NO20171470A1 - Use of HIF alpha stabilizers to increase erythropoiesis. - Google Patents

Abstract

The present invention relates to methods and compounds for the regulation or enhancement of erythropoiesis and iron metabolism and the pretreatment or prevention of iron deficiency and anemia associated with chronic disease.

Description

USE OF HIF ALPHA STABILIZERS FOR INCREASE OF

THE ERYTROPOIES

FIELD OF THE INVENTION

The present invention relates to methods and compounds for regulating or increasing erythropoiesis and iron metabolism and for treating or preventing iron deficiency and anemia associated with chronic disease.

BACKGROUND OF THE INVENTION

Anemia generally refers to any abnormality in hemoglobin or erythrocytes that leads to reduced oxygen levels in the blood. Anemia can also develop in connection with chronic diseases, for example chronic infection, neoplastic disorders and chronic inflammatory disorders, including disorders with subsequent inflammatory suppression of the bone marrow, etc. Anemia associated with chronic disease is one of the most frequently occurring syndromes in medicine.

Anemia associated with chronic disease (ACD) is often associated with iron deficiency. ACD can develop from insufficient availability of iron (for example, anemia associated with iron deficiency) or, if the body's total iron level is sufficient but the requirements for hemoglobin production are defective (for example, functional iron deficiency). Iron is necessary for the formation of hemoglobin for the red blood cells in erythropoietic precursor cells in the bone marrow.

A number of physiological deficits are observed in patients with anemia associated with chronic disease, including decreased erythropoietin (EPO) formation, decreased EPO response in the bone marrow, and decreased iron metabolism including decreased absorption of iron from the gut, decreased transport of iron through enterocytes, decreased iron oxidation to iron (III) level by hefaestin or ceruloplasmin, reduced binding and absorption of iron by transferrin and the transferrin receptor and reduced transport of iron to the bone marrow where the utilization of iron takes place, including synthesis of heme. Individually and together, these physiological deficiencies contribute to inefficient or impaired erythropoiesis, which can lead to microcytic anemia and hypochromic red blood cells associated with reduced hemoglobin formation and reduced oxygen transport.

Anemia associated with chronic disease is associated with elevated production of inflammatory cytokines (Means (1995) Stem cells 13: 32-37 and Means (1999) Int J Hematol 70: 7-12), including for example tumor necrosis factor-α (TNF-α ), interleukin-1 β (IL-1 β), IL-6 and interferon- γ (IFN- γ). In several in vitro and in vivo animal model systems, inflammatory cytokines negatively affect the ability to mediate EPO formation, EPO response and coordinated regulation of iron metabolism (Roodman et al. (1989) Adv Exp Med Biol 271: 185-196; Fuchs et al. et al (1991) Eur J Hematol 46: 65-70 Jelkmann et al (1994) Ann NY Acad Sci 718: 300-311;

Vannucchi et al. (1994) Br J Hematol 87: 18-23; and Oldenburg et al. (2001) Aliment Pharmacol Ther 15: 429-438). Administration of erythropoietin did not reverse anemia in mice continuously exposed to TNF-α (Clibon et al. (1990) Exp Hematol 18: 438-441). Elevated levels of inflammatory cytokines such as TNF-α, IL-1β and IFN-γ contribute to the failure of EPO formation and EPO resistance observed in patients with anemia associated with chronic disease (Jelkmann et al. (1991) Ann NY Acad Sci 718: 300-311 and Macdougall and Cooper (2002) Neprol Dial Transplant 17(11): 39-43). Accordingly, various cytokines, such as inflammatory cytokines and cytokines associated with inflammation, participate in many aspects of the pathogenesis of anemia associated with chronic disease, including inhibition of erythroid progenitor cells, inhibition of EPO formation, and impaired release and availability of iron for erythropoiesis.

There is thus a need in the field for methods for the treatment or prevention of anemia associated with chronic disease. There is a need in the art for methods that can overcome deficiencies in the current use of recombinant EPO for the treatment of anemia associated with chronic disease.

More specifically, there is a need for methods and compounds that effectively overcome the suppressed EPO formation and reduced EPO response associated with anemia associated with chronic disease, for methods and compounds that effectively improve the regulation of iron metabolism and overcome deficiencies associated with altered or abnormal iron metabolism and iron utilization and for methods and compounds that effectively enhance total or complete erythropoiesis by improving the metabolic pathways associated with EPO formation, EPO response and signaling, iron availability, iron utilization, uptake, transport, processing of iron, etc. There is a need in the art for methods that can overcome or alleviate the consequences of cytokine-induced effects in individuals with anemia associated with chronic disease.

Iron deficiency is one of the most common nutritional deficiencies worldwide and is globally the most important cause of anaemia. The iron balance is basically regulated by the rate of erythropoiesis and the size of the iron stores. Iron deficiency can occur with or without anemia and has been linked to impaired cognitive development.

Iron deficiency is defined as insufficient supply of iron (level or stores) or as insufficient availability or utilization of iron in the body. This can be due to deficiencies in the diet, for example a lack of iron in the diet; impaired absorption of iron, for example due to surgical treatment (post-gastrectomy) or disease (Crohn's disease); or a depletion of iron stores or increased iron loss due to chronic or acute blood loss as a result of injury or trauma, menstruation, blood donation, phlebotomy (such as due to various forms of medical treatment or surgical procedures); due to increased need for iron, for example due to rapid growth in childhood or adolescence, pregnancy, erythropoietin treatment, etc.

Iron deficiency can also be functional iron deficiency, for example iron deficiency due to the patient's inability to access and utilize the body's iron stores. Iron is not available to the extent necessary to allow normal hemoglobinization of erythrocytes, leading to reduced reticulocyte content and reduced hemoglobin content in the erythrocytes. Functional iron deficiency is often observed in healthy individuals with apparently normal, or even elevated iron stores, but with impaired availability of iron, measured for example by a low level of percent transferrin saturation. This type of iron deficiency is often associated with acute or chronic inflammation.

Iron deficiency of any kind can lead to iron-deficient or iron-limited erythropoiesis, in which the number of red blood cells decreases and the red blood cells in the circulation are smaller than normal (microcytic) and lack sufficient hemoglobin and are therefore light in color (they are hypochromic).

Individuals with iron deficiency, including functional iron deficiency, may develop impaired hemoglobin synthesis, decreased % saturated transferrin, and decreased levels of hemoglobin and hematocrit, leading to iron deficiency anemia. Iron deficiency anemia is the most frequently occurring anemia in the world. Iron is a necessary component of hemoglobin; without iron, the bone marrow is incapable of efficient hemoglobin formation.

Iron deficiency anemia can occur in individuals with depleted or weakened iron supply and can occur in individuals with functional iron deficiency where iron is present in the stores, but is not available for, for example, hemoglobin formation.

In light of the foregoing, there is a need in the art for methods of treating or preventing disorders associated with iron metabolism and a need in the art for methods of enhancing iron metabolism. There is a need for methods for the treatment or prevention of iron deficiency including functional iron deficiency, and for the treatment or prevention of conditions associated with iron deficiency such as microcytosis and iron deficiency anemia.

The present invention provides methods and compounds for enhancing the metabolic and physiological reaction pathways that contribute to complete and efficient erythropoiesis, and more specifically for the treatment of anemia associated with chronic disease. Methods and compounds for overcoming the suppressive/inhibitory effects of inflammatory cytokines on EPO formation and the EPO response are also provided. Additionally, the present invention provides methods and compounds for improving iron metabolism and for treating or preventing conditions associated with impaired iron metabolism such as iron deficiency, including functional iron deficiency, iron deficiency anemia, microcytosis, iron deficient erythropoiesis, etc.

SUMMARY OF THE INVENTION

The present invention relates to methods and compounds for the induction of improved or complete erythropoiesis in an individual. More specifically, the methods comprise inducing enhanced or complete erythropoiesis by stabilizing HIF α in a subject. Methods of inducing enhanced erythropoiesis by inhibiting HIF prolyl hydroxylase are particularly encompassed. In specific embodiments, the methods comprise administering to a subject a compound according to the invention. In various embodiments, the subject may be a cell, a tissue, an organ, an organ system or an entire organism.

The subject is, in various embodiments, a cell, a tissue, an organ, an organ system, or an entire organism. In certain embodiments, the organism is a mammal, preferably a human.

In one aspect, the method increases the production of factors necessary for the differentiation of erythrocytes from hematopoietic progenitor cells including, for example, hematopoietic stem cells (HSC), CFU-GEMM (colony forming unit granulocyte/erythroid/monocyte/megakaryocyte) cells, etc. Factors that stimulate erythropoiesis include, but are not limited to, erythropoietin. In another aspect, the methods increase the formation of factors necessary for the uptake, transport and utilization of iron. Such factors include, but are not limited to, erythroid aminolevulinate synthase, transferrin, transferrin receptor, ceruloplasmin, etc. In yet another aspect, the method increases the amount of factors necessary for erythrocyte differentiation and, in addition, factors necessary for uptake, transport and utilization of iron.

In another embodiment, the methods according to the invention increase the response of hematopoietic precursor cells to erythropoietin. As described above, such precursors include HSC; CFU-GEMM, etc. The progenitor cells' response can be enhanced by, for example, changing the expression of erythropoietin receptors, intracellular factors that participate in erythropoietin signaling and secreted factors that facilitate the interaction between erythropoietin and the receptors.

In another aspect, the methods can be used to overcome inhibition of erythropoiesis induced by inflammatory cytokines such as tumor necrosis factor-α (TNF-α), interleukin 1 β (IL-1 β) and the like. In certain aspects, the methods can be used to treat anemia that cannot be treated with exogenously administered erythropoietin. Such anemia can for example be due to chronic inflammatory disorders or autoimmune disorders including, but not limited to, chronic bacterial endocarditis, osteomyelitis, rheumatoid arthritis, gouty fever, Crohn's disease and ulcerative colitis.

In certain embodiments, the methods according to the invention can be used for the treatment of anemia associated with chronic disease. Methods of inducing enhanced or complete erythropoiesis in patients with anemia associated with chronic disease are particularly provided. In certain embodiments, the methods increase the amount of iron available for the formation of new red blood cells.

In another aspect, the present invention provides methods of enhancing bone marrow EPO responsiveness.

Methods for inhibiting TNF α suppression by EPO are particularly provided, as are methods for inhibiting IL-1 β suppression by EPO.

The present invention relates to methods for treating/preventing anemia associated with chronic disease and methods for regulating the processing of iron and treating/preventing conditions associated with iron deficiency and/or failure in iron processing.

In one aspect, the invention provides a method for treating anemia associated with chronic disease in an individual, the method comprising administering to the individual an effective amount of a compound that stabilizes the alpha subunit of hypoxia-inducible factor (HIF) such that anemia associated with chronic disease processed in the individual. Methods of achieving specific physiological effects in a subject with anemia associated with chronic disease are also provided; more specifically, methods of increasing the number of reticulocytes, increasing the average blood cell volume, increasing the average amount of hemoglobin in the blood cells, increasing the hematocrit, increasing the hemoglobin concentration and increasing the red blood cell count, etc., in the subject with anemia associated with chronic disease, wherein each of these methods comprise administering to a subject an effective amount of a compound that stabilizes the alpha subunit of hypoxia-inducible factor (HIF), thereby achieving the desired physiological effect. In various aspects, the anemia associated with chronic is associated with, for example, inflammation, autoimmune disease, iron deficiency, microcytosis, cancer, etc.

In various embodiments, the subject is a cell, tissue, or organ. In other embodiments, the subject is an animal, preferably a mammal, most preferably a human. If the subject is a cell, the invention includes in particular that the cell can be an isolated cell, either a prokaryotic or a eukaryotic cell. If the subject is a tissue, the invention particularly includes both endogenous tissues and in vitro tissues, for example tissues grown in culture. In preferred embodiments, the subject is an animal, preferably an animal from a mammalian species, including rat, rabbit, bovine, sheep, pig, mouse, horse and primate species. In a most preferred embodiment, the subject is a human.

Stabilization of HIF α can be achieved by any of the methods available to and known to those skilled 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 encompasses the provision of a constitutively stable HIF α variant, for example, stable HIF muteins, etc., or a polynucleotide encoding such a variant. In other aspects, the present invention comprises stabilizing HIF α comprising administering an agent that stabilizes HIF α. The agent may consist of polynucleotides, for example antisense sequences; polypeptides; antibodies; other proteins; carbohydrates; fat; lipids; and organic and inorganic compounds, for example small molecules, etc. In a preferred embodiment, the present invention comprises stabilization of HIF α, for example in a subject, by administering to the subject an agent that stabilizes HIF α, wherein the agent is a compound, for example a low molecular weight compound or the like which stabilizes HIF α.

In various aspects, HIFα is HIF1α, HIF2α, or HIF3α. In a preferred aspect, stabilizing HIF α comprises administering to the subject an effective amount of a compound that inhibits HIF hydroxylase activity. In certain aspects, the HIF hydroxylase is selected from the group consisting of EGLN1, EGLN2 and EGLN3.

In one embodiment, the invention provides a method of increasing the average blood cell volume in an individual, the method comprising administering to the individual an effective amount of a compound that stabilizes the alpha subunit of hypoxia-inducible factor (HIF). In yet another embodiment, the invention provides a method for increasing the average hemoglobin level in the blood cells of an individual, the method comprising administering to the individual an effective amount of a compound that stabilizes the alpha subunit of hypoxia-inducible factor (HIF). In another embodiment, the present invention comprises a method for reducing microcytosis in an individual, the method comprising administering to the individual an effective amount of a compound that stabilizes the alpha subunit of hypoxia-inducible factor (HIF).

The invention further provides a method for treating or preventing microcytic anemia, the method comprising administering to the individual an effective amount of a compound that stabilizes the alpha subunit of hypoxia-inducible factor (HIF).

In one aspect, the invention relates to a method for treating or preventing a condition associated with iron deficiency in a subject, wherein the method comprises administering to the subject an effective amount of a compound that stabilizes the alpha subunit of hypoxia-inducible factor (HIF). In a particular aspect, the invention provides a method of improving iron processing in a subject wherein the method comprises administering to the subject an effective amount of a compound that stabilizes the alpha subunit of hypoxia-inducible factor (HIF). Also provided is a method of treating or preventing a condition associated with impaired availability of iron in a subject, the method comprising administering to the subject an effective amount of a compound that stabilizes the alpha subunit of hypoxia-inducible factor (HIF).

In other embodiments, the invention relates to a method for overcoming cytokine-induced effects in a subject. More specifically, in one aspect, the invention provides a method of overcoming cytokine suppression of EPO formation in a subject, the method comprising administering to the subject an effective amount of a compound that stabilizes the alpha subunit of hypoxia-inducible factor (HIF). The invention further provides a method of overcoming cytokine suppression of iron availability in a subject, the method comprising administering to the subject an effective amount of a compound that stabilizes the alpha subunit of hypoxia-inducible factor (HIF). In another aspect, the present invention comprises a method for treating or preventing cytokine-associated anemia in an individual, the method comprising administering to the individual an effective amount of a compound that stabilizes the alpha subunit of hypoxia-inducible factor (HIF). Also provided are methods of increasing EPO formation in the presence of a cytokine in a subject, the methods comprising administering to the subject an effective amount of a compound that stabilizes the alpha subunit of hypoxia-inducible factor (HIF). In specific embodiments, the cytokine is selected from the group consisting of TNF-α and IL-1β.

In one aspect, the invention provides a method for reducing a cytokine-induced VCAM expression in a subject, the method comprising administering to the subject an effective amount of a compound that stabilizes the alpha subunit of hypoxia-inducible factor (HIF). In a specific aspect, the cytokine is TNF-α or IL-1β. In one aspect, the method relates to the reduction of cytokine-induced VCAM expression in endothelial cells in an individual. In another aspect, the individual has a condition selected from the group consisting of an inflammatory disease, an autoimmune disease, and anemia associated with chronic disease.

In another aspect, the invention provides a method for reducing cytokine-induced E-selectin expression in a subject, the method comprising administering to the subject an effective amount of a compound that stabilizes the alpha subunit of hypoxia-inducible factor. In one specific aspect, the cytokine is TNF-α or IL-1β. In one aspect, the method relates to the reduction of cytokine-induced E-selectin expression in endothelial cells in an individual. In another aspect, the individual has a condition selected from the group consisting of inflammatory disease, autoimmune disease, and anemia associated with chronic disease.

The invention provides various methods to regulate/enhance the processing and metabolism of iron. In one aspect, the invention provides methods of increasing the transport, uptake, utilization and absorption of iron in a subject, each of said methods comprising administering to the subject an effective amount of a compound that stabilizes the alpha subunit of hypoxia-inducible factor (HIF). In particular embodiments, the invention provides methods of increasing transferrin expression, transferrin receptor expression, IRP-2 expression, ferritin expression, ceruloplasmin expression, NRAMP2 expression, sproutin expression and ALAS-2 expression in a subject, each of these methods comprising administering to the subject an effective amount of a compound that stabilizes the alpha subunit of hypoxia-inducible factor (HIF). In other embodiments, the invention provides methods of reducing hepcidin expression, the method comprising administering to the subject an effective amount of a compound that stabilizes the alpha subunit of hypoxia-inducible factor (HIF). Methods of increasing heme synthesis in a subject by administering to the subject an effective amount of a compound that stabilizes the alpha subunit of hypoxia-inducible factor (HIF) are also provided.

In certain aspects, the invention includes methods of increasing serum iron levels, increasing transferrin saturation, increasing soluble transferrin receptor levels, and increasing serum ferritin levels in an individual, the methods comprising administering to the individual an effective amount of a compound that stabilizes the alpha subunit of hypoxia-inducible factor (HIF ). In yet another aspect, the invention provides a method of increasing iron transport to bone marrow in an individual, the method comprising administering to the individual an effective amount of a compound that stabilizes the alpha subunit of hypoxia-inducible factor (HIF).

In one aspect, the present methods are used for treating or manufacturing a drug for a subject, preferably a human, having one of the disorders or conditions discussed herein. It should be understood that various parameters associated with clinical conditions vary depending on age, sex, etc. In one aspect, the subject has a serum ferritin level that is below the normal range, for example below 50-200 μg/l; thus, an individual with a serum ferritin level that is below 200 ng/ml, below 150 ng/ml, below 100 ng/ml, below 75 ng/ml and below 50 ng/ml may be a suitable individual for treatment with the methods or use of the drugs provided by the present invention. Alternatively, a suitable individual can be demonstrated by demonstrating a total iron binding capacity (TIBC) that is below the normal range, for example less than TIBC 300-360 μg/dl.

In another embodiment, the individual has a serum iron level that is below the normal range, for example below a serum iron level of 50-150 μg/dl. Other suitable parameters for the detection of suitable individuals include measurements of transferrin saturation of less than 30-50%, measurement of a sideroblast level in bone marrow of less than 40-60% and a hemoglobin level of less than approximately 10 to 11 g/dl. Any of the parameters described above are measured, for example, as in standard hematological analyses, chemical blood analyzes and complete blood count (CBC) analysis, typically presented as a measurement of several blood parameters and obtained, for example, by analyzing blood in an automated instrument that for example, it measures the total red blood cell count, the total white blood cell count, the platelet count, and the relative proportion of red blood cells. The measurement can be performed by any standard means of measuring hematological and/or biochemical blood analysis, including, for example, automated systems such as the CELL DYN 4000 analyzer (Abbott Laboratories, Abbott Park, IL), the Coulter GenS analyzer (Beckman Coulter, Inc., Fullerton , CA) or the ADVIA 120 analyzer from Bayer (Bayer Healthcare AG, Leverkusen, Germany), etc.

In one aspect, the invention comprises a method for treating or preventing iron deficiency in a subject, where the method comprises administering to the individual an effective amount of a compound that stabilizes HIF α, whereby iron deficiency in the subject is treated or prevented. In further aspects, the iron deficiency is a functional iron deficiency; it is associated with anemia; it is associated with a disorder selected from the group consisting of an inflammation, an infection, an immunodeficiency disorder, and a neoplastic disorder; or it is associated with a disorder selected from the group consisting of anemia associated with chronic disease, iron deficiency anemia (IDA), and microcytic anemia.

An individual according to the invention can be an individual with any clinically accepted standard measurement that indicates iron deficiency or risk of developing iron deficiency. In certain embodiments, for example, the subject has a low serum ferritin level (<30 ng/ml) or reduced % transferrin saturation, for example less than 16% (for adults). Serum ferritin levels below 50 ng/ml, below 40 ng/ml, below 30 ng/ml and below 20 ng/ml are specifically included. It should be noted that if an individual has or is at risk of developing an iron deficiency that is functional iron deficiency, the serum ferritin level may be higher than the normal range, for example 200 ng/ml and higher. Iron deficiency can be observed in the presence of iron-limited/iron-deficient erythropoiesis (impaired hemoglobin synthesis, typically observed if % transferrin saturation is below 15 to 20%). These iron parameters can be measured using any standard CBC assay or biochemical assay described above and/or using automated devices more specifically aimed at iron analysis, such as the Unimate 5 Iron and Unimate 7 UIBC kits (Roche, Switzerland) .

An individual who may benefit from the present methods of treatment or prevention may be an individual who has or is susceptible to iron deficiency anemia, for example an individual with a transferrin saturation percentage of 10-15% or less than 10%.

In one aspect, the individual who has or is susceptible to iron deficiency will have or is susceptible to functional iron deficiency. A hemoglobin content in the reticulocytes of less than 28 picograms/cell will indicate such a condition. In another aspect, the subject who has or is susceptible to functional iron deficiency exhibits more than 5% hypochromic red blood cells.

In certain embodiments, the subject is an individual who has or is at risk of developing anemia associated with chronic disease. Such an individual may show a mild or moderate anaemia, for example a hemoglobin level of around 10-13 g/dl, more precisely 10-11 g/dl. In other embodiments, the individual exhibits a more acute anemia, for example, a hemoglobin level that is below 10 g/dl including a level below 5 g/dl and levels below 3 g/dl. In some embodiments, the subject who has or is susceptible to anemia associated with chronic disease exhibits abnormalities in iron distribution. Such abnormalities may be, for example, a serum iron level below about 60 μg/dl or a serum ferritin level above the normal range, for example above 200 ng/ml, above 300 ng/ml or above 400 ng/ml.

In certain aspects, the subject may have or be susceptible to microcytic anemia. Such an individual may, for example, show an average blood cell volume of less than 80 femtoliters, for example measured as part of a complete blood count. In other aspects, the individual has an average blood cell volume that is less than the normal value of 90 /- 8 femtoliters. The individual may in various aspects have a reduced average cellular hemoglobin level, for example an average cellular hemoglobin level of less than 30 /- 3 picograms of hemoglobin/cell; or a reduced mean cellular hemoglobin concentration, for example a mean cellular hemoglobin concentration of less than 33 /- 2%.

A method for treating or preventing functional iron deficiency in an individual comprising administering to the individual an effective amount of a compound that stabilizes HIF α, thereby treating or preventing functional iron deficiency, is also provided.

In one embodiment, the present invention provides a method for regulating or enhancing iron metabolism or a metabolic process involving iron in an individual, the method comprising administering to the individual an effective amount of a compound that stabilizes HIF α, whereby the iron metabolism or process that is associated with iron metabolism is regulated or enhanced in the individual. In another embodiment, the invention provides a method for regulating or enhancing a process included in iron metabolism selected from the group consisting of iron uptake, iron absorption, iron transport, iron storage, iron processing, iron mobilization and iron utilization, wherein the method comprises administering to the individual an effective amount of a compound that stabilizes HIF α, whereby the process associated with iron metabolism is regulated or enhanced in the individual.

Also provided herein is a method of increasing iron absorption in an individual, the method comprising administering to the individual an effective amount of a compound that stabilizes HIF α and thereby increases iron absorption in the individual. In certain aspects, iron absorption is in the intestine; the absorption is of iron in the diet; or it occurs in enterocytes in the duodenum.

The following methods are also included herein: A method for enhancing iron transport in an individual, where the method comprises administering to the individual an effective amount of a compound that stabilizes HIF α and thereby provides increased iron transport in the individual; a method of increasing iron stores in an individual, the method comprising administering to the individual an effective amount of a compound that stabilizes HIF α and thereby increases iron storage in the individual; a method for increasing iron uptake in an individual, wherein the method comprises administering to the individual an effective amount of a compound which stabilizes HIF α and thereby increases iron uptake in the individual; a method for increasing iron processing in an individual, the method comprising administering to the individual an effective amount of a compound that stabilizes HIF α and thereby provides increased iron processing in the individual; a method for increased iron mobilization in an individual wherein the method comprises administering to the individual an effective amount of a compound that stabilizes HIF α and thereby provides increased iron mobilization in the individual; and a method for increasing iron utilization in an individual, wherein the method comprises administering to the individual an effective amount of a compound which stabilizes HIF α and thereby provides increased iron utilization in the individual.

In one embodiment, the invention comprises a method for increasing the supply of iron for erythropoiesis in an individual, where the method comprises administering to the individual an effective amount of a compound that stabilizes HIF α and thereby provides an increased supply of iron for erythropoiesis in the individual. In various embodiments, the increased supply of iron for erythropoiesis is an increased supply of iron for heme synthesis; an increased supply of iron for hemoglobin production; or an increased supply of iron for the formation of red blood cells.

The invention further provides methods for regulating the expression of iron regulatory factors in an individual, where the method comprises administering to the individual an effective amount of a compound that stabilizes HIF α and thereby regulates the expression of metabolic factors for iron in the individual.

Methods of increasing the expression of certain iron regulatory factors are encompassed herein, including: A method of increasing transferrin receptor expression in an individual wherein the method comprises administering to the individual an effective amount of a compound that stabilizes HIF α so that the expression of transferrin expression is increased in the individual; a method of increasing transferrin expression in an individual, the method comprising administering to the individual an effective amount of a compound that stabilizes HIF α so that transferrin expression is increased in the individual; a method of increasing ceruloplasmin expression in an individual, the method comprising administering to the individual an effective amount of a compound that stabilizes HIF α so that ceruloplasmin expression is increased in the individual; a method of increasing NRAMP2 (slc11a2) expression in an individual, the method comprising administering to the individual an effective amount of a compound that stabilizes HIF α such that NRAMP2 expression is increased in the individual; a method for increasing the expression of cytochrome b reductase 1 in the duodenum of an individual, wherein the method comprises administering to the individual an effective amount of a compound that stabilizes HIF α so that the expression of cytochrome b reductase 1 in the individual's duodenum is increased; and a method for increasing 5-aminolevulinate synthase expression in an individual, the method comprising administering to the individual an effective amount of a compound that stabilizes HIF α so that 5-aminolevulinate synthase expression in the individual is increased.

In one embodiment, the invention provides a method for increasing the iron content in the serum of an individual, where the method comprises administering to the individual an effective amount of a compound that stabilizes HIF α and thereby increases the iron content in the individual's serum. In certain embodiments, the subject is a human and the serum iron level is elevated to a value between 50 and 150 μg/dl.

In another aspect, the present invention provides methods for increasing the total iron binding capacity (TIBC) in an individual. The method comprises administering to the subject an effective amount of a compound that stabilizes HIF α such that TIBC is elevated in the subject. In a preferred aspect, the individual is a human and the total iron-binding capacity is increased to a value of between 300 and 360 μg/dl.

Methods and compounds for modulating serum ferritin levels in an individual are provided. In a particular embodiment, the individual is a human and the serum ferritin level is increased to above 15 μg/l. In a further embodiment, the individual is an adult male and the serum ferritin level is increased to a value of approximately 100 μg/l. In another embodiment, the subject is an adult female and the serum ferritin level is elevated to a level of approximately 30 μg/l.

