Compositions and methods for treating heart disease

Abstract

The present disclosure relates to methods of treating diseases and disorders including heart disease using compositions comprising at least one of 3-indole propionic acid or nicotinamide, or pharmaceutically acceptable salts or prodrugs thereof.

Description

[0001] Attorney Docket No.: UCH-31725

[0002] UCLA Ref. No.: [UCLA 2022-142-3] WO

[0003] COMPOSITIONS AND METHODS FOR TREATING HEART DISEASE GOVERNMENT SUPPORT CLAUSE

[0004] This invention was made with government support under HL144651 and HL148577, awarded by the National Institutes of Health. The government has certain rights in the invention.

[0005] BACKGROUND

[0006] Heart failure with preserved ejection fraction (HFpEF) is an increasingly prevalent disorder that accounts for half of all cases of heart failure. HFpEF is characterized by diastolic dysfunction. HFpEF is distinct from heart failure with reduced ejection fraction (HFrEF). Unlike the proven therapies for HFrEF, trials of traditional heart failure medications have been unsuccessful in HFpEF and there are, to date, only the SGLT2 (sodium-glucose cotransporter 2) inhibitor, dapagliflozin, has been shown to improve symptoms in chronic HFpEF. HFpEF is associated with multiple comorbidities, including obesity, diabetes, and hypertension. HFpEF remains an unsolved biological and clinical problem of immense significance. Accordingly, new treatments for HFpEF are needed.

[0007] SUMMARY OF THE INVENTION

[0008] In certain aspects, the present disclosure provides methods of treating a heart disease, comprising administering a composition comprising 3 -indolepropionic acid (IPA) or a pharmaceutically acceptable salt or prodrug thereof to a subject in need thereof.

[0009] In certain aspects, the present disclosure provides methods of treating diastolic dysfunction, comprising administering a composition comprising at least one of 3-indolepropionic acid (IPA), nicotinamide, or a pharmaceutically acceptable salt or prodrug thereof to a subject in need thereof.

[0010] In certain aspects, the present disclosure provides methods of treating metabolic disease comprising administering a composition comprising at least one of 3-indolepropionic acid (IPA), nicotinamide, or a pharmaceutically acceptable salt or prodrug thereof to a subject in need thereof.

[0011] In certain aspects, the present disclosure provides methods of reducing cholesterol or treating hyperlipidemia comprising administering a composition comprising at least one of 3-indolepropionic acid (IPA), nicotinamide, or a pharmaceutically acceptable salt or prodrug thereof to a subject in need thereof.Attomey Docket No.: UCH-31725

[0012] UCLA Ref. No.: [UCLA 2022-142-3] WO

[0013] In certain aspects, the present disclosure provides methods of treating obesity comprising administering a composition comprising at least one of 3 -indolepropionic acid (IPA), nicotinamide, or a pharmaceutically acceptable salt or prodrug thereof to a subject in need thereof.

[0014] In certain aspects, the present disclosure provides methods of reducing vascular fibrosis comprising administering a composition comprising at least one of 3 -indolepropionic acid (IPA), nicotinamide, or a pharmaceutically acceptable salt or prodrug thereof to a subject in need thereof.

[0015] In some embodiments, administration of the composition reduces the weight, the adipose tissue mass (e.g., white or brown adipose tissue mass), or body mass index of the subject.

[0016] In some embodiments, the subject has heart disease. In some embodiments, the heart disease is Heart Failure with preserved Ejection Fraction (HFpEF). In some embodiments, the subject has insulin resistance. In some embodiments, the subject has diabetes.

[0017] In some embodiments, the subject has gut microbiota dysbiosis. In some embodiments, administration of the composition reduces intestinal epithelial barrier damage. In some embodiments, administration of the composition reduces gut dysbiosis.

[0018] In some embodiments, the composition comprises IPA or a pharmaceutically acceptable salt or prodrug thereof. In some embodiments, the composition comprises IPA or a pharmaceutically acceptable salt thereof. In some embodiments, the composition comprises nicotinamide (NAM) or a pharmaceutically acceptable salt or prodrug thereof. In some embodiments, the composition comprises tryptophan or a pharmaceutically acceptable salt thereof. In some embodiments, the composition comprises a bacterium that can convert tryptophan to IPA. In some embodiments, the composition comprises at least one of Lactobacillus reuteri, Akkermansia muciniphila, or Clostridium sporogenes.

[0019] In some embodiments, the composition is administered orally. In some embodiments, the composition further comprises a pharmaceutically acceptable excipient. In some embodiments, the composition is administered for between about 1 and 140 days. In some embodiments, the composition is administered for at least about 20 days (e.g., at least about 40, 60, 80, 100, 120, or 140 days). In some embodiments, the subject is a human.Attorney Docket No.: UCH-31725

[0020] UCLA Ref. No.: [UCLA 2022-142-3] WO

[0021] BRIEF DESCRIPTION OF THE DRAWINGS FIGs. 1A-U show that HFpEF mice exhibit metabolic remodeling which is associated with diastolic dysfunction. C57BL / 6J male mice were fed with chow diet or HFD + 1-NAME diet for 7 weeks (n = 8-10). FIG. 1A shows body weight at baseline, week 3, 5, and 7 (n=10).

[0022] FIG. IB shows heart weight / tibia length ratio measured after 7 weeks of chow diet or HFD + 1-NAME diet. FIG. 1C shows lung weight (wet / dry ratio) measured after 7 weeks of chow diet or HFD + 1-NAME diet. FIG. ID shows representative images of echocardiography measured in chow diet (n=8) or HFD+l-NAME (n=10) mice. FIG. IE shows E / A ratio measured in chow diet (n=8) or HFD+l-NAME (n=10) mice. FIG. IF shows E / e’ ratio measured in chow diet (n=8) or HFD+l-NAME (n=10) mice. FIG. 1G shows left ventricle ejection fraction measured in chow diet (n=8) or HFD+l-NAME (n=10) mice. FIG. 1H shows left ventricle mass / surface area measured in chow diet (n=8) or HFD+l-NAME (n=10) mice. FIG. II shows the results of an exercise tolerance test showing total running distance of chow diet (n=8) and HFD+l-NAME (n=l 3) fed mice in a treadmill. FIG. 1J shows fat mass at baseline, week 3, 5, and 7. FIG. IK shows lean mass at baseline, week 3, 5, and 7.

[0023] FIG. IL shows plasma total cholesterol measured after 7 weeks of chow diet or HFD + 1-NAME feeding. FIG. IM shows unesterified cholesterol measured after 7 weeks of chow diet or HFD + 1-NAME feeding. FIG. IN shows glucose tolerance test and quantification measured after 7 weeks of chow diet or HFD + 1-NAME feeding. FIG. IO shows plasma glucose measured after 7 weeks of chow diet or HFD + 1-NAME feeding. FIG. IP shows insulin measured after 7 weeks of chow diet or HFD + 1-NAME feeding. FIG. IQ shows calculated HOMA-IR measured after 7 weeks of chow diet or HFD + 1-NAME feeding. FIG.

[0024] 1R shows the correlation between metabolic traits (glucose tolerance as quantified by GTT AUC) and diastolic function traits (E / e’ ratio) in 30 inbred strains of male mice with a “Two-hit” HFpEF model. FIG. IS shows the correlation between metabolic traits (HOMA-IR) and diastolic function traits (E / e’ ratio) in 30 inbred strains of male mice with a “Two-hit” HFpEF model. FIG. IT shows the correlation between metabolic traits (plasma total cholesterol “TC”) and diastolic function traits (E / A ratio) in 30 inbred strains of male mice with a “Two-hit” HFpEF model. FIG. 1U shows the correlation between metabolic traits (insulin) and diastolic function traits (E / e’ ratio) in 30 inbred strains of male mice with a “Two-hit” HFpEF model. Each point represents a mouse. All data are presented as the mean + SEM. P<0.05 is statistically significant. Data were analyzed by Student’s t test (FIGs. IB, C, H, I, L-Q), by 2-way ANOVA (FIGs. 1A, E, F, G, J, K, N), or by Spearman Rank correlation (FIGs. 1R-Attomey Docket No.: UCH-31725

[0025] UCLA Ref. No.: [UCLA 2022-142-3] WO

[0026] U). AUC indicates area under the curve; GTT, glucose tolerance test; HW, heart weight; LV, left ventricular; TC, total cholesterol; and TL, tibia length.

[0027] FIGs.2A-I show decreased heart and plasma IPA levels in HFpEF mice. C57BL / 6J male mice were fed with chow diet or HFD+Nco-nitro-L-arginine methyl ester (HFD+1-NAME) diet for 7 weeks. Heart tissue and plasma were collected for metabolomics (n = 7-8).

[0028] FIG. 2A shows principal component analysis (PCA) plot of chow and HFD + 1-NAME (HFpEF) samples. FIG. 2B shows a heatmap depicting relative abundance of significantly differentiated metabolites in heart tissue from HFpEF (n=7) vs chow (n=8) mice. FIG.2C shows a heatmap depicting relative abundance of significantly differentiated metabolites in plasma from HFpEF (n=7) vs chow (n=8) mice. FIG.2D shows intermediates in tryptophan metabolism were decreased in the heart from HFpEF mice relative to control mice. Relative abundance of metabolites was shown, and the bar graphs denote chow (white) and HFpEF samples (grey). Decreased metabolites were denoted with grey arrows. FIG.2E shows IPA levels in heart tissue of male mice. FIG.2F shows IPA levels in plasma of male mice. FIGs.

[0029] 2G-I show the associations between plasma or cardiac IPA levels and diastolic parameters (E / A ratio, E / e’ ratio, or LVEF). Each point represents a mouse. All data are presented as the mean ± SEM. ns, not significant. P<0.05 is statistically significant. Data were analyzed by Student t test (FIGs.2D-F), or by Spearman rank correlation (FIGs.2G-I). cAMP indicates cyclic adenosine monophosphate; CoA, coenzyme A; GABA, y-aminobutyric acid; LVEF, left ventricular ejection fraction.

[0030] FIGs.3A-R show that indole-3-propionic acid (IPA) supplementation attenuates diastolic dysfunction and metabolic remodeling. C57BL / 6J male mice (8-week-old) were fed with chow diet or high-fat diet (HFD)+Nco-nitro-L-arginine methyl ester (1-NAME) containing control or high IPA (562.5 mg IPA / kg diet) for 7 weeks. FIG.3A shows experimental design. FIGs.3B-C show relative IPA levels in plasma (FIG.3B) and heart tissue (FIG.3C) of mice fed with HFD+l-NAME containing control (n=8) or high IPA (n=8). FIGs.3D-E show body weight (FIG.3D) and fat mass (FIG.3E) at baseline, weeks 2, 5, and 7. n=4 in Chow and Chow+IPA; n=8 in heart failure with preserved ejection fraction (HFpEF) and HFpEF+IPA. FIGs.3F-G show glucose tolerance test (FIG. 3F) and insulin tolerance test (FIG.3G) as well as the area under curve (AUC) of the mice after 7 weeks of chow diet or HFD+l-NAME containing control or high-IPA. *P<0.05 and **P<0.01 of glucose levels between HFpEF and HFpEF+IPA group. HFpEF and Chow group were significantly different (asterisks shown in area under the curve [AUC]). n=4 in Chow andAttomey Docket No.: UCH-31725

[0031] UCLA Ref. No.: [UCLA 2022-142-3] WO

[0032] Chow+IPA; n=8 in HFpEF and HFpEF+IPA. FIGs.3H-L show white adipose weights (FIG.

[0033] 3H), brown adipose weights (FIG.31), plasma total cholesterol (FIG.3J), unesterified cholesterol (FIG.3K), and free fatty acids (FIG.3L) of the mice fed with chow diet or HFD+l-NAME containing control or high-IPA for 7 weeks. n=4 in Chow and Chow+IPA; n=8 in HFpEF and HFpEF+IPA. FIGs.3M-R show heart weight / tibia length ratio (FIG. 3M), representative images of echocardiography (FIG.3N), E / A ratio (FIG.30), E / e’ ratio (FIG. 3P), LV mass / surface area (FIG.3Q), and exercise tolerance test (FIG.3R) were measured. Running distance was assessed at the end of feeding and echocardiography was determined at baseline, week 4, and week 7. n=4 in Chow and Chow+IPA; n=8 in HFpEF and HFpEF+IPA. Representative of 4 (FIGs.3A-R) experiments. Each point represents a mouse. All data are presented as the mean+SEM. P<0.05 are statistically significant, ns, not significant. Data were analyzed by Student t test (FIG.3B and FIG.3C), by Kruskal- Wallis test (FIGs.3H-M, Q-R), or by 2-way ANOVA (FIGs.3D-G, O-P). FIGs.3D-G and O-R, the P values indicate significance between HFpEF and HFpEF+IPA group.

[0034] FIGs.4A-W show indole- 3 -propionic acid (IPA) supplementation increases nicotinamide and SIRT3 (sirtuin 3) levels in the heart. FIG.4A shows a heatmap depicting relative abundance of significantly differentiated metabolites by IPA in the heart of male mice fed with high-fat diet (HFD)+Nco-nitro-L-arginine methyl ester (1-NAME) containing control or high-IPA for 7 weeks. n=8. FIG.4B shows relative abundance of indicated metabolites. n=8. FIG.4C shows heart nicotinamide (NAM) level in C57BL / 6J male mice fed with chow diet, HFD+l-NAME or HFD+l-NAME containing high IPA for 7 weeks. n=7 in Chow; n=16 in heart failure with preserved ejection fraction (HFpEF); n=8 in HFpEF+IPA. FIG.4D shows association between heart IPA and NAM levels in C57BL / 6J male mice fed with chow diet or HFD+l-NAME. n=8. FIG.4E shows nicotinamide adenine dinucleotide (NAD) salvage pathway. FIG.4F shows NAD+ / NADH ratio in the heart tissue of mice fed with chow diet, HFD+l-NAME, or HFD+l-NAME containing high IPA for 7 weeks. n=7 in Chow; n=16 in HFpEF; n=8 in HFpEF+IPA. FIGs.4G-J show plasma LPS levels (n=6; FIG.4G), protein carbonyl levels (n=8; FIG.4H), indicated protein levels (n=3; FIG.41), and protein quantification (FIG.4J) in the heart tissue of mice fed with chow diet, HFD+l-NAME or HFD+l-NAME containing high IPA for 7 weeks. FIGs.4K-M, Protein levels (FIG.4K), SIRT3 protein quantification (FIG.4L), and NNMT protein quantification (FIG.4M) in HL-1 cells treated with control or IPA (1 mmol / L) in the presence of 100 pmol / L phenylephrine (PE) with or without AhR antagonist CH-223191 (5Attomey Docket No.: UCH-31725

[0035] UCLA Ref. No.: [UCLA 2022-142-3] WO

[0036] pM) for 48 hours. FIGs.4N-P show protein levels (FIG.4N), SIRT3 protein quantification (FIG. 40), and RT-qPCR (FIG.4P) showing Sirt3 knockdown in the heart using AAV9-Szrt3-shRNA. The relative protein levels were determined by comparing them to the first sample in the control group. The relative mRNA levels were determined by comparing them to the average expression in the control group. n=6. FIGs.4Q-W, C57BL / 6J male mice (8 weeks old) were injected with control or AAV9-Szrt3-shRNA, and fed with HFD+l-NAME or HFD+l-NAME containing high IPA for 7 weeks (n=7). Body weight (FIG.4Q), fat mass (FIG. 4R), lean mass (FIG.4S), E / e’ ratio (FIG.4T), LV mass / surface area (FIG.4U), LVEF (left ventricular ejection fraction; FIG.4 V), and heart weight / tibia length (FIG.4W) were measured after the feeding. Representative of 2 (FIGs.4A-H, N-W), 4 (FIGs.41, J), and 5 (FIGs.4K-M) experiments. Each point represents a mouse. All data are presented as the mean+SEM. P<0.05 are statistically significant, ns, not significant. Data were analyzed by Student t test (FIGs.4B, O, P), by 1-way ANOVA (FIGs.4C, F-H), by Spearman rank correlation (FIG.4D), by Kruskal-Wallis test (FIGs.4L and M), or by 2-way ANOVA (FIGs. 4 J, Q-W).

