharmaceutical composition of any one of claims 1-4, wherein the complex further comprises an in situ formed composition.

14. The pharmaceutical composition of any one of claims 1-13, wherein the Eno1 is released from the complex when delivered to a muscle cell.

15. The pharmaceutical composition of any one of claims 6 to 10, wherein the dendrimer and the ENO1 are present in the complex at a ratio of 1:1 to 10:

1.

16. The pharmaceutical composition of any one of claims 6 to 10, wherein the dendrimer and the ENO1 are present in the complex at a ratio of 3:1 to 5:

1.

17. The pharmaceutical composition of any one of claims 6 to 10, wherein the muscle targeting peptide and the dendrimer are present in the complex at a ratio of 0.1:1 to 10:

1.

18. The pharmaceutical composition of any one of claims 6-10, wherein the muscle targeting peptide and the dendrimer are present in the complex at a ratio of 1:1 to 3:

1.

19. The pharmaceutical composition of any one of claims 6 to 10, wherein the Eno1 and the muscle targeting peptide are present in the complex at a ratio of 1:1 to 1:

30.

20. The pharmaceutical composition of any one of claims 1-19, wherein the composition is formulated for parenteral administration.

21. The pharmaceutical composition of any one of claims 1-19, wherein the composition is formulated for intramuscular administration, intravenous administration, or subcutaneous administration.

22. Use of the pharmaceutical composition of any one of claims 1 to 5 in the preparation of a medicament for lowering blood glucose in a subject with elevated blood glucose.

23. Use of the pharmaceutical composition of any one of claims 1 to 5 in the preparation of a medicament for improving glucose tolerance in a subject with impaired glucose tolerance.

24. Use of the pharmaceutical composition of any one of claims 1 to 5 in the preparation of a medicament for improving insulin sensitivity in a subject with reduced insulin sensitivity.

25. Use of the pharmaceutical composition of any one of claims 1-5 in the preparation of a medicament for treating diabetes in a subject.

26. The use of claim 25, wherein the diabetes is type 2 diabetes or type 1 diabetes.

27. The use of claim 25, wherein the diabetes is prediabetes.

28. Use of the pharmaceutical composition of any one of claims 1 to 5 in the preparation of a medicament for lowering HbA1c levels in a subject having elevated HbA1c levels.

29. Use of the pharmaceutical composition of any one of claims 1 to 5 in the preparation of a medicament for lowering blood glucose levels in a subject with impaired blood glucose control.

30. The use of any one of claims 22-29, wherein glucose flux in skeletal muscle cells of the subject is increased.

31. Use of the pharmaceutical composition of any one of claims 1-5 in the preparation of a medicament for increasing glucose flux in a subject.

32. Use of the pharmaceutical composition of any one of claims 1 to 5 in the preparation of a medicament for increasing the glycolytic activity or capacity in skeletal muscle cells of a subject.

33. Use of the pharmaceutical composition of any one of claims 1 to 5 in the preparation of a medicament for increasing mitochondrial free fatty acid oxidation in skeletal muscle cells of a subject.

34. The use according to any one of claims 22 to 33, wherein the Enol is administered parenterally.

35. The use of claim 34, wherein the Enol is administered by a route selected from intramuscular, intravenous and subcutaneous.

36. The use of any one of claims 22-35, wherein the subject is a human.

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