In one aspect, the invention comprises a method for increasing the transferrin saturation in an individual, where the method comprises administering to the individual an effective amount of a compound that stabilizes HIF α so that the transferrin saturation is increased in the individual. In one aspect, the transferrin loading is increased above a level selected from the group consisting of 10%, 15%, 20%, 30%, 40% and 50%. The present invention includes methods for increasing percent transferrin content in an individual. In one embodiment, the individual is a human and the percent transferrin content is increased to a value of over 18%. In another embodiment, the percent transferrin content is increased to a value of between 25 and 50%. Percent transferrin is typically calculated using the formula: (iron in serum)(100)/(TIBC).

Also provided are methods of increasing the level of soluble transferrin receptor in an individual, the methods comprising administering to the individual an effective amount of a compound that stabilizes HIF α so that the level of soluble transferrin receptor in the individual is increased. The invention further provides methods for increasing the total erythroid bone marrow mass, measured for example as the level of transferrin receptor in serum. In one aspect, the subject is a human and the serum transferrin receptor level is elevated to from 4 to 9 μg/l, as measured by an immunoassay.

A method for reducing hepcidin expression in an individual is provided, wherein the method comprises administering to the individual an effective amount of a compound that stabilizes HIF α so that hepcidin expression in the individual is reduced.

In one embodiment, the invention provides a method for treating or preventing a disorder associated with iron deficiency in an individual, wherein the method comprises administering to the individual an effective amount of a compound that stabilizes HIF α, whereby the disorder associated with iron deficiency in the individual is treated or prevented . In one embodiment, the iron deficiency is functional iron deficiency. In various embodiments, the disorder is selected from the group consisting of an inflammation, an inflammation, an immunodeficiency disorder, and a neoplastic disorder; or it is selected from the group consisting of anemia associated with chronic disease, iron deficiency anemia, and microcytic anemia.

The invention provides a method for enhancing erythropoiesis in an individual who has or is susceptible to iron deficiency, where the method comprises administering to the individual an effective amount of a compound that stabilizes HIF α so that erythropoiesis in the individual is enhanced. In a particular aspect, it is understood that the iron deficiency is functional iron deficiency.

The invention further provides a method for enhancing erythropoiesis in an individual in which the individual has or is susceptible to functional iron deficiency, where the method comprises administering to the individual an effective amount of a compound that stabilizes HIF α so that erythropoiesis in the individual is enhanced. In various aspects, the chronic disease is selected from the group consisting of an inflammation, an infection, an immunodeficiency disorder, and a neoplastic disorder.

A method for enhancing erythropoiesis in an individual where the individual has or is susceptible to anemia associated with chronic disease is also provided, wherein the method comprises administering to the individual an effective amount of a compound that stabilizes HIF α so that erythropoiesis in the individual is enhanced .

In one embodiment, the invention comprises a method for enhancing erythropoiesis in an individual in which the individual does not respond to EPO treatment, where the method comprises administering to the individual an effective amount of a compound that stabilizes HIF α so that erythropoiesis in the individual is enhanced.

A method for treating or preventing anemia associated with chronic disease in an individual wherein the method comprises administering to the individual an effective amount of a compound that stabilizes HIF α so that anemia associated with chronic disease is treated or prevented in the individual is also provided. It is encompassed in various aspects that the anemia associated with chronic disease is associated with a condition selected from the group consisting of an inflammation, an infection, an immunodeficiency disorder, and a neoplastic disorder.

The invention specifically includes the following: A method for increasing the reticulocyte content in an individual with a chronic disease, where the method comprises administering to the individual an effective amount of a compound that stabilizes HIF α so that the reticulocyte level in the individual is increased; a method of increasing the hematocrit in an individual with a chronic disease, the method comprising administering to the individual an effective amount of a compound that stabilizes HIF α so that the individual's hematocrit is increased; a method of increasing the hemoglobin content of an individual with a chronic disease, the method comprising administering to the individual an effective amount of a compound that stabilizes HIF α so that the hemoglobin content of the individual is increased; a method of increasing the number of red blood cells in an individual with a chronic disease, the method comprising administering to the individual an effective amount of a compound that stabilizes HIF α so that the number of red blood cells in the individual is increased; a method for increasing the average blood cell volume in an individual with a chronic disease, the method comprising administering to the individual an effective amount of a compound that stabilizes HIF α so that the average blood cell volume in the individual is increased; a method of increasing the average amount of hemoglobin in the blood cells of an individual with a chronic disease, the method comprising administering to the individual an effective amount of a compound that stabilizes HIF α so that the average amount of hemoglobin in the individual's blood cells is increased; a method of increasing the iron concentration in the serum of an individual with a chronic disease, the method comprising administering to the individual an effective amount of a compound that stabilizes HIF α so that the iron concentration in the individual's serum is increased; and a method for increasing the transferrin saturation in an individual with a chronic disease, the method comprising administering to the individual an effective amount of a compound that stabilizes HIF α so that the transferrin saturation in the individual is increased. In all of these methods, in certain embodiments, the chronic disease is selected from the group consisting of an inflammation, an infection, an immunodeficiency disorder, and a neoplastic disorder; or it is selected from the group consisting of anemia associated with chronic disease, anemia associated with iron deficiency, iron deficiency, functional iron deficiency and microcytic anemia.

The following methods are also provided: A method for increasing the reticulocyte level in an individual with iron deficiency, the method comprising administering to the individual an effective amount of a compound that stabilizes HIF α so that the reticulocyte level in the individual is increased; a method of increasing the hematocrit of an iron-deficient individual, the method comprising administering to the individual an effective amount of a compound that stabilizes HIF α so that the hematocrit of the individual is increased; a method of increasing the hemoglobin level in an individual with iron deficiency, the method comprising administering to the individual an effective amount of a compound that stabilizes HIF α so that the hemoglobin level in the individual is increased; a method of increasing the number of red blood cells in an individual with iron deficiency, the method comprising administering to the individual an effective amount of a compound that stabilizes HIF α so that the number of red blood cells in the individual in the individual is increased; a method of increasing the average blood cell volume in an individual with iron deficiency, the method comprising administering to the individual an effective amount of a compound that stabilizes HIF α so that the average blood cell volume of the individual is increased; a method of increasing the average hemoglobin content in the blood cells of an individual with iron deficiency, the method comprising administering to the individual an effective amount of a compound that stabilizes HIF α so that the average hemoglobin level in the individual's blood cells is increased; a method of increasing the iron concentration in the serum of an individual with iron deficiency, the method comprising administering to the individual an effective amount of a compound that stabilizes HIF α so that the iron concentration in the individual's serum is increased; and a method for increasing the transferrin saturation in an individual with iron deficiency, the method comprising administering to the individual an effective amount of a compound that stabilizes HIF α so that the transferrin saturation in the individual is increased. In all of these methods, in certain embodiments, the iron deficiency is a functional iron deficiency.

The following methods are also included: A method for increasing the reticulocyte level in an individual with functional iron deficiency, where the method comprises administering to the individual an effective amount of a compound that stabilizes HIF α so that the reticulocyte level in the individual is increased; a method of increasing the hematocrit in an individual with functional iron deficiency, the method comprising administering to the individual an effective amount of a compound that stabilizes HIF α so that the hematocrit of the individual is increased; a method for increasing the hemoglobin level in an individual with functional iron deficiency, the method comprising administering to the individual an effective amount of a compound that stabilizes HIF α so that the hemoglobin level in the individual is increased; a method of increasing the number of red blood cells in an individual with functional iron deficiency, the method comprising administering to the individual an effective amount of a compound that stabilizes HIF α so that the number of red blood cells increases in the individual; a method for increasing the average blood cell volume in an individual with functional iron deficiency, the method comprising administering to the individual an effective amount of a compound that stabilizes HIF α so that the average blood cell volume in the individual is increased; a method for increasing the average hemoglobin level in the blood cells of an individual with functional iron deficiency, the method comprising administering to the individual an effective amount of a compound that stabilizes HIF α so that the average hemoglobin level in the individual's blood cells is increased; a method for increasing the iron concentration in the serum of an individual with functional iron deficiency, the method comprising administering to the individual an effective amount of a compound that stabilizes HIF α so that the iron concentration in the individual's serum is increased; and a method for increasing the transferrin saturation in an individual with functional iron deficiency, wherein the method comprises administering to the individual an effective amount of a compound that stabilizes HIF α so that the transferrin saturation in the individual is increased.

In one aspect, the invention comprises a method for overcoming or alleviating the effects of a cytokine-induced impairment of erythropoiesis in an individual, wherein the method comprises administering to the individual an effective amount of a compound that stabilizes HIF α so that the effects of the cytokine-induced impairment of erythropoiesis in the individual overcome or alleviated. In certain aspects, the cytokine-induced impairment of erythropoiesis is suppression of EPO production or impaired iron metabolism. In any of the methods described above, the cytokine is an inflammatory cytokine. In other embodiments, the cytokine is selected from the group consisting of TNF-α, IL-1β and IFN-γ.

Methods of reducing cytokine induction of VCAM-1 expression and/or E-selectin expression are also provided wherein the method comprises administering to an individual in need thereof an effective amount of a compound that stabilizes HIF α such that cytokine induction of VCAM-1 expression and/or E-selectin expression is reduced.

In any of the methods described above, the cytokine is an inflammatory cytokine. In other embodiments, the cytokine is selected from the group consisting of TNF-α, IL-1β and IFN-γ.

Methods for treating or preventing a disorder associated with cytokine activity in an individual wherein the disorder is selected from the group consisting of iron deficiency, functional iron deficiency, iron deficiency anemia, anemia associated with chronic disease, and microcytic anemia are provided herein, wherein the methods comprise administering to the individual an effective amount of a compound that stabilizes HIF α so that the disorder associated with cytokine activity is treated or prevented. In any of the methods described above, the cytokine is an inflammatory cytokine. In further embodiments, the cytokine is selected from the group consisting of TNF-α, IL-1β and IFN-γ.

Methods of treating or preventing a disorder associated with cytokine activity in an individual wherein the disorder is associated with a condition selected from the group consisting of an inflammation, an infection, an immunodeficiency, and a neoplastic disorder, wherein the methods comprise administering to the individual an effective amount of a compound that stabilizes HIF α so that the disorder associated with cytokine activity is treated or prevented is also provided. In any of the methods described above, the cytokine is an inflammatory cytokine. In further embodiments, the cytokine is selected from the group consisting of TNF-α, IL-1β and IFN-γ.

In one aspect, the invention comprises a method for increasing EPO formation in the presence of a cytokine in an individual, where the method comprises administering to the individual an effective amount of a compound that stabilizes HIF α so that EPO formation in the individual is increased. A method for treating or preventing microcytosis in an individual where the method comprises administering to the individual an effective amount of a compound that stabilizes HIF α so that microcytosis in the individual is treated or prevented is also provided herein. In further aspects, the microcytosis is associated with a disorder selected from the group consisting of chronic disease, anemia associated with chronic disease, iron deficiency, functional iron deficiency, and anemia associated with iron deficiency. In any of the methods described above, the cytokine is an inflammatory cytokine. In further embodiments, the cytokine is selected from the group consisting of TNF-α, IL-1β and IFN-γ.

In any of the present methods for treatment or prevention, it is understood that a compound according to the invention can be administered as part of a combination treatment which additionally comprises the administration of another therapeutic agent, for example EPO, iron and vitamins, for example B- vitamins, etc.

A kit comprising a compound that stabilizes HIF α and at least one other agent is provided herein. In one aspect, there is provided an additional agent selected from the group consisting of erythropoietin, iron and B vitamins, and likewise a pharmaceutical composition comprising a compound that stabilizes HIF α and at least one additional agent selected from the group consisting of erythropoietin, iron and B vitamins .

The present invention provides compounds and methods for treating or preventing anemia associated with chronic disease, wherein the anemia associated with chronic disease is associated with an elevated cytokine level. More specifically, the invention provides methods and compounds for use in overcoming or ameliorating the consequences of cytokine-induced effects in an individual with elevated cytokine levels, for example, cytokine suppression of EPO formation, cytokine-induced expression of various cell adhesion factors, etc.

In one embodiment, the invention provides methods and compounds for overcoming cytokine suppression of EPO formation. These methods and compounds are useful for overcoming TNF α and/or IL-1 β suppression of EPO formation, as measured by, for example, the ability to overcome TNF α and/or IL-1 β suppression of EPO formation in cultured Hep3B cells.

In one embodiment, the invention provides methods and compounds for reducing cytokine-induced elevation of the expression of various cell adhesion factors. The methods and compounds can be used to overcome TNF α-, IL-1 β- and IFN-γ-induced increases in the expression of adhesion factors, for example VCAM-1 and E-selectin, in endothelial cells as measured by, for example, a decrease in the expression level of VCAM-1 or E-selectin in endothelial cells (HUVEC, etc).

The invention provides methods and compounds for treating or preventing iron deficiency in an individual. More specifically, the present methods and compounds can be used to enhance iron metabolism or treat or prevent diseases and disorders associated with impaired iron metabolism, such as impaired uptake, storage, processing, transport, mobilization and utilization of iron, etc.

In one aspect, the methods and compounds modulate the expression of factors involved in iron metabolism, such as transport, utilization, storage, etc. The methods and compounds, for example, increase the expression of transferrin receptor as measured by, for example, elevated expression of transferrin receptor in liver cells (e.g., Hep3B, HepG2 ), kidney cells (for example HK-2) or lymphocytes (for example THP-1), or by elevated levels of soluble transferrin receptor in humans. The present methods and compounds increase ceruloplasmin gene expression as measured by, for example, increased gene expression in mouse kidney cells and in Hep3B cells. In one aspect, the invention provides methods and compounds that reduce hepcidin expression, for example as measured by reduced gene expression of hepcidin in mouse liver. In yet another aspect, methods and compounds of the present invention are used to increase the expression of factors, including NRAMP2, cytochrome b reductase 1 in the duodenum, etc., as measured by, for example, elevated gene expression in the mouse intestine. The present methods and compounds increase the expression of 5-aminolevulinate synthase, the first enzyme in the heme synthesis pathway and the rate-limiting enzyme for heme synthesis, as measured by, for example, elevated gene expression in mouse intestine.

The present methods and compounds can be used to enhance iron metabolism. Specifically, the present methods and compounds enhance iron metabolism, as measured by, for example, elevated serum iron levels, elevated percent transferrin saturation, and decreased microcytosis in a rat model of impaired iron metabolism.

The present invention provides methods and compounds for the induction of enhanced erythropoiesis. More specifically, the present methods and compounds enhance erythropoiesis, as measured by, for example, elevated reticulocyte count, hematocrit and red blood cell count in a rat model of impaired erythropoiesis and in humans, or measured by, for example, elevated hemoglobin level in a rat model of impaired erythropoiesis.

The present methods and compounds reduce microcytosis as measured by, for example, increased mean hemoglobin level in blood cells and increased mean blood cell volume in a rat model of impaired erythropoiesis.

The present methods comprise administering to a subject an effective amount of a compound that stabilizes HIF α. Such stabilization can take place via, for example, inhibition of HIF hydroxylase activity. A preferred compound according to the invention is a compound which inhibits HIF-prolyl hydroxylase activity. The inhibition may be direct or indirect, competitive or non-competitive, etc. In various embodiments, a compound according to the invention is selected from the group consisting of 2-oxoglutarate-like compounds, iron-chelating compounds and proline analogues. In one aspect, a 2-oxoglutarate-like compound is a heterocyclic carbonyl glycine of formula I, Ia or Ib. In another aspect, an iron chelating compound is a hydroxamic acid of formula III. In particular embodiments as exemplified herein, the compound is compound D.

Examples of compounds according to the invention include [(1-chloro-4-hydroxyisoquinoline-3-carbonyl)amino]acetic acid (compound A), [(4-hydroxy-7-phenoxyisoquinoline-3-carbonyl)amino]acetic acid (compound B), [(4-hydroxy-7-phenylsulfanylisoquinoline-3-carbonyl)amino]acetic acid (compound C) and 3-{[4-(3,3-dibenzylureido)benzenesulfonyl]-[2-(4-methoxyphenyl)ethyl]amino} -N-Hydroxypropionamide (compound D). Additional compounds of the present invention and methods for detecting other compounds of the present invention are provided below.

BRIEF DESCRIPTION OF THE FIGURES

Figures 1A and 1B describe results showing that methods and compounds of the present invention overcome the suppressive effects of TNF-α on EPO formation.

Figures 2A and 2B show results showing that methods and compounds of the present invention overcome the suppressive effects of TNF-α on EPO formation in cells pretreated with TNF-α.

Figures 3A and 3B show results showing that methods and compounds of the present invention overcome the suppressive effects of IL-1β on EPO formation.

Figures 4A and 4B show results showing that methods and compounds of the present invention overcome the suppressive effects of IL-1β on EPO formation in cells pretreated with IL-1β.

Figure 5 shows results showing that methods and compounds according to the present invention reduce VCAM-1 expression associated with TNF-α.

Figures 6A, 6B and 6C show results showing elevated expression of transferrin receptor and iron transporter (Figure 6A), iron transport intestinal protein (Figure 6B) and 5-aminolevulinate synthase (Figure 6C) after treatment of mice with compounds according to the present invention.

Figure 7 shows results showing that methods and compounds according to the present invention produced an increased reticulocyte loss in an animal model for anemia associated with chronic disease.

Figure 8 shows results showing that methods and compounds according to the present invention increased hematocrit in an animal model for anemia associated with chronic disease.

Figure 9 shows results showing that methods and compounds according to the present invention produced elevated hemoglobin levels in an animal model for anemia associated with chronic disease.

Figure 10 shows results showing that methods and compounds of the present invention produced an elevated number of red blood cells in an animal model of anemia associated with chronic disease.

Figure 11 shows results showing that methods and compounds according to the present invention produced reduced microcytosis in an animal model for anemia associated with chronic disease.

Figure 12 shows results showing that methods and compounds according to the present invention increased the average hemoglobin content in blood cells and improved hypochromia in an animal model for anemia associated with chronic disease.

Figure 13 shows results showing that methods and compounds according to the present invention increased hematocrit in normal animals and in an animal model for anemia associated with chronic disease.

Figure 14 shows results showing that methods and compounds according to the present invention produced elevated hemoglobin levels in normal animals and in an animal model for anemia associated with chronic disease.

Figure 15 shows results showing that methods and compounds according to the present invention produced an increased number of red blood cells in normal animals and in an animal model for anemia associated with chronic disease.

Figure 16 shows results showing that methods and compounds according to the present invention increased average blood cell volume in normal animals and in an animal model for anemia associated with chronic disease.

Figure 17 shows results showing that methods and compounds according to the present invention produced an elevated hemoglobin level in blood cells in normal animals and in an animal model for anemia associated with chronic disease.

Figures 18A and 18B show results showing that methods and compounds of the present invention produced elevated serum iron levels (Figure 18A) and transferrin saturation (Figure 18B) in normal animals and in an animal model of anemia associated with chronic disease.

Figure 19 shows results showing that methods and compounds according to the present invention produced elevated gene expression of NRAMP2 (slc112a) and sproutin (CYBRD1, duodenal cytochrome b reductase 1) in normal animals and in an animal model of anemia associated with chronic disease.

Figure 20 shows results showing elevated numbers of reticulocytes after administration of a compound according to the present invention to healthy humans.

Figure 21 shows results showing increased numbers of red blood cells in healthy people administered a compound according to the present invention.

Figure 22 shows results showing elevated levels of soluble transferrin receptor after administration of a compound of the present invention to healthy humans.

Figure 23 shows results showing a reduced ferritin level in serum in healthy people administered a compound according to the present invention.

Figures 24A and 24B show results showing that methods and compounds of the present invention reduced the expression of VCAM-1 and E-selectin associated with TNF-α.

Figure 25 shows results showing that methods and compounds according to the present invention reduced VCAM-1 expression associated with TNF-α and IL-1β.

Figure 26 shows results showing that methods and compounds according to the present invention reduced E-selectin expression associated with TNF-α, IL-1β and IFN-γ.

Figures 27A and 27B show results showing that methods and compounds according to the present invention and IL-6 synergistically produced a higher EPO level in hepatocytes.

DESCRIPTION OF THE INVENTION

Before the present compositions and methods are described, it should be understood that the invention is not limited to the particular methodologies, methods, cell lines, assays and reagents described, as these may vary. It should also be understood that the terminology used herein is used to describe particular embodiments of the present invention, and is not intended in any way to limit the scope of the present invention, as described in the appended claims.

It must be noted that as used herein and in the appended claims, the singular forms "a" and "an" include multiple references, unless the context clearly indicates otherwise. Thus, for example, a reference to "a fragment" includes a large number of such fragments; a reference to "a compound" is a reference to one of several compounds and to equivalent compounds as described herein and known to those skilled in the art, etc.

Unless otherwise indicated, all technical and scientific terms used herein have the same meaning as is usually used by average persons skilled in the field to which the invention belongs. Although all methods and materials corresponding to or equivalent to those described herein may be used in carrying out or testing the present invention, the preferred methods, devices and materials are now described. All publications cited herein are incorporated herein by reference in their entirety for the purpose of describing the methodologies, reagents and tools reported in those publications that may be used in connection with the invention. Nothing herein shall be construed as an admission that the invention does not preempt such descriptions, due to earlier discovery.

The implementation of the present invention will, unless otherwise stated, use conventional methods in chemistry, biochemistry, molecular biology, cell biology, genetics, immunology and pharmacology which are within the state of the art. Such techniques are fully explained in the literature. See for example Gennaro, A.R., ed. (1990) Remington's Pharmaceutical Sciences, 18th ed., Mack Publishing Co.; Hardman, J.G., Limbird, L.E., and Gilman, A.G., eds. (2001) The Pharmacological Basis of Therapeutics, 10th ed., McGraw-Hill Co.; Colowick, S. et al., eds., Methods In Enzymology, Academic Press, Inc.; Weir, D. M., and Blackwell, C. C., eds. (1986) Handbook of Experimental Immunology, Volumes I-IV, Blackwell Scientific Publications; Maniatis, T. et al., eds. (1989) Molecular Cloning: A Laboratory Manual, 2nd Edition, Volumes I-III, Cold Spring Harbor Laboratory Press; Ausubel, F.M. et al., ed. (1999) Short Protocols in Molecular Biology, 4th edition, John Wiley &Sons; Ream et al., eds. (1998) Molecular Biology Techniques: An Intensive Laboratory Course, Academic Press; Newton, C.R., and Graham, A., eds. (1997) PCR (Introduction to Biotechniques Series), 2nd edition, Springer Verlag.

DEFINITIONS

The term "anemia associated with chronic disease" refers to any form of anemia that develops as a result of, for example, long-term infection, inflammation, neoplastic disorders, etc. The anemia that develops is often characterized by a shortened lifespan of red blood cells and sequestration of iron in macrophages , which means that a reduced amount of iron is available for the formation of new red blood cells. Conditions associated with anemia associated with chronic disease include, but are not limited to, chronic bacterial endocarditis, osteomyelitis, rheumatic fever, ulcerative colitis, and neoplastic disorders. Additional conditions include other diseases and disorders associated with infection, inflammation, and neoplasia including, for example, inflammatory infections (eg, pulmonary abscess, tuberculosis, osteomyelitis, etc.), non-infectious inflammatory disorders (eg, rheumatoid arthritis, systemic lupus erythematosus, Crohn's disease, hepatitis, inflammatory bowel disease, etc.) and various cancers, tumors and malignant conditions (eg carcinoma, sarcoma, lymphoma, etc.).

The terms "disorders" and "diseases" and "conditions" are used interchangeably and refer to any condition that deviates from the normal.

The term "erythropoietin" refers to any recombinant or naturally occurring erythropoietin including, for example, human erythropoietin (GenBank accession number AAA52400; Lin et al. (1985) Proc Natl Acad Sci USA 82: 7580-7584), the human recombinant erythropoietin EPOETIN (Amgen, Inc. ., Thousand Oaks, CA), the human recombinant erythropoietin ARANESP (Amgen), the human recombinant erythropoietin PROCRIT (Ortho Biotech Products, L.O., Raritan, NJ), etc.

The term "HIF α" refers to the alpha subunit of the protein hypoxia-inducible factor. HIF α can be any human protein or a protein from another mammal, or a 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 no. 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. CAB96612) and -HIF-3 α (Genbank accession no. CAB96611); and HIF-1 α from cattle (Genbank accession number BAA78675). HIF α can also be any non-mammalian protein or fragment thereof, including HIF-1 α from Xenopus laevis (Genbank accession number CAB96628), HIF-1 α from Drosophila melanogaster (Genbank accession number JC4851) and HIF-1 α from chicken (Genbank accession designation BAA34234). HIF α gene sequences can also be obtained by routine cloning techniques, for example by using all or part of a HIF α gene sequence described above as a probe for recovery and determination of the sequence of a HIF α gene in another species.

Fragments of HIF α include the regions defined by human HIF-1 α from amino acid 401 to 603 (Huang et al., supra), amino acid 531 to 575 (Jiang et al. (1997) J Biol Chem 272: 19253-19260) , amino acid 556 to 575 (Tanimoto et al., supra), amino acid 557 to 571 (Srinivas et al. (1999) Biochem Biophys Res Commun 260: 557-561) and amino acid 556 to 575 (Ivan and Kaelin (2001) Science 292 : 464-468). Furthermore, a fragment of HIF α includes any fragment containing at least one occurrence of the motif LXXLAP, for example as this occurs in the native HIF α sequence as L397TLLAP and L559EMLAP. In addition, a fragment of HIF α includes any fragment that retains at least one functional or structural property associated with HIF α.

The terms "HIF prolyl hydroxylase" and "HIF PH" refer to any enzyme that can hydroxylate a proline residue in the HIF protein. The proline residue that is hydroxylated by HIF PH preferably comprises the proline residue that is present in the motif LXXLAP, for example as this occurs in the native human HIF-1 α sequence as L397TLLAP and L559EMLAP. HIF PH comprises members of the Egl-Nine (EGLN) gene family described by Taylor (2001, Gene 275: 125-132) and characterized by Aravind and Koonin (2001, Genome Biol 2: RESEARCH007), Epstein et al. (2001, Cell 107: 43-54) and Bruick and McKnight (2001, Sciences 294: 1337-1349). Examples of HIF prolyl hydroxylase enzymes include human SM-20 (EGLN1) (GenBank accession number AAG33965; Dupuy et al. (2000) Genomics 69: 348-54), EGLN2 isoform 1 (GenBank accession number CAC42510; Taylor, supra), EGLN2 isoform 2 ( GenBank accession designation NP_060025) and EGLN3 (GenBank accession designation CAC42511; Taylor, supra); mouse EGLN1 (GenBank accession no. CAC42515), -EGLN2 (GenBank accession no. CAC42511) and -EGLN3 (SM-20) (GenBank accession no. CAC42517); and rat SM-20 (GenBank accession number AAA19321). In addition, HIF PH may include EGL-9 from Caenorhabditis elegans (GenBank accession number AAD56365) and the CG1114 gene product from Drosophila melanogaster (GenBank accession number AAF52020). HIF-prolyl hydroxylase also includes any fragment of the above-described full-length proteins that retains at least one structural or functional property.

The term "prolyl hydroxylase inhibitor" or "PHI" as used herein refers to any compound that reduces or otherwise modulates the activity of an enzyme that hydroxylates amino acid residues. Although enzymatic activity where proline residues are hydroxylated is preferred, hydroxylation of other amino acids including, but not limited to, arginine is also encompassed. Compounds which can be used in the methods according to the invention include, for example, iron-chelating compounds, 2-oxoglutarate-like compounds and modified amino acid analogues, for example proline analogues.

