WO2008156701A2

WO2008156701A2 - Hydroxamate inhibitors of insulin-degrading enzyme and uses thereof

Info

Publication number: WO2008156701A2
Application number: PCT/US2008/007447
Authority: WO
Inventors: Malcolm A. Leissring; Dennis Selkoe; Gregory D. Cuny; Sungwoon Choi; Ross L. Stein; Benjamin Turk; Lewis C. Cantley
Original assignee: The Brigham And Women's Hospital, Inc.; President And Fellows Of Harward College; Yale University
Priority date: 2007-06-13
Filing date: 2008-06-13
Publication date: 2008-12-24
Also published as: WO2008156701A3

Abstract

The present invention provides a novel therapeutic approach for the prevention and treatment of infections and for the treatment of diabetes wherein the degradation of insulin is reduced through inhibition of insulin-degrading enzyme (IDE) by hydroxamate inhibitors.

Description

HYDROXAMATE INHIBITORS OF INSULIN-DEGRADING ENZYME

AND USES THEREOF

FIELD OF THE INVENTION

The present invention relates to therapeutic approaches to the treatment of diabetes, the transport of molecules into cells and across cell layers, and prevention of varicella zoster virus infection and cell-to-cell spread.

BACKGROUND OF THE INVENTION

Diabetes is a highly debilitating and increasingly common disorder that is typically associated with impaired insulin signaling. A few types of diabetes are recognized including: Type 1, in which insulin production is impaired due to loss of pancreatic beta cells; Type 2, in which cellular sensitivity to circulating insulin is impaired; Hyperosmolar Hyperglycemic Nonketotic Syndrome; gestational diabetes; prediabetes. Clinical treatment of diabetes generally consists of elevating insulin signaling, either by intravenous injection of insulin or through administration of therapeutic compounds that increase insulin secretion or affect intracellular signaling pathways responsive to insulin. Current approved anti-diabetic therapeutic compounds are members of few different classes of drugs, including: insulin and insulin analogs, sulfonylureas, meglitinides, biguanides, thiazolidinediones, alpha-glucosidase inhibitors, DP rv inhibitors, and PPAR-g modulators. Some of these drugs have undesirable side- effects including hypoglycemia, diarrhea, and in some instances liver damage. Therefore, there is a need in the art for novel approaches towards treating and managing this disease.

SUMMARY OF THE INVENTION hi one aspect, the present invention provides a novel therapeutic strategy to the treatment of diabetes wherein the degradation of insulin is reduced through inhibition of insulin-degrading enzyme (IDE), hi one aspect, the invention provides a novel therapeutic strategy for delivering insulin or other therapeutic molecules into organisms and/or into intraorganismal or intracellular compartments by inhibiting IDE's ability to degrade such molecules.

In one aspect, the invention provides a novel strategy for facilitating the transport of pathogenic substances out of intraorganismal or intracellular compartments, where the accumulation of such substances is pathogenic, by inhibiting IDE's ability to degrade such molecules.

The ability of insulin, related or unrelated peptides, or other molecules to gain entry into cells and/or to be transported across cell layers such as epithelial cells or the blood-brain-barrier is normally limited by the degradation of such molecules by enzymes secreted from cells, on the surface of cells or within cells. Methods that inhibit these enzymes and thereby prevent the degradation of therapeutic molecules would facilitate their transport into organisms and/or into intraorganismal or intracellular compartments relevant to their therapeutic action. Conversely, methods that inhibit these enzymes could facilitate the transport of pathogenic molecules away from intraorganismal or intracellular compartments that are vulnerable to their pathogenic effects. hi one aspect, the invention provides hydroxamate-based compounds that are useful for inhibiting the activity of IDE. hi another aspect, the invention provides methods of treating or preventing diabetes comprising administering to a diabetic subject a compound that inhibits insulin- degrading enzyme (IDE).

In another aspect, the invention provides methods for treating a diabetic subject by administering a composition comprising an IDE inhibitor in a therapeutically effective amount. In some embodiments, the composition includes one or more IDE inhibitors disclosed herein, or one or more stereoisomeric forms, or pharmaceutically acceptable hydrates, or pharmaceutically acceptable acid or base addition salt forms thereof.

In another aspect, the invention provides methods for treating a diabetic subject by administering both an IDE inhibitor and a second therapeutic compound in therapeutically effective amounts. The two compounds can be administered as a combination composition comprising both compounds. Alternatively, the two compounds can be administered separately (e.g., as two different compositions) either simultaneously or sequentially as described herein. In some embodiments, the IDE inhibitor composition includes two or more IDE inhibitor compounds disclosed herein, or one or more stereoisomer^ forms or pharmaceutically acceptable acid or base addition salts thereof. In some embodiments, the second therapeutic composition includes one or more sulfonylureas and/or one or more meglinitides and/or one or more biguanides and/or one or more thiazolidinediones and/or one or more alpha-glucosidase inhibitors and/or one or more insulins. In other embodiments, the second therapeutic composition includes one or more other compounds that are useful to treat a diabetic subject. In other embodiments, the second therapeutic composition includes human or non-human insulin or analogs or derivatives thereof, including insulin mimicking compounds.

In one aspect, the invention provides IDE inhibitors developed through substrate- based rational design.

In another aspect, the invention provides a novel therapeutic strategy for treating chicken pox and/or shingles wherein varicella zoster virus (VZV) infection and/or cell- to-cell spread is prevented by inhibitors or other molecules that bind to IDE, which the virus uses to gain entry into cells.

Varicella zoster virus (VZV) causes chicken pox in young individuals and shingles in older individuals. Methods that can block entry of the virus into cells or its spread from infected cells to uninfected ones would be useful therapeutically.

In one aspect, the invention provides hydroxamate-based compounds that bind to IDE and thereby block the entry of VZV into cells or its transport between cells.

Accordingly, compositions of the invention may be administered in a therapeutically effective amount to treat patients infected with, or at risk of infection by, VZV or other virus that requires or uses IDE to enter a cell during a cycle of infection.

In one aspect, the invention provides peptide hydroxamate inhibitors of IDE that can be used in any of the methods described herein.

In one embodiment, the peptide hydroxamate inhibitors are conventional peptides with the formula:

wherein R₁, R₂ , R₃, R₄, R₅ and R₆ are independently selected and wherein

Ri is H, OH, O-alkyl or alkyl,

R₂ is aryl, heteroaryl, Ph, 1-naphthyl, 2-naphthyl, substituted Ph, substituted 1- naphthyl, or substituted 2-naphthyl,

R₃ is NHC(=NH)NH₂, NH₂, NHC(O)alkyl, or NHC(O)aryl,

R₄ is NH₂ or [NR₅CH(CH₂)_OR₆CC=O)]_PNH₂,

R₅ is H or Me,

R₆ is 4-hydroxyphenyl, CO₂H, indol-3-yl, or phenyl, m is 0 - 3, n is 0 - 3, o is 0 - 3, and p is 0 - 2.

In one embodiment of the invention

R₂ is 2-naphthyl, and

R₃ is NHCC=NH)NH₂.

In one embodiment of the invention R₂ is aryl, heteroaryl, substituted aryl, substituted heteroaryl, halo-substituted aryl such as described herein, for example 2- fluorophenyl, 3 -fluorophenyl, 4- fluorophenyl, halo-substituted heteroaryl such as described herein, alkyl or aryl-substituted aryl, such as described herein, alkyl or aryl- substituted heteroaryl as described herein, for example 2-tert-butyl, 3-tert-buty\, 4-tert- butyl, 4-(tert-butyl)-phenyl, 1-naphthyl, 2-naphthyl, 2-benzothiophene, -trans-CH=CH- phenyl, 2-phenyl, 3-phenyl, or 4-phenyl. hi one embodiment R₂ can be nitrile, CN, methoxy, CF₃, OCF₃, methyl, or CO₂H. hi one embodiment the 1-naphthyl, 2-naphthyl, and 2-benzothiophene are substituted with a halogen, nitrile, CN, methoxy, CF₃, OCF₃, methyl, or CO₂H.

In a certain embodiment R₁, R₂ , R₃, R₄, R₅ and R₆ are independently selected and any and every independent combination is contemplated.

In one embodiment the peptide hydroxamate inhibitors are retro-inverso peptides with the formula:

wherein R₁, R₂ , R₃, R₄, R₅ and R$ are independently selected and wherein

R₁ is H, OH, O-alkyl or alkyl,

R₂ is aryl, heteroaryl, Ph, 1-naphthyl, 2-naphthyl, substituted Ph, substituted 1- naphthyl, substituted 2-naphthyl, substituted aryl, or substituted heteroaryl,

R₃ is NHC(=NH)NH₂, NH₂, NHC(O)alkyl, or NHC(O)aryl,

R₄ is [C(=O)CH(CH₂)_oR₇NH]_pH or [C(=O)CH(CH₂)_oR₇NH]_pC(=O)Me,

R₅ is H or Me,

R₆ is H or Me,

R₇ is 4-hydroxyphenyl, CO₂H, indol-3-yl, or phenyl, m is 0 - 3, n is 0 - 3, o is 0 - 3, p is 0 - 2.

In one embodiment of the invention

R₂ is 2-naphthyl, and

R₃ is NHC(=NH)NH₂.

In one embodiment of the invention R₂ is aryl, heteroaryl, substituted aryl, substituted heteroaryl, halo-substituted aryl such as described herein, for example 2- fluorophenyl, 3-fluorophenyl, 4-fluorophenyl, halo-substituted heteroaryl such as described herein, alkyl or aryl-substituted aryl, such as described herein, alkyl or aryl- substituted heteroaryl as described herein, for example 2-tert-butyl, 3-tert-butyl, A-tert- butyl, 4-(tert-butyl)-phenyl, 1-naphthyl, 2-naphthyl, 2-benzothiophene, -trøw.s-CH=CH- phenyl, 2-phenyl, 3 -phenyl, or 4-phenyl. In one embodiment R₂ can be nitrile, CN, methoxy, CF₃, OCF₃, methyl, or CO₂H. In one embodiment the 1-naphthyl, 2-naphthyl, and 2-benzothiophene are substituted with a halogen, nitrile, CN, methoxy, CF₃, OCF₃, methyl, or CO₂H. In a certain embodiment R₁, R₂ , R₃, R₄, R₅ and R₆ are independently selected and any and every independent combination is contemplated.

In one aspect, the invention provides methods for rational design and optimization of peptide hydroxamate IDE inhibitors. hi one aspect of the invention IDE inhibitors are provided that increase secretion of insulin from a pancreatic beta cell line. hi one aspect of the invention IDE inhibitors are provided that potentiate the activity of insulin within cells. hi one aspect of the invention IDE inhibitors are provided that regulate the trafficking of insulin to the nucleus. hi one aspect of the invention IDE inhibitors are provided that induce transport of insulin across epithelial cell layers. hi one aspect of the invention IDE inhibitors are provided that inhibit binding of a varicella zoster virus (VZV) glycoprotein to IDE. hi one aspect of the invention methods are provided for analysis of IDE's cleavage-site specificity. hi one aspect of the invention methods are provided for the synthesis of conventional peptide hydroxamate IDE inhibitors. hi one aspect of the invention methods are provided for the synthesis of retro- inverso peptide hydroxamate IDE inhibitors.

Compounds of the invention can be prepared in different forms including but not limited to acid or base addition salts, hydrates, therapeutic compositions, formulations with one or more additional compounds such as other therapeutic compounds, stereoisomeric forms, single isomers, enantiomerically pure preparations, racemic mixtures, precipitates, crystals, single polymorphs, mixtures of polymorphs, and other forms that are useful (e.g. for storage such as long term storage, or for therapeutic administration). Therapeutic preparations of the invention are preferably sterile.

As described herein, compositions, preparations, formulations, and/or compounds of the invention can be administered as a single dose or in multiple doses administered at hourly, daily, weekly, monthly intervals, or at other shorter, longer, or intermediate intervals.

BRIEF DESCRIPTIONS OF THE DRAWINGS FIG. 1 shows (A) a schematic that illustrates the cleavage-site specificity of IDE and (B) examples of peptide hydroxamates derived therefrom and associated IC₅₀ values against IDE.

FIG. 2 shows conventional hydroxamic acids incorporating improvements at the Pl ' position and associated IC_5O values against IDE.

FIG. 3 shows a graph depicting the relative IC_5O values of various peptide hydroxamates against DDE vis-a-vis the canonical metalloprotease, neprilysin (NEP).

FIG. 4 illustrates examples of biological effects of peptide hydroxamate IDE inhibitors in cells.

FIG. 5 shows a graph depicting data from experiments showing that IDE inhibitors facilitate the transcellular transport of peptides across MDCK cell monolayers.

FIG. 6 shows a Western blot of IDE showing that IDE inhibitors disrupt the interaction between varicella zoster virus glycoprotein E and IDE.

DETAILED DESCRIPTION

In one aspect, the invention provides compounds, compositions and methods for treating diabetic subjects. Compounds and compositions of the invention are useful to inhibit the activity of insulin degrading enzyme (IDE), or to block the binding of VZV to it. IDE inhibitor compounds and compositions of the invention are particularly useful to prevent and/or manage diabetes and symptoms associated with diabetes. The invention provides methods for treating a diabetic subject, including the step of administering to the diabetic subject a therapeutically effective amount of a compound or therapeutic preparation. In preferred embodiments, the diabetic subject is a human diabetic subject.

