WO2015167567A1

WO2015167567A1 - Stat3 inhibitors for treatment of hyperglycemia and impaired spatial vision

Info

Publication number: WO2015167567A1
Application number: PCT/US2014/036314
Authority: WO
Inventors: Rafal Farjo; Phillip VANLANDINGHAM; Alexander B. QUIAMBAO; Didier J. NUNO; Ronald Andrew WASSEL; Jodi Green
Original assignee: Charlesson Llc
Priority date: 2014-05-01
Filing date: 2014-05-01
Publication date: 2015-11-05

Abstract

Provided are methods of treating hyperglycemia using a STAT3 inhibitor. Also provided is a STAT3 inhibitor for use in treating hyperglycemia. Further provided are methods of treating impaired spatial vision using a STAT3 inhibitor. Also provided is a STAT3 inhibitor for use in treating impaired spatial vision. In certain embodiments, the STAT3 inhibitor is administered systemically. In certain embodiments, the STAT3 inhibitor is administered orally. In certain embodiments, the STAT3 inhibitor is a small molecule. In certain embodiments, the STAT3 inhibitor is 1-acetyl-5 -hydroxyanthracene- 9,10-dione (CLT-005) or a pharmaceutically acceptable salt thereof. Also provided is a pharmaceutical composition comprising CLT-005, or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable carrier, formulated for oral administration.

Description

STAT3 INHIBITORS FOR TREATMENT OF HYPERGLYCEMIA AND

IMPAIRED SPATIAL VISION

BACKGROUND OF THE INVENTION

Signal transducer and activator of transcription 3 (STAT3) is a transcription factor that exerts a positive effect to promote expression of several angiogenic growth factors, such as vascular endothelial growth factor (VEGF) and platelet-derived growth factor (PDGF), and it has been shown to be constitutively active in several tumors and

transformed cell types. Upon phosphorylation at tyrosine 705 (Y705), STAT3 monomers dimerize and translocate to the cell nucleus to exert an effect on gene expression. Activated STAT3 (pSTAT3) is known to positively regulate VEGF through a STAT3-binding site on the VEGF promoter. Additionally, pSTAT3 also causes an upregulation of several proinflammatory molecules, such as tumor necrosis factor-alpha (TNF-a), interleukin 6 (IL-6), IL-10, IL-12, and monocyte chemotactic protein-1 (MCP-1). Several of the downstream targets of STAT3, including IL-6 and IL-10, are ligands for Jak/gpl30 receptors and produce positive feedback to amplify the inflammatory response.

Several studies have demonstrated that inflammation plays a crucial role in the pathogenesis of retinal diseases and age-related macular degeneration (AMD). These eye diseases have several characteristics of chronic inflammation, such as increased nitric oxide production, intracellular adhesion molecule- 1 (ICAM-1) up-regulation, leukostasis, and increased vascular permeability. It has been shown that patients with proliferative retinopathies have elevated serum pro-inflammatory markers, such as TNF-a, C-reactive peptide (CRP), interleukin-1 (IL-1), IL-6, IL-18, soluble ICAM-1, and circulating vascular cell adhesion molecule- 1 (VCAM-1). Induction of MCP-1, IL-8, and TNF-a is also implicated in ischemia-induced retinal neovascularization.

U.S. Patent Application Publication No. 2011/0195509 to Pardoll et al. discloses methods for treating autoimmune disease using an inhibitor of STAT3. STAT3 inhibitors are disclosed in this reference to include antisense RNA, RNAi, antibodies, and small molecules, although not a single example of any actual STAT3 inhibitor is provided.

Lin L et al. (2010) Neoplasia 12(l):39-50 discloses a small molecule LLL12 (5- hydroxy-9, 10-dioxo-8a,9, 10,1 Oa-tetrahydroanthracene- 1 -sulfonimidic acid) as an inhibitor of STAT3 phosphorylation that exhibits growth suppressive activity in human cancer cells. Zhang X et al. (2012) Proc Natl Acad Sci USA 109(24):9623-8 discloses another small molecule BP-1-102 (4-(N-(4-cyclohexylbenzyl)-2-(2,3,4,5,6-pentafluoro-N- methylphenylsulfonamido)acetamido)-2-hydroxybenzoic acid) also as an inhibitor of STAT3 phosphorylation that exhibits growth suppressive activity in human cancer cells.

U.S. Patent No. 8,058,316 to Farjo discloses yet another small molecule inhibitor of

STAT3 phosphorylation, CLT-005 (l-acetyl-5-hydroxyanthracene-9,10-dione) and its use to treat certain retinal diseases, characterized by at least one of inflammation, angiogenesis, or neovascularization, by intravitreal administration (i.e., direct injection into the eye).

In contrast to the foregoing, there are also reports in the literature indicating that STAT3 activation in the eye can be protective to photoreceptors. See, for example, Ueki Y et al. (2008) JNeurochem 105(3):784-96; and Chollangi S et al. (2009) Neurobiol Dis. 34(3):534-44.

SUMMARY OF THE INVENTION It has now been discovered, in accordance with the instant invention, that STAT3 inhibitors are useful for the treatment of hyperglycemia, as well as for the treatment of hyperglycemia-induced or diabetes-related complications such as nephropathy.

It has also now been discovered, in accordance with the instant invention, that STAT3 inhibitors are useful for the treatment of impaired spatial vision, e.g., to improve any one or more of spatial frequency threshold, contrast threshold, and contrast sensitivity. Surprisingly, it has been found that the beneficial effect of STAT3 inhibitors on these measures of spatial vision is independent of retinal inflammation, neovascularization, and/or vascular leakage.

It has also been discovered, in accordance with the instant invention, that STAT3 inhibitors are useful for the treatment of impaired spatial vision, even when administered systemically or orally.

It has further been discovered, in accordance with the instant invention, that, surprisingly, the small molecule STAT3 inhibitor, CLT-005, is useful in the treatment of a retinal disease, wherein the retinal disease is characterized by at least one of inflammation, angiogenesis, or neovascularization, when the small molecule STAT3 inhibitor is administered systemically.

It has further been discovered, in accordance with the instant invention, that, surprisingly, the small molecule STAT3 inhibitor, CLT-005, is useful in the treatment of a retinal disease, wherein the retinal disease is characterized by at least one of inflammation, angiogenesis, or neovascularization, when the small molecule STAT3 inhibitor is administered orally.

An aspect of the invention is a method of treating hyperglycemia. The method includes the step of administering to a subject in need thereof a therapeutically effective amount of an inhibitor of signal transducer and activator of transcription 3 (STAT3). In an embodiment, the inhibitor of STAT3 is a small molecule, i.e., a molecule having a molecular weight of less than or equal to about 1,500 Daltons.

An aspect of the invention is a method of treating hyperglycemia. The method includes the step of administering to a subject in need thereof an effective amount of 1- acetyl-5-hydroxyanthracene-9, 10-dione (CLT-005).

An aspect of the invention is a method of improving at least one measure of spatial vision selected from the group consisting of spatial frequency threshold, contrast threshold, and contrast sensitivity. The method includes the step of administering to a subject in need thereof a therapeutically effective amount of an inhibitor of signal transducer and activator of transcription 3 (STAT3). In an embodiment, the inhibitor of STAT3 is a small molecule.

An aspect of the invention is a method of improving at least one measure of spatial vision selected from the group consisting of spatial frequency threshold, contrast threshold, and contrast sensitivity. The method includes the step of administering to a subject in need thereof an effective amount of l-acetyl-5-hydroxyanthracene-9,l 0-dione (CLT-005).

In accordance with each of the aspects and embodiments above, in an embodiment the administering is systemically administering.

In accordance with each of the aspects and embodiments above, in an embodiment the administering is orally administering.

An aspect of the invention is a pharmaceutical composition. The pharmaceutical composition includes l-acetyl-5-hydroxyanthracene-9,l 0-dione (CLT-005), or a

pharmaceutically acceptable salt thereof, and a pharmaceutical carrier, wherein the composition is formulated for oral administration. BRIEF DESCRIPTION OF THE DRAWINGS

Fig. 1 is a collage of six photomicrographs depicting pSTAT nuclear translocation in human umbilical vein endothelial cells (HUVECs) treated in vitro with the reagents shown. Fig. 2 A is a bar graph depicting contrast sensitivity threshold in mice in a mouse model of dry age-related macular degeneration (dry AMD). CEP-MSA,

carboxyethylpyrrole-mouse serum albumin. Fig. 2B is a collage of Western blots of retina tissue from naive and CEP-MSA immunized mice, depicting pSTAT3, STAT3, or actin (control).

Fig. 3A is a collage of fundus photographs (top two rows) and fundus angiograms (bottom row) from BN rats with laser-induced retinal injury. Rats were treated with the indicated agents. Bubbles indicate successful laser application. Fig. 3B is a bar graph depicting lesion size (area) for rats treated orally with vehicle or the indicated

concentrations of CLT-005. *, p<0.05 (Student's t-test). Fig. 3C is a bar graph depicting vessel growth around lesion areas. *, p < 0.05; * *, p < 0.01 (Student's t-test).

Fig. 4A is a bar graph depicting spatial frequency threshold as a function of time (weeks) in streptozotocin (STZ)-diabetic and buffer control rats. Time 0 = administration of STZ or buffer. Significance represents comparison between controls and STZ diabetics at each time point using a one-way ANOVA. * = p < 0.05; * * *, p < 0.0001. Fig. 4B is a bar graph depicting grating contrast threshold as a function of time (weeks) in

streptozotocin (STZ)-diabetic and buffer control rats. Time 0 = administration of STZ or buffer. The y-axis represents the log of the raw threshold percentages of the grating contrast. Significance represents comparison between controls and STZ diabetics at each time point using a one-way ANOVA. * =/? < 0.05; ***, /? < 0.0001. Fig. 4C is a graph depicting contrast threshold as in Fig. 4B over indicated spatial frequencies. CTL, control; Db, diabetic. Fig. 4D is a bar graph depicting scotopic responses in diabetic and control rats. * * *, p < 0.0001 (Student's t-test). Fig. 4E is a bar graph depicting photopic b-wave responses in diabetic and control rats. p < 0.0001 (Student's t-test).

Fig. 5A is a graph depicting body weights of na^'ive and STZ-diabetic rats treated with the indicated agents by daily oral gavage over 16 weeks. Fig. 5B is a bar graph depicting plasma concentration of CLT-005 in STZ-diabetic rats after 16 weeks of daily oral gavage with the indicated doses of CLT-005. **, /? < 0.01 by one-way ANOVA. Fig. 5C is a graph depicting non- fasting blood glucose levels in STZ-diabetic rats at baseline and after 16 weeks of daily oral gavage with the indicated agents. Fig. 5D is a bar graph depicting cumulative average weekly non-fasting blood glucose levels in STZ-diabetic rats collected over 16 weeks of daily oral gavage with the indicated agents. p > 0.0001 by one-way ANOVA with Tukey post-test. Fig. 6A is a graph depicting spatial frequency threshold (SFT) over time in naive and STZ-diabetic rats treated with the indicated agents. Fig. 6B is a bar graph depicting average SFT values within each group at week 16. Comparison is made to the vehicle- treated group. *, p < 0.05; ***, p < 0.0001 by one-way ANOVA.