In particular embodiments, the invention provides for the use of structural mimics of 2-oxoglutarate. Such compounds can inhibit the target enzyme from the 2-oxoglutarate dioxygenase enzyme family competitively relative to 2-oxoglutarate and non-competitively relative to iron. (Majamaa et al. (1984) Eur J Biochem 138: 239-245; and Majamaa et al. (1985) Biochem J 229: 127-133). PHI that is specifically encompassed for use in the present methods is described, for example, in Majamaa et al., supra; Kivirikko and Myllyharju (1998) Matrix Biol 16: 357-368; Bickel et al. (1998) Hepatology 28: 404-411; Friedman et al. (2000) Proc Natl Acad Sci USA 97: 4736-4741; Franklin (1991) Biochem Soc Trans 19): 812 815; Franklin et al. (2001) Biochem J 353-333-338; and in the international patent publications WO 03/053977 and WO 03/049686 which are both incorporated herein by reference in their entirety. Examples of PHI include [(1-chloro-4-hydroxyisoquinoline-3-carbonyl)amino]acetic acid (compound A), [(4-hydroxy-7-phenoxyisoquinoline-3-carbonyl)amino]acetic acid (compound B), [ (4-hydroxy-7-phenylsulfanylisoquinoline-3-carbonyl)amino]acetic acid (compound C) and 3-{[4-(3,3-dibenzylureido)benzenesulfonyl]-[2-(4-methoxyphenyl)ethyl]amino} N-hydroxypropionamide (compound D) is used in the present examples to demonstrate the methods of the invention described herein.

INVENTION

The present invention relates to methods and compounds for the induction of enhanced or complete erythropoiesis in an individual. More specifically, the methods comprise the induction of enhanced or complete erythropoiesis by stabilizing HIF α in an individual. Methods of inducing enhanced erythropoiesis by inhibiting HIF-prolyl hydroxylase are specifically encompassed. In specific embodiments, the methods comprise administering to an individual a compound according to the invention. In various embodiments, the subject may be a cell, a tissue, an organ, an organ system or an entire organism.

Anemia associated with chronic disease is the most common form of anemia in hospitalized patients. Anemia associated with chronic disease occurs in patients with inflammatory disorders or cancers, including inflammatory infections (eg, pulmonary abscess, tuberculosis, osteomyelitis, etc.), non-infectious inflammatory diseases (eg, rheumatoid arthritis, systemic lupus erythrematosis, Crohn's disease, hepatitis, inflammatory bowel disease, etc) and various cancers, tumors and malignant conditions (eg carcinoma, sarcoma, lymphoma, etc), chronic bacterial endocarditis, osteomyelitis, rheumatic fever, ulcerative colitis and neoplastic disorders.

In one aspect, the invention provides methods of inducing enhanced or complete erythropoiesis for the treatment of anemia associated with chronic disease. Anemia associated with chronic disease is associated with a variety of chronic disorders, including, for example, rheumatoid arthritis, rheumatic fever, inflammatory bowel disease, ulcerative colitis, systemic lupus erythematosis, vasculitis, neoplastic disorders, etc., as well as chronic infection and chronic inflammation. Reduced or ineffective erythropoiesis is a common disease state in patients with anemia associated with chronic disease. Reduced or ineffective erythropoiesis may result from various metabolic abnormalities in the erythropoietic pathway, including, for example, suppressed EPO formation, reduced EPO response in bone marrow, and abnormal processing of iron, such as abnormal or inefficient uptake, mobilization, storage, and absorption of iron.

A physiological feature of disorders associated with anemia associated with chronic disease is elevated production of inflammatory cytokines (Means (1995) Stem Cells 13:32-37 and Means (1999) Int J Hematol 70: 7-12) including, for example, tumor necrosis factor-α (TNF-α), interleukin-1β (IL-1β) and interferon-γ (IFN-γ) which negatively affect the ability to mediate EPO formation, EPO response and coordinated regulation of iron metabolism (see for example Roodman et al (1989) Adv Exp Med Biol 271: 185-196; Fuchs et al (1991) Eur J Hematol 46: 65-70; Jelkmann et al (1991) Ann NY Acad Sci 718: 300-311; Vannucchi et al. al (1994) Br J Hematol 87: 18-23; and Oldenburg et al (2001) Aliment Pharmacol Ther 15: 429-438). The present invention provides methods for improving metabolic and physiological pathways associated with EPO formation, EPO signaling and iron utilization, leading to complete or enhanced erythropoiesis and reduction or amelioration of anemia associated with chronic disease.

The present invention offers advantages compared to existing forms of treatment for anemia associated with chronic disease, for example the administration of recombinant EPO. Reduced EPO formation is only one side of reduced erythropoiesis and it is known that the administration of recombinant EPO does not affect other defects associated with reduced erythropoiesis present in patients with anemia associated with chronic disease (see for example Clibon et al. (1990) Exp Hematol 18: 438-441 and Macdougall and Cooper (2002) Neprol Dial Transplant 17(11): 39-43). These deficiencies include, for example, reduced EPO response in the bone marrow as well as a number of aspects of iron metabolism that contribute to complete or total erythropoiesis including absorption of iron from the gut, transport through enterocytes, oxidation of iron to the ferric state by hepaestin or ceruloplasmin, binding and uptake of iron by transferrin and transferrin receptor and transport of iron to the bone marrow where iron utilization takes place, including synthesis of heme. Many patients do not respond to administration of recombinant EPO of the groups described above, where response to administration of recombinant EPO is reduced or absent, even at high doses of recombinant EPO.

The presence of inflammatory cytokines in anemia associated with chronic disease leads, among other things, to reduced iron levels in serum and increased storage of iron, mainly in macrophages, in a cell compartment that is not easily accessible to emerging erythroid precursor cells that require iron for the correct synthesis of heme. The invention provides methods for enhancing the metabolic reaction pathways that contribute to complete and total erythropoiesis. In one embodiment, the therapeutic agent is administered in combination with additional agents that further enhance the effect, for example iron and B vitamins.

Anemia associated with chronic disease is associated with elevated ferritin levels. Despite a high ferritin level, individuals with anemia associated with chronic disease are unable to utilize iron efficiently. A high ferritin level indicates reduced recirculation of iron to the bone marrow and elevated storage of iron, a functional iron deficiency often associated with anemia associated with chronic disease, and a pseudoinflammatory state often present in uremic patients with chronic kidney disease. By reducing the ferritin level, methods and compounds according to the present invention lead to reduced storage of iron and enhance recirculation of iron via transferrin and transferrin receptor. A reduced serum ferritin level would indicate improved utilization of iron and enhanced recirculation of iron to the bone marrow, which leads to increased availability of iron for the formation of heme and for erythropoiesis.

The genomic response to hypoxia includes changes in gene expression and cell physiology to alleviate the acute and chronic effects of oxygen deprivation.

Hypoxia-inducible factor (HIF) is a transcription factor consisting of an oxygen-regulated alpha subunit (HIF α) and a constitutively expressed beta subunit (HIF β). HIF α is destabilized under normoxic conditions due to hydroxylation of specific proline residues by HIF-specific proline hydroxylases (HIF-PH).

If oxygen becomes limiting, for example under hypoxic conditions, HIF-PH cannot hydrolyze HIF α, the subunit will not be degraded and active HIF complexes are formed, translocated to the nucleus and activate gene transcription.

In certain aspects, the present invention provides methods of treating anemia associated with chronic disease by a pharmaceutical mimic of hypoxia. In certain aspects, the methods enhance EPO formation in a manner that is resistant to the suppressive effects of inflammatory cytokines. EPO formation is normally induced by hypoxia or low oxygen concentration, but expression and secretion remain reduced in the presence of inflammatory cytokines such as TNF-α, IL-1β and IFN-γ, all of which frequently occur in patients with chronic disease. (See, for example, Means (1995) Stem Cells 13: 32-37; Means (1999) Int J Hematol 70: 7-12; Roodman et al. (1989) Adv Exp Med Biol 271: 185-196; Fuchs et al. (1991) Eur J Hematol 46: 65-70, Jelkmann et al (1991) Ann NY Acad Sci 718: 300-311, and Vannmucchi et al.

(1994) Br J Hematol 87: 18-23). Prolyl hydroxylase inhibitors overcome the suppressive effects of inflammatory cytokines on EPO formation, at least in part, as shown by the ability of Hep3B cells to secrete EPO at a level higher than that observed in the presence of inflammatory cytokines. (See, for example, Figures 1A, 1B, 2A, 2B, 3A, 3B, 4A and 4B). Agents such as the iron-chelating compound desferrioxamine have also shown some effect in studies of erythropoietin-resistant anemia, such as anemia associated with chronic disease. (See, for example, Salvarani et al. (1996) Rheumatol Int 16: 45-48 and Goch et al. (1995) Eur J Hematol 55: 73-77).

In other aspects, the present invention provides methods for enhanced signaling via the EPO receptor in the presence of inflammatory cytokines. The common occurrence of inflammatory cytokines in patients with chronic disease leads to a reduced efficiency of EPO signaling, shown by the inability of many patients to respond to recombinant EPO with enhanced erythropoiesis. This is believed to be due to a reduced sensitivity to the bioactivity of EPO as well as defects in the bone marrow architecture and/or the bone marrow microenvironment (see, for example, Clibon et al. (1990) Exp Hematol 18: 438-441 and Macdougall and Cooper (2002) Neprol Dial Transplant 17(11): 39-43). In certain embodiments, the present invention provides methods for inducing total and complete erythropoiesis and restoring the sensitivity of appropriate cells to signaling via the EPO receptor.

Iron deficiency is one of the most common dietary deficiencies worldwide and is the leading cause of anemia globally. The iron balance is basically regulated based on the rate of erythropoiesis and the size of the iron stores. Iron deficiency can occur with or without anemia and is associated with impaired cognitive development.

Iron deficiency is defined as insufficient iron supply (levels or stores) or as insufficient availability or utilization of iron in the body. This can be due to dietary deficiencies, for example a lack of iron in the diet; lack of absorption of iron, for example due to surgical treatment (postgastrectomy) or disease (Crohn's disease); or to a depletion of iron stores or increased iron loss due to chronic or acute blood loss as a result of injury or trauma, menstruation, blood donation, phlebotomy (such as due to various medical procedures, surgical interventions); due to increased iron requirements, for example due to rapid growth in childhood or adolescence, pregnancy, erythropoietin treatment, etc.

Iron deficiency can also be functional iron deficiency, for example iron deficiency characterized by the patient's impaired ability to access and utilize iron stores. Iron is not available to a degree sufficient to allow normal hemoglobinization of erythrocytes, leading to reduced hemoglobin content in reticulocytes and erythrocytes. Functional iron deficiency is often observed in healthy individuals with apparently normal or even elevated iron stores, but with impaired availability of iron, measured for example by a low level of percent transferrin saturation. This type of iron deficiency is often associated with acute or chronic inflammation.

Iron deficiency of all kinds can lead to iron-deficient or iron-limited erythropoiesis where the number of red blood cells decreases and the red blood cells in the circulation are smaller than normal (microcytic) and lack sufficient hemoglobin, and which consequently have a light color (hypochromic).

Individuals with iron deficiency, including functional iron deficiency, may develop impaired hemoglobin synthesis, decreased % transferrin saturation, and decreased hemoglobin and hematocrit levels, leading to iron deficiency anemia. German angel anemia is the most common form of anemia worldwide. Iron is an essential component of hemoglobin; without iron, the bone marrow is unable to efficiently form hemoglobin. Iron deficiency anemia can occur in individuals with depleted or weakened iron supply or in individuals with functional iron deficiency where iron is present in stores, but is not available for, for example, hemoglobin formation.

Iron metabolism generally includes the processes by which a cell, a tissue, an organ, an organ system or an entire organism maintains iron homeostasis by changing, for example enhancing or reducing, specific processes in iron metabolism.

Iron metabolism or processes involved in iron metabolism include processes involved in the processing, transport, uptake, utilization, storage, mobilization, absorption, etc. of iron. Specific aspects of iron metabolism and iron processing include expression of iron transporters and enzymes that facilitate transport of iron across a cell membrane and retention in or secretion of iron from a cell; altered expression of proteins involved in iron transport in the blood; altered expression of transferrin and transferrin receptors; altered expression and/or activity of proteins involved in iron absorption; altered expression and activity of iron-associated transcriptional and translational regulatory proteins; and altered distribution of iron in the body or in culture fluids, including for example the interstitial space (ie the extracellular space), the intracellular space, blood, bone marrow and the like.

In certain aspects, the present invention provides methods for improving the uptake, transport, processing and utilization of iron. Anemia associated with chronic disease is associated with defects in iron utilization that negatively affect heme synthesis and hemoglobin formation, leading to reduced erythropoiesis. (See, for example, Oldenburg et al. (2001) Aliment Pharmacol Ther 15: 429-438). Reduced iron levels in serum, reduced mobilization of iron and possible increases in iron storage associated with this in patients with chronic disease may be associated with a microbial defense mechanism of the macrophage under conditions of prolonged inflammation. (see Fuchs et al. (1991) Eur J Hematol 46: 65-70). In some aspects, the present invention provides methods of enhancing efficient iron metabolism by stabilizing HIF α.

A number of proteins participate in iron metabolism including proteins such as erythroid 5-aminolevulinic acid synthase (ALAS) (the first and rate-limiting step in heme synthesis) (Bottomley and Muller-Eberhard (1988) Semin Hematol 25: 282-302 and Yin et al. (1998 ) Blood, Cells, Molecules, and Diseases 24(3): 41-533), transferrin, transferrin receptor, iron transporters (participating in the transport of iron), ceruloplasmin, etc. Increased expression of transferrin and transferrin receptor stimulates iron uptake in erythroid precursor cells and facilitates uptake of iron and transport to the bone marrow by macrophages (Goswami et al. (2002) Biochem Cell Biol 80: 679-689). Ceruloplasmin leads to increased oxidation of iron(II) to iron(III) so that binding to transferrin can occur (Goswami et al. (2002) Biochem Cell Biol 80: 679-689). In certain aspects, methods of the present invention enhance iron metabolism by increasing the expression or activity of proteins involved in iron metabolism, including erythroid 5-aminolevulinate synthase, transferrin, transferrin receptor, NRAMP2, sproutin (duodenal cytochrome b reductase 1) and ceruloplasmin. In other aspects, methods of the present invention enhance iron metabolism by reducing the expression or activity of hepcidin and by modulating ferritin expression.

In one embodiment, the invention provides methods and compounds for increasing the expression of genes whose products participate in the metabolism and processing of iron, including the uptake, storage, transport and absorption of iron, etc. Such genes include, but are not limited to, transferrin receptor, ceruloplasmin , NRAMP2, 5-aminolevulinate synthase, sproutin (CYRBD1), etc. Therapeutic up-regulation of genes involved in iron metabolism and processing will effectively increase iron availability and thereby provide a beneficial effect in patients with anemia associated with chronic disease, anemia associated with iron deficiency, functional iron deficiency, etc. In another embodiment, the invention provides methods and compounds for reducing the expression of hepcidin, a protein associated with iron regulation.

A correct iron metabolism is partly regulated by iron response element-binding proteins (IRPs) that bind to iron response elements (IREs) present in the 5'- and/or 5'-UTRs of mRNAs that code for, for example, ferritin (iron storage), mitochondrial aconitase (energy metabolism), erythroid aminolevulinate synthase and transferrin receptor. IRP binding to a 5'-IRE, for example in the ferritin transcript, inhibits translation of the relevant mRNA; while binding to a 3'-IRE such as, for example, in the transferrin transcript, protects the relevant mRNA from degradation. IRP-2 is constitutively formed in cells but is consequently degraded and inactivated under conditions of more iron deficiency. However, IRP-2 is stabilized under conditions of iron deficiency and/or hypoxia (Hanson et al. (1999) J Biol Chem 274: 5047-5052). Since IRP-2 reduces the expression of ferritin which is responsible for the long-term storage of iron and increases the expression of transferrin and transferrin receptor, IRP-2 facilitates the uptake, transport and utilization of iron so that erythropoiesis is enhanced (Klausner et al. (1993) Cell 72: 19 -28). Recently, IRE has been described in other genes also required for erythropoiesis including 5-aminolevulinate synthase, the iron transporter NRAMP2 (also called Alc11a2, DCT1, DMT1, mk (microcytic anemia gene locus in mouse)) and the iron transporter that mediates absorption of iron from dietary sources in the duodenum ( Haile (1999) Am J Med Sci 318: 230-240 and Gunshin et al (2001) FEBS Lett 509: 309-316).

By mimicking hypoxic conditions, the methods of the present invention lead to a strong stabilization of IRP-2 in addition to HIF α, which provides a synergistic effect that includes both the endogenous EPO formation and enhanced uptake, transport and utilization of iron for formation of functional erythrocytes.

Among adults, the average absorption of iron from dietary iron is approximately 6% compared to 13% for non-pregnant women. NRAMP2 (also termed DMT1, DCT1, slc112) is a ubiquitously expressed divalent metal transporter that participates in the transmembrane transport of non-transferrin-bound iron. NRAMP2 is an iron transport protein associated with the transport of iron from the lumen of the gastrointestinal tract to enterocytes in the duodenum and from erythroblast endosomes to the cytoplasm. In animals deficient in the diet, the expression of NRAMP2 (slc11a2) was dramatically elevated at the apical pole of enterocytes in the absorptive columnar epithelium of the proximal duodenum. (See, for example, Canonne-Hergaux et al. (1999) Blood 93: 4406-4417). Genetic rodent models have linked this gene to anemias associated with iron deficiency, including mice with hypochromic and microcytic anemia (mk mice) with a mutated NRAMP2 gene. MK mice show severe defects in iron absorption and the erythroid utilization of iron.

In certain aspects, methods and compounds of the present invention are useful for increasing the absorption of iron from dietary iron. The present invention provides methods and compounds for increasing the expression of genes associated with iron transport and iron absorption.

More specifically, compounds of the present invention were effective in increasing the expression of NRAMP2 in intestine. An elevated NRAMP2 (slc11a2) expression would be desirable to increase intestinal absorption of iron, such as dietary iron.

In addition, the present invention provides results showing elevated sprouting gene expression in the intestine of animals treated with a compound of the present invention. The intestinal iron reductase sproutin, also called Dcytb and Cybrd1 (CYBRD1, duodenal cytochrome b reductase 1) is an iron(III) reductase and catalyzes the reduction of extracellular iron(III) to iron(II) which is associated with iron absorption. Sproutin is co-expressed with NRAMP2 in iron-starved animals in the apical region of duodenal villi (see, for example, KcKie et al. (2001) Science 291: 1755-1759).

Methods and compounds of the present invention are useful for increasing the expression of the ceruloplasmin gene. Ceruloplasmin, also called ferroxidase-1, transfers reduced iron released from storage sites (such as ferritin) to the oxidized form. Oxidized iron can bind to the plasma transport protein transferrin. Ceruloplasmin deficiency is associated with accumulation of iron in the liver and other tissues. There is material which suggests that ceruloplasmin promotes the outflow of iron from the liver and promotes the influx of iron into iron-deficient cells (see for example Tran et al. (2002) J Nutr 132: 351-356).

Compounds of the present invention reduced the expression of hepcidin mRNA in mouse liver. Inflammation leads to the formation of IL-6 which acts on hepatocytes and induces hepcidin formation. Hepcidin inhibits the release of iron from macrophages and the absorption of iron in the intestine, which leads to a reduced supply of iron and, for example, to hypoferremia. Reduced hepcidin expression is associated with increased release of iron from reticuloendothelial cells and increased absorption of iron from the gut. Accordingly, methods and compounds of the present invention are useful for reducing hepcidin expression, increasing the absorption of iron from the gut, and reducing hypoferremia.

Methods of treating anemia associated with hepatitis C virus (HCV) infection are specifically covered. Today's treatment of HCV infection includes interferon-α and ribaviron in combination. This combination treatment is associated with reduced hemoglobin concentration and anemia. In one aspect, methods and compositions for treating anemia associated with HCV infection are provided. In another aspect, methods and compositions are provided for treating anemia associated with interferon-α treatment of HCV infection. In another aspect, the present invention provides compounds and methods useful for treating anemia associated with ribavirin treatment of HCV infection.

Methods of increasing the production of factors necessary for differentiation into erythrocytes from hematopoietic progenitor cells including, for example, hematopoietic stem cells (HSC), CFU-GEMM (colony forming unit granulocyte/erythroid/monocyte/megakaryocyte) cells, etc., are also encompassed. Factors that stimulate erythropoiesis include, but are not limited to, erythropoietin. In another aspect, the methods provide increased formation of factors necessary for the uptake, transport and utilization of iron. Such factors include, but are not limited to, erythroid aminolevulinate synthase, transferrin, transferrin receptor, ceruloplasmin, ferritin, etc. In yet another aspect, the method provides increased concentration of factors necessary for differentiation into erythrocytes and additional factors necessary for uptake, transport and utilization of iron.

Methods of enhancing the response of hematopoietic progenitor cells to erythropoietin are also encompassed. As described above, such progenitor cells include HSC, CFU-GEMM, etc. The response of the progenitor cells can be enhanced, for example, by changing the expression of erythropoietin receptors, intracellular factors that participate in erythropoietin signaling and secreted factors that facilitate the interaction between erythropoietin and the receptors. The present invention provides methods for enhancing the EPO response in bone marrow by, for example, increasing the expression of the EPO receptor.

Procedures

Various methods are provided herein. In one aspect, the methods comprise administering to a subject an agent that stabilizes HIF α.

Stabilization of HIF α can be achieved by any of the methods available to and known to those skilled 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 encompasses the provision of a constitutively stable HIF α variant, for example, stable HIF α muteins, etc., or a polynucleotide encoding such a variant. (See, for example, U.S. Patent No.

6,562,799 and 6,124,131; as well as the U.S. patent document no. 5,432,927). In other aspects, the present invention comprises stabilizing HIF α comprising administering an agent that stabilizes HIF α. The agent may consist of polynucleotides, for example antisense sequences (see, for example, International Patent Publication No. WO 03/045440); polypeptides; antibodies; other proteins; carbohydrates; fat; lipids; and organic and inorganic compounds, such as small molecules, etc. In a preferred embodiment, the present invention comprises stabilizing HIF α, for example in an individual by administering to the individual an agent that stabilizes HIF α, wherein the agent is a compound, for for example, a low molecular weight compound, etc., which stabilizes HIF α.

In other embodiments, the methods according to the invention comprise stabilization of 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 enzyme, e.g., EGLN-1, EGLN-2, EGLN-3, etc. (see, e.g., Taylor (2001) Gene 275: 125-132; Epstein et al. (2001) Cell 107: 43-54; and Bruick and McKnight (2001) Science 294: 1337-1340). However, it is specifically understood that the enzyme can be any enzyme selected from the 2-oxoglutarate dioxygenase enzyme family including, for example, procollagen lysyl hydroxylase, procollagen prolyl 3-hydroxylase, procollagen prolyl 4-hydroxylase α(I) and - α(II), thymine 7-hydroxylase, aspartyl (asparaginyl) β-hydroxylase , ε-N-trimethyllysine hydroxylase and γ-burytobetaine hydroxylase, etc. (see for example Majamaa et al. (1985) Biochem J 229: 127-133; Myllyharju and Kivirikko (1997) EMBO J 16: 1173-1180; Thornburg et al. ( 1993) 32: 14023-14033; and Jia et al (1994) Proc Natl Acad Sci USA 91: 7227-7231).

In certain embodiments, the methods comprise treating anemia associated with chronic disease or regulating iron metabolism by administering to a subject an effective amount of an agent that stabilizes HIF α. In preferred embodiments, the agent is a compound of the present invention. In one aspect, the compound stabilizes HIF α by inhibiting the hydroxylation of certain amino acid residues in HIF α, for example proline residues, asparagine residues, etc. In a preferred embodiment, the amino acid residues are proline residues. In specific embodiments, the amino acid residues may be the P564 residue in HIF-1 α or a homologous proline residue in another HIF α isoform, or the P402 residue in HIF-1 α or a homologous proline residue in another HIF α isoform, etc. I in other embodiments, the present methods may comprise inhibition of hydroxylation of HIF α-asparagine residues, for example the N803 residue in HIF-1 α or a homologous asparagine residue in another HIF α isoform.

Connections

In preferred methods, the present methods comprise administering to a subject an effective amount of a compound that stabilizes HIF α. Examples of compounds are given in, for example, International Patent Publication No. WO 03/049686 and International Patent Publication No. WO 03/053997, both of which are incorporated herein by reference in their entirety. More specifically, compounds according to the invention include the following.

In certain embodiments, a compound of the invention is a compound that inhibits HIF hydroxylase activity. In various embodiments, the activity is due to a HIF prolyl hydroxylase, e.g., EGLN1, EGLN2, or EGLN3, etc. In other embodiments, the activity is due to a HIF asparaginyl hydroxylase, e.g., including, but not limited to, FIH. A preferred compound according to the invention is a compound which inhibits HIF-prolyl hydroxylase activity.

The inhibition may be direct or indirect, competitive or non-competitive, etc.

In one aspect, a compound of the invention is any compound that inhibits or otherwise modulates the activity of a 2-oxoglutarate dioxygenase enzyme. 2-oxoglutarate dioxygenase enzymes include, but are not limited to, hydroxylase enzymes. Hydroxylase enzymes hydroxylate amino acid residues in the target substrate and include, for example, prolyl hydroxylases, lysyl hydroxylases, asparaginyl (asparagyl, aspartyl) hydroxylases, etc. Hydroxylases are sometimes described based on the target substrate, for example, HIF hydroxylases, procollagen hydroxylases, etc. and/or based on target residues in the substrate , e.g., prolyl hydroxylases, lysyl hydroxylases, etc., or from 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 such as factor inhibiting HIF (FIH), etc.; procollagen hydroxylases, for example procollagen lysyl hydroxylases, procollagen prolyl hydroxylases, for example procollagen prolyl 3-hydroxylase, procollagen prolyl 4-hydroxylase α(I) and - α(II), etc.; thymine 7-hydroxylase; aspartyl (asparaginyl) β-hydroxylase; ε-N-trimethyllysine hydroxylase; γbutyrobetaine hydroxylase, etc. Although the enzymatic activity may include any activity associated with any 2-oxoglutarate dioxygenase, specifically includes hydroxylation of amino acid residues in a substrate. Although hydroxylation of proline and/or asparagine residues in a substrate is specifically included, hydroxylation of other amino acids is also included.

In one aspect, a compound according to the invention that shows inhibitory activity against one or more 2-oxoglutarate dioxygenase enzymes can also show inhibitory activity against one or more other 2-oxoglutarate dioxygenase enzymes, for example a compound that inhibits the activity of a HIF hydroxylase can additionally inhibit the activity of a collagen prolyl hydroxylase, a compound that inhibits the activity of a HIF prolyl hydroxylase may additionally inhibit the activity of a HIF asparaginyl hydroxylase, etc.

Since HIF α is modified by proline hydroxylation, a reaction that requires oxygen and Fe<2+>, the present invention in one aspect includes the enzyme responsible for HIF α hydroxylation being a member of the 2-oxoglutarate dioxygenase family. Such enzymes include, but are not limited to, procollagen lysyl hydroxylase, procollagen prolyl 3-hydroxylase, procollagen prolyl 4-hydroxylase α(I) and - α(II), thymine 7-hydroxylase, aspartyl (asparaginyl) β-hydroxylase, ε-N-trimethyllysine hydroxylase and γbutyrobetaine hydroxylase, etc. These enzymes require oxygen, Fe<2+>, 2-oxoglutarate and ascorbic acid for the hydroxylase activity (see for example Majamaa et al. (1985) Biochem J 229: 127-133; Myllyharju and Kivirikko (1997) EMBO J 16: 1173-1180; Thornburg et al. (1993) 32: 14023-14033; and Jia et al. (1994) Proc Natl Acad Sci USA 91: 7227-7231).

