Methods of treating diabetes, hepatitis and / or inflammatory liver disease

Abstract

The present invention discloses the use of an extracellular human metallothionein (MT) inhibitor for treating a disease selected from diabetes, prediabetes, impaired glucose tolerance, hepatitis and / or inflammatory liver disease, and a composition containing extracellular human MT.

Description

[0001] Cross-references

[0002] This application claims priority to U.S. Provisional Patent Application No. 62 / 618,332 filed on January 17, 2018, and its entire contents are incorporated herein by reference. Background Art

[0003] Diabetes is usually treated with insulin replacement. Research has been conducted to replace the islets that are damaged in diabetes, but unless the inflammatory process that destroys the islets' insulin production is stopped, islet transplantation or regeneration of islet stem cells in the pancreas will be a short-term treatment. Likewise, current methods for treating hepatitis and / or inflammatory liver disease are inadequate. Summary of the invention

[0004] On the one hand, the present disclosure provides a method for treating or limiting the development of a disease, the disease is selected from diabetes, prediabetes, impaired glucose tolerance, hepatitis and / or inflammatory liver disease, including administering an effective therapeutic amount of a composition to a subject suffering from the disease or at risk of the disease to treat or limit the development of the disease, the composition including an extracellular human metallothionein (MT) inhibitor. In one embodiment, the subject suffers from diabetes or is at risk of diabetes, and the method is used to treat or limit the development of diabetes. In another embodiment, the subject has a risk of type 1 diabetes, and the method is used to limit the development of type 1 diabetes in the subject. In one such embodiment, the subject may have one or more risk factors for type 1 diabetes, including, but not limited to, a parent or sibling with type 1 diabetes, pancreatic tumors, pancreatitis, islet cell autoantibodies, insulin autoantibodies, glutamate decarboxylase autoantibodies (GADA), insulinoma-associated (IA-2) autoantibodies, zinc transporter autoantibodies (ZnT8), and a variant of the IDDM1 gene selected from DRB1 0401, DRB1 0402, DRB1 0405, DQA 0301, DQB1 0302, and DQB1 0201; polyuria, polydipsia, dry mouth, polyphagia, fatigue, or weight loss.

[0005] In other embodiments, the subject is a patient with type 1 diabetes, and the method is used to treat type 1 diabetes in the subject. In one such embodiment, the treatment includes one or more of the following: reducing the frequency of insulin injections required; slowing the development or progression of complications of type 1 diabetes in the subject, including but not limited to destruction of pancreatic beta cells, hyperglycemia, hypoglycemia, polyuria, polyphagia, polydipsia, weight loss, blurred vision, fatigue, decreased wound healing ability, urinary tract infection, sexual dysfunction, dry mouth, diabetic ketoacidosis, cardiovascular disease, diabetic nephropathy, diabetic neuropathy, diabetic retinopathy, stroke, renal failure and foot ulcers; and delaying the need for pancreatic or islet cell transplantation.

[0006] In other embodiments, the subject has the risk of type 2 diabetes, and the method is used to limit the development of type 2 diabetes in the subject. In one such embodiment, the subject may have one or more risk factors for type 2 diabetes; the risk factors include but are not limited to obesity, smoking, sedentary lifestyle, parents or siblings with type 2 diabetes, prediabetes, parents or siblings with prediabetes, poor eating habits (e.g., too much fat, insufficient fiber, too much simple carbohydrates), age 50 years or older, high blood pressure, high cholesterol, testosterone deficiency, metallothionein 1A (MT1A) rs8052394 site (G changes) single nucleotide polymorphism, and history of gestational diabetes.

[0007] In other embodiments, the subject is a patient with type 2 diabetes and the method is used to treat type 2 diabetes in the subject. In one such embodiment, the treatment may include limiting one or more complications of type 2 diabetes and reducing the frequency of need for insulin or other treatments, including but not limited to hyperglycemia, hypoglycemia, insulin resistance, diabetic nephropathy, diabetic neuropathy, diabetic retinopathy, proteinuria, impaired glomerular clearance, diabetic circulatory disorders, renal failure, cardiovascular disease, polyuria, polydipsia, weight loss, stroke.

[0008] In other embodiments, the subject is a prediabetic patient, and the method is used to treat prediabetes in the subject. In one such embodiment, the treatment may include limiting or slowing the progression of one or more complications of prediabetes, including but not limited to type 2 diabetes, hyperglycemia, insulin resistance and / or cardiovascular disease.

[0009] In one embodiment, the subject is an impaired glucose tolerance patient, and the method is used to treat the impaired glucose tolerance of the subject. In such an embodiment, the treatment may include limiting or slowing down the progression of a variety of complications of impaired glucose tolerance, including but not limited to type 2 diabetes, hyperglycemia, insulin resistance and / or cardiovascular disease.

[0010] In a further embodiment, the subject is a hepatitis patient and the method is used to treat hepatitis in the subject. In one such embodiment, the treatment may include limiting or slowing the progression of one or more complications of hepatitis, including but not limited to yellowing of white spots on the skin and / or eyes, loss of appetite, vomiting, fatigue, abdominal pain, diarrhea, acute liver failure, liver scarring, liver failure, and liver cancer.

[0011] In other embodiments, the subject is an inflammatory liver disease patient, and the method is used to treat the inflammatory liver disease in the subject. In one such embodiment, the inflammatory liver disease includes non-alcoholic steatohepatitis (NASH) and / or non-alcoholic fatty liver disease (NAFLD). In other such embodiments, the treatment includes limiting or slowing the progression of one or more complications of inflammatory liver disease; the inflammatory liver disease complications include but are not limited to fatigue, discomfort, liver fibrosis, liver cancer and / or cirrhosis.

[0012] In one embodiment, the extracellular human MT inhibitor may include an anti-MT antibody or an antigen binding fragment thereof and / or an aptamer that specifically binds to extracellular human MT. In such an embodiment, the extracellular human MT inhibitor includes an anti-MT antibody or an antigen binding fragment thereof. The anti-MT antibody or its antigen binding fragment includes but is not limited to a monoclonal antibody or its antigen binding fragment, a humanized anti-MT antibody or its antigen binding fragment.

[0013] In other embodiments, the extracellular human MT inhibitor is linked to a pancreatic or hepatocyte targeting moiety. In one such embodiment, the pancreatic cell targeting moiety may include one or more peptides or other moieties that preferentially bind to pancreatic β cells, selected from glucagon-like peptide-1 (GLP-1), glucagon-like peptide-2 (GLP-2), peptide YY (PYY), neuropeptide Y (NPY), pancreatic peptide (PPY) and exendin-4. In other embodiments, the hepatocyte targeting moiety includes but is not limited to circumsporozoite protein (CSP), CSP region I, CSP I plus, milky human serum albumin, glycosylated lipoproteins and / or arabinogalactan.

[0014] In one embodiment, the subject is a mammal including but not limited to a human subject.

