TREATMENT AND PREVENTION OF DIABETIC EYE DISEASES WITH A BILE

ACID DERIVATIVES

BACKGROUND

The circadian clock impacts nutritional habits, sleeping patterns, and meal frequency which, in turn, have profound effects on maintaining human health. There are two key pacemakers in mammals, the suprachiasmatic nucleus (SCN), responsive to light signals through the retina-hypothalamic tract, and the peripheral pacemaker in the liver that is responsive to food and feeding cycles and resides in a non-light sensitive tissue. Feeding at specific times, or reducing the quantity of food, prolongs the lifespan of mice, in part by synchronizing the SCN. The peripheral clock in the liver is also reset by food availability.

Type 2 diabetes and metabolic dysfunction adversely impact circadian rhythms of rest and activity, body temperature, hormone secretion, and gene expression. Individuals with diabetes experience not only circadian dysfunction and altered glucose metabolism, but also changes in lipid metabolism, gut pathology, and decreased bile acid synthesis. In a rodent model of type 2 diabetes, circadian clock gene expression within the liver is corrected to a non-diabetic rhythm following short term (2 week) active phase (nighttime) restricted feeding.

People with diabetes have a higher risk of developing eye diseases which can cause blindness than people without diabetes. Diabetic retinopathy is the most common diabetic eye disease and a leading cause of blindness in American adults. Chronically high blood sugar from diabetes is associated with damage to the tiny blood vessels in the retina, leading to diabetic retinopathy.

There is a need for therapies for the treatment and prevention of diabetic eye diseases, such as diabetic retinopathy. The present application addresses the need.

SUMMARY

The present application relates to a method of treating or preventing an eye disease in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of a compound of Formula A:

or a pharmaceutically acceptable salt or amino acid conjugate thereof, wherein:

Ri is Ci-Ce alkyl;

R2, R3, R5, and R ₆ are each independently H or OH;

R7 is H or Ci-Ce alkyl.

The present application also relates to a compound of Formula A, or a

pharmaceutically acceptable salt or amino acid conjugate thereof, for treating or preventing an eye disease in a subj ect in need thereof.

The present application also relates to a compound of Formula A, or a

pharmaceutically acceptable salt or amino acid conjugate thereof, for use in the manufacture of a medicament for the treatment or prevention of an eye disease in a subject in need thereof.

The present application also relates to use of a compound of Formula A, or a pharmaceutically acceptable salt or amino acid conjugate thereof, in the manufacture of a medicament for the treatment or prevention of an eye disease in a subject in need thereof.

In one embodiment, a compound of Formula A is Compound 1 :

or a pharmaceutically acceptable salt or amino acid conjugate thereof.

In one embodiment, a pharmaceutically acceptable salt of Compound 1 is the sodium salt of Compound 1 (i.e., Compound 1-Na). In another embodiment, a pharmaceutically acceptable salt of Compound 1 is the triethylammonium salt of Compound 1 (i.e., Compound 1-TEA). Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs. In the case of conflict, the present specification, including definitions, will control. In the specification, the singular forms also include the plural unless the context clearly dictates otherwise. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present application, suitable methods and materials are described below. All publications, patent applications, patents, and other references mentioned herein are incorporated by reference. The references cited herein are not admitted to be prior art. In the case of conflict, the present specification, including definitions, will control. In addition, the materials, methods, and examples are illustrative only and are not intended to be limiting. Other features and advantages of the application will be apparent from the following detailed description and claims.

BRIEF DESCRIPTION OF THE DRAWINGS Figure 1 A is an illustration of the feeding protocols for the experimental animals.

Figure IB is a graph displaying the HbAlc level in mice subject to ad lib feeding or IF feeding. *p < 0.05.

Figure 1C is a graph displaying the survival rate in mice subject ad lib feeding or IF feeding as a function of days after the start of the IF feeding. *p < 0.05.

Figure 2A is an immunohistochemical image of acellular capillary in the retina of db/m mice subject to ad lib feeding. Arrows indicate acellular capillaries on trypsin digests of retinas.

Figure 2B is an immunohistochemical image of acellular capillary in the retina of db/m mice subject to IF feeding. Arrows indicate acellular capillaries on trypsin digests of retinas.

Figure 2C is an immunohistochemical image of acellular capillary in the retina of db/db mice subject to ad lib feeding. Arrows indicate acellular capillaries on trypsin digests of retinas.

Figure 2D is an immunohistochemical image of acellular capillary in the retina of db/db mice subject to IF feeding. Arrows indicate acellular capillaries on trypsin digests of retinas. Figure 2E is bar graph (left) and bar graph scatter plot (right) displaying the number of acellular capillaries per mm ² of retinal area in db/m or db/db mice subject to ad lib feeding or IF feeding.

Figure 2F is bar graph displaying the number of Iba positive cells per mm ² of retinal area in db/m or db/db mice subject to ad lib feeding or IF feeding during feeding and fasting.

Figure 2G is bar graph displaying the number of CD45 positive cells per mm ² of retinal area in db/m or db/db mice subject to ad lib feeding or IF feeding during feeding and fasting.

Figure 2H is bar graph scatter plot displaying the number of NF200 positive nerve fibers in db/m or db/db mice subject to ad lib feeding or IF feeding.

Figure 21 is an immunohistochemical image of F200 positive cells in the bone marrow of db/m mice subject to ad lib feeding. Arrows indicate NF200 positive cells.

Figure 2J is an immunohistochemical image of F200 positive cells in the bone marrow of db/m mice subject to IF feeding. Arrows indicate F200 positive cells.

Figure 2K is an immunohistochemical image of NF200 positive cells in the bone marrow of db/db mice subject to ad lib feeding. Arrows indicate NF200 positive cells.

Figure 2L is an immunohistochemical image of F200 positive cells in the bone marrow of db/db mice subject to IF feeding. Arrows indicate F200 positive cells.

Figure 3 A-3F are bar graphs showing comparison of Shanon diversity in the microbiota of select groups of mice. ANOVA-determined p-values are presented for each comparison.

Figure 4A-4C are graphs displaying the dimensional reduction of the Jaccard distance on presence/absence OTU table, using the PCoA ordination method in mice subject to ad lib feeding or IF feeding: db/m ad lib (circles) vs. db/db ad lib (triangles) (Figure 4A), db/m ad lib (triangles below the dashed line) vs. db/m IF (feeding: triangles above the dashed line; fasting: circles) (Figure 4B), and db/db ad lib (triangles to the left of the dashed line) vs. db/db IF (feeding: triangles to the right of the dashed line; fasting: circles) (Figure 4C).

Figure 4D is a plot displaying the relative abundance of bacteria present in at least 10% of the samples from db/m or db/db mice subject to ad lib feeding or IF feeding through principal component analysis. Figure 4E are pie graphs showing relative proportions of the 6 most abundant phyla (the aggregate relative abundance for each of the phyla not represented here is less than 0.1) of the gut microbiome samples from db/m or db/db mice subject to ad lib feeding or IF feeding (feeding and fasting) through principal component analysis.

Figure 5A is a plot displaying gut microorganism genera that show statistically significant changes in db/db ad lib mice vs db/m controls (db/md ad lib).

Figure 5B is a plot displaying gut microorganism genera that show statistically significant changes in db/db IF mice vs db/m controls during feeding (db/md IF-feeding).

Figure 5C is a plot displaying gut microorganism genera that show statistically significant changes in db/db IF mice vs db/m controls during fasting (db/md IF-fasting).

Figure 6A is a plot showing genera in the microbiome analysis that show statistically significant changes in db/m-IF fasting vs. db/m-IF feeding mice.

Figure 6B is a plot showing genera in the microbiome analysis that show statistically significant changes in db/db-IF fasting vs. db/db-IF feeding mice.

Figure 6C is a Venn diagram for the comparison of lists of bacterial species that were significantly increased from their perspective AL diets that identifies commonly or uniquely increased OTUs among the four data sets of db/m-IF feeding, db/m-IF fasting, db/db-IF feeding, and db/db-IF fasting from the microbiome analysis.

Figure 6D is a Venn diagram for the comparison of lists of bacterial species that were significantly decreased from their perspective AL diets that identifies commonly or uniquely decreased OTUs among the four data sets of db/m-IF feeding, db/m-IF fasting, db/db-IF feeding, and db/db-IF fasting from the microbiome analysis.

Figure 7A-7D graphs displaying the diurnal pattern of several gut microorganism at different time during the day in db/m and db/db mice subject to ad lib feeding or IF feeding during feeding and fasting: genus Bifidobacterium OTU 1259 (Figure 7A), genus Sutterella OTU 378 (Figure 7B), genus Coprococcus OTU 182 (Figure 7C), and genus Oscillospira OTU 13 (Figure 7D).

Figure 7E is a series of graphs displaying examples of operational taxonomic units (OTUs) that show diurnal variations in the ad lib regimen. Figure 8A is a graph displaying the relative abundance and diurnal pattern of the gut microorganism order Clostridiales in db/m and db/db mice subject to AL feeding or IF feeding during feeding and fasting.

Figure 8B is a graph displaying the relative abundance and diurnal pattern of the gut microorganism family Ruminococcaceae in db/m and db/db mice subject to AL feeding or IF feeding during feeding and fasting.

Figure 8C is a graph displaying the relative abundance and diurnal pattern of the gut microorganism family Lachnospiraceae in db/m and db/db mice subject to AL feeding or IF feeding during feeding and fasting.

Figure 8D is a graph displaying the relative abundance and diurnal pattern of the gut microorganism genus Lactobacillus in db/m and db/db mice subject to AL feeding or IF feeding during feeding and fasting.

Figure 8E is a graph displaying the relative abundance and diurnal pattern of the gut microorganism genus Bifidobacterium in db/m and db/db mice subject to AL feeding or IF feeding during feeding and fasting.

Figure 8F is a graph displaying the relative abundance and diurnal pattern of the gut microorganism genus Oscillospira in db/m and db/db mice subject to AL feeding or IF feeding during feeding and fasting.

Figure 8G is a graph displaying the relative abundance and diurnal pattern of the gut microorganism genus Ruminococcus in db/m and db/db mice subject to AL feeding or IF feeding during feeding and fasting.

Figure 8H is a graph displaying the relative abundance and diurnal pattern of the gut microorganism genus Bacteroides in db/m and db/db mice subject to AL feeding or IF feeding during feeding and fasting.

Figure 81 is a graph displaying the relative abundance and diurnal pattern of the gut microorganism genus Akkermansia in db/m and db/db mice subject to AL feeding or IF feeding during feeding and fasting.

Figure 8J is a graph displaying the relative abundance of the gut microorganism family Clostridiaceae in db/m and db/db mice subject to AL feeding or IF feeding during feeding and fasting. Figure 8K is a graph displaying the relative abundance of the gut microorganism genus Oscillospira in db/m and db/db mice subject to AL feeding or IF feeding during feeding and fasting.

Figure 8L is a graph displaying the relative abundance of the gut microorganism Lactobacillus reuteri in db/m and db/db mice subject to AL feeding or IF feeding during feeding and fasting.

Figure 8M is a graph displaying the relative abundance of the gut microorganism Akkermansia muciniphila in db/m and db/db mice subject to AL feeding or IF feeding during feeding and fasting.

