CN116615224A - Methods and compounds for treating diabetes and related metabolic disorders - Google Patents

Abstract

The present application provides compounds, compositions, and methods of use thereof for treating diabetes (e.g., type 1 diabetes, type 2 diabetes). In some aspects, the method comprises administering to the subject a first, second, third, fourth, and fifth daily dose of insulin-like growth factor 2 ("IGF-2"), or a variant thereof, at a first, second, third, fourth, and fifth different time, respectively, wherein each of the daily doses comprises at least 65 μg of IGF-2. In other aspects, compounds, compositions, and methods containing IGF-2 or variants thereof are used for treating disorders, such as type 1 or type 2 diabetes, in a patient in need thereof.

Description

Methods and compounds for treating diabetes and related metabolic disorders

Sequence listing

The present application contains a sequence listing submitted electronically in ASCII format and is incorporated herein by reference in its entirety. The ASCII copy, created at 10 months 12 of 2021, is named 1462-0017SeqListing. Txt and is 1,966 bytes in size.

Cross Reference to Related Applications

The present application claims priority and benefit from U.S. provisional patent application No. 63/090,943, filed on month 13 of 2020, and U.S. provisional patent application No. 63/234,862, filed on month 8 of 2021, which are incorporated herein by reference in their entireties.

Background

Diabetes mellitus (diabetes mellitus, DM), commonly referred to as diabetes (diabetes), is a major medical problem worldwide. By 2015, 4.15 million people worldwide were estimated to have diabetes mellitus, with DM type 2 accounting for about 90% of cases. This represents 8.3% of the adult population, where the ratio is equal for females and males. The incidence of DM increases in most of the world population.

Diabetes is a group of metabolic diseases in which there is a high blood glucose level over a long period of time. Symptoms of hyperglycemia include increased urinary frequency, thirst, and increased hunger. Diabetes may cause many complications if left untreated. Acute complications may include diabetic ketoacidosis, nonketotic hypertonic coma, or death. Serious long-term complications include heart disease, stroke, chronic renal failure, foot ulcers, and eye damage.

Diabetes is due, for example, to insufficient insulin production by the pancreas or to the inability of cells of the body to respond appropriately to the insulin produced. There are three main types of diabetes.

Type 1 DM is due to the inability of the pancreas to produce sufficient insulin. This form was previously known as "insulin dependent diabetes mellitus" (IDDM) or "juvenile diabetes. The reason is unknown.

Type 2 DM begins with insulin resistance, a condition in which cells do not respond appropriately to insulin. Insulin deficiency may also occur as the disease progresses. This form was previously known as "non-insulin dependent diabetes mellitus" (NIDDM) or "adult-onset diabetes. The main causes of type 2 DM are overweight and hypokinesia.

Gestational diabetes is the third major form and occurs when pregnant women without a history of diabetes develop high blood glucose levels.

Type 1 DM may be administered by insulin injection. Type 2 DM may be treated with drugs with or without insulin. Gestational diabetes usually resolves after birth of the infant.

Daily injections may be required for insulin administration, which is expensive and inconvenient for the patient. In addition, the use of insulin may result in hypoglycemia, headache, hunger, weakness, sweating, tremors, irritability, inattention, shortness of breath, tachycardia, syncope, or seizures. Insulin therapy requires continuous daily therapy to be effective.

Disclosure of Invention

Aspects described herein provide compositions and methods for treating diabetes and related conditions using insulin-like growth factor 2 ("IGF-2") or variants thereof. In some cases, the treatment provides long-term results, which eliminates the need for ongoing daily injections and the side effects and expense of daily insulin therapy.

Aspects described herein provide methods of treating diabetes (and related conditions) in a subject in need of treatment by administering to the subject a first, second, third, fourth, and fifth daily dose of IGF-2 or a variant thereof, respectively, on a first, second, third, fourth, and fifth different day, wherein each of the daily doses comprises at least 65 μg of IGF-2 or a variant thereof.

Further aspects provide methods of treating diabetes by administering IGF-2 or a variant thereof to a subject in need of treatment in an amount of about 65 μg/kg subject body weight to about 1626 μg/kg subject body weight.

Further aspects provide methods of reducing blood glucose levels in a subject by administering IGF-2 or a variant thereof to a subject in need of treatment in an amount of about 65 μg/kg subject body weight to about 813 μg/kg subject body weight.

Further aspects provide pharmaceutical compositions comprising IGF-2 or a variant thereof and a pharmaceutically acceptable excipient in an amount sufficient to reduce blood glucose levels in a subject to about normal levels, as compared to a subject not receiving IGF-2 or a variant thereof.

Aspects described herein provide methods of treating diabetes in a subject in need of treatment. The method comprises administering to the subject a daily dose of IGF-2 or a variant thereof on each of N different days. In this aspect, N is at least 5, and both (a) N and (b) the daily dose of IGF-2 or a variant thereof administered to the subject on each of the N different days are sufficiently high to (i) reduce the glucose level of the subject to about a normal level before the end of the N different days, and (ii) maintain the glucose level of the subject at about a normal level for at least 10 days after the end of the N different days.

Aspects described herein provide methods of treating type 2 diabetes in a subject in need of treatment and having body weight. The method comprises administering to the subject a first, second, third, fourth, and fifth daily dose of IGF-2 or a variant thereof on each day, wherein each of the daily doses comprises at least 244 μg of IGF-2 or a variant thereof per kilogram of body weight.

Aspects described herein provide methods of preventing the onset of type 1 diabetes in a subject having body weight. The method comprises administering to the subject a first, second, third, fourth, and fifth daily dose of IGF-2 or a variant thereof on each day, wherein each of the daily doses comprises at least 65 μg of IGF-2 or a variant thereof per kilogram of body weight.

Additional aspects described herein provide methods of increasing insulin levels in the blood stream of a subject having diabetes and having body weight. The method comprises administering to the subject a first, second, third, fourth, and fifth daily dose of IGF-2 or a variant thereof on each day, wherein each of the daily doses comprises at least 65 μg of IGF-2 or a variant thereof per kilogram of body weight.

Aspects described herein provide methods of increasing the number of functional beta cells in a subject having diabetes and having body weight. The method comprises administering to the subject a first, second, third, fourth, and fifth daily dose of IGF-2 or a variant thereof on each day, wherein each of the daily doses comprises at least 65 μg of IGF-2 or a variant thereof per kilogram of body weight.

Still further aspects described herein provide methods of preventing the onset of type 2 diabetes in a subject having body weight. The method comprises administering to the subject a first, second, third, fourth, and fifth daily dose of IGF-2 or a variant thereof on each day, wherein each of the daily doses comprises at least 65 μg of IGF-2 or a variant thereof per kilogram of body weight.

Brief Description of Drawings

FIGS. 1-4 depict blood glucose levels in four mice during an experiment in which diabetes is induced with Streptozotocin (STZ) and IGF-2 is provided to the mice at a daily dose of 3,000 μg/kg (1/1 dose) at a designated time point;

FIGS. 5A, 5B, 6A and 6B depict exemplary blood glucose levels in four mice during an experiment in which diabetes is induced with STZ and IGF-2 is provided to the mice at a daily dose of 800 μg/kg (1/4 dose) at a designated time point;

FIGS. 7-10 depict exemplary blood glucose levels in four mice during an experiment, wherein diabetes is induced with STZ at a specified time point and IGF-2 is provided to the mice at a daily dose of 300 μg/kg (1/10 dose) at the specified time point;

FIGS. 11A, 11B, 12A and 12B depict exemplary blood glucose levels in four mice during an experiment in which diabetes is induced with STZ and IGF-2 is provided to the mice at a daily dose of 12,000 μg/kg at a designated time point.