In one aspect, a compound of the invention is a compound that stabilizes HIF α. The compound preferably stabilizes HIF α by inhibiting HIF hydroxylase activity. It is thus specifically covered that a compound according to the invention can be selected from among previously discovered modulators of hydroxylase activity. For example, low molecular weight inhibitors of prolyl 4-hydroxylase have been demonstrated (see, for example, Majamaa et al. (1984) Eur J Biochem 138: 239-245; Majamaa et al. (1985) Biochem J 229: 127-133; Kivirikko and Myllyharju (1998) Matrix Biol 16: 357-368; Bickel et al (1998) Hepatology 28: 404-411; Friedman et al (2000) Proc Natl Acad Sci USA 97: 4736-4741; and Franklin et al (2001) ) Biochem J 353: 333-338; all of which are incorporated herein by reference in their entirety). The present invention encompasses the use of these compounds in the methods provided herein.

In some aspects, compounds of the present invention include, for example, structural mimics of 2-oxoglutarate. Such compounds can inhibit the desired member of the 2-oxoglutarate dioxygenase enzyme family competitively relative to 2-oxoglutarate and non-competitively relative to iron (Majamaa et al. (1984) Eur J Biochem 138: 239-245; and Majamaa et al. Biochem J 229: 127-133).

In certain embodiments, compounds used in the methods according to the invention are selected from compounds of formula (I)

in which

A is 1,2-arylidene, 1,3-arylidene, 1,4-arylidene; or (C1-C4)-alkylene, optionally substituted with one or two substituents selected from halogen, cyano, nitro, trifluoromethyl, (C1-C6)-alkyl, (C1-C6)-hydroxyalkyl, (C1-C6)-alkoxy, -O-[CH2]x-CfH(2f+1-g)Halg, (C1-C6)-Fluoroalkoxy, (C1-C8)-Fluoroalkenyloxy, (C1-C8)-Fluoroalkynyloxy, -OCF2Cl, -O-CF2- CHFCl; (C1-C6)-Alkylmercapto, (C1-C6)-Alkylsulfinyl, (C1-C6)-Alkylsulfonyl, (C1-C6)-Alkylcarbonyl, (C1-C6)-Alkoxycarbonyl, Carbamoyl, N-(C1-C4)- alkylcarbamoyl, N,N-di-(C1-C4)-alkylcarbamoyl, (C1-C6)-alkylcarbonyloxy, (C3-C8)-cycloalkyl, phenyl, benzyl, phenoxy, benzyloxy, anilino, N-methylanilino, phenylmercapto, phenylsulfonyl, phenylsulfinyl, sulfamoyl, N-(C1-C4)-alkylsulfamoyl, N,N-di-(C1-C4)-alkylsulfamol; or with substituted (C6-C12)-aryloxy, (C7-C11)-aralkyloxy, (C6-C12)-aryl, (C7-C11)-aralkyl radical which carries on the aryl group from one to five identical or different substituents selected from halogen, cyano, nitro, trifluoromethyl, (C1-C6)-alkyl, (C1-C6)-alkoxy, -O-[CH2]x-CfH(2f+1-g)Halg, -OCF2Cl, -O-CF2-CHFCl, (C1-C6)-Alkylmercapto, (C1-C6)-Alkylsulfinyl, (C1-C6)-Alkylsulfonyl, (C1-C6)-Alkylcarbonyl, (C1-C6)-Alkoxycarbonyl, Carbamoyl, N-(C1-C4)- alkylcarbamoyl, N,N-di-(C1-C4)-alkylcarbamoyl, (C1-C6)-alkylcarbonyloxy, (C3-C8)-cycloalkyl, sulfamoyl, N-(C1-C4)-alkylsulfamoyl, N,N-di- (C 1 -C 4 )-alkylsulfamoyl; or wherein A is -CR<5>R<6>and wherein R<5>and R<6> are independently selected from hydrogen, (C1-C6)-alkyl, (C3-C7)-cycloalkyl, aryl or a substituent on the α-carbon atom of an α-amino acid, wherein the amino acid is a naturally occurring L-amino acid or its D-isomer.

B is -CO2H, -NH2, -NHSO2CF3, tetrazolyl, imidazolyl, 3-hydroxyisoxazolyl, -CONHCOR''', -CONHSOR''', CONHSO2R''', where R''' is aryl, heteroaryl, (C3-C7 )-cycloalkyl or (C1-C4)-alkyl optionally monosubstituted with (C6-C12)-aryl, heteroaryl, OH, SH, (C1-C4)-alkyl, (C1-C4)-alkyl, (C1-C4)- thioalkyl, (C1-C4)-Sulfinyl, (C1-C4)-Sulfonyl, CF3, Cl, Br, F, I, NO2, -COOH, (C2-C5)-Alkoxycarbonyl, NH2, mono-(C1-C4- alkyl)-amino, di-(C1-C4-alkyl)-amino or (C1-C4)-perfluoroalkyl; or wherein B is a CO2-G carboxyl radical wherein G is a radical of an alcohol G-OH wherein G is selected from (C1-C20)-alkyl radical, (C3-C8)-cycloalkyl radical, (C2-C20)-alkenyl radical, (C3-C8)-cycloalkenyl radical, retinyl radical, (C2-C20)-alkynyl radical, (C4-C20)-alkenynyl radical, wherein the alkenyl, cycloalkenyl, alkynyl and alkenynyl radicals contain one or more double or triple bonds; (C6-C16)-carbocyclic aryl radical, (C7-C16)-carbocyclic aralkyl radical, heteroaryl radical or heteroaralkyl radical, wherein a heteroaryl radical or a heteroaryl group in a heteroaralkyl radical contains 5 or 6 ring atoms; and wherein the radicals defined for G are substituted with one or more substituents selected from hydroxyl, halogen, cyano, trifluoromethyl, nitro, carboxyl, (C1-C12)-alkyl, (C3-C8)-cycloalkyl, (C5-C8) -Cycloalkenyl, (C6-C12)-aryl, (C7-C16)-aralkyl, (C2-C12)-alkenyl, (C2-C12)-alkynyl, (C1-C12)-Alkoxy, (C1-C12)-Alkoxy -(C1-C12)-Alkyl, (C1-C12)-Alkoxy-(C1-C12)-Alkoxy, (C6-C12)-Aryloxy, (C7-C16)-Aralkyloxy, (C1-C8)-Hydroxyalkyl, - O-[CH2]x-CfH(2f+1-g)-Fg, -OCF2Cl, -OCF2-CHFCl, (C1-C12)-alkylcarbonyl, (C3-C8)-cycloalkylcarbonyl, (C6-C12)-arylcarbonyl, (C7-C16)-Aralkylcarbonyl, Cinnamoyl, (C2-C12)-Alkenylcarbonyl, (C2-C12)-Alkynylcarbonyl, (C1-C12)-Alkoxycarbonyl, (C1-C12)-Alkoxy-(C1-C12)-Alkoxycarbonyl, (C6-C12)-aryloxycarbonyl, (C7-C16)-aralkoxycarbonyl, (C3-C8)-cycloalkoxycarbonyl, (C2-C12)-alkenyloxycarbonyl, (C2-C12)-alkynyloxycarbonyl, acyloxy, (C1-C12)-alkoxycarbonyloxy, (C1-C12)-Alkoxy-(C1-C12)-Alkoxycarbonyloxy, (C6-C12)-Aryloxycarbonyloxy, (C7-C16)-Aralkyl oxycarbonyloxy, (C3-C8)-cycloalkoxycarbonyloxy, (C2-C12)-alkenyloxycarbonyloxy, (C2-C12)-alkynyloxycarbonyloxy, carbamoyl, N-(C1-C12)-alkylcarbamoyl, N,N-di(C1-C12)-alkylcarbamoyl , N-(C3-C8)-cycloalkylcarbamoyl, N-(C6-C16)-arylcarbamoyl, N-(C7-C16)-aralkylcarbamoyl, N-(C1-C10)-alkyl-N-(C6-C16)-arylcarbamoyl , N-(C1-C10)-alkyl-N-(C7-C16)-aralkylcarbamoyl, N-((C1-C10)-Alkoxy-(C1-C10)-alkyl)-carbamoyl, N-((C6-C12 )-aryloxy-(C1-C10)alkyl)-carbamoyl, N-((C7-C16)-aralkyloxy-(C1-C10)-alkyl)-carbamoyl, N-(C1-C10)-alkyl-N-(( C1-C10)-Alkoxy-(C1-C10)-alkyl)-carbamoyl, N-(C1-C10)-alkyl-N-((C6-C16)-aryloxy-(C1-C10)-alkyl)-carbamoyl, N-(C1-C10)-alkyl-N-((C7-C16)-aralkyloxy-(C1-C10)-alkyl)-carbamoyl, carbamoyloxy, N-(C1-C12)-alkylcarbamoyloxy, N,N-di- (C1-C12)-alkylcarbamoyloxy, N-(C3-C8)-cycloalkylcarbamoyloxy, N-(C6-C12)-arylcarbamoyloxy, N-(C7-C16)-aralkylcarbamoyloxy, N-(C1-C10)-alkyl-N- (C6-C12)-arylcarbamoyloxy, N(C1-C10)-alkyl-N-(C7-C16)-aralkylcarbamoyloxy, N-((C1-C10)-alk yl)-carbamoyloxy, N-((C6-C12)-aryloxy-(C1-C10)-alkyl)-carbamoyloxy, N-((C7-C16)-aralkyloxy-(C1-C10)-alkyl)-carbamoyloxy, N -(C1-C10)-Alkyl-N-((C1-C10)-Alkoxy-(C1-C10)-Alkyl)-Carbamoyloxy, N-(C1-C10)-Alkyl-N-((C6-C12)- aryloxy-(C1-C10)-alkyl)-carbamoyloxy, N-(C1-C10)-alkyl-N-((C7-C16)-aralkyloxy-(C1-C10)-alkyl)-carbamoyloxy, amino, (C1- C12)-alkylamino, di-(C1-C12)-alkylamino, (C3-C8)-cycloalkylamino, (C2-C12)-alkenylamino, (C2-C12)-alkynylamino, N-(C6-C12)-arylamino, N (C-C11)-Aralkylamino, N-Alkylaralkylamino, N-Alkylarylamino, (C1-C12)-Alkoxyamino, (C1-C12)-Alkoxy-N-(C1-C10)-Alkylamino, (C1-C12)-Alkylcarbonylamino, (C3-C8)-cycloalkylcarbonylamino, (C6-C12)arylcarbonylamino, (C7-C16)-aralkylcarbonylamino, (C1-C12)-alkylcarbonyl-N-(C1-C10)-alkylamino, (C3-C8)-cycloalkylcarbonyl-N -(C1-C10)-alkylamino, (C6-C12)-arylcarbonyl-N-(C1-C10)alkylamino, (C7-C11)-aralkylcarbonyl-N-(C1-C10)-alkylamino, (C1-C12)- alkylcarbonylamino-(C1-C8)-alkyl, (C3-C8)-cycloalkylcarbonylamino-(C1-C8)alk yl, (C6-C12)-arylcarbonylamino-(C1-C8)-alkyl, (C7-C12)-aralkylcarbonylamino(C1-C8)-alkyl, amino-(C1-C10)-alkyl, N-(C1-C10) alkylamino-(C1-C10)-alkyl, N,N-di-(C1-C10)-alkylamino-(C1-C10)-alkyl, (C3-C8)cycloalkylamino-(C1-C10)-alkyl, (C1- C12)-alkylmercapto, (C1-C12)-alkylsulfinyl, (C1-C12)-alkylsulfonyl, (C6-C16)-arylmercapto, (C6-C16)-arylsulfinyl, (C6-C12)-arylsulfonyl, (C7-C16) -aralkylmercapto, (C7-C16)-aralkylsulfinyl, (C7-C16)-aralkylsulfonyl, sulfamoyl, N-(C1-C10)-alkylsulfamoyl, N,N-di(C1-C10)-alkylsulfamoyl, (C3-C8)- cycloalkylsulfamoyl, N-(C6-C12)-alkylsulfamoyl, N-(C7-C16)-aralkylsulfamoyl, N-(C1-C10)-alkyl-N-(C6-C12)-arylsulfamoyl, N-(C1-C10)- alkyl-N-(C7-C16)-aralkylsulfamoyl, (C1-C10)-alkylsulfonamido, N-((C1-C10)-alkyl)-(C1-C10)-alkylsulfonamido, (C7-C16)-aralkylsulfonamido and N- ((C1-C10)-alkyl-(C7-C16)-aralkylsulfonamido; wherein radicals which are aryl or contain an aryl group may be substituted on the aryl group with from one to five identical or different substituents selected from hydroxyl, halogen, cyano, trifluoromethyl, nitro, carboxyl, (C1-C12)-alkyl, (C3-C8) -Cycloalkyl, (C6-C12)-aryl, (C7-C16)-aralkyl, (C1-C12)-Alkoxy, (C1-C12)-Alkoxy-(C1-C12)alkyl, (C1-C12)-Alkoxy- (C1-C12)Alkoxy, (C6-C12)-Aryloxy, (C7-C16)-Aralkyloxy, (C1-C8)-Hydroxyalkyl, (C1-C12)-Alkylcarbonyl, (C3-C8)-Cycloalkylcarbonyl, (C6- C12)-Arylcarbonyl, (C7-C16)aralkylcarbonyl, (C1-C12)-Alkoxycarbonyl, (C1-C12)-Alkoxy-(C1-C12)-Alkoxycarbonyl, (C6-C12)-Aryloxycarbonyl, (C7-C16)- aralkyloxycarbonyl, (C3-C8)-cycloalkylcarbonyl, (C2-C12)-alkenyloxycarbonyl, (C2-C12)-alkynyloxycarbonyl, (C1-C12)-alkylcarbonyloxy, (C3-C8)-cycloalkylcarbonyloxy, (C6-C12)-arylcarbonyloxy, (C7-C16)-Aralkylcarbonyloxy, Cinnamoyloxy, (C2-C12)-Alkenylcarbonyloxy, (C2-C12)-Alkynylcarbonyloxy, (C1-C12)-Alkoxycarbonyloxy, (C1-C12)-Alkoxy-(C1- C12)Alkoxycarbonyloxy, (C6-C12)-Aryloxycarbonyloxy, (C7-C16)-Aralkyloxycarbonyloxy, (C3-C8)-Cycloalkoxycarbonyloxy, (C2-C12)-Alkenyloxycarbonyloxy, (C2-C12)-Alkynyloxycarbonyloxy, Carbamoyl, N-(C1 -C12)-alkylcarbamoyl, N,N-di-(C1-C12)-alkylcarbamoyl, N-(C3-C8)-cycloalkylcarbamoyl, N-(C6-C12)-arylcarbamoyl, N-(C7-C16)-aralkylcarbamoyl, N-(C1-C10)-Alkyl-N-(C6-C12)-Arylcarbamoyl, N-(C1-C10)-Alkyl-N-(C7-C16)-Aralkylcarbamoyl, N-((C1-C10)-Alkoxy -(C1-C10)-alkyl)-carbamoyl, N-((C6-C12)-aryloxy-(C1-C10)-alkyl)-carbamoyl, N-((C7-C16)-aralkyloxy-(C1-C10) -alkyl)-carbamoyl, N-(C1-C10)-alkyl-N-((C1-C10)-Alkoxy-(C1-C10)-alkyl)-carbamoyl, N-(C1-C10)-alkyl-N- ((C6-C12)-aryloxy-(C1-C10)-alkyl)-carbamoyl, N-(C1-C10)-alkyl-N-((C7-C16)-aralkyloxy-(C1-C10)-alkyl)- carbamoyl, carbamoyloxy, N-(C1-C12)-alkylcarbamoyloxy, N,N-di-(C1-C12)-alkylcarbamoyloxy, N-(C3-C8)-cycloalkylcarbamoyloxy, N-(C6-C12)-arylcarbamoyloxy, N- (C7-C16)-aralkylcarbamoyloxy, N-(C1-C10)-alkyl-N-(C6-C12)-arylcarbamoy oxy, N(C1-C10)-alkyl-N-(C7-C16)-aralkylcarbamoyloxy, N-((C1-C10)-alkyl)-carbamoyloxy, N-((C6-C12)-aryloxy-(C1-C10 )-alkyl)-carbamoyloxy, N-((C7-C16)-aralkyloxy-(C1-C10)-alkyl)-carbamoyloxy, N-(C1-C10)-alkyl-N-((C1-C10)-alkyloxy- (C1-C10-alkyl)-carbamoyloxy, N-(C1-C10)-alkylN-((C6-C12)-aryloxy-(C1-C10)-alkyl)-carbamoyloxy, N-(C1-C10)-alkyl -N-((C7-C16)-aralkyloxy-(C1-C10)-alkyl)-carbamoyloxy, amino, (C1-C12)-alkylamino, di-(C1-C12)-alkylamino, (C3-C8)-cycloalkylamino , (C3-C12)-alkenylamino, (C3-C12)-alkynylamino, N-(C6-C12)-arylamino, N-(C7-C11)-aralkylamino, N-alkylaralkylamino, N-alkylarylmino, (C1-C12) -Alkoxyamino, (C1-C12)-Alkoxy-N-(C1-C10)-Alkylamino, (C1-C12)-Alkylcarbonylamino, (C3-C8)-Cycloalkylcarbonylamino, (C6-C12)-Arylcarbonylamino, (C7-C16) -alkylcarbonylamino, (C1-C12)-alkylcarbonyl-N-(C1-C10)-alkylamino, (C3-C8)-cycloalkylcarbonyl-N-(C1-C10)-alkylamino, (C6-C12)-arylcarbonyl-N-( C1-C10)-alkylamino, (C7-C11)-aralkylcarbonyl-N-(C1-C10)-alkylamino, (C1-C12)-alkylka rbonylamino-(C1-C8)-alkyl, (C3-C8)-cycloalkylcarbonylamino-(C1-C8)-alkyl, (C6-C12)-arylcarbonylamino-(C1-C8)-alkyl, (C7-C16)-aralkylcarbonylamino- (C1-C8)-alkyl, amino-(C1-C10)-alkyl, N-(C1-C10)-alkylamino-(C1-C10)alkyl, N,N-di-(C1-C10)-alkylamino-( C1-C10)-alkyl, (C3-C8)-cycloalkylamino-(C1-C10)-alkyl, (C1-C12)-alkylmercapto, (C1-C12)-alkylsulfinyl, (C1-C12)-alkylsulfonyl, (C6- C12)-arylmercapto, (C6-C12)-arylsulfinyl, (C6-C12)-arylsulfonyl, (C7-C16)-aralkylmercapto, (C7-C16)-aralkylsulfinyl or (C7-C16)-aralkylsulfonyl;

X is O or S;

Q is O, S, NR' or a bond;

where if Q is a bond, R<4> is halogen, nitrile or trifluoromethyl;

or where, if Q is O, S or NR', then R<4> is hydrogen, (C1-C10)-alkyl radical, (C2-C10)-alkenyl radical, (C2-C10)-alkynyl radical, wherein the alkenyl or alkynyl radical contains one or two C-C double or triple bonds; an unsubstituted fluoroalkyl radical of the formula -[CH2]x-CfH(2f+1-g)-Fg, (C1-C8)-Alkoxy-(C1-C6)-alkyl radical, (C1-C6)Alkoxy-(C1-C4) -Alkoxy-(C1-C4)-alkyl radical, aryl radical, heteroaryl radical, (C7-C11)-aralkyl radical or a radical of the formula X

-[CH2]v-[O]w-[CH2]t-E (Z)

where

E is a heteroaryl radical, a (C3-C8)-cycloalkyl radical or a phenyl radical of formula F

v is 0-6,

w is 0 or 1,

t is 0-3, and

R<7>, R<8>, R<9>, R<10> and R<11> are identical or different and are hydrogen, halogen, cyano, nitro, trifluoromethyl, (C1-C6)-alkyl, (C3 -C8)-Cycloalkyl, (C1-C6)-Alkoxy, -O-[CH2]x-CfH(2f+1-g)-Fg, -OCF3-Cl, -O-CF2-CHFCl, (C1-C6) -alkylmercapto, (C1-C6)-hydroxyalkyl, (C1-C6)- alkoxy-(C1-C6)-alkyl, (C1-C6)- alkoxy-(C1-C6)-alkyl, (C1-C6)-alkylsulfinyl , (C1-C6)-Alkylsulfonyl, (C1-C6)-Alkylcarbonyl, (C1-C8)-Alkoxycarbonyl, Carbamoyl, N-(C1-C8)-Alkylcarbamoyl, N,N-Di-(C1-C8)-Alkylcarbamoyl or (C7-C11)-aralkylcarbamoyl, optionally substituted with fluorine, chlorine, bromine, trifluoromethyl, (C1-C6)-alkoxy, N-(C3-C8)-cycloalkylcarbamoyl, N-(C3-C8)-cycloalkyl-(C1 -C4)-alkylcarbamoyl, (C1-C6)-alkylcarbonyloxy, phenyl, benzyl, phenoxy, benzyloxy, NR<Y>R<Z>in which R<y>and R<z> are independently selected from hydrogen, (C1 -C12)-Alkyl, (C1-C8)-Alkoxy-(C1-C8)-Alkyl, (C7-C12)-ArAlkoxy-(C1-C8)-Alkyl, (C6-C12)-Aryloxy-(C1-C8 )-alkyl, (C3-C10)-cycloalkyl, (C3-C12)-alkenyl, (C3-C12)-alkynyl, (C6-C12)-aryl, (C7-C11)-Aralkyl, (C1-C12)-Alkoxy, (C7-C12)Armethoxy, (C1-C12)-Alkylcarbonyl, (C3-C8)-Cycloalkylcarbonyl, (C6-C12)arylcarbonyl, (C7-C16 )-aralkylcarbonyl; or further wherein R<y>and R<z>together are -[CH2]h, wherein a CH2 group may be replaced by O, S, N-(C1-C4)-alkylcarbonylimino or N-(C1-C4) -Alkoxycarbonylimino; phenylmercapto, phenylsulfonyl, phenylsulfinyl, sulfamoyl, N-(C1-C8)-alkylsulfamoyl or N,N-di-(C1-C8)-alkylsulfamoyl; or wherein alternatively R<7>and R<8>, R<8>and R<9>, R<9>and R<10>or R<10>and R<11>together form a chain selected from -[ CH2]n- or -CH=CH-CH=CH-, where a CH2 group in the chain is optionally replaced by O, S, SO, SO2 or NR<Y>; and n is 3, 4 or 5; and if E is a heteroaryl radical, the radical may carry 1-3 substituents selected from those defined for R<7>-R<11>, or if E is a cycloalkyl radical, the radical may carry a substituent selected from the substituents defined for R< 7>-R<11>;

or where, if Q is NR', R<4> is alternatively R'', where R' and R'' are the same or different and are hydrogen, (C6-C12)-aryl, (C7-C11)-aralkyl, (C1-C8)-Alkyl, (C1-C8)-Alkoxy-(C1-C8)-Alkyl, (C7-C12)-ArAlkoxy-(C1-C8)-Alkyl, (C6-C12)-Aryloxy-(C1 -C8)-alkyl, (C1-C10)-alkylcarbonyl, optionally substituted (C7-C16)-aralkylcarbonyl or optionally substituted (C6-C12)-arylcarbonyl; or wherein R' or R'' together are -[CH2]wherein a CH2 group may be substituted with O, S, N-acylimino or N-(C1-C10)-Alkoxycarbonylimino and h is from 3 to 7.

Y is N or CR<3>;

R<1>, R<2> and R<3> are the same or different and are hydrogen, hydroxyl, halogen, cyano, trifluoromethyl, nitro, carboxyl, (C1-C20)-alkyl, (C3-C8)-cycloalkyl, (C3-C8)-Cycloalkyl-(C1-C12)-Alkyl, (C3-C8)-Cycloalkyloxy, (C3-C8)-Cycloalkyl-(C1-C12)Alkoxy, (C3-C8)-Cycloalkyloxy-(C1- C12)-Alkyl, (C3-C8)-Cycloalkyloxy-(C1-C12)-Alkoxy, (C3-C8)-Cycloalkyl-(C1-C8)-Alkyl-(C1-C6)-Alkoxy, (C3-C8) -Cycloalkyl-(C1-C8)-Alkoxy-(C1-C6)-Alkyl, (C3-C8)-Cycloalkyloxy-(C1-C8)-Alkoxy-(C1-C6)-Alkyl, (C3-C8)-Cycloalkoxy -(C1-C8)-Alkoxy-(C1-C8)-Alkoxy, (C6-C12)-Aryl, (C7-C16)-Aralkyl, (C7-C16)-Aralkenyl, (C7-C16)-Aralkynyl, ( (C2-C20)-Alkenyl, (C2-C20)-Alkynyl, (C1-C20)-Alkoxy, (C2-C20)-Alkenyloxy, (C2-C20)-Alkynyloxy, Retinyloxy, (C1-C20)-Alkoxy-( C1-C12)-alkyl, (C1-C12)-Alkoxy-(C1-C12)-Alkoxy, (C1-C12)-Alkoxy-(C1-C8)-Alkoxy-(C1-C8)-Alkyl, (C6- C12)-Aryloxy, (C7-C16)-Aralkyloxy, (C6-C12)-Aryloxy-(C1-C6)-Alkoxy, (C7-C16)-Aralkoxy-(C1-C6)-Alkoxy, (C1-C16) -hydroxyalkyl, (C6-C16)-aryloxy-(C1-C8)-a alkyl, (C7-C16)-aralkoxy-(C1-C8)-alkyl, (C6-C12)-aryloxy-(C1-C8)-alkoxy-(C1-C6)-alkyl, (C7-C12)-aralkyloxy- (C1-C8)-Alkoxy-(C1-C6)-Alkyl, (C2-C20)-Alkenyloxy-(C1-C6)-Alkyl, (C2-C20)-Alkynyloxy-(C1-C6)-Alkyl, Retinyloxy- (C1-C6)-alkyl, -O-[CH2]xCfH(2f+1-g)Fg, -OCF2Cl, -OCF2-CHFCl, (C1-C20)-alkylcarbonyl, (C3-C8)-cycloalkylcarbonyl, (C6 -C12)-Arylcarbonyl, (C7-C16)-Aralkylcarbonyl, Cinnamoyl, (C2-C20)-Alkenylcarbonyl, (C2-C20)-Alkynylcarbonyl, (C1-C20)-Alkoxycarbonyl, (C1-C12)-Alkoxy-(C1 -C12)-Alkoxycarbonyl, (C6-C12)-Aryloxycarbonyl, (C7-C16)-Aralkoxycarbonyl, (C3-C8)-Cycloalkoxycarbonyl, (C2-C20)-Alkenyloxycarbonyl, Retinyloxycarbonyl, (C2-C20)-Alkynyloxycarbonyl, (C6 -C12)-Aryloxy-(C1-C6)-Alkoxycarbonyl, (C7-C16)-Aralkoxy-(C1-C6)-Alkoxycarbonyl, (C3-C8)-Cycloalkyl-(C1-C6)-Alkoxycarbonyl, (C3-C8 )-CycloAlkoxy-(C1-C6)-Alkoxycarbonyl, (C1-C12)-Alkylcarbonyloxy, (C3-C8)-Cycloalkylcarbonyloxy, (C6-C12)-Arylcarbonyloxy, (C7-C16)-Aralkylcarbonyloxy, Cinnamoyloxy, (C2-C1 2)-Alkenylcarbonyloxy, (C2-C12)-Alkynylcarbonyloxy, (C1-C12)-Alkoxycarbonyloxy, (C1-C12)-Alkoxy-(C1-C12)-Alkoxycarbonyloxy, (C6-C12)-Aryloxycarbonyloxy, (C7-C16) -aralkyloxycarbonyloxy, (C3-C8)-cycloalkyloxycarbonyloxy, (C2-C12)-alkenyloxycarbonyloxy, (C2-C12)-alkynyloxycarbonyloxy, carbamoyl, N-(C1-C12)-alkylcarbamoyl, N,N-di-(C1-C12) -alkylcarbamoyl, N-(C3-C8)-cycloalkylcarbamoyl, N,N-dicyclo-(C3-C8)-alkylcarbamoyl, N-(C1-C10)-alkyl-N-(C3-C8)-cycloalkylcarbamoyl, N-( (C3-C8)-cycloalkyl-(C1-C6)-alkyl)-carbamoyl, N-(C1-C6)-alkyl-N((C3-C8)-cycloalkyl-(C1-C6)-alkyl)-carbamoyl, N-(+)-dehydroabiethylcarbamoyl, N-(C1-C6)-alkyl-N-(+)-dehydroabiethylcarbamoyl, N-(C6-C12)-arylcarbamoyl, N-(C7-C16)-aralkylcarbamoyl, N-(C1 -C10)-Alkyl-N-(C6-C16)-arylcarbamoyl, N-(C1-C10)-Alkyl-N-(C7-C16)aralkylcarbamoyl, N-((C1-C18)-Alkoxy-(C1-C10 )-alkyl)-carbamoyl, N-((C6-C16)-aryloxy-(C1-C10)-alkyl)-carbamoyl, N-((C7-C16)-aralkyloxy-(C1-C10)-alkyl)-carbamoyl , N-(C1-C10)-alkyl-N-((C1-C10)-Alkoxy-(C 1-C10)-alkyl)-carbamoyl, N-(C1-C10)-alkyl-N-((C6-C12)-aryloxy-(C1-C10)-alkyl)-carbamoyl, N-(C1-C10)- alkyl-N-((C7-C16)-aralkyloxy-(C1-C10)-alkyl)-carbamoyl; CON(CH2)h, in which a CH2 group may be substituted with O, S, N-(C1-C8)-alkylimino, N-(C3-C8)-cycloalkylimino, N-(C3-C8)-cycloalkyl-( C1-C4)-alkylimino, N-(C6-C12)-arylimino, N-(C7-C16)-aralkylimino, N-(C1-C4)-alkoxy-(C1-C6)-alkylimino, and h is from 3 to 7; a carbamoyl radical of formula R

