HK1087337B - Use of a hif-alpha stabilizing agent for the preparation of medicament for treatment of diabetes - Google Patents

Description

Application of preparation for stabilizing HIF alpha in preparing medicine for treating diabetes

The present application claims priority from U.S. provisional application serial No.60/431351 filed on 6/12/2002, U.S. provisional application serial No.60/476331 filed on 6/2003, and U.S. provisional application serial No.60/476726 filed on 6/2003, the contents of each of which are incorporated herein by reference.

Technical Field

The present invention relates to regulation and homeostasis of glucose, and more particularly, to treatment or prevention of diseases associated with impaired glucose regulation such as diabetes.

Back of body environment

Diabetes is a disease characterized by hyperglycemia due to defects in insulin secretion, insulin activity, or both. Type I diabetes, formerly known as insulin-dependent diabetes mellitus (IDDM), is an autoimmune disease in which langerhans cells act on the islets of langerhans and destroy the body's ability to produce insulin. Type I diabetes accounts for 10% of all cases of diabetes, affecting as many as 100 million people in the united states. Type II diabetes, formerly known as insulin independent diabetes mellitus (NIDDM), is a metabolic disease caused by the body's inability to produce sufficient insulin or to properly utilize insulin. Approximately 90% of all diabetics in the united states suffer from type II diabetes. Both type I and type II diabetes are metabolic diseases characterized by hyperglycemia due to defects in insulin secretion, insulin activity, or both.

The prevalence of diabetes increases at an alarm rate. Between 1976 and 1994, the diabetic population increased from 8.9% to 12.3% in adults in the United states. Approximately 1600 million (approximately 6% of the population) people in the united states currently suffer from diabetes, with 80 million newly diagnosed diabetic patients each year. (Harris et al, Diabetes Care, 21: 518-. Diabetes mellitus is associated with disorders and complications that widely affect various organs in the body before, during and after onset; such as retinopathy, nephropathy, neuropathy, etc., leading to blindness, renal failure, etc. Diabetes can seriously affect quality of life and can even lead to death. There is therefore a need in the art for effective methods for treating and preventing the development and progression of diabetes and its associated complications.

Several risk factors associated with diabetes, often type II diabetes, particularly family history of diabetes, certain ethnic groups or races, history of gestational diabetes, obesity, particularly high levels of visceral and abdominal fat, sedentary life types, age, hypertension, schizophrenia, etc.; and alterations in glucose metabolism, including Impaired Glucose Tolerance (IGT) or prediabetes. There is therefore a need in the art for effective methods for treating and preventing the development and progression of risk factors associated with diabetes.

Diabetes and other diseases are associated with uncontrolled glucose regulation. Diabetes and hyperglycemia can lead to the development, or in response, to a variety of diseases and conditions, including: atherosclerosis, vascular diseases such as stroke, etc.; obesity, cardiovascular diseases such as Congestive Heart Failure (CHF), Myocardial Infarction (MI), and downstream effects, among others; or risk factors associated with these diseases and conditions. There is therefore a need in the art for effective methods of treating and preventing, or minimizing the risk factors for developing, diabetes or hyperglycemia-related diseases. The present invention answers this need by providing a drug treatment that, unlike current treatments for different diseases and different aspects of different diseases, provides a compound that targets a family (member) of relevant physiological processes and achieves a synergistic effect that achieves the desired therapeutic effect.

Current treatments for diabetes are to control glucose levels in the blood (e.g., to achieve glycemic control) in an effort to regenerate natural physiological glucose homeostasis. This approach may have limited effectiveness due to changes in life style, such as recommendations for controlling diet and increasing exercise. This type of life change may not prevent or overcome the occurrence of the relevant physiological factors, and may delay, but not prevent, the progression of the disease. In addition, patient compliance with this method may limit its effectiveness.

Common treatment methods include treatment with insulin, for example by injections of the insulin to be administered often once a day or even multiple times a day in a carefully designed time regime. Such treatments requiring insulin injections may be associated with the risk of hypoglycemia and hyperinsulinemia. In addition, the success of such treatments is often compromised by the lack of patient compliance, i.e., failure to comply with the recommended treatment regimen. Other insulin-based therapies, such as administration of insulin secretagogues (compounds that stimulate insulin secretion by the pancreas), also have similar risks of inducing hypoglycemia, and the like. These therapies, and existing therapies such as PPAR-gamma antagonist therapy, are associated with other effects, such as diabetes and a risk factor of self: the body weight is increased. There is therefore a need for methods and compounds that are effective in treating or preventing diabetes, hyperglycemia, and diabetes-related risk factors, such as weight gain and obesity, that often improve the ease of administration without the attendant risk or occurrence of other diabetes-related factors, such as weight gain and the like. For diabetes and related diseases, there is a need for therapies that more closely mimic the body's own physiological mechanisms that achieve glucose homeostasis. Of course such treatments may require improved ease of administration, increased patient comfort and patient compliance. In addition, the treatment of diabetes or related diseases requires that such treatment not have an effect of exacerbating or worsening the disease being treated.

Glucose metabolism, such as synthesis, processing and utilization of glucose, is essential to maintain normal glucose balance and homeostasis. Disruption of glucose metabolism or regulation can lead to disruption of glucose homeostasis, resulting in inappropriately high (i.e., hyperglycemic) or low (i.e., hypoglycemic) levels of blood glucose. Hyperglycemia and hypoglycemia affect quality of life chronically or severely, producing nerve and vessel damage. There is therefore a need for effective methods of regulating blood glucose metabolism and homeostasis (achieving glycemic control), and treating or preventing diseases and disorders associated with altered or impaired glucose metabolism and homeostasis, such as diabetes, hyperglycemia, and the like.

Summary of The Invention

The present invention relates to methods and compounds for regulating glucose metabolism and achieving glucose homeostasis. Methods of lowering blood glucose levels, reducing insulin resistance, lowering glycated hemoglobin levels, and improving glycemic control in a subject are also provided. Methods for treating or preventing diabetes, hyperglycemia, and other diseases associated with elevated blood glucose levels, such as treating or preventing diabetes-related diseases, such as those that are risk factors for, or occur with or result from diabetes, are provided.

In various embodiments, the subject is a cell, tissue, or organ. In other embodiments, the subject is an animal, preferably a mammal, more preferably a human. When the subject is a cell, the invention specifically contemplates that the cell can be an isolated cell, prokaryotic or eukaryotic cell. When the subject is a tissue, the invention specifically contemplates endogenous tissues and in vitro tissues, such as cultured tissues. In a preferred embodiment, the subject is an animal, in particular a mammalian species, including rats, rabbits, cattle, sheep, pigs, mice, horses, and primates. Most preferably, the subject is a human.

The present invention provides methods of modulating glucose metabolism. In one aspect, the present methods comprise modulating glucose metabolism in a subject by stabilizing HIF α in the subject. In various aspects, HIF α is HIF1 α, HIF2 α, or HIF3 α. In a preferred aspect, stabilizing HIF α comprises administering to the subject an effective amount of a compound that inhibits HIF hydroxylase activity.

Stabilization of HIF α can be performed by methods known and known to those of skill in the art, and can include any agent that interacts with, or modifies HIF α or factors that interact with HIF α, e.g., enzymes for which HIF α is a substrate. In certain aspects, the present invention contemplates providing structurally stable HIF α variants, e.g., stable HIF muteins, etc., or polynucleotides encoding such variants. In other aspects, the present invention contemplates stabilizing HIF α, including administration of any agent that stabilizes HIF α. The agent may comprise a polynucleotide, such as an antisense sequence; a polypeptide; an antibody; other proteins; a carbohydrate; fat; a lipid; and organic and inorganic substances such as small molecules and the like. In a preferred embodiment, the present invention contemplates stabilizing HIF α in a subject, e.g., by administering to the subject any agent that stabilizes HIF α, wherein the agent is a compound, e.g., a small molecule compound, that stabilizes HIF α.

The present invention also relates to methods of modulating glucose metabolism in a subject by administering to the subject an effective amount of a compound of the invention. In a preferred aspect, a compound of the invention is a compound that inhibits HIF hydroxylase activity. In a most preferred aspect, a compound of the invention is a compound that inhibits HIF prolyl hydroxylase activity. In another preferred aspect, the HIF hydroxylase is selected from the group consisting of EGLN1, EGLN2, and EGLN 3.

The present invention also provides methods for regulating a glucose metabolic process in a subject by stabilizing HIF α in the subject, or by administering to the subject an effective amount of a compound of the invention, thereby regulating the glucose metabolic process in the subject. In various embodiments, the glucose metabolic process is selected from the group consisting of glucose uptake, glucose transport, glucose storage, glucose processing, glucose utilization, and glucose synthesis, among others.

In particular embodiments, the present methods alter expression of a glucose regulatory factor in a subject by stabilizing HIF α in the subject, or by administering to the subject an effective amount of a compound that alters expression of the glucose regulatory factor in the subject.

In one embodiment, the present invention provides methods for increasing expression of a glucose regulatory factor in a subject by stabilizing HIF α in the subject, or by administering to the subject an effective amount of a compound capable of increasing expression of the glucose regulatory factor in the subject. In other embodiments, the glucose regulating factor is selected from the group consisting of: PFK-P, PFK-L, enolase-1, GluT-1, lactate dehydrogenase, aldolase-1, hexokinase-1, IGFBP-1, and IGF. In a particular aspect, the increase in glucose regulatory factor is a sustained increase. In one aspect, the glucose regulatory factor is a glycolytic factor. In another aspect, the glycolytic factor is selected from the group consisting of: PFK-P, PFK-L, enolase-1, lactate dehydrogenase, aldolase-1 and hexokinase-1.

The present invention provides methods for achieving glucose homeostasis in a subject. In one aspect, the present methods comprise achieving glucose homeostasis in a subject by stabilizing HIF α in the subject, thereby achieving glucose homeostasis in the subject. In another aspect, the methods of the invention comprise achieving glucose homeostasis in a subject by administering to the subject an effective amount of a compound of the invention, such that glucose homeostasis in the subject is achieved.

The present invention provides methods for lowering blood glucose levels in a subject. In one aspect, the present methods comprise decreasing blood glucose levels in a subject by stabilizing HIF α in the subject, thereby decreasing blood glucose levels in the subject. In another aspect, the methods of the invention comprise reducing glycated hemoglobin levels in a subject by administering to the subject an effective amount of a compound of the invention, thereby reducing glycated hemoglobin levels in the subject.

This document relates to methods of treating or preventing diabetes in a subject having or at risk of developing diabetes. In one embodiment, the methods comprise treating or preventing diabetes in a subject having, or at risk for developing, diabetes by stabilizing HIF α in the subject, thereby preventing or treating diabetes. In another aspect, the methods of the invention comprise treating or preventing diabetes in a subject by administering to the subject an effective amount of a compound of the invention, thereby treating or preventing diabetes in the subject.

The invention also provides methods of treating or preventing a disease associated with elevated blood glucose levels in a subject. In one embodiment, the methods comprise treating or preventing a disorder associated with increased blood glucose levels in a subject by stabilizing HIF α in the subject, thereby preventing or treating the disorder associated with increased blood glucose levels. In another aspect, the methods of the invention comprise treating or preventing a disease associated with elevated blood glucose levels in a subject by administering to the subject an effective amount of a compound of the invention, thereby treating or preventing the disease associated with elevated blood glucose levels in the subject. In various embodiments, the disease is selected from: diabetes, hyperglycemia, obesity, impaired glucose tolerance, hypertension, retinopathy, neuropathy, nephropathy, hyperlipidemia, and vascular disease.

The invention also relates to methods of treating or preventing diabetes-related disorders in a subject. In one embodiment, the methods comprise treating or preventing a diabetes-related disorder in a subject by stabilizing HIF α in the subject, thereby preventing or treating the diabetes-related disorder in the subject. In another aspect, the methods of the invention comprise treating or preventing a diabetes-related disorder in a subject by administering to the subject an effective amount of a compound of the invention, thereby treating or preventing the diabetes-related disorder in the subject. In various embodiments, the disease is selected from: hypertension, obesity, hyperglycemia, impaired glucose tolerance, hyperlipidemia, nephropathy, neuropathy, retinopathy, atherosclerosis, and vascular disease. In one embodiment, the subject is a diabetic subject. In another embodiment, the subject is a subject at risk for diabetes.

The present invention provides methods for reducing blood triglyceride levels in a subject. In one aspect, the present methods comprise decreasing blood triglyceride levels in a subject by stabilizing HIF α in the subject, thereby decreasing blood triglyceride levels in the subject. In another aspect, the methods of the invention reduce blood triglyceride levels in a subject by administering to the subject an effective amount of a compound of the invention, thereby reducing blood triglyceride levels in the subject.

The present invention provides methods of reducing insulin resistance in a subject. In one aspect, the present methods comprise decreasing insulin resistance in a subject by stabilizing HIF α in the subject, thereby decreasing insulin resistance in the subject. In another aspect, the methods of the invention can reduce insulin resistance in a subject by administering to the subject an effective amount of a compound of the invention, thereby reducing insulin resistance in the subject.

The present invention provides methods for improving glycemic control in a subject. In one aspect, the present methods may favor glycemic control in a subject by stabilizing HIF α in the subject, thereby increasing glycemic control in the subject. In another aspect, the methods of the invention improve glycemic control in a subject by administering to the subject an effective amount of a compound of the invention, thereby improving glycemic control in the subject. In yet another aspect, the subject is a subject with hyperglycemia.

In various embodiments, the invention provides formulations or medicaments or pharmaceutical compositions containing the compounds of the invention, as well as methods of making and using such formulations or medicaments or pharmaceutical compositions.

Brief Description of Drawings

FIGS. 1A and 1B show aldolase and glucose transporter-1 (GluT-1) induced in cells treated with compounds of the invention.

FIGS. 2A, 2B and 2C show the expression of genes involved in glucose regulation in kidney, liver and lung, respectively, in animals treated with compounds of the invention.

FIGS. 3A and 3B show dose and temperature responses of genes encoding GluT-1 and IGFBP-1, respectively, in the kidney and liver of animals treated with a compound of the invention.

Figure 4 shows the reduction of blood glucose levels in animals treated with compounds of the invention.

Figures 5A and 5B show an increase in glucose tolerance in an animal model of diet-induced type 2 diabetes after treatment with a compound of the invention.

FIG. 6 shows the reduction of glycation of heme in db/db mice treated with a compound of the invention.

Fig. 7A and 7B show the change in body weight and heart weight of animals treated with the compounds of the present invention.

Figure 8 shows reduction of visceral fat in animals treated with compounds of the invention.

Figures 9A, 9B and 9C show that both body weight gain and abdominal fat pad weight were reduced in an animal model of diet-induced obesity following treatment with a compound of the invention.

FIGS. 10A and 10B show expression of genes encoding Inducible Nitric Oxide Synthase (iNOS) and adrenomedullin following treatment with a compound of the invention.

Figure 11 shows blood triglyceride levels in animals treated with compounds of the invention.

FIGS. 12A and 12B show the stability of HIF-1 α in cells treated with compounds of the invention.

Description of the invention

Before the present compositions and methods are described, it is to be understood that this invention is not limited to the particular methodologies, procedures, cell lines, assays, and reagents described, as these may vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments of the invention, and is not intended to limit the scope of the present invention, which will be limited only by the appended claims.

It must be noted that, as used herein and in the claims, the singular forms "a," "an," and "the" include plural references unless the context clearly dictates otherwise. Thus, for example, reference to a "fragment" includes a plurality of such fragments, as described herein and known to those of skill in the art; reference to a "compound" means a reference to one or more compounds and equivalents thereof.

