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 the treatment of a disease selected from diabetes mellitus, prediabetes mellitus, impaired glucose tolerance, hepatitis and / or inflammatory liver disease, and a composition containing an extracellular human MT.

Description

[0001] Cross-reference

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

[0003] Diabetes is typically treated with insulin replacement therapy. Studies have been conducted to replace the islets of Langerhans that are damaged in diabetes, but islet transplantation or regeneration of islet stem cells in the pancreas will be a short-term treatment unless the inflammatory process that destroys the islets' ability to produce insulin is halted. Similarly, current methods for treating hepatitis and / or inflammatory liver diseases are inadequate. Summary of the Invention

[0004] In one aspect, the present disclosure provides methods for treating or limiting the development of a disease selected from diabetes, prediabetes, impaired glucose tolerance, hepatitis, and / or inflammatory liver disease, comprising administering to a subject having the disease or at risk of having the disease an effective therapeutic amount of a composition to treat or limit the development of the disease, the composition comprising an extracellular metallothionein (MT) inhibitor. In one embodiment, the subject has diabetes or is at risk of developing diabetes, and the method is for treating or limiting the development of diabetes. In another embodiment, the subject is at risk of developing type 1 diabetes, and the method is for limiting 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 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 variants of the IDDM1 gene, the variants of the IDDM1 gene being selected from DRB10401, DRB10402, DRB10405, DQA0301, DQB10302, and DQB10201; 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 the subject's type 1 diabetes. 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 type 1 diabetes complications 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 infections, sexual dysfunction, dry mouth, diabetic ketoacidosis, cardiovascular disease, diabetic nephropathy, diabetic neuropathy, diabetic retinopathy, stroke, kidney failure, and foot ulcers; and delaying the need for pancreatic or islet cell transplantation.

[0006] In other embodiments, the subject is at 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, having a parent or sibling with type 2 diabetes, prediabetes, having a parent or sibling with prediabetes, poor eating habits (e.g., too much fat, insufficient fiber, too many simple carbohydrates), age 50 or older, high blood pressure, high cholesterol, testosterone deficiency, the single nucleotide polymorphism of metallothionein 1A (MT1A) rs8052394 locus (G change), and a history of gestational diabetes.

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

[0008] In other embodiments, the subject is a patient with prediabetes, and the method is used to treat the 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 a patient with impaired glucose tolerance, and the method is for treating the impaired glucose tolerance of the subject. In one such embodiment, the treatment can include restricting 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.

[0010] In a further embodiment, the subject is a patient with hepatitis, and the method is for treating the hepatitis of the subject. In one such embodiment, the treatment can include restricting or slowing the progression of one or more complications of hepatitis, including but not limited to yellowing of the skin and / or eyes, poor appetite, vomiting, fatigue, abdominal pain, diarrhea, acute liver failure, liver scarring, liver failure, and liver cancer.

[0011] In other embodiments, the subject is a patient with inflammatory liver disease, and the method is for treating 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 restricting or slowing the progression of one or more complications of the inflammatory liver disease; the complications of the inflammatory liver disease include but not limited to fatigue, discomfort, liver fibrosis, liver cancer, and / or cirrhosis.

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

[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 can 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 not limited to cyclophilin (CSP), CSP region I, CSPIplus, lactosylated human serum albumin, glycosylated lipoprotein, 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) an inhibitor of extracellular metallothionein (MT);

[0017] (b) a pancreatic or hepatocyte targeting moiety linked to the inhibitor of extracellular 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 monoclonal antibodies and / or humanized antibodies or antigen-binding fragments thereof. In other embodiments, the cell targeting moiety is a pancreatic cell targeting moiety, which is, for example, selected from glucagon-like peptide-1 (GLP-1), glucagon-like peptide-2 (GLP-2), peptide YY (PYY), neuropeptide Y (NPY), pancreatic polypeptide (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, CSPIplus, lactosylated human serum albumin, glycosylated lipoprotein, and / or arabinogalactan.

[0019] In one embodiment, the composition comprises a recombinant polypeptide. In other aspects, the present disclosure provides a recombinant nucleic acid encoding the 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, 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 the use of the composition, the recombinant nucleic acid, the recombinant host cell comprising the recombinant expression vector, and the pharmaceutical composition, the pharmaceutical composition comprising the composition, the recombinant nucleic acid, the recombinant expression vector, the recombinant host cell, or the drug of the present invention, or the pharmaceutical composition for treating or limiting the development of a disease in any embodiment or combination of embodiments disclosed herein, the disease being selected from diabetes, prediabetes, impaired glucose tolerance, hepatitis, and / or inflammatory liver disease.

[0021] In other embodiments, the inhibitor of extracellular MT comprises the composition, the recombinant nucleic acid, the recombinant host cell comprising the recombinant expression vector, and the pharmaceutical composition, the pharmaceutical composition being the pharmaceutical composition comprising the composition, the recombinant nucleic acid, the recombinant expression vector, and the recombinant host disclosed in the present disclosure, or the pharmaceutical composition in any embodiment or combination of embodiments disclosed herein. Brief Description of the Drawings

[0022] Figure 1Shows the results of glucose tolerance tests in mice that received an intraperitoneal injection of glucose at the end of treatment with MOPC21 or UC1MT;

[0023] Figure 2 Shows the change in the results of glucose tolerance tests normalized to Figure 1 the initial measurement of each animal in the group shown;

[0024] Figure 3 Shows the effects of MOPC21 or UC1MT on the RNA expression levels in epididymal white adipose tissue as revealed by qPCR studies. UC1MT treatment has an effect on the expression of certain genes in white epididymal adipose tissue (especially peroxisome proliferator-activated receptor γ coactivator 1-α (PGC-1α), a transcriptional coactivator that regulates energy metabolism, and peroxisome proliferator-activated receptor α (PPAR-γ));

[0025] Figure 4 shows the effects of antibody treatment on adipose tissue weight: (A) percentage of tissue weight to 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 Shows the effects of MOPC21 or UC1MT on the RNA expression levels in the liver as revealed by qPCR studies. In high-fat diet-treated mice, the anti-inflammatory IL-10 gene expression associated with UC1MT treatment was significantly increased compared to HFD mice treated with an isotype-matched control antibody of MOPC21;