[0015] In other aspects, the present disclosure provides a composition comprising:

[0016] (a) Inhibitors of extracellular human metallothionein (MT);

[0017] (b) Pancreatic or hepatocyte targeting moieties linked to inhibitors of extracellular human MT.

[0018] In one embodiment, the inhibitor comprises an anti-metallothionein antibody or a fragment thereof that specifically binds to human metallothionein (MT), including but not limited to a monoclonal antibody and / or a humanized antibody or an antigen-binding fragment thereof. In other embodiments, the cell targeting moiety is a pancreatic cell targeting moiety, and the pancreatic cell targeting moiety is, for example, selected from glucagon-like peptide-1 (GLP-1), glucagon-like peptide-2 (GLP-2), peptide YY (PYY), neuropeptide Y (NPY), pancreatic peptide (PPY) and exendin-4. In other embodiments, the cell targeting moiety is a hepatocyte targeting moiety, which may include but is not limited to circumsporozoite protein (CSP), CSP region I, CSP I plus, lactose-like human serum albumin, glycosylated lipoproteins and / or arabinogalactan.

[0019] In one embodiment, the composition includes a recombinant polypeptide. In other aspects, the present disclosure provides a recombinant nucleic acid encoding a recombinant polypeptide, a recombinant expression vector comprising the recombinant nucleic acid, a recombinant host cell comprising the recombinant expression vector, and a pharmaceutical composition, the pharmaceutical composition comprising (a) the composition, the recombinant nucleic acid and the recombinant expression vector or the recombinant host cell of the present invention; and (b) a pharmaceutically acceptable carrier.

[0020] In other embodiments, the present invention provides compositions, recombinant nucleic acids, recombinant host cells comprising recombinant expression vectors, and uses of the pharmaceutical compositions, which include the compositions, recombinant nucleic acids, recombinant expression vectors, recombinant host cells or drugs described in the present invention, or any embodiment or combination of embodiments disclosed herein for treating or limiting the development of a disease selected from diabetes, prediabetes, impaired glucose tolerance, hepatitis and / or inflammatory liver disease.

[0021] In other embodiments, the inhibitor of extracellular human MT includes a composition, a recombinant nucleic acid, a recombinant host cell comprising a recombinant expression vector, and a pharmaceutical composition, wherein the pharmaceutical composition is a pharmaceutical composition comprising the composition, recombinant nucleic acid, recombinant expression vector, and recombinant host described in the present disclosure, or is a pharmaceutical composition of any embodiment or combination of embodiments disclosed herein. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1Shown are the results of glucose tolerance tests in mice injected with glucose intraperitoneally at the end of the treatment course with MOPC21 or UC1MT;

[0023] Figure 2 shows the normalization to Figure 1 Changes in glucose tolerance test results from initial measurements for each animal in the groups shown;

[0024] Figure 3 The effects of MOPC21 or UC1MT on RNA expression levels in epididymal white adipose tissue by qPCR studies are shown. UC1MT treatment had an effect on the expression of certain genes in white epididymal adipose tissue (particularly peroxisome proliferator-activated receptor gamma-coenzyme activator 1-alpha (PGC-1a), a transcriptional coactivator regulating energy metabolism, and peroxisome proliferator-activated receptor alpha (PPAR-r));

[0025] Figure 4 shows the effect of antibody treatment on adipose tissue weight: (A) Tissue weight as a percentage of body weight; (B) Tissue weight (grams). Note the changes in epididymal white adipose tissue (eWAT). (BAT = brown adipose tissue; sWAT = subcutaneous white adipose tissue);

[0026] Figure 5 The effect of MOPC21 or UC1MT on liver RNA expression levels by qPCR studies is shown. Anti-inflammatory IL-10 gene expression associated with UC1MT treatment was significantly increased in HFD-treated mice compared to HFD mice treated with MOPC21 isotype-matched control antibody;

[0027] Figure 6 Effects of MOPC21 or UC1MT on hepatic triglycerides using the Thermo Fisher Trigylcerides kit (colorimetric) are shown. Means and standard errors are shown for male mice fed a HFD for 16 weeks given 10 UC1MT N=8 MOPC. *p=0.0453, two-tailed t-test;

[0028] Figure 7 Shown is the effect of daily intraperitoneal injection of MOPC21 or UC1MT (100ul per mouse for two weeks) on blood glucose levels in NOD mice after 30 weeks. UC1MT treatment inhibits T1D development in NOD mice;

[0029] Figure 8 shows the effect of intraperitoneal injection of UC1MT or MOPC21, 100ul (twice a week) on blood glucose levels in mice at week 3 (A) and week 6 (B). UC1MT treatment can inhibit the development of T1D in NOD mice;

[0030] Fig. 9 The effect of MOPC21 or UC1MT on the infiltration of inflammatory cells into the islets of Langerhans (insulinitis) is shown. DETAILED DESCRIPTION

[0031] As used herein, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise. As used herein, "and" is used interchangeably with "or" unless the context clearly dictates otherwise.

[0032] Unless the context clearly indicates otherwise, all embodiments of any aspect of the disclosure may be used in combination.

[0033] Unless the context clearly indicates otherwise, throughout the specification and claims, the words "include", "comprising", etc. should be understood to have an inclusive meaning, rather than an exclusive or exhaustive meaning; that is, the meaning of "including but not limited to". Words using the singular or plural number also include the plural and singular, respectively. In addition, when used in this application, the words "herein", "above" and "below" and words of similar meaning shall refer to the invention as a whole, rather than to any particular part of the invention. The composition and method of use thereof may "include", "consist essentially of", or "consist of" any component or step disclosed in the specification.

[0034] The description of the embodiments of the present disclosure is not intended to be exhaustive or to limit the present disclosure to the specific forms disclosed. Although specific embodiments and examples of the present disclosure are described herein for illustrative purposes, various equivalent modifications are possible within the scope of the present disclosure, as those skilled in the relevant art will recognize.

[0035] In one aspect, the present invention provides a method for treating or limiting the progression of a disease selected from diabetes, prediabetes, impaired glucose tolerance, hepatitis and / or inflammatory liver disease, the method comprising administering to an individual suffering from or at risk of the disease an effective therapeutic amount of a composition comprising an extracellular human metallothionein (MT) inhibitor to treat or limit the progression of the disease.

[0036] As disclosed in the examples below, inhibitors of extracellular human MT can be used to treat or limit the development of diabetes, prediabetes, impaired glucose tolerance, hepatitis and / or inflammatory liver diseases.

[0037] As used herein, "effective therapeutic amount" refers to an amount of the composition that is effective in treating and / or limiting the relevant symptoms.