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, the preferred methods, devices, and materials are now described. All publications cited herein are incorporated herein by reference for the purpose of describing and disclosing the methodologies, reagents, and tools reported in the publications that might be used in connection with the invention. Nothing herein is to be construed as an admission that the invention is not entitled to antedate such disclosure by virtue of prior invention.

The practice of the present invention will employ, unless otherwise indicated, conventional methods of chemistry, biochemistry, molecular biology, cell biology, genetics, immunology and pharmacology, which are within the skill of the art. These techniques are explained fully in the literature. See, e.g., Remington's pharmaceutical sciences, edited by Gennaro, A.R, 18 th edition, 1990, Mack Publishing co.; hardman, j.g., limpird, L.E and Gilman, a.g. eds, Pharmacological Basis of therapy (The Pharmacological Basis of Therapeutics), 10 th edition, 2001; McGraw-Hill Co; colowick, S et al, Methods In Enzymology, academic Press, Inc.; experimental Immunology manuals compiled by Weir, D.M and Blackwell, C.C (Handbook of experimental Immunology), Vol.I-IV, 1986, Blackwell Scientific Publications; molecular Cloning, A Laboratory Manual, second edition, Vol.I-III, 1989, Cold Spring Harbor Laboratory Press, compiled by Maniatis, T et al; ausubel, F.M et al eds, molecular biology techniques: detailed laboratory procedures (Molecular Biology Techniques: An Intensive laboratory Course), 1998, Academic Press; newton, c.r., and Graham, a. eds, Introduction to the biotechnology Series (Introduction to Biotechniques Series), second edition, 1997, Springer Verlag.

Definition of

The term "glucose regulation" or "regulation of glucose metabolism" as used herein refers to the process by which a cell, tissue, organ system or whole organism maintains glucose homeostasis by altering, for example, the increase or decrease of specific processes of glucose metabolism. Glucose metabolism or glucose metabolic processes include processes involving glucose synthesis, processing, transport, uptake, utilization, or storage, and include gluconeogenesis and glycolysis. Specific aspects of glucose metabolism and regulation include the expression of glucose transporters or enzymes that promote the passage of glucose across cell membranes and the retention or secretion of glucose by cells; alterations in the expression and/or activity of enzymes involved in glucose utilization and formation, such as glycolytic and gluconeogenic enzymes; and changes in glucose distribution in vivo or in culture, including, for example, in the interstitium (i.e., extracellular) and intracellular fluids, blood, urine, and the like.

The term "homeostasis of glucose" refers to the maintenance, restoration or attainment of normal or near-normal blood glucose levels. Control of blood glucose may also be referred to as normalization of glycated hemoglobin levels in an organism.

The terms "metabolic disease" and "metabolic disorder" are used interchangeably and refer to any disease associated with or exacerbated by impaired or altered glucose regulation or glycemic control (e.g., insulin resistance). Such disorders include, but are not limited to, diabetes, hyperglycemia, obesity, and the like.

The term "hyperglycemia" as used herein generally refers to higher than normal blood glucose levels. Hyperglycemia can be determined by any method accepted and used by those skilled in the art. Normal human blood glucose levels are currently considered to be between about 70-120mg/dl, but vary depending on the fasting state. Pre-prandial blood glucose may range from about 80-120mg/dl, while two hours post-prandial blood glucose may be about 180mg/dl or less. In addition, fasting subjects have normal blood glucose below about 110 mg/dl. Those with blood glucose levels of about 126mg/dl or higher are generally considered hyperglycemic, and those with blood glucose levels above about 200mg/dl are generally considered diabetic.

The term "obesity" refers to excess fat in the body. Obesity can be determined by any method accepted and used by those skilled in the art. The currently accepted measure of obesity is Body Mass Index (BMI), a measure of body weight in kilograms compared to the square of height in meters. Generally, the BMI of an adult over the age of 20 is considered normal between 18.5 and 24.9; BMI between 25.0 and 29.9 is considered overweight; BMI above 30.0 is considered obese and BMI at 40 or above 40 is considered morbidly obese (see, e.g., Gallagher et al (2000) Am J Clin Nurt 72: 694-701). These BMI ranges depend on the effect of body weight on the increased risk of disease. Some common diseases associated with overweight and obesity include cardiovascular disease, hypertension, osteoarthritis, cancer, and diabetes. Although BMI is associated with body fat, the relationship between BMI and exact body fat varies with age and gender. For example, women with the same BMI may have a higher percentage of body fat than men.

The body fat percentage is another measure of obesity various methods exist for indirectly measuring the body fat of poplars, including: skin wrinkle determination, water density determination, bio-resistance analysis (BIA), dual energy X-ray absorption determination, total body potassium determination, and in vivo nitrogen activation determination. Water density determination, or water (middle) static determination (HW) the total body volume is determined by determining the difference between the weight of the subject in water and in air. Similarly, air displacement plethysmographs (AP) determine total body volume by determining the reduction in chamber volume caused by placing a subject in a chamber with a fixed air volume. And then calculating the total density and composition of the organism by using a verified prediction formula. BIA estimates the resistance or current impedance as a voltage drop caused by a small current passing between the two electrodes. An indication of the water and electrolyte composition of the body, the level of impedance, can be used to estimate lean tissue content and body water volume from developed regression equations. Other regression equations may be used to estimate body meat and fat mass assuming a moisture content of the meat tissue. The percentage of body fat in women is usually about 17-27%, although up to 31% is considered acceptable. The percent of male body fat should generally be between 0-20%, although up to 25% is considered acceptable.

The term "HIF α" refers to the α subunit of hypoxia inducible factor protein. HIF α can be a human or other mammalian protein, or fragment thereof, including human HIF-1 α (Genbank accession No. Q16665), HIF-2 α (Genbank accession No. AAB41495), and HIF-3 α (Genbank accession No. AAD22668); mouse HIF-1 α (Genbank accession No. Q61221), HIF-2 α (Genbank accession Nos. BAA20130 and AAB41496), and HIF-3 α (Genbank accession No. AAC72734); rat HIF-1 α (Genbank accession No. CAA70701), HIF-2 α (Genbank accession No. CABAC96612), and HIF-3 α (Genbank accession No. CABAC96611); and bovine HIF-1 α (Genbank accession No. BAA78675). HIF α can also be any non-mammalian protein or fragment thereof, including Xenopus laevis HIF-1 α (Genbank accession No. CAB96628), Drosophila melanogaster HIF-1 α (Genbank accession No. JC4851), and chicken HIF-1 α (Genbank accession No. NoBAA 34234). The HIF α gene sequences may also be obtained by conventional cloning techniques, e.g., using all or a portion of the HIF α gene sequences described above as probes to find HIF α gene sequences in other species.

Fragments of HIF α comprise the amino acid 401-603 region of human HIF-1 α (Huang et al, supra), the amino acid 531-575 region (Jiang et al, J Bio chem.272: 19253-260, 1997), the amino acid 556-575 region (Tanimoto et al, supra), the amino acid 557-571 region (Srinivas et al, Biochem Biophys ResCommun.260: 557-561, 1999), the amino acid 556-575 region (Ivan and Kaelin, science.292: 464-468, 2001). In addition, fragments of HIF α include any fragment containing at least one LXXLAP motif, e.g., L found in the native sequence of HIF α₃₉₇TLLAP and L₅₅₉EMLAP. Furthermore, fragments of HIF α include any fragment that retains at least one of the characteristic functions and structures of HIF α. For example, the HIF peptide for use in the screening assay of example 14 may comprise DLDLEMLAPYIPMDDDFQL (SEQ ID NO: 5).

The term "HIF hydroxylase" refers to any enzyme that is capable of hydroxylating an amino acid residue in a HIF α protein, particularly the HIF α subunit. The residues are preferably proline and/or asparagine residues.

The term "HIF asparaginyl hydroxylase" refers to any enzyme that is capable of hydroxylating any asparagine residue in the HIF protein, and preferably asparagine residues hydroxylated by HIF asparaginyl hydroxylase include: for example, the N803 residue of HIF-1 α or a homologous asparagine residue in other HIF α isoforms. HIF asparaginyl hydroxylases include the inhibition of HIF Factor (FIH), the HIF asparaginyl hydroxylases responsible for the regulation of HIF α transactivation (Genbank accession No. NoAAL 27308; Mahon et al Gene Dev.15: 2675-.

The terms "HIF prolyl hydroxylase" and "HIF PH" refer to any enzyme that is capable of hydroxylating a proline residue in the HIF protein. Preferably by HThe hydroxylated proline residue of IF PH includes the proline in the motif LXXLAP, e.g., L in the native sequence of human HIF-1 α₃₉₇TLLAP and L₅₅₉Proline from EMLAP. HIF PH includes the Egl-Nine (EGLN) Gene family members described by Taylor (Gene 275: 125-132, 2001) and characterized by Aravin and Koonin (Genome Biol 2: RESEARCH0007, 2001), Epstein et al (cell.107: 43-54, 2001) and Bruick and McKnight (Science 294: 1337-1340, 2001). Examples of HIF PH enzymes include human SM-20(EGLN1) (Genbank accession No. AAG 33965; Dupuy et al. Genomics 69: 348-54, 2000), EGLN2 isoform 1(Genbank accession No. CACC42510; Taylor, supra), EGLN2(Genbank accession No. NP-060025) and EGLN3(Genbank accession No. CACC42511; Taylor, supra); mouse EGLN1H (Genbank accession No. CACACC42515), EGLN2(Genbank accession No. CACC42511) and EGLN3(SM-20) (Genbank accession No. CACC42517) and rat SM-20(Genbank accession No. AAA19321). In addition, HIF PH may include the C.elegans (Caenorhabditis elegans) EGL-9(Genbank, accession No. AAD5665) and Drosophila melanogaster (Drosophila melanogaster) CG1114 gene products (Genbank accession No. AAF52050). HIF PH also includes any active fragment of the full-length proteins described above.

As used herein, a "sample" may be produced from any source, such as a bodily fluid, an exudate, a tissue, a cell, or a cultured cell, including but not limited to: saliva, blood, urine, serum, plasma, vitreous fluid, synovial fluid, cerebrospinal fluid, amniotic fluid and organ tissue (e.g., biopsy tissue); chromosomes, organelles, or other membranes isolated from cells; genomic DNA, cDNA, RNA, mRNA, etc.; and cleared cells or tissues, or prints or printouts of such cells or tissues. The sample may be generated from any source, such as a human non-human mammalian subject. Samples from any animal model of disease are also contemplated. The sample may be liquid or may be immobilized or bound to a matrix. The sample may be any substance suitable for determining the presence or absence of transcripts or proteins involved in the regulation of metabolism, or determining the level of fat and glucose. The methods for obtaining such samples are within the level of skill in the art.

A "subject" as used herein may include isolated prokaryotic or eukaryotic cells, or cultured tissue. Preferably the subject comprises an animal, particularly a mammal, including rat, rabbit, cow, sheep, pig, horse and primate, particularly a human.

Disclosure of Invention

The present invention provides methods and compounds for treating or preventing diabetes, hyperglycemia, and other diseases associated with altered or impaired glucose metabolism and/or in vivo stability. Also provided are methods and compounds for treating, preventing, and delaying the onset and/or progression of diabetes and other diseases associated with altered or impaired glucose metabolism and/or in vivo stabilization, as such methods and compounds either modulate glucose metabolism and achieve in vivo stabilization of glucose.

The present invention relates to the discovery that stabilizing the alpha subunit of hypoxia inducible factor (HIF α) results in lowering blood glucose levels. The present invention also relates to the discovery that stabilizing HIF α regulates glucose metabolism. In addition, the present invention relates to the discovery that the compounds of the present invention can be used to lower blood glucose levels, regulate glucose metabolism, and achieve glucose homeostasis.

Hypoxia Inducible Factor (HIF) is a physiological factor involved in the activities of diverse biological pathways. HIF α is degraded in normal oxygen environments. Under hypoxic or low oxygen conditions, HIF α stabilizes and exerts several downstream effects. It has recently been determined that hydroxylation of specific residues in the HIF α subunit, which leads to directed degradation of HIF α and thus prevents the formation of stable HIF complexes in normoxic environments, is due to activation of certain HIF hydroxylases (see, e.g., Ivan and Kaelin. Science 292: 464-. These HIF hydroxylases belong to the 2-oxoglutarate dioxygenase family. These enzymes are oxygen-dependent, and hydroxylation of HIF α residues is inhibited under hypoxic or anoxic conditions. The therapeutic stabilization of HIF α and stabilization of HIF α by inhibition of hydroxylation of HIF α have been described (see International publication No. WO 03/049686, incorporated herein by reference).

Method

In one aspect, the invention provides methods for lowering blood glucose levels or regulating glucose metabolism by stabilizing the alpha subunit of hypoxia inducible factor (HIF α) in a subject. In another aspect, the methods reduce blood glucose levels or regulate glucose metabolism by inhibiting hydroxylation of HIF α in a subject. In a preferred aspect, the present methods comprise methods for decreasing blood glucose levels or regulating glucose metabolism by inhibiting HIF hydroxylase activity in a subject. In a most preferred aspect, the method comprises decreasing blood glucose levels or regulating glucose metabolism by inhibiting the activity of a HIF prolyl hydroxylase enzyme.

Stabilization of HIF α can be performed by any of the methods known and understood by those of skill in the art, and can include the use of any agent that interacts with, binds to, or modifies HIF α, or factors that interact with HIF α, including, for example, enzymes for which HIF α is a substrate. In certain aspects, the present invention provides structurally stable HIF α variants, such as stable HIF muteins and the like, or polynucleotides encoding certain variants (see, e.g., U.S. patent nos. 6,562,799 and 6,124,131 and 6,432,927). In other aspects, the present invention contemplates stabilizing HIF α comprising administering an agent that stabilizes HIF α. The agent may consist of a polynucleotide, such as an antisense sequence (see International publication WO 03/045440); a polypeptide; an antibody; other proteins; a carbohydrate; fat; a lipid; and organic and inorganic substances such as small molecules and the like. In a preferred embodiment, the present invention stabilizes HIF α by administering to the subject an agent that stabilizes HIF α, wherein the agent is a compound, e.g., a small molecule compound that stabilizes HIF α, etc.

In other embodiments, the methods of the invention comprise stabilizing HIF α by inhibiting the activity of at least one enzyme selected from the 2-oxoglutarate dioxygenase family. In a preferred embodiment, the enzyme is a HIF hydroxylase, such as EGLN-1, EGLN-2, EGLN-3 (see Taylor. Gene 275: 125-132, 2001; Epstein et al, Cell 107: 43-54, 2001; Bruick and McKnight scienne 294: 1337-1340, 2001). However, it is specifically contemplated that the enzyme is selected from the group consisting of enzymes of the 2-oxoglutarate dioxygenase family, including for example: procollagen lysyl hydroxylase, procollagen prolyl 3-hydroxylase, procollagen prolyl 4-hydroxylase α (I) and α (II), thymine 7-hydroxylase, asparagine β -hydroxylase, epsilon-N-trimethyllysine hydroxylase, and gamma-butyrobetaine hydroxylase, and the like. (see, e.g., Majamaa et al, Biochem J.229: 127-.

In certain embodiments, the methods comprise reducing blood glucose levels or regulating glucose metabolism by inhibiting the hydroxylation of certain residues of HIF α, such as proline residues, aspartic acid residues, and the like. In a preferred embodiment, the residue is a proline residue. In particular embodiments, the residue is P in HIF-1 α₅₆₄Residues or homologous proline in other HIF α isoforms, or P in HIF-1 α₄₀₂Residues or homologous proline in other HIF α isoforms, and the like. In other embodiments, the present methods may comprise inhibiting an asparagine residue of HIF α, e.g., N of HIF-1 α₈₀₃Hydroxylation of residues or homologous asparagine residues in other HIF α isoforms.