FIG. 13 depicts exemplary blood concentrations of IGF-2 over time in mice after Intraperitoneal (IP) injection of 40 μg of IGF-2 (total IGF-2 and free IGF-2);

FIG. 14 depicts exemplary blood glucose levels over time in an experiment comparing insulin and IGF-2 action;

figure 15 shows the average blood glucose levels for the four STZ treated mice experiments depicted in figures 1-4;

FIG. 16 shows an exemplary short-term effect of IGF-2 on glucose levels and IGF-2 levels after injection of IGF-2 in STZ treated mice;

FIG. 17 shows an exemplary long-term effect of IGF-2 on glucose levels in STZ-treated mice;

FIG. 18 shows glucose levels in four mice that did not exhibit a persistent response to treatment with IGF-2;

FIG. 19 shows the increase in insulin levels in four STZ-treated mice after four weeks of treatment with IGF-2;

FIG. 20 shows the results of an exemplary glucose tolerance test in STZ-treated mice treated with IGF-2;

FIG. 21 depicts pancreatic histology results of STZ-treated mice;

FIG. 22 (upper panel) shows results of islet immunohistochemical staining of insulin positive cells in mice treated with IGF-2 treated STZ, and associated glucose response results for permanently cured and non-permanently cured mice (lower panel);

FIG. 23 shows the results of an exemplary experiment performed on a first group of db/db mice to determine how IGF-2 affects blood glucose levels;

FIG. 24 shows the results of an exemplary experiment performed on a second group of db/db mice to determine how IGF-2 affects blood glucose levels;

FIG. 25 shows the results of exemplary experiments performed on db/db mice of the third and fourth groups to determine how IGF-2 affects blood glucose levels;

FIG. 26 shows the results of an exemplary experiment showing how long-term treatment with IGF-2 increases serum insulin levels in db/db mice;

FIG. 27 provides exemplary histopathological results showing the number of islet cells tested positive for insulin and glucagon after treatment of db/db mice with IGF-2;

FIG. 28 shows results of islet immunohistochemical staining of insulin positive cells in db/db mice treated with IGF-2;

FIG. 29 shows experimental results to determine how IGF-2 affects the onset of type 1 diabetes in NOD mice;

FIG. 30A shows the results of another experiment to determine how IGF-2 affects the onset of type 1 diabetes in NOD mice;

FIG. 30B shows serum insulin levels two weeks after treatment with IGF-2;

FIG. 31 shows the effect of different levels of IGF-2 on cell proliferation and insulin secretion following in vitro glucose induction;

FIG. 32 shows viability of STZ treated mice islet cells stained with MTT following treatment with IGF-2 compared to treatment with GLP-1; and

FIG. 33 shows how treatment with IGF-2 alters insulin response to glucose pulses in human islet cells.

Detailed Description

Aspects described herein provide methods of treating diabetes (and related conditions) in a subject in need of treatment by administering to the subject a first, second, third, fourth, and fifth daily dose of IGF-2 or a variant thereof, respectively, on a first, second, third, fourth, and fifth different day, wherein each of the daily doses comprises at least 65 μg of IGF-2 or a variant thereof per kilogram body weight. The term "normal level" means that at that level, if a glucose level is maintained (e.g., a glucose level of about 60 to about 110mg/dL in a human), the subject will not be considered in need of treatment.

Animal experiments described herein were performed in mice using IGF-2 doses suitable for use in mice. It is expected that Human Equivalent Doses (HEDs) will be used to treat humans with IGF-2. In this regard, HED doses of IGF-2 and variants thereof were calculated according to established U.S. food and drug administration guidelines (U.S. food and Drug Administration guidelines). Nair AB, jacob s., A simple practice guide for dose conversion between animals and human, J Basic Clin Pharma 2016;7:27-31. For example, a dose of HED IGF-2 based on a mouse IGF-2 dose is obtained by dividing the mouse dose by 12.3. In this aspect, a mouse IGF-2 dose of 800 μg/kg corresponds to a human dose of 65 μg/kg, a mouse IGF-2 dose of 3000 μg/kg corresponds to a human dose of 244 μg/kg, and a mouse IGF-2 dose of 12,000 μg/kg corresponds to a human dose of 976 μg/kg. As described herein, HED for IGF-2 and variants thereof can be calculated by dividing the mouse dose by 12.3. In another aspect, the dosage of IGF-2 and variants thereof may be at least 800, 3,000, or 12,000 μg/kg in a human, for example.

As described herein, IGF-2 and variants thereof provide a range of therapeutic options for maintaining "euglycemic" (i.e., blood glucose levels within a normal range) in subjects with hyperglycemia, type I and type II diabetes, and related autoimmune disorders. Without being bound by theory, and based on the data described herein, IGF-2 increases serum insulin levels and the number of functional beta pancreatic cells. Importantly, these effects can be used for short-term treatment (e.g., 30 days or less) or long-term treatment. In addition, even after stopping the treatment, the effect of normoglycemia is maintained in many cases. In this aspect, treatment with IGF-2 as described herein can be used to treat a condition such as type II diabetes and delay or prevent the onset of the condition such as type I diabetes. In addition, treatment of IGF-2 and variants thereof as described herein may be used to prevent the onset of type II diabetes. For example, IGF-2 treatment may be used in subjects at risk for diabetes or diagnosed with pre-diabetes to prevent or eliminate the onset of type II diabetes.

The term "diabetes" includes general diabetes, type I diabetes, type II diabetes and gestational diabetes. "diabetes-related disorders" include abnormal insulin resistance, abnormal blood glucose levels, abnormal insulin levels, hyperinsulinemia, glycosylated hemoglobin levels, metabolic syndrome, elevated blood pressure, hyperglycemia, excess body fat around the waist, or abnormal cholesterol or triglyceride levels, or combinations thereof. IGF-2 and variants thereof may be used to treat conditions associated with diabetes.

The term "IGF-2" refers to human insulin-like growth factor 2 and variants thereof. IGF-2 comprises SEQ ID NO.1 and variants having at least 95% homology to SEQ ID NO. 1.

In some cases, the first, second, third, fourth, and fifth different days occur on different consecutive days.

In some cases, a sixth, seventh, and eighth daily dose of IGF-2 or a variant thereof, or a sixth, seventh, eighth, ninth, and tenth daily dose of IGF-2 or a variant thereof, respectively, can be administered to the subject on a sixth, seventh, eighth, ninth, and tenth different day, wherein the first, second, third, fourth, fifth, sixth, seventh, eighth, ninth, and tenth different day occur on consecutive days.

The method may further comprise administering to the subject a sixth, seventh, eighth, ninth, and tenth daily dose of IGF-2, or a variant thereof, on a sixth, seventh, eighth, ninth, and tenth different day, respectively, wherein the first, second, third, fourth, fifth, sixth, seventh, eighth, ninth, and tenth different days occur on consecutive days.

In some cases, each of the daily doses comprises at least 163 μg of IGF-2 or a variant thereof per kilogram of subject body weight. In some cases, each of the daily doses comprises at least 244 μg IGF-2 or a variant thereof per kilogram subject body weight. In some cases, each of the daily doses comprises at least 813 μg IGF-2 or a variant thereof per kilogram body weight of the subject. In some cases, each of the daily doses comprises 163-1626 μg IGF-2 or a variant thereof per kilogram subject body weight.

Further aspects provide methods of treating diabetes by administering IGF-2 or a variant thereof to a subject in need of treatment in an amount of about 65 μg/kg subject body weight to about 1626 μg/kg subject body weight.

In some cases, administration is repeated for at least 5 days. In some cases, administration is repeated for at least 10 days. In some cases, administration in humans may be repeated more frequently than in animals such as mice. In some cases, the subject may receive daily doses of IGF-2 or variants thereof divided into one, two, three, or more injections (or another route of administration) in order to achieve a particular daily dose (e.g., at least 800 (65 HED), 3000 (244 HED) (referred to as 1X1 or X1 in the figures), 12,000 (976 HED) (referred to as 1X4 or X4 in the figures) μg/kg subject body weight). The subject may receive daily doses of IGF-2 or a variant thereof on consecutive days (e.g., at least 5, at least 10, at least 15, at least 20, at least 25, at least 30, at least 40, or at least 50 consecutive days). IGF-2 or variants thereof may be provided to a subject by any suitable route of administration (oral, injectable, subcutaneous, transdermal, etc.).

Further aspects provide methods of reducing blood glucose levels in a subject by administering IGF-2 or a variant thereof to a subject in need of treatment in an amount of about 65 μg/kg subject body weight to about 813 μg/kg subject body weight.

In some cases, blood glucose levels are reduced to about normal levels as compared to a subject that does not receive IGF-2 or a variant thereof.

In some cases, administration is repeated for at least 5 days. In some cases, administration is repeated for at least 10 days. In some cases, administration is repeated for at least 15 days. In some cases, administration is repeated for at least 20 days.

Further aspects provide pharmaceutical compositions comprising IGF-2 or a variant thereof and a pharmaceutically acceptable excipient in an amount sufficient to reduce blood glucose levels in a subject to about normal levels, as compared to a subject not receiving IGF-2 or a variant thereof.

In some cases, the amount of IGF-2 or variant thereof is from about 3.25mg to about 49mg. In some cases, the amount of IGF-2 or variant thereof is from about 8.13mg to about 41mg. In some cases, the amount of IGF-2 or a variant thereof is from about 24mg to about 33mg.