Fig. 7A is a graph depicting contrast thresholds over time in naive and STZ-diabetic rats treated with the indicated agents. STZ-diabetic animals receiving 500 mg/kg CLT-005 retained their ability to distinguish contrast over the course of 16 weeks at levels comparable to insulin rescued STZ-diabetic rats. Fig. 7B is a bar graph depicting average contrast threshold values within each group at week 16. Comparison is made to the vehicle-treated group. *, p < 0.05 by one-way ANOVA. Fig. 7C is a graph depicting contrast sensitivity measured over a range of spatial frequency values at 16 weeks post-STZ in STZ-diabetic rats treated with the indicated agents. Across all measured spatial frequencies, the group of animals receiving 500 mg/kg CLT-005 showed significantly higher contrast thresholds compared to vehicle treated animals.

Fig. 8 A is a collage of 6 photographic images of cataracts from naive and STZ- diabetic rats treated with the indicated agents. Fig. 8B is a graph depicting cataract score over time in naive and STZ-diabetic rats treated with the indicated agents. Fig. 8C is a bar graph depicting cataract scores and SFT at week 12 post-STZ for STZ-diabetic rats treated with the indicated agents. Numbers within each bar represent the average cataract score. Fig. 8 D is a bar graph depicting SFT of all eyes with a cataract score of 2 plotted independent of the remaining SFT values within each group at week 12. The data shows that even when the most severe cataracts are plotted, there is no direct effect on SFT values across groups. Fig. 8E is a scatter plot depicting each eye with a cataract score of 2 in either the vehicle or 500 mg/kg CLT-005 group. The data shows that even in animals with the same cataract scores, the group receiving 500 mg/kg CLT-005 maintain higher SFTs.

Fig. 9A is a bar graph depicting CLT-005 in retina after daily gavage for 16 weeks with the indicated dosing of CLT-005. Fig. 9B is a bar graph depicting CLT-005 in pigment epithelium, choroid, and sclera taken together (PECS) after daily gavage for 16 weeks with the indicated dosing of CLT-005.

Fig. 10 is a bar graph depicting kidney cortical tubular vacuolization in naive and

STZ-diabetic rats treated for 16 weeks with vehicle, insulin, or CLT-005 in the indicated doses. DETAILED DESCRIPTION OF THE INVENTION

STAT3 is a member of the STAT protein family. In response to cytokines and growth factors, STAT family members are phosphorylated by receptor-associated kinases, and then form homo- or heterodimers that translocate to the cell nucleus where they act as transcription activators. STAT3 is activated through phosphorylation in response to various cytokines and growth factors including IFNs, EGF, IL-5, IL-6, HGF, LIF, and BMP2. STAT3 mediates the expression of a variety of genes in response to cell stimuli, and thus plays a key role in many cellular processes such as cell growth and apoptosis. The small GTPase Racl has been shown to bind and regulate the activity of this protein. Three alternatively spliced transcript variants encoding distinct iso forms have been described.

Isoform 1 of human STAT3 is an 88 kDa, 770-amino acid polypeptide having an amino acid sequence provided by GenBank Accession No. NP 644805. Upon

phosphorylation of tyrosine 705 (Tyr705; Y705), activated pSTAT3 monomers dimerize and translocate to the cell nucleus, where they exert their transcription activator activity. The dimerization is believed to involve reciprocal pTyr-Src homology 2 (SH2) domain interaction.

STAT3 has been described as an important transcription factor in cancer. In this context, STAT3 induces the transcription and up-regulation of proliferation and

antiapoptotic proteins. It has also been reported to promote angiogenesis and impair immune responses.

STAT3 has also been described as an important transcription factor in a number of cell-mediated autoimmune diseases, including arthritis and multiple sclerosis.

Leptin is an adipocyte-derived cytokine that has been linked to obesity in both humans and other animal models. Activation of the leptin receptor (Lep-R), a member of the gpl30 receptor family, triggers a cascade of phosphorylation events that lead to changes in cellular gene expression. Leptin stimulation has been shown to exert a pro-angiogenic effect both in vitro and in vivo. Treatment of human vascular endothelial cells with leptin causes a rapid phosphorylation of STAT3, leading to angiogenesis.

An aspect of the invention is a method of treating hyperglycemia. The method includes the step of administering to a subject in need thereof a therapeutically effective amount of an inhibitor of STAT3. An inhibitor of STAT3 can include any compound that reduces the expression, activation, trafficking, or transcription factor activity of STAT3. In an embodiment, the inhibitor of STAT3 inhibits the activation of STAT3 monomers, for example by interfering with phosphorylation of Y705.

In an embodiment, the inhibitor of STAT3 is a small molecule, i.e., a molecule having a molecular weight of less than or equal to about 1 ,500 Daltons. In an embodiment, the small molecule inhibitor has a molecular weight less than or equal to about 1 ,000 Daltons. In an embodiment, the small molecule inhibitor has a molecular weight less than or equal to about 750 Daltons. In an embodiment, a small molecule inhibitor has a molecular weight less than or equal to about 500 Daltons. CLT-005 has a molecular weight of 266 Daltons.

Small molecule STAT3 inhibitors include, without limitation, compounds of formula I:

where R¹, R², R³, and R⁴ are independently selected from H, OH, alkyloxy (e.g., methoxy and ethoxy), acyl, and -S0₂NH₂.

In an embodiment, the small molecule STAT3 inhibitor is a compound of formula I wherein R¹ is H and R² is -C(0)CH₃.

In an embodiment, the small molecule STAT3 inhibitor is a compound of formula I wherein R¹ is H, R² is -C(0)CH₃, R³ is OH, and R⁴ is H (i.e., CLT-005).

In an embodiment, the small molecule STAT3 inhibitor is a compound of formula I wherein R¹ is H, R² is -C(0)CH₃, R³ is H, and R⁴ is OH.

In an embodiment, the small molecule STAT3 inhibitor is a compound of formula I wherein R¹ is H, R² is -C(0)CH₃, R³ is -OCH₃, and R⁴ is H.

In an embodiment, the small molecule STAT3 inhibitor is a compound of formula I wherein R¹ is H, R² is -C(0)CH₃, R³ is H, and R⁴ is -OCH₃.

In an embodiment, the small molecule STAT3 inhibitor is a compound of formula I wherein R¹ is H, R² is -S0₂NH₂, R³ is H, and R⁴ is OH (i.e., LLL12). Lin L et al. (2010) Neoplasia 12(l):39-50.

In an embodiment, the small molecule STAT3 inhibitor is the compound STA21 :

Lin L et al. (2010) Mo I Cancer 9:217-226.

In an embodiment, the small molecule STAT3 inhibitor is the compound XZH-5 :

Liu A et al. (2012) PLoS ONE 7(10):e46624.

In an embodim nt, the small molecule STAT3 inhibitor is the compound BP- 1-102

Zhang X et al. (2012) Proc Natl Acad Sci USA 109(24):9623-8.

For each of the foregoing, the invention also encompasses pharmaceutically acceptable salts of the compounds.

Hyperglycemia is abnormal elevation of glucose concentration in the blood. It is a hallmark feature of diabetes mellitus, both type 1 and type 2. Chronically elevated blood glucose levels are believed to give rise to a number of complications, including vascular and microvascular disease. Such complications may manifest as atherosclerotic and ischemic heart disease, retinopathy, and nephropathy, with significant associated morbidity and mortality. Conventional treatment for type 1 diabetes includes administration of exogenous insulin. Conventional treatment for type 2 diabetes includes calorie restriction, exercise, and various agents useful for increasing insulin secretion or increasing insulin sensitivity. Such agents include, for example, metformin, sulfonureas, nonsulfonurea secretagogues, alpha glucosidase inhibitors, thiazolidinediones, glucagon-like peptide- 1 analogs, and dipeptidyl peptidase IV inhibitors.

As used herein, the term "treat" means prevent, slow the progression of, halt the progression of, reduce, or eliminate at least one sign or symptom of a disease or condition of a subject. In an embodiment, the term "treat" means slow the progression of, halt the progression of, reduce, or eliminate at least one sign or symptom of a disease or condition of a subject.

The method is useful to treat hyperglycemia. In accordance with the invention, hyperglycemia is treated if the blood glucose level is at least 10 percent lower with treatment than it was or would have been without treatment. In an embodiment, the blood glucose level is 10 percent lower with treatment than it was or would have been without treatment. In various embodiments, the blood glucose level is 10 percent, 15 percent, 20 percent, 25 percent, 30 percent, 35 percent, 40 percent, 45 percent, 50 percent, or greater than 50 percent lower with treatment than it was or would have been without treatment. Blood glucose levels can be measured using any suitable method, many of which are known in the art. In an embodiment, blood glucose levels are measured using capillary blood, e.g., using so-called fingerstick monitoring.

As used herein, a "subject" is a living mammal. In an embodiment, a subject is a mouse, rat, hamster, guinea pig, rabbit, cat, dog, goat, sheep, pig, horse, cow, or non-human primate. In an embodiment, a subject is a human.

As used herein, an "effective amount" refers to an amount sufficient to achieve a desired biological effect.

As used herein, a "therapeutically effective amount" refers to an amount sufficient to achieve a desired therapeutic effect. For example, in an embodiment, a therapeutically effective amount is an amount sufficient to achieve at least a 10 percent decrease in blood glucose level compared to control. In an embodiment, the administering is systemically administering. Systemic administration can be accomplished by administering by any method to achieve clinically meaningful circulating amounts of an active pharmaceutical ingredient. For example, systemic administration includes oral administration and any of various parenteral routes of administration, including, without limitation, intravenous and intraperitoneal.

In an embodiment, the administering is orally administering. In an embodiment, orally administering encompasses administering by gavage.

In an embodiment, the subject has type 1 diabetes mellitus.

In an embodiment, the subject has type 2 diabetes mellitus.

In an embodiment, the administering is systemically administering.

In an embodiment, the subject has type 1 diabetes mellitus.

In an embodiment, the subject has type 2 diabetes mellitus.

In an embodiment, the subject is a human.

An aspect of the invention is a method of improving at least one measure of spatial vision selected from the group consisting of spatial frequency threshold, contrast threshold, and contrast sensitivity. The method includes the step of administering to a subject in need thereof a therapeutically effective amount of an inhibitor of signal transducer and activator of transcription 3 (STAT3).

Alternatively, an aspect of the invention is a method of treating impaired spatial vision. The method includes the step of administering to a subject in need thereof a therapeutically effective amount of an inhibitor of signal transducer and activator of transcription 3 (STAT3) to improve at least one measure of spatial vision selected from the group consisting of spatial frequency threshold, contrast threshold, and contrast sensitivity.

In an embodiment, the inhibitor of STAT3 is a small molecule.

In an embodiment, the administering is systemically administering.

In an embodiment, the subject has type 1 diabetes mellitus. In an embodiment, the subject has type 2 diabetes mellitus.

In an embodiment, the subject is a human.

In an embodiment, the subject does not have a retinal disease characterized by at least one of inflammation, angiogenesis, or neovascularization.

As used herein, the term "spatial vision" refers to the ability visually to resolve or discriminate spatially defined features. Measures of spatial vision include spatial frequency threshold, contrast threshold, and contrast sensitivity. For example, the familiar use of a letter or pattern eye chart with lines of characters of varying sizes measures spatial vision. The quality of spatial vision is sometimes reported in terms of the distance in feet from a target that a subject with normal vision can discriminate compared to what a test subject can discriminate at 20 feet; normal vision is then "20/20". An adult with 20/200 vision or worse is considered blind.