Compound (I)

In one aspect, the invention provides methods for lowering blood glucose levels or regulating glucose metabolism by administering to a subject a compound of the invention. The compounds of the present invention may be any compound that inhibits or modulates 2-oxoglutarate dioxygenase activity. 2-oxoglutarate dioxygenase enzymes include, but are not limited to, hydroxylases. Hydroxylases that hydroxylate target substrate residues include, for example: prolyl, lysyl, aspartyl (asparagine) hydroxylase, and the like. Hydroxylases are sometimes described by substrates, e.g., HIF hydroxylase, procollagen hydroxylase, and the like; and/or by a target residue in the substrate, such as prolyl hydroxylase, lysyl hydroxylase, and the like; or by both, e.g., HIF prolyl hydroxylases, procollagen prolyl hydroxylases, etc. Representative 2-oxoglutarate dioxygenase enzymes include, but are not limited to, HIF hydroxylases, including HIF prolyl hydroxylases, e.g., EGLN1, EGLN2, and EGLN3, HIF asparaginyl hydroxylases, e.g., HIF inhibitory Factor (FIH), and the like; procollagen hydroxylases such as procollagen lysyl hydroxylases, procollagen prolyl hydroxylases such as procollagen prolyl 3-hydroxylase, procollagen prolyl 4-hydroxylase α (I) and α (II), etc.; thymine 7-hydroxylase; asparagine beta-hydroxylase; epsilon-N-trimethyllysine hydroxylase; gamma-butyrobetaine hydroxylase, and the like. While the enzymatic activity may include activity associated with any 2-oxoglutarate dioxygenase enzyme, it is specifically contemplated to be capable of hydroxylating amino acid residues in the substrate. Although hydroxylation of proline and/or aspartic acid residues in the substrate is specifically included, hydroxylation of other amino acids is also contemplated.

In certain embodiments, the compounds of the invention are compounds that inhibit hydroxylase activity. In a preferred embodiment, a compound of the invention is a compound that inhibits HIF hydroxylase. In various embodiments, the activity is due to a HIF prolyl hydroxylase enzyme, e.g., EGLN1, EGLN2, or EGLN3, and the like. In other embodiments, the activity is due to a HIF asparagine hydroxylase, such as, but not limited to, FIH.

In one aspect, a compound of the invention that exhibits inhibitory activity against one or more 2-oxoglutarate dioxygenase enzymes may also exhibit inhibitory activity against one or more other 2-oxoglutarate dioxygenase enzymes, e.g., a compound that inhibits HIF hydroxylase activity may also inhibit collagen prolyl hydroxylase; a compound that inhibits HIF prolyl hydroxylase may also inhibit the activity of HIF asparaginyl hydroxylase.

In one aspect, the invention provides methods for lowering blood glucose levels or regulating glucose metabolism by administering to a subject a compound of the invention. The compounds of the invention are small molecule compounds that inhibit HIF hydroxylase activity. Preferred compounds of the invention are those that inhibit HIF prolyl hydroxylase activity. Can inhibit directly or indirectly, and can be competitive or non-competitive inhibition, etc. In particular, the compounds of the invention and methods for identifying other compounds of the invention are provided.

Glucose

Under normal conditions, glucose is the main energy source supplied by the body to peripheral tissues. Brain and other nervous tissues normally require glucose as the sole energy source, even in stressful situations, such as chronic fasting, where large amounts of glucose are required. The liver is a major organ that regulates blood glucose levels, preventing blood glucose level from decreasing during fasting, by decomposing stored glycogen or synthesizing glucose from precursors such as lactic acid, pyruvic acid, glycerol, and amino acids. Maintaining glucose homeostasis, such as glucose homeostasis, requires a balance between hepatic glucose production and peripheral glucose uptake and utilization.

The in vivo stability of blood glucose, the maintenance of glucose homeostasis, is closely controlled and influenced by a number of biochemical factors, including various physiological processes. To achieve glucose homeostasis, the body regulates glucose from several levels, including uptake, transport, storage, processing, synthesis, utilization, etc. of glucose. Glucose homeostasis is therefore affected by a number of factors, including, for example: extrinsic factors such as physiological need, food intake, etc.; and intrinsic factors such as circulating levels of insulin, glycogen, etc.

Elevated glucose levels

Disruption of normal regulation of glucose can lead to changes in blood glucose levels, i.e., an increase or decrease in blood glucose levels compared to normal blood glucose levels. For example, chronic elevated blood glucose levels are characteristic of hyperglycemia, diabetes, and the like, and can produce a variety of adverse effects on various organs, tissues, and systems of the body. Diabetes, hyperglycemia, or elevated blood glucose levels are associated with a number of diseases and conditions, including accelerated atherosclerosis, increased chronic heart disease, myocardial infarction, stroke, microangiopathy, vascular injury, peripheral vascular disease leading to decreased circulation in the arms and legs, macrovascular complications, ophthalmic diseases such as diabetic retinopathy handsome, macular degeneration, cataracts, and the like; renal diseases include diabetic nephropathy, renal injury, etc.; nerve damage and other neuropathies include diabetic neuropathy, peripheral neuropathy, damage to autonomic nervous system nerves, and the like, hyperinsulinemia, hyperlipidemia, insulin resistance, impaired glucose metabolism, impaired glucose tolerance, skin and connective tissue disease, foot wounds and ulcers, diabetic ketoacidosis, and the like.

Changes or impairment of glucose regulation, and the presence or risk of developing diabetes, hyperglycemia, etc., can be identified by measuring circulating glucose levels or measuring blood/plasma glucose levels. Blood glucose levels are most commonly determined by fasting blood glucose determinations, random blood glucose determinations, or oral glucose tolerance tests.

Blood glucose levels were determined by fasting blood glucose testing. In such an assay, the measured value reflecting fasting (i.e., no food or liquid other than water) blood glucose levels is typically maintained between about 70mg/dL and 110 mg/dL. If blood glucose levels rise above this typical range in the fasting state, e.g., to levels of 126mg/dL or higher, a diagnosis of diabetes can be made. In one embodiment, the present invention provides compounds and methods that maintain fasting blood glucose levels at or near normal fasting blood glucose levels, i.e., between 70mg/dL and 110 mg/dL. In another embodiment, the present invention provides compounds and methods that elevate or restore fasting low blood glucose levels (i.e., lower than normal fasting blood glucose levels) to between 70mg/dL and 110 mg/dL. In another embodiment, the present invention provides compounds and methods that lower (reduce) or restore elevated fasting blood glucose levels (i.e., blood glucose levels above normal fasting levels) to below about 126mg/dL and about 70mg/dL, more preferably about 120-70mg/dL, and most preferably about 110-70 mg/dL.

Another method of determining blood glucose levels is a random blood glucose test. Blood glucose levels determined in this manner typically exhibit values ranging from low to moderate 100 seconds (mg/dL). Random blood glucose levels of about 180mg/dL or greater are hyperglycemic, with levels of about 180mg/dL or greater indicating a risk of developing diseases associated with impaired glucose regulation, such as diabetes. Thus, in one aspect, the present invention provides methods and compounds that maintain or achieve normal blood glucose levels, i.e., low to moderate 100 seconds (mg/dL), as determined by a random blood glucose test. In another aspect, the methods and compounds of the invention are useful for restoring blood glucose levels that are lowered below normal levels, i.e., low to moderate 100 seconds (mg/dL), as determined by a random blood glucose test to normal levels. In yet another aspect, the methods and compounds of the present invention are useful for lowering/reducing blood glucose levels above normal, i.e., above low to mid 100 seconds (mg/dL). In various aspects, the methods and compounds are capable of lowering elevated blood glucose levels to levels of about 200-100mg/dL, more preferably to levels of 180-100mg/dL, and most preferably to levels of 150-100 mg/dL.

Oral glucose tolerance tests can also be used to identify whether a subject has or is at risk of having impaired glucose regulation, hyperglycemia, or diabetes. In this test, subjects were given a glass of sugar water overnight and blood glucose levels were measured after a few hours. People with normal glucose tolerance/regulation, such as those without diabetes, have elevated and then rapidly declining blood glucose levels measured after consumption of sugar water. Normal blood glucose levels were read two hours after the sugar water was consumed at less than about 140mg/dL, and all values read between 0 and 2 hours were less than 200 mg/dL. Impaired glucose tolerance is generally diagnosed if the blood glucose level measured in the oral glucose tolerance test is within the range of about 140-199 mg/dL. Diabetes is generally diagnosed if the measured blood glucose level is about 200mg/dL or greater. The methods and compounds of the invention are useful for maintaining, restoring or achieving normal blood glucose levels following oral glucose tolerance testing.

Expression of glucose regulatory factor

In one embodiment, the invention provides compounds and methods that increase the expression of genes whose products are involved in cellular glucose uptake and utilization. Such genes include, but are not limited to: glucose transporters, such as glucose transporter (GluT) -1 and GluT-3; glucose glycolytic enzymes such as aldolase-A, enolase-1, hexokinase-2, phosphofructokinase-L and phosphofructokinase-P. Therapeutic upregulation of glucose transporters and their utilization will be effective in reducing insulin resistance, lowering blood glucose levels, and thus producing a beneficial effect in patients with metabolic disorders, such as type 2 diabetes, hyperglycemia, impaired glucose control, impaired glucose tolerance, and the like.

In one aspect, the invention provides compounds and methods for treating or preventing hyperglycemia. Such compounds and methods are suitable for treating or preventing hyperglycemia-related diseases, such as elevated blood glucose levels due to increased glucose release, decreased glucose utilization, and/or impaired glucose uptake. These diseases are also associated with insulin resistance, impaired glucose resistance, type 2 diabetes and/or obesity.

In one aspect, the methods of the invention provide a means for activating gene expression libraries that modulate levels and activities of glucose and glucose metabolism, including systemic processing and utilization. The method can compensate for the body's deficiency in regulating the natural mechanisms of such processing (e.g., failure to produce insulin in response thereto). The methods provided herein can treat metabolic diseases or disorders associated with impaired glucose control. These diseases include, but are not limited to: impaired Glucose Tolerance (IGT) or pre-diabetes, hyperglycemia, and the like.

The present invention specifically contemplates the selective design of prodrug compounds that can be activated when ingested by a particular organ. For example, since the liver is capable of producing many proteins involved in fat homeostasis, the present invention contemplates selectively targeting the liver in the methods of the invention. Selective up-regulation of certain genes in the liver, such as the aldolase gene, can be achieved using compounds that can be converted from inactive to active by liver-specific enzymes. For example, the carboxylic acid which acts on an active compound can be replaced by the corresponding alcohol. Alcohol Dehydrogenase (ADH) activity in the liver can convert such compounds into an active form. Because other organs lack ADH activity, the compound is selectively activated only in the liver. Similarly, the compounds used in the methods of the invention may be targeted to other organs, such as adipose tissue, kidney, skeletal muscle, heart, and the like.

Insulin

Insulin is a key hormone that regulates the metabolic balance of most cells in the body and stimulates glucose uptake. In the presence of insulin, most cells use glucose as their metabolic fuel, adipocytes use glucose to synthesize fat, and hepatocytes convert glucose to glycogen and fat. Blood insulin rises immediately followed by blood glucose rises, and several activities associated with glucose uptake stimulate insulin secretion. When blood glucose levels subsequently decrease, insulin release rapidly decreases and glucose entry into cells outside the nervous system is inhibited. Cells use glycogen and fat as metabolic fuels when glucose is not supplied. The liver and adipocytes begin to break down stored glycogen and fat. As a result, the liver supplies glucose to the blood rather than ingesting glucose from the blood, and both the liver and adipose tissue supply fatty acids to the blood. Thus, low levels of insulin reduce glucose uptake by insulin-sensitive tissues, promote gluconeogenesis and glycogenolysis (glycogen breakdown) in the liver, reduce glycogen synthesis, and promote metabolism of stored glycogen and fat.

The body's failure to produce insulin, or an unresponsiveness to insulin, such as reduced insulin sensitivity, i.e., insulin resistance, etc., can lead to a variety of diseases, including diabetes and hyperglycemia.

Impaired glucose transport is responsible for insulin resistance. The methods of the invention may reduce insulin resistance to restore impaired glucose transport or increase glucose transport. The present invention provides methods of reducing or diminishing insulin resistance. In certain aspects, insulin resistance is reduced by stabilizing HIF α. In other aspects, methods of reducing insulin resistance by inhibiting HIFPH activity are provided.

Increased insulin sensitivity is positively correlated with high plasma levels of insulin-like growth factor binding protein-1 (IGFBP-1). IGFBP-1 plasma levels are also inversely correlated with body mass index in adolescents (Travers et al, J Clin Endocrinol Metab.83: 1935-. In addition, low levels of IGFBP-1 are associated with increased cardiovascular risk of type 2 diabetes (Gibson et al J Clin Endocrinol Metab 81: 860-863, 1996). Thus, there is a need to increase the level of IGFBP-1 in restoring or maintaining insulin sensitivity, i.e., treating insulin resistance.

In addition, gene defects affecting phosphofructo-1-kinase (PFK) can lead to insulin resistance and type 2 diabetes. Since PFK is a rate-limiting enzyme in the glycolytic cascade, the decrease in this enzymatic activity in the glycolytic cascade is often clearly associated with insulin resistance and type 2 diabetes. Thus, increasing the levels of PFK and other glycolytic factors, such as aldolase, enolase, hexokinase, and the like, is desirable to restore or maintain insulin sensitivity, such as decreasing insulin resistance.

The compounds of the invention were shown to increase the expression of IGFBP-1, PFK and other glycolytic enzymes (see example 4). Thus, in one embodiment, the invention provides methods and compounds for the synergistic expression of certain genes, the products of which, e.g., IGFBP-1, PFK, etc., are involved in glucose processing and utilization. In another embodiment, the present invention provides methods and compounds for increasing insulin sensitivity, such as decreasing insulin resistance, by co-expressing such genes. In one aspect, the methods comprise, e.g., stabilizing HIF α in the subject. The invention also provides methods for increasing insulin sensitivity by administering to a subject a compound of the invention, e.g., an agent that inhibits HIF hydroxylase activity.

Glycosylated hemoglobin

Results from the Diabetes Control and Complications Trial (DCCT) demonstrated that improvements in glycemic control reduced complications, including nonproliferative and proliferative retinopathy (47% reduction), microalbuminemia (39% reduction), clinical nephropathy (54% reduction), and neuropathy (60% reduction) (DCCT research group, N Engl J Med 329: 977-. In addition, the United Kingdom Prognostic of Diabetes Study (UKPDS) demonstrated that glycemic control was associated with reduced microvascular complications, and strict blood pressure control significantly reduced both macrovascular and microvascular complications (UKPDS group, Lancet 352: 837-.

Glycated hemoglobin (also known as glycohemoglobin, glycosylated hemoglobin, HbA1c, or HbA1) can be formed by binding various sugar molecules (most commonly glucose) to hemoglobin molecules, the rate of formation being proportional to blood glucose concentration. The measurement of glycated hemoglobin levels provides an accurate index of the mean blood glucose concentration for the first 2-3 months. Clinically glycated hemoglobin levels provide an assessment of glycemic control in diabetic patients. Normal (non-diabetic) glycated hemoglobin levels range from 4-6%. In a study of diabetic individuals, DCCT found that a reduction or maintenance of HbA1c levels to 7.2% resulted in 76% reduction in retinopathy, 60% reduction in neuropathy, 50% reduction in nephropathy, and 35% reduction in cardiovascular disease compared to diabetic individuals with higher levels of HbA1 c.