In some cases, the pharmaceutical composition is administered to a subject exhibiting abnormal insulin resistance, abnormal blood glucose levels, abnormal insulin levels, abnormal glycosylated hemoglobin levels, or a combination thereof.

In some cases, IGF-2 is human IGF-2 or a variant thereof. Optionally, human IGF-2 is recombinant.

In some cases, the pharmaceutical composition may be administered to the subject at least once daily for at least 5 days. In some cases, the pharmaceutical composition may be administered to the subject at least once daily for at least 8 days. In some cases, the pharmaceutical composition may be administered to the subject at least once daily for at least 10 days.

In one aspect, IGF-2 may be used in a composition for treating a patient in need thereof, wherein the patient has diabetes or type 2 diabetes, according to the compositions and methods described herein.

Aspects described herein provide methods of treating type 2 diabetes in a subject in need of treatment and having body weight. The method comprises administering to the subject a first, second, third, fourth, and fifth daily dose of IGF-2 or a variant thereof on each day, wherein each of the daily doses comprises at least 244 μg of IGF-2 or a variant thereof per kilogram of body weight.

In some cases, each of the daily doses comprises at least 976 μg of IGF-2 or a variant thereof per kilogram body weight. In some cases, the subject is treated with IGF-2 or a variant thereof for a period of at least 35 days, and the glucose concentration in the subject's blood stream measured after 14 hours of fasting does not exceed 200mg/dl measured after 35 days of the period and after 14 hours of fasting.

Aspects described herein provide methods of preventing the onset of type 1 diabetes in a subject having body weight. The method comprises administering to the subject a first, second, third, fourth, and fifth daily dose of IGF-2 or a variant thereof on each day, wherein each of the daily doses comprises at least 65 μg of IGF-2 or a variant thereof per kilogram of body weight.

In some cases, each of the daily doses comprises at least 976 μg of IGF-2 or a variant thereof per kilogram body weight. In these cases, the concentration of glucose in the blood of the subject measured at the fifth daily dose and after the at least 180 minutes is less than 300mg/dl within at least 180 minutes after the subject receives a glucose dose of 2 grams/kg of the subject's body weight.

Additional aspects described herein provide methods of increasing insulin levels in the blood stream of a subject having diabetes and having body weight. The method comprises administering to the subject a first, second, third, fourth, and fifth daily dose of IGF-2 or a variant thereof on each day, wherein each of the daily doses comprises at least 65 μg of IGF-2 or a variant thereof per kilogram of body weight.

In some cases, the concentration of insulin in the subject's blood stream is increased by at least 50% as compared to the initial concentration of insulin in the subject's blood stream measured prior to administration of IGF-2 or a variant thereof to the subject.

In some cases, each of the daily doses comprises at least 244 μg of IGF-2 or a variant thereof per kilogram body weight. In some cases, each of the daily doses comprises at least 976 μg of IGF-2 or a variant thereof per kilogram body weight.

Aspects described herein provide methods of increasing the number of functional beta cells in a subject having diabetes and having body weight. The method comprises administering to the subject a first, second, third, fourth, and fifth daily dose of IGF-2 or a variant thereof on each day, wherein each of the daily doses comprises at least 65 μg of IGF-2 or a variant thereof per kilogram of body weight.

In some cases, the number of functional β cells in the subject is increased by at least four times after at least 70 days of administration of IGF-2 or a variant thereof to the subject, as compared to the initial number of functional β cells in the subject measured prior to administration of IGF-2 or a variant thereof to the subject.

In some cases, each of the daily doses comprises at least 244 μg of IGF-2 or a variant thereof per kilogram body weight. In some cases, each of the daily doses comprises at least 976 μg of IGF-2 or a variant thereof per kilogram body weight.

Still further aspects described herein provide methods of preventing the onset of type 2 diabetes in a subject having body weight. The method comprises administering to the subject a first, second, third, fourth, and fifth daily dose of IGF-2 or a variant thereof on each day, wherein each of the daily doses comprises at least 65 μg of IGF-2 or a variant thereof per kilogram of body weight.

The methods and compositions described herein may further comprise reducing at least one of insulin resistance, blood glucose levels, obesity, hyperinsulinemia, glycosylated hemoglobin levels, or a combination thereof in the subject.

IGF-2 includes SEQ ID NO. 1 and variants thereof, including but not limited to human IGF-2 and recombinant IGF-2.

SEQ ID NO:	Human accession number	Gene name
			1	P01344	IGF2

The active ingredients for use described herein may be contained in a pharmaceutically suitable vehicle selected such that such compositions are suitable for delivery by oral, rectal, parenteral (e.g., intravenous, intramuscular, intraarterial, intraperitoneal, etc.), or inhalation routes, osmotic pumps, and the like.

The pharmaceutical compositions contemplated for use in the practice of the present invention may be used in the form of solids, solutions, emulsions, dispersions, micelles, liposomes, etc., wherein the resulting composition contains one or more of the active compounds contemplated for use herein as its active ingredient, in admixture with an organic or inorganic carrier or excipient suitable for nasal, enteral or parenteral application. The active ingredient may be compounded with, for example, conventional non-toxic pharmaceutical and physiologically acceptable carriers for tablets, pills, capsules, dragees, lozenges, aqueous or oily suspensions, dispersible powders or granules, suppositories, solutions, emulsions, suspensions, hard or soft capsules, caplets or syrups or elixirs and any other suitable form for use. Carriers that may be used include dextrose, lactose, gum acacia, gelatin, mannitol, starch pastes, magnesium trisilicate, talc, corn starch, keratin, colloidal silica, potato starch, urea, medium chain triglycerides, dextran and other carriers suitable for use in preparing formulations in solid, semi-solid, or liquid form. In addition, adjuvants, stabilizers, thickeners and colorants can be used. The active compounds contemplated for use herein are included in the pharmaceutical compositions in an amount sufficient to produce the desired effect on the process, condition or disease of interest.

In addition, such compositions may contain one or more agents selected from flavoring agents (such as peppermint, oil of wintergreen, or cherry), coloring agents, preservatives, and the like, to provide pharmaceutically elegant and palatable preparations. Tablets containing the active ingredient in admixture with non-toxic pharmaceutically acceptable excipients may also be prepared by known methods. The excipients used may be, for example, (1) inert diluents such as calcium carbonate, lactose, calcium phosphate, sodium phosphate, and the like; (2) Granulating and disintegrating agents, such as corn starch, potato starch, alginic acid and the like; (3) Binders such as tragacanth, corn starch, gelatin, acacia, and the like; and (4) lubricants such as magnesium stearate, stearic acid, talc, and the like. The tablets may be uncoated or they may be coated by known techniques to delay disintegration and absorption in the gastrointestinal tract and thereby provide a sustained action over a longer period. For example, a time delay material such as glyceryl monostearate or glyceryl distearate may be employed. Tablets may also be manufactured by the process described in U.S. Pat. nos. 4,256,108;4,160,452; and 4,265,874 to form osmotic therapeutic tablets for controlled release.

When the formulations for oral use are in the form of hard gelatin capsules, the active ingredient may be mixed with inert solid diluents such as calcium carbonate, calcium phosphate, kaolin and the like. They may also be in the form of soft gelatin capsules wherein the active ingredient is mixed with water or an oil medium, for example peanut oil, liquid paraffin, olive oil and the like.

The pharmaceutical composition may be in the form of a sterile injectable suspension. Such suspensions may be formulated according to known methods using suitable dispersing or wetting agents and suspending agents. The sterile injectable preparation may also be a sterile injectable solution or suspension in a non-toxic parenterally-acceptable excipient, diluent or solvent, for example as a solution in 1, 4-butanediol. Sterile, fixed oils are conventionally employed as a solvent or suspending medium. For this purpose, any bland fixed oil may be employed including synthetic mono-or diglycerides, fatty acids (including oleic acid), naturally occurring vegetable oils such as sesame oil, coconut oil, peanut oil, cottonseed oil and the like, or synthetic fatty vehicles such as ethyl oleate and the like. Buffers, preservatives, antioxidants and the like may be incorporated as required.

In addition, sustained release systems, including semipermeable polymer matrices in the form of shaped articles (e.g., films or microcapsules), can also be used in the administration of the active compound as employed herein.

Isolated nucleic acid molecules, variants and fragments thereof

In one aspect, the application provides an isolated or recombinant nucleic acid molecule comprising a nucleotide sequence encoding a protein described herein, e.g., SEQ ID NO. 1. In another aspect, the application provides an isolated or recombinant nucleic acid molecule comprising a nucleotide sequence encoding a protein described herein, e.g., SEQ ID NO. 1.