[0027] Figure 6 Shows the effects of MOPC21 or UC1MT on liver triglycerides using the Thermo Fisher Trigylcerides kit (colorimetric method). Shown are the mean and standard error of male mice on a 16-week HFD that received 10 UC1MT, N = 8 MOPC. *p = 0.0453, two-tailed t-test;

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

[0029] Figure 8 shows the effects of intraperitoneal injection of UC1MT or MOPC21, 100 μl (twice a week) in mice on blood glucose levels after 3 weeks (A) and 6 weeks (B). UC1MT treatment inhibits the development of T1D in NOD mice;

[0030] Figure 9 Show the effect of MOPC21 or UC1MT on the infiltration of inflammatory cells into Langerhans islets (insulitis). Detailed implementation mode

[0031] Unless the context clearly indicates otherwise, as used herein, the singular forms "a", "an" and "the" include plural referents. Unless otherwise clearly indicated, "and" as used herein may be used interchangeably with "or".

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

[0033] Unless the context clearly indicates otherwise, throughout the specification and claims, words such as "comprising", "including" and the like should be understood in an inclusive sense, rather than an exclusive or exhaustive sense; that is, the meaning of "including but not limited to". The use of singular or plural words also includes plural and singular respectively. Additionally, when used in this application, words such as "herein", "above" and "below" and words with similar meanings should refer to the present invention as a whole, rather than any specific part of the present invention. The compositions and methods of using them can "comprise", "consist essentially of" or "consist of" any of the ingredients or steps 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 can be made within the scope of the present disclosure as will be recognized by those skilled in the relevant art.

[0035] In one aspect, the present invention provides a method for treating or limiting the development 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 therapeutically amount of a composition comprising an extracellular metallothionein (MT) inhibitor to treat or limit the development of the disease.

[0036] As disclosed in the following examples, an inhibitor of extracellular MT. It can be used to treat or limit the development of diabetes, prediabetes, impaired glucose tolerance, hepatitis and / or inflammatory liver disease.

[0037] As used in the present invention, "effective therapeutically amount" refers to the amount of a composition effective to treat and / or limit the relevant condition.

[0038] As used in the present invention, human metallothionein refers to 18 metallothionein isomers and subtype isomers that have been identified in humans and classified as MT1 to MT4. MT1 and MT2 are highly inducible in many cell types and can be released from cells where MT3 and MT4 are selectively expressed (Lynes et al., 2006, Laukens et al., 2009). The inhibitors of the present disclosure specifically target or bind the released metallothionein. Exemplary subtypes include, but are not limited to:

[0039] HumanMT1-A:

[0040] MDPNCSCATGGSCTCTGSCKCKECKCTSCKKSCCSCCPMS CAKCAQGCIC

[0041] KGASEKCSCCA(SEQ ID NO:1)

[0042] HumanMT1-B

[0043] MDPNCSCTTGGSCACAGSCKCKECKCTSCKKCCCSCCPVGCAKCAQGCVCKGSSEKCRCCA(SEQ IDNO:2)

[0044] HumanMT1-E

[0045] MDPNCSCATGGSCTCAGSCKCKECKCTSCKKSCCSCCPVGCAKCAQGCVCKGASEKCSCCA(SEQ IDNO:3)

[0046] HumanMT1_F

[0047] MDPNCSCAAGVSCTCAGSCKCKECKCTSCKKSCCSCCPVG CSKCAQGCVCKGASEKCSCC D(SEQID NO:4)

[0048] HumanMT1-G:

[0049] MDPNCSCAAAGVSCTCASSC KCKECKCTSC KKSCCSCCPVGCAKCAQGCICKGASEKCSC CA(SEQID NO:5)

[0050] HumanMT2:

[0051] MDPNCSCAAGDSCTCAGSCKCKECKCTSCKKSCCSCCPVGCAKCAQGCICKGASDKCSCCA(SEQ IDNO:6)

[0052] HumanMT3:

[0053] MDPETCPCPS GGSCTCADSC KCEGCKCTSCKKSCCSCCPAECEKCAKDCVCKGGEAAEAEAEKCSCCQ(SEQ ID NO:7)

[0054] HumanMT-4

[0055] MDPRECVCMS GGICMCGDNCKCTTCNCKTYWKSCCPCCPPGCAKCARGCICKGGSDKCSCCP(SEQID NO:8)

[0056] In one embodiment, the inhibitors of the present disclosure specifically target or bind to release MT1 and / or MT2.

[0057] "Specifically" or "selectively" binding to metallothionein refers to a binding reaction that determines the presence of metallothionein in a heterogeneous population of proteins and other biological products. Thus, under specified immunoassay or other conditions, a particular antibody or aptamer of the present invention binds to metallothionein at least twice the background and does not substantially bind to proteins other than metallothionein present in the sample. Thus, specific binding to an antibody or aptamer under such conditions may involve the use of an inhibitor selected from the group consisting of antibodies or aptamers that have been selected for their specificity for metallothionein.

[0058] As used herein, "antibody" includes immunoglobulin molecules that immunoreact with human MT (preferably selective for human MT, or selective for one or more human MT isomers) or fragments thereof, and includes monoclonal antibodies. There are multiple antibody isotypes, such as IgG1, IgG2, IgG3, IgG4 and other Igs, such as IgM, IgA, IgE isotypes. The term also includes genetically engineered forms, such as chimeric antibodies (e.g., humanized murine antibodies) and heteroconjugate antibodies (e.g., bispecific antibodies), fully humanized antibodies and human antibodies. As used throughout this application, the term "antibody" includes fragments having 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 Edition, Freeman & Co., New York (1998), the term also refers to recombinant single-chain Fv fragments (scFv). The term antibody also includes bivalent or bispecific molecules, diabodies, triabodies and tetra-bodies. Bivalent and bispecific molecules are described in, for example, Kostelny et al. (1992) J. Immunol. 148:1547, Pack and Pluckthun (1992) Biochemistry 31:1579, Hollinger et al., 1993, Gruber et al., ibid (1994), J. Immunol.:5368, Zhu et al. (1997), Protein Sci. 6:781, Hu et al. (1996), Cancer Res. 56:3055, Adams et al. (1993), Cancer Res. 53:4026 and McCartney et al. (1995) Protein Eng. 8:301. Antibodies can include heterobifunctional antibodies, e.g., whose anti-MT arm blocks MT function while MT can be stabilized at a specific location by binding to a tissue-specific determinant through the other arm of the antibody. In one embodiment, the antibody includes a monoclonal antibody. The methods of the present disclosure are demonstrated by using the exemplary monoclonal antibody UC1MT, which is described in US2003 / 0007973 and is commercially available from Abcam Inc., Cambridge, MA. Clone UC1MT has also been described by Lynes et al. (Toxicology 1993(85):161-177).