The present invention provides compounds and methods for reducing glycated hemoglobin levels. In one embodiment, the methods and compounds of the present invention are used to maintain, restore or bring glycated hemoglobin levels to about 4-6%. In another embodiment, the methods and compounds of the present invention can reduce glycated hemoglobin levels by less than about 9%, more preferably less than about 8%, and most preferably less than about 7%.

Diabetes and obesity

Obesity is a risk factor and sometimes a malignant side effect of diabetes. Obesity is characterized by excessive fat accumulation, particularly by excessive visceral or central fat levels. Thus, treatment or prevention of obesity can minimize the risk of developing diabetes, and indeed weight loss is often prescribed for diabetic patients or individuals diagnosed as at risk for diabetes.

The compounds of the invention have been shown to prevent or slow down weight gain in vivo studies (see examples 9 and 10). Thus, in one aspect, the present invention provides methods for treating or preventing diabetes by reducing or preventing obesity. In one aspect, the methods comprise, e.g., stabilizing HIF α in the subject. The invention also provides methods for treating or preventing obesity in a subject by administering to the subject a compound of the invention, e.g., an agent that inhibits HIF hydroxylase activity.

As noted above, diabetes is associated with a variety of diseases and conditions. For example. Cardiovascular disease (CVD) is a leading cause of death in type 2 diabetes patients. Type 2 diabetics die 2-6 times more of CVD than non-diabetic patients. A significant number of these patients die from Chronic Heart Disease (CHD). Hyperlipidemia is common in type 2 diabetic patients and causes CHD. A common lipid profile in type 2 diabetic patients is higher triglycerides and lower HDL cholesterol compared to non-diabetic individuals. Similar triglycerides and HDL cholesterol abnormalities are common in obese (especially in individuals with increased visceral fat), hypertensive and insulin-resistant (e.g., metabolic syndrome) non-diabetic individuals. Elevated triglyceride levels have been shown to be a risk factor for cardiovascular disease. Elevated triglycerides are an important component of metabolic syndrome.

Diabetes and hypertension

Hypertension, or elevated blood pressure, is a risk factor for diabetes. In addition, the effects of hypertension and its related diseases may be accompanied or cause diabetes. Therefore, treatment or prevention of hypertension would minimize diabetes or the risk of developing diabetes, and a therapeutic approach that emphasizes this aspect of diabetes would be valuable.

The present invention provides such a method. In particular, the methods and compounds of the present invention are useful for treating hypertension associated with diabetes. For example, the compounds of the present invention can increase the expression of blood pressure regulators such as adrenomedullin and nitric oxide synthase (see example 12). Thus, in one aspect, the invention provides a method of treating or preventing diabetes by reducing or preventing hypertension. In one aspect, the methods comprise, e.g., stabilizing HIF α in the subject. The invention also provides methods for treating or preventing hypertension in a subject by administering to the subject a compound of the invention, e.g., an agent that inhibits HIF hydroxylase activity.

Synergistic therapeutic method

Diabetes is associated with a number of deleterious, simultaneous or progressive, overlapping and/or sequential diseases. For example, hypertension, vascular and circulatory impairment, obesity can lead to an increased risk of developing diabetes. Therefore, therapeutic approaches that simultaneously reduce such risk factors and symptoms would be valuable.

The present invention provides such a method. In particular, the methods and compounds of the present invention can be used to achieve a variety of effects. For example, as mentioned above, obesity is a risk factor for the development of diabetes. In addition, obesity can occur as a result of diabetes, for example, due to specific treatment regimens. In particular, insulin mediates lipid uptake into adipose tissue, aggravating obesity and, in turn, insulin resistance. The present invention therefore provides in one aspect a method for treating or preventing diabetes by treating or preventing obesity and increasing insulin sensitivity in a synergistic manner.

The compounds of the present invention can lower blood glucose levels (see example 6), reduce visceral and abdominal fat (see example 9), increase expression of glycation enzymes to increase insulin sensitivity (see example 4), and increase expression of blood pressure regulating factors (see example 12), thus regulating vascular tone of the body, maintaining normal blood pressure levels or acting on changes in anti-vascular tone such as hypertension, etc. Accordingly, in one aspect, the present invention provides methods for treating or preventing diabetes, the methods comprising stabilizing HIF α in a subject. To lower blood glucose levels and reduce visceral fat. Another method further comprises increasing insulin sensitivity to treat or prevent diabetes in the subject. Another method further comprises increasing the expression of a blood pressure regulatory factor to treat or prevent diabetes in the subject.

Metabolic diseases

The present invention provides compounds that modulate metabolic activity and methods of using the compounds to treat diseases or disorders associated with metabolic disorders. Such diseases include, but are not limited to: diabetes, hyperglycemia, and obesity.

In one aspect, the invention provides a method of preventing or treating diabetes using the compound, the method comprising administering to a patient in need thereof a therapeutically effective amount of the compound or a pharmaceutically acceptable salt thereof, alone or in combination with a pharmaceutically acceptable excipient. In one embodiment, the compound is administered based on a predicted condition, such as impaired glucose homeostasis, impaired glucose tolerance, hyperglycemia, diabetes, or obesity.

In another aspect, the invention provides a method of treating hyperglycemia with the compound, comprising administering to a patient in need thereof a therapeutically effective amount of the compound or a pharmaceutically acceptable salt thereof, alone or in combination with a pharmaceutically acceptable excipient. In one embodiment, the compound is administered to a patient diagnosed as having a disease associated with the development of hyperglycemia, such as diabetes.

The compounds can be administered in combination with various other therapeutic methods. In one embodiment, the compound is used in combination with exogenous insulin, such as synthetic human insulin.

Glucose regulation

The methods and compounds of the invention can enhance the expression of enolase. These results indicate that the methods and compounds of the invention are useful for modulating the expression of genes involved in glycolysis. Methods and compounds for regulating glucose metabolism by increasing the hydroxylation of a sugar are also provided.

The present invention provides methods and compounds for increasing GluT-1 expression. In one aspect, the methods of the invention modulate the expression of factors involved in glucose uptake. The methods and compounds of the invention provide therapeutic methods for increasing glucose transport into cells. Therapeutic upregulation of glucose transport factors will effectively reduce insulin resistance, increase insulin sensitivity, lower blood glucose levels, and thereby have a beneficial effect on hyperglycemic or diabetic patients.

The methods and compounds of the invention can increase the expression of glucose regulators in the kidney, liver and lung, including phosphofructokinase-P, phosphofructokinase-L, aldolase-1, GluT-1, lactate dehydrogenase, enolase-1 and hexokinase-1. In one embodiment, the methods of the invention can coordinate the regulation of the expression of genes whose products are involved in glucose uptake and utilization, thereby regulating glucose metabolism. Therapeutic upregulation of glucose transport and utilization will decrease insulin resistance, increase insulin sensitivity, decrease blood glucose levels, thereby providing a method of treating hyperglycemia or diabetes.

The expression of the gene encoding insulin-like growth factor binding protein-1 (IGFBP-1) in the kidney and liver can be increased using the methods and compounds of the invention. High plasma levels of IGFBP-1 are positively correlated with increased insulin sensitivity. The methods and compounds of the invention are therefore useful for increasing insulin sensitivity and increasing glucose transport, thereby modulating glucose metabolism. Therapeutically increasing insulin sensitivity and glucose transport will lower blood glucose levels, thereby providing a method of treating hyperglycemia or diabetes.

The methods and compounds provided herein can increase glucose uptake by cells. The methods and compounds of the invention increase glucose uptake in the presence of insulin, indicating that the methods and compounds of the invention increase insulin sensitivity of cells, resulting in increased glucose uptake and altered glucose regulation. Therapeutic increases in insulin sensitivity and glucose uptake can be used to treat individuals with decreased insulin sensitivity or pancreatic tolerance, thereby providing a method of treating hyperglycemia or diabetes.

The methods and compounds of the invention are also useful for enhancing insulin-stimulated glucose uptake into tissues. The methods and compounds of the invention are useful for increasing glucose uptake in tissues and increasing their sensitivity to insulin. Increased glucose intake provides a means for lowering blood glucose levels in individuals with elevated blood glucose levels, such as hyperglycemic or diabetic individuals, or individuals with impaired glucose homeostasis. Thus, the methods and compounds of the present invention can treat hyperglycemia and diabetes by increasing pancreatic sensitivity, increasing glucose intake, and lowering blood glucose levels.

Treatment of animals with the compounds of the invention showed a dose-dependent decrease in blood glucose levels. Blood glucose levels can be maintained at desired levels by varying the dosage of the compound. Thus, in one aspect, the methods and compounds of the invention are useful for modulating blood glucose levels. In another aspect, the methods and compounds of the invention are useful for lowering blood glucose levels. The methods and compounds of the invention are therefore useful for therapeutically lowering blood glucose levels. By lowering blood glucose levels, the present invention provides methods for treating hyperglycemia and diabetes. Administration of the compounds of the invention improves glucose clearance from circulation in animal models of diet-induced obesity and impaired glucose tolerance. Increasing glucose clearance lowers blood glucose levels. Thus, in one aspect, the methods of the invention can be used to modulate glucose metabolism in an individual with impaired glucose tolerance by increasing glucose clearance or decreasing blood glucose levels. Therapeutic enhancement of glucose clearance and lowering of blood glucose levels can be used to treat patients, including diabetic patients or patients at risk of developing diabetes.

Treatment of diabetes

The methods and compounds of the invention are capable of lowering blood glucose levels in animal models of diabetes. In addition, the methods and compounds of the invention restore and achieve glucose homeostasis in animal models of diabetes and impaired glucose tolerance. Glycated hemoglobin levels reflect glycemic control and maintenance of glucose homeostasis in diabetic patients or hyperglycemic individuals. Decreasing glycated hemoglobin levels indicates that the methods and compounds of the present invention may be used to alter glucose regulation in a subject, thereby restoring, achieving, or maintaining glucose homeostasis. Thus, the methods and compounds of the present invention are useful for treating hyperglycemia and diabetes by modulating glucose metabolism and restoring, achieving or maintaining glucose homeostasis.

Treatment of animals with the compounds and methods of the invention reduces the accumulation of glycated hemoglobin in animal models of diabetes. Glycated hemoglobin levels reflect the control of blood glucose levels and the maintenance of glucose homeostasis in diabetic or hyperglycemic patients. A decrease in the level of glycated hemoglobin indicates that the compounds and methods of the present invention may be used to alter glucose regulation in a subject, thereby restoring, achieving, or maintaining glucose homeostasis. Thus, the compounds of the present invention may be used in methods for treating hyperglycemia and obesity by modulating glucose metabolism and restoring, achieving, or maintaining glucose homeostasis.

Animals treated with the compounds of the invention show a dose-dependent delay in their weight gain. In particular, dose-dependent reduction of visceral fat pads was observed in animals treated with the compound. Thus, the methods and compounds of the present invention are useful for reducing fat storage and reducing visceral fat. Obesity, particularly obesity with excess visceral, abdominal or central fat, is associated with hyperglycemia and diabetes. Specifically, obesity accompanied by decreased insulin sensitivity, increased insulin resistance, etc. will lead to hyperglycemia and the development of diabetes. In one aspect, the methods and compounds of the invention reduce the risk of developing hyperglycemia or diabetes by reducing visceral fat. In another aspect, the methods and compounds of the present invention reduce the risk of developing hyperglycemia or diabetes by reducing obesity.

Pharmaceutical formulations and routes of administration

The compositions of the present invention may be delivered directly or in a pharmaceutical composition containing excipients, as is well known in the art. The treatment methods of the present invention may comprise administering to a subject suffering from, or at risk of, a metabolic disorder, particularly a glucose regulation related disorder, such as diabetes, hyperglycemia, and the like, a therapeutically effective amount of a compound of the present invention. In a preferred embodiment, the subject is a mammal, and in a most preferred embodiment, the subject is a human.

An effective amount, e.g., dose, of a compound or drug can be readily determined by routine experimentation and can be administered by an effective and routine route and in appropriate formulations. Various formulations and drug delivery systems are available in The field (see Gennaro eds., 2000, Remington's Pharmaceutical Sciences, supra and Hardman, The Pharmacological Basis of therapy compiled by Limbird and Gilman, supra).

Suitable routes of administration may include, for example, oral, rectal, topical, intranasal, intrapulmonary, intraocular, enteral and parenteral administration. The major parenteral routes of administration include intravenous, intramuscular and subcutaneous administration. The second route of administration includes intraperitoneal, intraarterial, intraarticular, intracardiac, intracisternal, intradermal, intralesional, intraocular, intrapleural, intratracheal, intraurethral and intraventricular administration. The indication to be treated should preferably be associated with the physical, chemical and biological properties of the drug, indicating the type of formulation and the route of administration used, and whether local or systemic delivery is desired.

The pharmaceutical dosage forms of the compounds of the present invention may provide for single-release, controlled-release, sustained-release or targeted drug delivery systems. Common dosage forms include, for example, solutions, suspensions, (micro) emulsions, ointments, gels and patches, liposomes, tablets, dragees, soft or hard gelatine capsules, lozenges, ovosomes (ovule), implants, amorphous or crystalline powders, aerosols and lyophilised preparations. Depending on the route of administration used, special devices may be required to apply or administer the drug, for example, syringes and needles, inhalers, pumps, injection pens, applicators or special purpose bottles. Pharmaceutical dosage forms often contain a drug, excipients, and a container/seal system. One or more excipients, referred to as inactive ingredients, may be added to the compounds of the present invention to improve or facilitate manufacturing, stability, drug administration, and safety, and may provide a means for achieving a desired release profile of the drug. Thus, the type of excipient added to the drug depends on various factors, such as the physicochemical properties of the drug, the route of administration, and the method of manufacture. Pharmaceutically acceptable excipients this field already has, including those listed in various pharmacopoeias (see USP, JP, EP and BP, fdah page: (see USP, JP, EP & BP, fdah:)www.fda.gov) Inert Ingredient Guide 1996, and Handbook of Pharmaceutical Additives, compiled by Ash; synapse Information resources inc.2002).

Pharmaceutical dosage forms of the compounds of the present invention may be prepared by any of the methods well known in the art, such as by conventional mixing, sieving, dissolving, melting, granulating, dragee-making, tableting, suspending, extruding, spray-drying, milling, emulsifying, (nano/micro) encapsulating, entrapping or lyophilizing processes. The compositions of the invention as described above may comprise one or more physiologically acceptable inactive ingredients to facilitate processing of the active molecule into preparations for pharmaceutical use.

The appropriate formulation depends on the desired route of administration. For intravenous injection, for example, the composition may be formulated as an aqueous solution, and physiologically compatible buffers, including, for example, phosphoric acid, histidine or citric acid, may be used to adjust the pH of the formulation, and tonicity adjusting agents such as sodium chloride or dextrose may be used, if desired. For transmucosal or intranasal administration, preferably a semisolid, liquid formulation or patch, may contain penetration enhancers, such penetration enhancers being generally known in the art. For oral administration, the compounds may be formulated in liquid or solid dosage forms, and as single-release or controlled/sustained release formulations. Dosage forms suitable for oral administration to a subject include tablets, pills, dragees, hard and soft shell capsules, liquids, gels, syrups, slurries of sugars, suspensions and emulsions. The compounds may also be formulated in rectal compositions such as lozenges or retention enemas, such as enemas containing conventional lozenge bases such as cocoa butter or other glycerin bases.