However, aspects of the invention may be used to treat other conditions associated with IDE activity.

According to the invention, IDE inhibitors may be useful for a very broad range of experimental and therapeutic applications. On the experimental side, IDE inhibitors, by virtue of their rapid onset of action, should permit the resolution of the discrepancy between the effects of acute inhibition of IDE activity, which has been shown to potentiate insulin action in vivo, and chronic impairments in IDE activity, which are associated with diabetic phenotypes in animal models. The development of cell- permeant and -impermeant inhibitors will also facilitate the resolution of the relative contribution of intracellular vs. extracellular pools of IDE to the action of insulin, in addition to that of Aβ, glucagon and other substrates.

On the therapeutic side, IDE inhibitors have significant value in a wide range of applications. Bacitracin, a widely used cyclic peptide antibiotic that is also an IDE inhibitor at very high (~1 mg/mL) concentrations, is present in topical preparations that are known to accelerate wound healing. Because insulin itself has been shown to accelerate wound healing, and because DDE is present in wound fluid, it seems reasonable that the beneficial properties of bacitracin may relate to its ability to inhibit IDE. The inhibitors that have been now developed are -1,000,000 times more potent than bacitracin. Accordingly, IDE inhibitors of the invention may be used to accelerate wound healing, hi some embodiments, IDE inhibitors of the invention may be used to further understand the process of wound healing and investigate the role of insulin and IDE in wound healing.

Studies incorporating the very poor and non-selective IDE inhibitors currently available have shown that inhibition of IDE can facilitate the transport of insulin across epithelial cells. Accordingly, IDE inhibitors of the invention may be used to assist in the delivery of insulin (e.g., to enhance the transport of insulin across epithelial cells. In some embodiments, more potent and selective IDE inhibitor compounds may be used to further understand the role of IDE in the transport of insulin across epithelial cells and may be used for the development of novel methods of delivering insulin.

Given that IDE is not inhibited by EDTA except after prolonged incubation, there is a distinct possibility that current methods of blood collection may be underestimating the true levels of insulin, glucagon or other IDE substrates. Accordingly, IDE inhibitors of the invention may be used as an adjuvant for numerous applications, for example within the practice of phlebotomy. In some embodiments, IDE inhibitors of the invention may be used to investigate further the role of IDE in sample degradation (e.g., the degradation of insulin etc. in a sample).

Furthermore, IDE was recently shown to be the cellular receptor for varicella zoster virus (VZV; Li et al., Cell, 2006). Given that bacitracin inhibits cell-to-cell spread, aspects of the invention may involve using IDE inhibitors described herein for treating chicken pox and/or shingles.

Finally, the finding that pharmacological inhibition of IDE potentiates both insulin secretion from pancreatic beta cells and also insulin action within cells suggests that orally bioavailable inhibitors of IDE could have therapeutic potential for the treatment of diabetes. Accordingly, aspects of the invention relate to oral administration of one or more IDE inhibitors described herein. However, IDE inhibitors may be administered via any other suitable route as described herein.

In one aspect the invention provides IDE inhibitors discovered through rational design, and variants thereof.

In one aspect the invention provides peptide hydroxamate inhibitors of IDE.

In one embodiment the peptide hydroxamate inhibitors are conventional peptides with the formula:

R₁ is H, OH, O-alkyl or alkyl,

R₃ is NHC(=NH)NH₂, NH₂, NHC(O)alkyl, or NHC(O)aryl,

R₄ is NH₂ or [NR₅CH(CH₂)_oR₆C(=O)]_pNH₂,

R₅ is H or Me,

In certain embodiments, m may be 0, 1, 2, or 3; n may be 0, 1, 2, or 3; o may be 0, 1, 2, or 3; and p may be 0, 1, or 2. It should be appreciated that the values of m, n, o, and p, may be independent of each other.

In one embodiment of the invention,

R₂ is 2-naphthyl, and R₃ is NHC(=NH)NH₂.

In one embodiment of the invention, R₂ is aryl, heteroaryl, substituted aryl, substituted heteroaryl, halo-substituted aryl such as described herein, for example 2- fluorophenyl, 3 -fluorophenyl, 4-fluorophenyl, halo-substituted heteroaryl such as described herein, alkyl or aryl-substituted aryl, such as described herein, alkyl or aryl- substituted heteroaryl as described herein, for example 2-tert-butyl, 3-tert-butyl, A-tert- butyl, 4-(tert-butyl)-phenyl, 1-naphthyl, 2-naphthyl, 2-benzothiophene, -trans-CH=CH- phenyl, 2-phenyl, 3-phenyl, or 4-phenyl. In one embodiment R₂ can be nitrile, CN, methoxy, CF₃, OCF₃, methyl, or CO₂H. In some embodiments, the 1-naphthyl, 2- naphthyl, and 2-benzothiophene independently may be substituted with a halogen, nitrile, CN, methoxy, CF₃, OCF₃, methyl, or CO₂H.

wherein R₁, R₂ , R₃, R₄, R₅ and R₆ are independently selected and wherein Ri is H, OH, O-alkyl or alkyl,

R₃ is NHC(=NH)NH₂, NH₂, NHC(O)alkyl, or NHC(O)aryl,

R₄ is [C(=O)CH(CH₂)_oR₇NH]_pH or [C(=O)CH(CH₂)_oR₇NH]_pC(=O)Me,

R₅ is H or Me,

R₆ is H or Me

R₇ is 4-hydroxyphenyl, CO₂H, indol-3-yl, or phenyl, m is O - 3, n is 0 - 3, o is 0 - 3, p is O - 2. In certain embodiments, m may be 0, 1, 2, or 3; n may be 0, 1, 2, or 3; o may be 0, 1, 2, or 3; and p may be 0, 1, or 2. It should be appreciated that the values of m, n, o, and p, may be independent of each other.

In one embodiment of the invention,

R₂ is 2-naphthyl, and

R₃ is NHC(=NH)NH₂.

In one embodiment of the invention, R₂ is aryl, heteroaryl, substituted aryl, substituted heteroaryl, halo-substituted aryl such as described herein, for example 2- fluorophenyl, 3 -fluorophenyl, 4-fluorophenyl, halo-substituted heteroaryl such as described herein, alkyl or aryl-substituted aryl, such as described herein, alkyl or aryl- substituted heteroaryl as described herein, for example 2-tert-butyl, 3-tert-butyl, 4-tert- butyl, 4-(tert-butyl)-phenyl, 1-naphthyl, 2-naphthyl, 2-benzothiophene, -trans-CH=CH- phenyl, 2-phenyl, 3 -phenyl, or 4-phenyl. hi one embodiment R₂ can be nitrile, CN, methoxy, CF₃, OCF₃, methyl, or CO₂H. In some embodiments, the 1-naphthyl, 2- naphthyl, and 2-benzothiophene independently may be substituted with a halogen, nitrile, CN, methoxy, CF₃, OCF₃, methyl, or CO₂H. hi one aspect the invention provides methods for rational design and optimization of peptide hydroxamate IDE inhibitors.

In one aspect of the invention IDE inhibitors are provided that increase secretion of insulin from a pancreatic beta cell line. hi one aspect of the invention IDE inhibitors are provided that potentiate the activity of insulin within cells. hi one aspect of the invention IDE inhibitors are provided that regulate the trafficking of insulin to the nucleus. hi one aspect of the invention IDE inhibitors are provided that induce transport of insulin across epithelial cell layers. hi one aspect of the invention IDE inhibitors are provided that inhibit varicella zoster infection.

In one aspect of the invention IDE activity assays are provided. hi one aspect of the invention methods are provided for analysis of IDE's cleavage-site specificity. hi one aspect of the invention methods are provided for the synthesis of conventional peptide hydroxamate IDE inhibitors. In one aspect of the invention methods are provided for the synthesis of retro- inverso peptide hydroxamate IDE inhibitors.

The term "diabetic subject" refers to a subject that is affected by, or at risk of developing, diabetes such as diabetes mellitus and/or any of a group of related disorders in which there is a defect in the regulation of circulating and/or intracellular glucose (sugar) levels. Diabetic subjects include subjects with abnormally high levels of blood sugar (hyperglycemia) or abnormally low levels of blood sugar (hypoglycemia). Diabetic subjects also include subjects with glucose intolerance. Non limiting examples of diabetic conditions are described in the following paragraphs.

Diabetes mellitus ("diabetes") is a highly debilitating and increasingly common disorder that is typically associated with impaired insulin signaling. There are ~18 million people in the United States, or -6.3% of the population, who have diabetes. The major types of diabetes are:

Type 1 diabetes - Results from the body's impairment of insulin production due to loss of pancreatic beta cells. It is estimated that 5-10% of Americans who are diagnosed with diabetes have type 1 diabetes. Type 1 diabetes is usually diagnosed in children and young adults, and was previously known as juvenile diabetes. Conditions associated with type 1 diabetes include hyperglycemia, hypoglycemia, ketoacidosis and celiac disease. Some complications of type 1 diabetes include: heart disease (cardiovascular disease), blindness (retinopathy), nerve damage (neuropathy), and kidney damage (nephropathy).

Type 2 diabetes - Results from insulin resistance (a condition in which the body fails to properly use insulin, i.e., cellular sensitivity to circulating insulin is impaired), combined with relative insulin deficiency. Approximately 90-95% (17 million) of Americans who are diagnosed with diabetes have type 2 diabetes. Type 2 diabetes increases the risk for many serious complications including heart disease (cardiovascular disease), blindness (retinopathy), nerve damage (neuropathy), and kidney damage (nephropathy).

Hyperosmolar Hyperglycemic Nonketotic Syndrome, or HHNS, is a serious condition most frequently seen in older persons. HHNS can happen to people with either type 1 or type 2 diabetes, but it occurs more often in people with type 2. HHNS is usually brought on by something else, such as an illness or infection. In HHNS, blood sugar levels rise (over 600 mg/dl), and the body tries to eliminate the excess sugar by passing it into urine. IfHHNS continues, the severe dehydration will lead to seizures, coma and eventually death. HHNS may take days or even weeks to develop.

Gestational diabetes - Gestational diabetes affects about 4% of all pregnant women - about 135,000 cases in the United States each year. Pregnant women who have never had diabetes before but who have high blood sugar (glucose) levels during pregnancy are said to have gestational diabetes.

Pre-diabetes - Pre-diabetes is a condition that occurs when a subject's blood glucose levels are higher than normal but not high enough for a diagnosis of type 2 diabetes. It is estimated that before subjects develop type 2 diabetes, they almost always have "pre-diabetes" — blood glucose levels that are higher than normal but not yet high enough to be diagnosed as diabetes. At least 20.1 million people in the United States (21.1% of the population ages 40 to 74), have pre-diabetes. Recent research has shown that some long-term damage to the body, especially the heart and circulatory system, may already be occurring during pre-diabetes.

There are tests routinely used by those of ordinary skill in the art to establish if a subject is a "diabetic subject". Two different tests that can be used to determine whether a subject is a "diabetic subject" are: the fasting plasma glucose test (FPG) or the oral glucose tolerance test (OGTT). The blood glucose levels measured after these tests can be used to determine whether a subject has a normal metabolism, or whether a subject is a "diabetic subject," in other words whether a subject has pre-diabetes or diabetes. If the blood glucose level is abnormal following the FPG, the subject has impaired fasting glucose (IFG); if the blood glucose level is abnormal following the OGTT, the subject has impaired glucose tolerance (IGT). In the FPG test, the subject's blood glucose is measured first thing in the morning before eating. In the OGTT, the subject's blood glucose is tested after fasting and again 2 hours after drinking a glucose-rich drink.

Normal fasting blood glucose is below 100 mg/dl. A subject with pre-diabetes has a fasting blood glucose level between 100 and 125 mg/dl. If the fasting blood glucose level rises to 126 mg/dl or above, the subject has diabetes. In the OGTT, the subject's blood glucose is measured after a fast and 2 hours after drinking a glucose-rich beverage. Normal blood glucose is below 140 mg/dl 2 hours after the drink. In prediabetes, the 2-hour blood glucose is 140 to 199 mg/dl. If the 2-hour blood glucose rises to 200 mg/dl or above, the subject has diabetes. The term "diabetic subject" also refers to a subject having ketoacidosis - a serious condition where the body has dangerously high levels of ketones or acids that build up in the blood, and it can lead to diabetic coma (passing out for a long time) or even death.

According to the invention, a subject at risk of developing diabetes or a related disorder is a subject that is predisposed to such the disease or disorder due to genetic or other risk factors. While diabetes and pre-diabetes occur in subjects of all ages and races, some groups have a higher risk for developing the disease than others. Diabetes is more common in African Americans, Latinos, Native Americans, and Asian Americans/Pacific Islanders, as well as the overweight and aged population. Most people diagnosed with type 2 diabetes are overweight. A healthy weight is determined by your body mass index (BMI), which can be calculated based on subjects height and weight. Overweight is defined as a BMI greater than/equal to 25; obesity is defined as a BMI greater than/equal to 30. Overweight and obese subjects are at increased risk for developing pre-diabetes and diabetes. A family history of diabetes is also a risk factor. Age can also be a risk factor. In some embodiments, a subject at risk is identified as a subject having one or more of these risk factors. These and other risk factors can be assessed using risk factor tests known in the art.