[0037] FIGs.5A-X show that nicotinamide (NAM) mitigates diastolic dysfunction in heart failure with preserved ejection fraction (HFpEF). C57BL / 6J male mice were fed with chow diet or high-fat diet (HFD)+Nco-nitro-L-arginine methyl ester (1-NAME) containing control or high NAM (550 mg / kg per d in drinking water) for 7 weeks. FIGs.5A-B show heart NAM (FIG. 5A) and NAD+(FIG.5B) levels in HFpEF or HFpEF+NAM groups were measured with hydrophilic-interaction chromatography liquid chromatography-mass spectrometry (LC-MS). The relative NAM levels were determined by comparing them to the average expression in the HFpEF group. n=6. FIGs.5C-F show cecum samples of Chow, Chow+indole-3-propionic acid (IPA), HFpEF, HFpEF+IPA, and HFpEF+NAM were collected and 16S rRNA-based gut microbial profiling was performed. Relative abundance of taxa at the phylum level (FIG.5C), relative abundance of Firmicutes (FIG.5D), Bacteroidetes (FIG. 5E), and Firmicutes / Bacteroidetes ratio (FIG.5F) in indicated groups were shown. n=4 in Chow and Chow+NAM; n=6 in HFpEF, HFpEF+IPA, and HFpEF+NAM. FIGs.5G-I show body weight (FIG.5G), fat mass (FIG.5H), and lean mass (FIG.51) of the mice were measured at baseline, 3, 5, and 7 weeks of feeding with chow diet or HFD+l-NAME containing control or high NAM. n=4 in Chow and Chow+NAM; n=8 in HFpEF, and HFpEF+NAM. FIGs.5J-Q show glucose tolerance test and quantification (FIG.5J), plasma-free fatty acids (FIG.5K), representative images of echocardiography (FIG.5L),Attorney Docket No.: UCH-31725

[0038] UCLA Ref. No.: [UCLA 2022-142-3] WO

[0039] E / A ratio (FIG.5M), E / e’ ratio (FIG.5N), LVEF (FIG.50), LV mass / surface area (FIG.

[0040] 5P), and running distance (FIG.5Q) were determined. Echocardiography was performed at baseline, weeks 4 and 7 of the feeding and other parameters were collected after 7 weeks of the diet. n=4 in Chow and Chow+NAM; n=8 in HFpEF, and HFpEF+NAM. FIGs.5R-X show C57BL / 6J male mice were fed with HFD+l-NAME containing control, high IPA, high NAM, or high IPA+NAM for 7 weeks. E / A ratio (FIG.5R), E / e’ ratio (FIG.5S), LV mass / surface area (FIG. 5T), LVEF (FIG.5U), heart total cholesterol (FIG.5V), unesterified cholesterol (FIG.5W), and free fatty acids (FIG.5X) were measured after feeding. n=5. Representative of 2 (FIGs.5R-X) and 3 (FIGs.5A-Q) experiments. Each point represents a mouse. All data are presented as the mean+SEM. P<0.05 are statistically significant, ns, not significant. Data were analyzed by Student t test (FIGs.5A and B), by 1-way ANOVA (FIGs.5R-X), by Kruskal- Wallis test (FIGs.5D-F, K, P, Q), or by 2-way ANOVA (FIGs.5G-J, M-O). FIGs.5G- J, M-O, P values indicate significance between HFpEF and HFpEF+NAM group.

[0041] FIGs.6A-Y show that Nnmt antisense oligonucleotide (ASO) alleviates diastolic dysfunction in heart failure with preserved ejection fraction (HFpEF). C57BL / 6J male mice were subjected to once weekly intraperitoneal injection of control or Nnmt ASO (an ASO) and were fed with high-fat diet (HFD)+Nco-nitro-L-arginine methyl ester (1-NAME) for 7 weeks. FIG.6A shows RT-qPCR (n=8) and Western blotting (n=3) showing NNMT (nicotinamide N-methyltransferase) levels in the heart tissue from mice treated with control (CON) ASO or Nnmt ASO. The relative mRNA levels were determined by comparing them to the average expression in the control group. The relative protein levels were determined by comparing them to the first sample in the control group. FIGs.6B-D show body weight (FIG. 6B), fat mass (FIG.6C), and lean mass (FIG.6D) were measured at baseline, weeks 3, 5, and 7 of the feeding in control (n=9) or Nnmt ASO mice (n=10). FIGs.6E-Q show glucose tolerance test (FIG.6E), insulin tolerance test (FIG.6F), plasma insulin (FIG.6G), homeostatic model assessment for insulin resistance (HOMA-IR; FIG.6H), plasma triglycerides (FIG.61), total cholesterol (FIG.6J), unesterified cholesterol (FIG.6K), running distance (FIG.6L), representative images of echocardiography (FIG.6M), E / A ratio (FIG. 6N), E / e’ ratio (FIG.60), LVEF (FIG.6P), and left ventricular (LV) mass / surface area (FIG.6Q) were measured in control (n=9) or Nnmt ASO mice (n=10).

[0042] Echocardiography was performed at baseline, week 4 and 7 of the feeding, and other parameters were collected after 7 weeks of feeding. FIG.6R, RT-qPCR (n=6) and WesternAttomey Docket No.: UCH-31725

[0043] UCLA Ref. No.: [UCLA 2022-142-3] WO

[0044] blotting (n=3) showing Nnmt overexpression in the heart using AAV9-cTnT-Vwnt. The relative mRNA levels were determined by comparing them to the average expression in the control group. The relative protein levels were determined by comparing them to the first sample in the control group. FIGs.6S-Y show C57BL / 6J male mice were injected with control or AAV9-cTnT-Vwnt and fed with HFD+l-NAME or HFD+l-NAME containing high indole-3-propionic acid (IPA) for 7 weeks (n=7). Body weight (FIG.6S), fat mass (FIG. 6T), lean mass (FIG.6U), E / e’ ratio (FIG.6V), LV mass / surface area (FIG.6W), LVEF (FIG. 6X), and heart weight / tibia length (FIG.6Y) were measured after the feeding. n=7. Representative of 2 (FIGs.6A-Y) experiments. Each point represents a mouse. All data are presented as the mean+SEM. P<0.05 are statistically significant. Data were analyzed by Student t test (FIGs.6A, E-L, Q, R), by 1-way ANOVA (FIGs.6B-F, N-P), or by 2-way ANOVA (FIGs.6S-Y). AAV-9 indicates adeno associated virus 9; AUC, area under the curve; ns, not significant; and RT-qPCR, reverse transcription-quantitative PCR.

[0045] FIGs.7A-Q show that therapeutic effects of indole- 3 -propionic acid (IPA) in heart failure with preserved ejection fraction (HFpEF). C57BL / 6J male mice were fed with chow diet or high-fat diet (HFD)+Nco-nitro-L-arginine methyl ester (1-NAME) for 4 weeks and followed by control (HFpEF) or high indole-3 -propionic acid (IPA) supplementation (HFpEF+IPA; 562.5 mg IPA / kg diet) for another 5 weeks. n=8 in chow, n=8 in HFpEF, and n=12 in HFpEF+IPA. FIG.7A shows experimental design. FIGs.7B-H show body weight over time (FIG.7B), insulin (FIG.7C), calculated homeostatic model assessment for insulin resistance (HOMA-IR; FIG.7D), plasma total cholesterol (FIG.7E), unesterified cholesterol (FIG. 7F), white adipose tissue weight (FIG.7G), and brown adipose tissue weight (FIG. 7H). FIGs.7I-Q shows representative images of echocardiography (FIG.71). E / A ratio (FIG. 7J), E / e’ ratio (FIG.7K), and left ventricular (LV) ejection fraction (LVEF; FIG.7 L) over time, LV mass / surface area (FIG.7M), heart weight / tibia length ratio (FIG.7N), lung weight (FIG.70), exercise tolerance in running distance (FIG.7P) and workload (FIG.7Q) were measured. Representative of 2 (FIGs.7A-Q) experiments. Each point represents a mouse. All data are presented as the mean+SEM. P<0.05 are statistically significant, ns, not significant. Data were analyzed by 1-way ANOVA (FIGs.7B-H, J-Q). For FIGs.

[0046] 7B and 7J-L, P values indicate significance between HFpEF and HFpEF+IPA group.

[0047] FIGs.8A-E show that indole- 3 -propionic acid (IPA) levels are reduced in patients with heart failure (HF) with preserved ejection fraction (HFpEF) but not patients with HF with reduced ejection fraction (HFrEF). FIGs.8A-B show the association of IPA levels withAttomey Docket No.: UCH-31725

[0048] UCLA Ref. No.: [UCLA 2022-142-3] WO

[0049] HFpEF in the Cleveland Clinic HFpEF case / control cohort. FIG.8A shows violin plots of IPA levels stratified by HFpEF status. Each point represents 1 patient. Medium is represented by dash line; P value was calculated by Wilcoxon rank-sum test. FIG.8B shows forest plots indicating the odds of HFpEF according to the quartiles of IPA levels using multivariable logistic regression models. Unadjusted odd ratio (grey), adjusted model 1 (age, sex, diabetes, hypercholesterolemia; black), adjusted model 2 (age, sex, diabetes, body mass index, and hypertension; grey), symbols represent odds ratios and the 5% to 95% Cis are indicated by line length. n=48. FIGs.8C-D, Left ventricular ejection fraction (LVEF; FIG.8C) and arterial abundance of IPA (FIG.8D) in non-HF controls (n=13), HFpEF (n=22), and HFrEF patients (n=20) from Alfred Hospital HFpEF study. P value was calculated by Wilcoxon rank-sum tests. P<0.05 are statistically significant. Each point represents 1 patient. FIG.8E shows an illustration summarizing how IPA mediates gut-heart cross talk in HFpEF. IPA feeding improved gut homeostasis by mitigating gut microbiota dysbiosis and intestinal epithelial barrier damage induced by HFpEF. Thus, metabolic remodeling by the diet was attenuated, including reduced body mass gain, improved glucose levels and lipid homeostasis. In the heart, IPA restored nicotinamide (NAM) and NAD+ / NADH levels, increased SIRT3 (sirtuin 3) levels and decreased NNMT (nicotinamide N-methyltransferase) levels, attenuating oxidative stress and inflammation in the heart. These beneficial effects collectively protect against diastolic dysfunction in HFpEF. ns indicates no significance.

[0050] FIGs.9A-O show that IPA attenuates diastolic dysfunction, metabolic remodeling, hypertrophy and fibrosis in HFpEF mice. C57BL / 6J male mice were fed with control or high IPA together with chow diet or HFD + LNAME for 7 weeks, n = 4 for chow diet groups and n = 8 for HFpEF groups. FIG.9A shows body lean mass measured at baseline, week 2, 5, and 7. FIGs.9B-D show food intake (FIG.9B), heart total cholesterol (FIG. 9C), and heart free fatty acids (FIG.9D) were measured, n = 4. FIGs.9E-L show representative images of echocardiography (FIG.9E), LVEF (FIG.9F), peak mitral E velocity (FIG.9G), peak mitral e' velocity (FIG.9H), lung weight (FIG.91), heart rate (FIG. 9J), exercise intolerance workload (FIG.9K) and V02max (FIG.9L), were measured after 7 weeks of feeding. FIG.9M shows representative images of haematoxylin and eosin (H&E), wheat germ agglutinin (WGA) with DAPI, and Masson's trichrome (MT) staining in transversal sections of left ventricle of mice under different experimental conditions (n = 4). Scale bars, 900 pm (H&E), 20 pm (WGA), and 90 pm (MT). FIG.9N shows WGA quantification of cardiomyocyte cross sectional area. FIG.90 shows percentage of fibrosisAttomey Docket No.: UCH-31725

[0051] UCLA Ref. No.: [UCLA 2022-142-3] WO

[0052] area in MT-stained transversal sections. Representative of 4 experiments. Each point represents a mouse. All data are presented as the mean ± SEM. p<0.05 are statistically significant, ns, not significant. Data were analyzed by 1-way ANOVA (FIGs.9N-O) or by Kruskal-Wallis test (FIGs.9A-L).

[0053] FIGs. 10A-N show that IPA attenuates diastolic and metabolic dysfunction in female mice. C57BL / 6J female mice were fed with chow diet (n = 8), HFD + LNAME (n = 12) or HFD + LNAME (n = 8) with high IPA for 7 weeks. FIGs. 10A shows experimental design.

[0054] FIGs. 10B-D shows body weight (FIG. 10B), fat mass (FIG. 10C), and lean mass (FIG. 10D) measured at baseline, week 3, 5, and 7. FIGs. 10E-N show E / A ratio (FIG. 10E), E / e' ratio (FIG. 10F), LVEF (FIG. 10G), LV mass (FIG. 10H), heart weight / tibia length ratio (FIG. 101), wet lung weight / dry lung weight ratio (FIG. 10J), white adipose weight (FIG.

[0055] 10K), brown adipose weight (FIG. 10L), plasma total cholesterol (FIG. 10M), and unesterified cholesterol (FIG. ION) measured after 7 weeks of feeding. Representative of 2 experiments. Each point represents a mouse. All data are presented as the mean ± SEM. p<0.05 are statistically significant, ns, not significant. Data were analyzed by 1-way ANOVA (FIGs. 10B-D) or 2-way ANOVA (FIGs. 10E-N).