In one aspect, the proteins of the application are encoded by a nucleotide sequence. In one aspect, the application provides a nucleotide sequence encoding an amino acid sequence having at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or more sequence identity to the nucleotide sequence encoding SEQ ID NO. 1.

The skilled person will further appreciate that changes may be introduced by mutation of the nucleotide sequences of the present application, resulting in a change in the amino acid sequence of the encoded protein without altering the biological activity of the protein. Thus, variant isolated nucleic acid molecules may be produced by introducing one or more nucleotide substitutions, additions, or deletions into the corresponding nucleotide sequences disclosed herein such that one or more amino acid substitutions, additions, or deletions are introduced into the encoded protein. Mutations can be introduced by standard techniques, such as site-directed mutagenesis and PCR-mediated mutagenesis. Such variant nucleotide sequences are also included in the present application.

For example, conservative amino acid substitutions may be made at one or more predicted nonessential amino acid residues. "nonessential" amino acid residues are residues that can be altered from the wild-type sequence of the proteins described herein without altering the biological activity, whereas "essential" amino acid residues are required for biological activity. A "conservative amino acid substitution" is a substitution in which an amino acid residue is replaced with an amino acid residue having a similar side chain. Families of amino acid residues with similar side chains have been defined in the art. These families include amino acids with basic side chains (e.g., lysine, arginine, histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), beta-branched side chains (e.g., threonine, valine, isoleucine) and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine).

Amino acid substitutions may be made in non-conserved regions of retained function. Typically, such substitutions will not be made to conserved amino acid residues, or amino acid residues that are located within a conserved motif, wherein such residues are essential for protein activity. Examples of residues that are conserved and may be necessary for protein activity include, for example, residues that are identical between all proteins contained in an alignment of similar or related sequences of the invention (e.g., residues that are identical in an alignment of homologous proteins). Examples of residues that are conserved but may allow conservative amino acid substitutions and still retain activity include, for example, residues that have only conservative substitutions between all proteins contained in an alignment of similar or related sequences of the invention (e.g., residues that have only conservative substitutions between all proteins contained in an alignment of homologous proteins). However, one skilled in the art will appreciate that functional variants may have minor conservative or non-conservative changes in conserved residues.

Isolated proteins, variants and fragments thereof

A "fragment" or "biologically active portion" includes a protein fragment comprising an amino acid sequence sufficiently identical to the amino acid sequence set forth in SEQ ID NO. 1, and which exhibits, for example, antidiabetic activity.

"variant" means a protein having an amino acid sequence which is at least 95% identical to the amino acid sequence of SEQ ID NO. 1. Variants include proteins that differ in amino acid sequence due to mutagenesis. Variant proteins encompassed by the present invention are biologically active, i.e., they continue to possess the desired biological activity of the native protein, i.e., retain antidiabetic activity.

In various embodiments of the invention, the anti-diabetic protein comprises an amino acid sequence that is shorter than the full-length sequence due to the use of an alternative downstream start site.

Variants with alterations or modifications

It is recognized that the DNA sequence of a protein may be altered by various methods, and that these alterations may result in a DNA sequence encoding a protein having an amino acid sequence different from SEQ ID NO. 1. The protein may be altered in various ways, including amino acid substitutions, deletions, truncations and insertions of one or more amino acids of SEQ ID NO. 1, including up to 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 100, 105, 110, 115, 120, 125, 130, 135, 140, 145, 150, 155, or more amino acid substitutions, deletions or insertions. Methods for such operations are generally known in the art. For example, amino acid sequence variants of proteins may be prepared by mutations in DNA. This can also be achieved by one of several mutagenesis formulae and/or directed evolution. Changes in the amino acid sequence encoded should not significantly affect the function of the protein. Such variants will have the desired antidiabetic activity.

Alternatively, changes may be made to the protein sequence of many proteins at the amino or carboxy terminus without substantially affecting activity. This may include insertions, deletions, or alterations introduced by modern molecular methods such as PCR, including PCR amplification by including amino acid coding sequences in oligonucleotides utilized in PCR amplification to alter or extend protein coding sequences. Alternatively, the added protein sequence may comprise the complete protein coding sequence, such as the sequences commonly used in the art to produce protein fusions. Such fusion proteins are typically used to (1) increase expression of a protein of interest, (2) introduce binding domains, enzymatic activities, or epitopes to facilitate protein purification, protein detection, or other experimental uses known in the art, (3) target secretion or translation of the protein to subcellular organelles, such as the periplasmic space of gram-negative bacteria, or the endoplasmic reticulum of eukaryotic cells, which typically result in glycosylation of the protein.

Theory of operation

In healthy subjects, insulin regulates glucose uptake. However, in diabetics, insulin no longer effectively exerts this effect (due to insufficient insulin levels or insulin resistance). IGF-2 has been identified as useful in the resolution of type II diabetes.

While not wanting to be bound by theory, the following is one possible explanation of the mechanism of action of the disclosed invention. The inventors theorize that certain cells in the body, referred to herein as "BLCs" (which represent β -like cells), may be induced to secrete insulin or insulin-like substances ("ILMs") in response to high levels of glucose. Note that while the location of BLCs within the body has not been confirmed, their location need not be known to obtain the results described herein. New BLCs may also be produced, for example, by proliferation or transdifferentiation, etc.

More specifically, BLCs are dormant or inactive before they are exposed to IGF-2, in which case they do not secrete insulin or ILM or secrete insufficient amounts of insulin or ILM. But after exposure to IGF-2, BLC is activated and will begin to secrete insulin or ILM in response to high levels of glucose. One possible mechanism of action is that exposure to IGF-2 results in BLC secreting insulin and/or ILM in response to high levels of glucose. Another possible mechanism of action is that the BLC is naturally programmed to secrete insulin and/or ILM in response to high levels of glucose, but there are typically unknown substances that inactivate the BLC. In this case, IGF-2 neutralizes (e.g., cleaves) the normally dominant inactivating agent.

In either case, once the BLC has been activated, the BLC will sense the glucose level in the blood and will initiate the production of insulin or ILM at a level corresponding to the glucose level in the blood (such that higher levels of glucose will result in more insulin or ILM production). This production of insulin or ILM can occur directly in the BLC itself or indirectly (e.g., by action of other cells). Insulin or ILM circulates in the blood.

Another possible explanation for the mechanism of action is that exposure to IGF-2 increases the ability of conventional beta cells to regulate glucose levels in a subject, or down-regulates/shuts down another mechanism that prevents conventional beta cells from properly regulating glucose levels. Where this theory is correct, it is believed that treatment with IGF-2 as disclosed herein can restore normal activity of residual beta cells.

Examples

Example 1 materials and methods

Male C57BL/6 mice (raised under conventional conditions and allowing laboratory food and water to be obtained ad libitum) of 8-10 weeks old were used in the experiments described in examples 3-4 below. In each experiment, animals were matched by age and weight (20-24 g) and randomized into groups to receive different treatments. Diabetes is induced by one or more doses of Streptozotocin (STZ).

Briefly, animals received 100mg/kg (b.w.) of STZ (Cayman Chemical, ann Arbor, MI) dissolved in citrate buffer at ph4.5 intraperitoneally (i.p.), if desired, and the procedure was repeated. Clinical diabetes is defined as hyperglycemia (blood glucose levels >300mg/dL in fasted animals). Fasting blood glucose levels were measured three times per week and sampled from the tail tip after 6 hours of starvation throughout the experiment.

Fasting blood glucose levels (mg/dL) were determined using an Accu-Chek Performance blood glucose meter (Roche Diagnostics, mannheim, germany). Approximately two weeks after stabilization of hyperglycemia, C57BL/6STZ mice received intraperitoneal injections (0.3-12 mg/kg/day injections) of recombinant human IGF-2 for 5-10 consecutive days. Mice were tested during and at the end of post-treatment follow-up period: fasting blood glucose, body weight, glucose tolerance test (IPGTT), serum C-peptide levels, serum insulin levels, whole blood and histological analysis (CBC, chemistry, insulin IHC and H & E).

EXAMPLE 2 IGF-2 3000 ug/kg/day dose

FIGS. 1-4 show the effect of IGF-2 at 3000 μg/kg/day in four different mice treated according to the description of example 1.

Mice C1 (fig. 1), C8 (fig. 2), and C6 (fig. 3) received STZ 25 days before starting treatment with IGF-2 and exhibited about four-fold increase in fasting glucose levels. IGF-2 was administered on day 0 (at 3000 μg/kg/day) and ten more times during the first ten days after initial treatment with IGF-2. During the course of ten days of treatment with IGF-2, fasting glucose levels returned to the normal range and remained within the normal range until the end of the experiment. Notably, the increase in fasting glucose levels appears to be permanent (or at least semi-permanent) because IGF-2 was not administered on days 11-82.