According to the invention, the term "treatment" may be prophylaxis and/or therapy. Therapy may include preventing, slowing, stopping, or reversing (e.g. curing) a diabetic disease or disorder or certain symptoms associated with the disease or disorder. Accordingly, treating a diabetic subject may include managing the serum glucose levels of a subject such as a subject with diabetes, pre-diabetes, or at risk for diabetes. In some embodiments, treating a diabetic subject involves preventing or delaying the onset of the disease or disorder or of one or more symptoms associated with the disease or disorder. In some embodiments, treating a diabetic subject involves halting or slowing the progression of the disease or disorder or of one or more symptoms associated with the disease or disorder. In some embodiments, treating a diabetic subject involves preventing, delaying, or slowing the onset or progression of long-term symptoms associated with the disease or disorder, hi one embodiment, compositions of the invention are useful for helping a subject regulate glucose transfer between their bloodstream and certain cells. Treatment also may encompass prophylaxis to prevent or slow the development of diabetes, and/or the onset of certain symptoms associated with diabetes in a subject with ~ or at risk of developing — diabetes, pre-diabetes or a related disorder. For example, in the case of a subject with pre-diabetes or a subject at-risk for diabetes, treatment means decreasing the likelihood that the subject will develop Type 2 diabetes. In important embodiments, diabetes, pre-diabetes, or the risk of developing diabetes is treated by inhibiting the insulin-degrading enzyme, thereby increasing a subject's insulin levels. In another embodiment, compositions of the invention that inhibit insulin degradation are useful to facilitate the administration of therapeutically effective amounts of insulin given orally, transdermally, subdermally, rectally, vaginally or via inhalation into the lungs or nasal passages. In yet another embodiment, compositions of the invention are useful to prolong the action of therapeutically administered insulin.

The phrase "therapeutically-effective amount" as used herein means that amount of a compound, material, or composition comprising a compound of the present invention which is effective for producing some desired therapeutic effect in a subject at a reasonable benefit/risk ratio applicable to any medical treatment. Accordingly, in some embodiments, a therapeutically effective amount prevents or minimizes disease progression associated with diabetes. Disease progression can be monitored relative to the amount of glucose in the bloodstream and other well known tests for diabetes, such as the oral glucose tolerance test or the AlC test. The AlC test measures the percentage of glycated hemoglobin in a subject, hi a person who does not have diabetes, about 5% of all hemoglobin is glycated. For someone with diabetes and high blood glucose levels, the AlC level is higher than normal. How high the AlC level rises depends on the subject's average blood glucose level during the past weeks and months. Levels can range from normal to as high as 25% if diabetes is badly out of control for a long time. Diabetes progression can also be evaluated by monitoring short-term and/or long-term symptoms associated with the disease such as cardiac and vision problems.

In some embodiments, an inhibitor of the invention is provided (e.g., administered) in an amount sufficient to inhibit IDE activity (e.g., by a dectectable amount). For example, IDE activity may be inhibited by about 5%, about 10%, about 25%, about 50%, about 75%, about 80%, about 90%, about 95%, or more. IDE activity may be assayed using any suitable direct assay (e.g., for measuring IDE enzyme activity in vivo or in vitro) or indirect assay (e.g., using a reporter molecule or physiological observation associated with IDE activity as a measure of IDE activity). In some embodiments a "diabetic subject" is a human subject. In other embodiments, a "diabetic subject" can be any animal in need of treatment, including primates and other mammals such as equines, cattle, swine and sheep, and pets in general including dogs and cats.

Compounds and pharmaceutical compositions of the invention can be used in combination with one or more alternative diabetic medications. Alternative diabetic medications include members of six classes of drugs: sulfonylureas, meglitinides, biguanides, thiazolidinediones, alpha-glucosidase inhibitors and insulin and various insulin analogues and mimics. Because these drugs act in different ways to lower blood glucose levels, they can be used together to effectively treat a diabetic subject. For example, a biguanide and a sulfonylurea may be used together with one or more compounds of the invention. Combinations of two or more compounds can be used, hi some embodiments, one or more compounds of the invention can be combined with one or more alternative medications. In some embodiments, two or more compounds of the invention can be used with or without the addition of alternative medication(s).

Sulfonylureas: Sulfonylureas stimulate the beta cells of the pancreas to release more insulin. Sulfonylurea drugs have been in use since the 1950s. Chlorpropamide (brand name Diabinese) is the only first-generation sulfonylurea still in use today. The second generation sulfonylureas are used in smaller doses than the first-generation drugs. There are six second-generation drugs: glipizide (brand names Glucotrol and Glucotrol XL), glyburide (Micronase, Glynase, and Diabeta), acetohexamide (Dymerol), tolbutamide (Orinase), tolazamide (Tolinase) and glimepiride (Amaryl). These drugs are generally taken one to two times a day, before meals. Sulfonylurea drugs generally have similar effects on blood glucose levels, but they differ in side effects, how often they are taken, and interactions with other drugs.

Meglitinides: Meglitinides are drugs that also stimulate the pancreatic beta cells to release insulin. Repaglinide (brand name Prandin) and nateglinide (Starlix) are meglitinides. They are taken before each of three meals. Because sulfonylureas and meglitinides stimulate the release of insulin, it is possible to have hypoglycemia (low blood glucose levels).

Biguanides: Metformin (brand name Glucophage) is a biguanide. Biguanides lower blood glucose levels primarily by decreasing the amount of glucose produced by the liver. Metformin also helps to lower blood glucose levels by making muscle tissue more sensitive to insulin so glucose can be absorbed. It is usually taken two times a day.

Thiazolidinediones: Rosiglitazone (Avandia), troglitazone (Rezulin), and pioglitazone (ACTOS) form a group of drugs called thiazolidinediones. These drugs help insulin work better in the muscle and fat and also reduce glucose production in the liver. Thiazolidinediones are taken once or twice a day with food. Although effective in lowering blood glucose levels, thiazolidinediones can have a rare but serious effect on the liver.

Alpha-glucosidase inhibitors: Acarbose (brand name Precose) and meglitol (Glyset) are alpha-glucosidase inhibitors. These drugs help the body to lower blood glucose levels by blocking the breakdown of starches, such as bread, potatoes, and pasta in the intestine. They also slow the breakdown of some sugars, such as table sugar. Their action slows the rise in blood glucose levels after a meal. They are typically taken at the beginning of a meal. These drugs may have side effects, including gas and diarrhea.

Insulin: In addition to anti-diabetic drugs, compounds of the present invention can be used in combination with insulin or compounds that mimic the action of insulin. Insulin is a hormone produced by the beta cells of the pancreas. With each meal, beta cells release insulin to help the body use or store the blood glucose it gets from food, hi subjects with type 1 diabetes, the pancreas no longer makes insulin. The beta cells have been destroyed and these subjects need insulin shots to use glucose from meals. Subjects with type 2 diabetes make insulin, but their bodies don't respond well to it. Some subjects with type 2 diabetes need diabetes drugs or insulin shots to help their bodies use glucose for energy. Insulin must be injected into the fat under the skin for it to get into the blood.

There are many different insulins useful for different situations and lifestyles. More than 20 types of insulin are sold in the United States. These insulins differ in how they are made, how they work in the body, and price. Insulin can be made in a laboratory to be identical to human insulin. Alternatively, it can be isolated from animals such as pigs. Rapid-acting insulin, such as insulin lispro (Eli Lilly, Humalog) or insulin aspart (Novo Nordisk, Novolog), begin to work about 5 minutes after injection, peak in about 1 hour, and continue to work for 2 to 4 hours. Regular or Short-acting insulin (human) usually reaches the bloodstream within 30 minutes after injection, peaks anywhere from 2 to 3 hours after injection, and is effective for approximately 3 to 6 hours. Intermediate-acting insulin (human) generally reaches the bloodstream about 2 to 4 hours after injection, peaks 4 to 12 hours later and is effective for about 12 to 18 hours. Long-acting insulin (ultralente) reaches the bloodstream 6 to 10 hours after injection and is usually effective for 20 to 24 hours. All insulins come dissolved or suspended in liquids, however, the solutions have different strengths. The most commonly used strength in the United States today is U-100 (100 units of insulin per milliliter). Insulin preparations typically have added ingredients. These prevent bacteria from growing and help maintain a neutral balance between acids and bases. In addition, intermediate and long-acting insulins also contain ingredients that prolong their actions. In some rare cases, additives can bring on an allergic reaction. hi the compounds and compositions of the invention, the term "alkyl" refers to the radical of saturated aliphatic groups, including straight-chain alkyl groups, branched- chain alkyl groups, cycloalkyl (alicyclic) groups, alkyl substituted cycloalkyl groups, and cycloalkyl substituted alkyl groups, hi preferred embodiments, a straight chain or branched chain alkyl has 12 or fewer carbon atoms in its backbone (e.g., Ci-C₁₂ for straight chain, C₃-Cj₂ for branched chain), and more preferably 6 or fewer, and even more preferably 4 or fewer. Likewise, preferred cycloalkyls have from 3-10 carbon atoms in their ring structure, and more preferably have 5, 6 or 7 carbons in the ring structure.

Unless the number of carbons is otherwise specified, "lower alkyl" as used herein means an alkyl group, as defined above, but having from one to ten carbons, more preferably from one to six carbon atoms in its backbone structure, and even more preferably from one to four carbon atoms in its backbone structure. Likewise, "lower alkenyl" and "lower alkynyl" have similar chain lengths. Preferred alkyl groups are lower alkyls. hi preferred embodiments, a substituent designated herein as alkyl is a lower alkyl.

As used herein, the term "halogen" designates -F, -Cl, -Br or -I; the term "sulfhydryl" means -SH; and the term "hydroxyl" means -OH.

The term "methyl" or "Me" refers to the monovalent radical -CH₃, and the term "methoxyl" or "OMe" refers to the monovalent radical -CH₂OH.

The term "aralkyl" or "arylalkyl", as used herein, refers to an alkyl group substituted with an aryl group (e.g., an aromatic or heteroaromatic group). The terms "alkenyl" and "alkynyl" refer to unsaturated aliphatic groups analogous in length and possible substitution to the alkyls described above, but that contain at least one double or triple bond respectively.

The term "aryl" as used herein includes 5-, 6- and 7-membered single-ring aromatic groups that may include from zero to four heteroatoms, for example, benzene or phenyl or "Ph", pyrrole, furan, thiophene, imidazole, oxazole, thiazole, triazole, pyrazole, pyridine, pyrazine, pyridazine and pyrimidine, and the like. Those aryl groups having heteroatoms in the ring structure may also be referred to as "aryl heterocycles" or "heteroaromatics." The aromatic ring can be substituted at one or more ring positions with such substituents as described above, for example, halogen, azide, alkyl, aralkyl, alkenyl, alkynyl, cycloalkyl, hydroxyl, alkoxyl, amino, nitro, sulfhydryl, imino, amido, phosphonate, phosphinate, carbonyl, carboxyl, -C(O)NHOH, silyl, ether, alkylthio, sulfonyl, sulfonamido, ketone, aldehyde, ester, heterocyclyl, aromatic or heteroaromatic moieties, -CF₃, -CN, or the like. The term "aryl" also includes polycyclic ring systems having two or more cyclic rings in which two or more carbons are common to two adjoining rings (the rings are "fused rings") wherein at least one of the rings is aromatic, e.g., the other cyclic rings can be cycloalkyls, cycloalkenyls, cycloalkynyls, aryls and/or heterocyclyls.

The terms "ortho", "meta" and "para" apply to 1,2-, 1,3- and 1,4-disubstituted benzenes, respectively. For example, the names 1 ,2-dimethylbenzene and ortho- dimethylbenzene are synonymous.

The terms "heterocyclyl" or "heterocyclic group" or "heteroaryl" refer to 3- to 10- membered ring structures, more preferably 3- to 7-membered rings, whose ring structures include one to four heteroatoms. Heterocycles can also be polycycles. Heterocyclyl groups include, for example, thiophene, benzothiophene, thianthrene, furan, pyran, isobenzofuran, chromene, xanthene, phenoxathiin, pyrrole, imidazole, pyrazole, isothiazole, isoxazole, pyridine, pyrazine, pyrimidine, pyridazine, indolizine, isoindole, indole, indazole, purine, quinolizine, isoquinoline, quinoline, phthalazine, naphthyridine, quinoxaline, quinazoline, cinnoline, pteridine, carbazole, carboline, phenanthridine, acridine, pyrimidine, phenanthroline, phenazine, phenarsazine, phenothiazine, furazan, phenoxazine, pyrrolidine, oxolane, thiolane, oxazole, piperidine, piperazine, morpholine, lactones, lactams such as azetidinones and pyrrolidinones, sultams, sultones, and the like. The heterocyclic ring can be substituted at one or more positions with such substituents as described above, as for example, halogen, alkyl, aralkyl, alkenyl, alkynyl, cycloalkyl, hydroxyl, amino, nitro, sulfhydryl, imino, amido, phosphonate, phosphinate, carbonyl, carboxyl, -C(O)NHOH, silyl, ether, alkylthio, sulfonyl, ketone, aldehyde, ester, a heterocyclyl, an aromatic or heteroaromatic moiety, -CF₃, -CN, or the like.