[0059] In one embodiment, the antibodies used in the methods described herein are humanized antibodies. A humanized antibody is a form of a non-human (e.g., murine) antibody that is a specific chimeric immunoglobulin, immunoglobulin chain, or antigen-binding fragment thereof that contains a minimal sequence derived from a non-human immunoglobulin. In most cases, a humanized antibody is a human immunoglobulin (recipient antibody) in which the residues of the complementarity-determining regions (CDRs) of the recipient are replaced with the CDR residues of a non-human species (donor antibody) (e.g., mouse, rat, or rabbit) that has the desired specificity, affinity, and capacity. In some instances, the framework region (FR) residues of the human immunoglobulin are replaced with the corresponding non-human residues. In addition, a humanized antibody may contain residues not found in the recipient antibody or the imported CDR or framework sequences, but these residues are incorporated to further refine and optimize antibody performance. Generally, a humanized antibody will contain substantially all of at least one variable domain and usually 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 optimally also contain at least a portion of the immunoglobulin constant region or domain (Fc), usually the constant region or domain (Fc) of a human immunoglobulin. The antibody may have a modified Fc region 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 of the original antibody. Humanized antibodies may also be involved in affinity maturation.

[0060] As used herein, "treating" refers to accomplishing one or more of the following in an individual suffering from one or more of the said diseases: (a) reducing the severity of the disease; (b) limiting or preventing the development of characteristic symptoms of the disease being treated; (c) inhibiting the worsening of characteristic symptoms of the disease being treated; (d) limiting or preventing the recurrence of the disease in a patient previously suffering from the disease; and / or (e) limiting or preventing the recurrence of symptoms in a patient previously symptomatic. Any amount of such "treatment" is highly beneficial to a subject having one of the said diseases.

[0061] As used in the present invention, "limiting" or "limiting its development" refers to accomplishing one or more of the following in an individual at risk of one or more of the said conditions: (a) slowing the progression of the disease and / or (b) limiting or preventing the evolution of symptomatic features into a disease. Any amount of such "limiting development" is highly beneficial to a subject at risk of one of the said diseases.

[0062] Such treatment or limiting its development may include using an extracellular MT inhibitor as a sole therapy or may include using it to supplement or enhance other therapeutic interventions, as deemed appropriate by the attending medical staff.

[0063] In one embodiment, the subject is at risk of developing diabetes or has diabetes, and the method is for treating or limiting the development of diabetes.

[0064] In one such embodiment, the subject is at risk of type 1 diabetes, and the method is for limiting 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 into type 1 diabetes. Thus, 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 the characteristic symptoms of T1D. In this embodiment, a subject at risk of T1D has one or more T1D risk factors, from which the attending medical staff determines 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 variants of the IDDM1 gene, the variants of the IDDM1 gene being selected from DRB10401, DRB10402, DRB10405, DQA0301, DQB10302, and DQB10201. Optionally or in combination, the subject may exhibit one or more T1D symptoms (but has not 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 understood by those skilled in the art, any limitation of the development of T1D or its symptoms provides great benefits to subjects at risk.

[0065] 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 tissue compatibility in type 1 diabetes. Such variants include DRB10401, DRB10402, DRB10405, DQA0301, DQB10302, 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, and zinc transporter autoantibodies (ZnT8), usually precedes type 1 diabetes before any hyperglycemia occurs. The risk of T1D increases with the number of antibody types present, and the time interval from the appearance of autoantibodies to clinically diagnosable T1D in infants and young children can be several months, but in some individuals it may take several years. Such autoantibodies can be detected by, for example, immunofluorescence or binding assays.

[0066] In another embodiment, the subject has type 1 diabetes, and the method is for treating the subject's type I diabetes. In this embodiment, the subject has been diagnosed with T1D, and the method can be used to treat T1D. T1D involves the autoimmune destruction of β-cells in the pancreas, little or no insulin production, and hyperglycemia. Thus, 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 ability to heal wounds, urinary tract infections, 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 require pancreas or islet transplantation. Thus, in various embodiments, the treatment can include one or more of the following: reducing the frequency of need for insulin injections; slowing the development or progression of the subject's type 1 diabetes complications, which include but are not limited to the destruction of pancreatic islet β-cells, hyperglycemia, hypoglycemia, polyuria, polyphagia, polydipsia, weight loss, blurred vision, fatigue, decreased ability to heal wounds, urinary tract infections, 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 can include reducing the blood glucose level (mg / dL) by 10%, 15%, 20% or more, for example within 20 - 120 minutes after administration of the inhibitor.

[0067] In another embodiment, the subject is at risk of type 2 diabetes, and the method is for limiting the development of type 2 diabetes (T2D) in the subject. T2D is a metabolic disease characterized by hyperglycemia, insulin resistance, and relative insulin deficiency. Symptoms and / or complications include, but are not limited to, hypoglycemia, insulin resistance, diabetic nephropathy, diabetic neuropathy, diabetic retinopathy, proteinuria, impaired glomerular filtration, diabetic circulatory diseases, renal failure, cardiovascular diseases, polyuria, polydipsia, weight loss, and stroke. In one embodiment, the limited development of type 2 diabetes can include, for example, within 20 to 120 minutes after administration of the inhibitor, the treatment including a decrease in blood glucose level (mg / dL) of 10%, 15%, 20% or more, for example, within 20 to 120 minutes after administration of the inhibitor.

[0068] 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 dietary habits (e.g., too much fat, insufficient fiber, too many simple carbohydrates, etc.), 50 years of age or older, hypertension, high cholesterol, testosterone deficiency, the 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 for slowing the progression to T2D and / or (b) limiting or preventing the development of symptoms characteristic of T2D.