Mouse F1 (fig. 4) was treated similarly to mouse C1, mouse C8 and mouse C6, except that STZ was provided 20 days prior to initial treatment with IGF-2. The fasting blood glucose results of mouse F1 were similar to those of mice C1, C8 and C6.

Although all four of the examples depicted in fig. 1-4 show long term increases in empty stomach glucose levels, in some mice (not shown), empty stomach glucose results return to high levels after the end of the 10 day course of treatment with IGF-2.

EXAMPLE 3 dose of IGF-21/4 (800. Mu.g/kg/day)

Mouse A8 (fig. 5A) received STZ 25 days before starting treatment with IGF-2 and exhibited an approximately four-fold increase in fasting glucose levels. IGF-2 was administered on day 0 (at 800 μg/kg/day) and ten more times during the first ten days after initial treatment with IGF-2. During the course of ten days of treatment with IGF-2, fasting glucose levels returned to the normal range and remained within the normal range until the end of the experiment. The increase in fasting glucose levels appears to be permanent or semi-permanent).

Mouse A6 (fig. 5B) received STZ 25 days before starting treatment with IGF-2 and exhibited an approximately four-fold increase in fasting glucose levels. IGF-2 was administered on day 0 (at 800 μg/kg/day) and ten more times during the first ten days after initial treatment with IGF-2. During the ten day course of treatment with IGF-2, fasting glucose levels returned to the normal range and remained within the normal range until STZ was again provided. After the second administration of STZ, fasting glucose levels increased back to the diabetic range, indicating that the mechanism responsible for returning glucose levels to the normal range is vulnerable to STZ.

Mice F3 and F4 (fig. 6A and 6B) received STZ 20 days before starting treatment with IGF-2 and exhibited about a four-fold increase in fasting glucose levels. IGF-2 was administered on day 0 (at 800 μg/kg/day) and ten more times during the first ten days after initial treatment with IGF-2. During the ten day course of treatment with IGF-2, fasting glucose levels returned to the normal range, but returned to about 400 after the end of the ten day course of injection. Thus, no long-term results were obtained for both mice.

In this example, the results for the 800. Mu.g/kg/day dose were variable. Half of the mice had full or nearly full resolution (i.e., blood glucose levels remained near 200mg/dl as in fig. 5A and 5B). The remaining mice had partial improvement (i.e., blood glucose levels remained around 400mg/dl as in fig. 6A and 6B).

EXAMPLE 4 IGF 300 ug/kg/day dose

Mice B5 (FIG. 7) were treated with STZ three times (25, 20 and 17 days) before initial 300 μg/kg/day IGF-2 followed by ten additional 300 μg/kg/day doses of IGF-2 over a ten day course. Unlike the higher dose case described above in connection with fig. 1-6, fasting glucose levels did not return to the normal range and no long term results were observed.

Mouse B6 was treated similarly to mouse B5 (fig. 8), except that mouse B6 received two doses of STZ 20 and 12 days prior to the course of treatment with IGF-2 at a dose of 300 μg/kg/day. The results were similar to those obtained for mouse B5.

Mouse B3 was treated similarly to mouse B5 (fig. 9). Although the fasting glucose levels of the mice underwent a temporary drop from day 10-25, the long-term results were similar to those of mouse B5.

Mouse B4 was treated similarly to mouse B5 (fig. 10) except that mouse B6 received a single dose of STZ 25 days prior to the course of treatment with IGF-2 at a dose of 300 μg/kg/day. The mice also experienced a temporary drop in fasting glucose levels from days 10-25, but long-term results were similar to those of mouse B5.

Example 5-repeated comparison

In some cases, the number of repetitions appears to be a factor in achieving long-term results. Fig. 11A, 11B, 12A and 12B depict exemplary blood glucose levels in four mice during the experiment, in which diabetes is induced with Streptozotocin (STZ) and IGF-2 is provided to the mice at a daily dose of 12,000 μg/kg at the indicated time points. More specifically, when IGF-2 was provided to mice daily over 12 consecutive days, a long-term increase in blood glucose levels was obtained (see fig. 11A and 11B). However, when IGF-2 was provided to mice for only 5 consecutive days, no long-term elevation of blood glucose levels was obtained (see fig. 12A and 12B).

In one aspect, the long-term return of blood glucose to normal levels depends on both the number of repetitions and the IGF-2 dose per repetition. The treatment regimen may take into account a combination of these two factors. In some cases, the glucose level may eventually return to its elevated value when the number of repetitions or the dose per repetition is too small. In some cases, long-term return of blood glucose to normal levels is achieved (e.g., as described above in connection with fig. 1-5 and 11) when both the number of repetitions and the dose in each repetition are large enough.

Example 6-pharmacokinetics of IGF-2 (40 μg intraperitoneal injection)

FIG. 13 shows the change over time in the level of total IGF-2 (referred to as "factor A" in the figure) in blood after intraperitoneal injection of 40 μg. The results show peak total concentrations of total IGF-2 (about 16 μg) and 1 μg free IGF-2 as determined by ELISA (enzyme Linked immunosorbent assay) over a 240 minute time frame. Without being bound by this theory, it is believed that IGF-2 binding proteins may initially inactivate the biological activity of free IGF-2. And over time, binding between IGF-2 and the binding protein may be released, increasing the bioavailability of IGF-2 and leading to a more long-term effective treatment.

EXAMPLE 7 kinetics of blood glucose concentration of IGF-2 relative to insulin

FIG. 14 shows the comparative blood glucose concentration kinetics between insulin and IGF-2 (referred to as "factor A" in the figure) in glucose tolerance testing in mice. Notably, glucose levels decrease after administration of insulin or IGF-2. Thus, IGF-2 provides an effect that mimics insulin in the body, and this effect is referred to herein as an "insulin mimicking" effect. Notably, however, as shown in fig. 14, the insulin mimetic effect of IGF-2 persisted significantly longer than the hypoglycemic effect of insulin. More specifically, when 1 unit of insulin/kg is administered, the recovery of glucose levels begins after two hours. However, when 800. Mu.g/kg IGF-2 was administered, the recovery of glucose levels began after six hours. Furthermore, in the latter case, glucose levels do not begin to rise until two hours after IGF-2 is no longer detectable in the blood (see fig. 13). In this example, administration of IGF-2 may provide better results than administration of insulin in terms of blood glucose levels, even when only a single dose is used. Without being bound by theory, it is possible that IGF-2 combines with many binding proteins in the blood and then its active free form is slowly released from the complex.

Example 8-discussion

Taken together, the data in FIGS. 13 and 14 show that insulin mimetic effects of IGF-2 are separate from the long-term effects of IGF-2 described above in connection with FIGS. 1-5.

Insulin mimetic action can be used to treat hyperglycemia, while long-term action can be used to cure diabetes either completely or partially for long periods of time. In addition, the presence of the high "insulin resistance" typical of type 2 diabetes mellitus treated with insulin does not reduce the glycemic effect of IGF-2.

Unlike conventional diabetes treatment with insulin, where the dosage must be precisely controlled to prevent hypoglycemia, surviving subjects can tolerate a very wide range of IGF-2 doses without causing hypoglycemia. More specifically, in the above examples, the 10:1 ratio dose (i.e., between 3000 μg and 300 μg) did not cause hypoglycemia. Accordingly, IGF-2 compositions and methods as described herein can be advantageously used to effectively treat hyperglycemia without the life threatening risks associated with insulin therapy (e.g., hypoglycemia and insulin resistance). In addition, IGF-2 may exert a long-term effect beyond the treatment period to reduce or even cure diabetes when used as described herein in connection with fig. 1-5.

EXAMPLE 9 STZ treated mice

Fig. 15-23 show experimental results of mice treated with STZ. STZ eliminates or reduces the secretory capacity of pancreatic beta cells. STZ-treated mice were used as models for both type 1 diabetes and advanced type 2 diabetes.

Figure 15 shows the average blood glucose levels of four STZ-treated mice experiments depicted in figures 1-4, before treatment with IGF-2 at a dose of 3000 μg/kg/day (left panel) and after treatment (right panel). As shown in FIG. 15, intraperitoneal treatment with 3000 μg/kg/day IGF-2 reduced blood glucose levels to the normal range (e.g., 100-200mg/dL glucose) for 3-5 days per day, and maintained normal levels for the remainder of the 10 day window for IGF-2 administration. Even when the IGF-2 dose was increased to 12,000 μg/kg/day, no hypoglycemia was observed.

