WO2009035534A2

WO2009035534A2 - Treatment of ischemic eye disease by the systematic pharmaceutical activation of hypoxia inducible factor (hif)

Info

Publication number: WO2009035534A2
Application number: PCT/US2008/010413
Authority: WO
Inventors: Jonathan E. Sears; George B. Hoppe; Quteba Ebrahem
Original assignee: The Cleveland Clinic Foundation
Priority date: 2007-09-07
Filing date: 2008-09-05
Publication date: 2009-03-19
Also published as: WO2009035534A3

Abstract

The present invention consists of a method of treating a subject with an ischemic eye disease associated with the down-regulation of HIF comprising administering to the subject an effective amount of a compound that induces HIF activity and/or induces EPO activity to induce and maintain growth or normal retinal blood vessels in the early stage of the disease.

Description

TREATMENT OF ISCHEMIC EYE DISEASE BY THE SYSTEMATIC PHARMACEUTICAL ACTIVATION OF HYPOXIA INDUCIBLE FACTOR

(HIF)

RELATED APPLICATION This application claims the benefit of U.S. Provisional Application No.

60/967,901, filed on September 7, 2007, the entire teachings of which are incorporated herein by reference.

BACKGROUND OF THE INVENTION

Retinopathy of prematurity (ROP) is a vasoproliferative disease affecting the retinas of premature infants. When infants are born prematurely, the blood vessels that support the retinas are not completely developed. These vessels may develop in an abnormal or disorganized pattern that can lead to bleeding, scarring of the retina, retinal detachment and blindness. ROP is characterized by two phases, phase I (hyperoxic) during which high oxygen tension promotes retinal capillary regression, or vasoobliteration, due to down-regulation of hypoxia inducible factors 1 and 2

(HIF), and phase II (normoxic), an ischemic phase, during which an over expression of HIF-inducible proteins creates pathologic over-exaggeration of the proteins critical to normal blood vessel growth. The lack of retinal blood vessel growth during phase I augments the relative hypoxia of phase II by producing large quantities of HIF-inducible proteins such as vascular endothelial growth factor (VEGF) and erythropoietin (Epo). Even children that have ROP as infants whose symptoms cease or regress spontaneously, have increased risk for eye abnormalities later in life - such as nearsightedness, misalignment of the eyes, and/or future retina detachment. In fact, ROP is the leading cause of childhood blindness. Any child born before term and/or born at a low birth weight is susceptible to ROP.

Only one treatment is approved for ROP, which is laser photocoagulation. Experimental treatments include anti-VEGF treatments (Avastin), and suppression of other HIF-inducible proteins such as (bFGF). These treatments of ROP are based on suppression of pathological angiogenesis during phase II, which is entirely dependent on the status of retinal capillary bed affected by phase I. However, these therapies target only the result of relative hypoxia in phase II rather than focusing on preventing relative hypoxia by preserving capillary bed in phase I. These current treatments do not prevent damage to the retinas from occurring. Instead they merely try to minimize the effects of the second phase of the disease to limit its severity. Significant risks are associated with the current treatment of ROP. For example, the long-term effects of peripheral laser ablation include possible retinal detachment and loss of visual field. Additionally, the long-term effects of anti- VEGF therapy on the developing premature infant are unknown. Clearly, new treatment options that avoid the retinal damage are needed.

Another vasoproliferative disease is diabetic retinopathy. Diabetic retinopathy is characterized by damage to the blood vessels of the eyes due to high blood sugar and/or high blood pressure. Initially, the blood vessels of the eye weaken and eventually develop small bulges that can burst and leak into the retina and the vitreous gel of the eye. As the disease progresses, new fragile blood vessels grow on the surface of the retina. These blood vessels also break easily and cause bleeding in the middle of the eye. The bleeding can cause scar tissue, which can pull on the retina and ultimately cause retinal detachment. The disease is progressive and can lead to severe vision loss or blindness.

Current treatment for diabetic retinopathy includes laser treatment (photocoagulation) and surgical removal of the vitreous gel of the eye. These treatments can not reverse any damage done to the retinas, but may stop the progression of the disease, in combination with adequate control of the underlying diabetes. Currently, there is no cure for the disease.

Venous Occlusive Disease, which includes both branched and central Retinal Vein Occlusion, is second only to diabetic retinopathy for causing visual loss due to a retinal vascular disease. Branched retinal vein occlusion is characterized by blockage of the veins that drain retinal blood. Blockage results in pressure build-up within the capillaries, which leads to hemorrhaging and fluid leakage on the retina. If the blockage is severe, the capillaries may cease to function and the retina blood supply can be closed off. When there is significant closure of the capillaries, neovascularization (growth of abnormal blood vessels) may result, leading to bleeding into the vitreous gel and eventually to retinal detachment. There is no known treatment for branched retinal vein occlusion. Central retinal vein occlusion is the closure of the final retinal vein which collects all of the blood from the capillaries. The ischemic form of central retinal vein occlusion causes areas of the eye to have no blood supply. Additionally, a particular type of neovascularization occurs on the iris of the eye, which can lead to high pressure in the eye, severe vision loss, and can lead to the loss of the eye itself. If the neovascularization occurs at the back of the eye, retinal detachment and vitreous hemorrhaging can occur. Some non-ischemic cases of central retinal vein occlusion can be treated using laser photocoagulation.

Age Related Macular Degeneration (ARMD) is the leading cause of legal blindness in the United States, and affects ten percent of the population over age 52, and thirty-three percent of the population over age 75. There are two types of ARMD, the "wet" form (85% of all cases) and the "dry" form (15% of all cases). All ARMD starts out as the dry form, which can progress into the wet form or progress through intermediate and advanced stages. The wet form of ARMD is considered an advanced stage upon diagnosis. Both forms are postulated to be caused by choriocapillaris vascular failure causing the retinal pigment epithelium to degenerate, resulting in photoreceptor loss. However, the wet form of ARMD progresses rapidly and involves neovascularization of the choriocapillaries, ultimately leading to bleeding and protein leakage beneath the macula. In either type, the photoreceptor loss leads to degeneration of the outer retinal layers, or the macula of the eye, which in turn leads to vision loss. There are no treatments for the dry form of ARMD; the wet form can be treated with anti-VEGF agents to slow down the progression of vision loss. However, no treatment option provides a cure for the disease.

SUMMARY OF THE INVENTION

It has been found that inducing HIF activity can be used to treat ischemic eye diseases, such as phase I ROP, and thus preventing or reducing the amount of damage that occurs to the eyes. Specifically, it has been found that the administration of dimethyloxalylglycine (DMOG), a known inducer of HIF activity, leads to the almost complete reversal of capillary regression in the ROP Murine mouse model (see Example 3 and FIGs. 2 through 5).