As used herein, the definition of each expression, e.g. alkyl, m, n, etc., when it occurs more than once in any structure, is intended to be independent of its definition elsewhere in the same structure.

It will be understood that "substitution" or "substituted with" includes the implicit proviso that such substitution is in accordance with permitted valence of the substituted atom and the substituent, and that the substitution results in a stable compound, e.g., which does not spontaneously undergo transformation such as by rearrangement, cyclization, elimination, etc. Furthermore, it should be appreciated that compounds of the invention (e.g., substituted compounds) may include radicals of the groups described herein where appropriate.

As used herein, the term "substituted" is contemplated to include all permissible substituents of organic compounds. In a broad aspect, the permissible substituents include acyclic and cyclic, branched and unbranched, carbocyclic and heterocyclic, aromatic and nonaromatic substituents of organic compounds. Illustrative substituents include, for example, those described herein above. The permissible substituents can be one or more and the same or different for appropriate organic compounds. For purposes of this invention, the heteroatoms such as nitrogen may have hydrogen substituents and/or any permissible substituents of organic compounds described herein which satisfy the valences of the heteroatoms. This invention is not intended to be limited in any manner by the permissible substituents of organic compounds.

Certain compounds of the present invention may exist in particular geometric or stereoisomeric forms. The present invention contemplates all such compounds, including cis- and trø/w-isomers, R- and S-enantiomers, diastereomers, (D)-isomers, (L)- isomers, the racemic mixtures thereof, and other mixtures thereof, as falling within the scope of the invention. Additional asymmetric carbon atoms may be present in a substituent such as an alkyl group. All such isomers, as well as mixtures thereof, are intended to be included in this invention, hi certain embodiments, the present invention relates to a compound represented by any of the structures outlined herein, wherein the compound is a single stereoisomer. If, for instance, a particular enantiomer of a compound of the present invention is desired, it may be prepared by asymmetric synthesis, or by derivation with a chiral auxiliary, where the resulting diastereomeric mixture is separated and the auxiliary group cleaved to provide the pure desired enantiomers. Alternatively, where the molecule contains a basic functional group, such as amino, or an acidic functional group, such as carboxyl, diastereomeric salts are formed with an appropriate optically- active acid or base, followed by resolution of the diastereomers thus formed by fractional crystallization or chromatographic means well known in the art, and subsequent recovery of the pure enantiomers.

As used herein retro-inverso refers to peptides in which the primary sequence is reversed and D- rather than L-amino acids are used (Chorev and Goodman, Ace. Chem. Res. (1993) 26, 266-273; Trends Biotech (1995) 13: 438-445.)

Contemplated equivalents of the compounds described above include compounds which otherwise correspond thereto, and which have the same general properties thereof (e.g., functioning as anti-diabetic compounds), wherein one or more simple variations of substituents are made which do not adversely affect the efficacy of the compound, hi general, the compounds of the present invention may be prepared by the methods illustrated in the general reaction schemes as, for example, described below, or by modifications thereof, using readily available starting materials, reagents and conventional synthesis procedures. In these reactions, it is also possible to make use of variants, which are in themselves known, but are not mentioned here.

For purposes of this invention, the chemical elements are identified in accordance with the Periodic Table of the Elements, CAS version, Handbook of Chemistry and Physics, 67th Ed., 1986-87, inside cover.

In another aspect, the present invention provides "pharmaceutically acceptable" compositions, which comprise a therapeutically effective amount of one or more of the compounds described herein, formulated together with one or more pharmaceutically acceptable carriers (additives) and/or diluents. As described in detail, the pharmaceutical compositions of the present invention may be specially formulated for administration in solid, liquid or aerosolized form, including those adapted for the following: oral administration, for example, drenches (aqueous or non-aqueous solutions or suspensions), tablets, e.g., those targeted for buccal, sublingual, and systemic absorption, boluses, powders, granules, pastes for application to the tongue; parenteral administration, for example, by subcutaneous, intramuscular, intravenous or epidural injection as, for example, a sterile solution or suspension, or sustained-release formulation; topical application, for example, as a cream, ointment, or a controlled- release patch or spray applied to the skin, lungs, oral cavity, or other mucosal surfaces; intravaginal or intrarectal administration, for example, as a pessary, cream or foam; ocular administration, for example, as a liquid applied to the eye; nasal administration, for example, as a nasal spray; or inhalation into the lungs or nasal cavities, for example, as provided by an inhalation aerosol.

The phrase "pharmaceutically acceptable" is employed herein to refer to those compounds, materials, compositions, and/or dosage forms which are, within the scope of sound medical judgment, suitable for use in contact with the tissues of human beings and animals without excessive toxicity, irritation, allergic response, or other problem or complication, commensurate with a reasonable benefit/risk ratio.

The phrase "pharmaceutically-acceptable carrier" as used herein means a pharmaceutically-acceptable material, composition or vehicle, such as a liquid or solid filler, diluent, excipient, or solvent encapsulating material, involved in carrying or transporting the subject compound from one organ, or portion of the body, to another organ, or portion of the body. Each carrier must be "acceptable" in the sense of being compatible with the other ingredients of the formulation and not injurious to the patient. Some examples of materials which can serve as pharmaceutically-acceptable carriers include: sugars, such as lactose, glucose and sucrose; starches, such as corn starch and potato starch; cellulose, and its derivatives, such as sodium carboxymethyl cellulose, ethyl cellulose and cellulose acetate; powdered tragacanth; malt; gelatin; talc; excipients, such as cocoa butter and suppository waxes; oils, such as peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, corn oil and soybean oil; glycols, such as propylene glycol; polyols, such as glycerin, sorbitol, mannitol and polyethylene glycol; esters, such as ethyl oleate and ethyl laurate; agar; buffering agents, such as magnesium hydroxide and aluminum hydroxide; alginic acid; pyrogen-free water; isotonic saline; Ringer's solution; ethyl alcohol; pH buffered solutions; polyesters, polycarbonates and/or polyanhydrides; and other non-toxic compatible substances employed in pharmaceutical formulations.

As set out herein, certain embodiments of the present compounds may contain a basic functional group, such as amino or alkylamino, and are, thus, capable of forming pharmaceutically-acceptable salts with pharmaceutically-acceptable acids. The term "pharmaceutically-acceptable salts" in this respect refers to the relatively non-toxic, inorganic and organic acid addition salts of compounds of the present invention. These salts can be prepared in situ in the administration vehicle or the dosage form manufacturing process, or by separately reacting a purified compound of the invention in its free base form with a suitable organic or inorganic acid, and isolating the salt thus formed during subsequent purification. Representative salts include the hydrobromide, hydrochloride, sulfate, bisulfate, phosphate, nitrate, acetate, valerate, oleate, palmitate, stearate, laurate, benzoate, lactate, phosphate, tosylate, citrate, maleate, fumarate, succinate, tartrate, napthylate, mesylate, glucoheptonate, lactobionate, and laurylsulphonate salts and the like. (See, for example, Berge et al. (1977) "Pharmaceutical Salts", J. Pharm. Sci. 66:1-19)

The pharmaceutically acceptable salts of the subject compounds include the conventional nontoxic salts or quaternary ammonium salts of the compounds, e.g., from non-toxic organic or inorganic acids. For example, such conventional nontoxic salts include those derived from inorganic acids such as hydrochloride, hydrobromic, sulfuric, sulfamic, phosphoric, nitric, and the like; and the salts prepared from organic acids such as acetic, propionic, succinic, glycolic, stearic, lactic, malic, tartaric, citric, ascorbic, palmitic, maleic, hydroxymaleic, phenylacetic, glutamic, benzoic, salicyclic, sulfanilic, 2-acetoxybenzoic, fumaric, toluenesulfonic, methanesulfonic, ethane disulfonic, oxalic, isothionic, and the like.

In other cases, the compounds of the present invention may contain one or more acidic functional groups and, thus, are capable of forming pharmaceutically-acceptable salts with pharmaceutically-acceptable bases. The term "pharmaceutically-acceptable salts" in these instances refers to the relatively non-toxic, inorganic and organic base addition salts of compounds of the present invention. These salts can likewise be prepared in situ in the administration vehicle or the dosage form manufacturing process, or by separately reacting the purified compound in its free acid form with a suitable base, such as the hydroxide, carbonate or bicarbonate of a pharmaceutically-acceptable metal cation, with ammonia, or with a pharmaceutically-acceptable organic primary, secondary or tertiary amine. Representative alkali or alkaline earth salts include the lithium, sodium, potassium, calcium, magnesium, and aluminum salts and the like. Representative organic amines useful for the formation of base addition salts include ethylamine, diethylamine, ethylenediamine, ethanolamine, diethanolamine, piperazine and the like. (See, for example, Berge et al., supra).

Wetting agents, emulsifiers and lubricants, such as sodium lauryl sulfate and magnesium stearate, as well as coloring agents, release agents, coating agents, sweetening, flavoring and perfuming agents, preservatives and antioxidants can also be present in the compositions.

Examples of pharmaceutically- acceptable antioxidants include: water soluble antioxidants, such as ascorbic acid, cysteine hydrochloride, sodium bisulfate, sodium metabisulfite, sodium sulfite and the like; oil-soluble antioxidants, such as ascorbyl palmitate, butylated hydroxyanisole (BHA), butylated hydroxytoluene (BHT), lecithin, propyl gallate, alpha-tocopherol, and the like; and metal chelating agents, such as citric acid, ethylenediamine tetraacetic acid (EDTA), sorbitol, tartaric acid, phosphoric acid, and the like.

Formulations of the present invention include those suitable for oral, nasal, bronchial, topical (including buccal and sublingual), rectal, vaginal and/or parenteral administration. The formulations may conveniently be presented in unit dosage form and may be prepared by any methods well known in the art of pharmacy. The amount of active ingredient which can be combined with a carrier material to produce a single dosage form will vary depending upon the host being treated, and the particular mode of administration. The amount of active ingredient that can be combined with a carrier material to produce a single dosage form will generally be that amount of the compound which produces a therapeutic effect. Generally, this amount will range from about 1% to about 99% of active ingredient, preferably from about 5% to about 70%, most preferably from about 10% to about 30%. hi certain embodiments, a formulation of the present invention comprises an excipient selected from the group consisting of cyclodextrins, liposomes, micelle forming agents, e.g., bile acids, and polymeric carriers, e.g., polyesters and polyanhydrides; and a compound of the present invention. In certain embodiments, an aforementioned formulation renders orally bioavailable a compound of the present invention.

Methods of preparing these formulations or compositions include the step of bringing into association a compound of the present invention with the carrier and, optionally, one or more accessory ingredients, hi general, the formulations are prepared by uniformly and intimately bringing into association a compound of the present invention with liquid carriers, or finely divided solid carriers, or both, and then, if necessary, shaping the product.

Formulations of the invention suitable for oral administration may be in the form of capsules, cachets, pills, tablets, lozenges (using a flavored basis, usually sucrose and acacia or tragacanth), powders, granules, or as a solution or a suspension in an aqueous or non-aqueous liquid, or as an oil-in-water or water-in-oil liquid emulsion, or as an elixir or syrup, or as pastilles (using an inert base, such as gelatin and glycerin, or sucrose and acacia) and/or as mouth washes and the like, each containing a predetermined amount of a compound of the present invention as an active ingredient. A compound of the present invention may also be administered as a bolus, electuary or paste.

In solid dosage forms of the invention for oral administration (capsules, tablets, pills, dragees, powders, granules and the like), the active ingredient is mixed with one or more pharmaceutically-acceptable carriers, such as sodium citrate or dicalcium phosphate, and/or any of the following: fillers or extenders, such as starches, lactose, sucrose, glucose, mannitol, and/or silicic acid; binders, such as, for example, carboxymethylcellulose, alginates, gelatin, polyvinyl pyrrolidone, sucrose and/or acacia; humectants, such as glycerol; disintegrating agents, such as agar-agar, calcium carbonate, potato or tapioca starch, alginic acid, certain silicates, and sodium carbonate; solution retarding agents, such as paraffin; absorption accelerators, such as quaternary ammonium compounds; wetting agents, such as, for example, cetyl alcohol, glycerol monostearate, and non-ionic surfactants; absorbents, such as kaolin and bentonite clay; lubricants, such as talc, calcium stearate, magnesium stearate, solid polyethylene glycols, sodium lauryl sulfate, and mixtures thereof; and coloring agents. In the case of capsules, tablets and pills, the pharmaceutical compositions may also comprise buffering agents. Solid compositions of a similar type may also be employed as fillers in soft and hard-shelled gelatin capsules using such excipients as lactose or milk sugars, as well as high molecular weight polyethylene glycols and the like.