[0038] As described herein, human metallothionein refers to 18 metallothionein isoforms and subtypes that have been identified in humans and classified as MT1 to MT4. Where MT3 and MT4 are selectively expressed, MT1 and MT2 are highly inducible in many cell types and can be released from cells (Lynes et al., 2006, Laukens et al., 2009). The inhibitors disclosed herein specifically target or bind to released metallothioneins. Exemplary subtypes include, but are not limited to;

[0039] Human MT1-A:

[0040]

[0041] Human MT1-B

[0042]

[0043] Human MT1-E

[0044]

[0045] Human MT1_F

[0046]

[0047] Human MT1-G:

[0048]

[0049] Human MT2:

[0050]

[0051] Human MT3:

[0052]

[0053] Human MT-4

[0054]

[0055] In one embodiment, the inhibitors of the present disclosure specifically target or bind to release MT1 and / or MT2.

[0056] "Specific" or "selective" binding to metallothioneins refers to a binding reaction that determines the presence of metallothioneins in a heterogeneous population of proteins and other biological products. Thus, under specified immunoassay or other conditions, a particular antibody or aptamer of the invention binds to metallothioneins at least twice the background level and does not substantially bind to proteins other than metallothioneins present in the sample. Thus, specific binding to an antibody or aptamer under such conditions may involve the use of an inhibitor selected from a group consisting of antibodies or aptamers selected for their specificity for metallothioneins.

[0057] As used herein, "antibodies" include immunoglobulin molecules that immunoreact with human MT (preferably selective for human MT, or selective for one or more human MT isoforms) or fragments thereof, and include monoclonal antibodies. There are a variety of antibody isotypes, such as IgG1, IgG2, IgG3, IgG4 and other Ig, such as IgM, IgA, IgE isotypes. The term also includes genetically engineered forms, such as chimeric antibodies (e.g., humanized mouse antibodies) and heterologous binding antibodies (e.g., bispecific antibodies), fully humanized antibodies and human antibodies. As used throughout this application, the term "antibody" includes fragments with antigen binding ability (e.g., Fab', F(ab')2, Fab, Fv and rIgG. See also Pierce Catalog and Handbook, 1994-1995 (Pierce Chemical Co., Rockford, IL). See also, e.g., Kuby, J., Immunology, 3rd ed., Freeman & Co., New York (1998), which term also refers to recombinant single chain Fv fragments (scFv). The term antibody also includes bivalent or bispecific molecules, diabodies, triabodies and tetrabodies. Bivalent and bispecific molecules are described, e.g., in Kostelny et al. (1992) J. Immunol. 148:1547, Pack and Pluckthun (1992) Biochem. 31:1579, Hollinger et al., 1993, Gruber et al., supra (1994), J. Immunol. 5368, Zhu et al., supra (1995), J. Immunol. 5369, (1997), Protein Science 6:781, Hu et al. (1996), Cancer Research 56:3055, Adams et al. (1993), Cancer Research. 53:4026 and McCartney et al. (1995) Protein Eng. 8:301. The antibody may comprise a heterobifunctional antibody, for example, whose anti-MT arm blocks MT function while stabilizing MT in a specific location by binding to a tissue-specific determinant through the other arm of the antibody. In one embodiment, the antibody comprises a monoclonal antibody. The methods of the present disclosure are demonstrated using the exemplary monoclonal antibody UC1MT, which is described in US 2003 / 0007973 and is commercially available from Abcam Inc., Cambridge, Massachusetts. Clone UC1MT has also been described by Lynes et al. (Toxicology 1993(85):161-177).

[0058] In one embodiment, the antibody used in the methods described herein is a humanized antibody. A humanized antibody refers to a form of a non-human (e.g., mouse) antibody, which is a specific chimeric immunoglobulin, an immunoglobulin chain, or an antigen-binding fragment thereof, which contains a minimal sequence derived from a non-human immunoglobulin. In most cases, a humanized antibody is a human immunoglobulin (receptor antibody), wherein the residues of the complementary determining region (CDR) of the receptor are replaced by CDR residues of a non-human species (donor antibody) (e.g., mouse, rat, or rabbit), and the non-human species has the desired specificity, affinity, and ability. In some cases, the Fv framework region (FR) residues of a human immunoglobulin are replaced by corresponding non-human residues. In addition, a humanized antibody may include residues that are not found in the receptor antibody or the imported CDR or framework sequence, but these residues are included to further improve and optimize antibody performance. Typically, a humanized antibody will include substantially all of at least one variable domain, and typically two variable domains, wherein all or substantially all of the CDR regions correspond to the CDR regions of a non-human immunoglobulin, and all or substantially all of the FR regions are sequences of human immunoglobulin consensus sequences. The humanized antibody will also optimally comprise at least a portion of an immunoglobulin constant region or domain (Fc), typically that of a human immunoglobulin. The antibody may have an Fc region modified as described in WO 99 / 58572. Other forms of humanized antibodies have one or more CDRs (one, two, three, four, five, six) that are altered relative to the original antibody, also referred to as one or more CDRs "derived from" one or more CDRs derived from the original antibody. Humanized antibodies may also involve affinity maturation.

[0059] As used herein, "treating" means accomplishing one or more of the following in an individual suffering from one or more of the diseases: (a) reducing the severity of the disease; (b) limiting or preventing the development of symptoms characteristic of the treated disease; (c) inhibiting the worsening of symptoms characteristic of the treated disease; (d) limiting or preventing the recurrence of the disease in a patient who previously had the disease; and / or (e) limiting or preventing the recurrence of symptoms in a patient who previously had symptoms. Any amount of such "treatment" is highly beneficial to a subject suffering from one of the diseases described.

[0060] As used herein, "limiting" or "limiting the development of" means accomplishing one or more of the following in an individual at risk of one or more of the conditions: (a) slowing disease progression and / or (b) limiting or preventing the progression of symptoms characteristic of the disease. Any amount of such "limiting development" is highly beneficial to a subject at risk of one of the diseases.

[0061] Such treatment or limitation of its development may include the use of the extracellular MT inhibitor as a sole treatment approach, or may include its use to supplement or augment other therapeutic interventions, as deemed appropriate by the attending medical practitioner.

[0062] In one embodiment, the subject is at risk for or has diabetes, and the method is for treating or limiting the development of diabetes.

[0063] In one such embodiment, the subject is at risk for type 1 diabetes, and the method is used to limit the development of type 1 diabetes in the subject. As shown in the following examples, human MT1 inhibitors prevent the NOD mouse model from developing type 1 diabetes. Therefore, the method of this embodiment can be used to limit the development of type 1 diabetes (T1D) in subjects at risk of T1D. Limiting the development of T1D may include, but is not limited to, slowing the progression to T1D and / or slowing the development of characteristic symptoms of T1D. In this embodiment, the subject at risk of T1D has one or more T1D risk factors, from which the attending medical staff believes that the treatment is appropriate. Such T1D risk factors include, but are not limited to, having a parent or sibling with type 1 diabetes, pancreatic tumors, pancreatitis, islet cell autoantibodies, insulin autoantibodies, glutamic acid decarboxylase autoantibodies (GADA), insulinoma-associated (IA-2) autoantibodies, zinc transporter autoantibodies (ZnT8), and / or a variant of the IDDM1 gene selected from DRB1 0401, DRB1 0402, DRB1 0405, DQA 0301, DQB1 0302, and DQB1 0201. Alternatively or in combination, the subject may exhibit one or more symptoms of T1D (but has not yet been diagnosed with T1D); such symptoms may include, but are not limited to, polyuria (increased urination), polydipsia (increased thirst), dry mouth, polyphagia (increased hunger), fatigue, and weight loss. As will be appreciated by those skilled in the art, any limitation of the development of T1D or its symptoms provides a great benefit to subjects at risk.