Based on this discovery, a new method of treatment for ischemic eye diseases associated with the down-regulation of HIF has been found. This discovery has lead to a method of treatment of ischemic eye diseases such as ROP during phase I, diabetic retinopathy, venous occlusive disease and/or age related macular degeneration. Unlike traditional therapies for these conditions, the present invention provides treatment in the initial stages of a disease, before the majority of permanent damage has occurred. HIF-I has a key role in cellular responses to hypoxia, including the regulation of genes involved in energy metabolism, angiogenesis and apoptosis. HIF-I is a heterodimeric transcription factor consisting of a constitutively expressed HIF- lβ subunit, and a hypoxia inducible HIF- lα or HIF -2α subunit. The alpha subunits of HIF are rapidly degraded by the proteasome under normal conditions, but are stabilized by hypoxia. Under normoxic conditions, the HIF- lα subunit is hydroxylated by prolyl or asparaginyl hydroxylases in the presence of oxygen and iron. Therefore, one way to induce HIF- lα levels is to inhibit its degradation by inhibiting the activity of the prolyl or asparaginyl hydroxylases through hypoxia or iron depletion with iron chelators. Representative iron chelators are listed in Table 1. (see Nagle, D.G, et al, Curr Pharm Des, 2006, 12:2673-2688; Thomas, R., et al, MoI Cell Biochem, 2006, 296:35-44; Ivan, M., et al, Proc Natl Acad Sci USA, 2002, 99(21): 13459- 13464; Whitnall, M., et al., Semin Pediatr Neurol 2006, 13: 86-197, Kalinowski, D.S., et al, Pharmacol Rev 2005, 57:547-583, the entire teachings of all of which are incorporated herein by reference). One embodiment of the present invention is a method of treating a subject with an ischemic eye disease associated with the down-regulation of HIF comprising administering to the subject an effective amount of a compound that induces HIF activity and/or Epo activity.

Another embodiment of the invention is a method of treating a subject with an ischemic eye disease selected from the group consisting of phase I retinopathy of prematurity, diabetic retinopathy, venous occlusive disease, dry form of age related macular degeneration (ARMD), comprising administering to the subject an effective amount of a compound selected from the group consisting of a compound according to Formulas I through XXIX or Formulas 1 through 5 and pharmaceutically acceptable salts thereof. Another embodiment of this invention is a pharmaceutical composition comprising a compound according to Formulas I through VII or Formulas 1 through 5, or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable carrier, diluent or excipient.

Stimulation of HIF provides concerted expression of HIF-dependent proteins such as vascular endothelial growth factor (VEGF) and erythropoietin (Epo) to stimulate blood vessel growth. This stimulation leads to effective treatment of ischemic eye disease. In contrast with current therapies, where any therapy is available, the present method of treatment is effective at an earlier stage of the ischemic eye disease, and may be associated with significantly fewer harmful side- effects.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a graph showing the effect of compounds I , II, III, IV, V, VI and VII on erythropoietin (Epo) secretion. DMSO (dimethyl sulfoxide) is a solvent for compounds I-VII. DFO (desferoxamine) and CoCl₂ (cobalt (II) chloride) are known upregulators of HIF and serve here as positive controls.

FIG. 2 is a graph showing the increased production of VEGF as a function of time after injection of DMOG during Phase I of ROP.

FIG. 3 is a graph detailing the increased production of VEGF as a function of dose of DMOG 6 hours after administration. FIG. 4 is a graph detailing the increase of Epo production as a function of time after injection of DMOG during Phase I of ROP.

FIG. 5 is a graph detailing the increased production of Epo as a function of dose of DMOG 6 hours after administration.

DETAILED DESCRIPTION OF THE INVENTION

One embodiment of the present invention is a method of treating a subject with an ischemic eye disease associated with the down-regulation of HIF comprising administering to the subject an effective amount of a compound that induces HIF activity and/or induces EPO activity. In one embodiment, said compound is selected the group consisting of a compound according to Formulae I through XXIX or Formulas 1 through 5 or pharmaceutically acceptable salts thereof.

In another embodiment, the method comprises administration of a compound according to Formula 1 :

wherein R , R , and R are each individually hydrogen or Ci-C₈ aliphatic group; or a pharmaceutically acceptable salt thereof. In a more particular embodiment, the method comprises administration of the compound according to Formula XIII:

, or a pharmaceutically acceptable salt thereof.

In one embodiment of the invention is a method of treating a subject with an ischemic eye disease associated with the down-regulation of HIF comprising administering to the subject an effective amount of a compound represented by the following structural Formula 2:

ⁱ , wherein each R¹ is independently hydrogen or (d-C₆)alkyl; Ari is phenyl or pyridinyl, each optionally substituted with one to four substituents selected from the group consisting of halogen, hydroxy, nitro, carboxy, cyano, amino, (Ci-C₆)alkyl, halo(C!-C₆)alkyl, (Ci-

C₆)alkoxy, halo(Ci-C₆)alkoxy, hydroxy(d-C₆)alkyl, (C₁- C₆)alkylcarbonyl, (Cj-C₆)alkoxycarbonyl, (Ci-C₆)alkylamino and di(Cp C₆)alkylamino;

Ar₂ is aryl or heteroaryl, each optionally substituted with one to four substituents selected from the group consisting of halogen, hydroxy, nitro, carboxy, cyano, amino, (C_!-C₆)alkyl, halo(Ci-C₆)alkyl, (Ci- C₆)alkoxy, halo(d-C₆)alkoxy, hydroxy(Ci-C₆)alkyl, (Ci- C₆)alkylcarbonyl, (Ci-C₆)alkoxycarbonyl, (Ci-C₆)alkylamino, di(Ci- C₆)alkylamino and -X-Ar₃; Ar₃ is phenyl, optionally substituted with one to four substituents selected from the group consisting of halogen, hydroxy, nitro, carboxy, cyano, amino, (Ci-C₆)alkyl, halo(Ci-C₆)alkyl, (Ci-C₆)alkoxy, halo(Ci- C₆)alkoxy, hydroxy(Ci-C₆)alkyl, (Ci-C₆)alkylcarbonyl, (Ci- C₆)alkoxycarbonyl, (Ci-C₆)alkylamino and di(Ci-C₆)alkylamino; and X is a bond or -NR¹-; or a pharmaceutically acceptable salt thereof.

In a specific embodiment, the compound is represented by the Formula 3:

, where the variables are as described for Formula 2 or have the following values: each R² is independently halogen, hydroxy, nitro, carboxy, cyano, amino,

(Ci-C₆)alkyl, halo(C₁-C₆)alkyl, (d-C^alkoxy, halo(Ci-C₆)alkoxy, hydroxy(Ci-C₆)alkyl, (C]-C₆)alkylcarbonyl, (Ci-C₆)alkoxycarbonyl, (C₁-

C₆)alkylamino or di(C₁-C₆)alkylamino; each R³ is independently halogen, hydroxy, nitro, carboxy, cyano, amino, (Ci-C₆)alkyl, halo(d-C₆)alkyl, (Ci-C₆)alkoxy, halo(Ci-C₆)alkoxy, hydroxy(C_!-C₆)alkyl, (d-C₆)alkylcarbonyl, (Ci-C₆)alkoxycarbonyl, (Cj- C₆)alkylamino or di(Ci-C₆)alkylamino; m is O, 1, 2, 3 or 4; and n is O, 1, 2, 3 or 4; or a pharmaceutically acceptable salt thereof.

In one embodiment, each R² is independently halogen, hydroxy, amino, (Ci- C₃)alkyl, halo(Ci-C₃)alkyl, (Ci-C₃)alkoxy, halo(Ci-C₃)alkoxy, hydroxy(Ci-C₃)alkyl, (Ci-C₃)alkylamino or di(Ci-C₃)alkylamino; each R³ is independently halogen, hydroxy, amino, (Ci-C₃)alkyl, halo(Ci-C₃)alkyl, (Ci-C₃)alkoxy, halo(Ci-C₃)alkoxy, hydroxy(Ci-C₃)alkyl, (Ci-C₃)alkylamino or di(Ci-C₃)alkylamino; m is 0, 1 or 2; and n is 0, 1 or 2, wherein values of the remaining variables are as for Formula 2. In another embodiment each R² is independently bromo, chloro hydroxy,

(Ci-C₃)alkyl, (Ci-C₃)alkoxy, or di(Ci-C₃)alkylamino; and each R³ is independently bromo, chloro, hydroxy, (Ci-C₃)alkyl,

or di(Ci-C₃)alkylamino, wherein values of the remaining variables are as for Formula 2. In a specific embodiment, each R² is independently hydroxy or methoxy; each R³ is methyl; and m and n are 2, wherein values of the remaining variables are as for Formula 2.