in which

R<x>and R<y> are independently selected from hydrogen, (C1-C6)-alkyl, (C3-C7)-cycloalkyl, aryl or the substituent on an α-carbon atom of an α-amino acid, to which L - and D-amino acids belong to,

s is 1-5,

T is OH, or NR*R** and R*, R** and R*** are the same or different and are selected from hydrogen, (C6-C12)-aryl, (C7-C11)-aralkyl, (C1 -C8)-Alkyl, (C3-C8)-Cycloalkyl, (+)-Dehydroabiethyl, (C1-C8)-Alkoxy-(C1-C8)-Alkyl, (C7-C12)-Aralkoxy-(C1-C8)- alkyl, (C6-C12)-aryloxy-(C1-C8)-alkyl, (C1-C10)-alkanoyl, optionally substituted (C7-C16)-aralkanoyl, optionally substituted (C6-C12)-aroyl; or R* and R** together are -[CH2]wherein a CH2 group may be substituted with O, S, SO, SO2, N-acylamino, N-(C1-C10)-Alkoxycarbonylimino, N-(C1-C8 )-alkylimino, N-(C3-C8)-cycloalkylimino, N-(C3-C8)-cycloalkyl-(C1-C4)-alkylimino, N-(C6-C12)-arylimino, N-(C7-C16)- aralkylimino or N-(C1-C4)-alkoxy-(C1-C6)-alkylimino and h is from 3 to 7;

carbamoyloxy, N-(C1-C12)-alkylcarbamoyloxy, N,N-di-(C1-C12)-alkylcarbamoyloxy, N-(C3-C8)-cycloalkylcarbamoyloxy, N-(C6-C12)-arylcarbamoyloxy, N-(C7 -C16)-aralkylcarbamoyloxy, N-(C1-C10)-alkyl-N-(C6-C12)-arylcarbamoyloxy, N-(C1-C10)-alkyl-N-(C7-C16)-aralkylcarbamoyloxy, N-(( C1-C10)-alkyl)-carbamoyloxy, N-((C6-C12)-aryloxy-(C1-C10)-alkyl)-carbamoyloxy, N-((C7-C16)-aralkyloxy-(C1-C10)-alkyl )-carbamoyloxy, N-(C1-C10)-alkyl-N-((C1-C10)-alkoxy-(C1-C10)-alkyl)-carbamoyloxy, N-(C1-C10)-alkyl-N-(( C6-C12)-aryloxy-(C1-C10)-alkyl)-carbamoyloxy, N-(C1-C10)-alkyl-N-((C7-C16)-aralkyloxy-(C1-C10)-alkyl)-carbamoyloxyamino, (C1-C12)-alkylamino, di-(C1-C12)-alkylamino, (C3-C8)-cycloalkylamino, (C3-C12)-alkenylamino, (C3-C12)-alkynylamino, N-(C6-C12)- arylamino, N-(C7-C11)-aralkylamino, N-alkylaralkylamino, N-alkylarylamino, (C1-C12)-Alkoxyamino, (C1-C12)-Alkoxy-N-(C1-C10)-Alkylamino, (C1-C12 )-alkanoylamino, (C3-C8)-cycloalkanoylamino, (C6-C12)-aroylamino, (C7-C16)-aralkanoylamino, (C1- C12)-alkanoyl-N-(C1-C10)-alkylamino, (C3-C8)cycloalkanoyl-N-(C1-C10)-alkylamino, (C6-C12)-aroyl-N-(C1-C10)-alkylamino, (C7-C11)-aralkanoyl-N-(C1-C10)-alkylamino, (C1-C12)-alkanoylamino-(C1-C8)-alkyl, (C3-C8)-cycloalkanoylamino-(C1-C8)-alkyl, (C6-C12)-aroylamino-(C1-C8)-alkyl, (C7-C16)-aralkanoylamono-(C1-C8)-alkyl, amino-(C1-C10)-alkyl, N-(C1-C10)- alkylamino-(C1-C10)-alkyl, N,N-di(C1-C10)-alkylamino-(C1C10)-alkyl, (C3-C8)-cycloalkylamino(C1-C10)-alkyl, (C1-C20)- alkylmercapto, (C1-C20)-alkylsulfinyl, (C1-C20)-alkylsulfonyl, (C6-C12)-arylmercapto, (C6-C12)-arylsulfinyl, (C6-C12)-arylsulfonyl, (C7-C16)-aralkylmercapto, (C7-C16)-aralkylsulfinyl, (C7-C16)-aralkylsulfonyl, (C1-C12)-alkylmercapto-(C1-C6)-alkyl, (C1-C12)-alkylsulfinyl-(C1-C6)-alkyl, (C1 -C12)-alkylsulfonyl-(C1-C6)-alkyl, (C6-C12)-arylmercapto-(C1-C6)-alkyl, (C6-C12)-arylsulfinyl-(C1-C6)-alkyl, (C6-C12 )-arylsulfonyl-(C1-C6)-alkyl, (C7-C16)-aralkylmercapto-(C1-C6)-alkyl, (C7-C16)-aralkylsulfinyl-(C1-C6)-alkyl, (C7-C16)- aralkyl sul phenyl-(C1-C6)-alkyl, sulfamoyl, N-(C1-C10)-alkylsulfamoyl, N,N-di-(C1-C10)-alkylsulfamoyl, (C3-C8)-cycloalkylsulfamoyl, N-(C6-C12 )-arylsulfamoyl, N-(C7-C16)-aralkylsulfamoyl, N-(C1-C10)-alkyl-N-(C6-C12)-arylsulfamoyl, H-(C1-C10)-alkyl-N-(C7-C16 )-aralkylsulfamoyl, (C1-C10)-alkylsulfonamido, N-((C1-C10)-alkyl)-(C1-C10)-alkylsulfonamido, (C7-C16)-aralkylsulfonamido and N-((C1-C10)-alkyl -(C7-C16)-aralkylsulfonamido; where an aryl radical can be substituted with from 1 to 5 substituents selected from hydroxyl, halogen, cyano, trifluoromethyl, nitro, carboxyl, (C2-C16)-alkyl, (C3-C8)cycloalkyl, (C3-C8)-cycloalkyl-( C1-C12)-Alkyl, (C3-C8)-Cycloalkyl, (C3-C8)-Cycloalkyl-(C1-C12)-Alkoxy, (C3-C8)-Cycloalkyloxy-(C1-C12)-Alkyl, (C3- C8)-Cycloalkyloxy-(C1-C12)-Alkoxy, (C3-C8)-Cycloalkyl-(C1-C8)-Alkyl-(C1-C6)-Alkoxy, (C3-C8)-Cycloalkyl(C1-C8)- Alkoxy-(C1-C6)-Alkyl, (C3-C8)-Cycloalkyloxy-(C1-C8)-Alkoxy-(C1-C6)Alkyl, (C3-C8)-Cyclo-Alkoxy-(C1-C8)-Alkoxy-( (C1-C8)-Alkoxy, (C6-C12)-Aryl, (C7-C16)-Aralkyl, (C2-C16)-Alkenyl, (C2-C12)-Alkynyl, (C1-C16)-Alkoxy, (C1- C16)-Alkenyloxy, (C1-C12)-Alkoxy-(C1-C12)-Alkyl, (C1-C12)-Alkoxy-(C1-C12)-Alkoxy, (C1-C12)-Alkoxy(C1-C8)- Alkoxy-(C1-C8)-Alkyl, (C6-C12)-Aryloxy, (C7-C16)-Aralkyloxy, (C6-C12)-Aryloxy-(C1-C6)-Alkoxy, (C7-C16)-Aralkyloxy- (C1-C6)-Alkoxy, (C1-C8)-hydroxyalkyl, (C6-C16)-aryloxy-(C1-C8)-alkyl, (C7-C16)-aralkoxy-(C1-C8)-alkyl, (C6 -C 12 )-aryl xy-(C1-C8)-Alkoxy-(C1-C6)-Alkyl, (C7-C12)-Aralkyloxy-(C1-C8)-Alkoxy-(C1-C6)-Alkyl, -O-[CH2]xCfH( 2f+1-g)Fg, -OCF2Cl, -OCF2-CHFCl, (C1-C12)-alkylcarbonyl, (C3-C8)-cycloalkylcarbonyl, (C6-C12)-arylcarbonyl, (C7-C16)-aralkylcarbonyl, (C1 -C12)-Alkoxycarbonyl, (C1-C12)-Alkoxy-(C1-C12)-Alkoxycarbonyl, (C6-C12)-Aryloxycarbonyl, (C7-C16)-Aralkoxycarbonyl, (C3-C8)-Cycloalkoxycarbonyl, (C2-C12 )-Alkenyloxycarbonyl, (C2-C12)-Alkynyloxycarbonyl, (C6-C12)-Aryloxy-(C1-C6)-Alkoxycarbonyl, (C7-C16)-Aralkoxy-(C1-C6)-Alkoxycarbonyl, (C3-C8)- cycloalkyl-(C1-C6)-Alkoxycarbonyl, (C3-C8)-Cycloalkyl-(C1-C6)-Alkoxycarbonyl, (C1-C12)-Alkylcarbonyloxy, (C3-C8)-Cycloalkylcarbonyloxy, (C6-C12)-Arylcarbonyloxy, (C7-C16)-Aralkylcarbonyloxy, Cinnamoyloxy, (C2-C12)-Alkenylcarbonyloxy, (C2-C12)-Alkynylcarbonyloxy, (C1-C12)-Alkoxycarbonyloxy, (C1-C12)-Alkoxy-(C1-C12)-Alkoxycarbonyloxy, (C6-C12)-aryloxycarbonyloxy, (C7-C16)-aralkyloxycarbonyloxy, (C3-C8)-cycloalkyloxycarbonyloxy, (C2-C12 )-alkenyloxycarbonyloxy, (C2-C12)-alkynyloxycarbonyloxy, carbamoyl, N-(C1-C12)-alkylcarbamoyl, N,N-di(C1-C12)-alkylcarbamoyl, N-(C3-C8)cycloalkylcarbamoyl, N,N- dicyclo-(C3-C8)-alkylcarbamoyl, N-(C1-C10)-alkyl-N-(C3-C8)-cycloalkylcarbamoyl, N-((C3-C8)-cycloalkyl-(C1-C6)-alkyl)carbamoyl , N-(C1-C6)-alkyl-N-((C3-C8)-cycloalkyl-(C1-C6)-alkyl)carbamoyl, N-(+)-dehydroabiethylcarbamoyl, N-(C1-C6)-alkyl- N-(+)-dehydroabiethylcarbamoyl, N-(C6-C12)-arylcarbamoyl, N-(C7-C16)-aralkylcarbamoyl, N-(C1-C10)-alkyl-N-(C6-C16)-arylcarbamoyl, N- (C1-C10)-Alkyl-N-(C7-C16)-Aralkylcarbamoyl, N-((C1-C16)-Alkoxy-(C1-C10)-Alkyl)carbamoyl, N-((C6-C16)-Aryloxy- (C1-C10)-alkyl)carbamoyl, N-((C7-C16)-aralkyloxy-(C1-C10)-alkyl)carbamoyl, N-(C1-C10)-alkyl-N-((C1-C10)- alkoxy-(C1-C10)-alkyl)carbamoyl, N-(C1-C10)-alkyl-N-((C6-C12)-aryloxy-(C1-C10)-alkyl)carbamoyl, N-(C1-C10) -alkyl-N-((C7-C16)-aralkyloxy-(C1-C10)-alkyl)-carbamoyl, CON(CH2)h, in which one CH2 group can be replaced by O, S, N-(C1-C8) -alkylimino, N-(C3-C8 )-cycloalkylimino, N-(C3-C8)-cycloalkyl-(C1-C4)-alkylimino, N-(C6-C12)-arylimino, N-(C7-C16)-aralkylimino, N-(C1-C4)- alkoxy-(C 1 -C 6 )alkylimino and h is from 3 to 7; carbamoyloxy, N-(C1-C12)-alkylcarbamoyloxy, N,N-di-(C1-C12)-alkylcarbamoyloxy, N-(C3-C8)-cycloalkylcarbamoyloxy, N-(C6-C16)-arylcarbamoyloxy, N-(C7 -C16)-aralkylcarbamoyloxy, N-(C1-C10)-alkyl-N-(C6-C12)-arylcarbamoyloxy, N-(C1-C10)-alkyl-N-(C7-C16)-aralkylcarbamoyloxy, N-(( C1-C10)-alkyl)carbamoyloxy, N-((C6-C12)-aryloxy-(C1-C10)-alkyl)carbamoyloxy, N-((C7-C16)-aralkyloxy-(C1-C10)-alkyl)carbamoyloxy -N-(C1-C10)-alkyl-N-((C1-C10)-Alkoxy-(C1-C10)-alkyl)carbamoyloxy, N-(C1-C10)-Alkyl-N-((C6-C12) -aryloxy-(C1-C10)-alkyl)carbamoyloxy, N-(C1-C10)-alkyl-N-((C7-C16)-aralkyloxy-(C1-C10)-alkyl)carbamoyloxy, amino, (C1-C12 )-alkylamino, di-(C1-C12)-alkylamino, (C3-C8)-cycloalkylamino, (C3-C12)-alkenylamino, (C3-C12)-alkynylamino, N-(C6-C12)-arylamino, N- (C7-C11)-Aralkylamino, N-Alkylaralkylamino, N-Alkylarylamino, (C1-C12)-Alkoxyamino, (C1-C12)-Alkoxy-N-(C1-C10)-Alkylamino, (C1-C12)-Alkanoylamino, (C3-C8)-cycloalkanoylamino, (C6-C12)-aroylamino, (C7-C16)-aralkanoylamino, (C1-C12)-a alkanoyl-N-(C1-C10)-alkylamino, (C3-C8)-cycloalkanoyl-N-(C1-C10)-alkylamino, (C6-C12)-aroyl-N-(C1-C10)-alkylamino, (C7 -C11)-aralkanoyl-N-(C1-C10)-alkylamino, (C1-C12)-alkanoylamino-(C1-C8)-alkyl, (C3-C8)-cycloalkanoylamino-(C1-C8)-alkyl, (C6 -C12)-aroylamino-(C1-C8)-alkyl, (C7-C16)-aralkanoylamino(C1-C8)-alkyl, amino-(C1-C10)-alkyl, N-(C1-C10)-alkylamino-( C1-C10)-alkyl, N,N-di-(C1-C10)-alkylamino-(C1-C10)-alkyl, (C3-C8)-cycloalkylamino-(C1-C10)-alkyl, (C1-C12) -alkylmercapto, (C1-C12)-alkylsulfonyl, (C1-C12)-alkylsulfonyl, (C6-C16)-arylmercapto, (C6-C16)-arylsulfinyl, (C6-C16)-arylsulfonyl, (C7-C16)-aralkylmercapto , (C7-C16)-aralkylsulfinyl or (C7-C16)-aralkylsulfonyl;

or in which R<1>and R<2>or R<2>and R<3>form a chain [CH2] that is saturated or unsaturated with a C=C double bond, in which 1 or 2 CH2 groups are optionally replaced with O, S, SO, SO2 or NR' and R' is hydrogen, (C6-C12)-aryl, (C1-C8)-alkyl, (C1-C8)-alkoxy-(C1-C8)-alkyl, (C7 -C12)-araloxy-(C1-C8)-alkyl, (C6-C12)aryloxy-(C1-C8)-alkyl, (C1-C10)-alkanoyl, optionally substituted (C7-C16)-aralkanoyl or optionally substituted ( (C 6 -C 12 )-aroyl; and o is 3, 4 or 5;

or in which the radicals R<1> and R<2> or R<2> and R<3> together with the pyridine or pyridazine to which they are attached form a 5,6,7,8-tetrahydroisoquinoline ring, a 5,6,7, 8-tetrahydroquinoline ring or a 5,6,7,8-tetrahydrocinnoline ring;

or wherein R<1> and R<2> or R<2> and R<3> form a carbocyclic or heterocyclic aromatic ring of from 5 to 6 members;

or in which R<1> and R<2> or R<2> and R<3> together with the pyridine or pyridazine to which they are attached form an optionally substituted heterocyclic ring system selected from thienopyridines, furanopyridines, pyridopyridines, pyrimidinopyridines, imidazopyridines, thiazolopyridines, oxazolopyridines, quinoline, isoquinoline and cinnoline; wherein quinoline, isoquinoline or cinnoline preferably satisfies formulas Ia, Ib and Ic:

and in which the substituents R<12> to R<23> in all cases independently of each other have the same meaning as R<1>, R<2> and R<3>;

or in which the radicals R<1> and R<2> together with the pyridine group to which they are bound form a compound of formula Id:

wherein V is S, O or NR<k> and R<k> is selected from hydrogen, (C1-C6) alkyl, aryl or benzyl; wherein an aryl radical may optionally be substituted with from 1 to 5 substituents as defined above; and

R<24>, R<25>, R<26> and R<27> in all cases independently of each other have the same meaning as R<1>, R<2> and R<3>;

f is from 1 to 8;

g is 0 or from 1 to (2f+1);

x is from 0 to 3; and

h is from 3 to 7;

including physiologically active salts and prodrugs derived therefrom.

Examples of compounds according to formula (I) are described in the European patent documents EP0650960 and EP0650961. All compounds listed in EP0650960 and EP0650961, particularly those listed in the compound claims and the final products in the working examples, are incorporated into the present patent application by reference. Examples of compounds of formula (I) include, but are not limited to, [(3-hydroxypyridine-2-carbonyl)amino]acetic acid and [(3-methoxypyridine-2-carbonyl)amino]acetic acid.

In addition, examples of compounds of formula (I) are described in U.S. Pat. Patent Document No. 5,658,933. All compounds listed in the U.S. Patent Document No. 5,658,933, particularly those listed in the connection claims and the final products in the working examples, are incorporated into the present patent application by reference. Examples of compounds of formula (I) include, but are not limited to, 3-methoxypyridine-2-carboxylic acid N-(((hexadecyloxy)carbonyl)methyl)amide hydrochloride, 3-methoxypyridine-2-carboxylic acid N-(((1- Octyloxy)carbonyl)methyl)-amide, 3-methoxypyridine-2-carboxylic acid N-(((1-octyloxy)carbonyl)methyl)amide, 3-methoxypyridine-2-carboxylic acid N-(((hexyloxy)carbonyl)methyl)amide , 3-methoxypyridine-2-carboxylic acid N-(((butyloxy)carbonyl)methyl)amide, 3-methoxypyridine-2-carboxylic acid N-(((2-nonyloxy)carbonyl)methyl)amide racemate, 3-methoxypyridine-2-carboxylic acid N-(((heptyloxy)carbonyl)methyl)amide, 3-benzyloxypyridine-2-carboxylic acid N-(((octyloxy)carbonyl)methyl)amide, 3-benzyloxypyridine-2-carboxylic acid N-(((butyloxy)carbonyl)methyl )amide, 5-(((-3-(1-butyloxy)propyl)amino)carbonyl)-3-methoxypyridine-2-carboxylic acid N-((benzyloxycarbonyl)methyl)amide, 5-(((3-(1- butyloxy)propyl)amino)carbonyl)-3-methoxypyridine-2-carboxylic acid N-(((1-butyloxy)carbonyl)methyl)amide and 5-(((3-lauryloxy)propyl)amino)carbo nyl)-3-methoxypyridine-2-carboxylic acid N-(((benzyloxy)carbonyl)methyl)amide.

Additional compounds of formula (I) are substituted heterocyclic carboxamides described in U.S. Pat. Patent Document No. 5,620,995; 3-Hydroxypyridine-2-carboxamido esters described in U.S. Pat. Patent Document No. 6,020,350; sulfonamidocarbonylpyridine-2-carboxamides described in U.S. Pat. patent document no.

5,607,954; and sulfonamidocarbonylpyridine-2-carboxamides and sulfonamidocarbonylpyridine-2-carboxamide esters described in U.S. Pat. U.S. Patent Nos. 5,610,172 and 5,620,996. All compounds listed in these patent documents, particularly the compounds listed in the compound claims and the final products in the working examples, are incorporated into the present patent application by reference.

Examples of compounds according to formula (Ia) are described in U.S. Pat. U.S. Patent Nos. 5,719,164 and 5,726,305. All compounds listed in these patent documents, particularly the compounds listed in the compound claims and in the final products in the working examples, are incorporated into the present patent application by reference. Examples of compounds of formula (Ia) include, but are not limited to, N-((3-hydroxy-6-isopropoxyquinoline-2-carbonyl)amino)acetic acid, N-((6-(1-butyloxy)-3- hydroxyquinolin-2-yl)carbonyl)glycine, [(3-hydroxy-6-trifluoromethoxyquinolin-2-carbonyl)amino]acetic acid, N-((6-chloro-3-hydroxyquinolin-2-yl)carbonyl)glycine, N- ((7-chloro-3-hydroxyquinolin-2-yl)carbonyl)glycine and [(6-chloro-3-hydroxyquinolin-2-carbonyl)amino]acetic acid.

Examples of compounds according to formula (Ib) are described in U.S. Pat. patent document no.

6,093,730. All compounds listed in the U.S. Patent Document No. 6,093,730, particularly those listed in the connection claims and the final products from the working examples, are incorporated into the present patent application by reference. Examples of compounds of formula (Ib) include, but are not limited to, N-((1-chloro-4-hydroxy-7-(2-propyloxy)isoquinolin-3-yl)carbonyl)glycine, N-((1 -chloro-4-hydroxy-6-(2-propyloxy)isoquinolin-3-yl)carbonyl)glycine, N-((1-chloro-4-hydroxyisoquinoline-3-carbonyl)amino)acetic acid (compound A), N- ((1-chloro-4-hydroxy-7-methoxyisoquinolin-3-yl)carbonyl)glycine, N-(1-chloro-4-hydroxy-6-methoxyisoquinolin-3-yl)carbonyl)glycine, N-((7 -butyloxy)-1-chloro-4-hydroxyisoquinolin-3-yl)carbonyl)glycine, N-((6-benzyloxy-1-chloro-4-hydroxyisoquinoline-3-carbonyl)amino)acetic acid, ((7-benzyloxy- 1-Chloro-4-hydroxyisoquinoline-3-carbonyl)amino)acetic acid methyl ester, N-((7-benzyloxy-1-chloro-4-hydroxyisoquinoline-3-carbonyl)amino)acetic acid, N-((8-chloro-4- hydroxyisoquinolin-3-yl)carbonyl)glycine, N-((7-butoxy-4-hydroxyisoquinolin-3-carbonyl)amino)acetic acid.

Further compounds according to formula (I) which can also be used in the methods according to the invention include, but are not limited to, 6-cyclohexyl-1-hydroxy-4-methyl-1H-pyridin-2-one, 7-(4-methylpiperazin- 1-ylmethyl)-5-phenylsulfanylmethylquinolin-8-ol, 4-nitroquinolin-8-ol, 5-butoxymethylquinolin-8-ol, [(4-hydroxy-7-phenoxyisoquinoline-3-carbonyl)amino]acetic acid (Compound B) and [(4-hydroxy-7-phenylsulfanylisoquinoline-3-carbonyl)amino]acetic acid (compound C). Furthermore, the invention provides further examples of compounds in which, for example, positions A and B together can be, for example, hexanoic acid, cyanomethyl, 2-aminoethyl, benzoic acid, 1H-benzoimidazol-2-ylmethyl, etc.

In other embodiments, compounds used in the methods according to the invention are selected from compounds of formula (III)

or pharmaceutically acceptable salts thereof, wherein:

a is an integer from 1 to 4;

b is an integer from 0 to 4;

c is an integer from 0 to 4;

Z is selected from the group consisting of (C3-C10)cycloalkyl, (C3-C10)cycloalkyl independently substituted with one or more Y<1>, heterocycloalkyl with 3-10 members and heterocycloalkyl with 3-10 members which independently of each other are substituted with one or more Y<1>; (C5-C20)aryl, (C5-C20)aryl which is independently substituted with one or more Y<1>, heteroaryl with 5-20 members and heteroaryl with 5-20 members which are independently substituted with one or more Y <1>;

Ar<1> is selected from the group consisting of (C5-C20)aryl, (C5-C20)aryl independently substituted with one or more Y<2>, 5-20 membered heteroaryl and 5-20 membered heteroaryl which are independently substituted with one or more Y<2>;

each Y<1> is independently of the others selected from the group consisting of a lipophilic functional group, (C5-C20)aryl, (C6-C26)alkaryl, 5-20 membered heteroaryl and 6-26 membered alk-heteroaryl ;

each Y<2> is independently of the others selected from the group consisting of -R', -OR', -OR'', -SR'', -NR'R', -NO2, -halogen, -trihalomethyl, trihalomethoxy , -C(O)R', -C(O)OR', -C(O)NR'R', -C(O)NR'OR', -C(NR'R')=NOR', - NR'-C(O)R', -SO2R', -SO2R'', -NR'-SO2-R', -NR'-C(O)-NR'R', tetrazol-5-yl, -NR '-C(O)-OR', -C(NR'R')=NR', -S(O)-R', -S(O)-R'' and -NR'-C(S)- NR'R'; and

each R' is independently of the others selected from the group consisting of -H, (C 1 -C 8 )alkyl, (C 2 -C 8 )alkenyl and (C 2 -C 8 )alkynyl; and

each R'' is independently of the others selected from the group consisting of (C5-C20)aryl and (C5-C20)aryl independently substituted with one or more -OR', -SR', -NR'R', -NO2, -CN, halogen atoms or trihalomethyl groups,

or wherein c is 0 and Ar<1> is an N'-substituted ureaaryl, where the compound has the structural formula (IIIa):

or pharmaceutically acceptable salts thereof, wherein:

a, b and Z are as defined above; and

R<35> and R<36> are independently selected from the group consisting of hydrogen, (C1-C8)alkyl, (C2-C8)alkenyl, (C2-C8)alkynyl, (C3-C10)cycloalkyl, ( C5-C20)aryl, (C5-C20) substituted aryl, (C6-C26)alkaryl, (C6-C26) substituted alkaryl, 5-20 membered heteroaryl, 5-20 membered substituted heteroaryl, 6-membered alk-heteroaryl 26 membered and 6-26 membered substituted alk-heteroaryl; and

R<37> is independently selected from the group consisting of hydrogen, (C1-C8)alkyl, (C2-C8)alkenyl and (C2-C8)alkynyl.