FIG. 16 shows an exemplary short-term effect of IGF-2 (referred to as "factor A" or FA in the figures) on glucose levels and IGF-2 levels over a 240 minute time course following injection of IGF-2 in STZ-treated mice. More specifically, the upper graph shows the decrease in blood glucose levels in three STZ-treated mice after receiving 800 μg/kg/day doses of IGF-2 over a 240 minute time course. The lower panel shows the increase in total IGF-2 (upper trace) over the same time course as compared to free IGF-2 (i.e., uncomplexed IGF-2). IGF-2 is part of a complex system that includes IGF-1 and IGF-2 and binding proteins, proteases and other interacting molecules. Intraperitoneal injection of a single 800 μg/kg dose of IGF-2 reduced the hyperglycemia levels of 300-500mg/dL to normal levels (100-200 mg/dL) for a period of time exceeding four hours. Normal blood glucose levels are maintained while total serum IGF-2 is reduced to very low levels. Free IGF-2 levels are a small fraction of total IGF-2 concentration over the time course of the experiment. Without being bound by this theory, it is believed that slow release of IGF-2 from the serum complex can maintain normal blood glucose levels.

FIG. 17 shows an exemplary long-term effect of IGF-2 (referred to as FA in the figures) on glucose levels in STZ-treated mice. More specifically, FIG. 17 shows a long-term 300-day follow-up study of four mice during and after a 10-day course of treatment with IGF-2 at a dose of 3000 μg/kg/day. The data indicate that normal blood glucose levels are maintained for at least 300 days after treatment even though no additional dose of IGF-2 is administered after the initial 10 day treatment history. STZ-induced diabetes in four mice was considered to be permanently cured by a single 10-day treatment history.

Compared to the results depicted in fig. 17, fig. 18 depicts experimental results for four STZ-treated mice treated with IGF-2 at a dose of 3000 μg/kg/day that received a 10-day treatment history. These mice initially responded to treatment with IGF-2 at a dose of 3000 μg/kg/day, but were not permanently cured. Although no data was collected, it is believed that continued treatment of these mice will maintain blood glucose levels within normal ranges. Thus, mice that are not permanently cured may continue to be treated with IGF-2 or variants thereof in order to control their diabetes.

Figure 19 shows an increase in insulin levels after four weeks of treatment with IGF-2. Treatment resulted in a significant increase in insulin concentration following treatment in permanently cured mice. More specifically, serum insulin was increased by 50% in permanently cured mice, four weeks after treatment, compared to STZ-treated control mice that did not receive IGF-2 treatment.

Other experimental data indicate that c-peptide levels were increased 12-fold four weeks after treatment in four STZ treated mice, as depicted in figures 18 and 19. c-peptide is a biomarker for assessing pancreatic β cell function and is typically produced in equimolar amounts to endogenous insulin. Leight on et al, A Practical Review of C-Peptide Testing in Diabetes,Diabetes Ther.2017, 6 months; 8 (3):475-487.

FIG. 20 shows the results of an intraperitoneal glucose tolerance test on STZ-treated mice treated with IGF-2. In this experiment, four STZ-treated mice were treated with 12,000 μg/kg/day IGF-2 (four injections of 3000 μg/kg/day) for 5 days, and two mice were treated for 10 days. Glucose tolerance testing was performed 50 days after treatment with IGF-2 by challenging the treated mice with a 2 g/kg dose of glucose and determining blood glucose levels over a 180 minute time course. Based on published literature (Jorgensen et al, J.Am assoc.Lab. Animal Sci 2017 56 (1): 95-97), blood glucose profiles of treated mice were compared to saline controls and the results of normal (non-diabetic) obese and normal (non-diabetic) lean mice. Five IGF-2 treated mice were permanently cured and their response to the glucose tolerance test fell between the glucose tolerance results from non-diabetic obese mice and non-diabetic lean mice from the literature. Mouse O1 was not permanently cured and its glucose level was higher than normal obese mice.

Fig. 21 depicts pancreatic histology results of STZ treated mice. These results indicate that treatment with IGF-2 resulted in a significant increase in the number of cells tested positive for insulin in permanently cured mice compared to non-permanently cured mice and control (i.e., saline injection) mice. Permanently cured mice showed an almost four-fold increase in functional β cell numbers. Non-control mice were treated once daily with IGF-2 at a dose of 3000 μg/kg for 10 days. Mice were sacrificed on day 35 and pancreatic cells were assessed as insulin positive or negative.

FIG. 22 (upper panel) shows results of islet immunohistochemical staining of insulin positive cells in STZ-treated mice and untreated mice (naive mouse) treated with IGF-2. Staining showed that permanently cured mice had higher levels of insulin in their islets (as compared to untreated mice), whereas non-permanently cured mice had lower levels of insulin in their islets (as compared to untreated mice). This suggests that treatment with IGF-2 may lead to restoration of insulin secretion from the islets.

Fig. 22 (lower panel) shows blood glucose levels of non-permanently cured mice and permanently cured mice. Mice were treated as described in fig. 21.

Example 10-db/db mice (Lep ^db )

The db/db mice were bred to have leptin deficiency, increasing susceptibility of the mice to obesity, insulin resistance, and type 2 diabetes (T2D).

FIG. 23 shows how treatment of db/db mice with IGF-2 affects blood glucose levels. In this experiment, one group of db/db mice was injected with 3000 μg/kg/day IGF-2 for 68 days, a second group of db/db mice was injected with 12000 μg/kg/day IGF-2 for 66 days, and a third group of db/db mice was injected with saline for 68 days, once daily. The results show that treatment with 3000 μg/kg/day or 12000 μg/kg/day IGF-2 reduced blood glucose levels (14 hours space time abdominal blood glucose levels) to the normal range even after the end of the 68 day treatment period.

Fig. 24 shows experimental results similar to those of fig. 23 in the second group of db/db mice. The results show that treatment with 12,000 μg/kg/day IGF-2 reduced blood glucose levels (14 hours space time abdominal blood glucose levels) to the normal range even after the end of the 68 day treatment period. However, in the iterations of this experiment, the blood glucose level in the 3000 μg/kg/day group was not reduced relative to the control. This indicates that a daily dose of greater than 3000 μg/kg may be preferred and that a daily dose of at least 12,000 μg/kg may provide better results.

FIG. 25 shows the results of two additional experiments in which db/db mice were treated with IGF-2. In one experiment (left panel), one group of db/db mice was injected with IGF-2 at a daily dose of 12000 μg/kg, divided into two injections per day for 70 days, while the other group of db/db mice was injected with saline. The results show that treatment with 12000 μg/kg/day IGF-2 reduced blood glucose levels (14 hours space time abdominal blood glucose levels) to the normal range even after the end of the 70 day treatment period. In another experiment (right panel), one group of db/db mice was injected with IGF-2 at a daily dose of 12000 μg/kg, divided into two injections per day for 35 days, while the other group of db/db mice was injected with saline. The results show that treatment with 12000 μg/kg/day IGF-2 reduced blood glucose levels (14 hours space time abdominal blood glucose levels) to the normal range even after the end of the 35 day treatment period.

FIG. 26 shows how long-term treatment with IGF-2 increases serum insulin levels in db/db mice. In this experiment, one group of db/db mice (labeled FA X1) was injected once daily with 3000 μg/kg IGF-2 for 68 days, a second group of db/db mice (labeled FA X4) was injected twice daily with 12000 μg/kg IGF-2 for 68 days, and a third group of db/db mice was injected with saline. Serum insulin levels were measured 6.5 weeks after the end of 68 days of treatment. Treatment with a daily dose of 12,000 μg/kg increased serum insulin levels by about 50% relative to the control.

Fig. 27 provides results of histopathological and immunohistochemical studies showing the number of islet cells tested as insulin positive (left panel) and glucagon positive (right panel) in db/db mice. In this experiment, one group of db/db mice was injected with 12000 μg/kg of IGF-2 per day over 70 consecutive days, a second group of db/db mice was injected with saline, and a third group of db/db mice was a placebo group. Mice were sacrificed 70 days after the end of the first 70 days of treatment for pathology examination. The results showed an increase in insulin positive cell number of over 50%, which indicates beta cell proliferation. Evidence obtained to date does not support the hypothesis that the increase in the number of insulin positives is caused by transdifferentiation of glucagon-secreting alpha cells into beta cells.