[0069] As disclosed in the following examples, these methods significantly improve glucose tolerance in a T2D mouse model. Thus, in another embodiment, the subject has T2D, and the method is for treating T2D in the subject. In this embodiment, the treatment can include limiting one or more complications of type 2 diabetes and reducing the frequency of insulin or other therapies, the type 2 diabetes complications including, but not limited to, hyperglycemia, hypoglycemia, insulin resistance, diabetic nephropathy, diabetic neuropathy, diabetic retinopathy, proteinuria, impaired glomerular clearance, diabetic circulatory diseases, kidney failure, cardiovascular diseases, polyuria, polydipsia, weight loss, stroke. Limitation of any degree of these symptoms / complications is highly beneficial to a subject having T2D. In one embodiment, the treatment includes a decrease in blood glucose level (mg / dL) of 10%, 15%, 20% or more, for example, within 20 to 120 minutes after administration of the inhibitor.

[0070] In another embodiment, the subject has prediabetes, and the method is for treating 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. Thus, a subject with prediabetes may: (a) have impaired fasting glucose tolerance, i.e., a condition in which the β-cells have an insufficient response to an oral glucose challenge (OGT), or (b) may have persistently elevated fasting blood glucose (IFG), i.e., a condition in which the fasting blood glucose is elevated to a level higher than normal but not high enough to be classified as diabetes. The prediabetic state may be associated with insulin resistance and an increased risk of cardiovascular pathologies. Individuals with a prediabetic state have a relatively high risk of developing T2D. The methods of the present disclosure can be used to treat patients with prediabetes, e.g., by restricting 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, the treatment may include a 10%, 15%, 20% or more reduction in blood glucose level (mg / dL), e.g., within 20 - 120 minutes after administration of the inhibitor.

[0071] In another embodiment, the subject has impaired glucose tolerance, and the method is for treating 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. A patient is said to be in an IGT state when the blood glucose level shows a moderate elevation after 2 hours but is below the level that meets the criteria for type 2 diabetes. The fasting blood glucose may be normal or mildly elevated. Impaired glucose tolerance is a hyperglycemic state of prediabetes and is associated with insulin resistance and an increased risk of cardiovascular disease. IGT can precede type 2 diabetes by many years. In the present embodiment, the treatment includes restricting or slowing the progression of one or more complications of impaired glucose tolerance, the complications including but not limited to type 2 diabetes, hyperglycemia, insulin resistance, and / or cardiovascular disease. In one embodiment, the treatment may include a 10%, 15%, 20% or more reduction in blood glucose level (mg / dL), e.g., within 20 - 120 minutes after administration of the inhibitor.

[0072] In another embodiment, the subject is a hepatitis patient and the method is used to treat the hepatitis of the subject. As disclosed in the following examples, MT inhibitors can effectively limit tissue inflammation and reduce the pro-inflammatory cytokines MCP-1 and TNF-α, while enhancing the anti-inflammatory IL-10 signal in liver tissue. Hepatitis is an inflammation of liver tissue. Symptoms include but are not limited to yellowing of the skin and the whites of the eyes, loss of appetite, vomiting, tiredness, 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 alcohol consumption, certain medications, toxins, other infections, autoimmune diseases and non-alcoholic steatohepatitis (NASH). Thus, in various embodiments, treatment can include limiting or slowing the progression of one or more complications of hepatitis, including but not limited to yellowing of the skin and / or the whites of the eyes, loss of appetite, vomiting, fatigue, abdominal pain, diarrhea, acute liver failure, liver scarring, liver failure and liver cancer.

[0073] In another embodiment, the subject is a patient with inflammatory liver disease and the method is for treating the inflammatory liver disease of the subject. As used herein, "inflammatory liver disease" is a condition associated with the accumulation of triglyceride fat in the cytoplasm of hepatocytes caused by steatosis (i.e., abnormal retention of lipids within cells). The liver plays an important role in whole-body metabolism, and energy imbalance is particularly associated with defects in hepatic lipid metabolism. Specifically, obesity and insulin resistance are generally associated with increased characteristic lipid deposition in the liver in non-alcoholic fatty liver disease (NAFLD). Although lipid metabolism is highly dynamic, chronic lipid overload can lead to liver tissue damage, which leads to the recruitment of liver-resident and non-resident immune cells, which may cause fibrosis in non-alcoholic steatohepatitis (NASH). Hepatic fibrosis can lead to cirrhosis, cancer and a significantly increased risk of developing cardiovascular disease. This increases the potential to block the recruitment of immune cells to the liver to reduce the risk of non-alcoholic fatty liver disease (NAFLD). As shown in the following examples, MT inhibitor treatment increased the wet tissue weight of epididymal white adipose tissue, decreased total triglyceride levels, and also decreased the pro-inflammatory cytokines MCP-1 and TNF-α, while enhancing anti-inflammatory IL-10 signaling. Inflammatory liver disease may be steatosis (non-alcoholic fatty liver (NAFL)). In another embodiment, the 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 (steatosis) for reasons other than excessive alcohol consumption. Symptoms of NASH and NAFLD may include but are not limited to fatigue, general malaise, dull discomfort in the upper right abdomen, mild jaundice, and abnormal liver function tests in routine blood tests; complications of NASH and NAFLD may include but are not limited to hepatic fibrosis, liver cancer and / or cirrhosis. Thus, in one embodiment, inflammatory liver disease includes non-alcoholic steatohepatitis (NASH) and / or non-alcoholic fatty liver disease (NAFLD). In another embodiment, the treatment includes limiting or slowing the progression of one or more complications of inflammatory liver disease, which inflammation includes but is not limited to fatigue, discomfort, hepatic fibrosis, liver cancer and / or cirrhosis.

[0074] In one embodiment, an MT inhibitor (including but not limited to an anti-MT antibody) can be linked to a pancreatic cell targeting moiety 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 polypeptide (PPY), and exendin-4.