A tablet may be made by compression or molding, optionally with one or more accessory ingredients. Compressed tablets may be prepared using binder (for example, gelatin or hydroxypropylmethyl cellulose), lubricant, inert diluent, preservative, disintegrant (for example, sodium starch glycolate or cross-linked sodium carboxymethyl cellulose), surface-active or dispersing agent. Molded tablets may be made in a suitable machine in which a mixture of the powdered compound is moistened with an inert liquid diluent.

The tablets, and other solid dosage forms of the pharmaceutical compositions of the present invention, such as dragees, capsules, pills and granules, may optionally be scored or prepared with coatings and shells, such as enteric coatings and other coatings well known in the pharmaceutical-formulating art. They may also be formulated so as to provide slow or controlled release of the active ingredient therein using, for example, hydroxypropylmethyl cellulose in varying proportions to provide the desired release profile, other polymer matrices, liposomes and/or microspheres. They may be formulated for rapid release, e.g., freeze-dried. They may be sterilized by, for example, filtration through a bacteria-retaining filter, or by incorporating sterilizing agents in the form of sterile solid compositions that can be dissolved in sterile water, or some other sterile injectable medium immediately before use. These compositions may also optionally contain opacifying agents and may be of a composition such that they release the active ingredient(s) only, or preferentially, in a certain portion of the gastrointestinal tract, optionally, in a delayed manner. Examples of embedding compositions that can be used include polymeric substances and waxes. The active ingredient can also be in micro-encapsulated form, if appropriate, with one or more of the above-described excipients.

Liquid dosage forms for oral administration of the compounds of the invention include pharmaceutically acceptable emulsions, microemulsions, solutions, suspensions, syrups and elixirs. In addition to the active ingredient, the liquid dosage forms may contain inert diluents commonly used in the art, such as, for example, water or other solvents, solubilizing agents and emulsifϊers, such as ethyl alcohol, isopropyl alcohol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propylene glycol, 1,3- butylene glycol, oils (in particular, cottonseed, groundnut, corn, germ, olive, castor and sesame oils), glycerol, tetrahydrofuryl alcohol, polyethylene glycols and fatty acid esters of sorbitan, and mixtures thereof.

Besides inert diluents, the oral compositions can also include adjuvants such as wetting agents, emulsifying and suspending agents, sweetening, flavoring, coloring, perfuming and preservative agents. Suspensions, in addition to the active compounds, may contain suspending agents as, for example, ethoxylated isostearyl alcohols, polyoxyethylene sorbitol and sorbitan esters, microcrystalline cellulose, aluminum metahydroxide, bentonite, agar-agar and tragacanth, and mixtures thereof.

Formulations of the pharmaceutical compositions of the invention for rectal or vaginal administration may be presented as a suppository, which may be prepared by mixing one or more compounds of the invention with one or more suitable nonirritating excipients or carriers comprising, for example, cocoa butter, polyethylene glycol, a suppository wax or a salicylate, and which is solid at room temperature, but liquid at body temperature and, therefore, will melt in the rectum or vaginal cavity and release the active compound.

Formulations of the present invention which are suitable for vaginal administration also include pessaries, tampons, creams, gels, pastes, foams or spray formulations containing such carriers as are known in the art to be appropriate.

Dosage forms for the topical or transdermal administration of a compound of this invention include powders, sprays, ointments, pastes, creams, lotions, gels, solutions, patches and inhalants. The active compound may be mixed under sterile conditions with a pharmaceutically-acceptable carrier, and with any preservatives, buffers, or propellants which may be required.

The ointments, pastes, creams and gels may contain, in addition to an active compound of this invention, excipients, such as animal and vegetable fats, oils, waxes, paraffins, starch, tragacanth, cellulose derivatives, polyethylene glycols, silicones, bentonites, silicic acid, talc and zinc oxide, or mixtures thereof.

Powders and sprays can contain, in addition to a compound of this invention, excipients such as lactose, talc, silicic acid, aluminum hydroxide, calcium silicates and polyamide powder, or mixtures of these substances. Sprays can additionally contain customary propellants, such as chlorofluorohydrocarbons and volatile unsubstituted hydrocarbons, such as butane and propane.

Transdermal patches have the added advantage of providing controlled delivery of a compound of the present invention to the body. Dissolving or dispersing the compound in the proper medium can make such dosage forms. Absorption enhancers can also be used to increase the flux of the compound across the skin. Either providing a rate controlling membrane or dispersing the compound in a polymer matrix or gel can control the rate of such flux.

Ophthalmic formulations, eye ointments, powders, solutions and the like, are also contemplated as being within the scope of this invention.

Pharmaceutical compositions of this invention suitable for parenteral administration comprise one or more compounds of the invention in combination with one or more pharmaceutically-acceptable sterile isotonic aqueous or nonaqueous solutions, dispersions, suspensions or emulsions, or sterile powders which may be reconstituted into sterile injectable solutions or dispersions just prior to use, which may contain sugars, alcohols, antioxidants, buffers, bacteriostats, solutes which render the formulation isotonic with the blood of the intended recipient or suspending or thickening agents.

Examples of suitable aqueous and nonaqueous carriers, which may be employed in the pharmaceutical compositions of the invention include water, ethanol, polyols (such as glycerol, propylene glycol, polyethylene glycol, and the like), and suitable mixtures thereof, vegetable oils, such as olive oil, and injectable organic esters, such as ethyl oleate. Proper fluidity can be maintained, for example, by the use of coating materials, such as lecithin, by the maintenance of the required particle size in the case of dispersions, and by the use of surfactants.

These compositions may also contain adjuvants such as preservatives, wetting agents, emulsifying agents and dispersing agents. Prevention of the action of microorganisms upon the subject compounds may be ensured by the inclusion of various antibacterial and antifungal agents, for example, paraben, chlorobutanol, phenol sorbic acid, and the like. It may also be desirable to include isotonic agents, such as sugars, sodium chloride, and the like into the compositions. In addition, prolonged absorption of the injectable pharmaceutical form may be brought about by the inclusion of agents which delay absorption such as aluminum monostearate and gelatin.

In some cases, in order to prolong the effect of a drug, it is desirable to slow the absorption of the drug from subcutaneous or intramuscular injection. This may be accomplished by the use of a liquid suspension of crystalline or amorphous material having poor water solubility. The rate of absorption of the drug then depends upon its rate of dissolution, which in turn, may depend upon crystal size and crystalline form. Alternatively, delayed absorption of a parenterally-administered drug form is accomplished by dissolving or suspending the drug in an oil vehicle.

Injectable depot forms are made by forming microencapsule matrices of the subject compounds in biodegradable polymers such as polylactide-polyglycolide. Depending on the ratio of drug to polymer, and the nature of the particular polymer employed, the rate of drug release can be controlled. Examples of other biodegradable polymers include poly(orthoesters) and poly(anhydrides). Depot injectable formulations are also prepared by entrapping the drug in liposomes or microemulsions, which are compatible with body tissue.

In certain embodiments, a compound or pharmaceutical preparation is administered orally. In other embodiments, the compound or pharmaceutical preparation is administered intravenously. Alternative routes of administration include sublingual, intramuscular, transdermal, intravenous, nasal, bronchial, rectal and vaginal administrations.

When the compounds of the present invention are administered as pharmaceuticals, to humans and animals, they can be given per se or as a pharmaceutical composition containing, for example, 0.1% to 99.5% (more preferably, 0.5% to 90%) of active ingredient in combination with a pharmaceutically acceptable carrier.

The preparations of the present invention may be given orally, parenterally, topically, rectally, vaginally, or via inhalation into the lungs or nasal cavities. They are of course given in forms suitable for each administration route, and may be combined with formulations containing one or more insulins or other second therapeutic compounds. For example, they are administered in tablets or capsule form, by injection, inhalation, eye lotion, ointment, suppository, etc. administration by injection, infusion or inhalation; topical by lotion or ointment; and rectal by suppositories. Oral administrations are preferred.

The phrases "parenteral administration" and "administered parenterally" as used herein means modes of administration other than enteral and topical administration, usually by injection, and includes, without limitation, intravenous, intramuscular, intraarterial, intrathecal, intracapsular, intraorbital, intracardiac, intradermal, transdermal, subdermal, intraperitoneal, transtracheal, subcutaneous, subcuticular, intraarticulare, subcapsular, subarachnoid, intraspinal and intrasternal injection and infusion. The phrases "systemic administration," "administered systemically," "peripheral administration" and "administered peripherally" as used herein mean the administration of a compound, drug or other material other than directly into the central nervous system, such that it enters the patient's system and, thus, is subject to metabolism and other like processes, for example, subcutaneous administration.

These compounds may be administered to humans and other animals for therapy by any suitable route of administration, including orally, nasally, as by, for example, a spray, rectally, intravaginally, parenterally, intracistemally and topically, as by powders, ointments or drops, including buccally and sublingually, and via inhalation into the lungs.

Regardless of the route of administration selected, the compounds of the present invention, which may be used in a suitable hydrated form, and/or the pharmaceutical compositions of the present invention, are formulated into pharmaceutically acceptable dosage forms by conventional methods known to those of skill in the art.

Actual dosage levels of the active ingredients in the pharmaceutical compositions of this invention may be varied so as to obtain an amount of the active ingredient that is effective to achieve the desired therapeutic response for a particular patient, composition, and mode of administration, without being toxic to the patient.

The selected dosage level will depend upon a variety of factors including the activity of the particular compound of the present invention employed, or the ester, salt or amide thereof, the route of administration, the time of administration, the rate of excretion or metabolism of the particular compound being employed, the duration of the treatment, other drugs, compounds and/or materials used in combination with the particular compound employed, the age, sex, weight, condition, general health and prior medical history of the patient being treated, and like factors well known in the medical arts.

A physician or veterinarian having ordinary skill in the art can readily determine and prescribe the effective amount of the pharmaceutical composition required. For example, the physician or veterinarian could start doses of the compounds of the invention employed in the pharmaceutical composition at levels lower than that required to achieve the desired therapeutic effect and then gradually increase the dosage until the desired effect is achieved. In some embodiments, a compound or pharmaceutical composition of the invention is provided to a diabetic subject chronically. Chronic treatments include any form of repeated administration for an extended period of time, such as repeated administrations for one or more months, between a month and a year, one or more years, or longer. In many embodiments, a chronic treatment involves administering a compound or pharmaceutical composition of the invention repeatedly over the life of the diabetic subject. Preferred chronic treatments involve regular administrations, for example one or more times a day, or one or more times a week. In general, a suitable dose such as a daily dose of a compound of the invention will be that amount of the compound that is the lowest dose effective to produce a therapeutic effect. Such an effective dose will generally depend upon the factors described above. Generally doses of the compounds of this invention for a patient, when used for the indicated effects, will range from about 0.0001 to about 100 mg per kg of body weight per day. Preferably the daily dosage will range from 0.001 to 50 mg of compound per kg of body weight, and even more preferably from 0.01 to 10 mg of compound per kg of body weight. However, lower or higher doses can be used, hi some embodiments, the dose administered to a subject may be modified as the physiology of the subject changes due to age, disease progression, weight, or other factors.

According to the invention, an IDE inhibitor compound can be administered at daily, weekly, or monthly intervals. In some embodiments, the compound can be administered at shorter, intermediate (e.g. every 2, 3, 4, 5, or 6 days, every 2 or 3 weeks, etc.) or at longer intervals (e.g. every 2, 3, 4, 5, 6, 7, 8, 9, 10 or 11 months, or every year or at greater intervals). In other embodiments, an IDE inhibitor compound can be administered once as a single dosage that is not repeated unless a subsequent treatment is required.

If desired, the effective daily dose of the active compound may be administered as two, three, four, five, six or more sub-doses administered separately at appropriate intervals throughout the day, optionally, in unit dosage forms.

While it is possible for a compound of the present invention to be administered alone, it is preferable to administer the compound as a pharmaceutical formulation (composition) as described above. The compounds according to the invention may be formulated for administration in any convenient way for use in human or veterinary medicine, by analogy with other pharmaceuticals.

EXAMPLES Example 1 : IDE inhibitors discovered through high-throughput compound screening

IDE is one of the first proteases ever discovered (Simkin et al, Arch. Biochem., 1949), yet there remains a surprising lack of inhibitors for this metalloprotease, or indeed for any members of the evolutionarily distinct superfamily to which it belongs. Known inhibitors of IDE (Table 1) all suffer from very low potency and poor selectivity, and most are toxic to cells at effective concentrations. Insulin itself is a relatively potent and selective competitive inhibitor of IDE (Table 1), but its use is limited to in vitro applications because it is a potent hormone with multiple biological effects.

Towards the goal of developing improved inhibitors of IDE, ~115,000 compounds were screened using a fluorescence polarization-based Aβ degradation assay (Leissring et al., J. Biol Chem., 2003). Although several inhibitors were discovered, including some with ICs₀S as low as 0.25 μM, virtually all were Michael acceptors or other thiol-alkylating compounds. As expected, these compounds proved to be toxic when tested in cell culture. Among the remaining inhibitors, one set of related compounds was found to inhibit IDE with IC₅₀ values <10 μM. However, despite extensive modifications to this series through medicinal chemistry, their potency could not be improved further, and these compounds worked poorly in cultured cells.