As used herein, the terms "improve" and "improving" refer to effecting any objectively measurable degree or amount of improvement over control, as measured by at least any one of spatial frequency threshold, contrast threshold, and contrast sensitivity. In an embodiment, the degree or amount of improvement is at least 5 percent better than control. In an embodiment, the degree or amount of improvement is at least 10 percent better than control. In an embodiment, the degree or amount of improvement is at least 15 percent better than control. In an embodiment, the degree or amount of improvement is at least 20 percent better than control. For example, an adult with baseline 20/100 vision would be deemed "improved" at 20/95 vision.

In the study of visual perception, sinusoidal gratings are frequently used to probe the capabilities of the visual system. In these stimuli, spatial frequency is expressed as the number of cycles per degree of visual angle. Sine -wave gratings also differ from one another in amplitude (the magnitude of difference in intensity between light and dark stripes), and angle.

As used herein, the term "spatial frequency threshold" refers to the highest spatial frequency distinguished by a subject. Spatial frequency refers to the number of cycles that fall within a given distance on the retina, for example, within 1° (one degree) of visual angle as viewed by a subject. A cycle refers to one dark bar and one light bar.

As used herein, the term "contrast threshold" refers to the lowest percentage contrast at which a subject can distinguish a bar grating. Contrast refers to the intensity difference between light and dark bars of a bar grating; 100 percent contrast is the greatest possible contrast between black and white bars.

As used herein, the term "contrast sensitivity" refers to a measure of the range of contrast thresholds over different spatial frequencies.

Optokinetic tracking provides quantifiable behavorial measurements of spatial vision in a virtual environment. Animals are placed on a platform in an enclosed box surrounded by four computer monitors forming a square. Visual stimuli are presented on the computer monitors as continuous sine wave grating calculated by computer software, and rotating at 12 degrees/second. Animals are monitored for their ability to track the stimuli as it either increases in frequency, or decreases in contrast. Tracking movements are identified as slow, steady head movements in the direction of the rotating grating.

To measure spatial frequency threshold, the virtual stimuli are presented in increasing frequencies, e.g., beginning at 0.064 cycles/degree, and increasing until the animal is no longer able to distinguish the gratings. The frequency at which the animal last tracks the rotating grating is defined as the spatial frequency threshold (SFT).

To measure contrast threshold, the stimuli are presented at a constant frequency, e.g., 0.064 cycles/degree. The contrast refers to the difference between the black bars representing the stimuli, and the white background. With each positive tracking movement, the contrast of the presented stimuli decreases until the animal can no longer track. The contrast at which the animal last tracks is defined as the contrast threshold.

Using this system, contrast sensitivity refers to the contrast threshold at which an animal tracks over a range of spatial frequencies (e.g., 0.064 cycles/deg).

An aspect of the invention is a method of improving at least one measure of spatial vision selected from the group consisting of spatial frequency threshold, contrast threshold, and contrast sensitivity. The method includes the step of administering to a subject in need thereof a therapeutically effective amount of l-acetyl-5-hydroxyanthracene-9,10-dione (CLT-005).

Alternatively, an aspect of the invention is a method of treating impaired spatial vision. The method includes the step of administering to a subject in need thereof a therapeutically effective amount of l-acetyl-5-hydroxyanthracene-9,10-dione (CLT-005) to improve at least one measure of spatial vision selected from the group consisting of spatial frequency threshold, contrast threshold, and contrast sensitivity.

In an embodiment, the administering is systemically administering. In an embodiment, the administering is orally administering. In an embodiment, orally administering encompasses administering by gavage.

In an embodiment, the subject has type 1 diabetes mellitus.

In an embodiment, the subject has type 2 diabetes mellitus.

In an embodiment, the subject is a human.

An aspect of the invention is a method of treating diabetic nephropathy. The method includes the step of administering to a subject in need thereof a therapeutically effective amount of an inhibitor of signal transducer and activator of transcription 3

(STAT3).

In an embodiment, the inhibitor of STAT3 is a small molecule.

In an embodiment, the administering is systemically administering.

In an embodiment, the administering is orally administering.

In an embodiment, the subject has type 1 diabetes mellitus.

In an embodiment, the subject has type 2 diabetes mellitus.

In an embodiment, the subject is a human.

An aspect of the invention is a method of treating diabetic nephropathy. The method includes the step of administering to a subject in need thereof a therapeutically effective amount of l-acetyl-5-hydroxyanthracene-9,10-dione (CLT-005).

In an embodiment, the administering is systemically administering.

In an embodiment, the administering is orally administering.

In an embodiment, the subject has type 1 diabetes mellitus.

In an embodiment, the subject has type 2 diabetes mellitus.

In an embodiment, the subject is a human.

In accordance with each and every one of the foregoing embodiments, in an embodiment, the subject does not have a cancer which is susceptible to treatment with a STAT3 inhibitor.

In accordance with each and every one of the foregoing embodiments, in an embodiment, the subject does not have a cancer.

An aspect of the invention is a pharmaceutical composition, comprising l-acetyl-5- hydroxyanthracene-9,10-dione (CLT-005), or a pharmaceutically acceptable salt thereof, and a pharmaceutical carrier, wherein the composition is formulated for oral administration. In an embodiment, the pharmaceutical composition is formulated as a solid.

In an embodiment, the pharmaceutical composition is formulated as a pill, tablet, capsule, powder, or troche.

In an embodiment, the pharmaceutical composition is formulated as a liquid solution or liquid suspension.

Dosages & Dosing Regimens

As stated above, an "effective amount" refers to any amount that is sufficient to achieve a desired biological effect. Combined with the teachings provided herein, by choosing among the various active compounds and weighing factors such as potency, relative bioavailability, patient body weight, severity of adverse side-effects and preferred mode of administration, an effective prophylactic or therapeutic treatment regimen can be planned which does not cause substantial unwanted toxicity and yet is effective to treat the particular subject. The effective amount for any particular application can vary depending on such factors as the disease or condition being treated, the particular compound of the invention being administered, the size of the subject, or the severity of the disease or condition. One of ordinary skill in the art can empirically determine the effective amount of a particular compound of the invention and/or other therapeutic agent without necessitating undue experimentation. It is preferred generally that a maximum dose be used, that is, the highest safe dose according to some medical judgment. Multiple doses per day may be contemplated to achieve appropriate systemic levels of compounds.

Appropriate systemic levels can be determined by, for example, measurement of the patient's peak or sustained plasma level of the drug. "Dose" and "dosage" are used interchangeably herein.

For any compound described herein the therapeutically effective amount can be initially determined from animal models. A therapeutically effective dose can also be determined from human data for compounds of the invention which have been tested in humans and for compounds which are known to exhibit similar pharmacological activities, such as other related active agents. Higher doses may be required for oral administration than for parenteral administration. The applied dose can be adjusted based on the relative bioavailability and potency of the administered compound. Adjusting the dose to achieve maximal efficacy based on the methods described above and other methods as are well- known in the art is well within the capabilities of the ordinarily skilled artisan. Generally, daily intravenous doses of active compound or compounds will be from about 0.001 milligrams/kg per day to 100 milligrams/kg per day. It is expected that intravenous doses in the range of 0.05 to 5 milligrams/kg, in one or several administrations per day, will yield the desired results. Intravenous dosing on other schedules is also contemplated by the invention, e.g., every-other day, semi-weekly, weekly, biweekly, and monthly. Similar dosing for other parenteral routes of administration are also contemplated by the invention.

Generally, daily oral doses of active compound or compounds will be from about 0.01 milligrams/kg per day to 1000 milligrams/kg per day. It is expected that oral doses in the range of 0.5 to 50 milligrams/kg, in one or several administrations per day, will yield the desired results. Oral dosing on other schedules is also contemplated by the invention, e.g., every-other day, semi-weekly, weekly, biweekly, and monthly.

Dosage may be adjusted appropriately to achieve desired drug levels, local or systemic, depending upon the mode of administration. For example, it is expected that intravenous administration would be from an order to several orders of magnitude lower dose per day. In the event that the response in a subject is insufficient at such doses, even higher doses (or effective higher doses by a different, more localized delivery route) may be employed to the extent that patient tolerance permits. Multiple doses per day are contemplated to achieve appropriate systemic levels of compounds.

Pharmaceutical Formulations & Modes of Administration

The formulations of the invention are administered in pharmaceutically acceptable solutions, which may routinely contain pharmaceutically acceptable concentrations of salt, buffering agents, preservatives, compatible carriers, adjuvants, and optionally other therapeutic ingredients.

As used herein, the term "pharmaceutically acceptable salt" refers to any relatively non-toxic inorganic or organic acid addition salt of the STAT3 inhibitor. These salts can be prepared in situ during the final isolation and purification of the STAT3 inhibitor, or by separately reacting a purified STAT3 inhibitor in its free base form with a suitable organic or inorganic acid, and isolating the salt thus formed. Representative salts include the hydrobromide, hydrochloride, sulfate, bisulfate, phosphate, nitrate, acetate, valerate, oleate, palmitate, stearate, laurate, benzoate, lactate, phosphate, tosylate, citrate, maleate, fumarate, succinate, tartrate, naphthylate, mesylate, glucoheptonate, lactobionate, and laurylsulphonate salts, and the like. See, for example, Berge et al. (1977) "Pharmaceutical Salts", J. Pharm. Sci. 66: 1-19.

In other cases, the compounds of the present invention may contain one or more acidic functional groups and, thus, are capable of forming pharmaceutically acceptable salts with pharmaceutically acceptable bases. The term "pharmaceutically acceptable salt" in these instances refers to any relatively non-toxic inorganic or organic base addition salts of the STAT3 inhibitor. These salts can likewise be prepared in situ during the final isolation and purification of the STAT3 inhibitor, or by separately reacting the purified STAT3 inhibitor in its free acid form with a suitable base, such as the hydroxide, carbonate, or bicarbonate of a pharmaceutically acceptable metal cation, with ammonia, or with a pharmaceutically acceptable organic primary, secondary, or tertiary amine. Representative alkali or alkaline earth salts include the lithium, sodium, potassium, calcium, magnesium, and aluminum salts, and the like. Representative organic amines useful for the formation of base addition salts include ethylamine, diethylamine, ethylenediamine, ethanolamine, diethanolamine, piperazine, and the like (see, for example, Berge et al., supra).

The phrase "pharmaceutically acceptable excipient" or "pharmaceutically acceptable carrier" as used herein means a pharmaceutically acceptable material, composition or vehicle, such as a liquid or solid filler, diluent, excipient, solvent or encapsulating material, involved in carrying or transporting the subject chemical from one organ or portion of the body, to another organ or portion of the body. Each carrier must be "acceptable" in the sense of being compatible with the other ingredients of the formulation, not injurious to the patient, and substantially non-pyrogenic. Some examples of materials which can serve as pharmaceutically acceptable carriers include: (1) sugars, such as lactose, glucose, and sucrose; (2) starches, such as corn starch and potato starch; (3) cellulose, and its derivatives, such as sodium carboxymethyl cellulose, ethyl cellulose, and cellulose acetate; (4) powdered tragacanth; (5) malt; (6) gelatin; (7) talc; (8) excipients, such as cocoa butter and suppository waxes; (9) oils, such as peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, corn oil, and soybean oil; (10) glycols, such as propylene glycol; (11) polyols, such as glycerin, sorbitol, mannitol, and polyethylene glycol; (12) esters, such as ethyl oleate and ethyl laurate; (13) agar; (14) buffering agents, such as magnesium hydroxide and aluminum hydroxide; (15) alginic acid; (16) pyrogen- free water; (17) isotonic saline; (18) Ringer's solution; (19) ethyl alcohol; (20) phosphate buffer solutions; and (21) other non-toxic compatible substances employed in pharmaceutical formulations. In certain embodiments, pharmaceutical compositions of the present invention are non- pyrogenic, i.e., do not induce significant temperature elevations when administered to a patient.