[0075] Glucagon-like peptide 1; (aa92-128)

[0076] HDEFERHAEGTFTSDVSSYLEGQAAKEFIAWLVKGRG (SEQ ID NO:9)

[0077] Glucagon-like peptide 2; GLP2 (aa146-178)

[0078] HADGSFSDEMNTILDNLAARDFINWLIQTKITD (SEQ ID NO:10)

[0079] Pancreatic polypeptide (PPY)

[0080] MAAARLCLSLLLLSTCVALLLQPLLGAQGAPLEPVYPGDNATPEQMAQYAADLRRYINMLTRPRYGKRHKEDTLAFSEWGSPHAAVPRELSPLDL (SEQ ID NO:11)

[0081] Neuropeptide Y (NPY)

[0082] MLGNKRLGLSGLTLALSLLVCLGALAEAYPSKPDNPGEDAPAEDMARYYSALRHYINLITRQRYGKRSSPETLISDLLMRESTENVPRTRLEDPAMW (SEQ ID NO:12)

[0083] Peptide YY (PYY)

[0084] MVFVRRPWPALTTVLLALLVCLGALVDAYPIKPEAPGEDASPEELNRYYASLRHYLNLVTRQRYGKRDGPDTLLSKTFFPDGEDRPVRSRSEGPDLW (SEQ ID NO:13)

[0085] exendin-4

[0086] MKIILWLCVFGLFLATLFPISWQMPVESGLSSEDSASSESFASKIKRHGEGTFTSDLSKQMEEEAVRLFIEWLKNGGPSSGAPPPSG (SEQ ID NO:14)

[0087] The linkage between the pancreatic cell targeting moiety and the MT inhibitor (including but not limited to an MT antibody or its fragment or an aptamer) can be accomplished by any chemical reaction capable of binding the two molecules, provided that 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 linkage between the pancreatic cell targeting moiety and the MT antibody or its fragment can include a number of chemical mechanisms such as covalent binding, affinity binding, intercalation, coordination binding, and complexation. Covalent binding can be achieved by direct condensation of existing side chains or by the binding of an external bridging molecule. Many divalent or multivalent linkers can be used to conjugate 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.

[0088] 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.

[0089] The composition for administration is generally formulated as a pharmaceutical composition comprising a pharmaceutically acceptable carrier. Suitable acids capable of forming 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 sulfamic acid. Suitable bases capable of forming such salts include inorganic bases such as sodium hydroxide, ammonium hydroxide, and potassium hydroxide, and organic bases such as mono-, di-, and trialkylamines and arylamines (such as triethylamine, diisopropylamine, methylamine, dimethylamine, etc.) and optionally substituted ethanolamines (such as ethanolamine and diethanolamine, etc.).

[0090] In addition to the composition and the carrier, the pharmaceutical composition may further include (a) a lyoprotectant; (b) a surfactant; (c) a filler; (d) an osmotic pressure 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 further include a lyoprotectant, such as sucrose, sorbitol, or trehalose. In certain embodiments, the pharmaceutical composition contains a preservative, such as benzalkonium chloride, acetophenone, chlorhexidine, phenol, m-cresol, benzyl alcohol, methyl paraben, propyl paraben, chlorobutanol, o-cresol, p-cresol, chlorocresol, phenylmercuric nitrate, thimerosal, benzoic acid, and various mixtures thereof. In other embodiments, the pharmaceutical composition contains a filler, such as glycine. In other embodiments, the pharmaceutical composition contains 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 further contains an osmotic pressure regulator, such as a compound that renders the formulation substantially isotonic or isotonic with human blood. Exemplary osmotic pressure regulators include sucrose, sorbitol, glycine, methionine, mannitol, dextrose, inositol, sodium chloride, arginine, and arginine hydrochloride. In other embodiments, the pharmaceutical composition further contains a stabilizer, such as 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.

[0091] The compositions of the dosage unit formulations can be administered by any suitable route, including orally, parenterally, by inhalation spray, rectally, or topically, and contain conventional pharmaceutically acceptable carriers, adjuvants, and excipients. The term parenteral as used herein includes subcutaneous, intravenous, intraarterial, intramuscular, intrasternal, intratendinous, intraspinal, intracranial, intrathoracic, infusion techniques, or intraperitoneal. The dosage regimen can be adjusted to provide the optimum desired response (e.g., a therapeutic or prophylactic response). Suitable dosage ranges can be, for example, from 0.1 μg / kg to 100 mg / kg body weight; alternatively, it can be from 0.5 μg / kg to 50 mg / kg; from 1 μg / kg to 25 mg / kg body weight, or from 5 μg / kg to 10 mg / kg body weight. The compositions can be delivered by a single bolus injection or can be administered more than once (e.g., 2, 3, 4, 5, or more times) as determined by the attending medical staff. The compositions can be a therapeutic agent administered alone or can be administered in combination 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 has T1D or is at risk of T1D, and the inhibitor can be used in combination with one or more of insulin, metformin, or pramlintide. In another embodiment, the subject is a T2D patient or at risk of prediabetes and / or impaired glucose tolerance, and the inhibitor can be used in combination with one or more of metformin, sulfonylureas (including but not limited to glyburide, glipizide, and glimepiride), meglitinides (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 hepatitis or is at risk of hepatitis, and the inhibitor can be used in combination with one or more of entecavir, tenofovir, lamivudine, adefovir, telbivudine,simeprevir, sofosbuvir, interferon, or ribavirin.

[0092] In another embodiment, the provided composition comprises:

[0093] (a) an inhibitor of extracellular human metallothionein (MT);

[0094] (b) One or more of insulin, metformin, pramlintide, sulfonylureas (including but not limited to glibenclamide, glipizide, and glimepiride), meglitinides (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), entecavir, tenofovir, lamivudine, adefovir, telbivudine, simeprevir, sofosbuvir, interferon, or ribavirin. For example, the composition 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 combinations 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.

[0095] In another aspect, the present invention provides a composition comprising:

[0096] (a) An inhibitor of extracellular human metallothionein (MT);

[0097] (b) A pancreatic or hepatocyte-targeting moiety linked to the inhibitor of extracellular human MT.

[0098] 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 combinations 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.