In an alternative embodiment, the compound is represented by the Formula 4:

, wherein each R² is independently halogen, hydroxy, nitro, carboxy, cyano, amino, (Ci-C₆)alkyl, halo(Ci-C₆)alkyl, (d-C₆)alkoxy, halo(CrC₆)alkoxy, hydroxy(Ci-C₆)alkyl, (Ci-C₆)alkylcarbonyl, (C]-C₆)alkoxycarbonyl, (C₁- C₆)alkylamino or di(CrC₆)alkylamino; each R⁴ is independently halogen, hydroxy, nitro, carboxy, cyano, amino, (Ci-C₆)alkyl, halo(C]-C₆)alkyl, (C_rC₆)alkoxy, halo(Ci-C₆)alkoxy, hydroxy(Ci-C₆)alkyl, (Ci-C₆)alkylcarbonyl,

(C_r C₆)alkylamino or di(CrC₆)alkylamino; m is 0, 1, 2, 3 or 4; and p is O, 1, 2, 3 or 4. or a pharmaceutically acceptable salt thereof, wherein values of the remaining variables are as defined for Formula 2.

In another embodiment, each R² is independently halogen, hydroxy, amino, (Cj-C₃)alkyl, halo(C,-C₃)alkyl, (Ci-C₃)alkoxy, halo(Ci-C₃)alkoxy, hydroxy(Ci- C3)alkyl, (Ci-C₃)alkylamino or di(Ci-C₃)alkylamino; each R⁴ is independently halogen, hydroxy, amino, (Ci-C₃)alkyl, halo(Ci-C₃)alkyl, (Ci-C₃)alkoxy, halo(Ci- C₃)alkoxy, hydroxy(C|-C₃)alkyl, (Ci-C₃)alkylamino or di(CrC₃)alkylamino; m is 0, 1 or 2 and p is 0, 1 or 2, wherein values of the remaining variables are as defined for Formula 2.

In another embodiment each R² is independently bromo, chloro hydroxy, (Ci-C₃)alkyl, (Ci-C₃)alkoxy, or di(Ci-C₃)alkylamino; and each R⁴ is independently bromo, chloro, hydroxy, (Ci-C₃)alkyl, (Ci-C₃)alkoxy, or (Ii(C₁ -C₃)alkylamino, wherein values of the remaining variables are as for Formula 2.

In yet another embodiment, R² is hydroxy; n is 2; and p is 0, wherein values of the remaining variables are as defined for Formula 2. In another alternate embodiment, compound is represented by Formula 5:

Z is N, CH or CR² each R² is independently halogen, hydroxy, nitro, carboxy, cyano, amino,

(C,-C₆)alkyl, halo(Ci-C₆)alkyl, (Ci-C₆)alkoxy, 1IaIo(C₁ -C₆)alkoxy, hydroxy(Ci-C₆)alkyl, (Ci-C₆)alkylcarbonyl, (Ci-C₆)alkoxycarbonyl, (C₁-

C₆)alkylamino or di(Ci-C₆)alkylamino; each R⁵ is independently halogen, hydroxy, nitro, carboxy, cyano, amino, (C_rC₆)alkyl, halo(d-C₆)alkyl, (Ci-C₆)alkoxy, 1IaIo(C₁ -C₆)alkoxy, hydroxy(Ci-C₆)alkyl,

(Ci-C₆)alkoxycarbonyl, (C₁- C₆)alkylamino or di(CrC₆)alkylamino; m is 0, 1, 2, 3 or 4; and q is O, 1, 2, 3 or 4; or a pharmaceutically acceptable salt thereof, wherein values of the remaining variables are as defined for Formula 2. In one embodiment, Z is N; each R² is halogen, hydroxy, amino, (C₁-

C₃)alkyl, halo(Ci-C₃)alkyl, (C,-C₃)alkoxy, halo(Ci-C₃)alkoxy, hydroxy(Ci-C₃)alkyl, (Ci-C₃)alkylamino or di(Ci-C₃)alkylamino; each R⁵ is halogen, hydroxy, amino, (Ci-C₃)alkyl, halo(C,-C₃)alkyl, (C,-C₃)alkoxy, halo(C_!-C₃)alkoxy, hydroxy(C,- C₃)alkyl, (Ci-C₃)alkylamino or di(Ci-C₃)alkylamino; m is 0, 1 or 2 and q is 0, 1 or 2, wherein values of the remaining variables are as defined for Formula 2; wherein values of the remaining variables are as defined for Formula 2.

In another embodiment each R² is independently bromo, chloro hydroxy, (Ci-C₃)alkyl, (Ci-C₃)alkoxy, or di(Ci-C₃)alkylamino; and each R⁵ is independently bromo, chloro, hydroxy, (Ci-C₃)alkyl, (C₁-C₃)EIkOXy, or di(Ci-C₃)alkylamino, wherein values of the remaining variables are as for Formula 2.

In another embodiment, R⁵ is methyl; m is 0; and q is 0 or 1, wherein values of the remaining variables are as defined for Formula 2.

Suitable compounds which induce HIF and/or Epo activity are those according to Formulae I through XXIX, inclusive, and are shown in Table 1.

Compounds I through VII upregulate HIF or Epo activity according to the assays described herein. Compound VIII is a known inducer of HIF activation. Other compounds that may increase HIF activity are well known in the art and include iron chelators, such as Compounds IX through XVII and as hydrazones, such as Compounds IX through XV. Compounds XVIII through XXIX are known HIF inducers and/or Epo inducers and/or HIF prolyl or asparaginyl hydroxylases. Other examples of compounds that can induce HIF activity are described in Nagle, D.G, et al, Curr Pharm Des, 2006, 12:2673-2688; Thomas, R., et al, MoI Cell Biochem, 2006, 296:35-44; Ivan, M., et al, Proc Natl Acad Sci USA, 2002, 99(21):13459-13464; Whitnall, M., et al., Semin Pediatr Neurol 2006, 13: 86-197, Kalinowski, D.S., et al, Pharmacol Rev 2005, 57:547-583, the entire teachings of all of which are incorporated herein by reference, and include compounds of the Formula IX through XXIX, inclusive.

Additional compounds can readily be determined by suitable screening techniques and assays, as described in Example 1 and in assays described in the citations recited in the prior paragraph.

In a particular embodiment of the present invention, the ischemic eye disease associated with the down-regulation of HIF is phase I of retinopathy of prematurity, diabetic retinopathy, venous occlusive disease or age-related macular degeneration (ARMD). These ischemic eye diseases are characterized by an initial reduction and breakdown of the capillaries of the retina. This initial phase is then followed by an overproduction of blood vessels that are fragile and prone to breaking and leaking into the retina. Another particular embodiment of the invention is the method of treatment wherein the compound is administered to the eye of the subject, where the condition is an eye condition such as phase I ROP, diabetic retinopathy, venous occlusive disease or age-related macular degeneration.