Table 1. Examples of known IDE inhibitors

Compound IC*n (uM) effect, cone. (uM) Mode of action Comments

1 ,10 phenanthroline 300 2000 zinc chelation broad spectrum

N-ethylmaleimide 220 1500 thiol alkylation irreversible, cytotoxic bacitracin 400 1500 (-2 mg/ml_)steric blockage nonselective, cyclic peptide insulin 0.12 5 competitive biologically active

Table 1 is a list of known inhibitors of IDE and their associated disadvantages. Known inhibitors of IDE suffer from low potency, lack of specificity, cytotoxicity, or have potent biological properties in addition to IDE inhibition. Examples of known IDE inhibitors and the concentrations at which they inhibit recombinant IDE by 50% (IC₅₀S) by in vitro activity assays. N-ethylmaleimide is a toxic compound that irreversibly alkylates cysteines in a non-selective fashion. 1,10 phenanthroline is a metal chelating compound that chelates the zinc within IDE and also numerous other zinc- metalloproteases or other zinc-containing biomolecules. Bacitracin is a mixture of related cyclic peptides produced by strains of Bacillis subtilis, which is known to inhibit several other proteases and other enzymes, making it non-selective. Insulin inhibits IDE strongly via competitive inhibition, but also potently mediates other biological activities.

Example 2: Existing peptide hydroxamates inhibit IDE, but far less potently than other targets

Hydroxamic acids are among the most potent zinc-metalloprotease inhibitors, but this class of compounds tend to be non-selective and are therefore regarded as poor drug candidates. Hydroxamate inhibitors that are relatively selective for individual matrix - metalloproteases (MMPs) have been developed, but only after considerable medicinal chemistry efforts. However, because IDE belongs to a distinct family of zinc- metalloproteases derived from convergent evolution (Makarova & Grishin, Protein Sci., 1999), it was reasoned that its active site may be sufficiently distinct to permit the development of selective hydroxamic acid inhibitors. To address this question, a range of commercially available peptidic and non-peptidic hydroxamic acids was tested for their ability to inhibit IDE (Table 2). Several peptidic hydroxamates exhibited IC₅₀S in the low μM range, making them among the best IDE inhibitors yet identified. However, most of these compounds inhibit other, canonical zinc-metalloproteases several orders of magnitude more potently, making them useless for selectively inhibiting IDE. Notably, a variety of non-peptidic hydroxamates that are in some cases potent and broad-spectrum MMP inhibitors showed no activity against IDE (Table 2). These results suggest that IDE's active site is indeed sufficiently distinct from conventional zinc-metalloproteases to permit the development of selective inhibitors.

Table 2. IDE inhibition by commercially available hydroxamic acids

Compound type IDE ICm (uM) best known target target IC_sn

(LlM)

GM6001 (Galardin) peptidic 6 MMP-8 0.0001

TAPI-O peptidic 9 MMP-13 0.0002 TAPM peptidic TACE 0.1

TAPI-2 peptidic 11 TACE 5

Nullscript nonpeptidic 1 N/A

MMP Inhibitor Il nonpeptidic >100 MMP-9 0.0027

MMP Inhibitor III nonpeptidic >100 MMP-13 0.0001

MMP Inhibitor IV nonpeptidic >100 multiple MMPs 0.5

MMP-2/9 Inhibitor Il nonpeptidic >100 MMP-2 0.017

MMP-2/9 Inhibitor IV nonpeptidic >100 MMP-2 0.014

MMP-3 Inhibitor Il nonpeptidic >100 MMP-3 0.13

MMP-3 Inhibitor VII nonpeptidic >100 MMP-3 0.025

MMP-8 Inhibitor I nonpeptidic >100 MMP-8 0.004

MMP-9 Inhibitor I nonpeptidic >100 MMP-9 0.005

MMP-9/13 Inhibitor I nonpeptidic >100 MMP-9 0.0009

MMP-9/13 Inhibitor Il nonpeptidic >100 MMP-13 0.0013

Table 2 is a list of commercially available hydroxamic acid-based inhibitors of canonical zinc-metalloproteases, tabulating their lack of efficacy in inhibiting IDE. Commercially available hydroxamic acids that potently inhibit matrix -metalloproteases have little to no effect on IDE. Table provides in vitro IC₅₀ values of peptidic and nonpeptidic hydroxamic acids and published IC50s against the indicated metalloproteases. Note that most compounds inhibit IDE many orders of magnitude more weakly than other metalloproteases, suggesting that the active site of IDE is very distinct from that within most other metalloproteases.

Example 3: Rational design and optimization of peptide hydroxamate IDE inhibitors To develop peptidic hydroxamates with improved potency and selectivity for IDE, a substrate-based rational design approach was developed. The basic strategy involves determining the protease's cleavage-site specificity (i.e., which amino acids are preferred at each substitute surrounding active site), and then generating short peptides based on the optimal sequence, to which a hydroxamic acid moiety is attached. IDE is unusual among proteases in showing a distinct lack of cleavage-site specificity due to the strong influence of the tertiary structure of substrates (Shen et al., Nature, 2006). To circumvent this problem, IDE's cleavage preferences were determined by analyzing the cleavage sites within combinatorial mixtures of short peptides (Turk et al, Nature Biotechnol., 2001). Briefly, a mixture of N-terminally blocked 12-mer peptides comprising all possible permutations of all amino acids (except cysteine) was partially digested with IDE; the resulting C-terminal fragments, containing unblocked N-termini, were analyzed by successive rounds of Edman degradation, thereby revealing the preferred amino acids at sites progressively more distal to the scissile bond (by convention, designated P₁', P₂', P₃' ...(Schechter & berger, Biochem., 1966)). Preferred amino acids N-terminal to the scissile bond were also probed using a related method described previously (Turk et al., Nature Biotechnol., 2001). This analysis revealed that IDE has a strong preference for Arg, Tyr and Phe at the Pl ' position, and for Arg at the P2' position, while showing relatively little preference at sites more distal to the scissile bond on the C-terminal side or for any sites on the N-terminal side (Fig. IA).

Figure 1 shows the cleavage-site specificity of IDE and peptide hydroxamic acids derived there from. A, To determine which naturally occurring amino acids are preferred by EDE at different sites relative to the scissile bond (i.e., the cleavage-site specificity), a two-step approach was taken. To determine the amino acids preferred by IDE at positions C-terminal to the scissile bond, EDE was allowed to partially hydrolyze a mixture of N-terminally acetylated 12-mer peptides (Ac-XXXXXXXXXXXX-CChH, where "X" refers to any amino acid except cysteine). The resulting C-terminal peptide fragments, containing unblocked N-termini, were analyzed by successive rounds of Edman degradation as described (Turk et al., Nat Biotechnol. 2001 Jul;19(7):661-7). To determine amino acids preferred at positions N-terminal to the scissile bond, the results of the above analysis were used to generate a second peptide mixture (MGXXXXYKPEDKK-biotin) (SEQ ED NO.: 1 Resigned to promote hydrolysis N- terminal to the sequence YKPE (SEQ ED NO.:2 ). Following partial hydrolysis with EDE, biotinylated species, comprising intact peptides and C-terminal fragments, were removed with an avidin column, and the remaining peptides, consisting of freshly cleaved N- terminal fragments, were analyzed by Edman degradation. Amino acids that were overrepresented in each successive round of Edman degradation (red) reflect IDE's amino acid preference at the Pl', P2', P3' ... etc., positions. B, C, First generation peptide hydroxamate EDE inhibitors derived from the cleavage site specificity analysis depicted in A. Phenylalanine was incorporated in the Pl ' position instead of the more strongly preferred amino acids (tyrosine or arginine) due to considerations of synthetic tractability of the corresponding chemical precursors. Pure diastereomers were generated comprising either L-amino acids at all positions (B) or a D-amino acid at the Pl ' positions with L-amino acids at the remaining positions (Q, yielding in vitro IC₅oS of 0.078 μM and 5.4 μM, respectively.

Based on this analysis, and considerations of synthetic tractability, inhibitors were generated based on the peptide Phe-Arg-Trp-Glu (SEQ ID NO.: 3). For zinc- binding, a hydroxamic acid moiety was added to the N-terminus, positioned appropriately by the inclusion of an α carbonyl moiety, which has been shown to improve the potency of hydroxamate inhibitors targeting many other zinc- metalloproteases (Fisher & Mobashery, Cancer Metastasis Rev., 2006). Pure diastereomers were generated containing either all L-amino acids or a D-amino acid at the P₁' position (Fig. IB), yielding IC₅₀ values of 0.078 and 5.4 μM, respectively.

While this initial series of compounds was far more potent than currently available IDE inhibitors, their potency was poorer than is typically achieved with peptide hydroxamates and they unfortunately inhibited other zinc-metalloproteases with nearly equal potency. These deficiencies were related to the incorporation of Phe at the Pj ' position, which was selected for reasons of synthetic tractability, but which was not as strongly preferred as Arg or Tyr (Fig. IA) and which can be accommodated by many other zinc-metalloproteases. To optimize the P₁ ' position, a focused library was generated of retro-inverso peptide hydroxamates. These compounds could be generated with facility using Fmoc-based solid phase peptide synthesis with a 2-chlorotrityl hydroxylamine resin. The retro-inverso configuration required the use of all D-amino acids, and the need to incorporate an additional α carbon between the hydroxamic acid and the peptide sequence required the use of β-amino acids at the P₁ ' position. Fortunately, a limited selection of Fmoc-protected β-D-amino acids was available commercially. A library was tested of retro-inverso peptide hydroxamates based on the sequence Xaa-Arg-Tyr, where Xaa represented a variety of natural and unnatural β-D- amino acids. Although variants incorporating Phe at the P₁ ' position proved to be relatively poor inhibitors relative to conventional versions, a 100-fold gain in potency was realized when Phe was substituted with 2-naphthylalanine (Table 3).

Table 3 is a list of modifications incorporated into the Pl' position of a retro-inverso peptide hydroxamate and corresponding IC₅Q values against IDE.

Table 3. IDE inhibition by a focused library of retro-inverso peptide hydroxamates with variations at the Pl ' position. To rapidly identify variations at the Pl ' position with improved potency, a focused library of retro-inverso peptide hydroxamates was generated. To generate these compounds, conventional Fmoc-based solid phase peptide synthetic chemistry (described below) was used to couple a variety of beta amino acids to a 2-chloryltrityl hydroxylamine resin, followed by attachment of arginine and tyrosine residues. To mimic the stereochemistry of conventional left-hand side peptide hydroxamates (e.g., Fig. IB), D-isomers of arginine and tyrosine were used for the P2' and P3' positions, respectively, and beta amino acids with appropriate chirality were used at the Pl' position.

Because the retro-inverso peptide hydroxamates were generally less potent than conventional versions, conventional hydroxamates incorporating 2-naphthylalanine at the P₁' position were generated (Fig. 2). As before, pure diastereomers were synthesized with either all L-amino acids or with a D-amino acid at the P₁' position, which in this case yielded IC₅₀ values of 1.7 nM and 75 nM, respectively.

Given the facility with which the retro-inverso peptide hydroxamates could be generated, we explored the possibility of developing photocrosslinkable, affinity labelled versions as activity-based probes useful for a variety of applications. Versions were generated containing benzophenylalanine at either the P₂' or P₃' position, in addition to a fluorescent EDANS label and a biotin group. Whereas the addition of extraneous functionalities typically results in reduced potency, these compounds proved to be surprisingly potent, with IC_5O values in the range of 0.3 to 3 nM (Table 4). Notably, certain versions of these compounds also proved to be highly selective, being at least 3000 times more potent against IDE than for other prototypical zinc-metalloproteases (Fig. 3). Truncated versions of the 2-naphthylalanine-containing retro-inverso peptide hydroxamic acids were also tested. Significantly, compounds containing as few as 2 amino acids were found to be relatively potent (Table 5). Given their small size, these compounds represent useful pharmacophores for the generation of non-pep ti die hydroxamic acid IDE inhibitors.

Table 4 is a list of retro-inverso peptide hydroxamates containing extraneous functional moieties and their associated IC_5Os against IDE

Compound ICsn (μM)

Hx-2Nap-Arg-Tyr-Glu-Ac (SEQ ID NO. : 4) 1 1 ^aHx-2Λ/ap-Λrg-Bpa-Glu(EDANS)-Glu(PEG-biotin)-Ac (SEQ ID NO.: 5) 0.0035

Hx-2Λ/ap-/4rg-βpa-Glu(PEG-biotin)-Glu(EDANS)-Ac(SEQ ID NO.: 6) 0.0017 ^bHx-2Nap-Bpa- 7yr-Glu(EDANS)-Glu(PEG-biotin)-Ac (SEQ ID NO.: 7) 0.0003

^aCompound 1. Compound 3.