[0064] The insulin-dependent (type I) diabetes 1 (IDDM1) gene is located in the MHC class II region on chromosome 6. Certain variants of this gene increase the risk of reduced histocompatibility characteristics of type 1 diabetes. Such variants include DRB1 0401, DRB1 0402, DRB1 0405, DQA 0301, DQB1 0302, and DQB10201. Similarly, the appearance of diabetes-related autoantibodies, such as islet cell autoantibodies, insulin autoantibodies, glutamic acid decarboxylase autoantibodies (GADA), insulinoma-associated (IA-2) autoantibodies, zinc transporter autoantibodies (ZnT8) usually precedes the appearance of type 1 diabetes before any hyperglycemia. The risk of T1D increases with the type of antibody, and the time interval from the appearance of autoantibodies to clinically diagnosable T1D in infants and young children may be several months, but it may take years in some people. Such autoantibodies can be detected by, for example, immunofluorescence or binding assays.

[0065] In another embodiment, the subject suffers from type 1 diabetes, and the method is used to treat type 1 diabetes in the subject. In this embodiment, the subject has been diagnosed with T1D, and the method can be used to treat T1D. T1D involves autoimmune destruction of beta cells in the pancreas, little to no insulin production, and hyperglycemia. Therefore, treating T1D involves administering insulin to the subject. Subjects with T1D may experience symptoms or complications, including but not limited to hypoglycemia, polyuria, polyphagia, polydipsia, weight loss, blurred vision, fatigue, decreased wound healing ability, urinary tract infection, sexual dysfunction, dry mouth, diabetic ketoacidosis, cardiovascular disease, diabetic nephropathy, diabetic neuropathy, diabetic retinopathy, stroke, renal failure, and foot ulcers. In some cases, subjects with T1D may need a pancreas or islet transplant. Thus, in various embodiments, treatment may include one or more of the following: reducing the frequency of the need for insulin injections; slowing the development or progression of type 1 diabetes complications in a subject, including but not limited to destruction of pancreatic beta cells, hyperglycemia, hypoglycemia, polyuria, polyphagia, polydipsia, weight loss, blurred vision, fatigue, decreased wound healing, urinary tract infection, sexual dysfunction, dry mouth, diabetic ketoacidosis, cardiovascular disease, diabetic nephropathy, diabetic neuropathy, diabetic retinopathy, stroke, renal failure, and foot ulcers; and delaying the need for pancreas or islet cell transplantation. In one embodiment, the treatment may include reducing blood glucose levels (mg / dL) by 10%, 15%, 20% or more, for example within 20 to 120 minutes after administration of the inhibitor.

[0066] In another embodiment, the subject is at risk of type 2 diabetes, and the method is used to limit the development of type 2 diabetes (T2D) in the subject. T2D is a metabolic disease characterized by hyperglycemia, insulin resistance and relative lack of insulin. Symptoms and / or complications include, but are not limited to, hypoglycemia, insulin resistance, diabetic nephropathy, diabetic neuropathy, diabetic retinopathy, proteinuria, impaired glomerular space, diabetic circulatory system disease, renal failure, cardiovascular disease, polyuria, polydipsia, weight loss and stroke. In one embodiment, the limited development of type 2 diabetes may include, for example, within 20 to 120 minutes after the administration of the inhibitor, and the treatment includes reducing blood sugar levels (mg / dL) by 10%, 15%, 20% or more, for example, within 20 to 120 minutes after the administration of the inhibitor.

[0067] Risk factors for developing T2D include, but are not limited to, obesity, smoking, sedentary lifestyle, having a parent or sibling with type 2 diabetes, prediabetes, having a parent or sibling with prediabetes, poor diet (e.g., too much fat, not enough fiber, too much simple carbohydrates, etc.), age 50 or older, high blood pressure, high cholesterol, testosterone deficiency, metallothionein 1A (MT1A) rs8052394 locus (G change) single nucleotide polymorphism, and gestational diabetes. Thus, in various embodiments, the subject has one or more of these risk factors, and the method is used to slow the progression to T2D and / or (b) limit or prevent the development of symptoms characteristic of T2D.

[0068] As disclosed in the following examples, these methods significantly improve glucose tolerance in T2D mouse models. Therefore, in another embodiment, the subject suffers from T2D, and the method is used to treat T2D in the subject. In this embodiment, treatment may include limiting one or more complications of type 2 diabetes and reducing the frequency of insulin or other therapies, and the type 2 diabetes complications include but are not limited to hyperglycemia, hypoglycemia, insulin resistance, diabetic nephropathy, diabetic neuropathy, diabetic retinopathy, proteinuria, impaired glomerular clearance, diabetic circulatory system disease, renal failure, cardiovascular disease, polyuria, polydipsia, weight loss, stroke. Any degree of limitation of these symptoms / complications is very beneficial to subjects with T2D. In one embodiment, the treatment includes reducing blood glucose levels (mg / dL) by 10%, 15%, 20% or more, for example, within 20 to 120 minutes after the inhibitor is administered.

[0069] In another embodiment, the subject has prediabetes, and the method is used to treat the subject with prediabetes. In this embodiment, the subject is a patient with prediabetes. As used herein, "prediabetes" refers to a state that meets some but not all of the diagnostic criteria for diabetes. Therefore, prediabetic subjects may: (a) impaired fasting glucose tolerance, a condition in which β cells are insufficiently responsive to oral glucose stimulation (OGT), or (b) fasting blood glucose (IFG) may be persistently elevated, i.e., fasting blood glucose is elevated to higher than normal levels, but not enough to be classified as a disease of diabetes. Prediabetic states may be associated with increased risk of insulin resistance and cardiovascular pathology. Individuals with prediabetes have a relatively high risk of T2D. The methods disclosed herein can be used to treat patients with prediabetes, for example, by limiting or slowing the progression of one or more complications of prediabetes, including but not limited to T2D, hyperglycemia, insulin resistance, and / or cardiovascular disease. In one embodiment, treatment may include a 10%, 15%, 20% or more reduction in blood glucose levels (mg / dL), for example, within 20 to 120 minutes after administration of an inhibitor.