As used herein the term "subject" typically means a human, e.g. a premature infant and/or low birth weight infant, a human with diabetes (type I or type II), a human over age 52, but can also be an animal in need of treatment, e.g., companion animals (e.g., dogs, cats, and the like), farm animals (e.g., cows, pigs, horses, sheep, goats and the like) and laboratory animals (e.g., rats, mice, guinea pigs and the like).

"Treatment" or "treating", as used herein, refers to partially or totally inhibiting, delaying, or reducing the severity of an ischemic eye disease associated with the down regulation of HIF. The terms "treatment" and "treating" also encompass the prophylactic administration of a compound of the invention to a subject susceptible to an ischemic eye disease associated with the down regulation of HIF in an effort to reduce the likelihood of a subject developing the disease. Additionally, the terms "treatment" and "treating" encompass slowing or preventing the progression of an ischemic eye disease. Prophylactic treatment includes suppression (partially or completely) of the ischemic eye disease associated with the down-regulation of HIF, and further includes reducing the severity of the ischemic eye disease, if onset occurs. Prophylactic treatment is particularly advantageous for administration to subjects at risk for developing an ischemic eye disease associated with the down regulation of HIF, such as diabetic patients at risk for developing diabetic retinopathy, premature infants in need of oxygen treatment, and/or patients over the age of 52 at risk for ARMD.

"Hypoxia Inducible Factors" or "HIF", including both type 1 and type 2, are transcription factors that respond to changes in available oxygen in the cellular environment, specifically to decreases in oxygen. HIF exists as a heterodimer of two basic-helix-loop-helix proteins, called the α- and β-subunits, and is strongly induced by hypoxia. However, HIF activity is controlled by the expression of the α- subunit. Under normal oxygen conditions, the α-subunit is subject to ubiquitination and proteasomal degradation. HIF-I and HIF-2, when stabilized by hypoxic conditions, upregulate several genes to promote survival in low oxygen conditions. These include glycolysis enzymes, which allow ATP synthesis in an oxygen- independent manner, vascular endothelial growth factor (VEGF), which promotes angiogenesis, and the hematopoietic growth factor erythropoietin (Epo).

"Erythropoietin" or "Epo", also called hematopoietin or hemopoietin, is a glycoprotein hormone that controls erythropoiesis, or red blood cell production. Erythropoietin promotes red blood cell survival by protecting these cells from apoptosis. Epo is one of many gene products, whose expression is regulated by HIF. Epo is a cytokine that is directly involved in angiogenesis and is considered a key regulator of vascular repair. It has a range of actions including vasoconstriction- dependent hypertension, stimulating angiogenesis, and inducing proliferation of smooth muscle fibers.

"Downregulation of HIF" means a decrease in HIF activity, whether by inhibition or by reduction in the gene expression of HIF, or by an increase in the proteolytic degradation of HIF.

A "premature infant" is one born before 36 weeks gestation. A "low birth weight infant" is one born weighing under five pounds.

"A compound that induces HIF activity" is a compound that in vivo or in cell culture increases the amount of HIF protein, a compound that increases HIF expression, a compound that increases expression levels of the genes that HIF regulates, and/or a compound that inhibits the degradation or decomposition of HIF. In one example, a compound which induces HIF activity increases HIF levels or expression in human hepatoma cell line Hep3B at least 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 250, 300% as compared with untreated controls. Procedures for assessing HIF levels in Hep3B cells are described in Example 1. Examples of such compounds are those of Formulae I through IV, and others that can be determined by the screening method described in Example 1. Typically, such compounds have a formula weight less than 1000 amu. Particularly, a compound that induces HIF activity has a formula weight less than 500 amu.

"A compound that induces Epo activity" is a compound that in vivo or in cell culture increases the amount of Epo protein, a compound that increases Epo expression, and/or a compound that inhibits the degradation or decomposition of Epo. In one example, a compound which induces Epo activity increases Epo protein levels in human hepatoma cell line Hep3B at least 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 250, 300% as compared with untreated controls. Procedures for assessing Epo levels in Hep3B cells are described in Example 1. Examples of such compounds are those of Formulae I through VII, and others that can be determined by the screening method described in Example 1. Typically, such compounds have a formula weight less than 1000 amu. Particularly, a compound that induces Epo activity has a formula weight less than 500 amu.

"Aliphatic group" or "Alkyl" means a saturated or unsaturated, branched or straight-chain mono- or divalent hydrocarbon radical having the specified number of carbon atoms. Thus, "(Ci-C₈) aliphatic group" or "(C₁-C₈)alkyl" means a saturated or unsaturated radical having from 1-8 carbon atoms in a linear or branched arrangement. Such groups includes methyl, ethyl, i-propyl, butyl, pentyl, ethenyl, propenyl, ethynyl, allyl, 2-butynyl, and the like. "Effective amount" means the amount of a compound of the invention that elicits the desired biological response in a subject. Such response includes alleviation, total or partial, or inhibition, total or partial, of the onset of the symptoms of the disease being treated. Typically an effective amount is from 1 to 1000 μg per Ig of body weight of the subject. More particularly, an effective amount is from 100 to 500 μg per Ig of a subject's body weight. In some embodiments of the invention, an effective amount is from 250 to 750 μg per 1 g of body weight of the subject.

"Ischemic eye disease" means a disease affecting a subject's eye which is associated with intraocular ischemia, or restriction of the blood supply. Often, as a result of ischemic conditions of the eye, abnormal new blood vessels may develop within the retina or other areas of the eye deficient in oxygen, called neovascularization. Ischemic eye diseases include, but are not limited to, retinopathy of prematurity, diabetic retinopathy, dry age-related macular degeneration, and venous occlusive disease, including branched retinal vein occlusion and central retinal vein occlusion.

Certain compounds of Formulas 1 through 5 and Formulas I through XXIX, may exist in various stereoisomer^ or tautomeric forms. The invention encompasses all such forms, including active compounds in the form of essentially pure enantiomers, racemic mixtures, and tautomers, including forms those not depicted structurally.

The compounds of the invention may be present in the form of pharmaceutically acceptable salts. For use in medicines, the salts of the compounds of the invention refer to non-toxic "pharmaceutically acceptable salts." Pharmaceutically acceptable salt forms include pharmaceutically acceptable acidic/anionic or basic/cationic salts.

Pharmaceutically acceptable acidic/anionic salts include, the acetate, benzenesulfonate, benzoate, bicarbonate, bitartrate, bromide, calcium edetate, camsylate, carbonate, chloride, citrate, dihydrochloride, edetate, edisylate, estolate, esylate, fumarate, glyceptate, gluconate, glutamate, glycollylarsanilate, hexylresorcinate, hydrobromide, hydrochloride, hydroxynaphthoate, iodide, isethionate, lactate, lactobionate, malate, maleate, mandelate, mesylate, methylsulfate, mucate, napsylate, nitrate, pamoate, pantothenate, phosphate/diphospate, polygalacturonate, salicylate, stearate, subacetate, succinate, sulfate, tannate, tartrate, teoclate, tosylate, and triethiodide salts.

The compounds of the invention include pharmaceutically acceptable anionic salt forms, wherein the anionic salts include the acetate, benzenesulfonate, benzoate, bicarbonate, bitartrate, bromide, calcium edetate, camsylate, carbonate, chloride, citrate, dihydrochloride, edetate, edisylate, estolate, esylate, fumarate, glyceptate, gluconate, glutamate, glycollylarsanilate, hexylresorcinate, hydrobromide, hydrochloride, hydroxynaphthoate, iodide, isethionate, lactate, lactobionate, malate, maleate, mandelate, mesylate, methylsulfate, mucate, napsylate, nitrate, pamoate, pantothenate, phosphate/diphospate, polygalacturonate, salicylate, stearate, subacetate, succinate, sulfate, tannate, tartrate, teoclate, tosylate, and triethiodide salts.