Figure 8N is a graph displaying the relative abundance and diurnal pattern of the gut microorganism family Ruminoccoccaceae in db/m and db/db mice subject to AL feeding or IF feeding during feeding and fasting.

Figure 80 is a graph displaying the relative abundance and diurnal pattern of the gut microorganism genus Lactobacillaceae in db/m and db/db mice subject to AL feeding or IF feeding during feeding and fasting.

Figure 8P is a graph displaying the relative abundance and diurnal pattern of the gut microorganism family Lachnospiraceae in db/m and db/db mice subject to AL feeding or IF feeding during feeding and fasting.

Figure 8Q is a graph displaying the relative abundance and diurnal pattern of the gut microorganism genus Allobaculum in db/m and db/db mice subject to AL feeding or IF feeding during feeding and fasting.

Figure 8R is a graph displaying the relative abundance and diurnal pattern of the gut microorganism genus Bifidobacterium in db/m and db/db mice subject to AL feeding or IF feeding during feeding and fasting.

Figure 8S is a graph displaying the relative abundance and diurnal pattern of the gut microorganism genus Bacteroides in db/m and db/db mice subject to AL feeding or IF feeding during feeding and fasting.

Figure 8T is a graph displaying the relative abundance and diurnal pattern of the gut microorganism genus Clostridum in db/m and db/db mice subject to AL feeding or IF feeding during feeding and fasting. Figure 8U is a graph displaying the relative abundance and diurnal pattern of the gut microorganism genus Turicibacter in db/m and db/db mice subject to AL feeding or IF feeding during feeding and fasting.

Figure 8V is a graph displaying the relative abundance and diurnal pattern of the gut microorganism genus Allobaculum in db/m and db/db mice subject to AL feeding or IF feeding during feeding and fasting.

Figure 9A-9D are immunohistochemical images showing goblet cells in colon samples from db/m or db/db mice subject to ad lib feeding or IF feeding.

Figure 9E is a bar graph displaying the counts of goblet cells in colon samples from db/m or db/db mice subject to ad lib feeding or IF feeding. *p < 0.05.

Figure 9F-9I are immunohistochemical images showing villi length in colon samples from db/m or db/db mice subject to ad lib feeding or IF feeding.

Figure 9J is a bar graph displaying the quantification of villi length in colon samples from db/m or db/db mice subject to ad lib feeding or IF feeding. *p < 0.05 and **p < 0.01.

Figure 9K-9N are immunohistochemical images showing muscular thickness in colon samples from db/m or db/db mice subject to ad lib feeding or IF feeding.

Figure 90 is a bar graph displaying the quantification of muscular thickness in colon samples from db/m or db/db mice subject to ad lib feeding or IF feeding.

Figure 10 is a bar graph displaying the quantification of peptidoglycan (ng/mL), a component of bacterial cell wall, measured in plasma samples from db/m or db/db mice subject to ad lib feeding or IF feeding during feeding and fasting.

Figure 11 A is a bar graph displaying the quantification of cholate measured in plasma sample analysis of bile acid metabolism from db/m or db/db mice subject to ad lib feeding or IF feeding during feeding and fasting.

Figure 1 IB is a bar graph displaying the quantification of deoxycholate measured in plasma sample analysis of bile acid metabolism from db/m or db/db mice subject to ad lib feeding or IF feeding during feeding and fasting.

Figure 11C is a bar graph displaying the quantification of taurochenodeoxy cholate (TCDCA) measured in plasma sample analysis of bile acid metabolism from db/m or db/db mice subject to ad lib feeding or IF feeding during feeding and fasting. Figure 1 ID is a bar graph displaying the quantification of tauroursodeoxycholate (TUDCA) measured in plasma sample analysis of bile acid metabolism from db/m or db/db mice subject to ad lib feeding or IF feeding during feeding and fasting.

Figure 12A is an immunohistochemical staining image showing TGR5 localized primarily in the ganglion cell layer of the retina.

Figure 12B-12E are immunohistochemical staining images showing the localization of TGR5 of retina samples from db/m mice subject to ad lib feeding (Figure 12B), db/m mice subject to IF feeding (Figure 12C), db/db mice subject to ad lib feeding (Figure 12D).

Figure 12F is a graph displaying the expression level of TGR5 in db/m and db/db mice subject to ad lib feeding or IF feeding during feeding and fasting.

Figure 12G is a graph displaying the level of TGR5 mRNA in db/m and db/db mice subject to ad lib feeding or IF feeding during feeding and fasting.

Figure 12H is a graph displaying the level of TNFa, a downstream target of TGR5, mRNA in db/m and db/db mice subject to ad lib feeding or IF feeding during feeding and fasting.

Figure 121 is a bar graph displaying the number of acellular capillaries per mm ² of retinal area in DBA/2J mice (C) or DBA/2J mice treated with streptozotocin and placed on a high fat diet (D) with (C+INT or D+INT)) or without treatment with Compound 1-Na.

Figure 12J is a bar graph displaying the number of CD45 positive leukocytes per section of the retina in DBA/2J mice (C) or DB A/2J mice treated with streptozotocin and placed on a high fat diet (D) with (C+INT or D+INT)) or without treatment with Compound 1-Na.

Figure 12K is a bar graph displaying the number of CD1 lb positive macrophages per section of the retina in DBA/2J mice (C) or DB A/2J mice treated with streptozotocin and placed on a high fat diet (D) with (C+INT or D+INT)) or without treatment with Compound 1-Na.

Figure 12L is a bar graph displaying the number of Ibal positive microglia per section of the retina in DBA/2J mice (C) or DBA/2J mice treated with streptozotocin and placed on a high fat diet (D) with (C+INT or D+INT)) or without treatment with Compound 1-Na. Figure 13A-13D are mass spectra of lipidomic analysis of liver samples from mice subject to different feeding protocols: db/m ad lib (Figure 13A), db/db ad lib (Figure 13B), db/m IF (Figure 13C), and db/db/ IF (Figure 13D).

Figure 14A-14H are bar graphs displaying the log ₂ fold change of various lipids (db/db ad lib vs. db/m ad lib or db/db/ IF vs. db/db ad lib, as indicated): triglycerides (TG) (Figures 14A and 14B), cholesteryl esters (CE) (Figures 14C and 14D), diglycerides (DG) (Figures 14E and 14F), and hexosylceramides (HexCer) (Figures 14G and 14H). *p<0.0001.

Figure 15A and 15B are graphs displaying changes in the level of triglycerides (Figure 15 A) and free fatty acids (Figure 15B) in the plasma of mice subject to ad lib feeding or IF feeding. *p < 0.05, **p < 0.01.

Figure 16A-16D are graphs displaying the expression level of Artnl in SCN (Figure 16 A), Artnl in liver (Figure 16C), Per2 in SCN (Figure 16B), and Per2 in SCN (Figure 16D) in mice subject to ad lib feeding or IF feeding. *p < 0.05 and **p < 0.01.

Figure 17A is a heatmap illustrating expression of various genes at different time during the day in mice subject to ad lib feeding or IF feeding.

Figure 17B is a graph showing clustering of genes that are differentially expressed in mice subject to ad lib feeding or IF feeding: 1 : ZT9_fast, 2: ZT21_fast, 3 : ZT21_feed, 4: ZT9_feed, 5: ZT21, and 6: ZT9.

Figure 17C is a graph displaying difference in the genes that are upregulated or downregulated at night or during the day in mice subject to ad lib feeding or IF feeding.

Figure 18A-18F are graphs displaying difference in the genes that are upregulated or downregulated at night or during the day in mice subject to ad lib feeding or IF feeding: Anxal (Figure 18A), Lcnl3 (Figure 18B), Plac8 (Figure 18C), Vldlr (Figure 18D), Plin4 (Figure 18E), and Smpd3 (Figure 18F).

Figure 19 is a schematic diagram for a model of IF-induced changes in the

microbiome and their potential impact on development of DR.

DETAILED DESCRIPTION

The present application is based at least in part on the discovery that a compound of Formula A or a pharmaceutically acceptable salt or amino acid conjugate thereof is effective in reversing an eye disease, such as a diabetic eye disease {e.g., diabetic retinopathy). Accordingly, the present application relates to a method of treating or preventing an eye disease in a subject in need thereof, comprising administering to the subject a

therapeutically effective amount of a compound of Formula A:

(A),

or a pharmaceutically acceptable salt or amino acid conjugate thereof, wherein:

Ri is Ci-Ce alkyl;

R2, R3, Rs, and R ₆ are each independently H or OH;

R7 is H or Ci-Ce alkyl.

In one embodiment, the eye disease is modulated by FXR (e.g., where the expression of FXR plays a role in the initiation and/or development of the eye disease). In one embodiment, the eye disease is modulated by TGR5 (e.g., where the expression of TGR5 plays a role in the initiation and/or development of the eye disease). In one embodiment, the eye disease is modulated by FXR and TGR5. In one embodiment, the eye disease is a diabetic eye disease that is modulated by FXR (e.g., where the expression of FXR plays a role in the initiation and/or development of the diabetic eye disease). In one embodiment, the eye disease is a diabetic eye disease that is modulated by TGR5 (e.g., where the expression of TGR5 plays a role in the initiation and/or development of the diabetic eye disease). In one embodiment, the eye disease is a diabetic eye disease that is modulated by FXR and TGR5. In one embodiment, the eye disease or the diabetic eye disease is associated with decreased expression of FXR. In one embodiment, the eye disease or the diabetic eye disease is associated with decreased expression of TGR5. In one embodiment, the eye disease or the diabetic eye disease is associated with decreased expression of FXR and TGR5.

In one embodiment, the eye disease starts to develop in a subject that suffers from a high level of blood glucose. In one embodiment, the eye disease starts to develop in a subject that suffers chronically from a high level of blood glucose. In one embodiment, the eye disease starts to develop in a subject that has diabetes. In one embodiment, the eye disease is caused by changes in the blood vessels of the eye. In one embodiment, the eye disease is caused by changes in the blood vessels of the retina. In one embodiment, the eye disease is caused by fluid leakage or hemorrhage from the blood vessels of the retina. In one embodiment, the eye disease is caused by abnormal growth of new blood vessels on the surface of the retina. In one embodiment, the eye disease is caused by poor blood flow and the pressure buildup in the blood vessels of the retina. In one embodiment, the changes in the blood vessels of the eye or retina, the fluid leakage or hemorrhage from the blood vessels of the eye or retina, the abnormal growth of new blood vessels on the surface of the retina, or poor blood flow and the pressure buildup in the blood vessels of the retina are associated with a high level of blood glucose.

In one embodiment, the eye disease is selected from retinopathy, macular edema, neovascularization in the eye, retinal vascular occlusion, retinal lipid or cholesterol accumulation and a diabetic eye disease. In one embodiment, the diabetic eye disease is selected from diabetic retinopathy, diabetic macular edema, glaucoma, and cataracts. In one embodiment, the eye disease is retinopathy. In one embodiment, the eye disease is diabetic retinopathy.

The present application also relates to a method of improving one or more retinal functions in a subject in need thereof, comprising administering to the subject a

therapeutically effective amount of a compound of Formula A, or a pharmaceutically acceptable salt or amino acid conjugate thereof.