[0070] In another embodiment, the subject has impaired glucose tolerance, and the method is used to treat the subject's impaired glucose tolerance. As used herein, impaired glucose tolerance is defined as a two-hour glucose level of 140 to 199 mg / dL (7.8 to 11.0 mmol / L) in a 75 gram oral glucose tolerance test. When a patient's blood sugar level rises moderately after 2 hours, but is lower than the level that meets the criteria for type 2 diabetes, it is said to be in an IGT state. Fasting blood sugar may be normal or slightly elevated. Impaired glucose tolerance is a hyperglycemic state in the prediabetes stage, which is associated with increased risk of insulin resistance and cardiovascular disease. IGT may precede type 2 diabetes for many years. In the present embodiment, the treatment includes limiting or slowing the progression of one or more complications of impaired glucose tolerance, including but not limited to type 2 diabetes, hyperglycemia, insulin resistance and / or cardiovascular disease. In one embodiment, treatment may include a 10%, 15%, 20% or more reduction in blood sugar levels (mg / dL), for example, within 20 to 120 minutes after the administration of the inhibitor.

[0071] In another embodiment, the subject is a hepatitis patient, and the method is used to treat hepatitis in the subject. As disclosed in the following examples, MT inhibitors can effectively limit tissue inflammation and reduce proinflammatory cytokines MCP-1 and TNF-α, while enhancing anti-inflammatory IL-10 signals in liver tissue. Hepatitis is an inflammation of liver tissue. Symptoms include, but are not limited to, yellowing of the whites of the skin and eyes, loss of appetite, vomiting, fatigue, abdominal pain, diarrhea, acute liver failure, liver scarring, liver failure or liver cancer. The most common causes of hepatitis are viral infections (types A, B, C, D and E), heavy drinking, certain drugs, toxins, other infections, autoimmune diseases and non-alcoholic fatty liver disease (NASH). Therefore, in various embodiments, treatment may include limiting or slowing the progression of one or more complications of hepatitis, including, but not limited to, yellowing of the skin and / or whites of the eyes, loss of appetite, vomiting, fatigue, abdominal pain, diarrhea, acute liver failure, liver scarring, liver failure and liver cancer.

[0072] In another embodiment, the subject is an inflammatory liver disease patient, and the method is used to treat the inflammatory liver disease of the subject. As used herein, "inflammatory liver disease" is a disorder associated with the accumulation of triglyceride fat cytoplasm in hepatocytes caused by fatty degeneration (i.e., abnormal retention of lipids in cells). The liver plays an important role in systemic metabolism, and energy imbalance is particularly related to defects in liver lipid metabolism. Specifically, obesity and insulin resistance are generally associated with increased lipid deposition in the liver characteristic of non-alcoholic fatty liver disease (NAFLD). Although lipid metabolism is highly dynamic, chronic lipid overload can lead to liver tissue damage, resulting in the recruitment of liver-resident and non-resident immune cells, which may cause fibrosis of non-alcoholic fatty liver disease (NASH). Liver fibrosis can lead to cirrhosis, cancer and significantly increase the risk of cardiovascular disease. This increases the potential for blocking immune cell recruitment to the liver to reduce the risk of non-alcoholic fatty liver disease (NAFLD). As shown in the following examples, MT inhibitor treatment increases the wet tissue weight of epididymal white adipose tissue, reduces total triglyceride levels, and also reduces proinflammatory cytokines MCP-1 and TNF-α, while enhancing anti-inflammatory IL-10 signals. Inflammatory liver disease may be fatty degeneration (non-alcoholic fatty liver (NAFL)). In another embodiment, fatty liver disease may be non-alcoholic fatty liver disease (NAFLD), including but not limited to non-alcoholic steatohepatitis (NASH), which is the most extreme form of NAFLD. NAFLD is an inflammatory liver disease that occurs due to fat deposition (fatty degeneration) due to reasons other than excessive drinking. Symptoms of NASH and NAFLD may include but are not limited to fatigue, malaise, dull discomfort in the right upper abdomen, mild jaundice, and abnormal liver function tests in routine blood tests; complications of NASH and NAFLD may include but are not limited to liver fibrosis, liver cancer and / or cirrhosis. Therefore, in one embodiment, inflammatory liver disease includes non-alcoholic steatohepatitis (NASH) and / or non-alcoholic fatty liver disease (NAFLD). In another embodiment, the treatment comprises limiting or slowing the progression of one or more complications of inflammatory liver disease including, but not limited to, fatigue, malaise, liver fibrosis, liver cancer, and / or liver cirrhosis.

[0073] In one embodiment, MT inhibitors (including but not limited to anti-MT antibodies) can be linked to pancreatic cell targeting moieties to specifically target pancreatic cells that produce MT, such as pancreatic β cells. This embodiment will be particularly useful for treating or limiting the development of T1D, T2D, prediabetes and / or impaired glucose tolerance. In one embodiment, the pancreatic cell-specific targeting moiety comprises one or more peptides or other moieties that preferentially bind to pancreatic β cells, including but not limited to glucagon-like peptide-1 (GLP-1), glucagon-like peptide-2 (GLP-2), peptide YY (PYY), neuropeptide Y (NPY), pancreatic peptide (PPY) and exendin-4.

[0074] Glucagon-like peptide 1; (aa92-128)

[0075]

[0076] Glucagon-like peptide 2; GLP2 (aa146-178)

[0077]

[0078] Pancreatic Peptide (PPY)

[0079]

[0080] Neuropeptide Y (NPY)

[0081]

[0082] Peptide YY (PYY)

[0083]

[0084] exendin-4

[0085]

[0086] The connection of the pancreatic cell targeting moiety to the MT inhibitor (including but not limited to MT antibody or its fragment or aptamer) can be completed by any chemical reaction that can bind two molecules, as long as the pancreatic cell targeting moiety and the MT antibody or its fragment or aptamer retain their respective activities. In one embodiment, wherein the extracellular MT inhibitor comprises an antibody or its fragment, the composition comprises a recombinant fusion protein. In other embodiments, the connection between the pancreatic cell targeting moiety and the MT antibody or its fragment can include many chemical mechanisms, such as covalent binding, affinity binding, embedding, coordination binding and complexing. Covalent binding can be achieved by direct condensation of existing side chains or by the combination of external bridging molecules. Many divalent or multivalent linking agents can be used to couple protein molecules (such as antibodies) to other molecules. For example, representative non-limiting examples of coupling agents can be organic compounds, such as thioesters, carbodiimides, succinimide esters, diisocyanates, glutaraldehyde, diazobenzene and hexamethylenediamine.

[0087] In all embodiments, the subject can be any subject that can benefit from the treatment methods provided by the present disclosure, including mammals, humans, cows, dogs, cats, horses, chickens, etc. In one embodiment, the subject is a human.