[0056] FIGs. 11A-K show that IPA mitigates gut microbiota dysbiosis and intestinal epithelial barrier damage in HFpEF mice. C57BL / 6J male mice (8 weeks old) were fed with chow diet or HFD + LNAME containing control or high IPA (562.5 mg IPA / kg diet) for 7 weeks. Mice were euthanized at the end of the diet and the ceca and intestines were harvested. Genomic DNA was extracted from cecum samples and 16S rRNA-based gut microbial profiling was performed, n = 4 in Chow and Chow + NAM; n = 6 in HFpEF, HFpEF + IPA. FIGs. 11A-D show relative abundance of taxa at the phylum level (FIG. HA), relative abundance of Firmicutes (FIG. 11B), Bacteroidetes (FIG. 11C), and Firmicutes / Bacteroidetes ratio (FIG. 11D) in indicated groups. FIG. HE shows representative hematoxylin and eosin staining of the ilea. FIGs. 11F-G show the width (FIG. HF) and height (FIG. 11G) of the villi were measured (n = 20 randomly selected areas from 4 mice). FIGs. 11H-I show representative immunostaining (FIG. 11H) and quantification (FIG. HI) of OCCLUDIN protein in the ilea (n = 20 randomly selected areas from 4 mice).

[0057] FIGs. HJ-K show western blotting (FIG. HJ) and quantification (FIG. 11K) showing OCCLUDIN protein level in the intestine (n = 6). The relative protein levels were determined by comparing them to the first sample in the control group. Representative of 2 experiments. Each point represents a sample or a mouse. All data are presented as the mean ± SEM .Attorney Docket No.: UCH-31725

[0058] UCLA Ref. No.: [UCLA 2022-142-3] WO

[0059] p<0.05 are statistically significant, ns, not significant. Data were analyzed by 1-way ANOVA (FIGs. 11F-G, I) or by Kruskal- Wallis test (FIGs. 11B-D, K).

[0060] FIGs. 12A-J show metabolic and diastolic changes of mice fed with IPA. C57BL / 6J male mice (8 weeks old) were fed with chow diet or HFD + LNAME containing control or high IPA (562.5 mg IPA / kg diet) for 7 weeks. FIGs. 12A-B show a heatmap (FIG. 12A) and relative abundance (FIG. 12B) showing relative abundance of significantly differentiated metabolites in the plasma from mice under control or high IPA with HFD + LNAME for 7 weeks, n = 8. FIG. 12C shows heart 8-OHdG levels in the mice fed with chow diet, HFD + I-NAME or HFD + LNAME supplemented with high IPA for 7 weeks, n = 6. FIG. 12D shows RT-qPCR showing the levels of inflammatory genes in the heart of mice fed with HFD + I-NAME with or without IPA supplementation for 7 weeks. The relative mRNA levels were determined by comparing them to the average expression in the control group, n = 8. FIGs.

[0061] 12E-J show oxygen consumption rates (OCR) of isolated mitochondria from C57BL / 6J male mice fed with HFD + LNAME or HFD + LNAME containing high IPA for 7 weeks were measured by Seahorse assay. The coupling assay measures basal respiration in presence of palmitoyl-carnitine (FIG. 12E), state 3 (ADP) (FIG. 12F), and state 3u (FCCP) (FIG. 12G).

[0062] The electron flow measures complex I (palmitoyl-carnitine) (FIG. 12H), complex II (succinate) (FIG. 121), and complex IV (TMPD) respiration (FIG. 12J). n = 5.

[0063] Representative of 2 experiments. Each point represents a mouse. All data are presented as the mean ± SEM. p<0.05 are statistically significant, ns, not significant. Data were analyzed by Student's t test.

[0064] FIGs. 13A-F show NAD metabolism changes of mice fed with IPA. FIGs. 13A-C show the abundance of NAD+ (FIG. 13A) and NADH (FIG. 13B) in the heart tissue from mice fed with chow diet or HFD + LNAME for 7 weeks. Concentrations of NAD+ and NADH were calculated by running samples with the standards in LC-MS analysis. The sum of the two forms of NAD were shown (FIG. 13C). n = 4. FIGs. 13D-F show the abundance of NAD+ (FIG. 13D) and NADH (FIG. 13E) in the heart tissue from mice fed with HFD + LNAME or HFD + LNAME containing high IPA for 7 weeks, n = 4. The sum of the two forms of NAD were shown (FIG. 13F). The relative protein levels were determined by comparing them to the first sample in the control group. Representative of 2 experiments. Each point represents a mouse. All data are presented as the mean ± SEM. p<0.05 are statistically significant, ns, not significant. Data were analyzed by Student's t test (FIGs. 13A-F).Attomey Docket No.: UCH-31725

[0065] UCLA Ref. No.: [UCLA 2022-142-3] WO

[0066] DETAILED DESCRIPTION HFpEF affects half of all patients with heart failure worldwide, confers substantial morbidity and mortality, and there are presently no effective therapies. Patients with HFpEF display a metabolic profile associated with diabetes, increased inflammation and oxidative stress, and impaired lipid metabolism compared with patients with HFrEF. The present disclosure demonstrates that indole-3-propionic acid (IPA) was reduced in patients with HFpEF, and that administration of IPA or nicotinamide can reduce HFpEF, diastolic dysfunction, adipose tissue mass, lipidemia, and other conditions associated with HFpEF. The present disclosure also reveals the relationship between IPA, nicotinamide, and cardiometabolic diseases, especially HFpEF, offering a new therapeutic approach to treating these conditions.

[0067] The effects of gut microbiota on the host are mediated in part by the produced microbe-derived metabolites. Gut microbes metabolize tryptophan into indole and its derivatives, such as indoleacrylic acid (IA), indole-3-acetic acid (IAA), indole-3-aldehyde (I3A), and indole- 3 -propionic acid (IPA). IPA is reported to be synthesized by the commensal Clostridium sporogenes and functions to ameliorate inflammation and cell oxidative damage. IPA can be synthesized in the gut from dietary tryptophan, an essential amino acid. In the gut, dietary tryptophan can be converted into IPA by bacteria such as Lactobacillus reuteri, Akkermansia muciniphila, and Clostridium sporogenes.

[0068] In certain aspects, the present disclosure provides methods of treating a heart disease, comprising administering a composition comprising 3 -indolepropionic acid (IPA) or a pharmaceutically acceptable salt or prodrug thereof to a subject in need thereof.

[0069] In certain aspects, the present disclosure provides methods of treating a heart disease, comprising administering a composition comprising nicotinamide (NAM) or a pharmaceutically acceptable salt or prodrug thereof to a subject in need thereof.

[0070] In certain aspects, the present disclosure provides methods of treating diastolic dysfunction, comprising administering a composition comprising 3 -indolepropionic acid (IPA) or a pharmaceutically acceptable salt or prodrug thereof to a subject in need thereof.

[0071] In certain aspects, the present disclosure provides methods of treating diastolic dysfunction, comprising administering a composition comprising nicotinamide (NAM) or a pharmaceutically acceptable salt or prodrug thereof to a subject in need thereof.Attomey Docket No.: UCH-31725

[0072] UCLA Ref. No.: [UCLA 2022-142-3] WO

[0073] In certain aspects, the present disclosure provides methods of treating metabolic disease comprising administering a composition comprising 3 -indolepropionic acid (IPA) or a pharmaceutically acceptable salt or prodrug thereof to a subject in need thereof.

[0074] In certain aspects, the present disclosure provides methods of treating metabolic disease comprising administering a composition comprising nicotinamide (NAM) or a pharmaceutically acceptable salt or prodrug thereof to a subject in need thereof.

[0075] In certain aspects, the present disclosure provides methods of reducing cholesterol or treating hyperlipidemia comprising administering a composition comprising 3-indolepropionic acid (IPA) or a pharmaceutically acceptable salt or prodrug thereof to a subject in need thereof.

[0076] In certain aspects, the present disclosure provides methods of reducing cholesterol or treating hyperlipidemia comprising administering a composition comprising nicotinamide (NAM) or a pharmaceutically acceptable salt or prodrug thereof to a subject in need thereof.

[0077] In certain aspects, the present disclosure provides methods of treating obesity comprising administering a composition comprising 3 -indolepropionic acid (IPA) or a pharmaceutically acceptable salt or prodrug thereof to a subject in need thereof.

[0078] In certain aspects, the present disclosure provides methods of treating obesity comprising administering a composition comprising nicotinamide (NAM) or a pharmaceutically acceptable salt or prodrug thereof to a subject in need thereof.

[0079] In certain aspects, the present disclosure provides methods of reducing adipose tissue mass comprising administering a composition comprising 3-indolepropionic acid (IPA) or a pharmaceutically acceptable salt or prodrug thereof to a subject in need thereof.

[0080] In certain aspects, the present disclosure provides methods of reducing adipose tissue mass comprising administering a composition comprising nicotinamide (NAM) or a pharmaceutically acceptable salt or prodrug thereof to a subject in need thereof.

[0081] In certain aspects, the present disclosure provides methods of reducing vascular fibrosis comprising administering a composition comprising 3-indolepropionic acid (IPA) or a pharmaceutically acceptable salt or prodrug thereof to a subject in need thereof.

[0082] In certain aspects, the present disclosure provides methods of reducing vascular fibrosis comprising administering a composition comprising nicotinamide (NAM) or a pharmaceutically acceptable salt or prodrug thereof to a subject in need thereof.Attomey Docket No.: UCH-31725

[0083] UCLA Ref. No.: [UCLA 2022-142-3] WO

[0084] In some embodiments, the subject has heart disease. In some embodiments, the heart disease is heart failure. In some embodiments, the heart disease is Heart Failure with preserved Ejection Fraction (HFpEF).

[0085] In some embodiments, administration of the composition reduces the weight of the subject. In some embodiments, administration of the composition reduces adipose tissue mass in the subject. In some embodiments, administration of the composition reduces white and / or brown adipose tissue mass in the subject. In some embodiments, administration of the composition reduces the body mass index of the subject.

[0086] In some embodiments, the composition comprises IPA or a pharmaceutically acceptable salt or prodrug thereof. In some embodiments, the composition comprises IPA or a pharmaceutically acceptable salt thereof.

[0087] In some embodiments, the composition comprises nicotinamide (NAM) or a pharmaceutically acceptable salt or prodrug thereof.

[0088] In some embodiments, the composition comprises tryptophan or a pharmaceutically acceptable salt thereof. In some embodiments, the composition comprises a bacterium that can convert tryptophan to IPA. In some embodiments, the composition comprises at least one of Lactobacillus reuteri, Akkermansia muciniphila, or Clostridium sporogenes

[0089] In some embodiments, the composition is administered orally. In some embodiments, the composition further comprises a pharmaceutically acceptable excipient.

[0090] In some embodiments, the composition is administered for between about 1 and 140 days. In some embodiments, the composition is administered for at least about 20 days. In some embodiments, the composition is administered for at least about 40 days. In some embodiments, the composition is administered for at least about 60 days. In some embodiments, the composition is administered for at least about 80 days. In some embodiments, the composition is administered for at least about 100 days. In some embodiments, the composition is administered for at least about 120 days. In some embodiments, the composition is administered for at least about 140 days.

[0091] In some embodiments, the subject has insulin resistance. In some embodiments, the subject has diabetes. In some embodiments, the subject has gut microbiota dysbiosis. In some embodiments, administration of the composition reduces intestinal epithelial barrier damage. In some embodiments, administration of the composition reduces gut dysbiosis.

[0092] In some embodiments, the subject is a mammal (e.g., a mouse or a human). In some preferred embodiments, the subject is a human.Attomey Docket No.: UCH-31725

[0093] UCLA Ref. No.: [UCLA 2022-142-3] WO

[0094] Pharmaceutical Compositions

[0095] The compositions and methods of the present invention may be utilized to treat an individual in need thereof. In certain embodiments, the individual is a mammal such as a human, or a non-human mammal. When administered to an animal, such as a human, the composition or the compound is preferably administered as a pharmaceutical composition comprising, for example, a compound of the invention and a pharmaceutically acceptable carrier. Pharmaceutically acceptable carriers are well known in the art and include, for example, aqueous solutions such as water or physiologically buffered saline or other solvents or vehicles such as glycols, glycerol, oils such as olive oil, or injectable organic esters. In preferred embodiments, when such pharmaceutical compositions are for human administration, particularly for invasive routes of administration (i.e., routes, such as injection or implantation, that circumvent transport or diffusion through an epithelial barrier), the aqueous solution is pyrogen-free, or substantially pyrogen-free. The excipients can be chosen, for example, to effect delayed release of an agent or to selectively target one or more cells, tissues or organs. The pharmaceutical composition can be in dosage unit form such as tablet, capsule (including sprinkle capsule and gelatin capsule), granule, lyophile for reconstitution, powder, solution, syrup, suppository, injection or the like. The composition can also be present in a transdermal delivery system, e.g., a skin patch. The composition can also be present in a solution suitable for topical administration, such as a lotion, cream, or ointment.

[0096] A pharmaceutically acceptable carrier can contain physiologically acceptable agents that act, for example, to stabilize, increase solubility or to increase the absorption of a compound such as a compound of the invention. Such physiologically acceptable agents include, for example, carbohydrates, such as glucose, sucrose or dextrans, antioxidants, such as ascorbic acid or glutathione, chelating agents, low molecular weight proteins or other stabilizers or excipients. The choice of a pharmaceutically acceptable carrier, including a physiologically acceptable agent, depends, for example, on the route of administration of the composition. The preparation or pharmaceutical composition can be a self-emulsifying drug delivery system or a self-microemulsifying drug delivery system. The pharmaceutical composition (preparation) also can be a liposome or other polymer matrix, which can have incorporated therein, for example, a compound of the invention. Liposomes, for example, which comprise phospholipids or other lipids, are nontoxic, physiologically acceptable and metabolizable carriers that are relatively simple to make and administer.Attomey Docket No.: UCH-31725

[0097] UCLA Ref. No.: [UCLA 2022-142-3] WO

[0098] The phrase "pharmaceutically acceptable" is employed herein to refer to those compounds, materials, compositions, and / or dosage forms which are, within the scope of sound medical judgment, suitable for use in contact with the tissues of human beings and animals without excessive toxicity, irritation, allergic response, or other problem or complication, commensurate with a reasonable benefit / risk ratio.

[0099] The phrase "pharmaceutically acceptable carrier" as used herein means a pharmaceutically acceptable material, composition or vehicle, such as a liquid or solid filler, diluent, excipient, solvent or encapsulating material. Each carrier must be "acceptable" in the sense of being compatible with the other ingredients of the formulation and not injurious to the patient. Some examples of materials which can serve as pharmaceutically acceptable carriers include: (1) sugars, such as lactose, glucose and sucrose; (2) starches, such as corn starch and potato starch; (3) cellulose, and its derivatives, such as sodium carboxymethyl cellulose, ethyl cellulose and cellulose acetate; (4) powdered tragacanth; (5) malt; (6) gelatin; (7) talc; (8) excipients, such as cocoa butter and suppository waxes; (9) oils, such as peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, corn oil and soybean oil; (10) glycols, such as propylene glycol; (11) polyols, such as glycerin, sorbitol, mannitol and polyethylene glycol; (12) esters, such as ethyl oleate and ethyl laurate; (13) agar; (14) buffering agents, such as magnesium hydroxide and aluminum hydroxide; (15) alginic acid; (16) pyrogen-free water; (17) isotonic saline; (18) Ringer's solution; (19) ethyl alcohol; (20) phosphate buffer solutions; and (21) other non-toxic compatible substances employed in pharmaceutical formulations.