The present application also relates to a method of regulating one or more

complementary or nonoverlapping signaling and/or metabolic pathways involved in diabetic retinopathy. In certain embodiments, the regulating includes, but is not limited to, restoring reverse cholesterol transport, preventing inflammation, reducing pro-inflammatory macrophage activity, and/or preventing the formation of acellular capillaries.

In one embodiment, one or more retinal functions, such as the functions described herein above, are improved by at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100%) after the administration of a compound of the present application, for example, as compared to the level of the one or more retinal functions before the administration or to the normal level of the one or more retinal functions. In one embodiment, one or more retinal functions in a subject are impaired (e.g., to 90%, 80%, 70%, 60%, 50%, 40%, or 30% of the normal level of the one or more retinal functions), and improving one or more retinal functions by administration of a compound of the present application restores the one or more retinal functions to 50%, 60%, 70%, 80%, 90%, or 100% of the normal level of the one or more retinal functions before the impairment. In one embodiment, one or more retinal functions in a subject are decreased as compared to a control subject (e.g., a control subject as described herein). In one embodiment, one or more retinal functions in a subject are decreased (e.g., to 90%, 80%, 70%, 60%, 50%, 40%, or 30% of the level of the one or more retinal functions in a control subject), and improving one or more retinal functions by administration of a compound of the present application restores the one or more retinal functions in the subject to 50%, 60%, 70%, 80%, 90%, or 100% of the level of the one or more retinal functions in the control subject.

In one embodiment, the methods of the application are achieved through increasing the level of FXR and/or TGR5 in a subject in need thereof.

In one embodiment, the level of FXR and/or TGR5 is increased by at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100% after the administration of a compound of the present application. In one embodiment, the level of FXR and/or TGR5 is decreased in a subject (e.g., to 90%, 80%, 70%, 60%, 50%, 40%, or 30% of the normal level of FXR and/or TGR5), and increasing the level of FXR and/or TGR5 by administration of a compound of the present application restores the level of FXR and/or TGR5 to 50%, 60%, 70%, 80%, 90%, or 100% of the normal level of FXR and/or TGR5 before the decrease. In one embodiment, the level of FXR and/or TGR5 in the subject is decreased as compared to a control subject (e.g., a control subject as described herein). In one embodiment, the level of FXR and/or TGR5 in the subject is decreased (e.g., to 90%, 80%, 70%, 60%, 50%, 40%, or 30% of the level of FXR and/or TGR5 in a control subject), and increasing the level of FXR and/or TGR5 by administration of a compound of the present application restores the level of FXR and/or TGR5 to 50%, 60%, 70%, 80%, 90%, or 100% of the level of FXR and/or TGR5 in the control subject.

In one embodiment, the methods of the present application further comprise measuring the level of one or more targets in the subject, wherein the target is selected from FXR, TGR5, synaptopodin, Nrf-1, pAMPK, Sirtl, Sirt3, ERRa, PGCla, MCAD, Cox4, LCAD, TGF-β, fibronectin, FSP-1, T F-a, TLR2, TLR4, and acetyl-IDH2. In one embodiment, the methods of present application comprise administering to a subject in need thereof a therapeutically effective amount of a compound of the present application, when the level of one or more targets is increased in the subject as compared to the normal level of the one or more targets in the subject or as compared to a control subject. In one embodiment, the target is selected from SREBP-1, SCD-1, SCD-2, Fit- 1 , kidney triglyceride, kidney cholesterol, p65, p50, FKB, NOX-2, NOX-4, Hifla, Hif2a, Glutl, p- EIF2a, collagen I, collagen III, collagen IV, p22-phox, CD68, ICAM-1, Cox2, CTGF, FSP-1, Snail, ZEB1, TGF-β, fibronectin, FSP-1, acetyl-IDH2, TNF-a, TLR2, and TLR4.

In one embodiment, the methods of present application comprise administering to a subject in need thereof a therapeutically effective amount of a compound of the present application, when the level of one or more targets is decreased in the subject as compared to the normal level of the one or more targets in the subject or as compared to a control subject. In one embodiment, the target is selected from CD36, LPL, FXR, TGR5, synaptopodin, Nrf-1, pAMPK, Sirtl, Sirt3, ERRa, PGCla, MCAD, Cox4, and LCAD.

In one embodiment, the subject has one or more diseases, disorders, or conditions in addition to the eye disease, disorder, or condition. In one embodiment, the additional disease, disorder, or condition is selected from a renal disease, a cardiovascular disease, hypertension, a metabolic syndrome, diabetes, obesity, and insulin resistance.

Compounds and compositions of the application can be administered in therapeutically effective amounts in a combinatorial therapy with one or more therapeutic agents

(pharmaceutical combinations) or modalities. Where the compounds of the application are administered in conjunction with other therapies, dosages of the co-administered compounds will of course vary depending on the type of co-drug employed, on the specific drug employed, on the condition being treated and so forth. For example, synergistic effects can occur with substances.

Combination therapy includes the administration to a subject in need thereof one or more other biologically active ingredients (including, but not limited to, a FXR agonist, a TGR5 agonist, a second compound of Formula A) and non-drug therapies (including, but not limited to, surgery or dietary treatment). For instance, the compounds of the application can be used in combination with other pharmaceutically active compounds, preferably compounds that are able to enhance the effect of the compounds of the application. The compounds of the application can be administered simultaneously (as a single preparation or separate preparation) or sequentially to the other drug therapy or treatment modality. In general, a combination therapy envisions administration of two or more drugs during a single cycle or course of therapy (e.g., within 10 minutes, 20 minutes, 30 minutes, 60 minutes, 2 hours, 3 hours, 4 hours, 6 hours, 12 hours).

In another embodiment, the compounds may be administered in combination with one or more separate pharmaceutical agents, e.g., a chemotherapeutic agent, an

immunotherapeutic agent, an anti-inflammatory agent, or an adjunctive therapeutic agent.

In one embodiment, the methods of the present application comprise administering to a subject in need thereof a therapeutically effective amount of a compound of the present application, in combination with reducing the calorie intake in the subject. In one

embodiment, reducing calorie intake comprises consuming a diet that has a reduced amount of calorie by the subject. In one embodiment, the amount of calorie is reduced by 10%, by 20%, by 30%), by 40%, by 50%, by 60%>, or by 70% as compared to the normal calorie intake by the subject.

In one embodiment, the methods of the present application comprise administering to a subject in need thereof a therapeutically effective amount of a compound of the present application, in combination with intermittent fasting in the subject. In one embodiment, the intermittent fasting comprises fasting in the subject every other day, once every three days, once every four days, once every five days, once every six days, or once every seven days. In one embodiment, the intermittent fasting lasts for a period of one week, two weeks, three weeks, four weeks, six weeks, two months, three months, four months, six months, eight months, ten months, twelve months, 2 years, 5 years, or more.

In one embodiment, the subject is a human. In one embodiment, the control subject is a human. In one embodiment, the subject and/or control subject is a human less than 70 years of age, less than 65 years of age, less than 60 years of age, less than 55 years of age, less than 50 years of age, less than 45 years of age, less than 40 years of age, or less than 35 years of age. In one embodiment, the subject and/or control subject is a human less than 65 years of age.

In one embodiment, a compound of Formula A is of Formula B or Formula C:

For any of Formula A, B, or C, Ri, R2, R3, R4, R5, R ₆ , and R7 can be selected from the groups, and combined, where applicable, as described below.

In one embodiment, Ri is Ci-C ₆ alkyl selected from methyl, ethyl, n-propyl, i-propyl, n-butyl, i-butyl, t-butyl, pentyl, and hexyl. In one embodiment, Ri is C1-C4 alkyl selected from methyl, ethyl, n-propyl, i-propyl, n-butyl, i-butyl, and t-butyl. In one embodiment, Ri is methyl, ethyl, n-propyl, or i-propyl. In one embodiment, Ri is methyl or ethyl. In one embodiment, Ri is methyl. In one embodiment, Ri is ethyl.

In one embodiment, R2 is H and R3 is OH. In one embodiment, R3 is H and R2 is OH. In one embodiment, R5 is H. In one embodiment, R5 is OH.

In one embodiment, R2 is H, R3 is OH, and R5 is H. In one embodiment, R2 is H, R3 is OH, and Rs is OH.

In one embodiment, R7 is H. In one embodiment, R7 is Ci-C ₆ alkyl selected from methyl, ethyl, n-propyl, i-propyl, n-butyl, i-butyl, t-butyl, pentyl, and hexyl. In one embodiment, R7 is C1-C4 alkyl selected from methyl, ethyl, n-propyl, i-propyl, n-butyl, i- butyl, and t-butyl. In one embodiment, R7 is methyl, ethyl, n-propyl, or i-propyl. In one embodiment, R7 is methyl or ethyl. In one embodiment, R7 is methyl.

In one embodiment, R2 is H, R3 is OH, and R7 is H. In one embodiment, R2 is H, R3 is OH, and R7 is methyl.

In one embodiment, R2 is H, R3 is OH, R5 is H, and R7 is H. In one embodiment, R2 is

H, R ₃ is OH, R ₅ is OH, and R7 is methyl.

In one embodiment, R ₆ is H. In one embodiment, R ₆ is OH.

In one embodiment, R2 is H, R3 is OH, and R ₆ is H. In one embodiment, R2 is H, R3 is OH, and Re is OH.

In one embodiment, R2 is H, R3 is OH, R5 is H, and R ₆ is H. In one embodiment, R2 is

H, R3 is OH, R5 is OH, and R ₆ is OH. In one embodiment, R2 is H, R3 is OH, R5 is OH, and R ₆ is H. In one embodiment, R2 is H, R3 is OH, R5 is H, and R ₆ is OH. In one embodiment, R ₄ is CO2H. In one embodiment, R ₄ is OSO3H.

In one embodiment, R2 is H, R3 is OH, R ₄ is CO2H, and R5 is H. In one embodiment, R2 is H, R3 is OH, R ₄ is OSO3H, and R5 is H. In one embodiment, R2 is H, R3 is OH, R ₄ is CO2H, and Rs is OH. In one embodiment, R ₂ is H, R ₃ is OH, R ₄ is OSO3H, and Rs is OH. In a further embodiment, Ri is ethyl.

In one embodiment, R2 is H, R3 is OH, R ₄ is CO2H, and R5 is OH. In one

embodiment, R2 is H, R3 is OH, R ₄ is CO2H, R5 is OH, and R7 is methyl. In a further embodiment, Ri is ethyl.

In one embodiment, R2 is H, R3 is OH, R ₄ is CO2H, and R5 is H. In one embodiment, R2 is H, R3 is OH, R ₄ is CO2H, R5 is H, and R ₆ is OH. In a further embodiment, Ri is ethyl.

In one embodiment, a compound of Formula A is Compound 1 :

(1),

or a pharmaceutically acceptable salt or amino acid conjugate thereof.

In one embodiment, a compound of Formula A is Compound 2:

or a pharmaceutically acceptable salt or amino acid conjugate thereof.

In one embodiment, a compound of Formula A is Compound 3 :

or a pharmaceutically acceptable salt or amino acid conjugate thereof. In one embodiment, a compound of Formula A is Compound 4:

or a pharmaceutically acceptable salt or amino acid conjugate thereof.