[0088] The composition for administration is usually formulated into a pharmaceutical composition comprising a pharmaceutically acceptable carrier. Suitable acids that can form pharmaceutically acceptable salts include inorganic acids such as hydrochloric acid, hydrobromic acid, perchloric acid, nitric acid, thiocyanic acid, sulfuric acid and phosphoric acid, and organic acids such as formic acid, acetic acid, propionic acid, glycolic acid, lactic acid, pyruvic acid, oxalic acid, malonic acid, succinic acid, maleic acid, fumaric acid, anthranilic acid, cinnamic acid, naphthalenesulfonic acid and sulfanilic acid. Suitable alkalis that can form such salts include inorganic alkalis such as sodium hydroxide, ammonium hydroxide and potassium hydroxide, and organic alkalis such as mono-, di- and trialkylamines and arylamines (e.g. triethylamine, diisopropylamine, methylamine, dimethylamine, etc.) and optionally substituted ethanolamines (e.g. ethanolamine and diethanolamine, etc.).

[0089] In addition to the composition and the carrier, the pharmaceutical composition may also include (a) a lyoprotectant; (b) a surfactant; (c) a filler; (d) a tension regulator; (e) a stabilizer; (f) a preservative and / or (g) a buffer. In some embodiments, the buffer in the pharmaceutical composition is a Tris buffer, a histidine buffer, a phosphate buffer, a citrate buffer, or an acetate buffer. The pharmaceutical composition may also include a lyoprotectant, such as sucrose, sorbitol, or trehalose. In certain embodiments, the pharmaceutical composition comprises a preservative, such as benzalkonium chloride, acetophenone, chlorhexidine, phenol, m-cresol, benzyl alcohol, methylparaben, propylparaben, chlorobutanol, o-cresol, p-cresol, chlorocresol, phenylmercuric nitrate, thimerosal, benzoic acid, and various mixtures thereof. In other embodiments, the pharmaceutical composition comprises a filler, such as glycine. In other embodiments, the pharmaceutical composition includes a surfactant, such as polysorbate-20, polysorbate-40, polysorbate-60, polysorbate-65, polysorbate-80, polysorbate-85, poloxamer-188, sorbitan monolaurate, sorbitan monopalmitate, sorbitan monostearate, sorbitan monooleate, sorbitan trilaurate, sorbitan tristearate, sorbitan trioleate or a combination thereof. The pharmaceutical composition also includes a tension regulator, such as a compound that makes the preparation substantially isotonic or isotonic with human blood. Exemplary tension regulators include sucrose, sorbitol, glycine, methionine, mannitol, dextrose, inositol, sodium chloride, arginine and arginine hydrochloride. In other embodiments, the pharmaceutical composition further comprises a stabilizer, e.g., a molecule that substantially prevents or reduces the chemical and / or physical instability of the protein in lyophilized or liquid form when combined with the protein-based composition. Exemplary stabilizers include sucrose, sorbitol, glycine, inositol, sodium chloride, methionine, arginine, and arginine hydrochloride.

[0090] The composition of the dosage unit formulation can be administered by any suitable route, including oral, parenteral, by inhalation spray, rectal or topical, and the dosage unit formulation contains conventional pharmaceutically acceptable carriers, adjuvants and excipients. The term parenteral as used in the present invention includes subcutaneous, intravenous, intraarterial, intramuscular, intrasternal, intratendinous, intraspinal, intracranial, intrathoracic, infusion techniques or intraperitoneal. The dosage regimen can be adjusted to provide the best desired response (e.g., therapeutic or preventive response). A suitable dosage range can be, for example, 0.1ug / kg to 100mg / kg body weight; or, it can be 0.5ug / kg to 50mg / kg; 1ug / kg to 25mg / kg body weight, or 5ug / kg to 10mg / kg body weight. The composition can be delivered as a single bolus, or it can be administered more than once (e.g., 2, 3, 4, 5 or more times) as determined by the attending medical staff. The composition can be a therapeutic agent administered alone, or it can be administered together with one or more other therapeutic agents (alone or in combination) deemed appropriate by the attending medical staff. In one non-limiting embodiment, the subject suffers from T1D or is at risk of T1D, and the inhibitor can be used with one or more of insulin, metformin or pramlintide. In another embodiment, the subject is a T2D patient or has a risk of prediabetes and / or impaired glucose tolerance, and the inhibitor can be used with one or more of metformin, sulfonylureas (including but not limited to glibenclamide, glipizide, and glimepiride), meglitinib (including but not limited to repaglinide and nateglinide), thiazolidinediones (including but not limited to rosiglitazone and pioglitazone), DPP-4 inhibitors (including but not limited to sitagliptin, saxagliptin and linagliptin), GLP-1 receptor agonists (including but not limited to exenatide, liraglutide and semaglutide), SGLT2 inhibitors (including but not limited to canagliflozin, dapagliflozin and empagliflozin) or insulin. In one non-limiting embodiment, the subject has or is at risk for hepatitis and the inhibitor can be used in combination with one or more of entecavir, tenofovir, lamivudine, adefovir, telbivudine, simeprevir, sofosbuvir, interferon, or ribavirin.

[0091] In another embodiment, a provided composition comprises:

[0092] (a) Inhibitors of extracellular human metallothionein (MT);

[0093] (b) insulin, metformin, pramlintide, sulfonylureas (including but not limited to glibenclamide, glipizide and glimepiride), meglitinib (including but not limited to repaglinide and nateglinide) and thiazolidinediones (including but not limited to rosiglitazone and pioglitazone), DPP-4 inhibitors (including but not limited to sitagliptin, saxagliptin and linagliptin), GLP-1 receptor agonists (including but not limited to exenatide, liraglutide and semaglutide), SGLT2 inhibitors (including but not limited to canagliflozin, dapagliflozin and empagliflozin), one or more of entecavir, tenofovir, lamivudine, adefovir, telbivudine, simeprevir, sofosbuvir, interferon or ribavirin. For example, the composition can be used in the method of the present disclosure. In one embodiment, the inhibitor includes an anti-metallothionein antibody or a fragment thereof that specifically binds to human metallothionein (MT). All embodiments and combinations of the above disclosed antibodies are suitable for inclusion in the composition of this aspect. In one embodiment, the anti-MT antibody or antigen-binding fragment thereof comprises a monoclonal antibody or antigen-binding fragment thereof. In another embodiment, the anti-MT antibody comprises a humanized anti-MT antibody or antigen-binding fragment thereof.

[0094] In another aspect, the present invention provides a composition comprising:

[0095] (a) Inhibitors of extracellular human metallothionein (MT);

[0096] (b) Pancreatic or hepatocyte targeting moieties linked to inhibitors of extracellular human MT.

[0097] For example, these compositions can be used in the methods of the present disclosure. In one embodiment, the inhibitor comprises an anti-metallothionein antibody or a fragment thereof that specifically binds to human metallothionein (MT). All embodiments and groups of embodiments of the antibodies disclosed above are suitable for inclusion in the compositions of this aspect. In one embodiment, the anti-MT antibody or its antigen-binding fragment comprises a monoclonal antibody or its antigen-binding fragment. In another embodiment, the anti-MT antibody comprises a humanized anti-MT antibody or its antigen-binding fragment.