Examples of compounds of formula (III) are described in international patent publication WO 00/50390. All compounds listed in WO 00/50390, particularly those listed in the compound claims and the final products in the working examples, are incorporated into the present patent application by reference. Examples of compounds of formula (III) include 3-{[4-(3,3-dibenzylureido)benzenesulfonyl]-[2-(4-methoxyphenyl)ethyl]amino}-N-hydroxypropionamide (compound D), 3-{ 4-[3-(4-chlorophenyl)ureido]benzenesulfonyl}-[2-(4-methoxyphenyl)ethyl]amino}-N-hydroxyproprionamide and 3-{{4-[3-(1,2-diphenylethyl)ureido] benzenesulfonyl}-[2-(4-methoxyphenyl)ethyl]amino}-N-hydroxypropionamide.

Methods for detecting compounds according to the invention are also provided. In certain aspects, a compound of the invention is a compound that stabilizes HIF α. A compound's ability to stabilize or activate HIF α can, for example, be measured by direct measurement of HIF α in a sample, indirect measurement of HIF α, for example by measuring a decrease in HIF α associated with the von Hippell Lindau protein (see for example international Patent Publication No. WO 00/69908) or activation of HIF-responsive target genes or reporter constructs (see, for example, U.S. Patent Publication No. 5,942,434). Measurement and comparison of the level of HIF and/or HIF-responsive target proteins in the absence and presence of the compound will detect compounds that stabilize HIF α and/or activate HIF.

In other aspects, a compound of the invention is a compound that inhibits HIF hydroxylase activity. Analyzes for hydroxylase activity are standard in the field. Such assays can measure hydroxylase activity directly or indirectly. An assay can for example measure hydroxylated amino acid residues, for example proline, asparagine, etc., present in the enzyme substrate, for example a target protein, a synthetic peptide similar to the target protein or a fragment thereof (see for example Palmerini et al. (1985) J Chromatogr 339: 285-292). A reduction in the amount of hydroxylated amino acid residue, for example proline or asparagine, in the presence of a compound is indicative of a compound that inhibits hydroxylase activity. Alternatively, the assays can measure other products of the hydroxylation reaction, for example formation of succinate from 2-oxoglutarate (see, for example, Cunliffe et al. (1986) Biochem J 240: 617-619). Kaule and Gunzler (1990; Anal Biochem 184: 291-297) describe an example of a method that measures the formation of succinate from 2-oxoglutarate.

Methods such as those described above can be used for the detection of compounds that modulate HIF hydroxylase activity. The target protein may comprise HIF α or a fragment thereof, for example HIF(556-575). The enzyme may comprise, for example, HIF-prolyl hydroxylase (see, for example, GenBank accession number AAG33965, etc.) or HIF-asparaginyl hydroxylase (see, for example, GenBank accession number AAL27308, etc.), obtained from any source.

The enzyme can also be present in an impure cell lysate or in partially purified form.

Methods that measure HIF hydroxylase activity described in, for example, Ivan et al. (2001, Science 292: 464-468; and 2002, Proc Natl Acad Sci USA 99:

13459-13464) and Hirsila et al. (2003, J Biol Chem 278: 30772-30780); further methods are described in international patent publication WO 03/049686. Measurement and comparison of enzyme activity in the absence and presence of the compound will detect compounds that inhibit hydroxylation of HIF α.

A compound according to the invention is a compound which further provides a measurable effect, measured in vitro or in vivo, demonstrated by increased erythropoiesis, increased iron metabolism or a therapeutic improvement of conditions which include, for example, iron deficiency, including functional iron deficiency; anemia associated with chronic disease, iron deficiency and microcytosis or microcytic anemia; or a condition associated with inflammation, infection, immunodeficiency or a neoplastic disorder.

The measurable effect can be any of the following parameters: Increased hemoglobin, hematocrit, reticulocyte, red blood cell count, plasma EPO, etc.; improved iron metabolism, measured by reduction of observed symptoms including, for example, relief of chronic fatigue, pallor, dizziness, etc. or by elevated serum iron level, altered serum ferritin level, % transferrin saturation, total iron binding capacity, improved reticulocyte count, hemoglobin or hematocrit, all measured by a standard blood count analysis.

Pharmaceutical preparations and routes of administration

The compositions according to the present invention can be administered directly or in pharmaceutical compositions comprising excipients which are well known in the art. Current methods of treatment may include administering an effective amount of a compound of the present invention to an individual who has or is susceptible to having a metabolic disorder; preferably a disorder associated with glucose regulation, for example diabetes, hyperglycemia, etc. In a preferred embodiment the subject is a mammal and in a most preferred embodiment the subject is a human.

An effective amount, such as a dose, of a compound or drug can be easily determined by routine experimentation, as can an effective and convenient route of administration and a suitable formulation. Different designs and drug delivery systems are available in the field. (See, for example, Gennaro, eds. (2000) Remington's Pharmaceutical Sciences, supra; and Hardman, Limbird and Gilman, eds. (2001) The Pharmacological Basis of Therapeutics, supra).

Suitable routes of administration may include, for example, oral, rectal, topical, nasal, pulmonary, ocular, intestinal and parenteral administration. Primary routes of parenteral administration include intravenous, intramuscular and subcutaneous administration. Secondary routes of administration include intraperitoneal, intraarterial, intraarticular, intracardiac, intracisternal, intradermal, intralesional, intraocular, intrapleural, intrathecal, intrauretinal and intraventricular administration. The condition to be treated, together with the drug's physical, chemical and biological properties, will determine the type of design and route of administration to be used, as well as whether local or systemic delivery will be preferred.

Pharmaceutical dosage forms of a compound according to the invention can be provided in a system for immediate release, controlled release, sustained release or targeted drug delivery. Commonly used dosage forms include, for example, solutions and suspensions, (micro-)emulsions, ointments, gels and plasters, liposomes, tablets, drops, capsules with soft or hard shells, suppositories, ovules, implants, amorphous or crystalline powders, aerosols and freeze-dried formulations . Depending on the route of administration used, special devices may be required for application or administration of the drug, such as syringes and needles, inhalers, pumps, injection pens, applicators or special bottles.

Pharmaceutical dosage forms often consist of the drug, one or more excipients and a container/a containment system. One or more excipients, also referred to as inactive ingredients, can be added to a compound according to the invention to improve or facilitate the manufacture, stability, administration and safety of the drug and can constitute means to achieve a desired drug release profile. Which or which types of excipients are to be added to the drug can therefore depend on various factors, for example the physical and chemical properties of the drug, the route of administration and the method of manufacture. Pharmaceutically acceptable excipients are available in the art and include those listed in various pharmacopoeias. (See, for example, USP, JP, EP and BP, FDA website (www.fda.gov), Inactive Ingredient Guide 1996 and Handbook of Pharmaceutical Additives, ed. Ash; Synapse Information Resources, Inc. 2002).

Pharmaceutical dosage forms of a compound according to the present invention can be prepared by any of the methods well known in the art, for example by conventional methods such as mixing, sieving, dissolving, melting, granulating, drop making, tablet making, suspending, extruding, spray drying, pulverization, emulsification, (nano/micro-) encapsulation, capture or freeze-drying. As noted above, the compositions according to the present invention may comprise one or more physiologically acceptable inactive ingredients which facilitate the processing of active molecules into preparations for pharmaceutical use.

Suitable design will depend on the desired administration route. For intravenous injection, for example, the composition can be formulated in aqueous solution, if necessary using physiologically compatible buffers, including, for example, phosphate, histidine or citrate to adjust the pH of the formulation and a tonicity agent such as, for example, sodium chloride or dextrose. For transmucosal or nasal administration, semi-solid or liquid preparations or plasters may be preferred, possibly containing penetration-promoting agents. Such penetration-promoting agents are generally known in the art. For oral administration, the compounds may be formulated in liquid or solid dosage forms and as immediate or controlled/sustained release formulations. Suitable dosage forms for oral intake include tablets, pills, drops, capsules with hard and soft shells, liquids, gels, syrups, porridges, suspensions and emulsions.

The compounds can also be formulated into rectal compositions such as suppositories or retention enemas, which for example contain conventional suppository materials such as cocoa butter or other glycerides.

Solid oral dosage forms can be obtained by using excipients which may include fillers, disintegrants, binders (dry and wet), dissolution retarders, lubricants, lubricants, anti-adhesives, cation exchange resins, wetting agents, antioxidants, preservatives, dyes and flavourings. These excipients can be synthetic or natural. Examples of such excipients include cellulose derivatives, citric acid, dicalcium phosphate, gelatin, magnesium carbonate, magnesium/sodium lauryl sulfate, mannitol, polyethylene glycol, polyvinylpyrrolidone, silicates, silicon dioxide, sodium benzoate, sorbitol, starches, stearic acid or a stearic acid salt, sugars (for example, dextrose, sucrose, lactose, etc.), talc, gum tragacanth, vegetable oils (hydrogenated) and waxes. Ethanol and water can act as granulation aids. In certain cases, coating tablets with, for example, a taste-masking film or stomach acid-resistant film or a release-retarding film is desirable. Natural and synthetic polymers in combination with dyes, sugars and organic solvents or water are often used to coat tablets, which leads to drops. If a capsule is preferred to a tablet, the drug powder, suspension or solution can be supplied in a compatible hard or soft shell capsule.

In one embodiment, the compounds of the present invention can be administered topically, such as through a skin patch, a semi-solid or liquid preparation, for example a gel, a (micro-) emulsion, an ointment, a solution, a (nano/micro-) suspension or a foam. The penetration of the drug into the skin and the underlying tissues can, for example, be regulated by using penetration-promoting agents; a suitable choice and a suitable combination of lipophilic, hydrophilic and amphiphilic excipients including water, organic solvents, waxes, oils, synthetic and natural polymers, surfactants and emulsifiers; by adjusting the pH; and when using complexing agents. Other techniques such as iontophoresis can be used to regulate the skin penetration of a compound according to the invention. Transdermal or topical administration will, for example, be preferred in situations where local delivery with minimal systemic exposure is desirable.

For administration by inhalation or nasal administration, the compounds for use according to the present invention are advantageously supplied in the form of a solution, a suspension, an emulsion or a semi-solid aerosol from pressurized containers or a nebuliser, usually using a propellant gas, for eg halogenated carbons derived from methane and ethane, carbon dioxide or any other suitable gas. For topical aerosols, hydrocarbons such as butane, isobutene and pentane are useful. In the case of a pressurized aerosol, the appropriate dosage unit can be determined by equipping the container with a valve that delivers a predetermined amount. Capsules and cartridges of, for example, gelatin for use in an inhaler or insufflator can be designed. These typically contain a powder mixture of the compound and a suitable powder base such as lactose or starch.

Compositions designed for parenteral administration by injection are usually sterile and may be in unit dose forms, for example in ampoules, syringes, injection pens or in multi-dose containers, the latter usually containing a preservative. The compositions can be designed as suspensions, solutions or emulsions in oil-based or aqueous carriers and can contain design agents such as buffers, tonicity agents, viscosity-increasing agents, surface-active agents, suspending and dispersing agents, antioxidants, biocompatible polymers, gelating agents and preservatives. Depending on the injection site, the carrier may contain water, a synthetic or vegetable oil and/or organic co-solvents. In certain cases such as with a lyophilized product or concentrate, the parenteral formulation will be reconstituted or diluted prior to administration. Deposit designs that provide controlled or sustained release of a compound according to the invention may comprise injectable suspensions of nano/microparticles, nano/microcrystals or non-micronized crystals. Polymers such as poly(lactic acid), poly(glycolic acid) or copolymers thereof can function as controlled/sustained release supports, in addition to others well known in the art. Other deposition delivery systems may be in the form of implants and pumps that require surgical intervention.

Suitable carriers for intravenous injection of the molecules according to the invention are well known in the art and include water-based solutions containing a base such as, for example, sodium hydroxide, so that an ionized compound is formed, sucrose or sodium chloride as a tonicity agent and a buffer containing phosphate or histidine. Additional solvents such as polyethylene glycols can be added. These water-based systems effectively dissolve compounds according to the invention and provide low toxicity when administered systemically. The quantity ratio between the components in a dissolved system can be varied considerably without destroying the solubility properties and the toxicity properties. Furthermore, the identity of the components can be varied. For example, surfactants with low toxicity such as polysorbates or polyoxamers can be used, and likewise polyethylene glycol or other additional solvents, biocompatible polymers such as polyvinylpyrrolidone can be added and other sugars and polyols can replace dextrose.

For compositions useful in the present methods of treatment, a therapeutically effective dose can initially be estimated using a variety of techniques well known in the art.

Initial doses for use in animal studies can be based on effective concentrations established in cell culture assays. Dosage ranges suitable for humans can be determined by, for example, using results obtained from animal studies and cell culture assays.

A therapeutically effective dose or amount of a compound, agent or drug according to the present invention refers to an amount or dose of the compound, agent or drug that leads to relief of symptoms or prolonged survival in an individual. The toxicity and therapeutic effect of such molecules can be determined by standard pharmaceutical methods in cell cultures or experimental animals, for example by determining the LD50 (the dose that is lethal to 50% of the population) and the ED50 (the dose that is therapeutically effective in 50% of the population). The dose ratio of toxic and therapeutic effects is the therapeutic index, which can be expressed as the ratio LD50/ED50. Agents showing a high therapeutic index are preferred.

The effective amount or therapeutically effective amount is the amount of the compound or pharmaceutical composition that will elicit the biological or medical response in a tissue, system, animal or human that is desired by the researcher, veterinarian, physician or other clinical personnel, for for example regulation of glucose metabolism, reduction of an elevated or increased glucose level in the blood, treatment or prevention of a disorder associated with altered glucose metabolism, such as diabetes, etc.

Doses are preferably within a range of concentrations in the circulation that include the ED50 with little or no toxicity. The doses may vary within this range depending on the dosage form used and/or the route of administration used. The exact formulation, route of administration and dose and the exact dosage schedule should be selected according to methods known in the art in light of the details of the patient's condition.

The dosage amount and dosage schedule can be individually adjusted to obtain a plasma level of the active group sufficient to achieve the desired effects, for example, regulation of glucose metabolism, reduction of blood glucose level, etc., i.e. the minimal effective concentration (MEC). . The MEC will vary for the individual compounds, but can, for example, be estimated based on in vitro results and animal experiments. The doses necessary to achieve MEC will depend on the characteristics of the patient and the route of administration. In the case of local administration or selective uptake, the effective local concentration of the drug is not necessarily related to the plasma concentration.

The amount of agent or composition that is administered may depend on a number of factors, including the gender, age and weight of the individual being treated, the extent of the condition, the method of administration and the judgment of the attending physician.

The present compositions can, if desired, be present in a package or dispensing device containing one or more unit dosage forms containing the active ingredient. Such a package or device can, for example, comprise metal or plastic foil such as a bubble pack or glass and rubber stoppers, for example as in a medicine bottle. The package or dispensing device may be accompanied by instructions for administration. Compositions comprising a compound according to the invention formulated in a compatible pharmaceutical carrier may also be prepared, placed in a suitable container and labeled for the treatment of a specified condition.

These and other embodiments of the present invention will be readily apparent to those of ordinary skill in the art from the present description.

EXAMPLES

The invention will be further understood by reference to the following examples which are only intended to be examples of the invention. These examples are given only to illustrate the invention according to the claims. The present invention is not limited in the area of the exemplified embodiments, which are only intended as illustrations of certain aspects of the invention. All methods which are functionally equivalent are within the scope of the invention. Various modifications of the invention in addition to those described herein will be obvious to those skilled in the art from the above description and the attached figures. Such modifications are intended to be within the scope of the attached requirements.

Example 1: Overcoming suppressive effects of TNF-α on EPO formation

Hep3B cells were treated with different concentrations (0, 0.4, 2, 10 ng/ml) of TNF-α in the absence or presence of compound A or compound B for 3 days. The secreted EPO level was determined using a commercially available ELISA kit (R&D Systems, catalog no. DEP00). In the absence of compound, treatment of Hep3B cells with TNF-α reduced EPO formation in a dose-dependent manner. Hep3B cells treated with different concentrations of either compound A (Figure 1A) or compound B (Figure 1B) in the absence of TNF-α showed a dose-dependent increase of EPO formation. Addition of any of these compounds in the presence of TNF-α produced a strong reduction in the inhibitory effects of TNF-α on EPO formation. An overcoming of the suppressive effect of TNF-α on EPO formation by inhibition of prolyl hydroxylase was observed in the presence of low (eg 0.4 ng/ml) and high (eg 10 ng/ml) concentrations of TNF-α . Accordingly, the inhibitory effects of the inflammatory cytokine TNF-α on EPO formation were overcome by inhibition of prolyl hydroxylase activity using the compounds and methods of the present invention. These results suggest that the compounds and methods of the present invention are useful for increasing EPO formation in the presence of the inflammatory cytokine TNF-α. Furthermore, the methods and compounds of the present invention are useful for increasing EPO formation, and consequently for, for example, treating anemia in an individual in which the individual has a disorder associated with TNF-α, such as acute or chronic inflammation or another anemia associated with chronic illness.

A series of experiments was performed to examine the effects of the compounds of the present invention on EPO formation after treatment of cells with the inflammatory cytokine TNF-α (that is, in cells that had already been exposed to TNF-α). In these experiments, therefore, TNF-α signaling was initiated before the addition of a prolyl hydroxylase inhibitor. The Hep3B cells were treated with different concentrations (0, 0.4, 2, 10 ng/ml) of TNF-α for 2 hours, after which different concentrations of compound A or compound B were added to the cultured cells. The secreted EPO level was determined as described above 3 days after compound addition.

As shown in Figures 2A and 2B, compound A and compound B overcame the suppressive effects of TNF-α on EPO generation after 2 hours of pretreatment of Hep3B cells with TNF-α. These results showed that compounds and methods according to the present invention are useful for increasing EPO formation in cells treated with TNF-α. These results also suggested that treatment with compounds of the present invention is a useful means of increasing EPO formation and treating anemia in a subject in which EPO formation is suppressed by TNF-α.

Addition of compounds according to the present invention greatly reduced the inhibitory effects of TNF-α on EPO formation. Accordingly, compounds and methods according to the present invention are applicable for the treatment or prevention of anemia associated with elevated TNF-α, for example in inflammatory disorders.

Example 2: Overcoming the suppressive effects of IL-1β on EPO formation

Hep3B cells were treated with different concentrations (0, 0.4, 2, 10 ng/ml) of IL-1 β in the absence or presence of compound A or compound B for 3 days. The secreted EPO level was determined using a commercially available ELISA kit (R&D Systems, catalog no. DEP00). In the absence of compound, treatment of Hep3B cells with IL-1β reduced EPO formation in a dose-dependent manner. Hep3B cells treated with different concentrations of either compound A (Figure 3A) or compound B (Figure 3B) in the absence of IL-1β showed a dose-dependent increase of EPO formation. Addition of any of these compounds in the presence of IL-1β greatly reduced the inhibitory effects of IL-1β on EPO formation. An overcoming of the suppressive effects of IL-1β on EPO formation by inhibition of prolyl hydroxylase was observed in the presence of low (eg 0.4 ng/ml) and high (eg 10 ng/ml) concentrations of IL-1 β. Accordingly, the inhibitory effects of the inflammatory cytokine IL-1β on EPO formation were overcome by inhibiting prolyl hydroxylase activity using the compounds and methods of the present invention. These results suggest that the compounds and methods of the present invention are useful for increasing EPO formation in the presence of the inflammatory cytokine IL-1β. Furthermore, the methods and compounds of the present invention are useful for increasing EPO formation and consequently for treating anemia in an individual, for example in which the individual has a disorder associated with IL-1β, for example acute or chronic inflammation or another anemia associated with chronic disease.

A series of experiments was performed to investigate the effects of compounds of the present invention on EPO formation after treatment of cells with the inflammatory cytokine IL-1β (ie in cells that had already been exposed to IL-1β). Accordingly, in these experiments, IL-1 β signaling would be initiated prior to the addition of a prolyl hydroxylase inhibitor. Hep3B cells were treated with different concentrations (0, 0.4, 2, 10 ng/ml) of IL-1 β for 2 h, after which different concentrations of compound A or compound B were added to the cultured cells. The secreted EPO level was determined as described above 3 days after addition of the compound.

As shown in Figures 4A and 4B, compound A and compound B overcame the suppressive effects of IL-1β on EPO generation after 2 h of pretreatment of Hep3B cells with IL-1β. These results showed that compounds and methods of the present invention are useful for increasing EPO formation in cells exposed to IL-1β. These results also indicated that treatment with compounds of the present invention is a useful means of increasing EPO formation and treating anemia in a subject in which EPO formation is suppressed by IL-1β.

Addition of compounds of the present invention greatly reduced the inhibitory effects of IL-1β on EPO formation. Compounds and methods according to the present invention are consequently applicable for the treatment or prevention of anemia associated with IL-1 β, for example in inflammatory disorders.

Example 3: Inhibition of TNF-α-induced VCAM-1 expression

The endothelial cells' ability to bind lymphocytes is partly due to the expression on the endothelial cells of vascular cell adhesion molecule (VCAM)-1. VCAM-1 expression in endothelial cells is induced by various inflammatory cytokines such as TNF-α. To investigate the effect of HIF-prolyl hydroxylase inhibition on TNF-α-induced VCAM-1 expression, HUVEC (human umbilical vein endothelial cells) were stimulated with TNF-α in the absence or presence of different concentrations of compound B or compound C for 1 day. VCAM expression was then measured.

As shown in Figure 5, TNF-α (1 ng/ml) induced the VCAM-1 expression in HUVEC cells. However, addition of compound B or compound C to TNF-α-stimulated cells led to a dose-dependent inhibition of the TNF-α-induced VCAM-1 expression. These results demonstrated that methods and compounds of the present invention effectively reduce VCAM-1 expression associated with the inflammatory cytokine TNF-α. The results further indicated that compounds and methods according to the present invention are useful for inhibiting VCAM-1 expression associated with various inflammatory diseases and autoimmune diseases such as, for example, anemia associated with chronic disease.

Example 4: Inhibition of IL-1β-induced VCAM-1 expression

VCAM-1 expression in endothelial cells is also induced by the inflammatory cytokine IL-1β. To investigate the effect of HIF-prolyl hydroxylase inhibition on IL-1β-induced VCAM-1 expression, HUVECs (human umbilical vein endothelial cells) were stimulated with IL-1β in the absence or presence of different concentrations of compound B or compound C in 1 day. VCAM expression was then measured.

IL-1β (1 ng/ml) induced VCAM-1 expression in HUVEC cells. However, addition of compound B or compound C to IL-1β-stimulated cells led to a dose-dependent inhibition of the IL-1β-induced VCAM-1 expression (results not shown). These results demonstrated that methods and compounds of the present invention effectively reduce VCAM-1 expression associated with the inflammatory cytokine IL-1β. The results further indicated that compounds and methods according to the present invention are useful for inhibiting VCAM-1 expression associated with various inflammatory diseases and autoimmune diseases such as, for example, anemia associated with chronic disease.

Example 5: Inhibition of TNF-α- and IL-1β-induced VCAM-1 expression on endothelial cells

HUVEC were treated with vehicle control or different concentrations (0, 20, 40, 80 μM) of compound B or compound C for 24 h. The cells were washed and then stimulated with either 1 ng/ml TNF-α or 1 ng/ml IL-1β for 4 hours. VCAM-1 expression on the cell surface was then measured by a cell-based ELISA.

As shown in Figure 25, pretreatment with prolyl hydroxylase inhibitors led to reduced induction of cell surface VCAM-1 expression induced by the inflammatory cytokines TNF-α and IL-1β. These results showed that compounds and methods according to the invention inhibited the inflammatory function of TNF-α and IL-1β and inhibited the expression of adhesion molecules on the cell surface which is necessary to mediate heterocellular leukocyte adhesion. Inhibition of leukocyte adhesion by treatment with the present compounds is an effective means of reducing inflammatory cascades so that inflammation is reduced and the inflammatory effect of limited EPO formation and suppressed erythropoiesis is reduced.

Example 6: Inhibition of TNF-α-induced E-selectin expression

Endothelial E-selectin belongs to the selectin family of cellular adhesion molecules that mediate the initial attachment of leukocytes to vascular endothelial cells during inflammatory situations. IL-1, TNF-α and lipopolysaccharides all induce the expression of E-selectin. (See, for example, Bevilacqua et al. (1987) Proc Natl Acad Sci USA 84: 9238-9242 and Bevilacqua and van Furth (1993) J Leukoc Biol 54: 363-378). To examine the effect of HIF-prolyl hydroxylase inhibition on TNF-α-induced E-selectin expression, HUVEC were stimulated with 1 ng/ml TNF-α in the absence or presence of different concentrations of compound B or compound C for 1 day. The expression of E-selectin and VCAM was then measured.

As shown in Figures 24A and 24B, compound B and compound C showed a dose-dependent inhibition of TNF-α-induced expression of VCAM and E-selectin in HUVEC. The results in Figures 24A and 24B are given as percent inhibition of the expression of VCAM and E-selectin observed in response to different concentrations of compound B (Figure 24A) or compound C (Figure 24B). More than 60% inhibition of the expression of VCAM and E-selectin was observed in HUVEC treated with 50 μM compound B or compound C. These results showed that the methods and compounds of the present invention effectively reduce the expression of VCAM and E-selectin associated with the the inflammatory cytokine TNF-α in endothelial cells. The results further suggested that the compounds and methods of the present invention are useful for inhibiting the expression of VCAM and E-selectin associated with various inflammatory and autoimmune disorders, for example anemia associated with chronic disease. In addition, inhibition of endothelial cell expression of adhesion molecules, including VCAM and E-selectin, by methods and compounds of the present invention, constitutes a means of reducing early events in vascular inflammation.

Example 7: Inhibition of IL-1β-induced E-selectin expression

To examine the effect of HIF-prolyl hydroxylase inhibition on IL-1 β-induced E-selectin expression, HUVEC were stimulated with 1 ng/ml IL-1 β in the absence or presence of different concentrations of compound B or compound C for 1 day. E-selectin expression was then measured.

Compound B and compound C showed a dose-dependent inhibition of IL-1β-induced E-selectin expression in HUVEC (results not shown). These results showed that methods and compounds of the present invention effectively reduce E-selectin expression in endothelial cells associated with the inflammatory cytokine IL-1β. The results further indicated that compounds and methods according to the present invention are useful for inhibiting the E-selectin expression associated with various inflammatory disorders and autoimmune disorders such as, for example, anemia associated with chronic disease. In addition, inhibition of endothelial cell expression of adhesion molecules, including VCAM and E-selectin, by methods and compounds of the present invention, constitutes a means of reducing early events in vascular inflammation.

Example 8: Inhibition of TNF-α-, IL-1β- and IFN-γ-induced E-selectin expression

HUVEC were treated with vehicle control or different concentrations of compound B or compound C for 24 h. The cells were washed and then stimulated with either 1 ng/ml TNF-α, 1 ng/ml IL-1β or a combination consisting of 1 ng/ml each of TNF-α, IL-1β and IFN-γ for 4 hours. The expression of E-selectin on the cell surface was measured by a cell-based ELISA.