Excipients may be employed including fillers, disintegrants, binders (dry or wet), dissolution retardants, lubricants, slip promoters, anti-adherents, cation exchange resins, wetting agents, antioxidants, preservatives, coloring agents and flavoring agents. These excipients may be of synthetic or natural origin. Examples of such excipients include cellulose derivatives, citric acid, dicalcium phosphate, gelatin, magnesium carbonate, magnesium/sodium laurate, mannitol, polyethylene glycol, polyvinylpyrrolidone, silicates, silica, sodium benzoate, sorbitol, starch, stearic acid and its salts, sugars (i.e., glucose, sucrose, lactose, etc.), talc, tragacanth mucilage, vegetable oils (hydrogenated), and waxes. Ethanol and water can be used as granulation aids. In some cases, it may be desirable to coat the tablets with an odor-masking film, a gastric acid-resistant film, or a release-retarding film. Tablets are often coated with a combination of natural and synthetic polymers with colorants, sugars, organic solvents, and water to produce dragees. When the coated tablet, powder, suspension or solution is preferably in the form of a capsule, it can be delivered in a compatible hard or soft shell capsule.

In one embodiment, the compounds of the invention may be administered topically, e.g. via a skin patch, a semi-solid open liquid formulation, such as a gel, (small) suspension, ointment, solution, (nano/micro) suspension, or foam formulation. For example, penetration enhancers; lipophilic, hydrophilic and amphoteric excipients, including water, organic solvents, waxes, oils, synthetic and natural polymers, surfactants, pro-emulsifiers, are suitably selected and combined to adjust the penetration of the drug into the skin and underlying tissues by adjusting the pH, using pro-mixing agents. Other techniques, such as iontophoresis, may be used to modulate the penetration of the compounds of the present invention into the skin. Transdermal or topical administration is preferred, for example, where minimal systemic contact is required to deliver the drug locally.

For inhalation or intranasal administration, the compounds of the invention may be conveniently delivered in the form of a solution, suspension, emulsion or semi-solid aerosol in a pressurized pack or as a halocarbon using a propeller-propelled nebulizer, such as methane and ethane, carbon dioxide or any other suitable gas-generating halocarbon. For topical application of the aerosol, butane, isobutane and pentane hydrocarbons may be used. In the case of a pressurized aerosol, the appropriate dosage unit can be determined by providing a valve to deliver a determined amount of aerosol. Capsules and gelatin plugs may be formulated for use in an inhaler or insufflator. These capsules or suppositories usually contain a mixed powder of the compound and a suitable powder base such as lactose and starch.

Sterile compositions formulated for parenteral injection are usually provided in unit dosage form, e.g., in ampoules, syringes, injection pens or multi-dose containers, often containing a preservative. The composition may take the form of a suspension, solution or oily or aqueous emulsion and may contain formulating agents such as buffers, enhancers, viscosity enhancers, surfactants, suspending and dispersing agents, antioxidants, biocompatible polymers, chelating agents and preservatives. Depending on the injection site, the carrier may contain water, synthetic or vegetable oils and/or organic solubilizing agents. In some cases, such as in the case of a lyophilized product or a concentrated product, the parenteral formulation can be reconstituted or diluted prior to administration. Depot formulations for controlled or sustained release of the compounds of the invention may be provided, which may comprise injectable suspensions of nano/micro sized particles or nano/micro sized or non-micronized crystals. Polymers other than those well known in the art, such as poly (lactic acid) poly (glycolic acid), or copolymers thereof or as controlled/sustained release matrices. Other storage and delivery systems may be provided in the form of implants and pumps that require cutting.

Suitable carriers for intravenous injection of the molecules of the invention are well known in the art and include alkaline solutions containing bases such as sodium hydroxide and sucrose or sodium chloride as an enhancer, e.g., buffers containing phosphate or histidine, capable of forming an ionized compound. A co-solvent, such as polyethylene glycol, may be added. These aqueous systems are effective in dissolving the compounds of the present invention and produce only low toxicity for systemic administration. The ratio of the components of the solution system can be varied considerably without destroying their solubility and toxicity characteristics. In addition, the identity of the components may vary. For example, low toxicity surfactants such as polysorbates or poloxamers may be used, polyethylene glycols or other co-solvents, biocompatible polymers such as polyvinylpyrrolidone, other sugars and polyalcohols may be added in place of glucose.

For the compositions used in current methods of treatment, the therapeutically effective dose may initially be estimated by a variety of techniques well known in the art. The initial dose used in animal studies may depend on the effective concentration determined in cell culture assays. The range of dosages suitable for human use can be determined using data obtained from animal studies and cell culture.

A therapeutically effective amount or dose of a compound or drug of the invention refers to an amount or dose of the compound or drug that results in remission or prolonged survival of the subject. Toxicity and therapeutic efficacy of such molecules can be determined by standard pharmaceutical procedures in cell cultures or experimental animals, e.g., by assaying LD50 (the dose that causes death in 50% of the population) and ED50 (the dose that is therapeutically effective in 50% of the population). The dose ratio of toxic to therapeutic effect is the therapeutic index and can be expressed as the ratio LD50/ED 50. Formulations exhibiting high therapeutic indices are preferred.

An effective or therapeutically effective amount is that amount of the compound or pharmaceutical composition that induces a biological or medical response in a tissue, system, animal or human that is believed to be of interest to a researcher, veterinarian, medical doctor or other clinician, e.g., that regulates glucose metabolism, lowers elevated or increased blood glucose levels, or treats or prevents a disease associated with altered glucose metabolism, such as diabetes.

Preferred dosages should fall within the circulating concentration range that includes ED50 with little or no toxicity. The dosage may vary within this range depending upon the dosage form employed and/or the route of administration. The precise formulation, route of administration, dosage and dosage interval will be selected according to the characteristics of the subject's condition according to methods known in the art.

The dosage amounts and intervals may be adjusted individually to provide active ingredients that are sufficient to achieve the desired effect, e.g., modulating glucose metabolism, lowering plasma levels of glucose, etc., i.e., minimizing the effective concentration (MEC). The MEC for each compound is different but can be estimated from, for example, in vitro data and animal experiments. The dose required to achieve MEC depends on the individual characteristics and the route of administration. For topical administration or selective ingestion, the effective local concentration of the drug may not be related to plasma concentrations.

The amount of the agent or composition to be administered may be determined according to various factors including sex, age and weight of the subject to be treated, severity of the disease, mode of administration and judgment of the physician.

If desired, the compositions of the present invention may be contained in a filling or dispensing device containing one or more unit dosage forms containing the active ingredient. For example, such a filling or device may comprise a metal or plastic foil, such as a blister filling or glass, and a rubber stopper, such as a vial. The filling or dispensing device may be provided with instructions for administration. Compositions containing the compounds of the present invention formulated with compatible pharmaceutical carriers may also be prepared and placed in an appropriate container and labeled for treatment of a designated disease.

Compound and method for screening same

The compounds of the present invention are compounds that inhibit hydroxylase activity, specifically 2-oxoglutarate dioxygenase activity. More preferably, the hydroxylase activity is HIF hydroxylase activity. Most preferably, the hydroxylase activity is HIF prolyl hydroxylase activity.

The methods of the present invention are methods that rely on stabilizing HIF α to achieve a particular effect in a subject. The methods of the invention are accomplished by administering to a subject a compound that stabilizes HIF α to achieve a particular effect in the subject. Most preferably, the method is carried out by administering a compound of the invention.

Compounds of the invention are illustratively used in the methods of the invention relating to stabilizing HIF α. In particular, the present invention provides compounds that inhibit hydroxylase activity and/or hydroxylate HIF α, stabilize HIF α, etc., and methods for screening and identifying other compounds. Compounds of the present invention include compounds that inhibit hydroxylase activity, wherein the preferred hydroxylase activity is 2-oxoglutarate dioxygenase activity. More preferably, the hydroxylase activity is HIF hydroxylase activity. HIF hydroxylases hydroxylate HIF proteins, preferably any amino acid in the HIF α subunit, including, for example, proline or aspartic acid residues, and the like. In a particularly preferred embodiment, the hydroxylase activity is the activity of a HIF prolyl hydroxylase and/or a HIF asparaginyl hydroxylase.

Inhibitors of 2-oxoglutarate dioxygenase are known in the art. For example, several small molecule inhibitors of procollagen prolyl 4-hydroxylase have been identified (see, e.g., Majamaa et al, Eur J Biochem 138: 239-. Small molecule inhibitors of HIF hydroxylase have also been identified (see International publication Nos. WO02/074981, WO 03/049686, and WO 03/080566, the contents of all of which are incorporated herein by reference). The present invention specifically contemplates the use of these and other compounds that can be identified using methods known in the art.

All enzymes in the 2-oxoglutarate dioxygenase family require oxygen, Fe2+, 2-oxoglutarate and ascorbic acid for their hydroxylase activity (see Majamaa et al, Biochem J229: 127-. Thus, compounds of the invention include, but are not limited to: iron chelators, 2-oxoglutarate mimetics and modified amino acids such as proline or aspartate congeners.

In a specific embodiment, the present invention provides structural mimetics using 2-oxoglutarate. This compound competitively inhibits the reaction of the target 2-oxoglutarate dioxygenase with 2-oxoglutarate, but not with iron (Majamaa et al 1984, supra; Majamaa et al 1985, supra). Particular consideration is given to using, for example, Majamaa et al, supra; kivirikko and mylyharju, Matrix Biol 16: 357 and 368, 1998; bickel et al, Hepatology 28: 404, 411, 1998; friedman et al, Proc Natl Acad Sci USA 97: 4736 and 4741, 2000; franklin, Biochem Soc Trans 19: 812, 815, 1991; franklin et al, Biochem J353: 333-338, 2001 and International publication number WO 03/049686, the contents of all of which are incorporated herein by reference.

Exemplary compounds include phenanthrolines, which include, but are not limited to, U.S. patents 5,916,898 and 6,200,974; those described in international publication WO 99/21860; heterocyclic carbonylglycerols including, but not limited to, substituted quinoline-2-amides and esters thereof as described in U.S. Pat. Nos. 5,719,164 and 5,726,305; substituted isoquinoline-3-amides and esters thereof as described in U.S. patent 6,093,730; 3-methoxypyridinocarbonylglycerols and esters thereof described in European patent EP0650961 and U.S. patent 5,658,933; 3-hydroxypyridinocarbonylglycerols and esters thereof described in U.S. Pat. Nos. 5,620,995 and 6,020,350; 5-sulfonamidocarbonylpyridine carboxylates and esters thereof described in U.S. Pat. Nos. 5,607,954, 5,610,1725 and 620,996. All compounds listed in these patents, particularly in the claims and in the working examples, are incorporated herein by reference.

Thus, preferred compounds of the invention include, for example, heterocyclic amides. Particularly preferred heterocyclic amides include, for example, isoquinoline, quinoline, pyridine, cinnoline, carboline, and the like. Further, the structural types of preferred compounds include anthraquinones, azafluorenes, azaphenanthrolines, benzimidazoles, benzofurans, benzopyrans, benzothiophenes, catechols, dihydrochromones, alpha-diketones, furans, N-hydroxyamides, N-hydroxyureas, imidazoles, indazoles, indoles, isothiazoles, isoxazoles, alpha-keto acids, alpha-keto amides, alpha-keto esters, alpha-keto imines, oxadiazoles, oxalamides, oxazoles, oxazolines, purines, pyrans, pyrazines, pyrazoles, pyrazolines, pyridazines, pyridines, quinazolines, phenanthrolines, tetrazoles, thiadiazoles, thiazoles, thiazolines, thiophenes, and triazoles.

The following exemplary compounds used in the examples of the invention demonstrate the inventive process described herein: [ (7-chloro-3-hydroxy-quinolyl-2-carbonyl) -amino ] -acetic acid (compound A), [ (1-chloro-4-hydroxy-isoquinolyl-3-carbonyl) -amino ] -acetic acid (compound B), [ (4-hydroxy-7-phenoxy-isoquinolyl-3-carbonyl) -amino ] -acetic acid (compound C), [ (4-hydroxy-7-phenoxy-isoquinolyl-3-carbonyl) -amino ] -acetic acid (compound D), [ (1-chloro-4-hydroxy-7-methoxy-isoquinolyl-3-carbonyl) -amino ] -acetic acid (compound E), [ (3-hydroxy-6-isopropoxy-quinolinyl-2-carbonyl) -amino ] -acetic acid (compound F), [ (3-hydroxy-pyridinyl-2-carbonyl) -amino ] -acetic acid (compound G), and [ (7-benzyloxy-1-chloro-4-hydroxy-isoquinolinyl-3-carbonyl) -amino ] -acetic acid methyl ester (compound H).

Various assays and screening techniques, including those described below, can be used to identify compounds of the invention, i.e., compounds that are capable of producing hydroxylase activity. These compounds are suitable for use in the process of the invention. Other compounds suitable for use in the methods of the present invention, i.e., compounds that stabilize HIF α, can be identified by one of skill in the art using established assays and screening methods.

Assays generally provide a detectable signal associated with the consumption of a reaction substrate, or with the production of a reaction product. The detection may include, for example: fluorophores, radioisotopes, enzyme conjugates, and other detectable labels well known in the art. The results may be quantitative or qualitative. The tag may facilitate isolation of the reaction product, e.g., the biotin or histidine tag may be used to purify the reaction product from other reaction components by precipitation or affinity chromatography.

Assays for measuring hydroxylase activity are standardized in the art. Such assays are capable of directly or indirectly measuring hydroxylase activity. For example, one assay can measure hydroxylated residues, such as proline, aspartic acid, etc., present in enzyme substrates, such as target proteins, synthetic peptidomimetics, or fragments thereof (see Palmernii et al, J Chromatogr 339: 285-292, 1985). Reduction of the presence of hydroxylated proline or aspartic acid in a compound indicates that the compound inhibits hydroxylase activity. Alternatively, the assay can determine other products of the hydroxylation reaction, such as succinic acid formed from 2-ketoglutarate (see Cunliffe et al, Biochem J240: 617-619). Kaule and Gunxler (Anal Biochem 184: 291. sup. 297, 1990) describe an exemplary method for determining the production of succinic acid from 2-ketoglutaric acid.

These methods are useful for identifying compounds that modulate HIF hydroxylase activity. An exemplary method is described in example 14 (see above). Target proteins may include HIF α or fragments thereof, e.g., HIF (556-575); typical substrates for the assay described in example 14 are: DLDLEMLAPYIPMDDDFQL (SEQ ID NO: 5). Such enzymes include, for example, HIF prolyl hydroxylases (see GenBank accession No. AAG33965, etc.), HIF aspartyl hydroxylases (see GenBank accession No. AAL27308, etc.) obtained from any source. The enzyme may be present in a crude lysate of the cells or in partially purified form. For example, methods for directly or indirectly determining HIF hydroxylase activity are described in Ivan et al (Science 292: 464-468, 2001 and ProcNil Acad Sci USA 99: 13459-13464, 2002) and Hirsila et al (J Bio Chem 278: 30772-30780, 2003); other methods are described in international publication number WO 03/049686. Assaying and comparing the enzymatic activity in the absence and presence of the compound identifies a compound that inhibits hydroxylation of HIF α.

Assays for HIF α stability and/or HIF activation can include direct determination of HIF α in a sample (see example 14, supra), indirect determination of HIF α by determining a decrease in HIF α associated with von Hippel Lindau protein (see International publication No. WO00/69908), or activation of a HIF-responsive target gene or reporter construct (see U.S. Pat. No.5,942,434). Determining and comparing the level of HIF and/or HIF-responsive target proteins in the absence and presence of the compound identifies a compound that stabilizes HIF α and/or activates HIF α.