[0098] In one embodiment, the cell targeting moiety is a hepatocyte targeting moiety. In an exemplary embodiment of this type, the hepatocyte targeting moiety includes, but is not limited to, circumsporozoite protein (CSP), CSP region I, CSP region I-plus, emulsified human serum albumin, glycosylated lipoprotein and / or arabinogalactan. In one embodiment, the hepatocyte binding moiety is a peptide or a hepatocyte binding fragment thereof, the peptide being selected from CSP, CSP region I, CSP region I plus. CSP targets Plasmodium sporozoites to the liver due to the presence of circumsporozoite protein (CSP) on the surface of Plasmodium sporozoites (Rathore D, et al. Journal of Biochemistry, 2005; 280(21): 20524-20529). CSP is approximately 400 amino acids long and is organized into three domains: an N-terminal domain containing a conserved KLKQP motif named “Region I,” a highly repetitive central domain, and a C-terminal domain containing another conserved sequence named “Region II” (Singh et al. Cell 2007;131(3):492-504). In addition to the conserved Region I-KLKQP sequence, the N-terminal region contains two consensus heparan sulfate binding sequences upstream of Region I. Peptides containing the conserved Region I amino acids and the two consensus heparin binding sequences from upstream of Region I are named “Region I-plus” ( et al., Nature Reviews Microbiology, 2006;4(11):849-856).

[0099] In another embodiment, the cell targeting moiety is a pancreatic cell targeting moiety, including but not limited to glucagon-like peptide-1 (GLP-1), glucagon-like peptide-2 (GLP-2), peptide YY (PYY), neuropeptide Y (NPY), pancreatic peptide (PPY) and exendin-4; exemplary amino acid sequences of such peptides are described above.

[0100] Any chemical reaction combining two molecules can be used to complete the combination of the cell targeting moiety and the MT inhibitor (including but not limited to MT antibody or its fragment or aptamer), as long as the cell targeting moiety and the MT antibody or its fragment or aptamer maintain their respective activities. In an embodiment where the extracellular MT inhibitor includes an antibody or its fragment and the cell targeting moiety is a peptide, the composition includes a recombinant fusion protein. In other embodiments, the connection between the cell targeting moiety and the MT antibody or its fragment can include many chemical mechanisms, such as covalent binding, affinity binding, intercalation, coordination binding and compounding. Covalent binding can be achieved by direct condensation of existing side chains or by the combination of external bridging molecules. Many divalent or multivalent linking agents can be used to couple protein molecules (such as antibodies) with other molecules. For example, representative non-limiting examples of coupling agents can be organic compounds, such as thioesters, carbodiimides, succinimide esters, diisocyanates, glutaraldehyde, diazobenzene and hexamethylenediamine.

[0101] In another aspect, the present disclosure provides recombinant nucleic acids encoding recombinant fusion polypeptides of anti-MT antibodies or fragments thereof fused to a peptide targeting moiety, including those specifically disclosed herein. The recombinant nucleic acid sequence may include single-stranded or double-stranded RNA or DNA and derivatives thereof. Such recombinant nucleic acid sequences may include additional sequences for promoting expression and / or purification of the encoded protein, including but not limited to polyA sequences, modified Kozak sequences, and sequences encoding epitope tags, export and secretion signals, nuclear localization signals, and plasma membrane localization signals.

[0102] On the other hand, a recombinant expression vector is disclosed, which includes a recombinant nucleic acid of any embodiment or combination of embodiments of the present invention, operably connected to a suitable control sequence. "Recombinant expression vector" includes a vector that operably connects a nucleic acid coding region or gene to any control sequence that can affect the expression of a gene product. A "control sequence" operably connected to a nucleic acid sequence of the present invention is a nucleic acid sequence that can affect the expression of a nucleic acid molecule. A control sequence does not have to be adjacent to a nucleic acid sequence, as long as they have the function of directing its expression. Thus, for example, there may be an inserted transcribed but untranslated sequence between a promoter sequence and a nucleic acid sequence, and the promoter sequence may still be considered to be "operably connected" to a coding sequence. Other such control sequences include, but are not limited to, polyadenylation signals, termination signals, and ribosome binding sites. Such expression vectors may be of any type, including, but not limited to, plasmids and virus-based expression vectors. The expression vector may be replicable in a host organism as an episome or integrated into a host chromosomal DNA. In non-limiting embodiments, the expression vector may include a plasmid or a viral vector.

[0103] In another aspect, the present invention provides a recombinant host cell comprising the recombinant expression vector, wherein the host cell may be prokaryotic or eukaryotic, such as a mammalian cell. The cell may be transiently or stably transfected.

[0104] In another embodiment, the use of a composition, a recombinant nucleic acid, a recombinant expression vector, a recombinant host cell or a pharmaceutical composition of any embodiment or combination of embodiments disclosed herein is disclosed to treat or limit the development of a disease including diabetes, prediabetes, impaired glucose tolerance, hepatitis and / or inflammatory liver disease. These uses are as described above.

[0105] In another embodiment, the present invention provides a method for treating or limiting the development of a disease by administering an effective amount to a subject, a recombinant nucleic acid, a recombinant expression vector, a recombinant host cell or a pharmaceutical composition of any embodiment or combination of facts disclosed herein, wherein the disease includes diabetes, prediabetes, impaired glucose tolerance, hepatitis and / or inflammatory liver disease.

[0106] Example

[0107] UC1MT for the treatment of type 2 diabetes

[0108] To mimic the insulin resistance characteristic of type II diabetes, we fed mice a high-fat diet (HFD). Before starting treatment, 5-week-old C57BL / 6J mice were fed a standard 60% high-fat diet for 14 weeks. Mice were injected IP twice a week with 100 μL of 1 mg / mL anti-MT antibody (UCIMT; purchased from Abcam, Cambridge, MA) for 8 weeks, and then the mice were killed at 27 weeks of age (a total of 22 weeks of HFD). There were no differences in body weight or food intake between the two treatment groups. Figure 1 Shown are the results of a glucose tolerance test in mice injected intraperitoneally with glucose at the end of a course of treatment with an IgG1 isotype control (MOPC21) or UC1MT. Figure 2 The normalized change of the glucose tolerance test results for each animal in the group is shown. Control animals developed fatty liver and glucose intolerance, but mice treated with UC1MT showed better glucose tolerance compared to controls. These data demonstrate that anti-MT antibodies can be used to treat impaired glucose tolerance. Since glucose tolerance can modulate the inflammatory state, these results also suggest that UC1MT can improve the inflammatory status of mice, thereby improving the ability of HFD-fed mice to handle large glucose challenges.