Commonly, a compound that induces HIF activity and/or Epo activity is administered as a composition which comprises a mixture one or more compounds of the invention and an optional pharmaceutically acceptable carrier.

"Pharmaceutically acceptable carrier" means compounds and compositions that are of sufficient purity and quality for use in the formulation of a composition of the invention and that, when appropriately administered to an animal or human, do not produce an adverse reaction.

The invention further includes the process for making the composition comprising mixing one or more of the present compounds and an optional pharmaceutically acceptable carrier; and includes those compositions resulting from such a process, which process includes conventional pharmaceutical techniques.

Administration methods include administering an effective amount (i.e., a therapeutically effective amount) of a compound or composition of the invention at different times during the course of therapy or concurrently in a combination form. The methods of the invention include all known therapeutic treatment regimens.

The compositions of the invention include ocular, oral, nasal, transdermal, topical with or without occlusion, intravenous (both bolus and infusion), and injection (intraperitoneally, subcutaneously, intramuscularly, intratumorally, or parenterally). The composition may be in a dosage unit such as a tablet, pill, capsule, powder, granule, liposome, ion exchange resin, sterile ocular solution, or ocular delivery device (such as a contact lens and the like facilitating immediate release, timed release, or sustained release), parenteral solution or suspension, metered aerosol or liquid spray, drop, ampoule, auto-injector device, or suppository; for administration ocularly, orally, intranasally, sublingually, parenterally, or rectally, or by inhalation or insufflation.

For ocular administration, the composition is preferably in the form of an ophthalmic composition. The ophthalmic compositions are preferably formulated as eye-drop formulations or formulated for ocular injection, and filled in appropriate containers to facilitate administration to the eye, for example a dropper fitted with a suitable pipette. Preferably, the compositions are sterile and aqueous based, using purified water. In addition to the compound of the invention, an ophthalmic composition may contain one or more of: a) a surfactant such as a polyoxyethylene fatty acid ester; b) a thickening agents such as cellulose, cellulose derivatives, carboxyvinyl polymers, polyvinyl polymers, and polyvinylpyrrolidones, typically at a concentration n the range of about 0.05 to about 5.0% (wt/vol); c) (as an alternative to or in addition to storing the composition in a container containing nitrogen and optionally including a free oxygen absorber such as Fe), an anti- oxidant such as butylated hydroxyanisol, ascorbic acid, sodium thiosulfate, or butylated hydroxytoluene at a concentration of about 0.00005 to about 0.1% (wt/vol); d) ethanol at a concentration of about 0.01 to 0.5% (wt/vol); and e) other excipients such as an isotonic agent, buffer, preservative, and/or pH-controlling agent. The pH of the ophthalmic composition is desirably within the range of 4-8. Compositions of the invention suitable for oral administration include solid forms such as pills, tablets, caplets, capsules (each including immediate release, timed release, and sustained release formulations), granules and powders; and, liquid forms such as solutions, syrups, elixirs, emulsions, and suspensions. Forms useful for ocular administration include sterile solutions or ocular delivery devices. Forms useful for parenteral administration include sterile solutions, emulsions, and suspensions.

The compositions of the invention may be administered in a form suitable for once-weekly or once-monthly administration. For example, an insoluble salt of the active compound may be adapted to provide a depot preparation for intramuscular injection (e.g., a decanoate salt) or to provide a solution for ophthalmic administration.

The dosage form containing the composition of the invention contains a effective amount of the active ingredient necessary to provide a therapeutic or prophylactic effect. The composition may contain from about 1 μg to about 1000 μg of a compound of the invention, or salt form thereof, per Ig of body weight of the subject, and may be constituted into any form suitable for the selected mode of administration. Preferably, the dosage is from about 100 μg to 500 μg, or from about 250 μg to about 750 μg per Ig of body weight of the subject. The composition may be administered about 1 to about 5 times per day. Daily administration or post-periodic dosing may be employed.

For oral administration, the composition is preferably in the form of a tablet or capsule the active compound. Dosages will vary depending on factors associated with the particular patient being treated (e.g., age, weight, diet, and time of administration), the severity of the condition being treated, the compound being employed, the mode of administration, and the strength of the preparation. The oral composition is preferably formulated as a homogeneous composition, wherein the active ingredient is dispersed evenly throughout the mixture, which may be readily subdivided into dosage units containing equal amounts of a compound of the invention. Preferably, the compositions are prepared by mixing a compound of the invention (or pharmaceutically acceptable salt thereof) with one or more optionally present pharmaceutical carriers (such as a starch, sugar, diluent, granulating agent, lubricant, glidant, binding agent, and disintegrating agent), one or more optionally present inert pharmaceutical excipients (such as water, glycols, oils, alcohols, flavoring agents, preservatives, coloring agents, and syrup), one or more optionally present conventional tableting ingredients (such as corn starch, lactose, sucrose, sorbitol, talc, stearic acid, magnesium stearate, dicalcium phosphate, and any of a variety of gums), and an optional diluent (such as water).

Binder agents include starch, gelatin, natural sugars (e.g., glucose and beta- lactose), corn sweeteners and natural and synthetic gums (e.g., acacia and tragacanth). Disintegrating agents include starch, methyl cellulose, agar, and bentonite.

Tablets and capsules represent an advantageous oral dosage unit form. Tablets may be sugarcoated or film-coated using standard techniques. Tablets may also be coated or otherwise compounded to provide a prolonged, control-release therapeutic effect. The dosage form may comprise an inner dosage and an outer dosage component, wherein the outer component is in the form of an envelope over the inner component. The two components may further be separated by a layer which resists disintegration in the stomach (such as an enteric layer) and permits the inner component to pass intact into the duodenum or a layer which delays or sustains release. A variety of enteric and non-enteric layer or coating materials (such as polymeric acids, shellacs, acetyl alcohol, and cellulose acetate or combinations thereof) may be used.

Compounds of the invention may also be administered via a slow release composition; wherein the composition includes a compound of the invention and a biodegradable slow release carrier (e.g., a polymeric carrier) or a pharmaceutically acceptable non-biodegradable slow release carrier (e.g., an ion exchange carrier). Biodegradable and non-biodegradable slow release carriers are well known in the art. Biodegradable carriers are used to form particles or matrices which retain an active agent(s) and which slowly degrade/dissolve in a suitable environment (e.g., aqueous, acidic, basic and the like) to release the agent. Such particles degrade/dissolve in body fluids to release the active compound(s) therein. The particles are preferably nanoparticles (e.g., in the range of about 1 to 500 nm in diameter, preferably about 50-200 nm in diameter, and most preferably about 100 nm in diameter). In a process for preparing a slow release composition, a slow release carrier and a compound of the invention are first dissolved or dispersed in an organic solvent. The resulting mixture is added into an aqueous solution containing an optional surface-active agent(s) to produce an emulsion. The organic solvent is then evaporated from the emulsion to provide a colloidal suspension of particles containing the slow release carrier and the compound of the invention.