In one embodiment, a pharmaceutically acceptable salt of Compound 1 is the sodium salt of Compound 1 (i.e., Compound 1-Na). In yet another embodiment, a pharmaceutically acceptable salt of Compound 1 is the tri ethyl ammonium salt of Compound 1 (i.e., Compound 1-TEA).

As used herein, the term "Compound 1" refers to

which is also known as 6a-ethyl-3a,7a,23-trihydroxy-24-nor-5p-cholan-23-hydrogen sulphate. "Compound 1-Na" or "1 -Na" which is also known as INT-767 or 6a-ethyl- 3a,7a,23-trihydroxy-24-nor-5p-cholan-23-sulphate sodium" are used interchangeably, and refer to the sodium salt of Compound 1. As used herein, "Compound 1 -TEA" or "1-TEA" is used interchangeably, and refer to the triethylammonium salt of Compound 1. The structures of Compound 1 ~Na and Compound 1 -TEA are provided below.

The phrase a "compound of the application" or "compound of the present application" as used herein encompasses a compound of Formula A, Formula B, or Formula C, Compound 1, 1-Na, 1-TEA, Compound 2, Compound 3, or Compound 4, or a pharmaceutically acceptable salt or amino acid conjugate thereof.

As used herein, the term "amino acid conjugate" refers to a conjugate of the compound of the present application with any suitable amino acid. For example, such a suitable amino acid conjugate of a compound of the present application will have the added advantage of enhanced integrity in bile or intestinal fluids. Suitable amino acids include but are not limited to glycine and taurine. Thus, the present application encompasses the glycine and taurine conjugates of the compound of the present application (e.g., Compound 1).

As used herein, FXR refers to Farnesoid X Receptor, which is a member of the nuclear receptor family of ligand-activated transcription factors that includes receptors for the steroid, retinoid, and thyroid hormones. FXR binds to DNA as a heterodimer with the 9-cis retinoic acid receptor (RXR).

As used herein, TGR5 refers to a G-protein-coupled receptor that is responsive to bile acids (BAs).

As used herein, a "subject in need thereof is a subject having an eye disease against which a compound of the application is effective, or a subject having an increased risk of developing an eye disease against which a compound of the application is effective relative to the population at large. A "subject" includes a mammal. The mammal can be any mammal, e.g., a human, primate, bird, mouse, rat, fowl, dog, cat, cow, horse, goat, camel, sheep or a pig. Particularly, the mammal is a human. In one embodiment, a subject in need thereof is a subject that has high level of blood glucose. In one embodiment, a subject in need thereof is a subject that has diabetes.

As used herein, "control" or "control subject" refers to an untreated sample or an untreated subject. The control subject may be the same subject before the effect of the treatment, for example, with a compound of Formula A, or a different untreated subject. A control may include a known value or may be a known sample or subject that is not treated, for example, with a compound of Formula A.

The term "treating" as used herein refers to any indicia of success in the treatment or amelioration of any of the diseases, disorders, or conditions described herein. Treating can include, for example, reducing or alleviating the severity of one or more symptoms of any of the diseases, disorders, or conditions described herein, or it can include reducing the frequency with which symptoms of any of the diseases, disorders, or conditions described herein are experienced by a patient. "Treating" can also refer to reducing or eliminating any of the diseases, disorders, or conditions described herein of a part of the body, such as a cell, tissue or bodily fluid, e.g., retina.

As used herein, the term "preventing" refers to the partial or complete prevention of any of the diseases, disorders, or conditions described herein in an individual or in a population, or in a part of the body, such as a cell, tissue or bodily fluid (e.g., retina). The term "prevention" does not establish a requirement for complete prevention of a disease, disorder, or condition in the entirety of the treated population of individuals or cells, tissues, or fluids of individuals.

The term "treat or prevent" is used herein to refer to a method that results in some level of treatment or amelioration of any of the diseases, disorders, or conditions described herein, and contemplates a range of results directed to that end, including but not restricted to prevention of any of the diseases, disorders, or conditions described herein entirely.

Unless specified or the context dictates otherwise, "ocular" and "eye" are used interchangeably.

As used herein, "pharmaceutically acceptable" refers to a material that is not biologically or otherwise undesirable, e.g., the material may be incorporated into a pharmaceutical composition administered to a patient without causing any significant undesirable biological effects or interacting in a deleterious manner with any of the other components of the composition in which it is contained. Pharmaceutically acceptable carriers or excipients have met the required standards of toxicological and manufacturing testing and/or are included on the Inactive Ingredient Guide prepared by the U.S. Food and Drug administration.

A "pharmaceutically acceptable excipient" means an excipient that is useful in preparing a pharmaceutical composition that is generally safe, non-toxic and neither biologically nor otherwise undesirable, and includes excipient that is acceptable for veterinary use as well as human pharmaceutical use. A "pharmaceutically acceptable excipient" as used in the specification and claims includes both one and more than one such excipient.

The phrase "therapeutically effective amount" as used herein refers to an effective amount comprising an amount sufficient to treat a disease, disorder, or condition described herein or to prevent or delay a disease, disorder, or condition described herein. In some embodiments, an effective amount is an amount sufficient to delay the development of the disease, disorder, or condition. In some embodiments, an effective amount is an amount sufficient to prevent or delay recurrence. An effective amount can be administered in one or more administrations.

A therapeutically effective amount can be estimated initially either in cell culture assays, e.g., in a cell from the retina, or animal models, usually rats, mice, rabbits, dogs, or pigs. The animal model may also be used to determine the appropriate concentration range and route of administration. Such information can then be used to determine useful doses and routes for administration in humans. Therapeutic/prophylactic efficacy and toxicity may be determined by standard pharmaceutical procedures in cell cultures or experimental animals, e.g., ED50 (the dose therapeutically effective in 50% of the population) and LD50 (the dose lethal to 50% of the population). The dose ratio between toxic and therapeutic effects is the therapeutic index, and it can be expressed as the ratio, LD50/ED50. Pharmaceutical compositions that exhibit large therapeutic indices are preferred.

The term "regimen" as used herein refers to a protocol for dosing and/or timing the administration a compound of the application. A regimen can include periods of active administration and periods of rest as known in the art. Active administration periods include administration of a compound of the application in a defined course of time, including, for example, the number of and timing of dosages of the compositions. In some regimens, one or more rest periods can be included where no compound is actively administered, and in certain instances, includes time periods where the efficacy of such compounds can be minimal.

In one embodiment, a compound of the present application is administered once daily, twice daily, three times daily, once every 6 hours, or once every 4 hours. In one embodiment, a compound of the present application is administered for one day, two days, three days, four days, five days, six days, or seven days a week. In one embodiment, a compound of the present application is not administered every day of the week. In one embodiment, a compound of the present application is administered every other day, once every three days, once every four days, once every five days, once every six days, or once every seven days.

In one embodiment, a compound of the present application is administered for a period of one week, two weeks, three weeks, four weeks, six weeks, two months, three months, four months, six months, or more. In one embodiment, the period in which a compound of the present application is administered comprises one or more segments (e.g., one or more days, one or more weeks, or one or more months) during which the compound is not administered. In one embodiment, the one or more segments during which the compound is not

administered are preceded by and followed by administration of the compound.

As used herein, "intermittent fasting" or "IF" refers to a diet that cycles between a period of fasting and non-fasting during a defined period of time. This includes whole-day fasting involving a regular one-day (24 hours) fast and time-restricting feeding (TRF) involving eating only during a certain number of hours each day (24 hours).

As used herein, "combination therapy" means that a compound of the application can be administered in conjunction with another therapeutic agent. "In conjunction with" refers to administration of one treatment modality in addition to another treatment modality, such as administration of a compound of the application as described herein in addition to

administration of another therapeutic agent to the same subject. As such, "in conjunction with" refers to administration of one treatment modality before, during, or after delivery of a second treatment modality to the subject.

Unless specified or the context dictates otherwise, a "pharmaceutical composition" or "pharmaceutical formulation" is used interchangeably, and refers to a formulation containing a compound of the present application in a form suitable for administration to a subject. In one embodiment, the pharmaceutical composition is in bulk or in unit dosage form. It can be advantageous to formulate compositions in dosage unit form for ease of administration and uniformity of dosage. The specification for the dosage unit forms is dictated by and directly dependent on the unique characteristics of the active reagent and the particular therapeutic effect to be achieved. The unit dosage form is any of a variety of forms, including, for example, a capsule, an IV bag, a tablet, a single pump on an aerosol inhaler, or a vial.

The term "about" as used herein when referring to a measurable value such as an amount, a temporal duration, and the like, is meant to encompass variations of ±20% or ±10%, in some embodiments ±5%, in some embodiments ±1%, and in some embodiments ±0.1%) from the specified value, as such variations are appropriate to practice the disclosed methods or to make and used the disclosed compounds and in the claimed methods. Possible formulations include those suitable for oral, sublingual, buccal, parenteral (e.g., subcutaneous, intramuscular, or intravenous), rectal, topical including transdermal, intranasal, and inhalation administration. Most suitable means of administration for a particular patient will depend on the nature and severity of the disease being treated, the nature of the therapy being used, and the nature of the active compound.

Formulations suitable for oral administration may be provided as discrete units, such as tablets, capsules, cachets, lozenges, each containing a predetermined amount of the active compound; as powders or granules; as solutions or suspensions in aqueous or non-aqueous liquids; or as oil-in-water or water-in-oil emulsions.

Formulations suitable for sublingual or buccal administration include lozenges comprising a compound of the application and typically a flavored base, such as sugar and acacia or tragacanth and pastilles comprising the active compound in an inert base, such as gelatin and glycerin or sucrose acacia.

Formulations suitable for parenteral administration typically comprise sterile aqueous solutions containing a predetermined concentration of the active compound; the solution may be isotonic with the blood of the intended recipient. Additional formulations suitable for parenteral administration include formulations containing physiologically suitable co-solvents and/or complexing agents such as surfactants and cyclodextrins. Oil-in-water emulsions are also suitable formulations for parenteral formulations. Although such solutions may be administered intravenously, they may also be administered by subcutaneous or intramuscular injection.

Formulations suitable for rectal administration may be provided as unit-dose suppositories comprising a compound of the application in one or more solid carriers forming the suppository base, for example, cocoa butter.

Formulations suitable for topical or intranasal application include ointments, creams, lotions, pastes, gels, sprays, aerosols, and oils. Suitable carriers for such formulations include petroleum jelly, lanolin, polyethyleneglycols, alcohols, and combinations thereof.