[0100] A pharmaceutical composition (preparation) can be administered to a subject by any of a number of routes of administration including, for example, orally (for example, drenches as in aqueous or non-aqueous solutions or suspensions, tablets, capsules (including sprinkle capsules and gelatin capsules), boluses, powders, granules, pastes for application to the tongue); absorption through the oral mucosa (e.g., sublingually); subcutaneously; transdermally (for example as a patch applied to the skin); and topically (for example, as a cream, ointment or spray applied to the skin). The compound may also be formulated for inhalation. In certain embodiments, a compound may be simply dissolved or suspended in sterile water. Details of appropriate routes of administration and compositions suitable for same can be found in, for example, U.S. Pat. Nos. 6,110,973, 5,763,493, 5,731,000, 5,541,231, 5,427,798, 5,358,970 and 4,172,896, as well as in patents cited therein.Attomey Docket No.: UCH-31725

[0101] UCLA Ref. No.: [UCLA 2022-142-3] WO

[0102] The formulations may conveniently be presented in unit dosage form and may be prepared by any methods well known in the art of pharmacy. The amount of active ingredient which can be combined with a carrier material to produce a single dosage form will vary depending upon the host being treated, the particular mode of administration. The amount of active ingredient that can be combined with a carrier material to produce a single dosage form will generally be that amount of the compound which produces a therapeutic effect. Generally, out of one hundred percent, this amount will range from about 1 percent to about ninety-nine percent of active ingredient, preferably from about 5 percent to about 70 percent, most preferably from about 10 percent to about 30 percent.

[0103] Methods of preparing these formulations or compositions include the step of bringing into association an active compound, such as a compound of the invention, with the carrier and, optionally, one or more accessory ingredients. In general, the formulations are prepared by uniformly and intimately bringing into association a compound of the present invention with liquid carriers, or finely divided solid carriers, or both, and then, if necessary, shaping the product.

[0104] Formulations of the invention suitable for oral administration may be in the form of capsules (including sprinkle capsules and gelatin capsules), cachets, pills, tablets, lozenges (using a flavored basis, usually sucrose and acacia or tragacanth), lyophile, powders, granules, or as a solution or a suspension in an aqueous or non-aqueous liquid, or as an oil-in-water or water-in-oil liquid emulsion, or as an elixir or syrup, or as pastilles (using an inert base, such as gelatin and glycerin, or sucrose and acacia) and / or as mouth washes and the like, each containing a predetermined amount of a compound of the present invention as an active ingredient. Compositions or compounds may also be administered as a bolus, electuary or paste.

[0105] To prepare solid dosage forms for oral administration (capsules (including sprinkle capsules and gelatin capsules), tablets, pills, dragees, powders, granules and the like), the active ingredient is mixed with one or more pharmaceutically acceptable carriers, such as sodium citrate or dicalcium phosphate, and / or any of the following: (1) fillers or extenders, such as starches, lactose, sucrose, glucose, mannitol, and / or silicic acid; (2) binders, such as, for example, carboxymethylcellulose, alginates, gelatin, polyvinyl pyrrolidone, sucrose and / or acacia; (3) humectants, such as glycerol; (4) disintegrating agents, such as agar-agar, calcium carbonate, potato or tapioca starch, alginic acid, certain silicates, and sodium carbonate; (5) solution retarding agents, such as paraffin; (6) absorption accelerators, such asAttomey Docket No.: UCH-31725

[0106] UCLA Ref. No.: [UCLA 2022-142-3] WO

[0107] quaternary ammonium compounds; (7) wetting agents, such as, for example, cetyl alcohol and glycerol monostearate; (8) absorbents, such as kaolin and bentonite clay; (9) lubricants, such a talc, calcium stearate, magnesium stearate, solid polyethylene glycols, sodium lauryl sulfate, and mixtures thereof; (10) complexing agents, such as, modified and unmodified cyclodextrins; and (11) coloring agents. In the case of capsules (including sprinkle capsules and gelatin capsules), tablets and pills, the pharmaceutical compositions may also comprise buffering agents. Solid compositions of a similar type may also be employed as fillers in soft and hard-filled gelatin capsules using such excipients as lactose or milk sugars, as well as high molecular weight polyethylene glycols and the like.

[0108] A tablet may be made by compression or molding, optionally with one or more accessory ingredients. Compressed tablets may be prepared using binder (for example, gelatin or hydroxypropylmethyl cellulose), lubricant, inert diluent, preservative, disintegrant (for example, sodium starch glycolate or cross-linked sodium carboxymethyl cellulose), surface-active or dispersing agent. Molded tablets may be made by molding in a suitable machine a mixture of the powdered compound moistened with an inert liquid diluent.

[0109] The tablets, and other solid dosage forms of the pharmaceutical compositions, such as dragees, capsules (including sprinkle capsules and gelatin capsules), pills and granules, may optionally be scored or prepared with coatings and shells, such as enteric coatings and other coatings well known in the pharmaceutical-formulating art. They may also be formulated so as to provide slow or controlled release of the active ingredient therein using, for example, hydroxypropylmethyl cellulose in varying proportions to provide the desired release profile, other polymer matrices, liposomes and / or microspheres. They may be sterilized by, for example, filtration through a bacteria-retaining filter, or by incorporating sterilizing agents in the form of sterile solid compositions that can be dissolved in sterile water, or some other sterile injectable medium immediately before use. These compositions may also optionally contain opacifying agents and may be of a composition that they release the active ingredient(s) only, or preferentially, in a certain portion of the gastrointestinal tract, optionally, in a delayed manner. Examples of embedding compositions that can be used include polymeric substances and waxes. The active ingredient can also be in micro-encapsulated form, if appropriate, with one or more of the above-described excipients.

[0110] Liquid dosage forms useful for oral administration include pharmaceutically acceptable emulsions, lyophiles for reconstitution, microemulsions, solutions, suspensions, syrups and elixirs. In addition to the active ingredient, the liquid dosage forms may containAttomey Docket No.: UCH-31725

[0111] UCLA Ref. No.: [UCLA 2022-142-3] WO

[0112] inert diluents commonly used in the art, such as, for example, water or other solvents, cyclodextrins and derivatives thereof, solubilizing agents and emulsifiers, such as ethyl alcohol, isopropyl alcohol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propylene glycol, 1,3-butylene glycol, oils (in particular, cottonseed, groundnut, corn, germ, olive, castor and sesame oils), glycerol, tetrahydrofuryl alcohol, polyethylene glycols and fatty acid esters of sorbitan, and mixtures thereof.

[0113] Besides inert diluents, the oral compositions can also include adjuvants such as wetting agents, emulsifying and suspending agents, sweetening, flavoring, coloring, perfuming and preservative agents.

[0114] Suspensions, in addition to the active compounds, may contain suspending agents as, for example, ethoxylated isostearyl alcohols, polyoxyethylene sorbitol and sorbitan esters, microcrystalline cellulose, aluminum metahydroxide, bentonite, agar-agar and tragacanth, and mixtures thereof.

[0115] Dosage forms for the topical or transdermal administration include powders, sprays, ointments, pastes, creams, lotions, gels, solutions, patches and inhalants. The active compound may be mixed under sterile conditions with a pharmaceutically acceptable carrier, and with any preservatives, buffers, or propellants that may be required.

[0116] The ointments, pastes, creams and gels may contain, in addition to an active compound, excipients, such as animal and vegetable fats, oils, waxes, paraffins, starch, tragacanth, cellulose derivatives, polyethylene glycols, silicones, bentonites, silicic acid, talc and zinc oxide, or mixtures thereof.

[0117] Powders and sprays can contain, in addition to an active compound, excipients such as lactose, talc, silicic acid, aluminum hydroxide, calcium silicates and polyamide powder, or mixtures of these substances. Sprays can additionally contain customary propellants, such as chlorofluorohydrocarbons and volatile unsubstituted hydrocarbons, such as butane and propane.

[0118] Transdermal patches have the added advantage of providing controlled delivery of a compound of the present invention to the body. Such dosage forms can be made by dissolving or dispersing the active compound in the proper medium. Absorption enhancers can also be used to increase the flux of the compound across the skin. The rate of such flux can be controlled by either providing a rate controlling membrane or dispersing the compound in a polymer matrix or gel.Attomey Docket No.: UCH-31725

[0119] UCLA Ref. No.: [UCLA 2022-142-3] WO

[0120] The phrases "parenteral administration" and "administered parenterally" as used herein means modes of administration other than enteral and topical administration, usually by injection, and includes, without limitation, intravenous, intramuscular, intraarterial, intrathecal, intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal, transtracheal, subcutaneous, subcuticular, intraarticular, subcapsular, subarachnoid, intraspinal and intrastemal injection and infusion. Pharmaceutical compositions suitable for parenteral administration comprise one or more active compounds in combination with one or more pharmaceutically acceptable sterile isotonic aqueous or nonaqueous solutions, dispersions, suspensions or emulsions, or sterile powders which may be reconstituted into sterile injectable solutions or dispersions just prior to use, which may contain antioxidants, buffers, bacteriostats, solutes which render the formulation isotonic with the blood of the intended recipient or suspending or thickening agents.

[0121] Examples of suitable aqueous and nonaqueous carriers that may be employed in the pharmaceutical compositions of the invention include water, ethanol, polyols (such as glycerol, propylene glycol, polyethylene glycol, and the like), and suitable mixtures thereof, vegetable oils, such as olive oil, and injectable organic esters, such as ethyl oleate. Proper fluidity can be maintained, for example, by the use of coating materials, such as lecithin, by the maintenance of the required particle size in the case of dispersions, and by the use of surfactants.

[0122] These compositions may also contain adjuvants such as preservatives, wetting agents, emulsifying agents and dispersing agents. Prevention of the action of microorganisms may be ensured by the inclusion of various antibacterial and antifungal agents, for example, paraben, chlorobutanol, phenol sorbic acid, and the like. It may also be desirable to include isotonic agents, such as sugars, sodium chloride, and the like into the compositions. In addition, prolonged absorption of the injectable pharmaceutical form may be brought about by the inclusion of agents that delay absorption such as aluminum monostearate and gelatin.

[0123] In some cases, in order to prolong the effect of a drug, it is desirable to slow the absorption of the drug from subcutaneous or intramuscular injection. This may be accomplished by the use of a liquid suspension of crystalline or amorphous material having poor water solubility. The rate of absorption of the drug then depends upon its rate of dissolution, which, in turn, may depend upon crystal size and crystalline form. Alternatively, delayed absorption of a parenterally administered drug form is accomplished by dissolving or suspending the drug in an oil vehicle.Attorney Docket No.: UCH-31725

[0124] UCLA Ref. No.: [UCLA 2022-142-3] WO

[0125] Injectable depot forms are made by forming microencapsulated matrices of the subject compounds in biodegradable polymers such as polylactide-polyglycolide. Depending on the ratio of drug to polymer, and the nature of the particular polymer employed, the rate of drug release can be controlled. Examples of other biodegradable polymers include poly(orthoesters) and poly(anhydrides). Depot injectable formulations are also prepared by entrapping the drug in liposomes or microemulsions that are compatible with body tissue.

[0126] For use in the methods of this invention, active compounds can be given per se or as a pharmaceutical composition containing, for example, 0.1 to 99.5% (more preferably, 0.5 to 90%) of active ingredient in combination with a pharmaceutically acceptable carrier.

[0127] Methods of introduction may also be provided by rechargeable or biodegradable devices. Various slow release polymeric devices have been developed and tested in vivo in recent years for the controlled delivery of drugs, including proteinaceous biopharmaceuticals. A variety of biocompatible polymers (including hydrogels), including both biodegradable and non-degradable polymers, can be used to form an implant for the sustained release of a compound at a particular target site.

[0128] Actual dosage levels of the active ingredients in the pharmaceutical compositions may be varied so as to obtain an amount of the active ingredient that is effective to achieve the desired therapeutic response for a particular patient, composition, and mode of administration, without being toxic to the patient.

[0129] The selected dosage level will depend upon a variety of factors including the activity of the particular compound or combination of compounds employed, or the ester, salt or amide thereof, the route of administration, the time of administration, the rate of excretion of the particular compound(s) being employed, the duration of the treatment, other drugs, compounds and / or materials used in combination with the particular compound(s) employed, the age, sex, weight, condition, general health and prior medical history of the patient being treated, and like factors well known in the medical arts.

[0130] A physician or veterinarian having ordinary skill in the art can readily determine and prescribe the therapeutically effective amount of the pharmaceutical composition required. For example, the physician or veterinarian could start doses of the pharmaceutical composition or compound at levels lower than that required in order to achieve the desired therapeutic effect and gradually increase the dosage until the desired effect is achieved. By “therapeutically effective amount” is meant the concentration of a compound that is sufficient to elicit the desired therapeutic effect. It is generally understood that the effective amount ofAttomey Docket No.: UCH-31725

[0131] UCLA Ref. No.: [UCLA 2022-142-3] WO

[0132] the compound will vary according to the weight, sex, age, and medical history of the subject. Other factors which influence the effective amount may include, but are not limited to, the severity of the patient's condition, the disorder being treated, the stability of the compound, and, if desired, another type of therapeutic agent being administered with the compound of the invention. A larger total dose can be delivered by multiple administrations of the agent. Methods to determine efficacy and dosage are known to those skilled in the art (Isselbacher et al. (1996) Harrison’s Principles of Internal Medicine 13 ed., 1814-1882, herein incorporated by reference).

[0133] In general, a suitable daily dose of an active compound used in the compositions and methods of the invention will be that amount of the compound that is the lowest dose effective to produce a therapeutic effect. Such an effective dose will generally depend upon the factors described above.

[0134] If desired, the effective daily dose of the active compound may be administered as one, two, three, four, five, six or more sub-doses administered separately at appropriate intervals throughout the day, optionally, in unit dosage forms. In certain embodiments of the present invention, the active compound may be administered two or three times daily. In preferred embodiments, the active compound will be administered once daily.

[0135] The patient receiving this treatment is any animal in need, including primates, in particular humans; and other mammals such as equines, cattle, swine, sheep, cats, and dogs; poultry; and pets in general.

[0136] In certain embodiments, compounds of the invention may be used alone or conjointly administered with another type of therapeutic agent.