For use in therapy, an effective amount of the compound of the invention can be administered to a subject by any mode that delivers the compound of the invention to the desired surface. Administering the pharmaceutical composition of the present invention may be accomplished by any means known to the skilled artisan. Routes of administration include but are not limited to oral, intravenous, intramuscular, intraperitoneal,

subcutaneous, direct injection (for example, into a tumor), mucosal, inhalation, and topical.

For oral administration, compounds (i.e., compounds of the invention, and other therapeutic agents) can be formulated readily by combining the active compound(s) with pharmaceutically acceptable carriers well known in the art. Such carriers enable the compounds of the invention to be formulated as tablets, pills, dragees, capsules, liquids, gels, syrups, slurries, suspensions and the like, for oral ingestion by a subject to be treated. Pharmaceutical preparations for oral use can be obtained as solid excipient, optionally grinding a resulting mixture, and processing the mixture of granules, after adding suitable auxiliaries, if desired, to obtain tablets or dragee cores. Suitable excipients are, in particular, fillers such as sugars, including lactose, sucrose, mannitol, or sorbitol; cellulose preparations such as, for example, maize starch, wheat starch, rice starch, potato starch, gelatin, gum tragacanth, methyl cellulose, hydroxypropylmethyl-cellulose, sodium carboxymethylcellulose, and/or polyvinylpyrrolidone (PVP). If desired, disintegrating agents may be added, such as the cross-linked polyvinyl pyrrolidone, agar, or alginic acid or a salt thereof such as sodium alginate. Optionally the oral formulations may also be formulated in saline or buffers, e.g., EDTA for neutralizing internal acid conditions or may be administered without any carriers.

Also specifically contemplated are oral dosage forms of the above component or components. The component or components may be chemically modified so that oral delivery of the derivative is efficacious. Generally, the chemical modification

contemplated is the attachment of at least one moiety to the component molecule itself, where said moiety permits (a) inhibition of acid hydrolysis; and (b) uptake into the blood stream from the stomach or intestine. Also desired is the increase in overall stability of the component or components and increase in circulation time in the body. Examples of such moieties include: polyethylene glycol, copolymers of ethylene glycol and propylene glycol, carboxymethyl cellulose, dextran, polyvinyl alcohol, polyvinyl pyrrolidone and polyproline. Abuchowski and Davis, "Soluble Polymer-Enzyme Adducts", In: Enzymes as Drugs, Hocenberg and Roberts, eds., Wiley-Interscience, New York, N.Y., pp. 367-383 (1981); Newmark et al, J Appl Biochem 4: 185-9 (1982). Other polymers that could be used are poly-l,3-dioxolane and poly-l,3,6-tioxocane. Preferred for pharmaceutical usage, as indicated above, are polyethylene glycol moieties.

For the component (or derivative) the location of release may be the stomach, the small intestine (the duodenum, the jejunum, or the ileum), or the large intestine. One skilled in the art has available formulations which will not dissolve in the stomach, yet will release the material in the duodenum or elsewhere in the intestine. Preferably, the release will avoid the deleterious effects of the stomach environment, either by protection of the compound of the invention (or derivative) or by release of the biologically active material beyond the stomach environment, such as in the intestine.

To ensure full gastric resistance a coating impermeable to at least pH 5.0 is essential. Examples of the more common inert ingredients that are used as enteric coatings are cellulose acetate trimellitate (CAT), hydroxypropylmethylcellulose phthalate (HPMCP), HPMCP 50, HPMCP 55, polyvinyl acetate phthalate (PVAP), Eudragit L30D, Aquateric, cellulose acetate phthalate (CAP), Eudragit L, Eudragit S, and shellac. These coatings may be used as mixed films.

A coating or mixture of coatings can also be used on tablets, which are not intended for protection against the stomach. This can include sugar coatings, or coatings which make the tablet easier to swallow. Capsules may consist of a hard shell (such as gelatin) for delivery of dry therapeutic (e.g., powder); for liquid forms, a soft gelatin shell may be used. The shell material of cachets could be thick starch or other edible paper. For pills, lozenges, molded tablets or tablet triturates, moist massing techniques can be used.

The therapeutic can be included in the formulation as fine multi-particulates in the form of granules or pellets of particle size about 1 mm. The formulation of the material for capsule administration could also be as a powder, lightly compressed plugs or even as tablets. The therapeutic could be prepared by compression.

Colorants and flavoring agents may all be included. For example, the compound of the invention (or derivative) may be formulated (such as by liposome or microsphere encapsulation) and then further contained within an edible product, such as a refrigerated beverage containing colorants and flavoring agents. One may dilute or increase the volume of the therapeutic with an inert material. These diluents could include carbohydrates, especially mannitol, a-lactose, anhydrous lactose, cellulose, sucrose, modified dextrans and starch. Certain inorganic salts may be also be used as fillers including calcium triphosphate, magnesium carbonate and sodium chloride. Some commercially available diluents are Fast-Flo, Emdex, STA-Rx 1500, Emcompress and Avicell.

Disintegrants may be included in the formulation of the therapeutic into a solid dosage form. Materials used as disintegrates include but are not limited to starch, including the commercial disintegrant based on starch, Explotab. Sodium starch glycolate,

Amberlite, sodium carboxymethylcellulose, ultramylopectin, sodium alginate, gelatin, orange peel, acid carboxymethyl cellulose, natural sponge and bentonite may all be used. Another form of the disintegrants are the insoluble cationic exchange resins. Powdered gums may be used as disintegrants and as binders and these can include powdered gums such as agar, Karaya or tragacanth. Alginic acid and its sodium salt are also useful as disintegrants.

Binders may be used to hold the therapeutic agent together to form a hard tablet and include materials from natural products such as acacia, tragacanth, starch and gelatin.

Others include methyl cellulose (MC), ethyl cellulose (EC) and carboxymethyl cellulose (CMC). Polyvinyl pyrrolidone (PVP) and hydroxypropylmethyl cellulose (HPMC) could both be used in alcoholic solutions to granulate the therapeutic.

An anti-frictional agent may be included in the formulation of the therapeutic to prevent sticking during the formulation process. Lubricants may be used as a layer between the therapeutic and the die wall, and these can include but are not limited to; stearic acid including its magnesium and calcium salts, polytetrafluoroethylene (PTFE), liquid paraffin, vegetable oils and waxes. Soluble lubricants may also be used such as sodium lauryl sulfate, magnesium lauryl sulfate, polyethylene glycol of various molecular weights, Carbowax 4000 and 6000.

Glidants that might improve the flow properties of the drug during formulation and to aid rearrangement during compression might be added. The glidants may include starch, talc, pyrogenic silica and hydrated silicoaluminate.

To aid dissolution of the therapeutic into the aqueous environment a surfactant might be added as a wetting agent. Surfactants may include anionic detergents such as sodium lauryl sulfate, dioctyl sodium sulfosuccinate and dioctyl sodium sulfonate. Cationic detergents which can be used and can include benzalkonium chloride and benzethonium chloride. Potential non-ionic detergents that could be included in the formulation as surfactants include lauromacrogol 400, polyoxyl 40 stearate, polyoxyethylene hydrogenated castor oil 10, 50 and 60, glycerol monostearate, polysorbate 40, 60, 65 and 80, sucrose fatty acid ester, methyl cellulose and carboxymethyl cellulose. These surfactants could be present in the formulation of the compound of the invention or derivative either alone or as a mixture in different ratios.

Pharmaceutical preparations which can be used orally include push- fit capsules made of gelatin, as well as soft, sealed capsules made of gelatin and a plasticizer, such as glycerol or sorbitol. The push-fit capsules can contain the active ingredients in admixture with filler such as lactose, binders such as starches, and/or lubricants such as talc or magnesium stearate and, optionally, stabilizers. In soft capsules, the active compounds may be dissolved or suspended in suitable liquids, such as fatty oils, liquid paraffin, or liquid polyethylene glycols. In addition, stabilizers may be added. Microspheres formulated for oral administration may also be used. Such microspheres have been well defined in the art. All formulations for oral administration should be in dosages suitable for such

administration.

For buccal administration, the compositions may take the form of tablets or lozenges formulated in conventional manner.

For administration by inhalation, the compounds for use according to the present invention may be conveniently delivered in the form of an aerosol spray presentation from pressurized packs or a nebulizer, with the use of a suitable propellant, e.g.,

dichlorodifluoromethane, trichlorofluoromethane, dichlorotetrafluoroethane, carbon dioxide or other suitable gas. In the case of a pressurized aerosol the dosage unit may be determined by providing a valve to deliver a metered amount. Capsules and cartridges of e.g., gelatin for use in an inhaler or insufflator may be formulated containing a powder mix of the compound and a suitable powder base such as lactose or starch.

Also contemplated herein is pulmonary delivery of the compounds of the invention (or derivatives thereof). The compound of the invention (or derivative) is delivered to the lungs of a mammal while inhaling and traverses across the lung epithelial lining to the blood stream. Other reports of inhaled molecules include Adjei et al, Pharm Res 7:565- 569 (1990); Adjei et al, Int J Pharmaceutics 63: 135-144 (1990) (leuprolide acetate);

Braquet et al, J Cardiovasc Pharmacol 13(suppl. 5): 143-146 (1989) (endothelin-1); Hubbard Qt al, Annal Int Med 3:206-212 (1989) (a 1 -antitrypsin); Smith et al, 1989, J Clin Invest 84: 1145-1146 (a- 1 -proteinase); Oswein et al, 1990, "Aerosolization of Proteins", Proceedings of Symposium on Respiratory Drug Delivery II, Keystone, Colorado, March, (recombinant human growth hormone); Debs et al., 1988, J Immunol 140:3482-3488 (interferon-gamma and tumor necrosis factor alpha) and Platz et al., U.S. Pat. No.

5,284,656 (granulocyte colony stimulating factor). A method and composition for pulmonary delivery of drugs for systemic effect is described in U.S. Pat. No. 5,451,569, issued Sep. 19, 1995 to Wong et al.

Contemplated for use in the practice of this invention are a wide range of mechanical devices designed for pulmonary delivery of therapeutic products, including but not limited to nebulizers, metered dose inhalers, and powder inhalers, all of which are familiar to those skilled in the art.

Some specific examples of commercially available devices suitable for the practice of this invention are the Ultravent nebulizer, manufactured by Mallinckrodt, Inc., St. Louis, Mo.; the Acorn II nebulizer, manufactured by Marquest Medical Products, Englewood, Colo.; the Ventolin metered dose inhaler, manufactured by Glaxo Inc., Research Triangle Park, North Carolina; and the Spinhaler powder inhaler, manufactured by Fisons Corp., Bedford, Mass.

All such devices require the use of formulations suitable for the dispensing of compound of the invention (or derivative). Typically, each formulation is specific to the type of device employed and may involve the use of an appropriate propellant material, in addition to the usual diluents, adjuvants and/or carriers useful in therapy. Also, the use of liposomes, microcapsules or microspheres, inclusion complexes, or other types of carriers is contemplated. Chemically modified compound of the invention may also be prepared in different formulations depending on the type of chemical modification or the type of device employed.