Oral formulations generally include an inert diluent or an edible pharmaceutically acceptable carrier. They can be enclosed in gelatin capsules or compressed into tablets. For the purpose of oral administration, the active ingredient can be incorporated with excipients and used in the form of tablets, troches, or capsules. Oral formulations can also be prepared using a fluid carrier for use as a mouthwash, wherein the active ingredient in the fluid carrier is applied orally and swished and expectorated or swallowed. Pharmaceutically compatible binding agents, and/or adjuvant materials can be included as part of the composition. The tablets, pills, capsules, troches and the like can contain any of the following ingredients, or compounds of a similar nature: a binder such as microcrystalline cellulose, gum tragacanth or gelatin; an excipient such as starch or lactose, a disintegrating agent such as alginic acid, Primogel, or corn starch; a lubricant such as magnesium stearate or Sterotes®; a glidant such as colloidal silicon dioxide; a sweetening agent such as sucrose or saccharin; or a flavoring agent such as peppermint, methyl salicylate, or orange flavoring. Pharmaceutically compatible diluents may also include starch, dextrin, sucrose, glucose, lactose, mannitol, sorbitol, xylitol, microcrystalline cellulose, calcium sulfate, calcium hydrogen phosphate, calcium carbonate, and the like. Pharmaceutically compatible wetting agents included water, ethanol, isopropanol, and the like. Pharmaceutically compatible binders may also include starch pulp, dextrin, syrup, honey, glucose solution, microcrystalline cellulose, mucilage of arabic gum, gelatin mucilage, sodium hydroxymethylcellulose, methylcellulose,

hydroxypropylmethylcellulose, ethyl cellulose, acrylic resin, carbomer, polyvinyl pyrrolidone, polyethylene glycol, and the like. Pharmaceutically compatible disintegrants may also include dry starch, microcrystalline cellulose, low-substituted hydroxypropylcellulose, cross-linked polyvinylpyrrolidone, croscarmellose sodium, sodium carboxymethyl starch, sodium bicarbonate and citric acid, polyoxyethylene sorbitol fatty acid esters, sodium dodecyl sulfonate and the like. Pharmaceutically compatible lubricants and glidants may also include talc powder, silica, stearate, tartaric acid, liquid paraffin, polyethylene glycol, and the like.

Pharmaceutical formulations suitable for injectable use (e.g., intravenous,

intramuscular) include sterile aqueous solutions (where water soluble),

dispersions/suspensions, and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions. Suitable carriers include physiological saline,

bacteriostatic water, Cremophor EL™ (BASF, Parsippany, N.J.) or phosphate buffered saline (PBS). The carriers can also be a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyethylene glycol, and the like), and suitable mixtures thereof. The composition must be sterile and should be fluid to the extent that easy syringeability exists. It must be stable under the conditions of manufacture and storage and must be preserved against contaminating by microorganisms such as bacteria and fungi. The proper fluidity can be maintained, for example, by the use of agents such as lecithin, by the maintenance of the required particle size in the case of dispersion and by the use of surfactants. Prevention of the action of

microorganisms can be achieved by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, ascorbic acid, thimerosal, and the like. In many cases, it will include isotonic agents, for example, sugars, polyalcohols such as mannitol, sorbitol, sodium chloride in the composition. Other excipients include, but are not limited to, antioxidants such as ascorbic acid or sodium bisulfite; chelating agents such as ethylenediaminetetraacetic acid; buffers such as acetates, citrates or phosphates; and agents for the adjustment of tonicity such as sodium chloride or dextrose. The pH can be adjusted with acids or bases, such as hydrochloric acid or sodium hydroxide. Prolonged absorption of the injectable compositions can be brought about by including in the composition an agent which delays absorption, for example, aluminum monostearate and gelatin. The preparation can be enclosed in ampoules, disposable syringes or multiple dose vials made of glass or plastic.

Sterile injectable solutions can be prepared by incorporating the active ingredient in the required amount in an appropriate solvent with one or a combination of ingredients enumerated above, as required, followed by filtered sterilization. Generally, dispersions are prepared by incorporating the active ingredient into a sterile vehicle that contains a basic dispersion medium and the required other ingredients from those enumerated above. In the case of sterile powders for the preparation of sterile injectable solutions, methods of preparation are vacuum drying and freeze-drying that yields a powder of the active ingredient plus any additional desired ingredient from a previously sterile-filtered solution thereof.

Formulations of the application may be prepared by any suitable method, typically by uniformly and intimately admixing a compound of the application with liquids or finely divided solid carriers or both, in the required proportions and then, if necessary, shaping the resulting mixture into the desired shape.

For example, a tablet may be prepared by compressing an intimate mixture comprising a powder or granules of the active ingredient and one or more optional ingredients, such as a binder, lubricant, inert diluent, or surface active dispersing agent, or by molding an intimate mixture of powdered active ingredient and inert liquid diluent. Suitable formulations for administration by inhalation include fine particle dusts or mists which may be generated by means of various types of metered dose pressurized aerosols, nebulizers, or insufflators.

In addition to the ingredients specifically mentioned above, the formulations of the present application may include other agents known to those skilled in the art of pharmacy, having regard for the type of formulation in issue. For example, formulations suitable for oral administration may include flavoring agents and formulations suitable for intranasal administration may include perfumes.

Dosage and administration may be adjusted to provide sufficient levels of the active agent(s) (e.g., a compound of the present application) to maintain a desired effect. Factors which may be taken into account include the severity of the disease state, general health of the subject, age, weight, and gender of the subject, diet, time and frequency of administration, drug combination(s), reaction sensitivities, and tolerance/response to therapy. Pharmaceutical compositions may be administered every 1-96 hours, preferably every 2-72 hours, preferably every 3-48 hours, preferably every 4-24 hours, preferably every 6-12 hours. Long-acting pharmaceutical compositions may be administered every 3 to 4 days, every week, or once every two weeks depending on half-life and clearance rate of the particular formulation.

In one embodiment, a pharmaceutical composition is administered in a dosage form which comprises a compound of the application in a daily total amount of from about 0.1- 1500 mg, 0.2-1200 mg, 0.3-1000 mg, 0.4-800 mg, 0.5-600 mg, or 0.6-500 mg. In one embodiment, a pharmaceutical composition is administered in a dosage form which comprises a compound of the application in a daily total amount of from 0.5-20 mg, preferably 1 -15 mg, preferably 2-14 mg, preferably 4-12 mg, preferably 5-10 mg.

In one embodiment, a pharmaceutical composition is administered in a dosage form which comprises a compound of the application in a daily total amount of about 50 mg. In one embodiment, a pharmaceutical composition is administered in a dosage form which comprises a compound of the application in a daily total amount of about 40 mg. In one embodiment, a pharmaceutical composition is administered in a dosage form which comprises a compound of the application in a daily total amount of about 30 mg. In one embodiment, a pharmaceutical composition is administered in a dosage form which comprises a compound of the application in a daily total amount of about 20 mg. In one embodiment, a pharmaceutical composition is administered in a dosage form which comprises a compound of the application in a daily total amount of about 15 mg. In one embodiment, a pharmaceutical composition is administered in a dosage form which comprises a compound of the application in a daily total amount of about 10 mg. In one embodiment, a pharmaceutical composition is administered in a dosage form which comprises a compound of the application in a daily total amount of about 5 mg. In one embodiment, a pharmaceutical composition is administered in a dosage form which comprises a compound of the application in a daily total amount of about 2.5 mg. In one embodiment, a pharmaceutical composition is administered in a dosage form which comprises a compound of the application in a daily total amount of about 1 mg.

In one embodiment, a pharmaceutical composition is administered in a dosage form which comprises a compound of the application in a daily total amount of less than 10 mg/kg, preferably less than 5 mg/kg, such as, for example 0.1-5.0 mg/kg, preferably 0.5-4.5 mg/kg, preferably 1.0-4.0 mg/kg, preferably 1.2-3.5 mg/kg, preferably 1.4-3.0 mg/kg, preferably 1.5- 2.5 mg/kg, preferably 1.6-2.4 mg/kg. EXAMPLES

Example 1. Synthesis of compounds of the present application

Compounds of the present application can be prepared by methods known in the art (e.g., those described in U.S. Patent Nos. 7, 138,390; 7,994,352; 7,932,244; 8, 1 14,862; and 9,61 1,289). For example, a compound of the present application can be prepared by a process as shown in Scheme 1 and disclosed in WO 2014/066819.

Scheme 1

l-Na I

Step 1 is the esterification of Compound 2 to obtain Compound 4. Step 2 is a reaction to form Compound 5 from Compound 4. Step 3 is the protection of the hydroxy group at the C3 position of Compound 5 to afford Compound 6. Step 4 is the oxidative cleavage of Compound 6 to afford Compound 7. Step 5 is the reduction of Compound 7 to afford Compound 8. Step 6 is the sulfonation of Compound 8 to afford the sodium salt of

Compound 1 (1-Na). The sodium salt of Compound 1 can be converted to its free acid form (i.e., Compound 1) or other salt forms (e.g., Compound 1-TEA or the triethylammonium salt of Compound 1) according to procedures known in the art.

Example 2. Methods of the present application

■ Animals and intermittent fasting

Male B6.BKS(D)-Lepr ^db /J (stock number: 000697) homozygous genotype + db/+ db mice were used (denoted as db/db herein), and heterozygotes (m +/ + db) were used as controls (denoted as db/m herein). All mice were obtained from The Jackson Laboratory (Bar Harbor, ME), and housed in the institutional animal care facilities with strict 12h: 12h ligh dark cycle.

Blood glucose levels were tested every two weeks in db/db mice. Animals were considered diabetic and used in the intermittent fasting (IF) experiment if the serum glucose level was above 250 mg/dL for at least two consecutive measurements. The animals were fed ad lib before the IF was initiated at 4 months of age. The db/m and db/db mice were each divided into two subgroups, ad lib feeding (control) and IF, wherein the animals were fasted for 24 hours every other day for 7 months, beginning at night (Fig. 1 A). Glycated hemoglobin was measured using the AlCNow+ kit (Bayer HealthCare, Sunnyvale, CA) on the day prior to euthanasia.

All studies were repeated.

The experiments of the present disclosure were performed according to methods of the art, for example as in, Beli E. et al., Diabetes (2018; published online April 30, 2018 ahead of print) - the entire contents of which is incorporated herein by reference in its entirety.

■ Compound 1-Na treatment of diabetic animals

8-week old male DBA2/J mice were obtained from The Jackson Laboratory and maintained on a 12h: 12h ligh dark cycle. Mice were injected with streptozotocin (STZ;

Sigma-Aldrich, St. Louis, MO) intraperitoneally (40 mg/kg made freshly in 50 mM sodium citrate buffer, pH 4.5) for five consecutive days, or with 50 mM sodium citrate solution (pH 4.5) only. Blood glucose levels were measured 1 week after the last STZ injection and mice with glucose levels > 250 mg/dL on two separate days were considered diabetic. DBA/2J mice were fed with a Western diet (WD, 21% milk fat, 0.15% cholesterol, TD88137) obtained from Harlan-Teklad (Madison, WI) after diabetes onset in the STZ groups and were treated for 8 weeks with either normal chow or chow containing Compound 1-Na (30 mg/kg bw/day). ■ Lipidomic analysis

Monophasic lipid extraction with methanol: chloroform: water (2: 1 :0.74, v:v:v) was applied to 5 mg of frozen liver tissue from each animal. For mass spectrometry analysis, liver lipid extracts were combined with the synthetic internal standards PC (14:0/14:0), PE

(14:0/14:0), and PS (14:0/14:0) from Avanti® Polar Lipids (Alabaster, AL), and subjected to sequential functional group selective modification of PE and PS lipids using ¹³ Ci-S,S'- dimethylthiobutanoylhydroxysuccinimide ester ( Ci-DMBNHS), and the O-alkenyl-ether double bond of plasmalogen lipids using iodine and methanol.