[0137] The present disclosure includes the use of pharmaceutically acceptable salts of compounds of the invention in the compositions and methods of the present invention. In certain embodiments, contemplated salts of the invention include, but are not limited to, alkyl, dialkyl, trialkyl or tetra-alkyl ammonium salts. In certain embodiments, contemplated salts of the invention include, but are not limited to, L-arginine, benenthamine, benzathine, betaine, calcium hydroxide, choline, deanol, diethanolamine, diethylamine, 2-(diethylamino)ethanol, ethanolamine, ethylenediamine, N-methylglucamine, hydrabamine, IH-imidazole, lithium, L-lysine, magnesium, 4-(2-hydroxyethyl)morpholine, piperazine, potassium, l-(2-hydroxyethyl)pyrrolidine, sodium, triethanolamine, tromethamine, and zinc salts. In certain embodiments, contemplated salts of the invention include, but are not limited to, Na, Ca, K, Mg, Zn or other metal salts. In certain embodiments, contemplated salts of theAttomey Docket No.: UCH-31725

[0138] UCLA Ref. No.: [UCLA 2022-142-3] WO

[0139] invention include, but are not limited to, l-hydroxy-2-naphthoic acid, 2,2-dichloroacetic acid, 2-hydroxyethanesulfonic acid, 2-oxoglutaric acid, 4-acetamidobenzoic acid, 4-aminosalicylic acid, acetic acid, adipic acid, 1-ascorbic acid, 1-aspartic acid, benzenesulfonic acid, benzoic acid, (+)-camphoric acid, (+)-camphor-10-sulfonic acid, capric acid (decanoic acid), caproic acid (hexanoic acid), caprylic acid (octanoic acid), carbonic acid, cinnamic acid, citric acid, cyclamic acid, dodecylsulfuric acid, ethane- 1,2-disulfonic acid, ethanesulfonic acid, formic acid, fumaric acid, galactaric acid, gentisic acid, d-glucoheptonic acid, d-gluconic acid, d-glucuronic acid, glutamic acid, glutaric acid, glycerophosphoric acid, glycolic acid, hippuric acid, hydrobromic acid, hydrochloric acid, isobutyric acid, lactic acid, lactobionic acid, lauric acid, maleic acid, 1-malic acid, malonic acid, mandelic acid, methanesulfonic acid , naphthalene- 1,5-disulfonic acid, naphthalene-2- sulfonic acid, nicotinic acid, nitric acid, oleic acid, oxalic acid, palmitic acid, pamoic acid, phosphoric acid, proprionic acid, 1-pyroglutamic acid, salicylic acid, sebacic acid, stearic acid, succinic acid, sulfuric acid, 1-tartaric acid, thiocyanic acid, p-toluenesulfonic acid, trifluoroacetic acid, and undecylenic acid salts.

[0140] The pharmaceutically acceptable acid addition salts can also exist as various solvates, such as with water, methanol, ethanol, dimethylformamide, and the like. Mixtures of such solvates can also be prepared. The source of such solvates can be from the solvent of crystallization, inherent in the solvent of preparation or crystallization, or adventitious to such solvent.

[0141] Wetting agents, emulsifiers and lubricants, such as sodium lauryl sulfate and magnesium stearate, as well as coloring agents, release agents, coating agents, sweetening, flavoring and perfuming agents, preservatives and antioxidants can also be present in the compositions.

[0142] Examples of pharmaceutically acceptable antioxidants include: (1) water-soluble antioxidants, such as ascorbic acid, cysteine hydrochloride, sodium bisulfate, sodium metabisulfite, sodium sulfite and the like; (2) oil-soluble antioxidants, such as ascorbyl palmitate, butylated hydroxyanisole (BHA), butylated hydroxytoluene (BHT), lecithin, propyl gallate, alpha-tocopherol, and the like; and (3) metal-chelating agents, such as citric acid, ethylenediamine tetraacetic acid (EDTA), sorbitol, tartaric acid, phosphoric acid, and the like.Attomey Docket No.: UCH-31725

[0143] UCLA Ref. No.: [UCLA 2022-142-3] WO

[0144] Definitions

[0145] Unless otherwise defined herein, scientific and technical terms used in this application shall have the meanings that are commonly understood by those of ordinary skill in the art. Generally, nomenclature used in connection with, and techniques of, chemistry, cell and tissue culture, molecular biology, cell and cancer biology, neurobiology, neurochemistry, virology, immunology, microbiology, pharmacology, genetics and protein and nucleic acid chemistry, described herein, are those well known and commonly used in the art.

[0146] The methods and techniques of the present disclosure are generally performed, unless otherwise indicated, according to conventional methods well known in the art and as described in various general and more specific references that are cited and discussed throughout this specification. See, e.g. “Principles of Neural Science”, McGraw-Hill Medical, New York, N.Y. (2000); Motulsky, “Intuitive Biostatistics”, Oxford University Press, Inc. (1995); Lodish et al., “Molecular Cell Biology, 4th ed.”, W. H. Freeman & Co., New York (2000); Griffiths et al., “Introduction to Genetic Analysis, 7th ed.”, W. H. Freeman & Co., N.Y. (1999); and Gilbert et al., “Developmental Biology, 6th ed.”, Sinauer Associates, Inc., Sunderland, MA (2000).

[0147] Chemistry terms used herein, unless otherwise defined herein, are used according to conventional usage in the art, as exemplified by “The McGraw-Hill Dictionary of Chemical Terms”, Parker S., Ed., McGraw-Hill, San Francisco, C.A. (1985).

[0148] All of the above, and any other publications, patents and published patent applications referred to in this application are specifically incorporated by reference herein. In case of conflict, the present specification, including its specific definitions, will control.

[0149] The term “agent” is used herein to denote a chemical compound (such as an organic or inorganic compound, a mixture of chemical compounds), a biological macromolecule (such as a nucleic acid, an antibody, including parts thereof as well as humanized, chimeric and human antibodies and monoclonal antibodies, a protein or portion thereof, e.g., a peptide, a lipid, a carbohydrate), or an extract made from biological materials such as bacteria, plants, fungi, or animal (particularly mammalian) cells or tissues. Agents include, for example, agents whose structure is known, and those whose structure is not known. The ability of such agents to inhibit AR or promote AR degradation may render them suitable as “therapeutic agents” in the methods and compositions of this disclosure.

[0150] A “patient,” “subject,” or “individual” are used interchangeably and refer to either a human or a non-human animal. These terms include mammals, such as humans, primates,Attomey Docket No.: UCH-31725

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[0152] livestock animals (including bovines, porcines, etc.), companion animals (e.g., canines, felines, etc.) and rodents (e.g., mice and rats).

[0153] “Treating” a condition or patient refers to taking steps to obtain beneficial or desired results, including clinical results. As used herein, and as well understood in the art, “treatment” is an approach for obtaining beneficial or desired results, including clinical results. Beneficial or desired clinical results can include, but are not limited to, alleviation or amelioration of one or more symptoms or conditions, diminishment of extent of disease, stabilized (i.e. not worsening) state of disease, preventing spread of disease, delay or slowing of disease progression, amelioration or palliation of the disease state, and remission (whether partial or total), whether detectable or undetectable. “Treatment” can also mean prolonging survival as compared to expected survival if not receiving treatment.

[0154] The term “preventing” is art-recognized, and when used in relation to a condition, such as a local recurrence (e.g., pain), a disease such as cancer, a syndrome complex such as heart failure or any other medical condition, is well understood in the art, and includes administration of a composition which reduces the frequency of, or delays the onset of, symptoms of a medical condition in a subject relative to a subject which does not receive the composition. Thus, prevention of cancer includes, for example, reducing the number of detectable cancerous growths in a population of patients receiving a prophylactic treatment relative to an untreated control population, and / or delaying the appearance of detectable cancerous growths in a treated population versus an untreated control population, e.g., by a statistically and / or clinically significant amount.

[0155] “Administering” or “administration of’ a substance, a compound or an agent to a subject can be carried out using one of a variety of methods known to those skilled in the art. For example, a compound or an agent can be administered, intravenously, arterially, intradermally, intramuscularly, intraperitoneally, subcutaneously, ocularly, sublingually, orally (by ingestion), intranasally (by inhalation), intraspinally, intracerebrally, and transdermally (by absorption, e.g., through a skin duct). A compound or agent can also appropriately be introduced by rechargeable or biodegradable polymeric devices or other devices, e.g., patches and pumps, or formulations, which provide for the extended, slow or controlled release of the compound or agent. Administering can also be performed, for example, once, a plurality of times, and / or over one or more extended periods.

[0156] Appropriate methods of administering a substance, a compound or an agent to a subject will also depend, for example, on the age and / or the physical condition of the subjectAttomey Docket No.: UCH-31725

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[0158] and the chemical and biological properties of the compound or agent (e.g., solubility, digestibility, bioavailability, stability and toxicity). In some embodiments, a compound or an agent is administered orally, e.g., to a subject by ingestion. In some embodiments, the orally administered compound or agent is in an extended release or slow release formulation, or administered using a device for such slow or extended release.

[0159] As used herein, the phrase “conjoint administration” refers to any form of administration of two or more different therapeutic agents such that the second agent is administered while the previously administered therapeutic agent is still effective in the body (e.g., the two agents are simultaneously effective in the patient, which may include synergistic effects of the two agents). For example, the different therapeutic compounds can be administered either in the same formulation or in separate formulations, either concomitantly or sequentially. Thus, an individual who receives such treatment can benefit from a combined effect of different therapeutic agents.

[0160] A “therapeutically effective amount” or a “therapeutically effective dose” of a drug or agent is an amount of a drug or an agent that, when administered to a subject will have the intended therapeutic effect. The full therapeutic effect does not necessarily occur by administration of one dose, and may occur only after administration of a series of doses. Thus, a therapeutically effective amount may be administered in one or more administrations. The precise effective amount needed for a subject will depend upon, for example, the subject’s size, health and age, and the nature and extent of the condition being treated, such as cancer or MDS. The skilled worker can readily determine the effective amount for a given situation by routine experimentation.

[0161] As used herein, the terms “optional” or “optionally” mean that the subsequently described event or circumstance may occur or may not occur, and that the description includes instances where the event or circumstance occurs as well as instances in which it does not. For example, “optionally substituted alkyl” refers to the alkyl may be substituted as well as where the alkyl is not substituted.

[0162] The term “modulate” as used herein includes the inhibition or suppression of a function or activity (such as cell proliferation) as well as the enhancement of a function or activity.

[0163] The phrase “pharmaceutically acceptable” is art-recognized. In certain embodiments, the term includes compositions, excipients, adjuvants, polymers and other materials and / or dosage forms which are, within the scope of sound medical judgment, suitable for use inAttorney Docket No.: UCH-31725

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[0165] contact with the tissues of human beings and animals without excessive toxicity, irritation, allergic response, or other problem or complication, commensurate with a reasonable benefit / risk ratio.

[0166] “Pharmaceutically acceptable salt” or “salt” is used herein to refer to an acid addition salt or a basic addition salt which is suitable for or compatible with the treatment of patients.

[0167] The term “pharmaceutically acceptable acid addition salt” as used herein means any non-toxic organic or inorganic salt of any base compounds. Illustrative inorganic acids which form suitable salts include hydrochloric, hydrobromic, sulfuric and phosphoric acids, as well as metal salts such as sodium monohydrogen orthophosphate and potassium hydrogen sulfate. Illustrative organic acids that form suitable salts include mono-, di-, and tricarboxylic acids such as glycolic, lactic, pyruvic, malonic, succinic, glutaric, fumaric, malic, tartaric, citric, ascorbic, maleic, benzoic, phenylacetic, cinnamic and salicylic acids, as well as sulfonic acids such as p-toluene sulfonic and methanesulfonic acids. Either the mono or di-acid salts can be formed, and such salts may exist in either a hydrated, solvated or substantially anhydrous form. In general, the acid addition salts of compounds are more soluble in water and various hydrophilic organic solvents, and generally demonstrate higher melting points in comparison to their free base forms. The selection of the appropriate salt will be known to one skilled in the art. Other non-pharmaceutically acceptable salts, e.g., oxalates, may be used, for example, in the isolation of compounds for laboratory use, or for subsequent conversion to a pharmaceutically acceptable acid addition salt.

[0168] The term “pharmaceutically acceptable basic addition salt” as used herein means any non-toxic organic or inorganic base addition salt of any acid compounds or any of their intermediates. Illustrative inorganic bases which form suitable salts include lithium, sodium, potassium, calcium, magnesium, or barium hydroxide. Illustrative organic bases which form suitable salts include aliphatic, alicyclic, or aromatic organic amines such as methylamine, trimethylamine and picoline or ammonia. The selection of the appropriate salt will be known to a person skilled in the art.

[0169] Many of the compounds useful in the methods and compositions of this disclosure have at least one stereogenic center in their structure. This stereogenic center may be present in an R or a S configuration, said R and S notation is used in correspondence with the rules described in Pure Appl. Chem. (1976), 45, 11-30. The disclosure contemplates all stereoisomeric forms such as enantiomeric and diastereoisomeric forms of the compounds,Attomey Docket No.: UCH-31725

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[0171] salts, prodrugs or mixtures thereof (including all possible mixtures of stereoisomers). See, e.g., WO 01 / 062726.

[0172] Some of the compounds may also exist in tautomeric forms. Such forms, although not explicitly indicated in the formulae described herein, are intended to be included within the scope of the present disclosure.

[0173] “Prodrug” or “pharmaceutically acceptable prodrug” refers to a compound that is metabolized, for example hydrolyzed or oxidized, in the host after administration to form the compound of the present disclosure. Typical examples of prodrugs include compounds that have biologically labile or cleavable (protecting) groups on a functional moiety of the active compound. Prodrugs include compounds that can be oxidized, reduced, aminated, deaminated, hydroxylated, dehydroxylated, hydrolyzed, dehydrolyzed, alkylated, dealkylated, acylated, deacylated, phosphorylated, or dephosphorylated to produce the active compound. Examples of prodrugs using ester or phosphoramidate as biologically labile or cleavable (protecting) groups are disclosed in U.S. Patents 6,875,751, 7,585,851, and 7,964,580, the disclosures of which are incorporated herein by reference. The prodrugs of this disclosure are metabolized to produce IPA or a salt thereof. The present disclosure includes within its scope, prodrugs of the compounds described herein. Conventional procedures for the selection and preparation of suitable prodrugs are described, for example, in “Design of Prodrugs” Ed. H. Bundgaard, Elsevier, 1985.

[0174] The phrase “pharmaceutically acceptable carrier” as used herein means a pharmaceutically acceptable material, composition or vehicle, such as a liquid or solid filter, diluent, excipient, solvent or encapsulating material useful for formulating a drug for medicinal or therapeutic use.

[0175] The term “Log of solubility”, “LogS” or “logS” as used herein is used in the art to quantify the aqueous solubility of a compound. The aqueous solubility of a compound significantly affects its absorption and distribution characteristics. A low solubility often goes along with a poor absorption. LogS value is a unit stripped logarithm (base 10) of the solubility measured in mol / liter.Attomey Docket No.: UCH-31725

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[0177] EXAMPLES

[0178] The invention now being generally described, it will be more readily understood by reference to the following examples which are included merely for purposes of illustration of certain aspects and embodiments of the present invention and are not intended to limit the invention.

[0179] Example 1: Indole-3-Propionic Acid Protects Against Heart Failure With Preserved Ejection Fraction in Mouse Model

[0180] HFpEF is a complex disorder with multiple comorbidities, including obesity, diabetes, and hypertension. It is highly heterogeneous and driven in large part by environmental factors, making human studies difficult.