[0099] In one embodiment, the cell targeting moiety is a hepatocyte targeting moiety. In exemplary such embodiments, 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 Iplus. CSP targets liver-resident malaria sporozoites because circumsporozoite protein (CSP) is present on the surface of malaria sporozoites (Rathore D, et al. Journal of Biological Chemistry, 2005; 280(21):20524-20529). CSP is approximately 400 amino acids long and is organized into three domains: an N-terminal domain that includes a conserved KLKQP motif called "Region I", a highly repetitive central domain, and a C-terminal domain that contains another conserved sequence called "Region II" (Singh et al., Cell 2007; 131(3):492-504). In addition to the conserved Region I-KLKQP sequence, the N-terminal region also contains two consensus heparin sulfate binding sequences upstream of Region I. A peptide containing the conserved Region I amino acids and the two consensus heparin binding sequences from upstream of Region I is named "Region I-plus" (Prudêncio et al., Nature Reviews Microbiology, 2006; 4(11):849-856).

[0100] 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 polypeptide (PPY), and exendin-4; exemplary amino acid sequences of such peptides are as described above.

[0101] Any chemical reaction that binds two molecules can be used to accomplish the binding of the cell targeting moiety to the MT inhibitor (including but not limited to an MT antibody or its fragment or an aptamer), provided that the cell targeting moiety and the MT antibody or its fragment or aptamer retain their respective activities. In one embodiment where the MT inhibitor extracellularly is an antibody or its fragment and the cell targeting moiety is a peptide, the composition comprises a recombinant fusion protein. In other embodiments, the linkage between the cell targeting moiety and the MT antibody or its fragment can include a number of chemical mechanisms such as covalent binding, affinity binding, intercalation, coordination binding, and complexation. Covalent binding can be achieved by direct condensation of existing side chains or by the binding of an external bridging molecule. Many divalent or multivalent linkers can be used to conjugate 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, succinimidyl esters, diisocyanates, glutaraldehyde, diazobenzenes, and hexamethylenediamine.

[0102] In another aspect, the present disclosure provides recombinant nucleic acids encoding recombinant fusion polypeptides of anti-MT antibodies or their fragments fused to peptide targeting moieties, including those specifically disclosed herein. The recombinant nucleic acid sequences can include single-stranded or double-stranded RNA or DNA and their derivatives. Such recombinant nucleic acid sequences can include additional sequences for facilitating the expression and / or purification of the encoded protein, including but not limited to polyA sequences, modified Kozak sequences, and sequences encoding epitope tags, export signals, and secretion signals, nuclear localization signals, and plasma membrane localization signals.

[0103] In another aspect, recombinant expression vectors are disclosed that comprise recombinant nucleic acids of any embodiment or combination of embodiments of the present invention, operably linked to suitable control sequences. A "recombinant expression vector" includes a vector that operably links a nucleic acid coding region or gene to any control sequence capable of influencing the expression of the gene product. A "control sequence" operably linked to the nucleic acid sequence of the present invention is a nucleic acid sequence capable of influencing the expression of the nucleic acid molecule. The control sequence need not be adjacent to the nucleic acid sequence so long as they have the function of directing its expression. Thus, for example, there can be intervening transcribed but untranslated sequences between the promoter sequence and the nucleic acid sequence, and the promoter sequence can still be considered to be "operably linked" to the coding sequence. Other such control sequences include but are not limited to polyadenylation signals, termination signals, and ribosome binding sites. Such expression vectors can be of any type, including but not limited to plasmids and virus-based expression vectors. The expression vector can exist as an episome or be integrated into the host chromosomal DNA and be replicable within the host organism. In non-limiting embodiments, the expression vector can include a plasmid or a viral vector.

[0104] On the other hand, the present invention provides a recombinant host cell comprising the recombinant expression vector described above, and the host cell can be prokaryotic or eukaryotic, such as mammalian cells. The cells can be transfected transiently or stably.

[0105] In another embodiment, the use of a composition, recombinant nucleic acid, recombinant expression vector, recombinant host cell or pharmaceutical composition of any embodiment or combination of embodiments disclosed in the present invention to treat or limit the development of a disease is disclosed, and the disease includes diabetes, prediabetes, impaired glucose tolerance, hepatitis and / or inflammatory liver disease. These uses are as described above.

[0106] 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 embodiments disclosed herein, and the disease includes diabetes, prediabetes, impaired glucose tolerance, hepatitis and / or inflammatory liver disease.

[0107] Examples

[0108] UC1MT Treatment of Type 2 Diabetes

[0109] To mimic the insulin resistance characteristics 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. The mice were injected IP twice a week with 100 μL of 1 mg / mL anti-MT antibody (UC1MT; purchased from Abcam, Cambridge, MA) for 8 weeks, and then the mice were sacrificed at 27 weeks of age (total 22 weeks of HFD). There were no differences in body weight or food intake between the two treatment groups. Figure 1 Shows the results of a glucose tolerance test in mice injected intraperitoneally with glucose at the end of the treatment with IgG1 isotype control (MOPC21) or UC1MT. Figure 2 Shows the normalized change in the results of the glucose tolerance test for each animal in the group. Control animals developed fatty liver and glucose intolerance, but mice treated with UC1MT showed better glucose tolerance compared to the control. These data demonstrate that anti-MT antibody can be used to treat impaired glucose tolerance. Since glucose tolerance can regulate the inflammatory state, these results also suggest that UC1MT can improve the inflammatory condition in mice, thus enhancing the ability of HFD-fed mice to handle a large glucose challenge.

[0110] Effect of UC1MT or MOPC21 Treatment on NASH (Non-alcoholic Steatohepatitis) Liver Inflammation in HFD-Fed Mice

[0111] The liver plays an important role in whole-body metabolism, and energy imbalance is particularly associated with defects in hepatic lipid metabolism. Specifically, obesity and insulin resistance are commonly associated with increased lipid deposition in the livers of patients with 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, which in turn leads to the recruitment of liver-resident and non-resident immune cells, potentially causing fibrosis in non-alcoholic steatohepatitis (NASH) (Narayanan S et al., Immunology Letters, 2016 PMID:27340383). Hepatic fibrosis can lead to cirrhosis, cancer, and significantly increase the risk of developing cardiovascular disease. This increases the potential to block the recruitment of immune cells to the liver to reduce the risk of non-alcoholic fatty liver (NAFLD).

[0112] mRNA expression was evaluated by qPCR studies, and the liver phenotypes of the same animals in the above type 2 diabetes studies were examined. 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 that regulates energy metabolism and peroxisome proliferator-activated receptor (PPARγ)) ( Figure 3 ).