Immediately prior to analysis, lipid extracts were dried and resuspended in

isopropanol: methanol: chloroform (4:2: 1 v:v:v) containing 20 mM ammonium formate. 10 μΐ. of each lipid sample were directly infused at approximately 250 nL/minute by

nanoelectrospray ionization (nESI) into a high resolution/accurate mass Thermo Scientific™ model LTQ Orbitrap Velos™ mass spectrometer (San Jose, CA) using an Advion Triversa Nanomate® nESI source (Advion, Ithaca, NY), with a spray voltage of 1.4 kV and a gas pressure of 0.3 psi. High-resolution mass spectra were acquired in positive ionization mode using the FT analyzer operating at 100,000 mass resolving power. Each mass spectrum was signal-averaged for 2 minutes over the range of 200-2000 m/z. Higher-Energy Collision Induced Dissociation (HCD-MS/MS) product ion spectra were acquired in positive ionization mode to confirm lipid headgroups and backbone/acyl chain compositions of selected ions of interest using the FT analyzer operating at 100,000 mass resolving power. Peak finding, lipid identification, and quantification were performed using the Lipid Mass Spectrum Analysis (LIMSA) v. l .O software.

^■ Microarray analysis of gene expression

Liver samples from db/db mice collected at zeitgeber (ZT) time ZT9 (daytime) and ZT21 (nighttime) from both the diabetic ad lib and IF groups were used for microarray analysis. Total RNA was extracted using the RNeasy® Mini kit (Qiagen®, Valencia, CA) and RNA quality control analysis was conducted using Agilent™ 2100 Bioanalyzer with an RNA 6000 Nano Labchip® (Agilent Technologies). Only RNA samples with an average RNA integrity number (RIN) greater than 7 were considered for array analysis. Further standard experimental procedures following manufacturer's instructions were used for hybridization, staining and scanning GeneChip® Mouse Gene 2.0 ST Arrays (Affymetrix, Santa Clara, CA).

For the data analysis, the files were analyzed using the Expression Console software (Affymetrix) via the robust multichip analysis (RMA) algorithm with quantile normalization method. The statistical analysis was done with Transcriptome Analysis Console (Affymetrix) and in R (version 2.15.3). JMP statistical software with Genomics 6.0 (SAS Institute, Cary, NC) was used to conduct the ANOVA test. The model with FDR less than 0.3 was selected for further analysis and genes with p < 0.05 and absolute fold-change > 1.5 were selected as statistically significantly expressed genes. In parallel, the Transcriptome Analysis Console (Affymetrix) was also used to analyze differentially expressed genes.

^■ Peptidoglycan ELISA

Plasma samples were dilute 1 : 10 and analyzed for peptidoglycan levels using the mouse peptidoglycan ELISA kit (MBS263268, Mybiosource Inc, San Diego, CA) according to manufacturer's instructions.

^■ Bile acid analysis

At the termination of the experiment, whole blood was collected by cardiac puncture and transferred in EDTA coated tubes. Plasma was separated and frozen at -80°C. Samples were sent to Metabolon Inc (Morrisville, NC) and global metabolic analysis was performed with their Metabolon Platform using a Waters ACQUITY ultra-performance liquid chromatography (UPLC) and a Thermo Scientific QExactive high resolution/accurate mass spectrometer interfaced with a heated electrospray ionization (HESI-II) source and Orbitrap mass analyzer operated at 35,000 mass resolution.

^■ Quantitative real-time-PCR (qRT-PCR)

Total RNA was extracted from mouse tissues using miRNeasy Mini kit (Qiagen®, Valencia, CA), and RNA quality and concentration were measured by Nanodrop (Thermo Scientific™, Wilmington, DE). Reverse transcription was done with cDNA by iScript™ cDNA Synthesis Kit (Bio-Rad, Pleasanton, CA). Primers for Arntl, Per2, Nos3, Anxal, Lcnl3, Plac8, Vldlr and Ppid were purchased from Thermo Scientific™/ Applied

Biosystems® (Carlsbad, CA) (Arntl: Mm00500226_ml; Perl: Mm00478113_ml; Anxal: Mm00440225_ml; Lcnl3: Mm00463682_gl; Plac8: Mm00507371_ml; Vldlr:

Mm00443298_ml; Ppid: Mm00500226_ml) and qRT-PCR was carried out using TaqMan® master mix (Applied Biosystems®). For Plin4, Smpd3, Rpll3, and Ppib, primers were purchased from Qiagen® (PPM05785A, PPM26420A, PPM40286A and PPM03728A, respectively), and qRT-PCR was carried out using SYBR® Green (Qiagen®). In all cases, results are expressed as 2 ^"ACq , relative to the endogenous controls. ■ Microbiome analysis 180 fecal samples were obtained (collected every 4 hours for 48 hours). Genomic DNA was isolated from -0.1 g using the PowerSoil® DNA Isolation Kit (MO Bio, Carlsbad, CA), according to manufacturer's protocol. DNA was sent for sequenced using 16S rRNA gene V4 sequencing with the MiSeq platform to Second Genome, Inc. (South San Francisco, CA).

^■ Circadian oscillation assessing and relative abundance comparisons

Using JTK cycle J Biol Rhythms 2010; 25:372-380), it was examined whether a circadian cycle exists for relative abundance in db/m ad lib and db/db ad lib for all operational taxonomic units (OTUs) present in >10 samples. An OTU was considered as having a circadian cycle if the p-value was <0.1. The relative abundance patterns were analyzed using Kolmogorov-Smirnov test (a nonparametric test of equality of probability distribution), for the following comparisons: (i) db/db IF vs. db/m IF; (ii) db/db ad lib vs. db/m ad lib; (iii) db/m IF vs. db/m ad lib; (iv) db/db IF vs. db/db ad lib.

^■ Colon morphometric analysis

Colon tissue was collected, fixed in 4% buffered formalin and paraffin-embedded according to standard procedures. 10 μπι sections were cut and stained by either hematoxylin and eosin or periodic acid Schiff (PAS), for mucin. Entire sections were scanned with a Zeiss Axio Scan Zl Digital Slide Scanner (Carl Zeiss Microscopy GmbH, Jena, Germany) using a 20X objective. Morphometric analysis of goblet cells, villi length and mucscularis thickness was done using a Lumenera Infinity 1-2C camera and NIS Elements software (Nikon

Instruments, Melville, NY). The number of goblet cells/villus were determined in 13-30 villi group of mice. Villi length, in pixels, was determined on 17-26 villi/group, avoiding villi that were sectioned obliquely. The muscularis width measurements were done in 20 places distributed evenly around the transversal section of the colon, and data represent averages per section in pixels, ±SD; n = 4-7 sections/group. In all cases, one section per mouse was analyzed, for 3-6 mice per group from corresponding ZT.

^■ NF200+ cells staining

The tibiae were fixed by formalin and the staining of the NF200 ⁺ cells was carried out as described J Exp Med 2009; 206:2897-2906). Briefly, decalcified rat humerus bones were embedded in paraffin. Immunocytochemistry was performed on 4 μπι sections, using standard Avidin-Biotin complex staining protocol on an Autostainer (Dako) and polyclonal rabbit anti- F200 (Sigma-Aldrich) (1 : 100), followed by biotinylated secondary goat anti- rabbit IgG (Vector Laboratories) (1 :400) and then by R.T.U. Elite Peroxidase reagent (Vector Laboratories). The slides were developed using Nova red (Vector Laboratories),

counterstained using Gill (Lerner) 2 Hematoxylin (Thermo Fisher Scientific™), dehydrated, cleared, and mounted using Permount (Thermo Fisher Scientific™).

^■ Acellular capillaries

The eyes were fixed in 2% formalin, and trypsin digest were performed for analysis of acellular capillaries as previously described {J Exp Med 2009; 206:2897-2906). The sensory retina was separated from pigment epithelium and choroid under a dissecting microscope, prepared using trypsin digestion, and stained with hematoxylin and periodic acid-Schiff. Acellular capillaries were quantified in a masked, randomized manner by three independent examiners.

^■ Immunocytochemistry of retina

Enucleated eyes were fixed in 4% paraformaldehyde freshly made in phosphate buffered saline (PBS) at 4°C overnight. Eyecups were cryoprotected in 30% sucrose/PBS for several hours or overnight prior to quick-freezing in optical cutting temperature (OCT) compound. Then 12 μιη-thick sections were cut at -20°C to -22°C. Sections were pre- incubated with 5% BSA for 10 min, followed by incubation with FITC-conjugated

monoclonal antibodies against mouse CD1 lb and CD45 (BD Biosciences, San Jose, CA) (1 : 100 in 1% BSA) overnight at 4°C. An alkaline phosphatase-conjugated anti-FITC antibody (Sigma) was used as the secondary antibody, and signal was detected using NBT/BCIP as substrate (Roche, IN). Levamisole (Vector Laboratory, CA) was added to the sections to remove the endogenous phosphatase activity. Positive cells were counted from at least 12 sections at 100 μπι intervals for each eye.

■ Immunofluorescence staining

Enucleated eyes were fixed in 4% paraformaldehyde overnight at 4°C. The next day, eyes were incubated with 30% sucrose for 48 hours and then quickly frozen in optical cutting temperature (OCT) compound. Retinal cross sections (12 Sm) were cut at -20oC. The following antibodies were used for immunostaining: rabbit monoclonal to Iba (Wako, 019- 19741, 1 :500), rat monoclonal to CD45 (clone 30-Fl 1, R&D Systems, 1 :50), rabbit polyclonal to GPCR TGR5 (ABCAM, ab72608, 1 :250), monoclonal murine -anti NeuN, (clone A60, EMD Millipore, MAB377MI, 1 :250). Sections were pre-incubated with 5% goat serum (Invitrogen) in PBS for lh, followed by incubation with primary antibodies (in 1% normal goat serum) overnight at 4°C. As secondary antibody, Alexa Fluor 488 was used for TGR5 and Ibal+ and Alexa Fluor 594 was used for NeuN and CD45 (Invitrogen). Positive cells were counted from 3-5 sections at 100 μιη intervals for each eye, on at least four images per section at 20X magnification. Retinal sections were imaged using a confocal scanning laser microscope (ZEISS LSM 700 confocal microscope system with Axio Observer; Carl Zeiss Mditec, Jena, Germany) and colocalization was analyzed by using Zen lite software.

■ Statistical analysis

The data were plotted as mean ± SD. Circadian effect on the mouse data was tested by a single cosine analysis. The data were considered as circadian oscillation by zero-amplitude test with p-value < 0.05. The expression level between different treatment groups was carried out by two-way ANOVA analysis (Gradph Prism, La Jolla, CA). A Student t-test was used for comparisons between two groups. Oneway ANOVA, followed by the Tukey post hoc test, was used for multiple comparisons. All values are expressed as mean ± SEM. A value of p < 0.05 was considered to be statistically significant. Statistical tests were performed using statistics software (GraphPad Software; La Jolla, CA).

Example 3. IF improved survival without impacting glycated hemoglobin levels

Four month-old db/m and db/db mice were fed either an ad-lib diet (AL) or were fasted every other 24-hour interval (IF) for 7 months (Figure 1 A). Glycated hemoglobin levels were not affected by IF regimen and remained higher in both db/db cohorts compared to controls (Figure IB). However, despite no change in glycated hemoglobin, the survival rate increased significantly in db/db mice on the IF regimen (db/db-IF) compared to db/db mice of identical age on the ad-lib (AL) feeding regimen (db/db-AL) (Figure 1C).