[0181] To enable genetic and molecular analysis, the present disclosure uses a mouse model of the disorder developed by Schiattarella and colleagues (Schiattarella GG, Altamirano F, Tong D, French KM, Villalobos E, Kim SY, Luo X, Jiang N, May HI, Wang ZV, et al.

[0182] Nitrosative stress drives heart failure with preserved ejection fraction. Nature. 2019;568:351-356). It involves feeding mice a high-fat diet (HFD) and a nitric oxide synthase inhibitor.

[0183] The present disclosure demonstrates that reduced IPA levels are accompanied by metabolic remodeling, gut dysbiosis, increased oxidative stress, and inflammation in the heart of HFpEF mice. IPA feeding increased IPA levels in the heart and circulation and attenuated diastolic dysfunction induced by the HFpEF diet. IPA also mitigated gut microbiota dysbiosis and intestinal epithelial barrier damage, and improved metabolic homeostasis. In the hearts of HFpEF mice, IPA restored the expression of SIRT3 by aryl hydrocarbon receptor (AhR) binding, and it decreased inflammation and NNMT (nicotinamide N-methyltransferase) levels, increasing nicotinamide and NAD+ / NADH. Increased levels of both NAD+and SIRT3 are associated with improved diastolic function.

[0184] Additionally, two separate cohorts of human patients with HF with HFpEF were examined. Indeed, in both cohorts, the levels of IPA were significantly reduced in patients with HFpEF. Together, these data identify IPA as a critical regulator of diastolic dysfunction in HFpEF by enhancing the NAD salvage pathway. Therefore, IPA supplementation is an effective therapy for management of HFpEF.

[0185] Further details of the mouse model used and human cohorts analyzed in these examples are provided in Wang, et al. “Indole-3-Propionic Acid Protects Against Heart Failure with Preserved Ejection Fraction.” Circulation Research. (2024) 134:4, the entire contents of which are incorporated by reference.Attomey Docket No.: UCH-31725

[0186] UCLA Ref. No.: [UCLA 2022-142-3] WO

[0187] HFpEF Mice Exhibit Metabolic Remodeling Which Is Associated With Diastolic Dysfunction

[0188] To investigate the metabolic remodeling of HFpEF, the 2-hit HFpEF model in C57BL / 6J male and female mice was produced by the combination of HFD and inhibition of nitric oxide synthase using Nco-nitrol-L-arginine methyl ester (1-NAME). After 7 weeks feeding of HFD+l-NAME feeding, male mice exhibited HF phenotypes that recapitulate clinical symptoms of HFpEF, which include diastolic dysfunction (increased E / A ratio, and E / e’ ratio), cardiac hypertrophy, lung congestion, left ventricular (LV) mass / surface area (FIGs. 1A-H), exercise intolerance with reduced running distance and workload (FIG. II), and metabolic remodeling, including increased body fat accumulation, plasma cholesterol, glucose, and insulin levels, and homeostatic model assessment for insulin resistance (FIGs.

[0189] 1J-Q) Female mice also developed HF phenotypes that recapitulate clinical symptoms of HFpEF, including diastolic dysfunction, cardiac hypertrophy, lung congestion, LV mass, LV mass / surface area, and metabolic remodeling.

[0190] The HFpEF model was also developed in 30 genetically diverse inbred strains of male and female mice, a subset of the hybrid mouse diversity panel, to examine trait-trait correlations across genetic perturbations. In both sexes, the metabolic traits glucose tolerance, homeostatic model assessment for insulin resistance, plasma cholesterol levels, and insulin levels were positively correlated with the diastolic parameters E / e’ ratio and E / A ratio (FIGs.

[0191] 1R-1U). These results reveal a clear metabolic remodeling induced by the HFpEF diet and a positive association between metabolic and diastolic dysfunction.

[0192] Reduced Systemic IPA Levels in Mice With HFpEF

[0193] Metabolomics of heart tissue and plasma from C57BL / 6J male and female mice after 7 weeks of chow diet or HFD+l-NAME feeding was performed to determine the metabolic signatures of HFpEF. PCA plots revealed distinct clusters of samples from chow and HFpEF mice (FIG.2A). The intermediates in the main metabolic pathways, including glycolysis, tricarboxylic acid cycle, ketone bodies, and amino acid metabolism, were significantly differentiated in heart tissue and plasma samples from HFpEF mice relative to control mice (FIGs. 2B-2C). Notably, HFpEF mice showed a decrease of the metabolites involved in tryptophan metabolism in both male and female mice (FIGs.2B-2D). In male HFpEF mice, indole-3-propionic acid (IPA) was decreased by 4.7- and 3.8-fold in the heart tissue and circulation, respectively (FIGs.2E-2F). Moreover, plasma and cardiac IPA levels in the mice were inversely correlated with E / A ratio and E / e’ ratio but not with left ventricular ejectionAttorney Docket No.: UCH-31725

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[0195] fraction (LVEF; FIGs.2G-2I). Female mice fed with HFD+l-NAME exhibited similar metabolic changes relative to controls, and IPA was consistently decreased in the plasma and heart tissue. IPA levels were also significantly decreased in the liver and the white adipose tissue (WAT) in the mice fed with HFD+l-NAME relative to controls, indicating a systemic response to HFpEF.

[0196] IPA Attenuates Diastolic and Metabolic Dysfunction in HFpEF Model

[0197] Next, the effect of IPA on HFpEF was tested. C57BL / 6J male mice were fed with chow diet or HFD+l-NAME containing control or high IPA for 7 weeks (FIG.3A).

[0198] Compared with control HFpEF mice, IPA-fed mice exhibited about a 20-fold increase of IPA levels in both plasma and heart tissue and about 4-fold increase compared with mice fed a chow diet (FIGs.3B-3C). Seven weeks of HFD+l-NAME feeding induced an increase in body weight and fat mass while IPA supplementation decreased body weight gain and body fat accumulation (FIGs.3D-3E). IPA supplementation attenuated glucose intolerance and insulin resistance in HFpEF mice (FIGs.3F-3G). In addition, IPA reduced white and brown adipose tissue mass without affecting lean body mass (FIGs.3H-3I; FIG.9A), indicating improved body composition. Food intake was not changed by IPA feeding, indicating no difference in caloric intake. Consistent with improved glucose metabolism, IPA improved lipid homeostasis as assessed by reduced cholesterol and free fatty acids in both the circulation and the heart (FIGs.3J-3L; FIGs.9C-9D). Importantly, mice fed with IPA exhibited improved diastolic function as assessed by heart weight, E / A ratio, E / e’ ratio, LV mass / surface area, lung congestion, and exercise tolerance (FIG.3M-3R; FIGs.9E-L). In contrast, LVEF was unchanged by IPA or HFD + 1-NAME (FIG.9F), highlighting the specificity of the models to HFpEF. IPA significantly mitigated hypertrophy and fibrosis of the heart tissue induced by HFpEF (FIGs.9M-O).

[0199] The epithelial layer of cells isolated from the small intestine of mice fed on HFD+l-NAME containing either control or high IPA were analyzed using lOx Genomics Chromium droplet single-cell RNA sequencing. Transporters for key nutrient families (fat and cholesterol, amino acid, peptide, and carbohydrate) were examined in each cell of the enterocyte cluster. Modulated signature scores indicate that expression of the carbohydrate transporters and the peptide transporters decreased in the high IPA feeding group. Further assessment indicated that expression of UCP1 (uncoupling protein 1) and NDUFS1 (NADH: ubiquinone oxidoreductase core subunit SI) increased slightly in the brown adiposeAttomey Docket No.: UCH-31725

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[0201] tissue of mice fed with high-IPA diet. However, the core body temperature between control and high-IPA feeding mice remained the same.

[0202] Furthermore, impact of IPA on diastolic function in C57BL / 6J female mice was tested (FIG. 10A). Consistent with the phenotypes observed in male mice, female mice fed with IPA displayed reduced body weight gain and reduced fat mass gain when subjected to the HFpEF diet (FIGs. 10B-D). Also, IPA improved diastolic dysfunction in HFpEF mice, as assessed by reduced E / A ratio, E / e’ ratio, LV mass, heart weight, and lung weight (FIGs. 10E-J). Moreover, total adipose mass and plasma cholesterol were also reduced (FIG. 10K-N). Thus, IPA protected against diastolic and metabolic dysfunction in the HFpEF mouse model.

[0203] IPA Mitigates Gut Microbiota Dysbiosis and Intestinal Epithelial Barrier Damage IPA has been reported to protect against gut microbiota dysbiosis and intestinal epithelial barrier damage induced by a high-fat died (HFD). The gut microbiota and intestinal epithelium in the mice fed with chow diet, HFD+l-NAME, or HFD+l-NAME containing high IPA for 7 weeks were examined. Ceca were collected, and 16S rRNA-based gut microbial profiling was performed. HFpEF mice exhibited substantial gut microbiota dysbiosis including a decrease in Bacteroidetes and an increase in Firmicutes / Bacteroidetes ratios. IPA reduced Firmicutes abundance and restored Bacteroidetes abundance, thus reversing Firmicutes / Bacteroidetes ratio induced by HFD+l-NAME (FIGs. 11A-D).

[0204] In accordance with induced dysbiosis, the HFpEF model exhibited abnormal morphological alterations of small intestines including increased villus width and reduced villus length (FIGs. 11E-G). IPA restored villus width and length (FIGs. 11E-G), suggesting that IPA has a beneficial effect on epithelial homeostasis. Furthermore, the expression of occludin, a tight junction protein in the epithelium, was examined by immuno staining and immunoblotting. Compared with normal controls, occludin was decreased in the intestines of HFpEF mice, and IPA supplementation significantly upregulated occludin expression (FIGs.

[0205] 11H-K). Together, these results demonstrate that IPA protects against gut microbiota dysbiosis and intestinal epithelial barrier damage in HFpEF mice.

[0206] IPA Restores SIRT3 and the NAD Salvage Pathway in the Heart of HFpEF Mice Next, the metabolic effect of IPA supplementation in HFpEF mice was examined. IPA reduced branched-chain amino acids, riboflavin, and tryptophan in the heart (FIGs.4A-B; FIGs. 12A-B). In contrast, IPA augmented ketone body levels and showed a tendency toAttomey Docket No.: UCH-31725

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[0208] increase intermediates in the tricarboxylic acid cycle (FIGs.4A-B; FIGs. 12A-B). Notably, nicotinamide (NAM) levels were decreased in the heart of HFpEF mice relative to controls. This was significantly reversed by IPA (FIG.4C) such that NAM levels were positively correlated with IPA levels in the heart (FIG.4D). As a precursor of NAD+, NAM has a substantive beneficial effects on cardiometabolic diseases by mediating NAD bioavailability (FIG. 4E). Consistent with the level of NAM, reduced NAD+ / NADH level in the heart of HFpEF mice were observed, and IPA administration restored the NAD+ / NADH level (FIG.

[0209] 4F). In addition, IPA significantly reduced plasma lipopolysaccharide levels, inflammatory gene expression, and oxidative stress in the heart (FIGs.4G-4H; FIGs. 12C-12D).

[0210] Mitochondrial activity in HFpEF mice was next examined. The results indicate that IPA intervention tends to increase overall mitochondrial activity in the heart in mice (FIGs. 12E-J), which could contribute to improved diastolic dysfunction. Moreover, the sum of the 2 forms of NAD indicates that the global pool of NAD decreased in the heart of HFpEF mice (FIGs. 13A-C), and IPA supplementation increased the total NAD (FIGs. 13D-F).

[0211] The key mediators involved in the NAD-NAM circuit were then examined. IPA supplementation increased the expression of SIRT3 (sirtuin 3) and decreased NNMT in the heart (FIG.4I-4J), normalizing regulation of NAD-NAM circuit in the heart. In accordance with NNMT protein, NNMT activity was decreased by IPA in the heart tissue of HFpEF. In accordance with SIRT3 protein, lysine acetylation was increased in the heart tissue of HFpEF mice, and IPA decreased acetylation.

[0212] A careful assessment of the methyl-NAM and its downstream metabolites was then performed to achieve a fuller characterization of NAD+metabolome. The results showed that methyl-NAM increased in the heart of HFpEF mice. IPA supplementation decreased the methyl-NAM abundance in the heart, as well as in the liver and plasma. The abundance of its downstream metabolites 2-PY / 4-PY also decreased in the heart, liver, plasma, and urine of mice fed with a high-IPA diet, in accordance with the decrease of NNMT and methyl-NAM in mice fed with a high-IPA diet.

[0213] To analyze this pathway more thoroughly and achieve a broader understanding of the impact of IPA on NAD metabolism, the expression of key genes involved in NAD synthesis and NAD hydrolysis were examined. Several key genes involved in NAD synthesis, including Nmrk2, Nmnatl , and Nmnat2, were increased in the hearts of mice fed with high-IPA diet. The expression levels of Nampt and Nmnat2 were increased by IPA in the liver. The genes involved in NAD hydrolysis were also examined. The results indicated that theAttomey Docket No.: UCH-31725

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[0215] expression Parpl was increased by IPA in all organs examined, including the heart, liver, and WAT. The expression of Cd38 was also increased in the liver upon IPA feeding. These results show that IPA increases the total NAD pool and increases the expression of genes involved in both NAD synthesis and NAD hydrolysis at the same time.

[0216] The expression of SIRT3 and NNMT in the liver and the WAT was also examined. The results indicate that the NNMT levels in both the liver and WAT dramatically increased in the HFpEF mice compared with control mice and were decreased by IPA supplementation. The SIRT3 levels were decreased in the liver and WAT of HFpEF mice and were upregulated by IPA supplementation. Overall, these results indicate IPA has a significant impact on the NAD metabolism and that these biological effects of IPA are systemic.

[0217] IPA Promotes SIRT3 Expression Through the Ligand Binding of AhR in the Heart Two IPA receptors have been previously reported: the pregnane X receptor and the AhR. Only AhR was present in the heart as pregnane X receptor was not detected. IPA treatment increased AhR levels and some upstream regulators of AhR transcription increased upon IPA treatment.

[0218] In the classical mechanism of AhR activation as a transcription factor, ligand binding stimulates AhR nuclear translocation and dimerization with other proteins, resulting in the formation of unique protein complexes that bind to their DNA binding sites and stimulate gene expression. To investigate whether IPA binding could promote AhR nuclear translocation, AhR protein levels in the nuclear and cytosolic compartments, respectively, was assessed in HL-1 cells treated with control or IPA in the presence of phenylephrine (PE). AhR protein levels were dramatically increased in the nucleus and decreased in the cytosol. To examine the activation status of AhR, several well-defined AhR target genes of the CYP1A1 and CYP1B family were examined. The results show that both the expression of Cyplal and Cyplbl significantly increased in the heart of mice fed with a high-IPA diet compared with the controls. Similar levels of activation were observed in the liver and WAT. These results indicate that the high-IPA feeding group exhibits increased AhR activation status.