Table 4. The incorporation of additional functional amino acid derivatives to retro- inverso peptide hydroxamates yields unexpectedly large increases in potency against IDE. A retro-inverso peptide hydroxamate (Hx-2Nap-Arg-tyr-Glu-Ac, SEQ ED NO.: 4) designed to mimic the conventional peptide hydroxamate in Fig. IB yielded a comparatively high IC_5O values in vitro (11 μM). However, the addition of amino acids linked to a fluorescent group (GIu(EDANS)) and a biotin group (Glu(PEG-biotin)) together with substitution of the P2' or P3' amino acids with a D-isomer of benzophenylalanine (Bpa) yielded large improvements in potency. To facilitate comparison with conventional peptide hydroxamates, peptides are described with the C- terminal end on the left-hand side and the N- terminal end on the right-hand side. D- amino acids are depicted in italics. Hx=hydroxamic acid moiety (HONHCO-); Ac=acetyl group; 2Nap=beta-D-2-naphthylalanine.

Table 5. EDE inhibition by truncated retro-inverso hydroxamic acids

N-terminal modification Peptide sequence acetylated free amine.

Hx-2NapRWE 0.09 ND

Hx-2NapRYE 0.11 0.11

Hx-2NapRY 0.82 0.34 Hx-2NapR 2.8 0.63

Hx-2Nap 1.9 ND

Table 5. IDE inhibition by truncated retro-inverso hydroxamic acids. Truncated versions of retro-inverso peptide hydroxamates show relatively low IC_5O values against IDE, suggesting they represent good pharmacophores for the developments of non- peptidic IDE inhibitors. Listed are truncated versions of retro-inverso peptide hydroxamates and their corresponding IC₅₀ values against IDE. To facilitate comparison with conventional peptide hydroxamates, peptides are described with the C-terminal end on the left-hand side and the N-terminal end on the right-hand side. D-amino acids are depicted in italics. Hx=hydroxamic acid moiety (HONHCO--); Ac=acetyl group; 2Nap=beta-D-2-naphthylalanine; NH₂=amine. Figure 3 illustrates that IDE inhibitors show selectivity for IDE. The graph depicts IC_5O values for various peptide hydroxamate IDE inhibitors against IDE versus against the canonical metalloprotease, neprilysin (NEP). IC₅₀ values for both proteases were determined using a beta-amyloid degradation assay (Leissring et al., J. Biol. Chem., 2003). Note that some inhibitors inhibit IDE many orders of magnitude more potently than NEP.

Example 4: IDE inhibitors increase secretion of insulin from a pancreatic beta cell line

The effects of novel IDE inhibitors were examined on a range of endpoints relevant to insulin metabolism and activity. Disruption of the balance between the anabolism and catabolism of insulin would be expected to be particularly important in cells that synthesize insulin. Confirming this, it was found that a range of IDE inhibitors increased basal and glucose-stimulated insulin secretion from Min6 cells, a pancreatic beta cell line (Fig. 4A). Supporting the specificity of this effect, insulin secretion was unaffected by control compounds consisting of analogous peptides lacking the zinc- binding hydroxamic acid moiety (not shown). Moreover, RNAi-mediated downregulation of IDE yielded a similar potentiation of insulin secretion in the same cell line (not shown), confirming that the effect was due to EDE and not to other zinc- metalloproteases.

Example 5: IDE inhibitors potentiate the activity of insulin within cells It was also investigated whether pharmacological inhibition of IDE could affect the action of insulin within cells. To this end, HeLa cells were treated (shown to express the insulin receptor) with or without an IDE inhibitor, and downstream insulin- stimulated tyrosine phosphorylation was examined. Inhibition of IDE produced a marked potentiation of tyrosine phosphorylation of classical insulin-signalling targets such as IRS 1/2 (Fig. 4B).

Example 6: IDE inhibitors regulate the trafficking of insulin to the nucleus

Numerous studies have shown that extracellular insulin is trafficked to the cytosol, where it interacts with IDE. Though controversial, insulin has also been reported to be present in the nucleus, where it is implicated in gene regulation. Supporting the physiological relevance of insulin in these compartments, elegant work in Xenopus oocytes showed that microinjection of as little as 17 fmol insulin into the cytosol, or 0.017 finol insulin into isolated nuclei, was sufficient to increase RNA synthesis above that stimulated by extracellular insulin (Miller et al., Science, 1988). Li principle, inhibition of cytosolic IDE activity should facilitate trafficking of insulin to the nucleus.

Confocal microscopy was used to monitor the subcellular localization of fluorescently labelled insulin in HepG2 cells treated with or without different IDE inhibitors. A special preparation of insulin labelled with fluorescein isothiocyanate (FITC) was used exclusively on the amino terminus of the B chain, which has been shown not to affect binding to the insulin receptor, hi control cells, FITC-insulin accumulated principally in the cytoplasm and was largely excluded from nuclei (Fig. 4C). Treatment of cells with Compound 1, (the IDE inhibitor illustrated in Figure 2A), by contrast, led to a significant accumulation of FITC-insulin in the nucleus (11 + 3.7% vs. 34 + 4.0% without and with IDE inhibitor treatment, respectively; Fig. 4C). Because the inhibitor used was not predicted to be cell permeant, the effect of excess unlabeled insulin was also tested, which is expected to be trafficked together with FITC-insulin, and which has been shown previously to inhibit intracellular IDE. Excess insulin led to a striking accumulation of FITC-insulin within the nucleus, localized primarily to intensely fluorescent spherical structures. Under this condition, the vast majority (83 + 6.4%) of FITC insulin was present within the nucleus, with lower amounts within the cytoplasm (Fig. 4C). These findings are consistent with the idea that insulin can be trafficked to the nucleus in a manner that is regulated by IDE activity.

Example 7: IDE inhibitors induce transport of insulin and other peptides across epithelial cell layers

The effects of pharmacological inhibition of IDE on the transcellular transport of peptides was examined using a well-established model of the epithelial cell layer comprised of cultured Madin-Darby canine kidney (MDCK) cells, which form tight- junctions in culture (Madin & Darby, Proc. Soc. Exp. Biol. Med., 1958). MDCK cells were grown on specialized filters that permit the separation of culture medium into two compartments (apical and basolateral) according to established methods (Haass et al., J. Biol. Chem., 1995). After verifying that the cells had successfully formed tight junctions by measurement of the electrical resistance between the apical and basolateral compartments, radioiodonated insulin or beta-amyloid were added to the basolateral compartment, and incubated under normal cell culture conditions (37 ⁰C, 5% CO₂) for various in the absence or presence of IDE inhibitors, including 1,10 phenanthroline (2 mM) or Compound 1 (10 μM; Inhibitor shown in Fig. 2A). In the presence of either inhibitor, radiolabeled beta-amyloid and insulin were each found to accumulate in a time-dependent fashion within the apical compartment, as detected by a gamma counter sensitive to the presence of radioiodinated peptides, indicative of transcellular transport (Fig. 5 A,B). In contrast, no such transcellular transport was observed in the absence of inhibitor (Fig. 5 A,B), even at times as long as 24 hr (not shown).

Figure 5 shows that IDE inhibitors facilitate the transcellular transport of compounds across MDCK cell monolayers, a model of epithelial cells. Graphs show the percentage of radiolabeled insulin (A) or beta-amyloid (B) accumulating in the apical side of MDCK cell monolayers as a function of time following the addition of the radiolabeled compounds to the basolateral side. Note that transcellular transport is only observed in the presence of IDE inhibitors. cpm=counts per minute.

Example 8: IDE inhibitors inhibit the binding of varicella zoster viral glycoproteins to IDE

IDE was recently shown to be the cellular receptor for varicella zoster virus (VZV), the agent that causes chicken pox and shingles in humans (Li et al., Cell, 2006). VZV infection and cell-to-cell spread depend on the interaction of a viral protein, glycoprotein E (gE), with IDE, suggesting that compounds that block this interaction could have therapeutic value (Li et al., Cell, 2006). The efficacy of a subset of the IDE inhibitors described herein was tested for their potential to disrupt the gE-IDE interaction using a pull-down assay. Briefly, a fusion protein was generated, comprised of gE fused to the constant domain of immunoglobulin (Fc), designated gE:Fc. Purified gE:Fc was allowed to interact with IDE present in cell lysate, and the complex consisting of gE:Fc and IDE was isolated using protein A sepharose beads, which interact selectively with the Fc domain. The amount of IDE isolated by this process was evaluated by Western blotting. Multiple IDE inhibitors described herein, including Compound 1 and the three biotin-containing inhibitors described in Table 5, were found to disrupt the interaction between gE:Fc and DDE (Fig. 6). Based on the fact that the interaction between gE and IDE is essential for VZV infection and cell-to-cell spread, these results suggest that IDE inhibitors will have therapeutic value in the prevention of VZV-related disorders.

Figure 6 further shows that IDE inhibitors disrupt the interaction between IDE and glycoprotein E of VZV, an interaction essential for VZV infection and cell-to-cell transmission. Figure shows a Western blot of the amounts of IDE pulled down from cell lysates by a glycoprotein E:Fc fusion protein in the absence or presence of IDE inhibitors (20 mM). Lane 1= Compound 2 (Hx-2Nap-Arg-Bpa-G\u(ED ANS)-GIu(PEG- biotin)-Ac (SEQ ID NO.: 5)). Lane 2= compound 3 (Hx-2Nap-Bpa-Tyr-Glu(EDANS)- Glu(PEG-biotin)-Ac (SEQ ID NO.: 7); Lane 3-Compound 1 (see Fig. 2A). Lane 4=Hx- 2Nap-Arg-Bpa-G\u(?EG-biotin)-Glu(EOANS)-Ac) (SEQ ID NO.: 6); Lane 5=no compound.

Example 9: Activity assays

IDE activity was routinely quantified by monitoring changes in fluorescence (λex=340, λem = 420) induced by the hydrolysis of Abz-GGFLRKVGQ-Dnp (SEQ ID NO.: 8) (Song et al., J.Biol Chem, 2001), or by any of several other methods described previously, including trichloroacetic acid precipitation of ¹²⁵I-labeled insulin or Aβ(l-40) (Leissring et al, Neuron, 2003), or changes in fluorescence polarization induced by hydrolysis of fluorescein-β-amyloid(l-40)-lys(LC-biotin) (Leissring et al., J. Biol. Chem., 2003). hi vitro activity assays incorporated recombinant human IDE purified from bacteria (Farris et al., Biochemistry, 2005) or insect cells (Leissring et al., J. Biol. Chem., 2003).

Example 10: Analysis of IDE's cleavage-site specificity

To determine the amino acids preferred by IDE at positions C-terminal to the scissile bond, IDE was allowed to partially hydro lyze a mixture of N- terminally acetylated 12-mer peptides (AC-XXXXXXXXXXXX-CO₂H, where "X" refers to any amino acid except cysteine). The resulting C-terminal peptide fragments, containing unblocked N-termini, were analyzed by successive rounds of Edman degradation as described (Turk et al., Nature Biotechnol., 2001). To determine amino acids preferred at positions N-terminal to the scissile bond, the results of the above analysis were used to generate a second peptide mixture (MGXXXXYKPEoKK-biotin (SEQ ID NO.: I)) designed to promote hydrolysis N-terminal to the sequence YKPE (SEQ ID NO.: 2). Following partial hydrolysis with IDE, biotinylated species, comprising intact peptides and C-terminal fragments, were removed with an avidin column, and the remaining peptides, consisting of freshly cleaved N-terminal fragments, were analyzed by Edman degradation.

Example 11 : Synthesis of conventional peptide hydroxamic acids Table 6. Synthesis of conventional peptide hydroxamic acids

yl

6 R = Ph 6a R = Ph

7 R = 2-naphthyl 7a R = 2-naphthyl

8 R = Ph, R₁ = -NHOtBu 8a R = Ph, R₁ = -NHOtBu

9 R = 2-naphthyl, R₁ = -NHOtBu 9a R = 2-naphthyl, R₁ = -NHOtBu

1O R = Ph, R₁ = -N(CH₃J₂ 10a R = Ph, R1 = -N(CH₃J₂

ester

*£££££ ^^

12 R = 2-naphthyl, R₁ = -NHOtBu 13 R = Ph₁ R₁ = ^(CH₃J₂

14 R= Ph, R₁ = -NHOtBu, X = R 14a R = Ph, R₁ = -NHOtBu, X=R 15 R = Ph, R₁ = -NHOtBu, X = S 15a R = Ph, R₁ = -NHOtBu, X = S 16 R = 2-naphthyl, R₁ = -NHOtBu, X=R 16a R = 2-naphthyl, R₁ = -NHOtBu, X = R 17 R = 2-naphthyl, R₁ = -NHOtBu, X=S 17a R = 2-naphthyl, R₁ = -NHOtBu, X = S

18 R = Ph, R₁ = -N(CH₃J₂, X = R 18a R = Ph, R₁ = -N(CH₃)₂, X = R

19 R=Ph, R₁=^(CH₃J₂, X = S 19a R = Ph, R₁ = -N(CH₃)₂, X = S

HBTU, DIPEA 10% Pd/C, H₂ Cbz-Trp(tBOC)-COOH Cbz-Trp(tBOC)-Glu(tBu)-CONH₂ ►

EtOH/MeOH

20 HCI NH₂-Glu(tBu)-CONH₂ 21

20a

NH₂-Trp(tBO

23 R= Ph, R₁ = -NHOtBu, X = R 24 R= Ph, R₁ = -NHOtBu, X = S 25 R = 2-naphthyl, R₁ = -NHOtBu, X = R 26 R = 2-naphthyl, R₁ = -NHOtBu, X = S 27 R = Ph, R₁ = -N(CH₃J₂, X = R 28 R = Ph, R₁ = -N(CH₃J₂, X = S

1 )

2)

29 R = Ph₁R₁ = -NHOH, X= R 30 R = Ph, R₁ = -NHOH, X = S 31 R = 2-naphthyl, R₁ = -NHOH, X=R 32 R = 2-naphthyl, R₁ = -NHOH, X = S 33 R= Ph, R₁ = -N(CH₃)₂, X=R 34 R= Ph, R₁ = -N(CH₃J₂, X = S

Hydrocinnamic acid 2 was purchased from Sigma- Aldrich.