FIG. 28 shows immunohistochemical staining of islet cells from db/db mice. In this experiment, one group of db/db mice (labeled X4) was injected with 12000 μg/kg IGF-2 per day for 70 consecutive days, a second group of db/db mice (labeled X1) was injected with 3000 μg/kg IGF-2 per day for 70 consecutive days, and a third group of db/db mice (labeled control) was injected with saline. Mice were sacrificed 70 days after the end of the first 70 days of treatment for pathology examination. These images show that insulin positive cells increase in a dose dependent manner. The control plot shows a positive staining for insulin that increases in intensity in 3000 μg/kg/day mice and increases again in intensity in 12,000 μg/kg/day mice. Taken together, these data indicate that IGF-2 can be used to treat type 2 diabetes or to prevent the onset of type 2 diabetes in pre-diabetic patients.

EXAMPLE 11 non-obese diabetic (NOD) mice

Non-obese diabetic (NOD) mice are a polygenic model of spontaneous autoimmune type 1 diabetes (T1D). NOD mice are at increased risk of developing autoimmune type 1 diabetes. Thus, NOD mice were used to determine whether treatment of IGF-2 reduced spontaneous development of type 1 diabetes.

FIG. 29 shows the effect of IGF-2 treatment on the incidence of spontaneous autoimmune attacks/type 1 diabetes in NOD mice. In this experiment, one group of NOD mice (right panel) was injected with 3000 μg/kg IGF-2 per day for 76 consecutive days, and a second group of NOD mice (left panel) was injected with saline. The results indicate that IGF-2 treatment significantly reduced the incidence of spontaneous autoimmune attacks. More specifically, at the end of 76 days of treatment, only two treated mice developed high glucose levels.

Figure 30A depicts how many NOD mice develop autoimmune type I diabetes during the first 66 days of treatment with IGF-2 and at different time intervals after treatment. In this experiment, one group of NOD mice was injected with 3000 μg/kg IGF-2 per day for 66 consecutive days, and a second group of NOD mice was injected with saline. The incidence of spontaneous autoimmune type 1 diabetes in treated mice was significantly reduced relative to control.

FIG. 30B depicts serum insulin levels of NOD mice measured two weeks after 66 days of treatment with IGF-2. Mice treated with 3000 μg/kg/day IGF-2 had serum insulin levels approximately 4-fold higher than control mice. Taken together, these results indicate that IGF-2 can be used to prevent the onset of type I diabetes.

In another experiment, NOD mice were untreated or treated with IGF-2. In this experiment untreated NOD mice showed complete destruction of islet cells due to autoimmune attack, as evidenced by a complete lack of histological staining for insulin and a relatively small amount of histological staining for glucagon. In contrast, NOD mice treated with IGF-2 for 13 weeks had fully functional islet cells as shown by the prominent histological staining of glucagon and insulin.

The results described in the previous paragraph were confirmed by comparing the number of insulin-staining positive cells in NOD mice treated with IGF-2 relative to untreated NOD mice.

Example 12 in vitro experiments in beta-MIN 6 cells

beta-MIN 6 cells were used as an in vitro model of mouse islets. The effect of treatment with IGF-2 was measured using β -MIN6 cells as an in vitro model.

FIG. 31 shows the effect of IGF-2 on cell count (e.g., cell proliferation) and insulin secretion of beta-MIN 6 cells at three different concentrations (5 nM, 20nM, 80 nM) compared to control, untreated beta-MIN 6 cells.

The left panel of FIG. 31 shows that IGF-2 increased cell proliferation in a dose-dependent manner after 1 week of treatment at three measured concentrations. The right panel of fig. 31 shows that IGF-2 also increases insulin secretion in a dose-dependent manner after 1 week of treatment (after glucose induction). GLP-1 (satiety hormone) does not increase insulin secretion (right panel). The results confirm the in vivo results discussed above and indicate that IGF-2 can increase cell number and insulin secretion.

FIG. 32 shows the effect of IGF-2 on islet cell viability in normal mice in STZ treated mice islets using MTT [3- (4, 5-dimethylthiazol-2-yl) -2, 5-diphenyltetrazolium bromide ] dye. In living cells, the yellow dye MTT is converted to a purple dye by mitochondrial reductase. Thus, the presence of the violet dye, as determined by measuring the optical density of the cells at 570nm, is used as a measure of cell viability. As shown in fig. 32, with increasing IGF-2 concentration, mouse islet viability increased in a dose-dependent manner. In contrast, GLP-1 (satiety hormone) did not significantly increase islet viability in mice.

Another experiment was performed to show the effect of IGF-2 on insulin secretion from islets of STZ-treated mice 48 hours after IGF-2 treatment. Mouse islet cells were treated with 2.5mM STZ and subsequently treated with 50nM and 500nM IGF-2 (FA) or with 100nM and 1000nM GLP-1. The results were as follows: insulin secretion increases in a dose-dependent manner with increasing IGF-2 concentration. In contrast, GLP-1 (satiety hormone) does not significantly increase insulin secretion.

FIG. 33 shows the effect of IGF-2 on human islet cells in vitro. As shown in fig. 33, treatment of human islet cells with IGF-2 at a concentration of 50nM lasted four days, with an increase in insulin secretion in response to glucose pulses of approximately 50% compared to untreated human islet cells.

EXAMPLE 13 management and treatment of diabetes Using IGF-2

As described herein, IGF-2 and variants thereof may be used to manage or cure diabetes. Short term effects include lowering blood glucose in hyperglycemic subjects and supplementing insulin secretion due to the lack of sufficient functional beta cell mass.

IGF-2 and variants thereof may also be used to provide at least the following long-term benefits: (1) lowering blood glucose levels in a patient diagnosed with type 2 diabetes, (2) alleviating beta cell insulin secretory stress, (3) delaying or preventing the onset of type 1 diabetes, and (4) maintaining normoglycemia.

EXAMPLE 14 treatment of NOD with IGF-2

In one exemplary experiment NOD mice were treated with IGF-2 at a daily dose of 3000 μg/kg for 150 days. 4/5 of the treated mice maintained normoglycemia compared to 1/4 of the control mice. In another exemplary experiment NOD mice were treated with IGF-2 at a daily dose of 3000 μg/kg for 75 days followed by a glucose measurement for 90 days (during which IGF-2 was not administered). 5/8 of the treated mice maintained normoglycemia compared to the 2/11 control mice. The average insulin secretion in the treated mice was five times higher than in the control mice in both experiments.

Example 15 treatment of db/db mice

In one exemplary experiment, db/db mice were treated with a daily dose of 12,000 μg/kg for 70 days with a follow-up of 70 days (during which IGF-2 was not administered). All treated mice maintained normoglycemia for at least 50 days after treatment. In another exemplary experiment, db/db mice were treated with a daily dose of 12,000 μg/kg for 35 days with a 35 day follow-up (during which IGF-2 was not administered). All treated mice maintained normoglycemia for 35 days after treatment.

Example 16-summary of safety/toxicity

No pathology associated with treatment was confirmed in blood samples and tissue samples from thirty organs (pancreas, liver, etc.) at the end of 10 days of treatment with IGF-2, 24 days after termination of IGF-2 treatment and 100 days after termination of 30 days of treatment with IGF-2.

Although the present invention has been disclosed with reference to certain embodiments, numerous modifications, alterations and changes to the described embodiments are possible without departing from the sphere and scope of the present invention, as defined in the appended claims. Accordingly, the invention is not intended to be limited to the embodiments described, but is to be accorded the full scope defined by the language of the following claims and equivalents thereof.