2-oxidation [1-¹⁴C]Glutarate hydroxylation-coupling decarboxylation assays to identify compounds that modulate HIF-specific prolyl hydroxylase activity (see Hirsila et al, J Bio Chem 278: 30772-30780, 2003). The reaction is carried out at 25 ℃ in a reaction volume of 1.0ml, containing 10-100 microliters of a detergent such as Triton-X-100, an extract obtained from lysed cells expressing an endogenous or recombinant HIF prolyl hydroxylase, 0.05. mu. M DLDLEMLAPYIPMDDDFQL (SEQ ID NO: 5), 0.005. mu.M FeSO₄0.16 μ M2-Oxidation [1-¹⁴C]Glutaric acid, 2. mu.M ascorbic acid, 60. mu.g catalase, 0.1. mu.M dithiothreitol and 50. mu.M Tris-hydrochloric acid buffer, and the pH was adjusted to 7.8. The reaction was carried out at 37 ℃ for 20 minutes. Produced by the reaction¹⁴CO₂Captured on filter paper suspended in the atmosphere above the reaction mixture and soaked in lye and counted in a liquid scintillation counter.

These and other embodiments of the present invention will be readily apparent to those of ordinary skill in the art upon reading the present disclosure.

Examples

The invention will be further understood by reference to the following examples, which are intended to be purely illustrative of the invention. These examples are provided only to illustrate the claims of the present invention. The present invention is not limited in scope by the exemplary embodiments which are intended to illustrate one aspect of the present invention. Any method that is functionally equivalent is within the scope of the invention. Those skilled in the art will appreciate that various modifications of the invention, other than those described herein, can be made in light of the above description and with reference to the accompanying drawings, and that such modifications are within the scope of the invention as claimed.

Example 1: experimental Material

Generally, the prolyl hydroxylase inhibitor compounds for use in the methods of the invention are synthesized using standard chemical methods known to those skilled in the art. The purity of the compound is analyzed by high performance liquid chromatography, and the compound is stored at room temperature in a dark place. In the preparation of formulations for different applications, the compounds were suspended and ultrafinely pulverized for 20 minutes at 750rpm using a PULVERISETTE 7 planetary micronizer (Fritsh GMBH, Germany) to form uniformly sized particles.

Suspensions of the micronized compound for oral gavage were prepared immediately prior to use. For administration, the compounds were suspended in an aqueous solution containing 0.5% carboxymethylcellulose (CMC; Spectrum Chemical Gardena CA), 0.1% polysorbate 80(Mallinckrodt Baker, Inc., Phillipsburg NJ) and stirred continuously with a magnetic stirrer or with rotational shaking. The concentration of the suspension is calculated to achieve a given volume and desired dosage level. In another method, the compound is weighed and placed in gelatin capsules of appropriate size for oral administration, and control animals receive empty capsules of the same size; or the compound was dissolved in 100mM histidine (Mallinckrodt Baker) solution instead of water as desired.

For injection administration, the compound is first mixed with an equimolar amount of sodium hydroxide in a 10% aqueous solution of glucose (Spectrum) or 25mM histidine plus isotonic sodium chloride (Mallinckrodt Baker).

Example 2: increased in vitro expression of glucose regulatory factor

Compounds of the invention on proteins and substrates involved in glucose regulation and metabolismThe effect of the expression of factor (II) is as follows. Human 293A cells (adenovirus-transformed fetal kidney epithelial cells) were plated in 35mm dishes at 37 ℃ with 10% CO in DMEM medium containing 5% FBS and 1% penicillin-streptomycin₂Culturing under the condition for 1 day. The medium was changed to Opti-MemI followed by 18-24 hours. Vehicle control or compound B was added to the medium and incubated for 24, 48 or 72 hours. Plates were placed on ice and the culture supernatant discarded, and lysis buffer-1 (LB-1: 10Mm TrispH7.4, lm MEDTA, 150mM sodium chloride, 0.5% IGEPAL) was added. Cell lysates were collected by scraping, incubated on ice for 15 minutes, and then fractionated by centrifugation at 3000Xg for 5 minutes at 4 ℃. Supernatants representing cytoplasmic fractions were collected and cytoplasmic proteins were separated under reducing conditions by SDS-polyacrylamide gel electrophoresis (SDS-PAGE) and equal amounts of protein were added per lane.

SDS-PAGE was performed at 150V for 2 hours, and the proteins were transferred to PVDF membrane at 4 ℃ for 400mA1.5 hours. Wash once with T-TBS and add anti-aldolase antibody diluted to working concentration with blocking buffer. Incubate overnight at 4 ℃ with gentle shaking. The membrane was washed 4 times with T-TBS and then incubated with conjugated secondary antibody diluted with blocking buffer for 1 hour at room temperature. The membranes were washed 4 times with T-TBA and then developed and observed with X-ray film and ECL SUPERSIGNAL WESTFEMTO or PICO chemiluminescent substrate (Pierce Vnominal Co. Rockford IL) according to the manufacturer's instructions.

As shown in FIG. 1A, aldolase expression increased with time in cells treated with compound B for 24, 48, and 72 hours, but no increase was seen in aldolase expression in cells treated with the vector control. Compound B treated cultures showed no increase in β -tubulin expression, indicating that the increase in aldolase is specific and not associated with an overall increase in protein expression.

These results indicate that the compounds and methods of the invention are useful for modulating the expression of genes involved in glycolysis and suggest that treatment with the compounds of the invention can increase glucose utilization and metabolism by enhancing glycolysis.

Example 3: increased in vitro expression of glucose transporter (GluT) -1

10% CO in 100mm dishes at 37 ℃₂Human SSC-25 (squamous cell carcinoma) or rat H9c2 (ventricular cardiomyocytes) was cultured in DMEM medium containing 10% fetal bovine serum under conditions to confluence. Cells were washed 2 times with PBS and incubated with vehicle control, Compound D (10 and 25. mu.M) or Compound C (5, 10 and 20. mu.M) for 16 hours. The plate was placed on ice, the culture supernatant was discarded, and lysis buffer-1 (LB-1: 10mM Tris pH7.4, 1mM EDTA, 150mM sodium chloride, 0.5% IGEPAL) was added. Cell lysates were collected by scraping, incubated on ice for 15 min, and then centrifuged at 3000Xg for 5 min at 4 ℃. Supernatants representing cytoplasmic fractions were collected and cytoplasmic proteins were separated under reducing conditions by SDS-polyacrylamide gel electrophoresis (SDS-PAGE) and equal amounts of protein were added per lane.

SDS-PAGE was performed at 150V for 2 hours, followed by 4 ℃ 400mA for 1.5 hours or overnight, and the proteins were transferred to PVDF membrane, washed once with T-TBS, and anti-GluT-1 antibody (Alpha Diagnostics) diluted to working concentration with blocking buffer was added. After incubation overnight at 4 ℃ with gentle shaking, the membrane was washed 4 times with T-TBS and then incubated with conjugated secondary antibody diluted in blocking buffer for 1 hour at room temperature. The membranes were washed 4 times with T-TBA and then developed and observed with X-ray film and ECL SUPERSIGNAL WEST FEMTO or PICO chemiluminescent substrate (Pierce VchemicalCo. Rockford IL) according to the manufacturer's instructions.

The data shown in FIG. 1B indicate that Compound D and Compound C increased the levels of the major inducible glucose transporter, GluT-1 protein, that mediates glucose uptake in SSC-25 and H9C2 cells, respectively. The ability of the compounds and methods of the invention to increase the expression of GluT-1 by cells provides a tool for studying the effect of compounds on glucose uptake in vitro. These results show that the compounds and methods of the present invention are useful for increasing the expression of proteins involved in glucose uptake, thus providing therapeutic methods for enhancing glucose uptake and lowering blood glucose levels in patients with hyperglycemia, diabetes, or other diseases in which regulation of glucose homeostasis is deficient.

Example 4: increased in vitro expression of glucose regulatory factor

To determine the induction pattern of the gene over time, 24 male Swiss Webster mice (30-32 grams) were obtained from simonsen, inc and treated with oral 4ml/kg volumes of 0.5% carboxymethylcellulose (CMC: Sigma-Aldrich, st. louis MO) (0 mg/kg/day) or 1.25% compound B (25mg/ml 0.5% CMC) (100mg/kg) using a oral feeding tube. Anesthesia was performed with isoflurane at 4, 8, 16, 24, 48 and 72 hours after the last dose. The mice were then sacrificed and tissue samples of the kidney, liver, brain, lung and heart were isolated and stored in RNARATER fluid (Ambion) at-80 ℃.

To study the dose response of the compounds of the invention, 12 male Swiss Webster mice (30-32 grams, obtained from Simonsen, inc., Gilroy CA) were treated once daily with oral gavage for 4ml/kg volumes of 0.5% carboxymethylcellulose (CMC: Sigma-Aldrich, st. louis MO), compound D (25mg/ml, dosed with 0.5% CMC) (100 mg/kg/day), or compound B (7.5 and 25mg/ml, dosed with 0.5% CMC) (30 and 100 mg/kg/day, respectively) for 4 days. 4 hours after the last dose, the mice were anesthetized and sacrificed, and approximately 150mg of liver and each kidney were isolated and stored in RNAALATER solution (Ambion) at-20 ℃.

RNA from the tissues obtained in the above experiment was isolated by the following method: each organ was made into 50mg cubes, 875 microliters of RLT buffer (RNEARY kit: Qiagen Inc., Valencia CA) was added, and the sections were homogenized for 20 seconds using a rotary POLYTRON homogenizer (Kinematica, Inc., Cincinnati OH). The homogenate was microcentrifuged for 3 minutes to settle insoluble material, the supernatant was transferred to a new tube and RNA was isolated using the RNEASY kit (Qiagen) according to the manufacturer's instructions. The RNA was eluted in 80. mu.l of water and quantified using RIBOGREEN reagent (Molecular Probes, Eugene OR). Genomic DNA from the RNA was then removed using the DNA-FREE kit (Ambion Inc., Austin TX) according to the manufacturer's instructions. Absorbance values of 260 and 280 were measured to determine purity and concentration of RNA.

Alternatively, tissue samples were cut into small squares and homogenized in TRIZOL reagent (Invitrogen Life Technologies, CarlsbadCA) using a rotary POLYTRON homogenizer (Kinematica). The homogenate was left at room temperature and 0.2 volume of chloroform was added to stir the sample vigorously. The mixture was incubated at room temperature for several minutes and then centrifuged at 12,000g at 4 ℃ for 15 minutes. The aqueous phase was collected and 0.5 volume of isopropanol was added. The samples were mixed for 10 minutes at room temperature and centrifuged at 12,000g for 10 minutes at 4 ℃. The supernatant was discarded, and the pellet was washed with 75% EtOH and centrifuged at 7,500g for 5 minutes at 4 ℃. Genomic DNA from RNA was removed using the DNA-FREE kit (Ambion Inc., Austin TX) according to the manufacturer's instructions. Absorbance values of 260 and 280 were measured to determine purity and concentration of RNA.

RNA was precipitated with 0.3M sodium acetate (pH5.2), 50ng/ml glycogen and 2.5 volumes of ethanol at-20 ℃ for one hour. The centrifuged sample is washed with 80% cold ethanol, dried and resuspended in water. Double-stranded cDNA was synthesized using T7- (dT)24 first strand primer (Affymetrix, Inc., Santa Vlara CA) and SUPERSCRIPT CHOICE system (Invitrogen) according to the manufacturer's instructions. The final cDNA was extracted with equal volumes of 25: 14: 1 phenol/chloroform/isoamyl alcohol and PHASE LOCKGEL insert (brinkman, Inc., Westbury NY). The aqueous phase was collected and cDNA was precipitated with 0.5 volumes of 7.5M ammonium acetate and 2.5 volumes of ethanol. Alternatively, the cDNA was purified using GENECHIP sample clean-up module (Affymetrix) following the manufacturer's instructions.

Biotin-labeled cRNA was synthesized from cDNA in an in vitro translation reaction using BIOARRAY HIGHYIELD RNA transcription labeling kit (Enzo Diagnostics, Inc., Farmingdale NY) according to the manufacturer's instructions. The final labeled product was purified and fragmented using GENECHIP sample clearance module (Affymetrix) following the manufacturer's instructions.

Hybridization mixtures were prepared by adding 5. mu.g of probe, 100. mu.g/ml of herring sperm DNA, 500. mu.g/ml of acetylated BSA, 0.03nM of control oligo B2(Affymetrix) and 1 XGENECHIP eukaryotic hybridization control (Affymetrix) to 1 Xhybridization buffer (100mM MES, 1M [ Na + ], 20mM EDTA, 0.01% Tween 20). The hybridization mixture was incubated at 99 ℃ and 45 ℃ for 5 minutes in this order, followed by centrifugation for 5 minutes. The murine genomic U74AV2 array (MG-U74AV 2; Affymetrix) was placed at room temperature and then prehybridized with 1 Xhybridization solution at 45 ℃ for 10 minutes with shaking. The buffer was replaced with 80. mu.l of hybridization mixture and the array was hybridized with a metered equilibration solution at 60rpm at 45 ℃ for 16 hours. After hybridization the array was washed once with 6XSSPE, 0.1% Tween 20, then washed and stained with R-phycoerythrin-conjugated streptavidin (Molecular Probes, Eugene OR), biotinylated goat anti-streptavidin antibody (vector laboratories, Burlingame CA), and GENECHIP Fluidics Station 400 instrument (Affymetrix) according to the manufacturer's micro _ lvl program (Affymetrix). The arrays were analyzed using GENEARRAY scanner (Affymetrix) and microarray suite software (Affymetrix).

The murine genome U74AV2 array (MG-U74AV 2; Affymetrix) provided all sequences (about 6000) in Mouse UniGene database build 74 (National Center for Biotechnology Information, Bethesda MD) that had been functionally characterized and had about 6000 uninnotated Expressed Sequence Tag (EST) clusters.

As shown in fig. 2A, 2B and 2C, the expression of the genes encoding enzymes involved in glucose regulation increased in a synergistic manner after treatment with compound B. FIGS. 2A, 2B and 2C provide transcription patterns including Phosphofructokinase (PFK) -P (A) of platelet type, PFK-L (B) of liver type, enolase-1 (C), glucose transporter (GluT) -1(D), lactate dehydrogenase-1 (E), aldolase-1 (F) and hexokinase-1 (G). In the time curve, most mRNA levels peak early after compound administration and return to control levels after 24-48 hours. In addition, gene expression encoding glycolytic enzymes is similar between different organs, but the kidney (FIG. 2A) liver (FIG. 2B) and lung (FIG. 2C) behave differently in terms of the increase in relative expression levels of specific mRNAs and the time of the increase. These different parts are associated with different degrees of glycolytic activity that provide important energy to various tissues, particularly during stress. These results indicate that the compounds of the invention can specifically induce glycolysis, which can vary from tissue to tissue.

As shown in FIG. 3A, treatment with either low (30mg/ml) or high (100mg/ml) doses of the compounds resulted in dose-dependent changes in the expression of the GluT-1 encoding gene in the kidney. The increase in GluT-1 expression in this example provides a mechanism for regulating blood glucose levels by promoting glucose uptake by the cells. As shown in FIG. 3B, treatment with Compound B resulted in nearly identical transient induction of IGFBP-1 expression in the kidney and liver, but the expression levels varied quantitatively in one tissue compared to another. IGFBP-1 promotes the transport of insulin-like growth factors, increases the circulating levels of IGFBP-1, and increases insulin sensitivity and glucose utilization effects, which are associated with, for example, durable training (see Manetta et al, Metabolism 52: 821-826, 2003). Thus, the compounds of the invention can specifically induce direct mediators of blood glucose uptake and act indirectly on humoral regulators of blood glucose and glucose regulation.