Formulations suitable for use with a nebulizer, either jet or ultrasonic, will typically comprise compound of the invention (or derivative) dissolved in water at a concentration of about 0.1 to 25 mg of biologically active compound of the invention per mL of solution. The formulation may also include a buffer and a simple sugar (e.g., for compound of the invention stabilization and regulation of osmotic pressure). The nebulizer formulation may also contain a surfactant, to reduce or prevent surface induced aggregation of the compound of the invention caused by atomization of the solution in forming the aerosol. Formulations for use with a metered-dose inhaler device will generally comprise a finely divided powder containing the compound of the invention (or derivative) suspended in a propellant with the aid of a surfactant. The propellant may be any conventional material employed for this purpose, such as a chlorofluorocarbon, a

hydrochlorofluorocarbon, a hydrofluorocarbon, or a hydrocarbon, including

trichlorofluoromethane, dichlorodifluoromethane, dichlorotetrafluoroethanol, and 1,1,1,2- tetrafluoroethane, or combinations thereof. Suitable surfactants include sorbitan trioleate and soya lecithin. Oleic acid may also be useful as a surfactant.

Formulations for dispensing from a powder inhaler device will comprise a finely divided dry powder containing compound of the invention (or derivative) and may also include a bulking agent, such as lactose, sorbitol, sucrose, or mannitol in amounts which facilitate dispersal of the powder from the device, e.g., 50 to 90% by weight of the formulation. The compound of the invention (or derivative) should advantageously be prepared in particulate form with an average particle size of less than 10 micrometers (μιη), most preferably 0.5 to 5 μιη, for most effective delivery to the deep lung.

Nasal delivery of a pharmaceutical composition of the present invention is also contemplated. Nasal delivery allows the passage of a pharmaceutical composition of the present invention to the blood stream directly after administering the therapeutic product to the nose, without the necessity for deposition of the product in the lung. Formulations for nasal delivery include those with dextran or cyclodextran.

For nasal administration, a useful device is a small, hard bottle to which a metered dose sprayer is attached. In one embodiment, the metered dose is delivered by drawing the pharmaceutical composition of the present invention solution into a chamber of defined volume, which chamber has an aperture dimensioned to aerosolize and aerosol formulation by forming a spray when a liquid in the chamber is compressed. The chamber is compressed to administer the pharmaceutical composition of the present invention. In a specific embodiment, the chamber is a piston arrangement. Such devices are commercially available.

Alternatively, a plastic squeeze bottle with an aperture or opening dimensioned to aerosolize an aerosol formulation by forming a spray when squeezed is used. The opening is usually found in the top of the bottle, and the top is generally tapered to partially fit in the nasal passages for efficient administration of the aerosol formulation. Preferably, the nasal inhaler will provide a metered amount of the aerosol formulation, for administration of a measured dose of the drug.

The compounds, when it is desirable to deliver them systemically, may be formulated for parenteral administration by injection, e.g., by bolus injection or continuous infusion. Formulations for injection may be presented in unit dosage form, e.g., in ampoules or in multi-dose containers, with an added preservative. The compositions may take such forms as suspensions, solutions or emulsions in oily or aqueous vehicles, and may contain formulatory agents such as suspending, stabilizing and/or dispersing agents.

Pharmaceutical formulations for parenteral administration include aqueous solutions of the active compounds in water-soluble form. Additionally, suspensions of the active compounds may be prepared as appropriate oily injection suspensions. Suitable lipophilic solvents or vehicles include fatty oils such as sesame oil, or synthetic fatty acid esters, such as ethyl oleate or triglycerides, or liposomes. Aqueous injection suspensions may contain substances which increase the viscosity of the suspension, such as sodium

carboxymethylcellulose, sorbitol, or dextran. Optionally, the suspension may also contain suitable stabilizers or agents which increase the solubility of the compounds to allow for the preparation of highly concentrated solutions.

Alternatively, the active compounds may be in powder form for constitution with a suitable vehicle, e.g., sterile pyrogen- free water, before use.

The compounds may also be formulated in rectal or vaginal compositions such as suppositories or retention enemas, e.g., containing conventional suppository bases such as cocoa butter or other glycerides.

In addition to the formulations described above, the compounds may also be formulated as a depot preparation. Such long acting formulations may be formulated with suitable polymeric or hydrophobic materials (for example as an emulsion in an acceptable oil) or ion exchange resins, or as sparingly soluble derivatives, for example, as a sparingly soluble salt.

The pharmaceutical compositions also may comprise suitable solid or gel phase carriers or excipients. Examples of such carriers or excipients include but are not limited to calcium carbonate, calcium phosphate, various sugars, starches, cellulose derivatives, gelatin, and polymers such as polyethylene glycols.

Suitable liquid or solid pharmaceutical preparation forms are, for example, aqueous or saline solutions for inhalation, microencapsulated, encochleated, coated onto microscopic gold particles, contained in liposomes, nebulized, aerosols, pellets for implantation into the skin, or dried onto a sharp object to be scratched into the skin. The pharmaceutical compositions also include granules, powders, tablets, coated tablets, (micro)capsules, suppositories, syrups, emulsions, suspensions, creams, drops or preparations with protracted release of active compounds, in whose preparation excipients and additives and/or auxiliaries such as disintegrants, binders, coating agents, swelling agents, lubricants, flavorings, sweeteners or solubilizers are customarily used as described above. The pharmaceutical compositions are suitable for use in a variety of drug delivery systems. For a brief review of methods for drug delivery, see Langer R, Science 249: 1527- 33 (1990), which is incorporated herein by reference.

The compounds of the invention and optionally other therapeutics may be administered per se (neat) or in the form of a pharmaceutically acceptable salt. When used in medicine the salts should be pharmaceutically acceptable, but non-pharmaceutically acceptable salts may conveniently be used to prepare pharmaceutically acceptable salts thereof. Such salts include, but are not limited to, those prepared from the following acids: hydrochloric, hydrobromic, sulphuric, nitric, phosphoric, maleic, acetic, salicylic, p-toluene sulphonic, tartaric, citric, methane sulphonic, formic, malonic, succinic, naphthalene-2- sulphonic, and benzene sulphonic. Also, such salts can be prepared as alkaline metal or alkaline earth salts, such as sodium, potassium or calcium salts of the carboxylic acid group.

Suitable buffering agents include: acetic acid and a salt (1-2% w/v); citric acid and a salt (1-3% w/v); boric acid and a salt (0.5-2.5%) w/v); and phosphoric acid and a salt (0.8- 2%> w/v). Suitable preservatives include benzalkonium chloride (0.003-0.03%) w/v);

chlorobutanol (0.3-0.9% w/v); parabens (0.01-0.25% w/v) and thimerosal (0.004-0.02% w/v).

Pharmaceutical compositions of the invention contain an effective amount of a compound of the invention and optionally therapeutic agents included in a

pharmaceutically-acceptable carrier. The term "pharmaceutically-acceptable carrier" means one or more compatible solid or liquid filler, diluents or encapsulating substances which are suitable for administration to a human or other vertebrate animal. The term "carrier" denotes an organic or inorganic ingredient, natural or synthetic, with which the active ingredient is combined to facilitate the application. The components of the pharmaceutical compositions also are capable of being commingled with the compounds of the present invention, and with each other, in a manner such that there is no interaction which would substantially impair the desired pharmaceutical efficiency.

The therapeutic agent(s), including specifically but not limited to the compound of the invention, may be provided in particles. Particles as used herein means nanoparticles or microparticles (or in some instances larger particles) which can consist in whole or in part of the compound of the invention or the other therapeutic agent(s) as described herein. The particles may contain the therapeutic agent(s) in a core surrounded by a coating, including, but not limited to, an enteric coating. The therapeutic agent(s) also may be dispersed throughout the particles. The therapeutic agent(s) also may be adsorbed into the particles. The particles may be of any order release kinetics, including zero-order release, first-order release, second-order release, delayed release, sustained release, immediate release, and any combination thereof, etc. The particle may include, in addition to the therapeutic agent(s), any of those materials routinely used in the art of pharmacy and medicine, including, but not limited to, erodible, nonerodible, biodegradable, or nonbiodegradable material or combinations thereof. The particles may be microcapsules which contain the compound of the invention in a solution or in a semi-solid state. The particles may be of virtually any shape.

Both non-biodegradable and biodegradable polymeric materials can be used in the manufacture of particles for delivering the therapeutic agent(s). Such polymers may be natural or synthetic polymers. The polymer is selected based on the period of time over which release is desired. Bioadhesive polymers of particular interest include bioerodible hydrogels described in Sawhney H S et al. (1993) Macromolecules 26:581-7, the teachings of which are incorporated herein. These include polyhyaluronic acids, casein, gelatin, glutin, polyanhydrides, polyacrylic acid, alginate, chitosan, poly(methyl methacrylates), poly(ethyl methacrylates), poly(butylmethacrylate), poly(isobutyl methacrylate), poly(hexylmethacrylate), poly(isodecyl methacrylate), poly(lauryl methacrylate), poly(phenyl methacrylate), poly(methyl acrylate), poly(isopropyl acrylate), poly(isobutyl acrylate), and poly(octadecyl acrylate).

The therapeutic agent(s) may be contained in controlled release systems. The term "controlled release" is intended to refer to any drug-containing formulation in which the manner and profile of drug release from the formulation are controlled. This refers to immediate as well as non-immediate release formulations, with non-immediate release formulations including but not limited to sustained release and delayed release formulations. The term "sustained release" (also referred to as "extended release") is used in its conventional sense to refer to a drug formulation that provides for gradual release of a drug over an extended period of time, and that preferably, although not necessarily, results in substantially constant blood levels of a drug over an extended time period. The term "delayed release" is used in its conventional sense to refer to a drug formulation in which there is a time delay between administration of the formulation and the release of the drug there from. "Delayed release" may or may not involve gradual release of drug over an extended period of time, and thus may or may not be "sustained release."

Use of a long-term sustained release implant may be particularly suitable for treatment of chronic conditions. "Long-term" release, as used herein, means that the implant is constructed and arranged to deliver therapeutic levels of the active ingredient for at least 7 days, and preferably 30-60 days. Long-term sustained release implants are well- known to those of ordinary skill in the art and include some of the release systems described above.

Having now described the present invention in detail, the same will be more clearly understood by reference to the following examples, which are included herewith for purposes of illustration only and are not intended to be limiting of the invention.

EXAMPLES Example 1

CLT-005 Prevents STAT3 Nuclear Translocation in Human Vascular Endothelial Cells (HUVECs)

An established model of STAT3 activation was used to validate the mechanism of action of CLT-005. This model employs the stimulation of HUVEC cells with leptin in order to produce STAT3 phosphorylation (pSTAT3) and subsequent nuclear translocation. Once pSTAT3-dimers translocate to the nucleus, they promote expression of numerous proliferative, pro-angiogenic, and pro-inflammatory genes. This model was first demonstrated in seminal papers that demonstrated the pro-angiogenic properties of leptin. As CLT-005 was designed to bind the SH2 domain of STAT3, and prevent dimerization, we hypothesized that co-treatment with leptin and CLT-005 would inhibit STAT3 nuclear translocation. HUVEC cells were cultured and stimulated with 5 ng of leptin to induce STAT3 in the absence or presence of CLT-005. Following treatment, cells were probed an antibody recognizing pSTAT3, counterstained with DAPI, and examined with fluorescence microscopy. In untreated cells, minimal levels of pSTAT3 were observed, but upon treatment with 5 ng of leptin, there was a marked increase in nuclear pSTAT3 (Fig. 1). At a concentration of 2 μΜ, CLT-005 inhibited nuclear translocation of STAT3 (Fig. 1).