[0219] Next, it was examined whether AhR promotes SIRT3 expression as a transcription factor. AhR was inhibited using its antagonist CH- 223191 (“CH”) to test if it mediates the molecular regulation of IPA on cardiomyocytes (FIGs.4K-4M). A total of 6 groups, including control, control with IPA only, PE, PE with IPA only, PE with CH inhibitor, and PE with CH inhibitor plus IPA, were included in this study, and both protein levels andAttomey Docket No.: UCH-31725

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[0221] mRNA levels were examined. The results indicate that CH-223191 suppressed the upregulation of SIRT3 by IPA treatment, confirming that IPA promotes SIRT3 expression through its binding with AhR and subsequently nuclear translocation (FIG.4K-4M).

[0222] Moreover, the putative AhR-binding sites within the Sirt3 promoter region (TGCGTG) were identified. Subsequently, a ChIP assay was conducted to test the interaction between the AhR protein and the Sirt3 promoter. Our results indicate that AhR binds to promoter region of Sirt3. Collectively, these results demonstrate that IPA regulates Sirt3 through AhR.

[0223] In regard to the downregulation of cardiac NNMT by IPA, proinflammatory cytokines, such as IL-6 and TNF-a, elicit significant induction of NNMT expression. Since IPA has whole-body anti-inflammatory effects in multiple disease models, it was examined whether IPA could downregulate cardiac NNMT through an anti-inflammatory response. The expression of proinflammatory cytokine TNF-a is increased in the mice heart upon HFpEF diet feeding and IPA supplementation decreased TNF-a expression, in accordance with the expression of NNMT (FIGs.41-4 J). Also, IPA treatment in vitro of cardiomyocytes alone did not significantly decrease the NNMT levels (FIGs.4K-M). To determine whether proinflammatory cytokines could promote the expression of NNMT, NNMT expression in HL-1 cells incubated with different concentrations of TNF-a in the presence of PE was examined. The results show that TNF-a does induce the expression of NNMT in cardiomyocytes. Furthermore, it was examined whether IPA has a comprehensive impact on proinflammatory cytokines in HFpEF mice. The results indicate that IPA suppresses the expression of proinflammatory cytokines in the mice heart, such as TNF-a, IL-6, and IL-ip. These results demonstrate that IPA at least partially decreases the cardiac NNMT through the inhibition of proinflammatory cytokine expression.

[0224] Next, AhR siRNA knockdown was conducted to study the impact of IPA stimulation. The results showed that AhR knockdown suppressed the upregulation of SIRT3 by IPA treatment, indicating that IPA promotes SIRT3 expression through its binding with AhR. RT-qPCR analysis in all these 6 groups was performed, and the mRNA expression levels were in accordance with the protein levels (FIG. S17D through SUF). Furthermore, to test if SIRT3 mediates the effects of IPA in vivo, mRNA encoding SIRT3 in the hearts of C57BL / 6J male mice was knocked down using adeno-associated virus 9 delivery of shRNA (FIG.4N-P). Sirt3 knockdown diminished the effects of IPA on body mass and diastolic dysfunction in the HFpEF model (FIG.4Q-W), suggesting that SIRT3 plays a critical role in the protective effects of IPA on diastolic dysfunction.Attomey Docket No.: UCH-31725

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[0226] NAM Mitigates Diastolic Dysfunction in HFpEF

[0227] NAM was reported to protect against diastolic dysfunction in the models of obesity and HFpEF. C57BL / 6J male mice were fed with NAM to assess its effect on heart function. NAM feeding significantly increased heart NAM and NAD+ levels (FIGs. 5A-5B). The results indicate that NAM supplementation dramatically increased levels of methyl-NAM and its downstream metabolites 2-PY / 4-PY in the plasma, liver, heart, and urine. Surprisingly, NAM feeding reversed the Bacteroidetes and Firmicutes / Bacteroidetes ratio induced by HFpEF, in common with IPA feeding (FIGs.5C-F). The mice receiving NAM exhibited reduced body weight gain and fat accumulation with comparable food intake relative to controls in the progression to HFpEF (FIGs.5G-I). Accordingly, NAM improved glucose tolerance and plasma-free fatty acids (FIGs.5J-5K). Consistent with these results, mice receiving NAM exhibited attenuated diastolic dysfunction and cardiac hypertrophy as assessed by mitigated E / A ratio, E / e’ ratio, LV mass / surface area, lung congestion, and improved exercise tolerance (FIGs.5L-5Q). Further, mice were fed with the combination of IPA and NAM, together with HFD+l-NAME. The combination of IPA and NAM mitigated diastolic dysfunction and metabolic disorders to a similar extent as IPA or NAM treatment alone, suggesting they target the same pathway affecting diastolic function, although this does not eliminate the possibility that other pathways are involved (FIG.5R-X).

[0228] Furthermore, HFD+l-NAME feeding increased the systolic BP and diastolic BP in the mice, and high NAM supplementation decreased systolic BP in mice, comparable with high-IPA supplementation.

[0229] Nnmt Knockdown Mitigates Diastolic Dysfunction in HFpEF

[0230] To further test if reducing NAM breakdown protects against diastolic dysfunction in HFpEF, mRNA encoding NNMT, the enzyme that catalyzes the N-methylation of NAM, was knocked down with an antisense oligonucleotide (ASO). Nnmt ASO or control ASO were injected weekly into C57BL / 6J male mice maintained on HFD+l-NAME for 7

[0231] weeks. Nnmt ASO dramatically decreased heart NNMT expression (FIG.6A). Consistently, mice receiving Nnmt ASO exhibited decreased body weight gain and fat mass accumulation with no changes in lean mass or food intake relative to those receiving control ASO (FIG. 6B-6D). The Nnmt ASO improved glucose homeostasis and insulin resistance, in common with IPA feeding (FIG.6E-6F). In addition, plasma insulin, homeostatic model assessment for insulin resistance, plasma triglycerides, total cholesterol, and unesterified cholesterol were consistently decreased in the mice receiving the Nnmt ASO (FIG.6G through 6K).Attomey Docket No.: UCH-31725

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[0233] Consistent with the effects of NAM feeding, the Nnmt ASO significantly improved diastolic function as measured by improved exercise tolerance, reduced E / A ratio, E / e’ ratio, LV mass / surface area, and lung congestion compared with control ASO (FIG.6L-6Q). These results collectively demonstrate the protective effect of Nnmt knockdown in the HFpEF mouse model.

[0234] Additionally, Nnmt was overexpressed in the model of HFpEF to test if it mediates the effects of IPA on diastolic function. Nnmt overexpression using adeno-associated virus 9-cTnT-A z / dramatically increased heart NNMT expression (FIG.6R). Nnmt overexpression in the heart diminished the effects of IPA on body mass and diastolic dysfunction in HFpEF (FIGs. 6S-Y), demonstrating that IPA protects against diastolic dysfunction through the NAD-NAM circuit.

[0235] Therapeutic Effects of IPA in HFpEF

[0236] To test if IPA is beneficial after diastolic dysfunction has developed, C57BE / 6J male mice were fed with chow diet or HFD+l-NAME to induce HFpEF phenotypes, followed by IPA supplementation (FIG.7A). Four weeks of HFD+l-NAME induced HFpEF phenotypes as confirmed by increased body weight, plasma cholesterol, E / A ratio, and E / e’ ratio (FIG.

[0237] 1; FIGs.7B-K). The mice fed with HFD+l-NAME diet were then fed with HFD+l-NAME containing control or high IPA for another 5 weeks. Mice fed with IPA exhibited significantly lower body weight, adipose tissue weight, and metabolic remodeling compared with the mice from control group (FIG.7B-H). In addition, IPA significantly blunted the progression of diastolic dysfunction, as measured by lower E / A ratio, E / e’ ratio, heart weight, EV mass / surface area, preserved EVEF, lung congestion, and increased exercise tolerance (FIG. 7I-Q). These observations support the therapeutic effect of IPA in the HFpEF model.

[0238] IPA Levels Are Reduced in Patients With HFpEF

[0239] To determine the clinical relevance of IPA, plasma IPA levels were quantified in Cleveland Clinic participants without HF (n=48) and ambulatory patients with chronic HFpEF (n=48) using liquid chromatography-mass spectrometry / MS analysis. Chronic HFpEF subjects were clinically diagnosed with HF for at least 6 months before enrollment with a EVEF >50%. In this human HFpEF cohort with matched age and sex, IPA levels were significantly reduced in patients with HFpEF compared with non-HF controls (FIG.8A). Elevated IPA levels were inversely associated with HFpEF independent of age, sex, diabetes, and hypercholesterolemia (fourth quartile; unadjusted odds ratio [OR], 0.30 [95% CI, 0.088-Attomey Docket No.: UCH-31725

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[0241] 0.96]; P<0.05; adjusted OR [model 1], 0.28 [95% CI, 0.078-0.92]; P<0.05]. When BMI and hypertension were included in the multivariable logistic regression model (adjusted model 2), the inverse association between IPA levels and HFpEF was attenuated (adjusted OR [model 2], 0.33 [95% CI, 0.085-1.16]; P=0.09; FIG.8B). When IPA was treated as a continuous variable in the logistic regression models, similar results were observed (OR for HFpEF per interquartile range increase of IPA; unadjusted OR, 0.41 [95% CI, 0.16-0.93]; P<0.05; adjusted OR [model 1], 0.39 [95% CI, 0.15-0.90]; P<0.05; adjusted OR [model 2], 0.48

[0242] [95% CI, 0.18-1.18]; P=0.13).

[0243] In a separate human cohort from Alfred Hospital at Melbourne, patients with HFpEF (n=22) were collected for investigation of symptoms consistent with a diagnosis of HF (NYHA II-III) in the presence of a LVEF>50%, and a HFrEF cohort (n=20) had LVEF<40% (FIG. 8C). The diagnosis of HFpEF was confirmed by the presence of an end-expiratory pulmonary capillary wedge pressure >15 mm Hg at rest or >25 mm Hg during symptomlimited exercise. In this cohort, arterial IPA abundance was significantly decreased in HFpEF patients relative to non-HF controls (FIG.8D). Their results collectively reveal a significant reduction of IPA in patients with HFpEF, consistent with the results in the mouse model. Therefore, the present disclosure demonstrates that IPA reduces the signs and symptoms of HFpEF.

[0244] HFpEF accounts for half of all HF worldwide, conferring substantial morbidity and mortality. Compared with patients with HFrEF, patients with HFpEF display a metabolic profile characterized by insulin resistance, inflammation, oxidative stress, and impaired lipid metabolism. Using the HFpEF mouse model, both males and females of 30 diverse inbred strains of mice were examined, a subset of the hybrid mouse diversity panel, confirming the association between diastolic and metabolic dysfunction. Tryptophan metabolism was reduced in HFpEF mice, and IPA levels were decreased in the heart and circulation. IPA supplementation protected against diastolic dysfunction in the progression of HFpEF and therapeutic management after the development of HFpEF phenotypes.

[0245] Mechanistically, IPA exerts both direct and indirect impacts on the heart. Specifically, IPA enhances intestinal barrier function, reduces body weight, and promotes glucose and lipid homeostasis. In the context of the heart, IPA directly restored the NAD salvage pathway through activation of the AhR and expression of SIRT3, thereby improving metabolic and diastolic dysfunction associated with HFpEF.Attomey Docket No.: UCH-31725

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[0247] Further, two separate cohorts of human patients with HF with HFpEF were examined. Indeed, in both cohorts, the levels of IPA were significantly reduced in patients with HFpEF, suggesting that IPA levels exert a major impact on the disease (FIG.8E). Moreover, IPA ameliorates inflammation and cell oxidative damage. Elevated concentrations of IPA in human blood plasma are correlated with a lower risk of type 2 diabetes and reduced proportions of IPA-producing bacteria are found in patients with type 2 diabetes.

[0248] IPA appears to be acting in the heart by at least 2 pathways. One of these is mediated by AhR binding and subsequently nuclear translocation to activate the SIRT3 expression. On the other hand, NNMT suppression by IPA appears to be secondary to suppression of inflammation. The evidence indicating the involvement of the NAD salvage pathway (from NAM to NMN to NAD) is strong. Administration of IPA restored heart NAM, NAD+ / NADH levels, and the expression of SIRT3. Mechanistically, IPA reduces NAM catabolism and elevates NAD+levels in the heart. In addition to the effects on NAM and NAD+levels, IPA reduced intestinal dysbiosis, contributing to improved diastolic and metabolic functions in HFpEF mice.

[0249] Experimental animals

[0250] All animal experiments were approved by the University of California Los Angeles (UCLA) Animal Care and Use Committee, in accordance with Public Health Service guidelines. All mice were purchased from the Jackson Laboratory. Mice were maintained on a 12-h light / dark cycle from 6 am to 6 pm. 8 weeks old mice were fed with chow diet or HFD (Research Diet, Cat# D12492). Nco-Nitro-L-arginine methyl ester hydrochloride (1-NAME) (Sigma, Cat# N5751-25G) was dissolved in drinking water (0.5 g / L, pH = 7.4). HFpEF was induced by HFD + 1-NAME feeding for 7 weeks. NAM was delivered in drinking water (550mg / kg / day). IPA was delivered in food (562.5 mg IPA / kg diet). Nnmt ASO and CON ASO (50mg / kg / week) were obtained from lonis Pharmaceuticals (oligo number 407074 or 407019 for Nnmt ASO; oligo number 141923 for CON ASO) and intraperitoneally injected weekly during the feeding of HFD + 1-NAME.

[0251] Body composition was determined using nuclear magnetic resonance (NMR, Bruker Minispec). Echocardiography was performed at baseline, 4 weeks and 7 weeks after the indicated diet to assess cardiac function. Upon sacrifice, tissues were weighed and instantly frozen in liquid nitrogen. Blood was collected in a BD Microtainer (Tubes with K2EDTA,Attomey Docket No.: UCH-31725

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[0253] Cat# 365974) and plasma was collected after 10,000 rpm for 5 min at 4°C. Dry lung weight was determined after 48h of incubation in an oven at 37 °C.

[0254] Human Studies

[0255] The Cleveland Clinic GeneBank cohort was generated from patients undergoing elective diagnostic coronary angiography with extensive clinical and laboratory characterization and longitudinal observation. The study was approved by the Institutional Review Board of the Cleveland Clinic and all participants gave written informed consent. Cell Culture

[0256] Neonatal rat ventricular myocytes (NRVMs) were isolated 2-day-old Wistar rat pups (Harlan Laboratories) using enzymatic digestion method. Briefly, rat left ventricles were isolated in ADS buffer (6.8g NaCl, 4.76g HEPES, 0.12g NaH2PO2, 1g glucose, 0.4g KC1 and 0.1g MgSO4 in IL ddH2O) and digested with collagenase (Worthington, Lakewood, NJ, Cat# LS004177), and the resulting cell slurry was fractionated on a discontinuous Percoll gradient (GE Healthcare) by centrifugation at 3000 rpm for 30 min at room temperature. The myocyte-rich fraction was isolated, washed with ADS buffer and plated in Dulbecco’s modified Eagle’s medium (DMEM; Gibco) supplemented with 5% horse serum, 15 mM HEPES and 1% penicillin / streptomycin. Cells were plated at 1 million / well in 6-well plate.