Example 5: increased uptake of glucose

Insulin resistance reduces the body's ability to respond to insulin. Insulin resistance or decreased insulin sensitivity is often associated with hyperglycemia, diabetes and hyperinsulinemia. Decreased insulin resistance or increased insulin sensitivity can be determined by measuring glucose uptake following administration of a compound of the invention.

To determine the effect of the compounds of the invention on glucose intake in vivo, DIO (diet induced obesity) rats (Charles River) were fed a high fat diet for 4 weeks to induce obesity and insulin resistance. Alternatively, ZDF rats or any other genetic animal model of type 2 diabetes may be used. Animals were divided into treatment and control groups. The treated animals received the compound for 10 days while the rest received a high fat diet. Fasted animals were cannulated for the carotid and femoral arteries under general anesthesia 10 days later. Continuous infusion of insulin and 10% glucose infusion was performed using a reference method for quantitative determination of insulin resistance, the hyperinsulinemic-euglycemic maintenance procedure, at a rate sufficient to maintain plasma glucose levels at basal concentrations. Blood samples were collected to monitor blood glucose levels and glucose perfusion rates were adjusted appropriately.

Under normoglycemic steady state conditions, the rate of glucose perfusion in all tissues of the body is equal to the rate of glucose uptake, and is therefore a measure of tissue insulin sensitivity. Perfusion before and at steady state in this maintenance procedure [3-³H]The glucose canBasal and insulin-stimulated systemic glucose flux was estimated. Once a steady state glucose level is reached (about 60-75 minutes), 2-deoxy-D- [1-¹⁴C]Glucose was used to estimate insulin-stimulated glucose uptake in each tissue. Blood samples were assayed for glucose and tracer concentrations at 1, 3, 5, 10, 20, 30 and 45 minutes after bolus injection. Tissue samples were collected after 120 minutes euthanasia to determine specific tissue uptake of glucose. Those skilled in the art will not be aware of these methods.

Animals treated with the compounds of the present invention, whose tissues such as muscle and liver, have increased glucose uptake through increased sensitivity to insulin and decreased tolerance to insulin. The increase in glucose uptake following administration of the compounds of the invention indicates that the compounds of the invention may be used in insulin resistant patients, and in patients with reduced or impaired insulin sensitivity to therapeutically reduce their insulin resistance and increase their insulin sensitivity. Thus, the methods and compounds of the present invention increase glucose uptake and decrease blood glucose levels in hyperglycemia, diabetes, or other patients with impaired glucose homeostasis.

Example 6: dose-dependent reduction of blood glucose levels

The effect of the administered compounds on blood glucose levels was investigated as follows. 50 male Spraque Dawley rats (6-7 weeks old) obtained from Simonsen, Inc. were administered 0.5% CMC (Sigma-Aldrich) or 20, 60, 100, 200mg/kg body weight of Compound B once daily for 14 consecutive days by oral administration through a gavage tube. Animals were monitored for weight change and visible signs of toxicity and death. On day 15, animals were fasted overnight but allowed free access to water, anesthetized with isoflurane, the abdominal cavity was opened, and the inferior vena cava blood was collected. About 1ml of one sample was collected in EDTA-containing tubes for hematological analysis and about 1ml of a second sample was collected in anticoagulated tubes for serum chemical analysis. Blood sample analysis was performed by IDEXX (West sacrmento, CA).

As shown in figure 4, two separate experiments showed that animals treated with the compounds of the present invention showed a dose-dependent decrease in blood glucose levels. The relationship between compound dose and blood glucose levels suggests that blood glucose can be maintained at a desired level using the methods and compounds of the invention at appropriate doses. Thus, the methods and compounds of the present invention are useful for modulating, in particular lowering, blood glucose levels. Thus, the methods and compounds of the invention are useful for therapeutically reducing blood glucose levels in a subject, e.g., reducing blood glucose levels in a subject suffering from a glucose regulating disease, such as hyperglycemia or diabetes.

Example 7: glucose tolerance enhancement in diet-induced type 2 diabetes animal models

C57BL/6J mice fed a high fat diet, which resulted in severe obesity, hyperglycemia and hyperinsulinemia, were used as a model for diet-induced obesity, type 2 diabetes and glucose tolerance. 40 male C57BL/6J mice obtained from Jackson Laboratory (Barharbor ME) were divided into the following experimental groups: group 1: vehicle control animals, fed standard mouse chow (n-10); group 2: compound E (n-10) was fed to a standard mouse diet and given at 75 mg/kg/day via oral feeding tube; group 3: and (5) a carrier control. Animals were fed a high fat mouse diet (study diet 45% fat) (n ═ 10); group 4: animals were fed a high-fat mouse diet and compound E (n-10) was administered at 75 mg/kg/day via oral feeding tubes. The feeding regimen was continued for 14 days, and body weight and food consumption were measured daily. Animals were fasted for 4 hours prior to the intraperitoneal glucose tolerance test (IPGTT) with a glucose load of 2 grams per kg body weight. Blood samples were taken at 0, 15, 30, 60 and 90 minutes after glucose administration for blood glucose level determination.

As shown in fig. 5A, the glucose clearance rate was nearly identical and essentially identical for mice taking standard diet with (RxChow) compound administered or without (VeChow) compound, and for mice treated with high fat diet compound (RxHF). However, the glucose clearance rate was lower in mice on a high fat diet without compound (VeHF) than in other groups of mice. Differences in glucose area were calculated for each animal IPGTT curve (AUC) and compared to statistically significant differences between animals treated with standard diet plus compound (RxChow) and without compound (VeChow) versus animals treated with high fat diet without (VeHF) (t-test, P < 0.001). Furthermore, when comparing the glucose AUC of animals on a high-fat diet without compound (VeHF) with animals treated with a high-fat diet with compound (RxHF), differences in blood glucose levels were found to be statistically significant (t-test, P < 0.05). (see FIG. 5B). However, as shown in fig. 5B, there was no statistical difference in glucose AUC between standard diet (RxChow) or high fat diet (RxHF) treated mice and standard diet compound-free (VeChow) mice.

These data indicate improved glucose clearance, decreased blood glucose levels, normalization of glucose tolerance, and restoration of glucose homeostasis in animals treated with the compounds of the invention in animal models of diet-induced obesity and impaired glucose tolerance. These results suggest improved glucose utilization and regulation in the treated animals. The compounds of the invention restore normal glucose tolerance in animal models of impaired diet-induced glucose utilization and regulation (e.g., impaired glucose tolerance), indicating that the methods and compounds of the invention are useful for therapeutically restoring glucose homeostasis in patients with impaired glucose tolerance.

The improvement in glucose utilization and regulation and the restoration of glucose homeostasis following administration of a compound of the invention can also be measured by the Oral Glucose Tolerance Test (OGTT). A similar experiment as described in example 7 above was performed to determine the effect of the administered compound on blood glucose levels. The 40C 57BL/6J mice were divided into the following experimental groups: group 1: vehicle control animals, fed standard mouse chow (n-10); group 2: vehicle control animals, fed high fat mouse chow (study diet 45% fat) (n ═ 10); group 3: animals were fed a high fat mouse diet and given 75 mg/kg/day of compound E (n-10) via oral feeding tube; group 4: animals were fed a high-fat mouse diet and compound a (n-10) was administered at 75 mg/kg/day via oral feeding tubes. The feeding regimen was continued for 28 days and body weight was measured weekly. Animals were fasted overnight prior to OGTT with a glucose load of 1 gram per kg body weight. Blood samples were taken at 0, 30, 60, 9, 120 and 180 minutes after glucose administration for glucose level determination.

Example 8: reduced glycation of hemoglobin

Various sugars (most commonly glucose) form glycated hemoglobin by binding to hemoglobin molecules, the rate of formation of which is proportional to the glucose concentration. Measurement of glycated hemoglobin levels provides an accurate index of the mean blood glucose concentration for the first 2-3 months. Clinically, glycated hemoglobin levels can be used to assess glycemic control in diabetic or hyperglycemic patients.

The effect of the administered compounds on glycated hemoglobin levels was studied in a diabetic mouse model as follows. 20 male db/db mice (Harlan) received drinking water containing either vehicle (100. mu.M histidine) or Compound A (0.5mg/ml) for 8 weeks. Tail vein blood samples were collected before study initiation and 4 and 8 weeks post-treatment, and HbA1c levels were measured using the HbA1cNOW kit (metrikainc., Sunnyvale CA).

HbA1c levels y increased significantly from baseline at 8 weeks in the control group (P < 0.05). As shown in FIG. 6, HbA1c increased from 7.5% at week 0 to 10% at week 8. HbA1c did not increase with time in the compound-treated group, and was significantly lower at 8 weeks than in the non-treated group. The HbA1c level of animals treated with the compounds of the invention was about 7.5% at 8 weeks.

These data show that treatment with the compounds of the invention reduces the accumulation of glycated hemoglobin in animal models of type 2 diabetes. HbA1c reflects overall glycemic control in diabetic patients. Compounds lowering this model HbA1c indicates that the compounds of the invention are useful for therapeutically improving glycemic control in patients with diabetes or hyperglycemia.

Example 9: weight gain and reduced fat storage loss

The effect of the compounds of the present invention on body weight loss and fat storage was investigated as follows. Male Spraque Dawley rats (6-7 weeks old) obtained from Simonsen, inc. 40 were given 0.5% CMC (Sigma-Aldrich) or compound B at 20, 60, 100, 200mg/kg body weight orally via oral feeding tubes once daily for 14 consecutive days. Animals were monitored for weight change and visible signs of toxicity and death. On day 15, animals were fasted overnight but allowed free access to water and were anesthetized with isoflurane. An approximately 1ml portion of the whole blood sample was collected in EDTA-containing tubes for hematological analysis, and a second approximately 1ml portion was collected in anticoagulated tubes for serum chemical analysis. Blood sample analysis was performed by IDEXX (West sacrmento, CA). After blood samples were collected, the animals were sacrificed by cutting the septum. Microscopic observations of each animal were recorded and liver, kidney, heart, spleen, lung, stomach, small intestine and large intestine were collected for histological evaluation.

As shown in figure 7A, the animals treated with the compounds of the present invention showed a dose-dependent decrease in weight gain. Animal examination showed no overall delay in growth, as the absolute weights of most organs in treated animals were not significantly different from the weights of organs in untreated control animals. For example, the heart weight of compound-treated animals was not statistically significantly different compared to controls (fig. 7B). However, the relative weight of the organs in the treated animals was significantly increased compared to the untreated control. For example, the relative weight of the heart, expressed as a total body weight fraction, was significantly increased in animals treated with 100mg/kg compound compared to controls (p 0.036, one-way ANOVA/Tukey' test).

Since the absolute weight of the organ, e.g. the heart, is not significantly reduced, the overall growth process of the treated animal is not delayed. In addition, there is a loss of selectivity of other tissues due to a significant increase in organ weight relative to the total weight of the body. When treated with the compounds, a dose-dependent reduction in visceral fat was shown in figure 8. The arrows in the upper panel show visceral fat pad present in animals treated with low dose compounds, while the lower panel shows animals treated with high dose compounds completely lack fat pad.

These results indicate that the methods and compounds of the present invention are useful for regulating body weight, inducing loss or reduction of body weight without concomitant loss of muscle, and reducing visceral fat. Taken together, these results indicate that the methods and compounds of the present invention are useful for effectively controlling weight gain, particularly reducing visceral fat. These methods and compounds are advantageous for treating or preventing obesity and, therefore, are useful for treating or preventing obesity-related diabetes.

Example 10: weight gain reduction in diet-induced obese animal models

C57BL/6J mice fed high fat food that develop severe obesity, hyperglycemia, and hyperinsulinemia are a model for diet-induced obesity, type 2 diabetes, and impaired glucose tolerance. 40 male C57BL/6J mice purchased from Jackson Laboratory (Barharbor ME) were divided into the following experimental groups: group 1: vehicle control animals, fed standard mouse chow (n-10); group 2: vehicle control animals, fed high fat mouse chow (study diet 45% fat) (n ═ 10); group 3: animals were fed a high fat mouse diet and given 75 mg/kg/day of compound E (n-10) via oral feeding tube; group 4: animals were fed a high-fat mouse diet and compound a (n-10) was administered at 75 mg/kg/day via oral feeding tubes. This feeding regimen was continued for 28 days, and body weight was measured weekly. Animals were sacrificed and their organs and fat pads were collected and weighed.

As shown in fig. 9A, animals fed the high fat diet (group 2) had significantly higher body weights than animals fed the standard ration (group 1) (P < 0.05). However, animals fed a high fat diet but treated with compound E or compound a (groups 3 and 4, respectively) showed significantly less weight gain (P < 0.05). In fact, despite a high fat diet, the body weight of animals treated with the compound was essentially the same as that fed normal diet animals (group 3 and group 4 compared to group 1). Similarly, as shown in fig. 9B, the abdominal fat pads were significantly increased in animals fed high fat diet (group 2) compared to animals fed standard diet (group 1) and animals fed high fat diet and treated with the compound of the present invention (groups 3 and 4). As shown in fig. 9B, the fat pad weight of animals fed high fat diet and treated with compound was substantially the same as that of normal diet animals.

The weight of each organ of the animal was also determined 28 days after the study. As shown in fig. 9C, the weights of kidney and liver heart were not different between the experimental groups. These results indicate that the observed body weight differences are due to a reduction in fat storage, rather than a reduction in growth rate. These data show that treatment of animals with the compounds of the present invention abrogates the weight gain associated with high fat diets. This body weight gain prevention effect of the compounds of the present invention indicates that the compounds of the present invention may be used for the therapeutic reduction of body weight gain even at poor dietary intake. In addition, the methods and compounds of the invention are useful for modifying weight gain, suggesting that such compounds may be useful for therapeutically modulating weight loss in obese patients.

Example 11: weight loss in obese mice

The effect of administering the compounds of the present invention on weight loss was investigated as follows. C57BL/6J mice were obtained from Jackson Laboratory (Bar Harbor ME). C57BL/6J mice fed high fat food that develop severe obesity, hyperglycemia, and hyperinsulinemia are a model for diet-induced obesity, type 2 diabetes, and impaired glucose tolerance. Mice become obese after 8 weeks of high fat diet. Obese mice were divided into two experimental groups: group 1 animals were control obese mice, and group 2 animals were obese mice treated with the compounds of the invention. This will not be understood to be 3 also including another group of age matched non-obese mice. Animals were treated daily with a compound of the invention or with vehicle control. Mouse body weights were measured twice weekly over 21 days. Mice were weighed and sacrificed on day 21. Abdominal fat pads, liver, kidney and heart were isolated and weighed for analysis.

Weight loss following administration of the compounds indicates that the compounds of the invention are useful for the therapeutic reduction of body weight in obese patients.