These results clearly demonstrate the high potency of CLT-005 to inhibit signaling events initiating with leptin through the inhibition of STAT3.

Example 2

CLT-005 Prevents Loss of Contrast Sensitivity in a Mouse Model of Dry AMD

Age-Related Macular Degeneration (AMD) is a severe blinding disease that affects at least 30% of humans over the age of 70. Classic presentation of AMD shows a loss of central vision that progressively worsens with age. Patients initially present with a form of the disease called Dry AMD, which is characterized by the appearance of deposits (drusen) behind the retina and retinal pigment epithelial (RPE) cells. These drusen are thought to form as a result of oxidative stress, and some patients will progress to a later stage of the disease, called Wet AMD, where vascular leakage and abnormal neovascularization (NV) are present. Current lines of evidence suggest that drusen cause an unnatural inflammatory response in the retina and RPE. This inflammation contributes to the breakdown of the blood-retinal barrier and vascular leakage in the retina, which leads to edema, angiogenesis, and visual loss.

Hollyfield and colleagues have established a unique and innovative animal model for Dry AMD whereby mice are immunized with a carboxyethylpyrrole (CEP) which is covalently bound to mouse serum albumin (MSA). It has been demonstrated that immunization with CEP-MSA can lead to an ophthalmic phenotype very similar to Dry AMD, including deposition of complement molecule in the RPE, thickening of the Bruch's membrane, upregulation of inflammatory cytokines, and immune cell influx into the eye. Therefore, to examine the effect of CLT-005 in a mouse model of AMD, C57B1/6 mice were immunized with 200 g of CEP-MSA on Day 1 and received booster immunizations of 100 g on Days 10 and 40. The mice were administered topical CLT-005 at 1%, or vehicle alone on Days 1-60, QID. No adverse ophthalmic events were observed throughout the 60 days of QID dosing of 1% CLT-005 or the vehicle. Using optomotor tracking, the mean grating contrast threshold percentage distinguished by mice was observed to be significantly reduced in the CEP-MSA immunized group as compared to normal naive controls at Day 60 (p=0.0091, t-test). Daily eyedrop application of 1% CLT-005 prevented the loss of contrast threshold detection in CEP-MSA immunized mice (p=0.0284, t-test; Fig. 2B). Retention of contrast sensitivity in CLT-005 treated CEP-MSA immunized mice correlated with drug inhibition of STAT3 phosphorylation (Fig. 2C), thereby providing a likely mechanism of action for CLT-005 in a mouse model of dry AMD. Furthermore, as measurements of visual acuity and contrast sensitivity are the only acceptable primary endpoints by the FDA in a human clinical trial to treat AMD, these results are particularly exciting.

Example 3

CLT-005 Reduces Neovascular Lesion Size Following Oral or Topical Eyedrop Dosing in a Rat Model of Laser-Induced Choroidal Neovascularization

The anti-angiogenic effect of CLT-005 was evaluated in a rat model of laser- induced choroidal neovascularization (CNV). The laser CNV model has been a reliable animal surrogate in the development of therapeutics to treat retinal diseases, such as Wet AMD. In this model, a laser is used to create a lesion in Bruch's membrane, which is followed by neovascularization from the choroid into the retina (Fig. 3A). To examine the effect of topical CLT-005 on neovascular lesion size and leakage, a series of experiments were performed in Brown Norway (BN) rats. On Days 2-22 post-laser, BN rats were administered varying doses of CLT-005, or vehicle alone, via eye drops with a QID schedule (9 am, 12 pm, 3 pm, 6 pm). Animals were also examined on a daily basis to assess hyperemia, chemosis, and discharge following the last dose. No adverse ophthalmic findings were observed throughout entire study. On Day 22, color fundus photographs and fluorescein angiograms were acquired and used to quantify lesion size. Topical administration of 1% CLT-005 or intravitreal application of an anti-VEGF antibody significantly reduced lesion size at Day 22 (Figs. 3A & B). Notably, the application of 1% CLT-005 profoundly reduced central lesion leakage (Fig. 3B).

In a second study, the effect of orally delivered CLT-005 was examined. Rats were assigned to cohorts of 5 and received bilateral placement of three laser lesions on Day 1, and received oral administration of CLT-005 at 125, 250, or 500 mg/kg in soybean oil, or soybean oil alone once daily on Days 2-21. CLT-005 displayed a dose-dependent reduction in lesion size relative to the vehicle treated group (Fig. 3C), with a significant effect in rats receiving either 250 or 500 mg/kg CLT-005 (p = 0.0143 and 0.0076, respectively). Together these studies support the anti-angiogenic effect of CLT-005 mediated inhibition of STAT3 phosphorylation.

Example 4

Streptozotocin (STZ) -Diabetic BN Rats Have a Progressive Loss in Visual Function

To examine the potential therapeutic effects of CLT-005 on diabetic retinopathy, we first set out to establish a model whereby we could monitor the longitudinal effects of streptozotocin (STZ)-induced hyperglycemia on visual function in BN rats. Female BN (BN) rats were treated with either 50 mg/kg STZ, or sodium citrate buffer. Control and STZ-diabetic rats were assigned to paired cohorts, and each cohort was separately analyzed for visual function in two-week intervals beginning at 4 weeks post-STZ treatment. Non- fasting blood glucose levels were monitored at a minimum of one week intervals to ensure sustained glucose levels >250 mg/dL in the STZ-diabetic rats.

In the citrate buffer control groups of animals, there was no change in visual acuity as indicated by almost identical spatial frequency thresholds over time (Fig. 4A).

Conversely, in STZ-diabetic rats, there was an initial significant decline in visual acuity at 4 weeks post-STZ compared to controls (p < 0.05), with a progressive drop beginning at 8 weeks, and continuing through 16-weeks post-STZ (Fig. 4A). Although the decreased spatial frequency threshold for the 6-week post STZ diabetic group was not significant compared to controls, all subsequent time points (8, 10, 12, and 16 week post-STZ) showed a significantly diminished ability to distinguish the frequency of visual acuity stimuli (p < 0.001, Fig. 4A). Therefore, STZ-diabetic BN rats showed a significant and progressive loss in visual acuity beginning one month post STZ treatment and continuing through 16 weeks post-STZ.

We also analyzed the ability of STZ-diabetic rats to distinguish stimuli presented in increments of decreasing contrast at a spatial frequency of 0.064 cycles/degree. While the control groups displayed a consistent contrast threshold measurement over time, the diabetic groups showed a progressive temporal decline beginning at week 8, with a continued significant reduction at all subsequent measurements relative to controls (Fig. 4B). To better understand the loss in STZ-diabetic rats' ability to distinguish contrast, contrast threshold was measured over a range of spatial frequencies. The contrast thresholds at each spatial frequency remained consistent in the citrate buffer groups across all time points, whereas the contrast threshold at all measured frequencies showed dramatic reductions by 10 weeks post-STZ in the diabetic rats (Fig. 4C). The data shows temporal deviations in contrast sensitivity first occured at contrast thresholds measured at higher spatial frequencies (0.197 c/d and 0.272 c/d), and by 10 weeks post diabetic onset, contrast sensitivity was dramatically reduced at all observed spatial frequencies relative to time- matched controls (Fig. 4C).

To further confirm the loss in visual function is a due to hyperglycemia in STZ- treated rats, the effect of lowering blood sugar levels in STZ-treated rats via insulin treatment on visual function was analyzed. At 10 weeks post-STZ, the insulin treated animals displayed visual acuity and contrast measurements comparable to citrate buffer controls (Figs. 4 A & B). This data further supports the conclusion that vision loss in STZ- diabetic animals is related to continuous hyperglycemia.

We next recorded the electroretinography (ERG) profiles at 16 weeks post-STZ to determine if there is a deficit in retinal function corresponding to the visual deficits in STZ- diabetic rats. STZ-diabetic rats had a significant reduction in their Scotopic and Photopic B waves (Figs. 4 D & E). Therefore, one mechanism of visual loss in STZ-diabetic rats is an underlying loss of retinal function.

Example 5

Systemic Delivery of CLT-005 Ameliorates Vision Loss in STZ-Diabetic Rats

We next set out to examine the therapeutic effects of STAT3 inhibition via the small molecule inhibitor, CLT-005, on the loss of visual function in STZ-diabetic rats. BN rats with STZ-induced diabetes were given CLT-005 at 125, 250, or 500 mg/kg/animal, or vehicle alone, via daily oral gavage for 16 consecutive weeks. Additional controls included a non-diabetic group, and STZ-diabetic rats treated with insulin. For all groups, visual acuity and contrast threshold values were determined at 2 week intervals beginning at 4 weeks post-STZ and continuing through 16 weeks post-STZ.

Over the course of the study, no adverse indicators specific to CLT-005 tolerability such as abnormal changes in behavior, adverse clinical observations, or mortality, were observed. Fig. 5A shows all groups of STZ-diabetic animals gained weight over the course of the study, albeit to a lesser extent than non-diabetic animals. There was no significant difference in the average weight gain across STZ treated groups, including animals receiving insulin, indicating that even at high daily doses, CLT-005 did not exacerbate the diminished weight gain of diabetic animals. Non- fasting blood glucose levels in STZ treated animals at weekly intervals were also measured. All groups receiving either vehicle or CLT-005 displayed sustained hyperglycemia (blood glucose > 250 mg/dL). However, animals receiving either 250 or 500 mg/kg CLT-005 had significantly reduced blood glucose levels compared to vehicle- treated animals (Fig. 5B). Consistent with the possibility that CLT-005 may be positively regulating blood glucose levels in STZ-diabetic rats, the groups receiving the two highest doses of CLT-005 had the highest average levels at the beginning of the study, and showed a sharp downward trend over the duration of treatment (Fig. 5C). Pharmacokinetic analysis of whole blood also showed systemic levels of CLT-005 were dose-dependent (Fig. 5D). Therefore, oral gavage of CLT-005 is an effective means of systemic delivery and is well tolerated by STZ-diabetic rats.

Example 6

Systemic Delivery of CLT-005 Ameliorates Diabetic Visual Defects in a Dose-Dependent Manner

Optokinetic tracking was used to determine the effect of CLT-005 on vision loss in STZ-diabetic rats. Figure 6 shows there was a gradual decline in visual acuity in vehicle- treated STZ-diabetic rats (Fig. 6A), and, similar to non-treated STZ-diabetic animals (Fig. 4A), the loss in visual acuity was most dramatic from week 12 to week 16 post-STZ. STZ- diabetic animals receiving the lowest dose of CLT-005 (125 mg/kg) showed a declining trend in visual acuity similar to vehicle treated animals, with no significant difference in spatial frequency thresholds between the two groups. However, STZ-diabetic animals receiving the two highest doses of CLT-005 (250 and 500 mg/kg) were delayed in their loss of visual acuity (Fig. 6A). Further, both high dose groups had significantly higher spatial frequency thresholds at 16 weeks post-STZ relative to vehicle treated animals (Fig. 6B, p < 0.05 and p < 0.001, respectively). Strong evidence for the positive therapeutic impact of CLT-005 mediated STAT3 inhibition of diabetic-related vision loss comes from the observation that STZ-diabetic animals receiving 500 mg/kg CLT-005 maintained visual acuity levels through 16 weeks post-STZ similar to diabetic animals supplemented with insulin (Figs. 6A & B). Example 7

CLT-005 mediated inhibition of STAT3 also delayed the loss of STZ-diabetic rat ability to distinguish contrast in virtual stimuli (Fig. 7A). Similar to the time-dependent loss in visual acuity, both vehicle-treated and low dose CLT-005 treated STZ-diabetic animals showed comparable declines in contrast thresholds. Also, while the onset of decline was delayed, STZ-diabetic animals receiving 250 mg/kg CLT-005 did not have contrast threshold levels significantly different from vehicle at 16 weeks post-STZ (Figs. 7A & B). However, STZ-diabetic animals receiving the highest dose of CLT-005 showed both delayed onset and sustained higher contrast threshold values compared to all other groups, including insulin-treated diabetic rats (Figs. 7A & B). In agreement with the visual acuity data, the high dose CLT-005 group had higher contrast threshold values at all observed spatial frequencies (Fig. 7C). Taken together, the acuity and contrast visual threshold data show CLT-005 ameliorates vision loss in STZ-diabetic rats in a dose- dependent manner.