As shown in Figure 26, pretreatment of HUVECs with Compound B or Compound C inhibited the induction of cell surface E-selectin expression induced by the inflammatory cytokines TNF-α and IL-1β. In addition, pretreatment with any of these compounds reduced E-selectin expression in the presence of three inflammatory cytokines known to increase E-selectin expression (TNF-α, IL-1β, and IFN-γ). These results showed that the present compounds blocked the inflammatory action of TNF-α, IL-1β and IFN-γ on endothelial cells, exemplified by inhibition of the expression of the cell surface adhesion molecules that mediate the rolling of leukocytes on activated endothelium. Since leukocyte adhesion to activated endothelium via E-selectin is an early step in the continuation of inflammatory cascades, inhibition of leukocyte rolling by inhibition of E-selectin expression provides a means to reduce inflammatory cascades that further limit EPO formation and suppress erythropoiesis.

Example 9: Synergistic increase of EPO formation

Hep3B cells were treated with different concentrations (0, 0.1, 1, 10 ng/ml) of IL-6 in the absence or presence of different concentrations (3 μM, 10 μM, 30 μM) of compound A or compound B in 1 or 3 days. The secreted EPO level was determined using a commercially available ELISA kit (R&D Systems, catalog no. DEP00). In the absence of compound, treatment of Hep3B cells with IL-6 had minimal effect on EPO formation. As shown in Figures 27A and 27B, Hep3B cells treated with IL-6 had a slightly elevated EPO expression compared to untreated cells. Specifically, the EPO level in the control cells was approximately 20 mIU/ml, while the level in cells treated with 10 ng/ml IL-6 was approximately 50 mIU/ml.

Hep3B cells treated with compound A or compound B without IL-6 showed elevated EPO level in a dose-dependent manner. However, Hep3B cells treated with compound A or compound B in the presence of IL-6 showed a significant increase of the EPO level (see Figures 27A and 27B). The effect of compound treatment on EPO formation in the presence of IL-6 was synergistic. For example, Hep3B cells treated with 10 ng/ml IL-6 showed an EPO level of approximately 50 mIU/ml. Treatment of Hep3B cells with 10 mM compound A or compound B in the absence of IL-6 resulted in approximately 60 mIU/ml EPO and 220 mIU/ml EPO, respectively. In the presence of 10 ng/ml IL-6, addition of compound A and compound B produced an EPO level elevated to approximately 270 mIU/ml and greater than 400 mIU/ml, respectively.

Thus, compounds of the present invention acted synergistically with IL-6 to induce EPO expression in hepatocytes.

Example 10: Overcoming cytokine-induced suppression of EPO receptor signaling

The cell line TF-1 (human erythroleukemia; ATCC catalog designation CRL-2003) is stimulated to proliferate in response to the addition of EPO. In the presence of various proinflammatory cytokines, the EPO-mediated increase of TF-1 cell proliferation is attenuated. To determine the effects of prolyl hydroxylase inhibition on TF-1 cell proliferation, TF-1 cells are treated with different concentrations of the proinflammatory cytokines IL-1 β, TNF- α or IFN- γ in the absence or presence of prolyl hydroxylase inhibitors, and the EPO-mediated cell proliferation is measured as follows. Triplicate wells of cells grown in 96-well microtiter plates are incubated with serum-free medium in the absence or presence of EPO for 24 hours. During the last 4 hours of culture, 1 μCi of tritiated thymidine (<3>H-TdR; Amersham) is added to each well. The response of cells to EPO receptor signaling is determined by measuring cell proliferation.

Cell proliferation is measured by quantifying the amount of <3>H-TdR that is incorporated into the cells by first lysing the cells with water and then capturing the DNA on nylon filters in a cell harvester.

Alternatively, single cell suspensions of spleen cells obtained from phenylhydrazine-treated animals are used, which leads to an increased occurrence of EPO-responsive precursor cells in the spleen, as a source for EPO-responsive cells. EPO-mediated proliferation is then measured ex vivo as described above.

Treatment of TF-1 cells with EPO leads to increased cell proliferation, as determined by increased incorporation of tritiated thymidine. Addition of the proinflammatory cytokines IL-1β, TNF-α or IFN-γ to EPO-treated TF-1 cells leads to a reduced ability to respond to EPO, leading to reduced cell proliferation. The effect of addition of the present compounds on the inhibitory effects of proinflammatory cytokines on EPO-mediated cell proliferation in TF-1 cells is determined. Increased cell proliferation, as measured by increased incorporation of tritiated thymidine, in TF-1 cells treated with EPO and proinflammatory cytokines demonstrates that compounds and methods of the present invention overcome the suppressive effects of proinflammatory cytokines on the EPO-mediated increase in cell proliferation.

Example 11: Elevated expression of transferrin receptor

The effect of compounds according to the invention on the expression of transferrin receptor was investigated as follows. Different cells (Hep3B, HepG2, HK-2) were incubated with compound A or compound B for 1 day. The cells were then analyzed for transferrin receptor expression by FACS analysis and using the antibody CD71-PE (Ancell, catalog no. 223-050). The results are shown below in Table 1.

TABLE 1

As shown above in Table 1, addition of various compounds of the present invention to cells resulted in increased expression of transferrin receptor.

Inhibition of HIF prolyl hydroxylation using prolyl hydroxylase inhibitors according to the present invention gave increased expression of transferrin receptor in the cells. Elevated expression of transferrin receptor using prolyl hydroxylase inhibitors according to the present invention was observed in liver cells (eg Hep3B, HepG2), kidney cells (eg HK-2) and lymphocytes (eg THP-1). Accordingly, methods and compounds of the present invention are useful for increasing transferrin receptor expression in various cell types. In addition, increased expression of transferrin receptor will lead to increased transferrin receptor-mediated endocytosis of transferrin with iron(III) so that the transport, utilization, storage and metabolism of iron increases. Accordingly, methods and compounds of the present invention are useful for increasing erythropoiesis by providing increased transport, utilization, storage and metabolism of iron.

Example 12: Increased expression of transferrin receptor and increased iron uptake in vitro

The effect of compounds on iron uptake into cells is determined as follows. Primary monocytes and macrophages and monocyte and macrophage cell lines (eg THP-1) are treated for one, two or three days with different concentrations of prolyl hydroxylase inhibitors. The cells are then examined for the presence of transferrin receptor on the cell surface using fluorescence immunostaining and flow cytometry. Results showing that the addition of prolyl hydroxylase inhibitors gives increased expression of transferrin receptor on the cell surface, show the effect of prolyl hydroxylase inhibition in terms of increased transferrin binding and consequently iron binding to cells. An altered iron uptake in cells treated with prolyl hydroxylase inhibitors is determined as follows. The cells are treated with compound in the presence of<59>Fe. Increased iron uptake by cells treated with prolyl hydroxylase inhibitors is determined by measuring cell-associated<59>Fe. An increased amount of cell-associated<59>Fe indicates elevated iron uptake in the cells.

Example 13: Increased level and activity of iron regulatory protein 2

The regulation of the uptake, storage and utilization of iron occurs in part by the expression and activity of key proteins that participate in iron metabolism, including trans-acting proteins known as iron regulatory proteins (IRPs). IRP-1 and IRP-2 control stability and translation of mRNA by binding to specific iron-responsive elements in different mRNAs for proteins that participate in iron metabolism so that almost all aspects of iron metabolism are affected.

Iron deficiency results in increased IRP activity, which leads to elevated expression of transferrin receptor and reduced ferritin expression. Likewise, IRP activity decreases in the presence of iron, leading to reduced transferrin receptor expression and elevated ferritin expression.

In order to investigate the effect of the present compounds on different aspects of iron metabolism, the following experiment is performed. The mouse Hepa-1 cells are treated with prolyl hydroxylase inhibitors for up to 48 hours. The cells are then harvested and cell lysates are analyzed for IRP-2 expression by immunoblotting using an antibody specific for IRP-2 (Alpha Diagnostic International, Inc., San Antonio, TX). Results showing elevated levels of cytoplasmic IRP-2 after addition of compound demonstrate that the methods and compounds of the present invention are useful for providing an elevated IRP level and consequently elevated iron metabolism.

The effect of compounds according to the invention on IRP-2 activity, as measured by altered ferritin and transferrin expression, is determined as follows. The mouse macrophage cell line RAW 264.1 is treated with prolyl hydroxylase inhibitors for up to 48 hours. The cells are then harvested and analyzed for ferritin and transferrin protein expression by immunoblotting (ADI, catalog no. IRP21-S). Reduced level of ferritin expression and increased level of transferrin expression after prolyl hydroxylase inhibition show that methods and compounds according to the present invention are useful for stabilizing and increasing the activity of IRP-2. Increased expression of IRP-2 results in reduced expression of ferritin, which is responsible for the long-term storage of iron, and results in increased expression of transferrin and transferrin receptor, which facilitates the uptake, transport and utilization of iron and consequently results in increased erythropoiesis. By increasing the expression and activity of IRP-2, the methods and compounds of the present invention are useful for reducing the expression of ferritin and the long-term storage of iron associated with ferritin and increased expression of transferrin and transferrin receptor. Accordingly, the methods and compounds of the present invention are useful for providing increased uptake, transport and utilization of iron and are therefore useful for increasing erythropoiesis.

Example 14: Improved utilization of iron

Rats are administered either vehicle control or HIF-prolyl hydroxylase inhibitor before intravenous injection of<59>Fe-labeled ferric citrate (Amersham). Serial blood samples are drawn from the tail vein and total free radioactivity in plasma and erythrocyte-associated radioactivity are measured in a scintillation counter to detect transport of iron and incorporation into heme in the erythrocytes and hemoglobin synthesis. An elevated amount of erythrocyte-associated <59>Fe shows that the present compounds are useful for enhancing the utilization of iron necessary for heme synthesis, hemoglobin formation and erythropoiesis.

Example 15: Elevated expression of erythropoiesis genes in vitro

Hep3B cells (ATCC No. HB-8064) were grown in DMEM supplemented with 8% fetal calf serum. Hep3B cells were seeded in 6-well culture plates with �500,000 cells per well. After 8 hours, the medium was replaced with DMEM supplemented with 0.5% fetal calf serum and the cells were incubated for a further 16 hours. Compound B or compound D was added to the cells (final concentration 25 μM) and the cells were incubated for different periods of time. Control cells (not treated with compound, only addition of DMSO alone) were harvested after 0, 6 and 48 hours. The harvested cells were analyzed for cell viability (GUAVA) or added to RNA extraction buffer (RNeasy, Qiagen) and stored at -20 ºC for subsequent RNA purification. Replicate microarrays were prepared using RNA isolated from replicate experiments performed on different days. Total RNA was isolated from the cells using the RNeasy kit (Qiagen).

RNA was precipitated in 0.3 M sodium acetate (pH 5.2), 50 ng/ml glycogen and 2.5 volumes of ethanol for one hour at -20 ºC. The samples were centrifuged and centrifuged material was washed with cold 80% ethanol, dried and resuspended in water. Double-stranded cDNA was synthesized using a T7-(dT)24 first-strand primer (Affymetrix, Inc., Santa Clara, CA) and the SUPERSCRIPT CHOICE system (Invitrogen) according to the manufacturer's instructions. The final cDNA was extracted with an equal volume of 25:24:1 phenol:chloroform:isoamyl alcohol using a PHASE LOCK GEL insert (Brinkman, Inc., Westbury, NY). The aqueous phase was collected and cDNA precipitated using 0.5 volumes of 7.5 M ammonium acetate and 2.5 volumes of ethanol.

Alternatively, cDNA was purified using the GENECHIP sample purification module (Affymetrix) according to the manufacturer's instructions.

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

A hybridization mixture was prepared by diluting 5 μg probe to 100 μl with 1x hybridization buffer (100 mM MES, 1 M [Na<+>], 20 mM EDTA, 0.01% Tween 20), 100 μg/ml herring milk DNA, 500 μg/ml acetylated BSA, 0.03 nM control oligonucleotide B2 (Affymetrix) and 1x GENECHIP eukaryotic hybridization control (Affymetrix). The mixture was first incubated at 99 ºC for 5 minutes, then at 45 ºC for 5 minutes and then centrifuged for 5 minutes. The matrix "The Human Genome 133A" (Affymetrix) was warmed to room temperature and then prehybridized with 1x hybridization buffer at 45 ºC for 10 minutes while rotating. The buffer was then replaced with 80 μl of hybridization mixture and the matrix was hybridized for 16 h at 45 ºC at 60 rpm with a counterweight. After the hybridization, the arrays were washed once with 6x SSPE, 0.1% Tween 20 and then washed and stained using R-phycoerythrin-conjugated streptavidin (Molecular Probes, Eugene, OR), goat antistreptavidin antibody (Vector Laboratories, Burlingame, CA) and a GENECHIP Fluidics Station 400 instrument (Affymetrix) according to the manufacturer's micro_1v1 procedure (Affymetrix). The arrays were analyzed using a GENEARRAY scanner (Affymetrix) and the Microarray Suite software (Affymetrix).

The matrix "The Human Genome 133A" (Affymetrix) represents all sequences in the human unigene database version 133 (National Center for Biotechnology Information, Bethesda, MD) and comprises approximately 14,500 well-characterized human genes.

The RNA quality was analyzed by capillary electrophoresis (Agilent Bioanalyzer). Hybridization mixes were prepared as described (Affymetrix) and hybridized to human U133A arrays from Affymetrix comprising 22283 probe sets. Array performance was analyzed with the Affymetrix MicroArray Suite (MAS) software and individual probe sets were designated as "present", "marginal" and "absent" based on the software's default parameters. Statistical analysis and filtered probe set lists were generated using GeneSpring software (Silicon Genetics). The cutoff for "expressed" probe sets used a combination of Affymetrix "P" values and absolute expression values derived from the inherent data error model in Genespring. The values were normalized relative to average control samples.

As shown in Table 2 below, the expression of genes (fold increase of mRNA level relative to control) encoding erythropoietic proteins was elevated in Hep3B cells treated with compound of the present invention. (Two data points for ceruloplasmin for each condition are provided below in Table 2).

More specifically, the expression of ceruloplasmin and transferrin receptor 2 was elevated in Hep3B cells treated with various compounds of the present invention.

TABLE 2

Example 16: Dosage for animals

Animals used in the following examples include male Swiss Webster mice (30-32 g), male Sprague-Dawley rats (200-350 g), and female Lewis rats, obtained from Simonsen, Inc. (Gilroy, CA), Charles River (Hollister, CA ) or Harlan. The animals were housed using standard procedures and feed and water were available to the animals ad libitum. During treatment, the animals were analyzed for changes in body weight and signs of obvious toxicity and mortality.

The compounds were generally administered orally by gavage or by intravenous administration. The animals treated by oral gavage were given a volume of 4-10 ml/kg of either 0.5% carboxymethylcellulose (CMC; Sigma-Aldrich, St. Louis, MO) with or without 0.1% Polysorbate 80 (0 mg /kg/day) or varying doses of a compound according to the present invention (for example a HIF-prolyl hydroxylase inhibitor) in 0.5% CMC with or without 0.1% Polysorbate 80 using different dosing schedules. Blood samples were collected at suitable intervals during the treatment from, for example, the tail vein (rats), from the abdominal vein or by cardiocentesis (mice or rats). In general, animals were anesthetized with isoflurane and blood samples were collected in MICROTAINER serum separation tubes (Becton-Dickinson, Franklin Lakes, NJ). For measurement of serum constituents, the tubes were incubated at room temperature for 30 minutes and then centrifuged at 8000 rpm at 4 ºC for 10 minutes. The serum fraction was then processed and analyzed for the presence of specific constituents, such as iron (analysis performed by Quality Clinical Labs, Mountain View, CA). For determination of hematocrit, blood samples were collected in MICROTAINER EDTA-2K tubes (Becton-Dickinson); EDTA blood was then drawn into 75 mm x 1.1-1.2 mm LD capillary tubes (Chase Scientific Glass, Inc., Rockwood, TN) to approximately 3⁄4 of the length, one end of which was sealed with CRITOSEAL sealant (Sherwood Medical Company), and the tubes were centrifuged in a J-503M MICROHEMATOCRIT centrifuge (Jorgensen Laboratories, Inc., Loveland, CO) at 12,000 rpm for 5 min. The hematocrit was read against a reading card. Where indicated, a complete blood count (CBC), including blood hemoglobin level, reticulocyte count, and hematocrit, was performed by Quality Clinical Labs (Mountain View, CA).

At the end of each study, the animals were euthanized, for example by drawing blood under general anesthesia or by CO2 suffocation, and organ samples and tissue samples were collected. The tissues were either fixed in neutral buffered formalin or stored frozen at -70 ºC. Tissue for genomic analysis was placed in RNAlater.

Example 17: Elevated expression of genes encoding iron processing protein in vivo

Male Swiss Webster mice were treated as described above with a single dose of 0.5% CMC (Sigma-Aldrich) (0 mg/kg) or 100 mg/kg Compound A. 4, 8, 16, 24, 48, or 72 h after administration, the animals were anesthetized and killed, and tissue samples from kidney, liver, brain, lung and heart were isolated and stored in RNALATER solution (Ambion) at -80 ºC. Alternatively, the animals were treated with 4 consecutive daily doses of 0.5% CMC (0 mg/kg/day), 7.5 mg/ml compound A in 0.5% CMC (30 mg/kg/day) or 25 mg/ml compound in 0.5% CMC (100 mg/kg/day). Four hours after administration of the last dose, the animals were anesthetized and euthanized and approximately 150 mg of liver and each kidney were isolated and stored in RNALATER solution (Ambion) at -20 ºC.

RNA isolation was performed using the following procedure. A piece of each organ was cut into pieces, 875 μl of RLT buffer (RNEASY kit; Qiagen Inc., Valencia, CA) was added, and the pieces were homogenized for approximately 20 seconds using a rotor-stator POLYTRON homogenizer (Kinematica, Inc., Cincinnati, OH). The homogenate was microcentrifuged for 3 min to decant insoluble material, the supernatant was transferred to a new tube and RNA was isolated using an RNEASY kit (Qiagen) according to the manufacturer's instructions. RNA was eluted in 80 μl of water and quantified with RIBOGREEN reagent (Molecular Probes, Eugene, OR). The absorbance at 260 and 280 nm was measured to determine purity and concentration of RNA.

Alternatively, the tissue samples were cut and homogenized in TRIZOL reagent (Invitrogen Life Technologies, Carlsbad, CA) using a rotor-stator POLYTRON homogenizer (Kinematica). The homogenates were warmed to room temperature, 0.2 volumes of chloroform were added and the samples were thoroughly mixed. The mixtures were incubated at room temperature for several minutes and then centrifuged at 12,000 g for 15 minutes at 4 ºC. The aqueous phase was collected and 0.5 volumes of isopropanol was added. The samples were mixed, incubated at room temperature for 10 minutes and centrifuged for 10 minutes at 12000g at 4 ºC. The supernatant was removed and centrifuged material was washed with 75% EtOH and centrifuged at 7500g for 5 minutes at 4 ºC. The absorbance at 260 and 280 nm was measured to determine purity and concentration of RNA.

RNA was precipitated in 0.3 M sodium acetate (pH 5.2), 50 ng/ml glycogen and 2.5 volumes of ethanol for one hour at -20 ºC. The samples were centrifuged and centrifuged material was washed with cold 80% ethanol, dried and resuspended in water. Double-stranded cDNA was synthesized using a T7-(dT)24 first-strand primer (Affymetrix, Inc., Santa Clara, CA) and the SUPERSCRIPT CHOICE system (Invitrogen) according to the manufacturer's instructions. The final cDNA was extracted with an equal volume of 25:24:1 phenol:chloroform:isoamyl alcohol using a PHASE LOCK GEL insert (Brinkman, Inc., Westbury, NY). The aqueous phase was collected and the cDNA was precipitated using 0.5 volumes of 7.5 M ammonium acetate and 2.5 volumes of ethanol. Alternatively, cDNA was purified using the GENECHIP sample purification module (Affymetrix) according to the manufacturer's instructions.

Biotin-labeled cRNA was synthesized from cDNA in an in vitro translation (IVT) reaction using a BIOARRAY HighYield RNA Transcript Labeling Kit (Enzo Diagnostics, Inc., Farmingdale, NY) according to the manufacturer's instructions. The final labeled product was purified and fragmented using the GENECHIP sample purification module (Affymetrix) according to the manufacturer's instructions.

A hybridization mixture was prepared by diluting 5 μg probe to 100 μl in 1x hybridization buffer (100 mM MES, 1 M [Na<+>], 20 mM EDTA, 0.01% Tween 20), 100 μg/ml herring milk DNA, 500 μg/ml acetylated BSA, 0.03 nM control oligonucleotide B2 (Affymetrix) and 1x GENECHIP eukaryotic hybridization control (Affymetrix). The mixture was first incubated at 99 ºC for 5 minutes and then at 45 ºC for 5 minutes and then centrifuged for 5 minutes. The mouse genome array MOE430Aplus2 (Affymetrix) was warmed to room temperature and then prehybridized with 1x hybridization buffer at 45 ºC for 10 minutes while rotating. The buffer was then replaced with 80 μl of hybridization mixture and the matrix was hybridized for 16 hours at 45 ºC and 60 rpm with counterweight. After the hybridization, the arrays were washed once with 6x SSPE, 0.1% Tween 20 and then washed and stained using R-phycoerythrin-conjugated streptavidin (Molecular Probes, Eugene, OR), goat antistreptavidin antibody (Vector Laboratories, Burlingame, CA) and a GENECHIP Fluidics Station 400 instrument (Affymetrix) according to the manufacturer's EukGE-WS2v4 procedure (Affymetrix). The arrays were analyzed using a GENEARRAY scanner (Affymetrix) and the Microarray Suite software (Affymetrix).

The mouse genome array MOE430Aplus2 (Affymetrix) represents all sequences in the UniGene mouse database version 107 (National Center for Biotechnology Information, Bethesda, MD) including approximately 14,000 well-characterized mouse genes.

Table 3 below shows the ceruloplasmin mRNA expression in mouse kidney after administration of compound A. The results were normalized relative to the mean value observed in untreated control animals.

TABLE 3

The results as shown in Table 3 above showed that the methods and compounds of the present invention are useful for increasing the expression of the ceruloplasmin gene. Ceruloplasmin, also called ferroxidase-1, transfers reduced iron released from storage sites (such as ferritin) to the oxidized form.

Oxidized iron can bind to the plasma transport protein transferrin.

Ceruloplasmin deficiency is associated with accumulation of iron in the liver and other tissues. Available material suggests that ceruloplasmin promotes iron efflux from the liver and iron influx into iron-deficient cells (see, for example, Tran et al. (2002) J Nutri 132: 351-356).

Table 4 below shows the hepcidin mRNA expression in mouse liver after administration of compound A. The results were normalized relative to the level observed in untreated control animals.

TABLE 4

As shown above in Table 4, administration of compound A led to decreased expression of hepcidin mRNA in mouse liver. Reduced hepcidin expression is associated with increased release of iron from reticuloendothelial cells and increased absorption of iron in the gut. Accordingly, methods and compounds of the present invention are useful for reducing hepcidin expression and increasing intestinal iron absorption.

Figure 6A shows relative expression levels of the transferrin receptor (gray bars) in the kidney and the duodenal iron transporter NRAMP2 (natural resistance-associated macrophage protein 2) (also termed Slc11a2 (soluble carrier family 11, proton-coupled divalent metal ion transporter, member 2), alternatively termed DCT1 (divalent cation transporter 1), DMT1 (divalent metal transporter 1)) in intestine (black bars).

In another experiment, mRNA was isolated from small intestine harvested 4 hours after intravenous administration of 60 mg/kg compound A, compound B and compound C to mice. Probes were prepared from each of two animals from 5 treatment groups and hybridized to the mouse microarrays MOE430Aplus2 from Affymetrik (one animal per array). A statistical comparison of results obtained from matrices from treated animals and matrices from untreated animals was performed. Figure 6B shows the relative expression level of NRAMP2 mRNA in the small intestine of animals treated with compound A, compound B and compound C. The expression level is shown as the number of fold induction above the level in untreated control animals for each expressed gene. The results of these experiments showed that the methods and compounds of the present invention are useful for increasing the expression of NRAMP2 in the intestine. These results further indicated that methods and compounds according to the present invention are useful for increasing iron absorption so that the availability of iron for heme synthesis, hemoglobin synthesis, red blood cell formation and erythropoiesis is increased.

Figure 6C shows the number of folds of induction of 5-aminolevulinic synthase (ALAS-2) expression in treated animals compared to the vehicle-only animals. The results showed that treatment of normal animals with prolyl hydroxylase inhibitors led to elevated expression of genes involved in iron metabolism, including genes involved in absorption of iron from the intestine and transport of iron to peripheral tissues via transferrin receptors. The expression of these genes returned to the basal level (control) 16 hours after dosing. The results also showed a coordinated expression of ALAS-2, the first enzyme in the heme synthesis pathway and the rate-limiting enzyme for heme synthesis in the indicated tissues after treatment with prolyl hydroxylase inhibitor. Taken together, these results showed that compounds of the present invention produced a coordinated increase in the expression of genes encoding proteins involved in the promotion of erythropoiesis, including iron absorption, iron transport and heme synthesis.

Alternatively, flow cytometry analysis is used to measure the macrophage cell surface marker CD11c and the transferrin receptor level in immunostained mononuclear cells from peripheral blood. The activity of compound treatment is demonstrated by demonstrating elevated expression of transferrin receptor on macrophages. Furthermore, plasma can be collected and the transferrin level measured using a commercially available ELISA kit (see for example KomaBiotech, Korea).

Example 18: Elevated erythropoiesis in vivo

The effect of administration of the present compounds on erythropoiesis is determined as follows. Normal mice are rendered anemic and maintained in an anemic state by chronic administration of TNF-α, a treatment known to inhibit erythropoiesis due to lack of EPO formation and EPO signaling in response to TNF-α. After induction of anemia over a period of one to four weeks, the animals are administered prolyl hydroxylase inhibitors. The tissues are examined for formation of BFU-E and CFU-E and blood samples are analyzed for composition.

Results showing increased numbers of BFU-E and CFU-E in bone marrow, spleen and peripheral tissues and/or elevated hemoglobin level in serum, increased number of reticulocytes and elevated hematocrit in animals treated with PHI demonstrate efficacy.

Another experimental animal model has been used to investigate the effect of administration of prolyl hydroxylase inhibitors on erythropoiesis. In this model, transgenic mice developed anemia associated with chronic disease as a result of constitutive overexpression of TNF-α. After the onset of anemia in these mice, prolyl hydroxylase inhibitors are administered over different periods of time and using different dosing strategies. Samples of tissue and blood are then collected and analyzed. As described above, results showing increased numbers of PFU-E and CFU-E in bone marrow, spleen and peripheral tissues and/or elevated hemoglobin level in serum, increased number of reticulocytes and elevated hematocrit will effectively demonstrate that anemia associated with TNF-α overproduction in transgenic animals are treated by administration of prolyl hydroxylase inhibitors using methods and compounds of the present invention.

Example 19: Elevated iron level in serum

Male and female mice were treated twice weekly (Monday and Thursday) with different concentrations (0, 20, 60 or 150 mg/kg) of compound A for 93 days. The total serum iron level was determined.

TABLE 5

As shown in Table 5, administration of compound A increased serum iron levels in both male and female rats. (The results in Table 5 are given as iron level in serum /- standard deviation. * indicates a significant difference in the iron level in serum compared to untreated animals). These results showed that methods and compounds according to the present invention are useful for increasing iron levels in serum and are consequently useful for treating disorders associated with iron deficiency.