Example 4. IF prevented development of acellular capillaries and infiltration of

inflammatory cells in the retina of db/db mice

Acellular capillaries are the hallmark histological feature of DR (Figure 2E). The impact of IF on development in DR in db/db mice was evaluated by enumerating acellular capillaries (Figure 2A, Figure 2B, Figure 2C, and Figure 2D). The db/db-AL mice of eleven months of age showed increased numbers of acellular capillaries (Figure 2C) compared to age-matched db/m-AL control mice (Figure 2A). However, db/db-IF mice (Figure 2D and Figure 2E) did not demonstrate an increase in acellular capillaries.

As increases in retinal levels of inflammatory cells are another feature of DR, cryosections were examined for changes in activated microglia using Iba-1 staining and differences in infiltrating hematopoietic cells using CD45 staining. The db/db-AL mice showed a significant increase in Iba-1 ⁺ cells compared to db/m-AL mice and db/db-IF mice (Figure 2F). The CD45 ⁺ cell infiltration was significantly increased in the db/db-AL mice compared to the db/m-AL mice and db/db-IF mice (Figure 2G). Overall, IF protected db/db mice from developing histological features of DR.

It was previously found that neuropathic changes in the bone marrow preceded the development of diabetic retinopathy in Type 1 and Type 2 diabetes in rats. To examine whether IF prevented the bone marrow of dysfunction typically seen in diabetes,

immunohistochemical studies of the neuronal marker F200 in long bones from db/m and db/db mice were performed (Figure 2H). A reduced level of F200 ⁺ staining was observed in db/db mice as compared to age-matched controls, supporting the presence of neuropathic changes in the bone marrow (Figure 21 and Figure 2K). IF feeding preserved F200 ⁺ staining in the bone marrow of db/db mice to levels similar to control non-diabetic mice (Figure 2J and Figure 2L).

Example 5. Long-term IF altered the gut microbiome composition

The composition and diversity of the microbiome is integrated with the

pathophysiology of diabetes. Certain bacteria are shown to induce diabetes, while others alleviate it. Thus, the beneficial effects of IF on DR may, at least in part, be mediated through changes in microbiota composition. Fecal samples were collected and genomic DNA sequencing was used to elucidate and distinguish various bacterial taxa. A total of 1,363 filtered operational taxonomic units (OTUs) were detected. Sample richness and Shannon diversity (Figure 3A-F) were not changed by the implementation of IF but beta diversity, a measure of the between-sample diversity, showed clear differences between db/db-AL and db/m-AL mice (Figure 4 A), db/m-IF vs. db/m-AL (Figure 4B), and db/db-IF vs. db/db-AL

(Figure 4C). Principal component analysis of bacteria also demonstrated a clear separation of the four main groups (db/m-AL, db/db-AL, db/m-IF and db/db-IF). The AL groups were closer to each other while the IF groups diverged more (Figure 4D). This suggests that IF restructured a distinct microbiome in the diabetic compared to control mice.

The most represented phyla were Bacteroidetes, Firmicutes, Verrucomicrobia, Tenericutes, Actinobacteria, and Proteobacteria. The relative proportions of these varied dramatically among the experimental groups but the majority of bacteria belonged to the Bacteroidetes, Firmicutes and Verrucomicrobia phyla (Figure 4E). The ratio of

Firmicutesl Bacteroidetes (F/B) has been used as an indicator of changes in the microbiome with obesity. The F/B ratio was not altered in the db/m-IF mice; however, it was dramatically increased in the db/db-IF mice due to a significant increase of Firmicutes and a significant reduction of Bacteroidetes (Figure 4E). Apart from changes observed in the F/B ratio, Verrucomicrobia also changed significantly with IF showing an increase in db/m-IF and a reduction in db/db-IF (Figure 4E). Overall, these data indicate that IF resulted in a more dramatic restructuring of the microbiota composition in the diabetic mice with a significant expansion of Firmicutes at the expense of Bacteroidetes and Verrucomicrobia.

To understand how the microbiota composition was altered at the genus level, the differential taxa in each group were assessed (Figure 5A, Figure 5B, and Figure 5C). Among the differential taxa between db/m-AL and db/db-AL cohorts, the db/db-AL mice were enriched in Lactobacillus, Bifidobacterium, Bacteroides, and Akkermansia, and were deficit in species of Oscillospira, and Ruminococcus (Figure 5 A). The IF regimen in the db/db mice caused enrichment of species of the genus Lactobacillus, Oscillospira and Ruminococcus and, reduction of species of the genus Akkermansia, Bacteroides, and Bifidobacterium (Figure 5B and Figure 5C). The difference in the microbiota between fed vs. fasting state in the IF cohorts was very small, limited to only 24 bacteria in db/db-IF mice and 69 bacteria in db/m- IF mice (Figure 6A and Figure 6B). Certain common species were identified that were increased by fasting in both db/m-IF and db/db-IF cohorts, mostly species in the

Lachinospiraceae and Ruminococcaceae families with representatives from Oscillospira and Ruminococcus genera. Additionally, species of the genus Bifidobacterium were commonly reduced by the IF diet. The common and unique enriched bacterial species are shown in Figure 6C. The common or uniquely reduced bacteria are shown in Figure 6D. IF is known to influence diurnal rhythms and selected microbes in the gut exhibit diurnal changes. The taxa which displayed diurnal patterns in healthy db/m-AL mice and whether these taxa were affected by either the presence of diabetes or IF were examined. Only 34 OTUs showed diurnal cycling. Among these were members of Lachnospiraceae and Ruminococcaceae families and specifically, species of Oscillospira and Ruminococcus. Bacteria with diurnal regulation are shown in Figures 7A-7D.

Although there were few taxa differentially regulated by feeding/fasting or diurnal cycling, the evolutionary pressure of fasting and refeeding in combination with the host metabolic response to fasting led to the distinct restructuring of the microbiota in the db/m-IF and the db/db-IF mice. Members of the order Clostridiales (Figure 8 A) and members of the Ruminococcaceae (Figure 8B) and Lachnospiraceae families (Figure 8C) were uniquely equipped to adapt, expand and survive under IF conditions in db/db but not in db/m mice. Lactobacillus (Figure 8D), was enriched in both db/m-IF and db/db-IF, while the genus Bifidobacterium (Figure 8E) and Clostridum (Figure 8T) was reduced in both groups.

However, Oscillospira (Figure 8F), Ruminococcus (Figure 8G), and Turicibacter (Figure 8U) were enriched specifically in the db/db-IF, while Bacteroides (Figure 8H), Akkermansia (Figure 81) and Allobaculum (Figure 8V) were reduced only in the db/db-IF mice. These unique changes in the microflora of the db/db-IF mice may promote the integrity of the gut barrier.

Example 6. Long-term IF corrected diabetes-induced dysbiosis

Recent studies demonstrated that the composition of the microbiota is under circadian regulation and is entrained by feeding cycles. Thus, it was examined whether the IF regimen could impact the circadian rhythms of the gut microbiota expression and could correct diabetes-induced dysbiosis.

Fecal samples were collected and genomic DNA sequencing was used to discover and distinguish various bacterial taxa. A total of 1,363 filtered operational taxonomic units (OTUs) were detected, many of which showed differential abundance in the various conditions tested. The db/db mice fed ad lib regimen showed 166 OTUs differentially abundant when compared to controls, of which 109 were overabundant and 57 reduced

(Figure 5A). When the db/db IF were compared to db/m IF regardless of the eating phase, 397 OTUs differentiated IF diabetes and IF control samples, of which 204 OTUs were significantly over abundant and 193 were reduced in the diabetic samples compared to control. Principal component analysis of bacteria demonstrated that there was clear separation of the four main groups (db/m and db/db, each ad lib and IF). The two ad lib groups were closer to each other, while the two IF groups diverged more (Figure 4D).

Diabetic and control mice were not differentially affected by IF in terms of sample richness and Shannon diversity (Figure 3 A-F). In contrast, beta diversity (measure of the between-sample diversity) showed clear differences between (i) db/db and db/m mice fed ad lib (Figure 4A), (ii) db/m mice under IF vs. ad lib (Figure 4B), and (iii) db/db IF vs. ad lib (Figure 4C). This suggested that both diabetes and IF are characterized by different microbiota OTUs. The samples from db/db ad lib showed the smallest degree of variation in microbiome beta diversity (Figure 4C, triangles to the left of the dashed line).

The most represented phyla were Actinobacteria, Bacteroidetes, Firmicutes,

Proteobacteria, Tenericutes and Verrucomicrobia. The relative proportions of these varied dramatically among the experimental groups, most notably the BacteroideteslFirmicutes ratio. The ratio decreased slightly in favor of Firmicutes in db/db mice. Firmicutes were decreased in db/m IF, especially in the feeding phase, leading to an increase in the ratio. However, in the db/db mice, IF drastically reduced Bacteroidetes, while increasing Firmicutes, leading to the ratios of 0.52 and 0.43 in the feeding and fasting phases, respectively. Some primary bile acids are converted to secondary bile acids by enzyme activities in gut bacteria and expansion of Firmicutes could result in significant expansion of deoxycholic acid-producing bacteria.

At higher taxonomic levels (class, order, family), diurnal patterns were often obscured by the majority of OTUs that did not show daily variations. Only 34 OTUs showed diurnal cycling, as determined by JTK cycle program (Figure 7E). Bacteria whose relative abundance was differentially affected in diabetes vs. controls, and whether the IF regimen could bring this abundance back to control levels, were studied. Members of the Clostridiaceae family had increased relative abundance and diurnal variations in db/db ad lib, and their levels decreased in both IF groups of mice (Figure 8J). Conversely, members of the genus

Oscillospira had lower levels in db/db ad lib and diurnal variations were increased by IF in db/db mice (Figure 8K), a pattern also found in the Ruminococcaceae family (Figure 8N). Lactobacillus reuteri and the entire Lactobacillaceae family exhibited a different behavior: very low abundance and no diurnal oscillations in controls, and slightly increased levels with stronger oscillation in db/db ad lib. The IF regimen resulted in oscillations with greater amplitude in both db/m and db/db mice (Figure 8L and Figure 80). In another species, Akkermansia muciniphila, levels increased in db/db ad lib and were dramatically reduced in IF (Figure 8M). In the family Lachnospiraceae, diabetes dramatically dampened the diurnal variations while IF enhanced them (Figure 8P). At the same time, a paradoxical effect was found in three genera (Allobaculum, Bifidobacterium, and Bacteroides, Figure 8Q, Figure 8R, and Figure 8S) where the IF regimen drastically decreased their relative abundance. Example 7. IF promoted restoration of diabetes-induced gut pathology

Disruption of the gut barrier integrity is an important mechanism of disease, thus whether IF beneficially impacted colon morphology as measured by goblet cell number and villi length was examined (Figure 9A-0). The db/db-AL mice had reduced numbers of goblet cells (Figure 9B and Figure 9E) and reduced villi length (Figure 9G and Figure 9J). However, IF resulted in preservation of colon morphology in the db/db-IF mice, as goblet cell number (Figure 9D and Figure 9E) and villi length (Figure 91 and Figure 9J) were similar to nondiabetic mice. No significant changes were observed in the thickness of the muscularis (Figure 9K-N and Figure 90).