Sequence listing

<110> Bei Dawei Co., ltd

Y. Pa Erdi

D Zu Li

<120> methods and compounds for treating diabetes and related metabolic diseases

<130> 1462-0017

<150> US 63/090,943

<151> 2020-10-13

<150> US 63/234,862

<151> 2021-08-19

<160> 1

<170> PatentIn version 3.5

<210> 1

<211> 180

<212> PRT

<213> Chile person

<400> 1

Met Gly Ile Pro Met Gly Lys Ser Met Leu Val Leu Leu Thr Phe Leu

1 5 10 15

Ala Phe Ala Ser Cys Cys Ile Ala Ala Tyr Arg Pro Ser Glu Thr Leu

20 25 30

Cys Gly Gly Glu Leu Val Asp Thr Leu Gln Phe Val Cys Gly Asp Arg

35 40 45

Gly Phe Tyr Phe Ser Arg Pro Ala Ser Arg Val Ser Arg Arg Ser Arg

50 55 60

Gly Ile Val Glu Glu Cys Cys Phe Arg Ser Cys Asp Leu Ala Leu Leu

65 70 75 80

Glu Thr Tyr Cys Ala Thr Pro Ala Lys Ser Glu Arg Asp Val Ser Thr

85 90 95

Pro Pro Thr Val Leu Pro Asp Asn Phe Pro Arg Tyr Pro Val Gly Lys

100 105 110

Phe Phe Gln Tyr Asp Thr Trp Lys Gln Ser Thr Gln Arg Leu Arg Arg

115 120 125

Gly Leu Pro Ala Leu Leu Arg Ala Arg Arg Gly His Val Leu Ala Lys

130 135 140

Glu Leu Glu Ala Phe Arg Glu Ala Lys Arg His Arg Pro Leu Ile Ala

145 150 155 160

Leu Pro Thr Gln Asp Pro Ala His Gly Gly Ala Pro Pro Glu Met Ala

165 170 175

Ser Asn Arg Lys

180

Claims (43)

1. A method of treating diabetes in a subject in need of treatment, the subject having a body weight, the method comprising: administering to the subject a first, second, third, fourth, and fifth daily dose of IGF-2 or a variant thereof, respectively, on a first, second, third, fourth, and fifth different day, wherein each of the daily doses comprises at least 65 μg of IGF-2 or a variant thereof per kilogram of body weight.

2. The method of claim 1, wherein the first, second, third, fourth, and fifth different days occur on consecutive days.

3. The method of claim 1, further comprising administering to the subject a sixth, seventh, and eighth daily dose of IGF-2 or variant thereof on a sixth, seventh, and eighth different day, respectively.

4. The method of claim 1, further comprising administering sixth, seventh, eighth, ninth, and tenth daily doses of IGF-2 or a variant thereof to the subject on a sixth, seventh, eighth, ninth, and tenth different day, respectively, wherein the first, second, third, fourth, fifth, sixth, seventh, eighth, ninth, and tenth different days occur on consecutive days.

5. The method of any one of claims 1-4, wherein each of the daily doses comprises at least 163 μg of IGF-2 or a variant thereof per kilogram body weight.

6. The method of any one of claims 1-4, wherein each of the daily doses comprises at least 244 μg of IGF-2 or a variant thereof per kilogram body weight.

7. The method of any one of claims 1-4, wherein each of the daily doses comprises at least 813 μg of IGF-2 or a variant thereof per kilogram body weight.

8. The method of any one of claims 1-4, wherein each of the daily doses comprises 163-1626 μg IGF-2 or variant thereof per kilogram body weight.

9. A method of treating diabetes comprising administering IGF-2 or a variant thereof to a subject in need of treatment in an amount of about 65 μg/kg subject body weight to about 1626 μg/kg subject body weight.

10. The method of claim 9, wherein the administration is repeated for at least 5 days.

11. The method of claim 9, wherein the administration is repeated for at least 10 days.

12. A method of reducing blood glucose levels in a subject comprising administering IGF-2 or a variant thereof to a subject in need of treatment in an amount of about 65 μg/kg of the subject's body weight to about 813 μg/kg of the subject's body weight.

13. The method of claim 12, wherein the blood glucose level is reduced to about normal levels as compared to a subject not receiving the IGF-2 or variant thereof.

14. The method of claim 12, wherein the subject has diabetes.

15. The method of claim 12, wherein the subject has a disorder associated with diabetes.

16. The method of claim 12, wherein the administration is repeated for at least 5 days.

17. The method of claim 12, wherein the administration is repeated for at least 10 days.

18. The method of claim 12, wherein the administration is repeated for at least 15 days.

19. The method of claim 12, wherein the administration is repeated for at least 20 days.

20. A pharmaceutical composition comprising IGF-2 or a variant thereof and a pharmaceutically acceptable excipient, the IGF-2 or variant thereof being in an amount sufficient to reduce the blood glucose level of the subject to about normal levels compared to a subject that does not receive the IGF-2 or variant thereof.

21. The pharmaceutical composition of claim 20, wherein the amount of IGF-2 or a variant thereof is about 3.25mg to about 49mg.

22. The pharmaceutical composition of claim 20, wherein the amount of IGF-2 or a variant thereof is about 8.13mg to about 41mg.

23. The pharmaceutical composition of claim 20, wherein the amount of IGF-2 or a variant thereof is from about 24mg to about 33mg.

24. The pharmaceutical composition of claim 20, wherein the pharmaceutical composition can be administered to the subject at least once daily for at least 5 days.

25. The pharmaceutical composition of claim 20, wherein the pharmaceutical composition can be administered to the subject at least once daily for at least 8 days.

26. The pharmaceutical composition of claim 20, wherein the pharmaceutical composition can be administered to the subject at least once daily for at least 10 days.

27. A method of treating diabetes in a subject in need of treatment, the method comprising: administering to the subject a daily dose of IGF-2 or a variant thereof on each of N different days, wherein N is at least 5, and wherein (a) N and (b) the daily dose of IGF-2 or a variant thereof administered to the subject on each of the N different days are both high enough to (i) reduce the glucose level of the subject to about a normal level before the end of the N different days, and (ii) maintain the glucose level of the subject at about a normal level for at least 10 days after the end of the N different days.

28. The method of claim 27, wherein the N different days are consecutive days.

29. A method of treating type 2 diabetes in a subject in need of treatment, the subject having a body weight, the method comprising: administering to the subject a first, second, third, fourth, and fifth daily dose of IGF-2 or a variant thereof on each day, wherein each of the daily doses comprises at least 244 μg of IGF-2 or a variant thereof per kilogram of body weight.

30. The method of claim 29, wherein each of the daily doses comprises at least 976 μg IGF-2 or variant thereof per kilogram body weight.

31. The method of claim 29, wherein the subject is treated with IGF-2 or a variant thereof for a course of at least 35 days, and wherein the concentration of glucose in the subject's blood stream measured after 14 hours of fasting does not exceed 200mg/dl measured after 35 days of course and after 14 hours of fasting.

32. A method of preventing the onset of type 1 diabetes in a subject, the subject having a body weight, the method comprising: administering to the subject a first, second, third, fourth, and fifth daily dose of IGF-2 or a variant thereof on each day, wherein each of the daily doses comprises at least 65 μg of IGF-2 or a variant thereof per kilogram of body weight.

33. The method of claim 32, wherein each of the daily doses comprises at least 976 μg IGF-2 or variant thereof per kilogram body weight.

34. The method of claim 32, wherein the subject is treated with IGF-2 or a variant thereof, and wherein the concentration of glucose in the subject's blood measured at the fifth daily dose and after the at least 180 minutes is less than 300mg/dl for at least 180 minutes after the subject receives a glucose dose of 2 grams/kg of the subject's body weight.

35. A method of increasing insulin levels in the blood stream of a subject having diabetes, the subject having a body weight, the method comprising: administering to the subject a first, second, third, fourth, and fifth daily dose of IGF-2 or a variant thereof on each day, wherein each of the daily doses comprises at least 65 μg of IGF-2 or a variant thereof per kilogram of body weight.

36. The method of claim 35, wherein the concentration of insulin in the subject's blood stream is increased by at least 50% as compared to the initial concentration of insulin in the subject's blood stream measured prior to administration of IGF-2 or a variant thereof to the subject.

37. The method of claim 35, wherein each of the daily doses comprises at least 244 μg IGF-2 or variant thereof per kilogram body weight.

38. The method of claim 35, wherein each of the daily doses comprises at least 976 μg IGF-2 or variant thereof per kilogram body weight.

39. A method of increasing the number of functional β cells in a subject having diabetes, the subject having a body weight, the method comprising: administering to the subject a first, second, third, fourth, and fifth daily dose of IGF-2 or a variant thereof on each day, wherein each of the daily doses comprises at least 65 μg of IGF-2 or a variant thereof per kilogram of body weight.

40. The method of claim 39, wherein the number of functional beta cells in the subject is increased by at least four times after administering IGF-2 or a variant thereof to the subject for at least 70 days as compared to the initial number of functional beta cells in the subject measured prior to administering IGF-2 or a variant thereof to the subject.

41. The method of claim 39, wherein each of the daily doses comprises at least 244 μg of IGF-2 or a variant thereof per kilogram of body weight.

42. The method of claim 39, wherein each of the daily doses comprises at least 976 μg of IGF-2 or a variant thereof per kilogram of body weight.

43. A method of preventing the onset of type 2 diabetes in a subject, the subject having a body weight, the method comprising: administering to the subject a first, second, third, fourth, and fifth daily dose of IGF-2 or a variant thereof on each day, wherein each of the daily doses comprises at least 65 μg of IGF-2 or a variant thereof per kilogram of body weight.