2-naphthalenepropanoic acid (3): A mixture of 3-(2-naphthyl)acrylic acid (1, 1 g, 0.54 mmol, Lancaster- Alfa Aesar) and 10% Pd-C (100 mg) in ethanol (20 mL) was treated with 20% aqueous NaOH (5ml) and stirred under hydrogen (1 atm) for 48 h. The mixture was filtered, the filtrate was acidified with IN HCl, and extracted with EtOAc (250 ml) to afford compound 3 (710mg, 70%) which was used for next step without further purification: ¹H NMR (500 MHz, DMSO-d₆): δ 2.64 (t, 2H, J= 7.8), 2.99 (t, 2H, J= 7.8), 7.41 - 7.49 (m, 3H), 7.72 (s, IH), 7.82 - 7.87 (m, 3H), 12.16 (br s, IH).

Benzyl ester 4 — 5 were prepared following literature procedure in Eur. J. Org. Chem. 2002, 3986-3994 except the resultant oil was purified by silica gel column chromatography with 5% EtOAc in hexane.

Tert-Butoxycarbonyl ester 6 - 7 were prepared following literature procedure in J. Chem. Soc, Perkin Trans. 1, 1998, 2629-2634 as a racemic mixture.

Acids 6a - 7a were prepared following literature procedure in J. Chem. Soc, Perkin Trans. 1, 1998, 2629-2634.

Hydroxamate and amides 8 - 10 were prepared following literature procedure in J. Chem. Soc, Perkin Trans. 1, 1998, 2629-2634.

Acids 8a - 10a were prepared following literature procedure in J Chem. Soc, Perkin Trans. 1, 1998, 2629-2634.

Amides 11 - 13 were prepared following literature procedure in J. Chem. Soc, Perkin Trans. 1, 1998, 2629-2634 as a diastereomeric mixture and purified by silica gel column chromatography with 3% MeOH in dichloromethane to give each pure diastereomer 14 - 19.

Acids 14a - 19a were prepared following literature procedure in J. Org. Chem. 1989, 54, 751-756 and purified by silica gel column chromatography with 3 to 5% MeOH in dichloromethane to give each acid 14a — 19a.

Dipeptide 22 was prepared as described for 3 with 20% MeOH in EtOH for 5h except NaOH/water and used for next step without further purification after washing with ether.

General Procedure for the preparation of amides (21, 23-28): A mixture of acid (20, 14a - 19a), HBTU (1.05 equiv.), and DIPEA (1.05 equiv.) in DMF was treated with amine (20a, or 22, 1.01 equiv., for HCl salt of amine 20a an additional 1.01 equiv. DIPEA was added) and stirred for 16 h. The mixture was diluted with EtOAc, washed with saturated sodium hydrogen carbonate, brine, dried over sodium sulfate, filtered, concentrated in vacuo to afford a white waxy solid, which was purified by silica gel column chromatography with 3 to 5% MeOH in dichloromethane to give protected di- or tetra- peptide(21, 23-28). Deprotected tetrapeptide hydroxamic acid (29-34) were prepared following literature procedure in J. Chem. Soc, Perkin Trans. 1, 1998, 2629-2634 with 5% anisole in TFA. After concentrated in vacuo, the resultant solid or oil was dissolved in water, filtered, and the filtrate was lyophilized to give a white powder that was further purified by reverse- phase HPLC.

Example 12: Synthesis of retro-inverso peptide hydroxamic acids

9-fluorenylmethyleneoxy-carbonyl (Fmoc)-based solid-phase peptide synthesis was carried out manually using a 2-chlorotritylhydroxyl amine resin (Sigma- Aldrich). Following deprotection of the resin with 20% piperidine in dichloromethane (DCM), Fmoc-protected β-D-amino acids (5 eq.) were coupled in a 16-hr reaction using HATU (5 eq.), HOAt (5 eq.) and DIEA (10 eq.) in DCM, and subsequent amino acids (4 eq.) were coupled using HBTU (4 eq.), HOBt (4 eq.) and DIEA (4 eq.) in dimethylformamide (DMF). Cleavage from the resin and removal of protecting groups was effected by a mixture of trifluoroacetic acid (95%), triisopropylsilane (2.5%) and water (2.5%). Peptides were purified by reverse-phase liquid chromatography using a linear acetonitrile:water gradient and masses verified by matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry.

Having thus described several aspects this invention, it is to be appreciated that various alterations, modifications, and improvements will readily occur to those skilled in the art. Such alterations, modifications, and improvements are intended to be part of this disclosure, and are intended to be within the spirit and scope of the invention. Accordingly, the foregoing description and drawings are by way of example only.

The disclosures of all the patents, patent publications, scientific publications and other documents or information listed herein are incorporated by reference herein in their entirety.

Claims

1. A compound of the formula:

wherein R₁, R₂ , R₃, R₄, R₅ and R₆ are independently selected and wherein R, is H, OH, O-alkyl or alkyl,

R₃ is NHC(=NH)NH₂, NH₂, NHC(O)alkyl, or NHC(O)aryl,

R₄ is NH₂ or [NR₅CH(CH₂)_OR₆CC=O)]_PNH₂,

R₅ is H or Me,

R₆ is 4-hydroxyphenyl, CO₂H, indol-3-yl, or phenyl, m is O - 3, n is O - 3, o is O - 3, and p is O - 2.

2. The compound of claim 1, wherein R₂ is 2-naphthyl, and

R₃ is NHCC=NH)NH₂.

3. A pharmaceutical composition comprising a compound of the formula:

wherein R₁, R₂ , R₃, R₄, R₅ and R₆ are independently selected and wherein R₁ is H, OH, O-alkyl or alkyl,

R₃ is NHC(=NH)NH₂, NH₂, NHC(O)alkyl, or NHC(O)aryl,

R₅ is H or Me,

4. The pharmaceutical composition of claim 3, wherein R₂ is 2-naphthyl, and

R₃ is NHC(=NH)NH₂.

5. A pharmaceutical preparation comprising the pharmaceutical compositions of any of claims 3-4 and a pharmaceutically acceptable carrier.

6. The pharmaceutical preparation of claim 5, wherein the pharmaceutical preparation is sterile.

7. The pharmaceutical preparation of claim 6, wherein the pharmaceutically acceptable carrier is chosen from a sugar, a starch, cellulose, powdered tragacanth, malt, gelatin, talc, an oil, a glycol, a polyol, an ester, an agar, a buffering agent, alginic acid, pyrogen free water, isotonic saline, Ringer's solution, a pH buffered solution, a polyester, a polyanhydride, and a polycarbonate.

8. The pharmaceutical composition of claim 7, wherein the pharmaceutical composition is in solid unit dosage form.

9. The composition, preparation, or compound of any one of claims 1-8, comprising one or more compounds in a pharmaceutically acceptable hydrate form, and/or a pharmaceutically acceptable stereoisomeric form, and/or a pharmaceutically acceptable acid or base addition salt form.

10. A method of treating diabetes in a subject in need thereof by administering to the subject a compound comprising the formula:

R₃ is NHC(=NH)NH₂, NH₂, NHC(O)alkyl, or NHC(O)aryl,

R₄ is NH₂ or [NR₅CH(CH₂)_OR₆CC=O)]_PNH₂,

R₅ is H or Me,

R₆ is 4-hydroxyphenyl, CO₂H, indol-3-yl, or phenyl, m is O - 3, n is 0 — 3, o is 0 - 3, p is 0 — 2, and wherein the compound is administered in an amount effective to treat said subject.

11. The method of claim 10 wherein R₂ is 2-naphthyl, and

R₃ is NHC(=NH)NH₂.

12. The method of any of claims 10-11, wherein the compound is administered via a route chosen from oral, parenteral, topical, ocular, transdermal, bronchial and nasal routes.

13. The method of any of claims 10-11, wherein the compound is administered by subcutaneous, intramuscular, intravenous, or epidural injection.

14. A method of use of composition, preparation, or compound of any one of claims 1-9 for topical treatment for wound healing.

15. A method of use of composition, preparation, or compound of any one of claims 1-9 as an adjuvant to improve the delivery of insulin or other therapeutic molecules into organisms and/or into intraorganismal or intracellular compartments and/or across mucous membranes.

16. A method of use of composition, preparation, or compound of any one of claims 1-9 for the treatment of chicken pox and/or shingles.

17. A method of use of composition, preparation, or compound of any one of claims 1-9 for preventing varicella zoster virus infection and/or cell-to-cell spread.

18. A method of use of composition, preparation, or compound of any one of claims 1-9 for facilitating the transport of pathogenic substances out of intraorganismal or intracellular compartments.

19. A compound of the formula:

R₃ is NHC(=NH)NH₂, NH₂, NHC(O)alkyl, or NHC(O)aryl,

R₄ is [C(=O)CH(CH₂)_oR₇NH]_pH or [C(=O)CH(CH₂)_oR₇NH]pC(=O)Me,

R₅ is H or Me,

R₆ is H or Me

20. The compound of claim 19, wherein R₂ is 2-naphthyl, and

R₃ is NHC(=NH)NH₂.

21. A pharmaceutical composition comprising a compound of the formula:

wherein Ri, R₂ , R₃, R₄, R₅ and R₆ are independently selected and wherein R₁ is H, OH, O-alkyl or alkyl,

R₃ is NHC(=NH)NH₂, NH₂, NHC(O)alkyl, or NHC(O)aryl,

R₄ is [C(=O)CH(CH₂)oR₇NH]_pH or [C(=O)CH(CH₂)_oR₇NH]_pC(=O)Me,

R₅ is H or Me,

R₆ is H or Me

22. The pharmaceutical composition of claim 21 , wherein R₂ is 2-naphthyl, and

R₃ is NHC(=NH)NH₂.

23. A pharmaceutical preparation comprising the pharmaceutical compositions of any of claims 21-22 and a pharmaceutically acceptable carrier.

24. The pharmaceutical preparation of claim 23, wherein the pharmaceutical preparation is sterile.

25. The pharmaceutical preparation of claim 24, wherein the pharmaceutically acceptable carrier is chosen from a sugar, a starch, cellulose, powdered tragacanth, malt, gelatin, talc, an oil, a glycol, a polyol, an ester, an agar, a buffering agent, alginic acid, pyrogen free water, isotonic saline, Ringer's solution, a pH buffered solution, a polyester, a polyanhydride, and a polycarbonate.

26. The pharmaceutical composition of claim 25, wherein the pharmaceutical composition is in solid unit dosage form.

27. The composition, preparation, or compound of any one of claims 19-26, comprising one or more compounds in a pharmaceutically acceptable hydrate form, and/or a pharmaceutically acceptable stereoisomeric form, and/or a pharmaceutically acceptable acid or base addition salt form.

28. A method of treating diabetes in a subject in need thereof by administering to the subject a compound comprising the formula:

R₁ is H, OH, O-alkyl or alkyl,

R₃ is NHC(=NH)NH₂, NH₂, NHC(O)alkyl, or NHC(O)aryl,

R₄ is [C(=O)CH(CH₂)_oR₇NH]_pH or [C(=O)CH(CH₂)_oR₇NH]_pC(=O)Me,

R₅ is H or Me,

R₆ is H or Me

R₇ is 4-hydroxyphenyl, CO₂H, indol-3-yl, or phenyl, m is 0 — 3, n is 0 - 3, o is 0 - 3, p is 0 - 2, and wherein the compound is administered in an amount effective to treat said subject.

29. The method of claim 28 wherein R₂ is 2-naphthyl, and R₃ is NHC(=NH)NH₂.

30. The method of any of claims 28-29, wherein the compound is administered via a route chosen from oral, parenteral, topical, ocular, transdermal, bronchial and nasal routes.

31. The method of any of claims 28-29, wherein the compound is administered by subcutaneous, intramuscular, intravenous, or epidural injection.

32. A method of use of composition, preparation, or compound of any one of claims 19-27 for topical treatment for wound healing.

33. A method of use of composition, preparation, or compound of any one of claims 19-27 as an adjuvant to improve the delivery of insulin or other therapeutic molecules into organisms and/or into intraorganismal or intracellular compartments and/or across mucous membranes.

34. A method of use of composition, preparation, or compound of any one of claims 19-27 for the treatment of chicken pox and/or shingles.

35. A method of use of composition, preparation, or compound of any one of claims 19-27 for preventing varicella zoster virus infection and/or cell-to-cell spread.

36. A method of use of composition, preparation, or compound of any one of claims 19-27 for facilitating the transport of pathogenic substances out of intraorganismal or intracellular compartments.