Example 20: Effect in an animal model for anemia associated with chronic disease/impaired erythropoiesis/impaired iron metabolism

Anemia associated with chronic disease (ACD) is associated with various inflammatory conditions including arthritis, neoplastic disease and other disorders associated with chronic inflammation. A rat model of ACD was used to investigate the effects of HIF stabilization using the methods and compounds of the present invention for the treatment of anemia associated with chronic disease. In this animal model, ACD is induced in rats by peptidoglycan polysaccharide polymers. (See, for example, Sartor et al. (1989) Infection and Immunity 57: 1177-1185). In this model, the animals develop severe acute anemia in the initial stages followed by moderately severe chronic microcytic anemia in the later stages.

Animal model for ACD - experimental series 1:

Female Lewis rats with a body weight of approximately 160 grams were administered PG-PS 10S (Lee Laboratories, 15 μg/g body weight, intraperitoneally). PG-PS 10S contains purified peptidoglycan polysaccharide polymers isolated from the cell wall of Streptococcus pyogenes, group A, strain D58. Arthritis and anemia were allowed to develop for 35 days. On day 35, blood samples (approximately 400 μl) were drawn from the tail vein under general anesthesia (isoflurane) for CBC and reticulocyte count (performed by Quality Clinical Labs). Animals with a centrifuged hematocrit level of 45% or higher were considered non-anemic and were removed from the study.

On day 35 after the PG-PS injection, anemic animals were treated with vehicle alone or with compound A (60 mg/kg, PO) on two consecutive days per week for two weeks. Automated complete blood counts (CBC) were measured on days 35 (see above), 39, 42 and 49; and serum iron levels were measured on day 49.

Reticulocyte count

As shown in Figure 7, administration of compound A to anemic animals resulted in an increased reticulocyte count on day 39 (ie, 5 days after initiation of compound administration). The reticulocyte level was approximately 2% and 4% of the number of red blood cells in control animals (not anemic) and anemic animals (PG-PS-treated) respectively. However, the reticulocyte level in treated animals was approximately 10% of the number of red blood cells.

Treatment with compound A produced an increase in the reticulocyte count in anemic animals. Accordingly, compound A stimulated erythropoiesis in a rat model of ACD.

Hematocrit

The hematocrit level was elevated in anemic animals treated with compound A.

The hematocrit level (measured in a Baker 9000 instrument at Quality Clinical Labs) in anemic animals (PG-PS treated) was lower than 35% compared to 41% in non-anemic control animals (see Figure 8). Administration of compound A to anemic animals produced an increase in the hematocrit level to approximately 37% already 5 days after starting treatment with the compound. After a second dose of Compound A, the hematocrit level was elevated to approximately 40%, close to the hematocrit level observed in non-anemic control animals. Compound A produced increased hematocrit in anemic animals using a rat model of ACD. Accordingly, the methods and compounds of the present invention are useful for raising the hematocrit and treating anemia associated with chronic disease.

Hemoglobin

Administration of compound A also produced an increase in hemoglobin levels in anemic animals. As shown in Figure 9, non-anemic control animals at day 35 had a hemoglobin level of approximately 15 g/dl, while the hemoglobin level in PG-PS treated animals (ie, anemic animals) was approximately 13 g/dl. As shown in Figure 9, compound A produced an increase in hemoglobin levels in anemic animals as early as 5 days (day 39) after compound administration. The hemoglobin level was still elevated on day 49 and reached a level corresponding to the level of non-anaemic control animals, showing that a compound of the present invention restored a normal hemoglobin level in anemic animals. These results showed that compound A produced an increase in hemoglobin levels in anemic animals using a rat model of ACD. Accordingly, methods and compounds of the present invention are useful for increasing hemoglobin levels and treating anemia associated with chronic disease.

Number of red blood cells

Administration of compound A produced an increased number of red blood cells in anemic animals. As shown in Figure 10, red blood cell counts were elevated in anemic animals treated with Compound A as early as 5 days after the start of compound administration (ie, day 39 in Figure 10) compared to untreated anemic animals.

Compound A produced an increase in the number of red blood cells in anemic animals using a rat model of ACD. Accordingly, methods and compounds of the present invention are useful for increasing the number of red blood cells and treating anemia associated with chronic disease.

Average volume of blood cells

Anemic animals showed a reduced mean volume of blood cells compared to non-anemic control animals (see Figure 11). Anemic animals treated with compound A showed an elevated mean volume of the blood cells as early as 5 days after treatment (day 39 in Figure 11) compared to untreated anemic animals. The mean blood cell volume in treated animals remained elevated compared to untreated anemic animals throughout the experiment. These results showed that compound A improved (that is, reduced) the level of microcytosis (that is, microcythemia, the presence of many microcytes, abnormally small red blood cells associated with various forms of anemia). Accordingly, methods and compounds of the present invention improve/reduce microcytosis in anemia associated with chronic disease.

Average hemoglobin level in blood cells

Anemic animals also showed a reduced mean hemoglobin level in the blood cells. As shown in Figure 12, treatment of anemic animals with compound A produced an increase in the mean hemoglobin level in the blood cells compared to untreated anemic animals. These results showed that the methods and compounds of the present invention are useful for increasing the average hemoglobin level in the blood cells.

Animal model for ACD - experimental series 2:

Female Lewis rats (approximately 150-200 g) were injected with PG-PS (intraperitoneally). Arthritis and anemia were allowed to develop for 28 days. The animals were administered compound A by oral gavage twice weekly (Monday and Thursday) for six weeks, corresponding to days 28, 31, 35, 38, 42, 45, 49, 52, 56, 59, 63, 66 and 70 after PG- PS injection.

Whole blood was collected via the tail vein for CBC analysis on days 28, 42, 56 and 70. In addition, serum was collected on day 70 for iron binding analysis. CBC and iron binding assay were performed by Quality Clinical Labs (Mountain View, CA).

Hematocrit

The hematocrit level was reduced in animals 28 days after treatment with PG-PS. Figure 13 shows that animals injected with PG-PS were anemic and had a hematocrit that was 85% of the level in untreated (ie, non-anaemic) animals. (Week 0 in Figure 13 corresponds to day 28 in this experimental setup). Untreated (ie, non-anaemic) animals treated with Compound A (40 mg/kg) showed an increase in hematocrit level with time, up to more than 110% of the level in untreated animals. As shown in Figure 13, administration of Compound A to anemic animals resulted in elevated hematocrit levels.

Hemoglobin

Administration of compound A produced an increase in hemoglobin levels in both anemic and non-anaemic animals. As shown in Figure 14, the hemoglobin level in non-anaemic animals treated with Compound A (40 mg/kg) increased to approximately 110% of the level in untreated control animals. (Week 0 in Figure 14 corresponds to day 28 in this experimental setup). In anemic animals, hemoglobin levels increased with twice weekly administrations of 10 mg/kg, 20 mg/kg, or 40 mg/kg Compound A. Hematocrit levels continued to increase for at least 4 weeks.

Number of red blood cells

Anemic animals had a lower number of red blood cells than non-anaemic animals.

Specifically, the number of red blood cells in anemic animals was less than 90% of the level observed in non-anemic animals at day 28 after PG-PS injection. As shown in Figure 15, the number of red blood cells in anemic animals treated with compound A increased compared to untreated animals. (Week 0 in Figure 15 corresponds to day 28 in this experimental setup). An increased number of red blood cells was observed 2 weeks after compound administration, and the number continued to increase throughout the 6 weeks the experiment lasted.

Average volume of blood cells

Anemic animals showed a reduced mean volume of the blood cells compared to non-anaemic (untreated) animals. As shown in Figure 16, mean blood cell volume continued to decrease over time in animals treated with PG-PS, demonstrating that anemia associated with chronic disease led to microcytic anemia (characterized in part by lower red blood cell counts and smaller red blood cells) and inability to form hemoglobin due to lack of access to iron stores. (Week 0 figure 16 corresponds to day 28 in this experimental setup). Administration of Compound A to anemic animals resulted in a reduction of the decline in mean blood cell volume. Accordingly, inhibition of prolyl hydroxylase using the compounds and methods of the present invention effectively reduced the decline in mean blood cell volume associated with anemia associated with chronic disease and anemia associated with iron deficiency, such that the mean blood cell volume was restored, the mean blood cell volume was maintained, etc. These results further demonstrated that the methods and compounds of the present invention are useful for increasing the availability of iron from stores for use in hemoglobin formation.

Average hemoglobin level in blood cells

Anemic animals had a reduced mean hemoglobin level in their blood cells compared to control animals, showing that anemia associated with chronic disease affected hemoglobin formation. As shown in Figure 17, anemic animals administered Compound A showed a reduction in the decrease in the mean hemoglobin level in the blood cells with time. (Week 0 in Figure 17 corresponds to day 28 in this experimental setup).

Iron status - serum iron level and transferrin saturation

Patients with anemia associated with chronic disease are characterized clinically by reduced plasma iron concentration and reduced transferrin saturation. The effect of the present compounds on serum iron levels and transferrin saturation in normal and anemic animals was determined. Using an animal model of anemia associated with chronic disease, anemia was induced in rats by intraperitoneal injection of peptidoglycan polysaccharide polymers as described above. Arthritis and anemia were allowed to develop for 28 days. The animals were then treated with different concentrations of compound A twice weekly for 6 weeks. Serum iron levels and transferrin saturation were determined by Quality Clinical Labs.

As shown in Figure 18A, anemic animals (PG-PS) had a lower serum iron level than non-anemic animals (mock-treated). Administration of compound A led to elevated serum iron levels, both in anemic (PG-PS) animals and in non-anaemic control (sham) animals. Animals treated with compound A had higher transferrin saturation than untreated non-anaemic animals and untreated anemic animals (see Figure 18B). These results demonstrated that the methods and compounds of the present invention are useful for increasing serum iron levels and percent transferrin saturation.

Iron absorption

At week 6 after administration of compound A to anemic animals (40 mg/kg twice weekly), a microarray analysis was performed to examine the expression of genes encoding proteins involved in intestinal iron transport and absorption.

The microarray analysis was performed using methods described above using the rat genome array 230A (Affymetrix) representing all sequences in the Unigene rat database version 99 (National Center for Biotechnology Information, Bethesda, MD), including approximately 4699 well-characterized rat genes, approximately 10467 ESTs -sequences and approximately 700 non-EST sequences.

As shown in Figure 19, administration of Compound A to control animals led to increased intestinal expression of mRNA for NRAMP2 (open bars) and sproutin (filled bars). Untreated anemic animals (PG-PS) had reduced mRNA expression levels for both NRAMP2 and sproutin. These results showed that anemia associated with chronic disease is associated with reduced expression of proteins involved in the absorption of iron. However, anemic animals treated with compound A showed elevated expression of both NRAMP2 and sproutin in the gut (Figure 19). These results demonstrated that the methods and compounds of the present invention are useful for increasing the expression of genes associated with the transport and absorption of iron. In addition, these results indicated that compounds of the present invention provide increased absorption and transport of iron in healthy individuals and in individuals with anemia associated with chronic disease.

Example 21: Elevated erythropoiesis in humans

The effect of prolyl hydroxylase inhibition on human erythropoiesis was investigated as follows. An oral dose of 20 mg/kg of compound A was administered either twice or three times weekly for four weeks to healthy volunteer subjects. At various times after compound administration, blood was drawn for analysis of EPO, hemoglobin, hematocrit, red blood cell count, soluble transferrin receptor, and serum ferritin level.

Reticulocyte count

As shown in Figure 20, administration of Compound A to humans resulted in a reticulocyte count higher than that of the placebo control subjects. An elevated reticulocyte count occurred in subjects administered the compound twice or thrice weekly. The reticulocyte count increased to more than approximately 1.7% of the red blood cell count in treated subjects, compared to a level of approximately 1.4% in untreated subjects. Administration of compound A produced an increased reticulocyte count in humans. Accordingly, methods and compounds of the present invention are useful for increasing erythropoiesis and thereby increasing reticulocyte levels.

Hematocrit

The hematocrit level was elevated in humans treated with Compound A. In humans administered Compound A twice weekly for three weeks, the hematocrit level was greater than 46% compared to approximately 44% in subjects treated with placebo. Compound A produced elevated hematocrit in humans. Consequently, compounds and methods according to the present invention are useful for increasing erythropoiesis and thereby providing an increased hematocrit.

Number of red blood cells

Administration of compound A produced an increased number of red blood cells in humans. As shown in Figure 21, red blood cell counts were elevated in humans treated with 20 mg/kg of Compound A either twice or thrice weekly compared to untreated placebo control subjects. These results showed that methods and compounds according to the present invention are useful for increasing erythropoiesis and thereby providing an increased number of red blood cells.

Iron status - soluble transferrin receptor and serum ferritin level

Results shown above demonstrated that methods and compounds of the present invention effectively increase reticulocyte count, red blood cell count, hemoglobin level and hematocrit in humans. As shown in Figure 22, administration of Compound A to humans produced an increased level of soluble transferrin receptor compared to the level observed in untreated control subjects. An increased level of soluble transferrin was observed in humans treated 2 or 3 times weekly. A maximum response of 35% and 31% was observed on day 21 in patients treated 2 and 3 times weekly, respectively. The mean plasma concentration of sTfR in placebo patients was unchanged. In addition, serum ferritin levels were reduced by approximately 46% in humans treated with compound A, indicating increased utilization of iron in these individuals (see Figure 23).

Taken together, these results showed that HIF stabilization using compounds and methods according to the present invention led to increased mobilization of iron stores, increased transport of iron to bone marrow and improved utilization of iron for hemoglobin synthesis, erythropoiesis and formation of red blood cells.

Various modifications of the invention in addition to those shown and described herein will be apparent to those skilled in the art from the above description. Such modifications are intended to be within the scope of the attached requirements.

All references cited herein are incorporated herein by reference in their entirety.

Claims (90)

PATENT CLAIMS 1. Method for induction of enhanced or complete erythropoiesis in an individual, characterized in that it comprises administering to an individual an effective amount of a compound that stabilizes HIF α such that enhanced or complete erythropoiesis is induced in the individual 2. Method according to claim 1, is characterized by the fact that it is used to enhance erythropoiesis in an individual who has or is at risk of getting a chronic disease. 3. Method according to claim 2, characterized in that the chronic disease is selected from the group consisting of an inflammation, an infection, an immunodeficiency disorder and a neoplastic disorder. 4. Method according to any one of claims 1-3, is characterized by the fact that it is used to enhance erythropoiesis in an individual who has or is at risk of getting anemia associated with chronic disease. 5. Method according to any one of claims 1-4, is characterized by the fact that it is used to enhance erythropoiesis in an individual with erythropoietin resistance. 6. Method according to any one of claims 1-4, It is characterized by the fact that it is used to enhance erythropoiesis in an individual who has or is at risk of iron deficiency. 7. Method according to claim 6, c a r a c t e r i s t h a t iron deficiency is a functional iron deficiency. 8. Method according to any one of the preceding claims, characterized in that it further comprises the administration of at least one additional agent selected from the group consisting of erythropoietin, iron and B vitamins. 9. Method according to any one of the preceding claims, it is characterized by the fact that it regulates or enhances iron metabolism or a process associated with iron metabolism in the individual. 10. Method according to claim 9, is characterized by the process associated with iron metabolism being selected from the group consisting of iron uptake, iron absorption, iron transport, iron storage, iron processing, iron mobilization and iron utilization. 11. Method according to claim 10, c a r a c t e r i s t h a t it increases the absorption of iron in the individual. 12. Method according to claim 10, c a r a c t e r i s t h a t it gives increased absorption of iron in the individual. 13. Method according to claim 12, c a r a c t e r i s t h a t iron absorption takes place in the intestine. 14. Method according to claim 12, c a r a c t e r i s t h a t iron absorption is an absorption of iron from the diet. 15. Method according to claim 12, c a r a c t e r i s t h a t iron absorption is in enterocytes in the duodenum. 16. Method according to claim 10, c a r a c t e r i s t h a t it provides increased iron transport in the individual. 17. Method according to claim 10, c a r a c t e r i s t h a t it gives increased iron storage in the individual. 18. Method according to claim 10, c h a r a c t e r i s t h a t it gives increased iron processing in the individual. 19. Method according to claim 10, c h a r a c t e r i s t h a t it provides increased iron mobilization in the individual. 20. Method according to claim 10, c h a r a c t e r i s t h a t it provides increased iron utilization in the individual. 21. Method according to any one of the preceding claims, c a r a c t e r i s t h a t it provides increased availability of iron for erythropoiesis in the individual. 22. Method according to claim 21, c h a r a c t e r i s t h a t the increased availability of iron for erythropoiesis is increased availability of iron for heme synthesis. 23. Method according to claim 21, c a r a c t e r i s due to the increased availability of iron for erythropoiesis and increased availability of iron for hemoglobin formation. 24. Method according to claim 21, c h a r a c t e r i s t h a t the increased availability of iron for erythropoiesis is increased availability of iron for the formation of red blood cells. 25. Method according to any one of the preceding claims, c a r a c t e r i s t h a t it gives increased expression of transferrin receptor in the individual. 26. Method according to any one of the preceding claims, it is characterized by increased transferrin expression in the individual. 27. Method according to any one of the preceding claims, it is characterized by increased ceruloplasmin expression in the individual. 28. Method according to any one of the preceding claims, characterized by increased expression of NRAMP2 (slc11a2) in the individual. 29. Method according to any one of the preceding claims, c a r a c t e r i s t h a t it gives increased expression of duodenal cytochrome b reductase 1 in the individual. 30. Method according to any one of the preceding claims, It is characterized by increased expression of 5-aminolevulinate synthase in the individual. 31. Method according to any one of the preceding claims, c a r a c t e r i s t h a t it overcomes or relieves cytokine-induced impairment of erythropoiesis in the individual. 32. Method according to claim 31, c a r a c t e r i s t h a t the cytokine-induced impairment of erythropoiesis is a suppression of erythropoietin formation. 33. Method according to claim 31, c a r a c t e r i s that the cytokine-induced impairment of erythropoiesis is an impairment of iron metabolism. 34. Method according to any one of claims 31-33, c a r a c t e r i s t h a t the cytokine is an inflammatory cytokine. 35. Method according to claim 34, c h a r a c t e r i s t h a t the cytokines are selected from the group consisting of TNF- α, IL-1 β and IFN- γ. 36. Method for treating or preventing iron deficiency in an individual, characterized in that it comprises administering an effective amount of a compound that stabilizes HIF α to an individual so that iron deficiency in the individual is treated or prevented. 37. Method according to claim 36, c a r a c t e r i s t h a t iron deficiency is associated with anemia. 38. Method according to claim 36 or claim 37, characterized in that the iron deficiency is associated with a disorder selected from the group consisting of an inflammation, an infection, an immunodeficiency disorder and a neoplastic disorder. 39. Method according to any one of claims 36-38, c a r a c t e r i s t h a t iron deficiency is a functional iron deficiency. 40. Method according to any one of claims 36-39, c a r a c t e r i s t h a t it increases the iron level in the individual's serum. 41. Method according to any one of claims 36-40, c a r a c t e r i s t h a t it gives increased transference input in the individual. 42. Method according to any one of claims 36-41, is characterized by the fact that it produces an increased level of soluble transferrin receptor in the individual. 43. Method according to any one of claims 36-42, c a r a c t e r i s t h a t it produces reduced hepcidin expreion in the individual. 44. Method according to any one of claims 36-43, c a r a c t e r i s t h a t it gives an increased reticulocyte count in the individual. 45. Method according to any one of claims 36-44, c a r a c t e r i s t h a t it gives an increased hematocrit in the individual. 46. Method according to any one of claims 36-45, is characterized by the fact that it increases the hemoglobin level in the individual. 47. Method according to any one of claims 36-46, It is characterized by the fact that it gives an increased number of red blood cells in the individual. 48. Method according to any one of claims 36-47, it is characterized by the fact that it gives an increased average blood cell volume in the individual. 49 Method according to any one of claims 36-48, it is characterized by the fact that it gives an increased level of hemoglobin in the individual's blood cells. 50. Method according to any one of claims 36-49, It is characterized by the fact that it increases the level of iron in the individual's serum. 51. Method according to any one of claims 36-50, it is characterized by the fact that it increases the transference input in the individual. 52. Method according to any one of claims 36-51, characterized in that it further comprises administering to the individual an effective amount of at least one additional agent selected from the group consisting of erythropoietin, iron and B vitamins. 53. Procedure for treatment or prevention of a disorder associated with iron deficiency in an individual, characterized in that it comprises administering an effective amount of a compound that stabilizes HIF α to an individual such that a disorder associated with iron deficiency is treated or prevented in the individual. 54. Method according to claim 53, c a r a c t e r i s t h a t iron deficiency is a functional iron deficiency. 55. Method according to claim 53, characterized in that the disorder is selected from the group consisting of an inflammation, an infection, an immunodeficiency disorder and a neoplastic disorder. 56. Method according to claim 53, characterized by the disorder being selected from the group consisting of anemia associated with chronic disease, iron deficiency anemia and microcytic anemia. 57. Method for treating or preventing a disorder associated with cytokine activity in an individual, characterized in that it comprises administering to the individual an effective amount of a compound that stabilizes HIF α so that the disorder associated with cytokine activity is treated or prevented. 58. Method according to claim 57, is characterized by the disorder being selected from the group consisting of iron deficiency, functional iron deficiency, iron deficiency anemia, anemia associated with chronic disease and microcytic anemia. 59. Method according to claim 57, characterized in that the disorder is associated with a condition selected from the group consisting of an inflammation, an infection, an immunodeficiency disorder and a neoplastic disorder. 60. Method according to any one of claims 57-59, characterized by overcoming or alleviating a cytokine-induced increase in VCAM-1 expression or E-selectin expression. 61. Method for increasing erythropoietin formation in the presence of a cytokine in an individual, characterized in that it comprises administering to the individual an effective amount of a compound that stabilizes HIF α so that erythropoietin formation in the individual is increased. 62. Method for treating or preventing anemia associated with chronic disease in an individual, characterized in that it comprises administering to the individual an effective amount of a compound which stabilizes HIF α such that anemia associated with chronic disease is treated or prevented in the individual. 63. Method according to claim 62, characterized in that it further comprises administering to the individual at least one additional agent selected from the group consisting of erythropoietin, iron and B vitamins. 64. Method for treating or preventing microcytosis in an individual, characterized in that it comprises administering to the individual an effective amount of a compound that stabilizes HIF α so that microcytosis is treated or prevented in the individual. 65. Method according to claim 64, characterized in that the microcytosis is associated with a disorder selected from the group consisting of chronic disease, anemia associated with chronic disease, iron deficiency, functional iron deficiency and iron deficiency anemia. 66. Use of a compound that stabilizes HIF α for the manufacture of a medicament for the induction of enhanced or complete erythropoiesis in an individual; for the treatment or prevention of a disorder associated with iron deficiency; for regulation or enhancement of iron metabolism or a process associated with iron metabolism; for increasing iron absorption in an individual; for increasing iron transport in an individual; for increasing iron uptake in an individual; for increasing iron uptake in an individual; for increasing iron processing in an individual; for increasing iron mobilization in an individual; for increasing iron utilization in an individual; for increasing the availability of iron for erythropoiesis in an individual; for increasing transferrin receptor expression in an individual; for increasing transferrin expression in an individual; for increasing ceruloplasminekspresjonen in an individual; for increasing the expression of NRAMP2 (slc11a2) in an individual; for increasing the expression of duodenal cytochrome b reductase 1 in an individual; for increasing the expression of 5-aminolevulinic synthase in an individual; for increasing the serum iron level of an individual; for increase of transference in an individual; for increasing the level of soluble transferrin receptor in an individual; for reduction of hepcidin expression in an individual; for increasing erythropoiesis in an individual who has or is prone to iron deficiency; for enhancing erythropoiesis in an individual who has or is at risk of having a chronic disease; for enhancing erythropoiesis in an individual who has or is at risk of having anemia associated with chronic disease; for enhancing erythropoiesis in an individual resistant to erythropoietin therapy; for increasing the reticulocyte count in an individual; for increasing the hematocrit in an individual; for increasing the hemoglobin level in an individual; for increasing the number of red blood cells in an individual; for increasing the average blood cell volume in an individual; for increasing the average hemoglobin level in an individual's blood cells; for increasing the serum iron level of an individual; for increasing the hematocrit in an individual; for increase of transference in an individual; for overcoming or ameliorating cytokine-induced impairment of erythropoiesis in a subject: for overcoming or alleviating cytokine-induced increase in VCAM-1 expression in a subject; for overcoming or alleviating cytokine-induced increases in E-selectin expression in the subject; for the treatment or prevention of a disorder associated with cytokine activity in an individual; for increasing erythropoietin formation in the presence of a cytokine in an individual; for treating or preventing a microcytosis in an individual; for the treatment or prevention of iron deficiency in an individual; or for the treatment or prevention of anemia associated with chronic disease in an individual. 67. Application according to claim 66, wherein the disorder associated with iron deficiency is anemia, anemia associated with chronic disease, iron deficiency anemia, or microcytic anemia. 68. Application according to claim 64, where the suffering associated with iron deficiency or the chronic disease is selected among; or the anemia associated with chronic disease or the disorder associated with cytokine activity is associated with a condition selected from the group consisting of an inflammation, an infection, an immunodeficiency disorder and a neoplastic disorder. 69. Application according to any one of claims 66-68, where the iron deficiency is functional iron deficiency. 70. Application according to claim 66, wherein the process associated with iron metabolism is selected from the group consisting of iron uptake, iron absorption, iron transport, iron storage, iron processing, iron mobilization and iron utilization. 71. Application according to claim 70, in which iron absorption takes place in the intestine. 72. Application according to claim 70, where iron absorption is the absorption of iron from the diet. 73. Application according to claim 70, in which the iron absorption is in enterocytes in the duodenum. 74. Application according to claim 66, wherein the increased availability of iron for erythropoiesis is increased availability of iron for heme synthesis. 75. Application according to claim 66, in which the increased availability of iron for erythropoiesis is increased availability of iron for hemoglobin formation. 76. Application according to claim 66, in which the increased availability of iron for erythropoiesis is increased availability of iron for the formation of red blood cells. 77. Application according to any one of claims 66-76, wherein the medication further comprises at least one additional agent selected from the group consisting of erythropoietin, iron and B vitamins. 78. Application according to any one of claims 66-76, where the medication is for simultaneous, separate or sequential use with at least one additional agent selected from the group consisting of erythropoietin, iron and B vitamins. 79. Application according to claim 66, wherein the cytokine-induced impairment of erythropoiesis is suppression of erythropoietin formation. 80. Application according to claim 66, wherein the cytokine-induced impairment of erythropoiesis is an impairment of iron metabolism. 81. Use according to any one of claims 66, 79 or 80, wherein the cytokine is an inflammatory cytokine. 82. Application according to any one of claims 66 or 79-81, where the cytokine is selected from the group consisting of TNF-α, IL-1β and IFN-γ. 83. Application according to claim 66, wherein the disorder associated with cytokine activity is selected from the group consisting of iron deficiency, functional iron deficiency, iron deficiency anemia, anemia associated with chronic disease, and microcytic anemia. 84. Application according to claim 66, wherein the microcytosis is associated with a disorder selected from the group consisting of chronic disease, anemia associated with chronic disease, iron deficiency, functional iron deficiency, and iron deficiency anemia. 85. Application according to any one of claims 66-84, where the treatment of patients with elevated cytokine levels. 86. Application according to claim 85, wherein the cytokines are inflammatory cytokines. 87. Application according to claim 86, wherein the cytokine is selected from the group consisting of TNF-α, IL-1β and IFN-γ. 88. Application according to any one of claims 66-87, in which the treatment of patients with EPO resistance. 89. Seen, characterized in that it comprises a compound that stabilizes HIF α and at least one additional agent selected from the group consisting of erythropoietin, iron and B vitamins. 90. Pharmaceutical composition, characterized in that it comprises a compound that stabilizes HIF α and at least one additional agent selected from the group consisting of erythropoietin, iron and B vitamins.