Increases in bacterial products in plasma are indicative of intestinal barrier dysfunction or disruption. Thus, peptidoglycan in plasma samples from the four mice cohorts were measured. As shown in Figure 10, db/db-AL demonstrated a significant increase in plasma peptidoglycan levels supportive of increased gut permeability. IF resulted in a decrease in gut permeability when db/db-IF mice were in the fasted state as there was a significant reduction of circulating peptidoglycan levels. However, when the db/db-IF mice were in the fed state this potentially beneficial change was lost.

Example 8. IF altered bile metabolism

The ability of the microbial changes induced by IF to generate beneficial metabolites was evaluated. Bile acids are known to alter the gut-microbiome compositions, and conversely, the microbiome is known to alter the bile acid pool. Thus, the observed microbiome changes induced by IF could impact bile acid (BA) metabolism in the db/db mice. Metabolic analysis of plasma from the experimental cohorts identified the primary bile acid cholate but not chenodeoxycholate. Table 1 A-C is a table providing a list of bile acids found in the plasma. Table 1 A. Anal sis of bile acid content in plasma from ad-lib cohort

Table IB. Anal sis of bile acid content in plasma from IF cohort feeding)

Table 1C. Analysis of bile acid content in plasma from IF cohort (fasting)

Cholate was increased in the db/db-AL mice compared to db/m-AL mice and was reduced in the db/db-IF mice (Figure 11 A). Deoxycholate, the secondary bile acid that is produced by the activities of 7a-dehydroxylase, an enzyme found in intestinal bacteria, was moderately increased with IF especially during the fasting period (Figure 1 IB) supporting intestinal transformation of Bas. However, the ratio of conjugated to unconjugated BA was increased in the db/db-IF mice compared to db/m-IF during feeding, suggesting that there may be a reduction of bacteria that express bile salt hydrolase (BSH) activity required to deconjugate primary and secondary bile acids in the db/db-IF mice. A significant reduction of Bifidobacterium (Figure 8E), Clostridium (Figure 8T), and Bacteroides (Figure 8H) was observed and these bacteria are known to express BSH, which may be responsible, in part, for these observed changes in levels of conjugated to unconjugated secondary BAs. An increase in BA products indicative of 7a- and 7P-hydroxysteroid dehydrogenase (HSDH) enzymatic activity was also found. Taurochenodeoxycholate (TCDCA) was identified and significantly increased in the db/db-IF mice (Figure 11C). TCDCA is converted to tauroursodeoxycholate (TUDCA) by the actions of both 7a- and 7P-HSDH and TUDCA was significantly increased in the plasma of db/db-IF mice (Figure 1 ID). As taurine conjugated secondary bile acids are potent activators of TGR5, the beneficial effects observed following IF may be due to an increase in TUDCA synthesis in the gut.

Example 9. TGR5 activation reduces acellular capillaries and inflammation in the diabetic retina

TGR5 expression was examined to determine whether TUDCA could mediate beneficial effects in the retina. TGR5 was localized primarily to the ganglion cell layer of the retina (Figure 12A). Neither diabetes nor the IF regimen changed the localization (Figure 12B-E) or expression of TGR5 protein (Figure 12F) as assessed by immunohistochemistry nor changed TGR5 mRNA levels (Figure 12G). However, TNF-a, a downstream target of TGR5 was reduced significantly by IF in the diabetic cohort (Figure 12H). To further support the role of retinal TGR5, prevention of DR by pharmacological activation of TGR5 was investigated.

IF increased key bacteria that generate bile acids, which exert protective effects on metabolic disease by activation of nuclear hormone receptors, in particular the farnesoid X receptor (FXR), and the G-protein coupled receptor TGR5. The novel highly potent semisynthetic bile acid derivative and dual TGR5/FXR agonist Compound 1-Na was tested in a second diabetes model, the DB A/2J mice treated with streptozotocin and placed on a high fat diet. This model exhibits a more rapid progression of the vascular and inflammatory endpoints associated with DR and diabetic nephropathy, and replicates the natural history and metabolic characteristics of human metabolic syndrome and Type 2 diabetes. Compound 1- Na treatment of diabetic mice resulted in a marked reduction in the number of acellular capillaries (Figure 121) and reduced numbers of CD1 lb+ macrophages (Figure 12K), CD45+ leukocytes (Figure 12J), and activated Ibal+ microglia within the retina (Figure 12L). These data suggest a relationship between changes in the microbiota, IF and increases in TUDCA. IF likely provides a way to change bile acid metabolism in favor of increasing the endogenous generation of TUDCA (Figure 31).

Example 10. Lipidomic analysis of db/db mouse livers following IF

Abnormal hepatic lipogenesis represents a key feature of Type 2 diabetes, leading to dyslipidemia and contributing to development of diabetic complications. The effect of IF on liver lipids was assessed in the diabetic cohorts. Figures 13A-13D show high

resolution/accurate normalized mass spectra obtained from mouse liver at ZT9. Livers of db/db mice fed ad lib (Figure 13B) contained strikingly increased levels of neutral lipids, such as triglycerides (TG) and sterols including cholesteryl esters (CE), compared to ad lib-fed db/m mice (Figure 13A). In both db/db and db/m mice, IF significantly (p < 0.0001) corrected TG levels, including the abundant triglyceride species TG(50:2), TG(52:2), TG(52:3), TG(52:4) and TG(54:3) (Figure 13B, Figure 13D, Figure 14A, and Figure 14B). CE and diglycerides also decreased in db/db mice under IF (Figure 13C, Figure 13D, Figure 14C, Figure 14D, Figure 14E, and Figure 14F). IF normalized hepatic sphingolipid metabolism in db/db mice by preventing accumulation of glycosylated ceramide metabolites, particularly those containing C24:0 and C24: l fatty acids (Figure 14G and Figure 14H). Together these results show that IF reversed the aberrant accumulation of hepatic neutral lipids, sphingolipids and sterols induced by Type 2 diabetes. Plasma TG levels were found to be reduced during times of reduced food intake, such as daytime when the animals were resting and during the fasting phase of IF in both the db/m and db/db mice (Figure 15 A). Plasma free fatty acids levels were higher in the db/db mice and levels fell during the rest phase of the day when the mice reduced their intake (Figure 15B).

Example 11. Abnormal clock gene expression in db/db mice was corrected by IF initiated at night

Circadian rhythm is established by clock proteins, among which DMAL and PER2 play a central role. These proteins are crucial for proper cellular metabolism and general vascular health, as mutations in either gene result in vascular phenotypes similar to those seen in diabetes. In the four groups of mice, Arntl (Bmal) and Per2 mRNAs expression were determined in the SCN and liver.

In the SCN, Arntl and Per2 expression were in anti-phase. Arntl in db/m mice exhibited circadian oscillation that peaked at ZT21 (near the end of active phase) while in the db/db mice there was a phase shift in the peak expression (near the beginning of the rest phase, ZT1). Long term IF did not change Arntl mRNA expression in either db/m or the db/db mice, suggesting that IF did not influence the positive arm of the clock in the SCN (Figure 16 A).

Per 2 expression in SCN also exhibited circadian oscillation, with a peak at ZT13 in db/m ad lib, which was phase advanced at ZT9 in db/db ad lib. Under the IF regimen, no significant change was observed in the Per2 expression in db/m mice compared to mice on ad lib feeding. However, IF restored the peak time in the db/db mice to ZT13 on the deeding day and ZT9 on the fasting day. The level of Per2 mRNA expression was significantly increased at ZT5 on the fasting day and significantly decreased on the feeding day (Figure 16B).

Overall, the SCN Arntl mRNA expression was more influenced by the presence or absence of diabetes while Per2 mRNA was more influenced by IF and the feeding (decreased) or fasting (increased) phase.

In the liver, the expression of Arntl and Per2 mRNA was in antiphase. Towards the end of the active period, Arntl mRNA peaked (Figure 16C) whereas Per 2 mRNA at the beginning of the active phase (Figure 16D). In db/db ad lib Arntl expression was elevated compared to db/m mice. With IF, the expression of Arntl was higher in the db/m mice during the feeding period, while in the db/db mice it was slightly reduced compared to the ad lib diet (Figure 16C).

Per2 showed oscillations in the db/m mice on the ad lib regimen, with the peak of expression at the beginning of the active period. The expression increased in db/db ad lib at the end of the rest period. IF feeding regimen in control db/m mice greatly increased Per2 expression during the feeding period, but not to the same degree in the db/db mice which showed a similar oscillation whether in the feeding or fasting stage of IF (Figure 16D).

Collectively, these data suggest that diabetes affects both Arntl and Per2 distinctively in different tissues, by reducing their expression levels in SCN but increasing them in liver, and inducing phase shifts. The IF regimen brought both of these parameters closer to control (db/m ad lib) levels, in most instances.

Example 12. Microarray analysis of liver gene expression following IF

To better understand the impact of IF regimen on the liver of db/db mice, gene expression profiling was performed in mice on the IF regimen. The response from both the "feeding day" and "fasting day" was compared to ad lib feeding at two time points (ZT9 and ZT21), times associated with the greatest excursions. Among the 20267 annotated genes from the microarray, after FDR correction for multiple comparisons, 52 genes were differentially expressed: 35 genes were up-regulated for db/db mice under IF, while 17 genes were down- regulated. Expression pattern of each individual gene is illustrated in the heatmap shown in Figure 17 A.

To examine the clustering of the samples with the selected 52 genes, clustering by t- distributed stochastic neighbor embedding (t-S E) was conducted. Ad-lib samples clustered together and were clearly separated from the IF group, suggesting that the 52 genes are a molecular signature of the long term IF effects (Figure 17B).

Genes that showed differential regulation (p < 0.05) regardless the multiple comparisons correction were analyzed. When compared to ad lib feeding, it was observed that night (ZT21) and day (ZT9) changes in gene expression were modified by the feeding phase (Figure 17C). During the night (ZT 21), when mice are active, a few genes involved in lipid and cholesterol metabolism and immune response, were upregulated. In contrast, at ZT 21 numerous genes specific to the lymphoid and myeloid linages and to immune response were down-regulated, especially during the feeding phase. Conversely, during the day (ZT9), and almost complete reversal of these changes took place.

Validation was conducted with qRT-PCR of several of these genes. Anxal, a glucocorticoid-regulated gene which mediates anti-inflammatory effects, Lcnl3, a member of the lipocalin superfamily and an insulin sensitizer secreted by the liver, and Plac8 were upregulated by the IF feeding regimen during the feeding phase (Figure 18 A, Figure 18B, and Figure 18C). Vldlr was reduced by the IF regimen (Figure 18D), further supporting that IF impacted lipid metabolism in the db/db mice. Two other genes involved in lipid metabolism, Plin4 and Smpd3 showed significant changes in db/db IF as compared to ad lib, and as with the other genes the effect depended on food availability (Figure 18E and Figure 18F).

EQUIVALENTS

Those skilled in the art will recognize, or be able to ascertain, using no more than routine experimentation, numerous equivalents to the specific embodiments described specifically herein. Such equivalents are intended to be encompassed in the scope of the following claims.