The compounds of the invention may be incorporated for administration orally or by injection in a liquid form such as aqueous solutions, suitably flavored syrups, aqueous or oil suspensions, flavored emulsions with edible oils such as cottonseed oil, sesame oil, coconut oil or peanut oil and the like, or in elixirs or similar pharmaceutical vehicles. Suitable dispersing or suspending agents for aqueous suspensions, include synthetic and natural gums such as tragacanth, acacia, alginate, dextran, sodium carboxymethylcellulose, methylcellulose, polyvinylpyrrolidone, and gelatin. The liquid forms in suitably flavored suspending or dispersing agents may also include synthetic and natural gums. For parenteral administration, sterile suspensions and solutions are desired. Isotonic preparations, which generally contain suitable preservatives, are employed when intravenous administration is desired.

The compounds may be administered parenterally via injection. A parenteral formulation may consist of the active ingredient dissolved in or mixed with an appropriate inert liquid carrier. Acceptable liquid carriers usually comprise aqueous solvents and other optional ingredients for aiding solubility or preservation. Such aqueous solvents include sterile water, Ringer's solution, or an isotonic aqueous saline solution. Other optional ingredients include vegetable oils (such as peanut oil, cottonseed oil, and sesame oil), and organic solvents (such as solketal, glycerol, and formyl). A sterile, non-volatile oil may be employed as a solvent or suspending agent. The parenteral formulation is prepared by dissolving or suspending the active ingredient in the liquid carrier whereby the final dosage unit contains from 0.005 to 10% by weight of the active ingredient. Other additives include preservatives, isotonizers, solubilizers, stabilizers, and pain-soothing agents. Injectable suspensions may also be prepared, in which case appropriate liquid carriers, suspending agents and the like may be employed.

The following examples are intended to illustrate various embodiments of the present invention and are not intended in any way to restrict the scope thereof.

EXEMPLIFICATION

EXAMPLE 1

Screening Assays

Compounds and Cells. Compounds from Chembridge and Lopac 1280 small molecule libraries (designated Compound I- VII) were subjected to two screening assays. Screening included the measurement of the induction of endogenous was HIF- lα measured and the secretion of erythropoietin (a prototypical HIF-dependent gene product and a key regulator of angiogenesis).

Procedure. Human hepatoma cell line Hep3B cultures were treated with 10 microgram/mL of each compound for 20 hrs followed by harvesting cells and collecting conditioned media. Cellular protein lysates were subjected to gradient PAGE and Western blot analysis for HIF- lα using monoclonal antibody (BD Transduction Labs). Culture media were analyzed separately for levels of erythropoietin using commercially available ELISA kit from R&D Systems. The results of the screening are shown in FIG. 1.

EXAMPLE 2 Chemical Synthesis

The compounds of the invention are prepared by processes known in the art for the synthesis of hydrazones. Detailed descriptions about the preparation and purification of hydrazone compounds suitable for the invention can be found in PCT Application Number PCT/EP/2005/009902 (International Publication NumberWO/2006/029850) and PCT/US/2002/14106 (International Publication NumberWO/2002/089809), the entire teachings of which are incorporated herein by reference.

For example, the hydrazone derivative of general formula C can be obtained by the reaction of an aldehyde or ketone of Formula A with a compound of Formula B, whereby the substituents Ar₁, Ar₂ and R¹ are defined as in the foregoing.

A B C

A representative synthesis of compounds of Formulas I-IV is the synthesis of Compound VI, which follows the above general scheme, and has been disclosed in WO/2002/089809, page 33, Example 15. However, other synthetic pathways are not excluded and are also within the knowledge of a person skilled in the art.

In the compounds I, II, II and VI of the reaction equation Ar₁ is preferably selected from the group consisting of substituted or unsubstituted phenyl or pyridinyl groups and Ar₂ is preferably selected from the group consisting of substituted or unsubstituted [l,2,5]oxadiazolo[3,4-b]pyrazine, 5H- [1 ,2,4]triazino[5,6-b]indole and triazine groups.

EXAMPLE 3

Reversal of Phase I ROP Using DMOG in Mouse Model Murine ROP Model:

All animal experiments were performed in strict adherence to the ARVO Statement for the Use of Animals in Ophthalmic and Vision Research and in accordance with the National Institutes of Health guidelines. Animal protocols have been reviewed and approved by the Cleveland Clinic Animal Research Committee. Oxygen-induced retinopathy was obtained in mouse (C57BL6) pups according to a protocol previously established for the mouse model of ROP (Smith, Wesolowski et al. 1994). In brief, at P7, rodent pups and their nursing mothers were exposed to hyperoxic conditions (75% oxygen) for 5 days in an plexiglass incubator with adjustable oxygen sensor and feedback system (Pro-Ox, Redfield, N. Y.). On P 12, the pups returned to room-air (normoxic) conditions for 5 days until P 17. Age- matched animals were also maintained in room air (normoxic conditions) for the duration of the experiment. DMOG injections were administered

using a stock solution of DMOG prepared in PBS, sterile filtered and maintained in lOmg s/ml concentration. Injections were given 24h prior to hyperoxia (P6) and again at P8. PBS injections were given in equal volumes as the DMOG animals. Litters were divided randomly between control and experimental groups. Control animals were prepared each time for each experiment so that all litters were divided equally and had the same nursing dam.

Quantification of Vasobliteration and Angiogenesis Animals were anesthetized day P17 by intraperitoneal injection of Ketanest (1%), Rompun (0.1%) and sodium chloride (0.9%) in a concentration of 0.1 ml/1 Og mouse bodyweight. The heart was exposed through the diaphragm, and the left ventricle injected with 0.5cc of 0.1% Evans Blue dye through a 30 gauge needle and allowed to perfuse tissues for 5 minutes. The systemic spread of dye was confirmed by the presence of blue color throughout the skin and tail of the mice. Animals were euthanized with a lethal dose of intracardiac anesthetic and the eyes enucleated and placed in fresh 4% paraformaldehyde overnight. Eyes cups were dissected and retinal flatmounts created and examined under fluorescent microsopy. The relative avascular to vascular area was calculated using an Image Pro computer program (ProFormv5.0). Hyperfluorescent tufts confirmed to be neovascularization above the internal limiting lamina were counted using an image program for each eye. Six eyes (three animals) were used at each dose of PHD inhibitor. RESULTS

Known inducers of HIF activation, desferoxamine (DFO), and dimethyloxalylglycine (DMOG), Formula VIII, were tested in the mouse model of ROP.

Intraperitoneal injection of DMOG, at 100 ug/kg one day prior to hyperoxic phase I, almost completely prevented capillary regression. Furthermore, intraperitoneal injection of DMOG resulted in the rescue of retinal vasculature during phase I, which lead to the normalization of ROP in phase II, e.g., no exaggerated neovascularization, tufting, or vasodilatation (plus disease).

FIGs 2 through 5 which graphically depict the rise in VEGF and Epo in response to injections of DMOG. As shown in FIG. 2, the concentration of VEGF after 6 hours was nearly double the initial amount. The concentration of VEGF increase is proportional to the increase in dose of DMOG (FIG. 3). Even more surprising is the increase in Epo concentration after the administration of DMOG. As seen in FIGs. 4 and 5, the concentration of Epo dramatically increases with the increased dose of DMOG. With a single injection of DMOG, the increased concentrations of VEGF and/or Epo resulted in the rescue of the retinal vasculature.

Intraperitoneal injection of DFO, at 100 ug/kg one day prior to hyperoxic phase I, did not prevent capillary regression.