Example 12: increased expression of genes involved in blood pressure regulation

Hypertension is a risk factor for diabetes and diabetes-related diseases and conditions. The effect of the compounds of the present invention on the regulation of blood pressure was investigated as follows. Animals were treated with compound B or compound D and RNA samples were prepared as described in example 4. iNOS mRNA levels were determined by the following method. cDNA synthesis was performed using 1 μm random hexamer primers, 1 μ g total RNA and OMNISCRIPT reverse transcriptase (Qiagen) as per the manufacturer's instructions. The resulting cDNA was diluted 5-fold with water to a final volume of 100. mu.L. Analysis of relative levels of gene expression was performed by quantitative PCR using the FASTSTART DNAMASTER SYBR GREENI kit (Roche) and gene specific primers using the LIGHTCYCLER system (Roche) according to the manufacturer's instructions. The sample was heated to 90 ℃ for 6 minutes, then a total of 42 cycles: 95 ℃ for 15 seconds, 60 ℃ for 5 seconds, and 72 ℃ for 10 seconds. Inducible Nitric Oxide Synthase (iNOS) -specific primers are as follows:

m-iNOS-F2 CCCAGGAGGAGAGAGATCCGATT(SEQ ID NO：1)

m-iNOS-R2 AGGTCCCTGGCTAGTGCTTCAGA(SEQ ID NO：2)

the relative level of 18S ribosomal RNA gene expression was determined as a control. Quantitative PCR was performed using the QUANTITECT SYBRGREEN PCR kit (Qiagen) and gene-specific primers, using the LIGHTCYCLER System (Roche), according to the manufacturer's instructions. The sample was heated to 90 ℃ for 15 minutes, then a total of 42 cycles: 95 ℃ for 15 seconds, 60 ℃ for 20 seconds, and 72 ℃ for 10 seconds. Ribosomal RNA-specific primers were as follows:

18S-rat-2B TAGGCACGGCGACTACCATCGA(SEQ ID NO：3)

18s-rat-2A CGGCGGCTTTGGTGACTCTAGAT(SEQ ID NO：4)

each PCR run included a standard curve and a water blank. In addition, the melting cup was run after each PCR run was completed to assess the specificity of the amplification. The iNOS gene expression of the sample was normalized with respect to the RNA expression level of 18S ribosome.

As shown in FIG. 10A, the expression of the gene encoding iNOS was elevated after 8 hours of treatment with the compound of the present invention, and thereafter dropped back to the control level. In addition, the induction of iNOS is dose-dependent and can be achieved with both compounds of the invention (compounds B and D). These two compounds are two different pharmacophores, one phenanthroline derivative and one heterocyclic amide, and may be used in the process of the present invention. These results indicate that treatment with the compounds of the invention increases the expression of iNOS, a protein involved in vasodilation regulation. Thus, the methods and compounds of the present invention are useful for inducing vasodilation and lowering blood pressure.

To determine expression of adrenomedullin mRNA, samples were prepared and assayed by hybridization as described in example 4 above. As shown in FIG. 10B, the increase in adrenomedullin (representing the gene coding for a protein involved in vasodilation) in various tissues of animals treated with Compound B of the present invention. The adrenal medullary hormone mRNA levels in the heart, kidney and lung showed a rapid increase in expression over time following treatment with compound B, and then fell back to control levels within 16-24 hours.

Taken together, these results indicate that the methods and compounds of the invention can be used to provide a tool for activation of genes involved in regulating blood pressure expression via the diastolic mechanism. Such genes include, but are not limited to: inducible nitric oxide synthase and desmin. Therapeutic upregulation of vasodilating factors provides an effective means of lowering blood pressure, thereby providing a benefit to patients with metabolic disorders such as diabetes. By lowering blood pressure, the methods and compounds of the present invention may be used to provide a means for treating or preventing diabetes, hyperglycemia, and other diabetes-related metabolic disorders, among others.

Example 13: serum triglyceride reduction

The effect of the administered compound on triglyceride levels was studied in a diabetic mouse model as follows: 20 male db/db mice (Harlan, Indianapolis, Indiana) containing homozygous loss-of-function mutations in the leptin receptor were used for this study. Triglyceride levels are generally 1.5-2 fold higher in db/db mice compared to normal mice (Nishina et al, Metabolism 43: 549-553, 1994). Triglyceride levels progressively increase with age in db/db mice (Tuman and Doisy. Diabetologia 13: 7-11, 1977). Mice received drinking water containing either vehicle (100. mu.M histidine) or Compound A (0.5mg/ml, dosed with 100. mu.M histidine) for 8 weeks. At the end of the study, animals were fasted overnight and blood samples were taken from the tail vein cannula under general anesthesia and placed in serum separation tubes. Blood samples were sent to Quqlite clinical labs (Mountain View, Calif.) for analysis.

As shown in FIG. 11, the level of triglyceride in db/db mice from the end-of-experiment controls was approximately 120 mg/dL. However, triglyceride levels in animals treated with the compounds of the invention were about 85mg/dL, significantly lower than controls. Elevated triglyceride levels are associated with an increased risk of cardiovascular disease, and elevated triglyceride is a component of the metabolic syndrome. Because the methods and compounds of the present invention are effective in reducing or maintaining triglyceride levels in diseases associated with elevated triglycerides in general, such as diabetes, syndrome X, macrovascular disease or other dyslipidemias, the methods of the present invention are useful in treating individuals having or at risk of having such diseases.

Example 14: identification of Compounds and HIF α stabilization

2-oxidation [1-¹⁴C]Glutarate hydroxylation-coupling decarboxylation assays to identify compounds that modulate HIF-specific prolyl hydroxylase activity (see Hirsila et al, J Bio Chem 278: 30772-30780, 2003). The reaction is carried out at 25 ℃ in a reaction volume of 1.0ml, containing 10-100 microliters of a detergent such as Triton-X-100, an extract obtained from lysed cells expressing an endogenous or recombinant HIF prolyl hydroxylase, 0.05. mu. M DLDLEMLAPYIPMDDDFQL (SEQ ID NO: 5), 0.005. mu.M FeSO₄0.16 μ M2-Oxidation [1-¹⁴C]Glutaric acid, 2. mu.M ascorbic acid, 60. mu.g catalase, 0.1. mu.M dithiothreitol and 50. mu.M Tris-HCl buffer, and the pH was adjusted to 7.8. The enzyme reaction was carried out at 37 ℃ for 20 minutes. Produced by the reaction¹⁴CO₂Captured on filter paper suspended in the atmosphere above the reaction mixture and soaked in lye and counted in a liquid scintillation counter.

Stabilization of HIF α with the methods and compounds of the invention is examined as follows. Human cells derived from adenovirus-transformed fetal kidney epithelium (239A), cervical epithelial adenocarcinoma (Hela), hepatocellular carcinoma (Hep3B), squamous epithelial carcinoma (SSC-25) and lung fibroblast (HLF) (see American type culture Collection, Manassas VA and Qbiogene, Carlsbad CA) were plated in 100mm dishes at 37 deg.C and 20% O, respectively₂、10％CO₂The culture medium is as follows: HeLa cells were Dulbecco's Modified Eagle Medium (DMEM), 2% Fetal Bovine Serum (FBS); the HLF cells are DMEM and the HLF cells are,10% FBS; 293 cells were DMEM, 5% FBS; hep3B cells were Minimal Essential Medium (MEM), Earle's BSS (Mediatech Inc. Herndon VA), 2mM L-glutamine, 0.1mM non-essential amino acids, 1mM sodium pyruvate, 10% FBS. When the cell layer reached confluency, the original medium was replaced with OPTI-MEM medium (Invitrogen Life Technologies, Carlsbad CA) at 37 ℃ with 20% O₂、10％CO₂The cell layer was cultured for about 24 hours. Then, the compound of the present invention (one of the compounds B, F, G and H) or DMSO (0.5-1.0%) was added to the current medium, and the culture was continued overnight.

After incubation, the medium is removed, centrifuged and stored for analysis. The cells were washed twice with cold Phosphate Buffered Saline (PBS) and then lysed for 15 minutes on ice with 1.0ml of 10mM Tris (pH7.4), 1mM EDTA, 150mM NaCl, 0.5% IGEPAL (Sigma-Aldrich, St. Louis MO) and protease inhibitor cocktail (Roche Molecular Biochemicals). The cell lysate was centrifuged at 3,000Xg for 5 minutes at 4 ℃ and the cytoplasmic fraction (supernatant) was collected. The resuspended nuclei were lysed with 100. mu.l of 20mM HEPES (pH7.2), 400mM NaCl, 1mM EDTA, 1mM dithiothreitol and protease mixture (Roche Molecular biochemicals), centrifuged at 13000Xg for 5 minutes at 4 ℃ and the nuclear protein fraction (supernatant) was collected.

The core fractions were normalized for protein concentration, applied to 4-12% TG gels, and fractionated under reducing conditions. Proteins were transferred to PVDF membranes (Invitrogen corp., Carlsbad CA) at 500mA for 1.5 hours. The membrane was blocked with T-TBS, 2% milk for 1 hour at room temperature and incubated overnight with mouse anti-HIF-1. alpha. antibody (BD Biosciences, Bedford MA) diluted 1: 250 with T-TBS, 2% milk. The blot was developed with SUPERSIGNAL WEST chemiluminescent substrate (Pierce, Rockford IL). As shown in FIG. 12A, representative compounds of the invention (Compound D) stabilized HIF α in various cell types in a dose-dependent manner, allowing accumulation of HIF α in the cells.

Alternatively, nuclear fractions were prepared using a nuclear extraction kit and HIF-1 α was analyzed using TRANSAM HIF-1ELISA kit (Active Motif) according to the manufacturer's instructions. As shown in fig. 12B, Hep3B cells showed dose-dependent stable HIF α when treated with the compounds of the invention. FIG. 12B also shows that epithelial cells (293A) and hepatocellular carcinoma (Hep3B) treated with various compounds of the invention (compounds B, F, G and H) showed stabilization and accumulation of HIF α compared to vehicle-treated control cells.

In addition to those shown and described herein, it will be appreciated by those skilled in the art that various modifications may be made to the invention in light of the above teachings. Such modifications are intended to fall within the scope of the appended claims.

The contents of all references cited herein are incorporated herein by reference.

Sequence listing

<110> Fabroso Gen Ltd (FibroGen, Inc.)

V. Jingzel-Pukal (Guenzler-Pukall, Volkmar)

S.J. Claus (Klaus, Stephen J.)

I. Lantsem Palo Bok (Langsetmo Parabok, Ingrid)

T.W. Xili (Seeley, Todd W.)

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<151>2003-06-06

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Claims (16)

1. Use of a formulation that stabilizes HIF α, wherein the formulation is selected from the group consisting of [ (7-chloro-3-hydroxy-quinolinyl-2-carbonyl) -amino ] -acetic acid, [ (1-chloro-4-hydroxy-isoquinolinyl-3-carbonyl) -amino ] -acetic acid, [ (4-hydroxy-7-phenoxy-isoquinolinyl-3-carbonyl) -amino ] -acetic acid, 4-oxo-1, 4-dihydro- [1, 10] phenanthrolinyl-3-carboxylic acid, [ (1-chloro-4-hydroxy-7-methoxy-isoquinolinyl-3-carbonyl) -amino ] -acetic acid, a pharmaceutically-acceptable salt thereof, or a pharmaceutically-acceptable salt thereof, for the manufacture of a medicament for the treatment or prevention of a condition associated, [ (3-hydroxy-6-isopropoxy-quinolinyl-2-carbonyl) -amino ] -acetic acid, [ (3-hydroxy-pyridinyl-2-carbonyl) -amino ] -acetic acid and [ (7-benzyloxy-1-chloro-4-hydroxy-isoquinolinyl-3-carbonyl) -amino ] -acetic acid methyl ester.

2. Use of a formulation that stabilizes HIF α, wherein the formulation is selected from the group consisting of [ (7-chloro-3-hydroxy-quinolinyl-2-carbonyl) -amino ] -acetic acid, [ (1-chloro-4-hydroxy-isoquinolinyl-3-carbonyl) -amino ] -acetic acid, [ (4-hydroxy-7-phenoxy-isoquinolinyl-3-carbonyl) -amino ] -acetic acid, 4-oxo-1, 4-dihydro- [1, 10] phenanthrolinyl-3-carboxylic acid, [ (1-chloro-4-hydroxy-7-methoxy-isoquinolinyl-3-carbonyl) -amino ] -acetic acid, for the manufacture of a medicament for the treatment or prevention of diabetes in a subject at risk for developing diabetes, [ (3-hydroxy-6-isopropoxy-quinolinyl-2-carbonyl) -amino ] -acetic acid, [ (3-hydroxy-pyridinyl-2-carbonyl) -amino ] -acetic acid and [ (7-benzyloxy-1-chloro-4-hydroxy-isoquinolinyl-3-carbonyl) -amino ] -acetic acid methyl ester.

3. The use according to claim 1, wherein the disease associated with elevated blood glucose levels is selected from the group consisting of hyperglycemia, obesity, hypertension, hyperlipidemia, nephropathy, retinopathy, impaired glucose tolerance and vascular disease.

4. The use according to claim 3, wherein the vascular disease is atherosclerosis.

5. Use of a formulation that stabilizes HIF α in the manufacture of a medicament for:

a. regulating glucose metabolism or glucose metabolic processes;

b. glucose homeostasis is achieved;

c. lowering blood glucose levels;

d. reducing glycated hemoglobin levels;

e. increasing the expression of glucose regulatory factor;

f. altering expression of a glycolytic factor;

g. reducing insulin resistance in the subject; or

h. Enhancing subject blood glucose level control;

wherein the agent is selected from the group consisting of [ (7-chloro-3-hydroxy-quinolinyl-2-carbonyl) -amino ] -acetic acid, [ (1-chloro-4-hydroxy-isoquinolinyl-3-carbonyl) -amino ] -acetic acid, [ (4-hydroxy-7-phenoxy-isoquinolinyl-3-carbonyl) -amino ] -acetic acid, 4-oxo-1, 4-dihydro- [1, 10] phenanthrolinyl-3-carboxylic acid, [ (1-chloro-4-hydroxy-7-methoxy-isoquinolinyl-3-carbonyl) -amino ] -acetic acid, [ (3-hydroxy-6-isopropoxy-quinolinyl-2-carbonyl) -amino ] -acetic acid, and, [ (3-hydroxy-pyridinyl-2-carbonyl) -amino ] -acetic acid and [ (7-benzyloxy-1-chloro-4-hydroxy-isoquinolinyl-3-carbonyl) -amino ] -acetic acid methyl ester.

6. The use of claim 5, wherein the glucose metabolic process is selected from the group consisting of glucose uptake, glucose transport, glucose storage, glucose processing, and glucose utilization.

7. The use according to claim 5, wherein the glucose regulatory factor is selected from the group consisting of phosphofructokinase-P, phosphofructokinase-L, enolase, glucose transporter-1, lactate dehydrogenase, aldolase-1, hexokinase-1, insulin-like growth factor binding protein-1 and insulin-dependent growth factor.

8. The use according to claim 5, wherein the glycolytic factor is selected from phosphofructokinase-P, phosphofructokinase-L, enolase-1, lactate dehydrogenase, aldolase-1, hexokinase-1.

9. The use of claim 1, 2 or 5, wherein the agent is [ (7-chloro-3-hydroxy-quinolinyl-2-carbonyl) -amino ] -acetic acid.

10. The use of claim 1, 2 or 5, wherein the agent is [ (1-chloro-4-hydroxy-isoquinolinyl-3-carbonyl) -amino ] -acetic acid.

11. The use of claim 1, 2 or 5, wherein the agent is [ (4-hydroxy-7-phenoxy-isoquinolinyl-3-carbonyl) -amino ] -acetic acid.

12. The use according to claim 1, 2 or 5, wherein the formulation is 4-oxo-1, 4-dihydro- [1, 10] phenanthrolinyl-3-carboxylic acid.

13. The use of claim 1, 2 or 5, wherein the agent is [ (1-chloro-4-hydroxy-7-methoxy-isoquinolinyl-3-carbonyl) -amino ] -acetic acid.

14. The use of claim 1, 2 or 5, wherein the agent is [ (3-hydroxy-6-isopropoxy-quinolinyl-2-carbonyl) -amino ] -acetic acid.

15. The use of claim 1, 2 or 5, wherein the agent is [ (3-hydroxy-pyridyl-2-carbonyl) -amino ] -acetic acid.

16. The use of claim 1, 2 or 5, wherein the formulation is [ (7-benzyloxy-1-chloro-4-hydroxy-isoquinolinyl-3-carbonyl) -amino ] -acetic acid methyl ester.