[0257] 24 hours after resting in plating medium, the NRVMs were then changed to serum-free medium and treated with 100 pM phenylephrine (PE, Sigma Cat# P6126-10G) or IPA for 48 hours.

[0258] Transthoracic Echocardiography

[0259] Trans-thoracic echocardiography was conducted with Vevo 2100 high-frequency, high-resolution digital imaging system (VisualSonics) equipped with a MS400 MicroScan Transducer. The mice were anesthetized and maintained with 1-2% isoflurane in 95% oxygen. A parasternal short axis view as indicated by the presence of papillary muscles was used to obtain M-mode images for analysis of fractional shortening, ejection fraction, and other cardiac functional parameters. Apical four-chamber view was used to obtain tissue Doppler imaging (TDI) mode and Pulse-wave Doppler (PWD) mode for analysis of myocardial velocity and blood flow velocity, respectively. All parameters were measured at least three times. After the test all mice recovered from anesthesia without difficulties and were returned to original cages immediately.Attorney Docket No.: UCH-31725

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[0261] Metabolic Cage

[0262] Indirect calorimetry was performed with a Columbus Instruments Comprehensive Lab Animal Monitoring System (Columbus Instruments). C57BL / 6J male mice (8 weeks old) were fed with HFD + 1-NAME diet containing control or high IPA (562.5 mg IPA / kg diet) for 7 weeks. After feeding, animals were placed individually in metabolic chambers at ambient temperature (26.5 °C) for 3 consecutive days with 12 h light / dark cycles. Animals had free access to food and water. After initial 7-9 h acclimation period, respiratory was measured in 20 min intervals. Energy expenditure (EE) was calculated from VO2 and RER (respiratory exchange ratio) using the Lusk equation, EE in Kcal / hr = (3.815 + 1.232 x RER) x VO2 in ml / min. Results were normalized to lean mass.

[0263] Exercise Exhaustion Test

[0264] After three days of acclimatization to treadmill, an exhaustion test was performed in the mice to determine exercise tolerance. Mice ran on the treadmill (20°) at warming-up speed of 5 m / min and the speed was increased gradually to 18 m / min. The speed was kept at 18 m / min until the mice were exhausted. Exhaustion was defined as the inability of the mice to return to running within 10 s of direct contact with an electric- stimulus grid. Running time was recorded and running distance was calculated. Anesthesia was not required and mice were returned to original cages immediately after the test.

[0265] Food Intake

[0266] After 5 weeks of HFD + 1-NAME feeding, cage averaged daily food intake was calculated by subtracting remaining food from the initially premeasured food for 3 consecutive days.

[0267] RNA Extraction and Reverse Transcription

[0268] Upon sacrifice, tissues were instantly frozen in liquid nitrogen. Cells or frozen tissue were homogenized in QIAzol Lysis Reagent (Qiagen, ImL per 20 mg tissue) and mixed with chloroform (200 pL per 1 mL QIAzol) for phase separation. RNA extraction was carried out using miRNeasy Mini Kit (Qiagen, Cat# 217004) as recommended by the manufacturer. Total RNA was eluted in ~50 pL RNase-free water and assessed for purity. RNA concentration was examined using a Nanodrop ND- 100 Spectrophotometer and 2 pg of total RNA per sample was reverse transcribed using a MultiScribe Reverse Transcriptase kit (Applied Biosystems, Cat# 4311235) with random primers. Reverse-transcribed cDNA was then diluted in water (lOx dilution) for qPCR analysis.Attomey Docket No.: UCH-31725

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[0270] Quantitative PCR

[0271] Quantitative PCR was carried out using a PowerTrack SYBR Green Master Mix (100029284). Samples were run on a LightCycler 480 II (Roche) and analyzed with the Roche LightCycler 1.5.0 Software. qPCR targets were normalized to actin and quantified using the delta Ct method. All qPCR primer sequences were obtained from PrimerBank.

[0272] Western Blotting

[0273] Heart tissues were lysed in whole cell extraction buffer containing protease and phosphatase inhibitor (Fisher, Cat# 78441). Protein content was measured using BCA protein assay kit (Pierce) and samples were denatured in 4x LDS loading buffer (Life Technologies) at 99°C for 5 min. Samples were loaded into 4%-12% Bis-Tris gels (Invitrogen) and separated out at 80 volts. Protein was then transferred to PVDF membranes (Immobilon) for 2 hours at 100 volts. Following transfer, membranes were blocked in 5% milk (Gibco) in TBST for 1 hour at room temperature. Membranes were then placed in primary antibodies on a shaker overnight at 4 C. Primary antibodies were used as follows: rabbit Occludin (Abeam, Cat# abl67161, 1:1000), rabbit ACTIN (Cell Signaling, Cat# 4967S, 1:2000). The following day, membranes were washed in TBST then placed in secondary antibodies (1:5000) for 1.5 hours at room temperature. Blots were then washed in TBST and placed in Amersham ECL detection solution (GE health sciences). Blots were imaged using IMAGER and bands were quantified using ImageJ software.

[0274] Intraperitoneal Glucose Tolerance Test

[0275] Intraperitoneal glucose tolerance tests (i.p. GTT) were performed by injection of glucose (2g / kg body weight in sterile saline) after 16-h fasting. Tail blood glucose levels (mg / dL) were measured with a glucometer before (0 min) and at 15, 30, 60, and 120 min after glucose administration. Anesthesia was not required and mice were returned to original cages immediately after the test.

[0276] Intraperitoneal Insulin-Tolerance Test

[0277] Intraperitoneal insulin tolerance tests (i.p. ITT) were performed by injection of insulin (1 U / kg body weight, Humulin R) after 4-h fasting. Tail blood glucose levels (mg / dL) were measured with a glucometer before (0 min) and at 15, 30, 60, and 120 min after glucose administration. Anesthesia was not required and mice were returned to original cages immediately after the test.Attomey Docket No.: UCH-31725

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[0279] Plasma and Heart Lipid Measurement

[0280] Plasma lipids were measured as described previously. Mice were fasted for 4 hours prior to retroorbital bleeding using isoflurane. Plasma cholesterol, triglyceride concentrations were determined by enzymatic procedures using a Biomek 1000 Automated Laboratory Workstation (Beckman). Plasma insulin was measured with Alpco Diagnostics Mouse ultrasensitive ELISA kit and glucose was measured with Stanbio Laboratory procedure (Cat# 1070). Free fatty acids were measured with Wako Diagnostics HR series NEFA-HR (2) kit. Samples were measured at a wavelength of 490 nm with a Vmax Microplate Reader (Molecular Devices, Inc.) in triplicate.

[0281] For heart lipid measurement, about 50mg heart tissue was homogenized in methanol and chloroform was added into each tube (Fisher, Cat# 14-959-35AA) overnight at 4°C. The next day, solution was filtered with sharkskin filter paper (Fisher, Cat# 09-0924-389, 110 mm) and 0.043% Magnesium Chloride was added to each tube. Then the tubes were shaken and centrifuged at 1800rpm for 15min at 4°C (Beckman J6-B Type centrifuge). After aspirating off all supernatant and white precipitate, resulting solution was dried with stream of nitrogen gas and re-suspended in 1% TritonX-100 in chloroform. After vortex, samples were dried again with stream of nitrogen gas and re- suspended with 1.8% TritonX-100 in water. After 15min of incubation at 65°C, lipids were measured with the same methods as plasma samples. Results were normalized to tissue weight.

[0282] Protein Carbonyl Content Assay

[0283] Protein Carbonyl Content was measured using Protein Carbonyl Content Assay Kit (Abeam, Cat# abl26287). Heart tissue was homogenized in dH2O and 100 pl of sample containing 1 mg protein was used in the assay. 100 pl DNPH was added to each sample and incubated for 10 min at room temperature. Then 30 pl of TCA was added to each sample and centrifuged at maximum speed for 2 min. After removing the supernatant, the pellet was washed with 500 pl of cold acetone. After centrifuge, the pellet was resolubilized with 200 pl of Guanidine solution. 100 pl of each sample was transferred to the 96-well plate and measured OD at -375 nm in a microplate reader. The carbonyl content was determined as follows: Carbonyl content = [(OD 375 nm) / 6.364) x (100)] nmol / well.

[0284] Targeted metabolomics

[0285] After 4 hours of fasting, mice were euthanized with isoflurane and blood was collected immediately by retroorbital bleeding in a BD Microtainer (Tubes with K2EDTA, Cat# 365974). Plasma was collected after 10,000 rpm for 5 min at 4°C. Heart tissues wereAttomey Docket No.: UCH-31725

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[0287] frozen in liquid nitrogen until analysis. Metabolites were extracted using methanol / chloroform / water method. Metabolites were determined by liquid chromatography -tandem mass spectrometry (LC-MS / MS) analysis. Targeted metabolite profiling was established using reference standards to determine MS multiple reaction-monitoring transitions, declustering potentials, collision energies, and chromatographic retention time. LC-MS / MS system composed of a Shimadzu Nexera LC-30AD UHPLC (Shimadzu Corporation, Kyoto, Japan) system coupled to a QTRAP6500 mass spectrometer (AB Sciex, Foster City, CA) was used to measure hydrophilic metabolites in positive and negative ionization modes. The polar metabolites in both positive and negative ionization mode were separated in hydrophilic interaction liquid chromatography (HILIC) mode using an Atlantis® HILIC column (Waters) and a XBridgeTM Amide column (Waters), respectively, which allow the separation of metabolites of different properties. Samples were analyzed in a randomized order in duplicate. Data was acquired in the same batch on the same day. MRM Q1 / Q3 peak integration of the raw data files was analyzed with software Sciex OS 2.0 (AB Sciex). The abundance of each metabolite was indicated by the peak area and normalized to the bookended pooled tissue extracts.

[0288] Intracellular NAD NADH Assay

[0289] Intracellular NAD+ / NADH levels were determined with the EnzyChrom™ NAD+ / NADH assay kit (BioAssay Systems, Cat# E2ND-100) following the manufacturer's instructions. About 105cells per sample were washed with cold PBS and cell pellets were stored at -80°C until analysis. Cell pellets were resuspended with either NAD+extraction buffer for NAD+determination or NADH extraction buffer for NADH determination. After heat at 60°C, assay buffer and opposite buffer were added to neutralize the extracts. Then the samples were centrifuged at 14,000 rpm for 5 min. The supernatant was used for NAD+and NADH assays. The concentrations of NAD+and NADH in each extract were then quantified based on a lactate dehydrogenase cycling reaction, in which the formed NADH reduces a formazan (MTT) reagent. The samples and NAD standards were transferred into a 96-well plate in duplicate. Working reagents were added quickly and measured at optical density 565 nm. The intensity of the reduced product color was proportional to the NAD+ / NADH concentration in the sample.

[0290] Histology

[0291] After mice sacrifice, intestines were harvested and fixed in 4% paraformaldehyde overnight and processed for routine paraffin histology. 5- pm sections were stained withAttomey Docket No.: UCH-31725

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[0293] hematoxylin and eosin (H&E). Villus width and length were assessed with ImageJ software. Sections were incubated with first antibody against Occludin (1:100; Thermo Fisher, Cat# 40-4700) overnight and then incubated with anti-Rabbit secondary antibody. Images were obtained using Leica Aperio ImageScope.

[0294] Gut Microbiota Analysis

[0295] Cecum samples were collected and snap frozen in liquid nitrogen immediately after mice sacrifice. Genomic DNA was extracted using the ZymoBIOMICS DNA Microprep Kit (Zymo Research, USA) according to the manufacturer’s protocol. The V4 region of the 16S ribosomal RNA gene was amplified by PCR and underwent paired-end sequencing on an Illumina HiSeq (Illumina, San Diego, USA). Sequence depths are about 50,000 sequences per sample. The sequences were processed using the DADA2 pipeline in R which assigns taxonomy with the SILVA 132 database. The data were incorporated into QIIME 2 version 2019.10 after processing in R.Attomey Docket No.: UCH-31725

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[0297] INCORPORATION BY REFERENCE

[0298] All publications and patents mentioned herein are hereby incorporated by reference in their entirety as if each individual publication or patent was specifically and individually indicated to be incorporated by reference. In case of conflict, the present application, including any definitions herein, will control.

[0299] EQUIVALENTS

[0300] While specific embodiments of the subject invention have been discussed, the above specification is illustrative and not restrictive. Many variations of the invention will become apparent to those skilled in the art upon review of this specification and the claims below. The full scope of the invention should be determined by reference to the claims, along with their full scope of equivalents, and the specification, along with such variations.

Claims

Attomey Docket No.: UCH-31725UCLA Ref. No.: [UCLA 2022-142-3] WOCLAIMSWe claim:

1. A method of treating a heart disease, comprising administering a composition comprising 3-indolepropionic acid (IPA) or a pharmaceutically acceptable salt or prodrug thereof to a subject in need thereof.

2. The method of claim 1, wherein the heart disease is heart failure.

3. A method of treating diastolic dysfunction, comprising administering a composition comprising 3-indolepropionic acid (IPA) or a pharmaceutically acceptable salt or prodrug thereof to a subject in need thereof.

4. The method of claim 3, wherein the subject has heart disease.

5. The method of any one of claims 1-2 and 4, wherein the heart disease is Heart Failure with preserved Ejection Fraction (HFpEF).

6. The method of any one of claims 1-5, wherein the composition comprises IPA or a pharmaceutically acceptable salt or prodrug thereof.

7. The method of any one of claims 1-6, wherein the composition comprises IPA or a pharmaceutically acceptable salt thereof.

8. The method of any one of claims 1-7, wherein the composition comprises tryptophan.

9. The method of any one of claims 1-8, wherein the composition comprises nicotinamide.

10. The method of any one of claims 1-9, wherein the composition is administered for between about 1 and 140 days.

11. The method of any one of claims 1-10, wherein the composition is administered for at least about 20 days.

12. The method of any one of claims 1-11, wherein the composition is administered for at least about 40 days.Attomey Docket No.: UCH-31725UCLA Ref. No.: [UCLA 2022-142-3] WO13. The method of any one of claims 1-12, wherein the composition is administered for at least about 60 days.

14. The method of any one of claims 1-13, wherein the composition is administered for at least about 80 days.

15. The method of any one of claims 1-14, wherein the composition is administered for at least about 100 days.

16. The method of any one of claims 1-15, wherein the composition is administered for at least about 120 days.

17. The method of any one of claims 1-16, wherein the composition is administered for at least about 140 days.

18. The method of any one of claims 1-17, wherein the subject has insulin resistance.

19. The method of any one of claims 1-18, wherein the subject has diabetes.

20. The method of any one of claims 1-19, wherein the subject has gut microbiota dysbiosis.

21. The method of any one of claims 1-20, wherein the composition is administered orally.

22. The method of any one of claims 1-21, wherein the composition further comprises a pharmaceutically acceptable excipient.

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