Table 1

XVI 5 -chloro-7-iodoquinolin- 8-ol

XVII quinolin-8-ol

XVIII ciclopirox olamine

XIX 8-methyl-pyridoxatin

XX N-oxalylglycine

XXI dimethyl-N-oxalylglycine

XXII N-oxalyl-2 S -alanine

Compounds according to Formulae I through VII can be purchased from ChemBridge Corporation (http://chembridge.com/chembridge/), 16981 Via Tazon, Suite G; San Diego, CA 92127. The compound according to Formula VIII can be purchased from Frontier Scientific, Inc.; P.O. Box 31; Logan, UT 84323-0031.

While this invention has been particularly shown and described with references to example embodiments thereof, it will be understood by those skilled in the art that various changes in form and details may be made therein without departing from the scope of the invention encompassed by the appended claims.

Claims

CLAIMSWhat is claimed is:

1. A method of treating a subject with an ischemic eye disease associated with the down-regulation of HIF comprising administering to the subject an effective amount of a compound that induces HIF activity.

2. The method of claim 1, wherein the compound that induces HIF activity is selected from the group consisting of compounds according to Formulae I through IV, or a pharmaceutically acceptable salt thereof.

3. The method of claim 1, wherein the compound that induces HIF activity is a compound according to the following formula:

wherein: R ₅ R , and R are each independently hydrogen or Ci-C₈ aliphatic group; or a pharmaceutically acceptable salt thereof.

4. The method of claim 3, wherein the compound that induces HIF activity is according to Formula VIII:

or a pharmaceutically acceptable salt thereof.

5. The method of Claim 1 , wherein the compound is represented by the following structural formula:

₅ wherein each R¹ is independently hydrogen or (Ci-C₆)alkyl; Ar₁ is phenyl or pyridinyl, each optionally substituted with one to four substituents selected from the group consisting of halogen, hydroxy, nitro, carboxy, cyano, amino, (Ci-C₆)alkyl, halo(Ci-C₆)alkyl, (Ci- C₆)alkoxy, halo(Ci-C₆)alkoxy, hydroxy(C_rC₆)alkyl, (Ci- C₆)alkylcarbonyl, (Ci-C₆)alkoxycarbonyl, (Ci-C₆)alkylamino and di(Cp C₆)alkylamino;

Ar₂ is aryl or heteroaryl, each optionally substituted with one to four substituents selected from the group consisting of halogen, hydroxy, nitro, carboxy, cyano, amino, (C]-C₆)alkyl, halo(Ci-C₆)alkyl, (Ci- C₆)alkoxy, halo(Ci-C₆)alkoxy, hydroxy(Ci-C₆)alkyl, (Ci- C₆)alkylcarbonyl, (Ci-C₆)alkoxycarbonyl, (Ci-C₆)alkylamino, di(Ci-

C₆)alkylamino and -X-Ar₃;

Ar₃ is phenyl, optionally substituted with one to four substituents selected from the group consisting of halogen, hydroxy, nitro, carboxy, cyano, amino, (Ci-C₆)alkyl, halo(d-C₆)alkyl, (d-C₆)alkoxy, halo(Ci- C₆)alkoxy, hydroxy(C i -C₆)alkyl, (C i -C₆)alkylcarbonyl, (C , -

C₆)alkoxycarbonyl, (Ci-C₆)alkylamino and di(Ci-C₆)alkylamino; and X is a bond or -NR¹-; or a pharmaceutically acceptable salt thereof.

6. The method of Claim 5, wherein the compound is represented by the following structure:

, wherein each R² is independently halogen, hydroxy, nitro, carboxy, cyano, amino, (Ci-C₆)alkyl, halo(Ci-C₆)alkyl, (d-C₆)alkoxy, halo(Ci-C₆)alkoxy, hydroxy(Ci-C₆)alkyl, (Ci-C₆)alkylcarbonyl, (Ci-C₆)alkoxycarbonyl, (C₁- C₆)alkylamino or di(Ci-C₆)alkylamino; each R³ is independently halogen, hydroxy, nitro, carboxy, cyano, amino, (d-C₆)alkyl, halo(Ci-C₆)alkyl, (Ci-C₆)alkoxy, halo(Ci-C₆)alkoxy, hydroxy(C]-C₆)alkyl, (Ci-C₆)alkylcarbonyl, (Ci-C₆)alkoxycarbonyl, (Ci- C₆)alkylamino or di(Ci-C₆)alkylamino; m is 0, 1, 2, 3 or 4; and n is O, 1, 2, 3 or 4; or a pharmaceutically acceptable salt thereof.

7. The method of Claim 6, wherein each R² is independently halogen, hydroxy, amino, (Ci-C₃)alkyl, halo(Ci-

C₃)alkyl, (Ci-C₃)alkoxy, halo(Ci-C₃)alkoxy, hydroxy(d-C₃)alkyl, (C _r C3)alkylamino or di(Ci-C₃)alkylamino; each R³ is independently halogen, hydroxy, amino, (Ci-C₃)alkyl, halo(Ci- C₃)alkyl, (Ci-C₃)alkoxy, halo(d-C₃)alkoxy, hydroxy(d-C₃)alkyl, (Ci- C₃)alkylamino or di(Ci-C₃)alkylamino; m is 0, 1 or 2; and n is 0, 1 or 2.

8. The method of Claim 7, wherein each R² is independently bromo, chloro hydroxy, (Ci-C₃)alkyl, (Ci-

C3)alkoxy, or di(Ci-C₃)alkylamino; and each R³ is independently bromo, chloro, hydroxy, (Ci-C₃)alkyl, (Ci-

C₃)alkoxy, or di(C]-C₃)alkylamino.

9. The method of Claim 8, wherein each R² is independently hydroxy or methoxy; each R³ is methyl; and m and n are 2.

10. The method of Claim 9, wherein the compound is represented by the following structural formula:

or a pharmaceutically acceptable salt thereof.

11. The method of Claim 5, wherein the compound is represented by the following structure:

_{j wherein} each R² is independently halogen, hydroxy, nitro, carboxy, cyano, amino, (Ci-C₆)alkyl, halo(Ci-C₆)alkyl, (Ci-C₆)alkoxy, halo(Ci-C₆)alkoxy, hydroxy(Ci-C₆)alkyl, (d-C₆)alkylcarbonyl, (Ci-C₆)alkoxycarbonyl, (Cp C₆)alkylamino or di(Ci-C₆)alkylamino; each R⁴ is independently halogen, hydroxy, nitro, carboxy, cyano, amino, (C₁-C₆)alkyl, halo(Ci-C₆)alkyl, (Ci-C₆)alkoxy, halo(Ci-C₆)alkoxy, hydroxy(Ci-C₆)alkyl, (Ci-C₆)alkylcaτbonyl, (Ci-C₆)alkoxycarbonyl, (Ci- C₆)alkylamino or di(C₁-C₆)alkylamino; m is 0, 1, 2, 3 or 4; and p is O, 1, 2, 3 or 4.

12. The method of Claim 11 , wherein each R² is independently halogen, hydroxy, amino, (Ci-C₃)alkyl, halo(Ci- C₃)alkyl, (d-C₃)alkoxy, halo(d-C₃)alkoxy, hydroxy(Ci-C₃)alkyl, (C₁-

C₃)alkylamino or di(Cj-C₃)alkylamino; each R⁴ is independently halogen, hydroxy, amino, (Ci-C₃)alkyl, halo(Ci- C₃)alkyl, (Ci-C₃)alkoxy, halo(Ci-C₃)alkoxy, hydroxy(Ci-C₃)alkyl, (C i- C₃)alkylamino or di(Ci-C₃)alkylamino; m is 0, 1 or 2 and p is 0, 1 or 2.