Example 8

Cataract Formation Correlates with Visual Deficits in Diabetic BN Rats

It has been reported that cataract formation contributes to visual deficit in STZ- diabetic Long Evans rats. Aung et al, Invest Ophthalmol Vis Sci 54: 1370-7 (2013). In our initial study of STZ-diabetic animals not receiving therapeutic intervention, we observed cataract formation beginning between 6 and 10 weeks post STZ, with total opacity of the lens occurring around week 12 post-STZ. These time points of progressive cataract formation correlate with the visual decline in these animals. However, the cataracts were not systematically scored in these animals, so we were unable to directly correlate the data.

Therefore, to correlate cataract severity and visual loss in the CLT-005 and vehicle treated groups, we imaged and scored cataracts immediately following optokinetic tracking measurements. Fig. 8A shows representative images of cataracts from each group. In both vehicle- and CLT-005 treated STZ-diabetic rats, initial cataract formation was observed at 6 weeks post-STZ, whereas cataract onset was delayed in insulin-treated rats (Fig. 8B). At 10 weeks post-STZ, the relative severity of cataracts in the vehicle- and low dose groups began to diverge from the two high dose groups, and the cataract scores remained significantly higher on average at all subsequent time points for the vehicle- and low dose groups (Fig. 8A and Table 1). Therefore, systemic delivery of CLT-005 diminishes the severity of cataract formation in STZ-diabetic rats.

Table 1: Statistical Comparison of Cataract Scores in STZ-Diabetic Rats

To analyze whether the improved visual function in STZ-diabetic animals receiving 250 and 500 mg/kg CLT-005 is due to lower grade cataracts in these animals, we correlated individual cataract scores with the corresponding visual acuity thresholds. We analyzed the relationship between cataract severity and visual function within groups at 12 weeks post- STZ. This time point offers the highest degree of variability in cataract scores within groups, thus allowing us to separate high grade and low grade cataracts within each group at a single time point. When we separated the data within groups based on cataract score, we did not observe a statistical difference between animals with high cataract scores versus low cataract scores (Fig. 8C). We also compared animals with a cataract score of 2 across groups at 12 weeks post-STZ, and found the SFT values correlate more strongly with drug dose than with cataract score (Fig. 8D). This data supports the conclusion that CLT-005 rescues visual acuity loss independent of its effect on cataract formation.

To examine whether CLT-005 is present locally within the eye following systemic delivery, we performed pharmacokinetic analysis in retina and Pigment Epithelium, Choroid, and Sclera (PECS) tissue collected separately. We found that in both tissues, CLT-005 concentration follows a dose-dependent trend such that the highest drug levels in tissue are found in rats receiving the highest dose (Figs. 8 A & B). To determine whether CLT-005 is functioning as predicted to regulate inflammatory and pro-angiogenic pathways, we used multiplex analysis to examine the expression profiles of cytokines whose levels indicate upregulation of these pathways. As shown in Table 2, inflammatory cytokines were upregulated in STZ-diabetic rats (Table 2, vehicle column). Significantly, CLT-005 had a profound, dose-dependent effect on downregulating all examined cytokines in the retina. Therefore, orally administered CLT-005 acts locally in the eye to prevent vision loss, and a critical mechanism of this action is thought to be through a dramatic positive regulation of the local inflammatory response.

Table 2: Multiplex Analysis of Inflammatory Proteins in S TZ-Diabetk Rat Retina

Hme Vehicte 12S mg¾g 250 mg/kg SO mg/kg Irtsuln pg/mg SEM pg/mg pg mg SEM pg/mg SEM pg mg SEfJt pgimg SEM

Protein

!L-1a 33.186 ± 2.788 45 W + 9,935 2S.1G0 ± .698 27.S48 ± 1.947 31003 i ft 77:1 27.415 ± 6.442 iL4 §.283 ± 0.17Ξ 12,4m 2.982 8.6S3 i 0.711 8.848 i 0.306 8.348 ± 0.106 8.883 ± 0.691 iL-Ιβ 383.138 ± 17.112 SIS.868 ± 222.729 328.035 * 25.55 281.858 ± 23.923 234,735 ± 12.804 273.418 ± 7.309

74.830 ± 5.221 S3. 58 t 8345 74.878 i.5.252 73.333 .t 8.226 81330 ± 1 892 88.648 .± 1.990

± 1.445 i 9.555 23.578 ± 1.662 22.1 S3 ± 2.005 19.445 ± 2.241 22.443 ± 2.509

6,685 ± 0.482 9.888 + 3.247 4.825 i 0.7Q7 4.855 + 0.768 2.803 ± 0 G8f 4.155 .± 0.337

SL-10 184.063 f. 6-386 «s.s*s ± 23.218 mm 16.823 102,088 t 8.564 S1855 + 4.008 91.788 113.698 iL-12 ip70i 16.388 ± 2.504 29.803 i 7.881 18.363 ± 1.878 14.888 ± 3.718 ίθ.803 ± a ?6ci 15.718 ± 0.951

!F 27.483 + 2.414 46.173 ± 14.042 3δ.8δ0 ί 3.223 24.245 ± 4 :984 ns 24.148 ± 0.877 iL-δ 34.888 12.421 41,28$ t 8.658 28.840 ± 0.833 33.288 ± 1.773 28.258 ± f.825 28.81Θ ± 2.594

SL-17 8.370 t 0.240 13.333 ± 4MB 7.878 ± 0.538 8.243 ± 0.581 7.008 ± 0335 7.725 ± 0.600

SL-18 783.68S ± 22.481 771.100 47MB 720.743 ± 63.026 748.233 ± 37.609 MB JIB ± 4S5J 718.113 ± 35.921

MCP-1 «1.075 ± 2.190 70.778 + 11 4m m, t 4.425 48.888 ± 3.985 42.285^" ± 1 2m 48.818 .i 1341

GRG-KC 2.118 ± 0.483 «J?« + 1M2 2.638 ± 0.S3? 2.243 ± 0 305 1453 ± Q.m 2,133 ± 0.353

VEGF 33.118 ± 6.040 42.230 7.35$ 28.180 ± 3,986 24.188 ± 4.008 24.133 ± 1,792 27.855 ± 4.740 A TIS 47.453 ± 3.279 78,810 ± 27.567 44.818 £ COS 42.323 ± 2.739 34.JS5 ± 4.027 37.420 ± 2705

EPQ 138.783 1 8.480 1H.7SS t 29.454 120.543 ± 8.681 123.878 ± 41)08 122J33 t 14.677 111.328 ± 7.485 iL-7 87.338 ± 11.42? 128.555 t 24.969 88.788 ± 10.348 85.488 ± 12.501 €8645 ± s 73.318 * 5.241 -CSF 3.253 ± 1 33?. 13.723 ± 2.m 6.6B8 ± 0.963 8.588 ± 1.155 4.775 ± Q.237 5.765 ± 1.096

HIP-30 8J33 * 0.257 10.878 2.754 7.868 ± Q.m 8.753 * 0.329 8.453 ± o. m 7.083 ± 0.626

Table 3: Multiplex Analysis of inflammatory Proteins i STZ-Diabetk Rat PECS

Naive Vehicle 125mg kg 250 mg,¾g 6 0 mg¾g

Protsiri

TNFtt 5*¾.¾¾ t 63.443 704,268 i 63.281 649, 73 ± 24.763 ¾1MS¾ ± 32.838 aim t 33.429 6e¾,465 ± 36.504 IF y 42,3425 i 5.0814 50.4&5 ί 4.2817 4m t 1.1808 47.3025 i 2.0580 44.1475 i 4.4208 S2.67 ± 2.7174

Example 9

Materials and Methods

STZ Injection and Glucose Measurement

Age-matched female BN rats weighing between 126-137 grams were obtained from Charles River Laboratories, Inc. (Wilmington, MA). Animals were acclimated for 1 week prior to STZ treatment. On the day prior to injection, the rats were weighed and baseline blood glucose measurements were taken via tail vein bleed using a OneTouch Glucose Monitoring System (Johnson & Johnson). Following an overnight fast, rats were administered 50 mg/kg of STZ in 10 mM sodium citrate, pH 4.5, intraperitoneally. Non- fasting blood glucose was again measured 3 days post STZ administration to check for hyperglycemia (blood glucose concentration of 250 mg/dL or greater). Non-fasting blood glucose levels were monitored weekly for the duration of the study. All blood glucose measurements were individually recorded according to an established animal identification system.

Formulation Preparation and Storage

Drug and vehicle formulations were prepared weekly and stored protected from light at room temperature. The drug concentration was prepared to ensure the gavage volume did not exceed 10 mL/kg/animal. CLT-005 was weighed and added to corn oil to achieve the appropriate concentration. The mixture was then sonicated continuously for 5 min at a setting of 40% using a probe sonicator (Vibra Cell Model VCX 750 from Sonics & Materials, Inc.). To ensure drug stability, all formulations were monitored daily for potential precipitation.

Oral Gavage

Animals were gavaged once daily beginning at 3 days post STZ-injection and continuing for the duration of the study. For each group, the gavage volume was 10 xLlg of body weight. An 18 G 3" curved feeding needle (Cadence Science, Cranston, RI) attached to a 3-mL Luer lock syringe was placed in the animal's mouth and advanced via the esophagus to the stomach where the drug was dispensed. The animals were monitored for signs of any possible adverse reactions to gavage.

Insulin Implantation

Linplant sustained release insulin implants (Linshin Canada, Inc., Toronto, Ontario) were used to treat STZ-induced diabetic rats with exogenous insulin. Hair was removed from the surgical area and skin was cleaned with a 10% povidone-iodine solution. A 16G disposable hypodermic needle was used to pierce the skin, and the insulin implant was placed subcutaneously on the upper abdomen via a trocar. Glucose was monitored weekly, and if the blood glucose elevated above 250 mg/dL, a second round of surgical insulin implantation was performed.

Optokinetic Tracking

Visual function was measured using a virtual optokinetic tracking system

(OptoMotry, CerebralMechanics) designed for rapid, quantifiable behavioral measurements of spatial vision in a virtual environment. Prusky GT et al, Invest Ophthalmol Vis Sci. 45 :461 1-6 (2004). The animals were placed on a platform surrounded by four computer monitors forming a square inside an enclosed box. The monitors display continuous vertical sine wave gratings rotating across the monitors at 12 degrees/s which appear to the animal as a virtual three-dimensional rotating sphere. The animal's ability to visualize the sine wave was monitored via a video camera positioned directly above the animal to display an image perpendicular to the animal's field of vision. The rotation of the virtual cylinder was constantly centered at the animal's viewing position to ensure a consistent viewing distance. Tracking movements were identified as slow, steady head movements in the direction of the rotating grating.