[0113] At the time of animal sacrifice, the liver, pancreas, inguinal white adipose tissue, eWAT, interscapular 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, and the pancreas was fixed with Z-FIX TM (Fisher Scientific). The data showed that the effects of the anti-MT antibody were:

[0114] · Increased wet tissue weight of epididymal white adipose tissue ( Figure 4A -B);

[0115] · Decreased total triglyceride levels in the liver ( Figure 6 );

[0116] · Decreased expression of certain pro-inflammatory cytokines (MCP-1 and TNF-a) ( Figure 5 );

[0117] · Enhanced expression of anti-inflammatory IL-10 signaling ( Figure 5 ).

[0118] The anti-metallothionein antibody did not alter the body weight or weight gain of mice treated with a high-fat diet, nor did it significantly alter liver histology.

[0119] Consistent with the above-improved glucose tolerance, the hepatic triglyceride levels in mice treated with anti-MT antibody were decreased. These data indicate that MT inhibitors can improve whole-body glucose metabolism, thereby reducing the burden of hepatic nutrient excess and limiting the development of hepatic steatosis.

[0120] Type 1 diabetes research

[0121] The NOD / ShiLtJ mouse strain (commonly known 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 islets of Langerhans. The pancreatic insulin content in female rats at approximately 12 weeks of age is significantly decreased, while that in male rats shows a marked decrease several weeks later. 0% of females and 45% of males develop diabetes by 30 weeks; the median incidence in females is 17 weeks. The immunophenotypes in the NOD background include antigen presentation, T lymphocyte repertoire, NK cell function, macrophage cytokine production, wound healing, and C5 complement deficiency. These deficiencies make the NOD background a common choice for immunodeficient mouse strains.

[0122] We used female NOD (non-obese diabetic) mice for our type 1 DM prevention study because NOD mice have been found to spontaneously develop type 1 diabetes since they were described in 1980. The pathogenic events start at least 3 weeks after birth with the appearance of islet antigens in the pancreatic lymph nodes. Insulitis begins with APCs and then lymphocytes at approximately 4 - 6 weeks of age and gradually develops over the next 15 weeks. Frank diabetes with BS > 250 mg / dL starts within 18 to 20 weeks. In addition, the incidence of spontaneous diabetes in NOD mice is 60 - 80% in females and 20 - 30% in males. Therefore, we decided to divide the NOD mice into 3 groups (10 mice per treatment group): (1) negative control treatment with PBS; (2) treatment with non-specific IgG at 0.1 mg / mL; using MOPC as an isotype control; (3) intraperitoneal injection of 0.1 mg / ml UCIMT per mouse per day to study the potential role of anti-MT monoclonal antibody in type 1 diabetes. Each group received the designated treatment starting at 5 weeks of age. The total treatment course 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 sacrificed. The remaining non-diabetic NOD mice were sacrificed at 30 weeks of age. As Figure 7 shown in Figure 8, compared with the isotype control MOPC21 or the PBS vehicle control group, anti-MT antibody treatment significantly alleviated glucose intolerance in mice treated with a high-fat diet. UC1MT also led to a marked reduction in insulitis (i.e., inflammatory cell infiltration of the islets) ( Figure 9 ).

Claims

1. A method for treating or limiting the development of a disease, wherein, The disease is selected from diabetes, prediabetes, impaired glucose tolerance, hepatitis, and / or inflammatory liver disease, wherein the method comprises administering to a subject having the disease or at risk of having the disease an effective therapeutically amount of a composition to treat or limit the development of the disease, the composition comprising extracellular human metallothionein (MT).

2. The method according to claim 1, wherein The subject is a patient with diabetes or at risk of developing diabetes, and the method is for treating or limiting the development of diabetes.

3. The method according to claim 2, wherein, The subject is a patient at risk of type 1 diabetes, and the method is for limiting the development of type 1 diabetes in the subject.

4. The method according to claim 3, wherein The subject has one or more risk factors for type 1 diabetes, including but 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, and zinc transporter autoantibodies (ZnT8), IDDM1 gene variants selected from DRB10401, DRB10402, DRB10405, DQA 0301, DQB10302, and DQB10201, polyuria, polydipsia, dry mouth, polyphagia, fatigue, or weight loss.

5. The method according to claim 2, wherein The subject has type 1 diabetes, and the method is for treating the subject's type 1 diabetes.

6. The method according to claim 5, wherein The treatment includes one or more of reducing the frequency of insulin injections needed, slowing the development or progression of the subject's type 1 diabetes complications, and delaying the need for pancreatic or islet cell transplantation, wherein the complications include but are not limited to destruction of pancreatic beta cells, hyperglycemia, hypoglycemia, polyuria, polyphagia, polydipsia, weight loss, blurred vision, fatigue, decreased wound healing ability, urinary tract infections, sexual dysfunction, dry mouth, diabetic ketoacidosis, cardiovascular disease, diabetic nephropathy, diabetic neuropathy, diabetic retinopathy, stroke, renal failure, and foot ulcers.

7. The method according to claim 2, wherein, The subject is at risk of type 2 diabetes, and the method is for limiting the development of type 2 diabetes in the subject.

8. The method according to claim 7, wherein, The subject has one or more of the risk factors for type 2 diabetes, the risk factors including but 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 eating habits (e.g., too much fat, insufficient fiber, too many simple carbohydrates), age 50 or older, hypertension, high cholesterol, testosterone deficiency, single nucleotide polymorphism at the rs8052394 locus (G change) of metallothionein 1A (MT1A), and a history of gestational diabetes.

9. The method according to claim 2, wherein The subject is a patient with type 2 diabetes, and the method is for treating the subject's type 2 diabetes.

10. The method according to claim 9, wherein, The treatment includes restricting one or more complications of type 2 diabetes and reducing the frequency of insulin or other treatment needs, and the complications include but are not limited to hyperglycemia, hypoglycemia, insulin resistance, diabetic nephropathy, diabetic neuropathy, diabetic retinopathy, proteinuria, impaired glomerular clearance, diabetic circulatory disorders, renal failure, cardiovascular diseases, polyuria, polydipsia, weight loss, stroke.

11. The method according to claim 1, wherein, The subject is a pre-diabetic patient, and the method is used to treat pre-diabetes in the subject.