[0109] Effects of UC1MT or MOPC21-treated HFD-fed mice on liver inflammation in NASH (non-alcoholic steatohepatitis)

[0110] The liver plays an important role in systemic metabolism, and energy imbalance is particularly associated with defects in hepatic lipid metabolism. Specifically, obesity and insulin resistance are often associated with increased lipid deposition in the liver in non-alcoholic fatty liver disease (NAFLD) (Katsiki N et al., Metabolism, 2016 PMID: 27237577). Although lipid metabolism is highly dynamic (Sakaguchi M et al., Cell Metabolism, 2017 PMID: 28065828), chronic lipid overload can lead to liver tissue damage, resulting in the recruitment of liver-resident and non-resident immune cells, which may cause fibrosis in non-alcoholic steatohepatitis (NASH) (Narayanan S et al., ImmunoNetwork, 2016 PMID: 27340383). Liver fibrosis can lead to cirrhosis, cancer, and significantly increase the risk of cardiovascular disease. This raises the potential to block the recruitment of immune cells to the liver to reduce the risk of non-alcoholic fatty liver disease (NAFLD).

[0111] The hepatic phenotype of the same animals used in the above type 2 diabetes study was examined by evaluating mRNA expression through qPCR studies. UC1MT treatment had an effect on the expression of certain genes in white epididymal adipose tissue (specifically peroxisome proliferator-activated receptor gamma coactivator 1-alpha (PGC-1a), a transcriptional coactivator regulating energy metabolism, and peroxisome proliferator-activated receptor (PPARγ)) ( Figure 3 ).

[0112] At sacrifice, liver, pancreas, squamous white adipose tissue, eWAT, intrascapular brown adipose tissue, and serum were collected. Half of each sample was used for RNA and half for histology (all tissues were fixed in formalin, pancreas was fixed with Z-FIX TM (Fisher Scientific) fixation. The data show that the anti-MT antibody has the following effects:

[0113] Increased wet tissue weight of epididymal white adipose tissue ( Figure 4A -B);

[0114] Decreased levels of total triglycerides in the liver ( Figure 6 );

[0115] Decreased expression of certain pro-inflammatory cytokines (MCP-1 and TNF-a) Figure 5 );

[0116] Increased expression of anti-inflammatory IL-10 signaling ( Figure 5 ).

[0117] Anti-metallothionein antibodies did not alter body weight or weight gain in mice treated with a high-fat diet, nor did they significantly change liver histology.

[0118] Consistent with the improved glucose tolerance described above, hepatic triglyceride levels were reduced in mice treated with anti-MT antibodies. These data suggest that MT inhibitors can improve systemic glucose metabolism, thereby alleviating the liver's burden of nutrient excess and limiting the development of hepatic steatosis.

[0119] Type 1 Diabetes Research

[0120] The NOD / ShiLtJ mouse strain (commonly referred to as NOD) is a polygenic model of autoimmune type 1 diabetes. Diabetes in NOD mice is characterized by hyperglycemia and insulitis, i.e., leukocyte infiltration of the pancreatic islets. Female rats show a significant decrease in pancreatic insulin content at approximately 12 weeks of age, while male rats show a significant decrease after a few weeks. 0% of females and 45% of males develop diabetes after 30 weeks; the median incidence in females is 17 weeks. The immune phenotype in the NOD background includes defects in antigen presentation, T lymphocyte repertoire, NK cell function, macrophage cytokine production, wound healing, and C5 complement. These defects make the NOD background a common choice for immunodeficient mouse strains.

[0121] We used female NOD (non-obese diabetic) mice for our type 1 DM prevention studies because NOD mice have been found to spontaneously develop type 1 diabetes since they were described in 1980. The pathogenic events begin at least 3 weeks after birth with the appearance of islet antigens in the pancreatic lymph nodes. Insulitis, first of APCs and then of lymphocytes, begins at approximately 4 to 6 weeks of age and progresses gradually over the next 15 weeks. Frank diabetes with BS > 250 mg / dL begins within 18 to 20 weeks. In addition, the incidence of spontaneous diabetes in NOD mice is 60 to 80% in females and 20 to 30% in males. Therefore, we decided to divide NOD mice into 3 groups (10 mice in each treatment group): (1) treated with PBS as a negative control; (2) treated with 0.1 mg / mL nonspecific IgG; MOPC was used as an isotype control; (3) each mouse was injected intraperitoneally with 0.1 mg / ml UCIMT every day to study the potential role of anti-MT monoclonal antibodies in T1 diabetes. Each group received the designated treatment starting at 5 weeks of age. The total course of treatment was 2 weeks. Blood glucose levels were checked once a week, and mice with blood glucose greater than 250 mg / dL for 2 consecutive weeks were killed. The remaining non-diabetic NOD mice were killed at 30 weeks of age. Figure 7 As shown in Figure 8, anti-MT antibody treatment significantly reduced glucose intolerance in mice treated with a high-fat diet compared to the isotype control MOPC21 or PBS vehicle control group. UC1MT also resulted in a significant reduction in insulin-like inflammation (i.e., inflammatory cell infiltration into the islets) ( Fig. 9 ).

Claims

1. Use of an extracellular human metallothionein (MT) inhibitor in the preparation of a medicament for treating or limiting the progression of a disease in a subject, wherein: The disease is selected from type 2 diabetes and non-alcoholic steatohepatitis (NASH), the extracellular human metallothionein (MT) inhibitor includes an anti-MT antibody or an antigen-binding fragment thereof that specifically binds to extracellular human MT, and the extracellular human metallothionein (MT) inhibitor is UC1MT.

2. The use according to claim 1, wherein The subject is a patient with type 2 diabetes or a patient at risk of developing type 2 diabetes.

3. The use according to claim 2, wherein The subject is at risk for type 2 diabetes.

4. The use according to claim 3, wherein The subject has one or more of the risk factors for type 2 diabetes selected from obesity, smoking, a sedentary lifestyle, a parent or sibling with type 2 diabetes, prediabetes, a parent or sibling with prediabetes, poor dietary habits, age 50 years or older, high blood pressure, high cholesterol, testosterone deficiency, a G-change single nucleotide polymorphism at metallothionein 1A (MT1A) rs8052394, and a history of gestational diabetes.

5. The use according to claim 2, wherein The subject is a patient with type 2 diabetes.

6. The use according to claim 5, wherein The treatment includes limiting one or more complications of type 2 diabetes and reducing the frequency of the need for insulin or other treatment, and the complications are selected from hyperglycemia, hypoglycemia, insulin resistance, diabetic nephropathy, diabetic neuropathy, diabetic retinopathy, proteinuria, impaired glomerular clearance, diabetic circulatory disorders, renal failure, cardiovascular disease, polyuria, polydipsia, weight loss, stroke.

7. The use according to claim 1, wherein The disease is non-alcoholic steatohepatitis (NASH).

8. The use according to claim 7, wherein The treatment includes limiting or slowing the progression of one or more complications of NASH selected from fatigue, malaise, liver fibrosis, liver cancer and / or liver cirrhosis.

9. The use according to claim 1, wherein The subject is a mammal.

10. The use according to claim 9, wherein The subject is a human.

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