Anesthesia and Euthanasia

An anesthesia cocktail was prepared at a ratio of 1 part Ketamine (100 mg/mL) and 5 parts Xylazine (20 mg/mL), and administered at 1 \lL/g of body weight via intraperitoneal (IP) injection using a 0.3 cc insulin syringe, 12 mm 30 G needle (rats), or an 8 mm 30G needle (mice).

Cataract Scoring

Following anesthesia and eye dilation, images of the rat lens were taken using an Amscope 0.3MP Aptima Camera attached to an upright brightfield Amscope microscope (Amscope, Irvine, CA), and Topoview software. Cataracts were scored on a scale established by Muranov et al. (Muranov 2004; Aung 2013), as shown in Table 4. Table 4: Cataract Scoring System

Electroretinography

Following overnight dark adaptation, retinal function was measured by

electroretinography (ERG). Dilation of the eyes and anesthesia were performed under a dim red light (< 50 lux). ERG analyses were performed using an Espion system from Diagnosys. For the assessment of scotopic response, stimulus intensity of 40 (S) cd.s/m² was presented to dark-adapted dilated eyes. The amplitude of the scotopic a-wave was measured from the prestimulus baseline to the a-wave trough. The amplitude of the b-wave was measured from the trough of the a-wave to the crest of the b-wave. To evaluate photopic response, animals were light adapted for 7 minutes then presented a strobe flash to the dilated eyes with intensity of 10 (S) cd.s/m². A total of 15 repeated flashes and measurements were averaged to produce the final waveform. The amplitude of the photopic b-wave was measured from the trough of the a-wave to the crest of the b-wave.

Tissue and Plasma Collection

A 1 cc syringe with a 26 G 5/8" needle was used to make a cardiac puncture on sedated animals. Whole blood was immediately placed in a BD Microtainer tube with K₂EDTA (BD #365974) and inverted several times. The samples were allowed to sit at room temperature for 15 min., and then centrifuged at 1500 x g for 15 min. at room temperature. The resultant supernatant (plasma) was transferred to a 1.5 mL screwcap tube and stored at -80 °C. For tissue collection, eyes were dissected to collect the retina alone, or the Pigment Epithelium, Choroid, and Sclera together (PECS). The dissected tissue was placed in a sterile screw cap microfuge tube (VWR), snap frozen in liquid nitrogen, and stored at -80 °C.

Pharmacokinetic Analysis

Eye tissue (retina and PECS) was weighed out in labeled 1.7 mL eppendorf tubes. One scoop of small (0.9-2.0 mm in diameter) stainless steel beads was added to each tube, and an appropriate volume of PBS was added to obtain a final concentration of 175 mg tissue homogenate/mL. The tissue was homogenized at a setting of 10 for 3 minutes in a bullet blender storm 24 (Next Advance, Averill Park, NY). A small volume of the tissue homogenate was then diluted in acetone and an internal standard (ketoprofen). The mixture was vortexed for 5 min at room temperature, and centrifuged at 5,000 rpm for 2 minutes. The supernatant was collected and transferred to an HPLC vial and run on an HPLC Agilent 1100 system. Separation of CLT-005 and Ketoprofen was carried out using a EpicC18MS (ES Industries) column (4.6 x 50 mm, 5 mm) with a 7 min isocratic elution consisting of 40% water in 0.1% formic acid and 60% 50:50 AcetonitrileTsopropyl alcohol in 0.1% formic acid at a flow rate of 500 mL/min. The injection volume was 10 mL.

Positive ion electrospray ionization mass spectrometric analysis was carried out using an Applied Biosystems API 3200 QTrap mass spectrometer at unit resolution with collision- induced dissociation and multiple reaction monitoring (MRM). The source temperature was 700 °C, the electrospray voltage was 5500 V, and the declustering potential was 70 V. Nitrogen was used as the collision gas at 60 eV, and the dwell time was 150 ms/ion.

During MRM, both CLT-005 and Ketoprofen were measured by recording the signal for the transition of the deprotonated molecules of m/z 267.1 to the most abundant fragment ion of m/z 165.2. The MRM transition of m/z 255.12 to 105.1 was monitored for the ketoprofen internal standard. Data were acquired and analyzed using Analyst software version 1.4 (Applied Biosystems).

Multiplex Analysis

Eye tissue (retina and PECS) was homogenized in cell lysis buffer (Bio Rad) using a bullet blender storm 24. Protein concentration was measured using a BCA assay (Pierce), and reconstituted to 5 mg/mL in cell lysis buffer. Cytokine levels were analyzed using a Bio Rad Bio-plex Pro Rat Cytokine Group I 23-Plex Assay according to protocol (cat. # L8001V11S5).

Eye Drop Formulation Preparation and Storage

Formulations were made up in 10 mL glass bottles. After the addition of the chemicals listed in Table 5, the samples were vortexed, sonicated for 2 min @ 40%>, and then homogenized @ 20 kPSI for 5 passes.

Table 5: Chemical Composition of CLT-005 Formulation

Soybean lecithin Alfa Aesar 36486

Pluronic F68 Sigma P1300-500G

Alpha tocopherol Sigma T3251-25G

HV CMC CalBioChem 217274

CLT-005 Norac XL-005-1 12-11

TABLE 6: Reagent Concentration for CLT-005 Formulation

Immunization

Ocular pathology was induced by immunization with CEP-Mouse Serum Albumin (CEP-MSA). CEP-MSA was prepared in an emulsion in Montanide ISA 720 VG (Seppic Inc.). Prior to immunization, animals were sedated with ketamine/xylazine at 85/14 mg/kg, respectively. Following sedation, on Day 1, 50 μg of the CEP-MSA emulsion was applied via intramuscular injection into each hind leg, with an additional 100 g applied via intramuscular injection at the base of the tail (200 g total/animal). On Days 11 and 41, animals were sedated with ketamine/xylazine and a total of 100 g of the CEP-MSA emulsion was applied via subcutaneous injection on the dorsal side of the animal at the base of the neck. All injections for sedation and immunization were performed utilizing a 0.3 cc insulin syringe attached to an 8 mm 31-gauge needle (BD#328438).

Laser Application to Produce CNV Lesions

Animals were dilated with 1% Cyclogyl solution and protected from light.

Following observable dilation, the animals were sedated with ketamine/xylazine. The fundus of sedated animals was observed and recorded using a Micron III small animal funduscope (Phoenix Research). Laser treatments were performed using a thermal laser which is connected through the Micron III custom laser attachment. A total of 3 lesions per eye were placed using a wavelength of 520 nm. Fundus images were recorded to confirm that the laser had successfully produced a bubble through the Bruch's membrane.

Fluorescein Angiography

Animals were anesthetized with ketamine/xylazine and then received an IP injection of 10% Fluorescein Sodium at 1 \L I gram of body weight. Fundus images were then captured as 8-bitt TIFF files using the Micron III and exciter/barrier filters for a target wavelength of 488 nm. Standard color fundus photos were also captured for each eye. Imaging and Lesion Quantification

All TIFF images were quantified using computerized image-analysis software

(ImageJ, NIH, USA). Lesions were then individually traced free-hand in order to quantify the area in pixels. The corresponding color fundus photos were used as a reference for lesion location. Areas of avascularization in the center of lesions were excluded from area calculations. In the case of a hemorrhage or two lesions overlapping, the lesions were excluded from analysis.

Fundoscopy

Animals were dilated with 1% Cyclogyl solution and protected from light. Animals were anesthetized with ketamine/xylazine. The fundus of sedated animals was observed and captured as 8-bitt TIFF files using a Micron III small animal funduscope (Phoenix Research). Standard fundus photos were also captured for each eye.

Animal Housing

All animals were housed under standard animal care conditions. Animals were maintained under normal cyclical light conditions consisting of 12 hours of light (< 500 lux) followed by 12 hours of darkness. Animals were housed so that each treatment group resided within a single cage to avoid cross-contamination of the various formulations. All animals received an ear tag with a 4 digit ID number for tracking and all animal information was stored in a local MS Access database.

Statistical Analysis

Statistical analyses were performed with Graphpad Prism software (version 5).

INCORPORATION BY REFERENCE

All patents and published patent applications mentioned in the description above are incorporated by reference herein in their entirety. EQUIVALENTS

Having now fully described the present invention in some detail by way of illustration and example for purposes of clarity of understanding, it will be obvious to one of ordinary skill in the art that the same can be performed by modifying or changing the invention within a wide and equivalent range of conditions, formulations and other parameters without affecting the scope of the invention or any specific embodiment thereof, and that such modifications or changes are intended to be encompassed within the scope of the appended claims.

Claims

1. A method of treating hyperglycemia, comprising administering to a subject in need thereof a therapeutically effective amount of an inhibitor of signal transducer and activator of transcription 3 (STAT3).

2. The method of claim 1, wherein the inhibitor of STAT3 is a small molecule.

3. The method of claim 1 or 2, wherein the administering is systemically

administering.

4. The method of claim 1 or 2, wherein the administering is orally administering.

5. The method of any one of claims 1-4, wherein the subject has type 1 diabetes mellitus.

6. The method of any one of claims 1-4, wherein the subject has type 2 diabetes mellitus.

7. The method of any one of claims 1-7, wherein the subject is a human.

8. A method of treating hyperglycemia, comprising administering to a subject in need thereof an effective amount of l-acetyl-5-hydroxyanthracene-9,10-dione (CLT-005).

9. The method of claim 8, wherein the administering is systemically administering.

10. The method of claim 8, wherein the administering is orally administering.

11. The method of any one of claims 8-10, wherein the subject has type 1 diabetes mellitus.

12. The method of any one of claims 8-10, wherein the subject has type 2 diabetes mellitus.

13. The method of any one of claims 8-12, wherein the subject is a human.

14. A method of improving at least one measure of spatial vision selected from the group consisting of spatial frequency threshold, contrast threshold, and contrast sensitivity, comprising administering to a subject in need thereof a therapeutically effective amount of an inhibitor of signal transducer and activator of transcription 3 (STAT3).

15. The method of claim 14, wherein the inhibitor of STAT3 is a small molecule.

16. The method of claim 14 or 15, wherein the administering is systemically administering.

17. The method of claim 14 or 15, wherein the administering is orally administering.

18. The method of any one of claims 14-17, wherein the subject is a human.

19. The method of any one of claims 14-18, wherein the subject does not have a retinal disease characterized by at least one of inflammation, angiogenesis, or neovascularization.

20. A method of improving at least one measure of spatial vision selected from the group consisting of spatial frequency threshold, contrast threshold, and contrast sensitivity, comprising administering to a subject in need thereof an effective amount of l-acetyl-5- hydroxyanthracene-9, 10-dione (CLT-005).

21. The method of claim 20, wherein the administering is systemically administering.

22. The method of claim 20, wherein the administering is orally administering.

23. The method of any one of claims 20-22, wherein the subject is a human.

24. The method of any one of claims 20-23, wherein the subject does not have a retinal disease characterized by at least one of inflammation, angiogenesis, or neovascularization.

25. A pharmaceutical composition, comprising l-acetyl-5-hydroxyanthracene-9,10- dione (CLT-005), or a pharmaceutically acceptable salt thereof, and a pharmaceutical carrier, wherein the composition is formulated for oral administration.

26. The pharmaceutical composition of claim 25, formulated as a solid.

27. The pharmaceutical composition of claim 26, formulated as a pill, tablet, capsule, powder, or troche.

28. The pharmaceutical composition of claim 25, formulated as a liquid solution or liquid suspension.