13. The method of Claim 12, wherein each R is independently bromo, chloro hydroxy, (C₁-C₃)alkyl, (Ci- C₃)alkoxy, or di(C_!-C₃)alkylamino; each R⁴ is independently bromo, chloro, hydroxy, (Ci-C₃)alkyl, (Ci- C₃)alkoxy, or di(Ci-C₃)alkylamino.

14. The method of Claim 13, wherein R² is hydroxy; n is 2; and p is 0.

15. The method of Claim 14, wherein the compound is represented by the following structure:

or a pharmaceutically acceptable salt thereof.

16. The method of Claim 5, wherein the compound is represented by the following structure:

, wherein

Z is N, CH or CR² each R is independently halogen, hydroxy, nitro, carboxy, cyano, amino, (Ci-C₆)alkyl, halo(Cj-C₆)alkyl, (C_rC₆)alkoxy, halo(Ci-C₆)alkoxy, hydroxy(Ci-C₆)alkyl, (Ci-C₆)alkylcarbonyl, (Ci-C₆)alkoxycarbonyl, (Ci- C₆)alkylamino or di(Ci-C₆)alkylamino; each R⁵ is independently halogen, hydroxy, nitro, carboxy, cyano, amino, (Ci-C₆)alkyl, halo(Ci-C₆)alkyl, (C_rC₆)alkoxy, halo(C]-C₆)alkoxy, hydroxy(Ci-C₆)alkyl, (Ci-C₆)alkylcarbonyl, (Ci-C₆)alkoxycarbonyl, (Ci- C₆)alkylamino or di(Ci-C₆)alkylamino; m is O, 1, 2, 3 or 4; and q is O, 1, 2, 3 or 4; or a pharmaceutically acceptable salt thereof.

17. The method of Claim 16, wherein

Z is N; each R² is halogen, hydroxy, amino, (Ci-C₃)alkyl, halo(Ci-C₃)alkyl, (Cj-

C₃)alkoxy, halo(Cj-C₃)alkoxy, hydroxy(Ci-C₃)alkyl, (Ci-C₃)alkylamino or di(Ci-C₃)alkylamino; each R⁵ is halogen, hydroxy, amino, (Ci-C₃)alkyl, halo(Ci-C₃)alkyl, (Cr

C₃)alkoxy, halo(Ci-C₃)alkoxy, hydroxy(Ci-C₃)alkyl, (Ci-C₃)alkylamino or di(Ci-C₃)alkylamino; m is 0, 1 or 2 and q is 0, 1 or 2.

18. The method of Claim 17, wherein each R² is independently bromo, chloro hydroxy, (Ci-C₃)alkyl, (Ci-

C₃)alkoxy, or di(C_!-C₃)alkylamino; and each R⁵ is independently bromo, chloro, hydroxy, (Ci-C₃)alkyl, (Ci-

C₃)alkoxy, or di(Ci-C₃)alkylamino.

19. The method of Claim 18, wherein R⁵ is methyl; m is 0; and q is 0 or 1.

20. The method of Claim 19, wherein the compound is represented by the following structure:

or a pharmaceutically acceptable salt thereof.

21. A method of treating a subject with an ischemic eye disease selected from the group consisting of phase I retinopathy of prematurity, diabetic retinopathy, venous occlusive disease, dry form of age related macular degeneration (ARMD), comprising administering to the subject an effective amount of a compound represented by the following structural formula:

wherein

Z is N, CH or CR² each R² is independently halogen, hydroxy, nitro, carboxy, cyano, amino, (Ci-C₆)alkyl, halo(d-C₆)alkyl, (C,-C₆)alkoxy, halo(C,-C₆)alkoxy, hydroxy(Ci-C₆)alkyl, (C]-C₆)alkylcarbonyl, (Ci-C₆)alkoxycarbonyl, (Ci- C₆)alkylamino or di(Ci-C₆)alkylamino; each R⁵ is independently halogen, hydroxy, nitro, carboxy, cyano, amino, (Ci-C₆)alkyl, halo(C_rC₆)alkyl, (C,-C₆)alkoxy, halo(d-C₆)alkoxy, hydroxy(C_!-C₆)alkyl, (Ci-C₆)alkylcarbonyl, (Ci-C₆)alkoxycarbonyl, (Ci- C₆)alkylamino or di(Cι-C₆)alkylamino; m is 0, 1, 2, 3 or 4; and q is O, 1, 2, 3 or 4; or a pharmaceutically acceptable salt thereof.

22. The method of Claim 21 , wherein Z is N; each R² is halogen, hydroxy, amino, (Ci-C₃)alkyl, halo(Ci-C₃)alkyl, (Ci- C₃)alkoxy, halo(C₁-C₃)alkoxy, hydroxy(Ci-C₃)alkyl, (Ci-C₃)alkylamino or di(Ci-C₃)alkylamino; each R⁵ is halogen, hydroxy, amino, (Ci-C₃)alkyl, halo(Ci-C₃)alkyl, (Ci- C₃)alkoxy, halo(Ci-C₃)alkoxy, hydroxy(Ci-C₃)alkyl, (Ci-C₃)alkylamino or di(Ci-C₃)alkylamino; m is 0, 1 or 2 and q is 0, 1 or 2.

23. The method of Claim 22, wherein each R² is independently bromo, chloro hydroxy, (Ci-C₃)alkyl, (Ci-

C₃)alkoxy, or di(Ci-C₃)alkylamino; and each R⁵ is independently bromo, chloro, hydroxy, (Ci-C₃)alkyl, (Ci-

C₃)alkoxy, or di(Ci-C₃)alkylamino.

24. The method of Claim 23, wherein m and q are 0.

25. The method of Claim 24, wherein the compound is represented by the following structure:

or a p armaceutically acceptable salt thereof.

26. The method of Claim 1 , wherein the compound is represented by the following structure:

or a pharmaceutically acceptable salt thereof.

27. A method of treating a subject with an ischemic eye disease selected from the group consisting of phase I retinopathy of prematurity, diabetic retinopathy, venous occlusive disease, dry form of age related macular degeneration (ARMD), comprising administering to the subject an effective amount of a compound selected from the group consisting of the following structural formulas:

or a pharmaceutically acceptable salt thereof.

28. The method of Claim 1 or 27, wherein the ischemic eye disease is phase I retinopathy of prematurity.

29. The method of Claim 1 or 27, wherein the ischemic eye disease is diabetic retinopathy.

30. The method of Claim 1 or 27, wherein the ischemic eye disease is venous occlusive disease.

31. The method of Claim 1 or 27, wherein the ischemic eye disease is the dry form of age related macular degeneration (ARMD).

32. The method of any one of Claims 28 to 31, wherein an effective amount of the compound is administered to the eye of the subject.

33. The method of Claim 28, wherein the subject is a premature infant, at risk for developing ROP.

34. The method of Claim 28, wherein the subject is a low-birth weight infant, at risk for developing ROP.

35. The method of Claim 29, wherein the subject is a human with diabetes.

36. The method of Claim 29, wherein the subject is a human at risk for developing diabetes.

37. The method of Claim 31 , wherein the subject is a human over age 52, at risk for developing ARMD.

38. A pharmaceutical composition comprising a compound according to Formulae I through VII or Formulas 1-5, or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable carrier, diluent or excipient.

39. The method of Claim 1 or 27, wherein the compound is administered intravenously, orally or as an ophthalmic composition.

40. A method of treating a subject with an ischemic eye disease associated with the down-regulation of HIF comprising administering to the subject an effective amount of a compound that induces EPO activity.