12. The method according to claim 11, wherein The treatment includes restricting or slowing the progression of one or more complications of pre-diabetes, including but not limited to type 2 diabetes, hyperglycemia, insulin resistance, and / or cardiovascular diseases.

13. The method according to claim 1, wherein, The subject is a patient with impaired glucose tolerance, and the method is used to treat the impaired glucose tolerance of the subject.

14. The method according to claim 13, wherein, The treatment includes restricting 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 diseases.

15. The method according to claim 1, wherein, The subject is a hepatitis patient, and the method is used to treat the hepatitis of the subject.

16. The method according to claim 15, wherein, The treatment includes restricting or slowing the progression of one or more complications of hepatitis, including but not limited to yellowing of the skin and / or eyes, poor appetite, vomiting, fatigue, abdominal pain, diarrhea, acute liver failure, liver scarring, liver failure, and liver cancer.

17. The method according to claim 1, wherein, The subject is a patient with inflammatory liver disease, and the method is used to treat the inflammatory liver disease in the subject.

18. The method according to claim 17, wherein, The inflammatory liver disease includes non-alcoholic steatohepatitis (NASH) and / or non-alcoholic fatty liver disease (NAFLD).

19. The method according to claim 17 or 18, wherein The treatment includes restricting or slowing the progression of one or more complications of inflammatory liver disease, including but not limited to fatigue, discomfort, liver fibrosis, liver cancer, and / or cirrhosis.

20. The method according to any one of claims 1 to 19, wherein, The extracellular human metallothionein (MT) inhibitor includes an anti-MT antibody or its antigen-binding fragment that specifically binds to extracellular human MT and / or an aptamer.

21. The method according to any one of claims 1 to 20, wherein, The extracellular human MT inhibitor includes an anti-MT antibody or its antigen-binding fragment.

22. The method according to claim 21, wherein, The anti-MT antibody or its antigen-binding fragment includes a monoclonal antibody or its antigen-binding fragment.

23. The method according to any one of claims 21 to 22, wherein, The anti-MT antibody includes a humanized anti-MT antibody or its antigen-binding fragment.

24. The method according to any one of claims 1 to 23, wherein The extracellular human MT inhibitor is linked to a pancreatic or hepatocyte targeting moiety.

25. The method according to claim 24, wherein, The cell targeting moiety is a pancreatic cell targeting moiety; the pancreatic cell targeting moiety includes one or more peptides or other moieties that preferentially bind to pancreatic beta 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 polypeptide (PPY), and exendin-4.

26. The method according to claim 24, wherein, The cell targeting moiety is a hepatocyte targeting moiety; the hepatocyte targeting moiety includes but is not limited to cyclophilin (CSP), CSP region I, CSPIplus, lactosylated human serum albumin, glycosylated lipoprotein, and arabinogalactan.

27. The method according to any one of claims 1 to 26, wherein The subject is a mammal.

28. The method according to any one of claims 1 to 27, wherein, The subject is a human.

29. A composition, comprising: (a) an inhibitor of extracellular human metallothionein (MT); (b) A pancreatic or hepatocyte targeting moiety linked to an extracellular human MT inhibitor.

30. The composition according to claim 29, wherein, The inhibitor includes an anti-metallothionein antibody or a fragment thereof that specifically binds to human metallothionein (MT).

31. The composition according to claim 30, wherein, The anti-MT antibody or its antigen-binding fragment includes a monoclonal antibody or its antigen-binding fragment.

32. The composition according to any one of claims 30 to 31, wherein, The anti-MT antibody includes a humanized anti-MT antibody or its antigen-binding fragment.

33. The composition according to any one of claims 29 to 32, wherein, The cell targeting moiety is a pancreatic cell targeting moiety 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.

34. The composition according to any one of claims 29 to 32, wherein, The cell targeting moiety is a hepatocyte targeting moiety including, but not limited to, circumsporozoite protein (CSP), CSP region I, CSPIplus, lactosylated human serum albumin, glycosylated lipoprotein, and arabinogalactan.

35. The composition according to any one of claims 29 to 34, wherein, The cell targeting moiety is a peptide, and the composition includes a recombinant polypeptide.

36. A recombinant nucleic acid encoding the recombinant polypeptide according to claim 35.

37. A recombinant expression vector comprising the recombinant nucleic acid according to claim 36.

38. A recombinant host cell comprising the recombinant expression vector according to claim 37.

39. A composition comprising: (a) An inhibitor of extracellular human metallothionein (MT); (b) One or more of insulin, metformin, pramlintide, sulfonylureas (including but not limited to glibenclamide, glipizide, and glimepiride), meglitinides (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), entecavir, tenofovir, lamivudine, adefovir, telbivudine, simtuzumab, sofosbuvir, interferon, or ribavirin.

40. The composition according to claim 39, wherein, The inhibitor includes an anti-metallothionein antibody or a fragment thereof that specifically binds to human metallothionein (MT).

41. The composition according to claim 40, wherein, The anti-MT antibody or its antigen-binding fragment includes a monoclonal antibody or its antigen-binding fragment.

42. The composition according to any one of claims 39 to 41, wherein, The anti-MT antibody includes a humanized anti-MT antibody or its antigen-binding fragment.

43. A pharmaceutical composition comprising: (a) The composition according to any one of claims 29-35 or 39-42, the recombinant nucleic acid according to claim 36, the recombinant expression vector according to claim 37, or the recombinant host cell according to claim 38; and (b) A pharmaceutically acceptable carrier. Use of the composition according to any one of claims 29 to 35 or 39 to 42, the recombinant nucleic acid according to claim 36, the recombinant expression vector according to claim 37, the recombinant host cell according to claim 38 or the pharmaceutical composition according to claim 43 in treating or limiting the progression of a disease selected from diabetes, prediabetes, impaired glucose tolerance, hepatitis and / or inflammatory liver disease.

45. The method according to any one of claims 1 to 28, wherein The extracellular human MT inhibitor comprises the composition according to any one of claims 29 to 35 or 39 to 42, the recombinant nucleic acid according to claim 36, the recombinant expression vector according to claim 37 or the recombinant host cell according to claim 38 or the pharmaceutical composition according to claim 43.

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