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WO2024182489A1 - Compositions et leurs procédés d'utilisation - Google Patents

Compositions et leurs procédés d'utilisation Download PDF

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Publication number
WO2024182489A1
WO2024182489A1 PCT/US2024/017626 US2024017626W WO2024182489A1 WO 2024182489 A1 WO2024182489 A1 WO 2024182489A1 US 2024017626 W US2024017626 W US 2024017626W WO 2024182489 A1 WO2024182489 A1 WO 2024182489A1
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WO
WIPO (PCT)
Prior art keywords
oxalate
panel
disease
nash
agxt
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Ceased
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PCT/US2024/017626
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English (en)
Inventor
Oren ROM
Mónica Díaz Gavilán
José Antonio GÓMEZ VIDAL
Fabio ARIAS BORDAJANDI
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Granada, University of
Louisiana State University
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Granada, University of
Louisiana State University
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Priority to IL323014A priority Critical patent/IL323014A/en
Publication of WO2024182489A1 publication Critical patent/WO2024182489A1/fr
Anticipated expiration legal-status Critical
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/335—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin
    • A61K31/34—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin having five-membered rings with one oxygen as the only ring hetero atom, e.g. isosorbide
    • A61K31/341—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin having five-membered rings with one oxygen as the only ring hetero atom, e.g. isosorbide not condensed with another ring, e.g. ranitidine, furosemide, bufetolol, muscarine
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/12—Ketones
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/335—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin
    • A61K31/35—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin having six-membered rings with one oxygen as the only ring hetero atom
    • A61K31/352—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin having six-membered rings with one oxygen as the only ring hetero atom condensed with carbocyclic rings, e.g. methantheline 
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/335—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin
    • A61K31/35—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin having six-membered rings with one oxygen as the only ring hetero atom
    • A61K31/352—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin having six-membered rings with one oxygen as the only ring hetero atom condensed with carbocyclic rings, e.g. methantheline 
    • A61K31/353—3,4-Dihydrobenzopyrans, e.g. chroman, catechin

Definitions

  • the present invention relates to compositions and methods of treating disease.
  • NASH nonalcoholic fatty liver disease
  • CVD atherosclerotic cardiovascular disease
  • R 1 is a hydrogen, -Ph, a furan, a thiophene, a pyridine, a halogen, -CF 3 , -CN, -N(R 4 )(R 5 ), -OR 4 , -SR 4 , -CH 2 -NH-(CH 2 )n-N(R 4 )(R 5 ), -COOH, -COOR 4 , i Pr, t Bu, -(CH 2 ) n CH 3 , -(CH 2 ) n i Pr, -(CH 2 ) n t Bu, -(CH 2 ) n OR 4 , -(CH 2 ) n N(R 4 )(R 5 ), -(CH 2 ) n COOR
  • the halogen is fluorine, chlorine, bromine, or iodine.
  • the compound is:
  • the compound is:
  • compositions comprising a compound described herein, or a combination thereof, and a pharmaceutically acceptable carrier, excipient, or diluent.
  • R1 is a hydrogen, -Ph, a furan, a thiophene, a pyridine, a halogen, -CF3, -CN, -N(R4)(R5), -OR4, -SR4, -CH2-NH-(CH2)n-N(R4)(R5), -COOH, -COOR4, i Pr, t Bu, -(CH2)nCH3, Docket No.: 2932719-000218-WO1 Date of Filing: February 28, 2024 -(CH2)n i Pr,
  • n is 0-10.
  • R3 is -COOH, -COOR6, or -NO2.
  • R4 is hydrogen, acyclic alkyl, C1-C9 alkyl, or aryl.
  • R5 is hydrogen, acyclic alkyl, C1-C9 alkyl, or aryl.
  • R6 is -CH3 or -CH2CH3.
  • the halogen is fluorine, chlorine, bromine, or iodine.
  • the compound is a salicylic acid derivative of Formula (II):
  • the oxalate production-related disease comprises a cardiometabolic disease, a cardiovascular disease, a metabolic disease, a liver disease, a renal disease, or a combination thereof.
  • the oxalate production-related disease comprises a GO-associated disease and/or an LDHA-associated disease.
  • the cardiometabolic disease, cardiovascular disease, metabolic disease, liver disease, renal disease, or combination thereof comprises heart failure, myocardial infarction, stroke, diabetes, dyslipidemia, hyperoxaluria, primary hyperoxaluria (PH), hypertension, obesity, hepatitis, cirrhosis, hepatocellular carcinoma, chronic kidney disease, nonalcoholic steatohepatitis (NASH), nonalcoholic fatty liver disease (NAFLD), atherosclerotic cardiovascular disease (ASCVD), or a combination thereof.
  • the therapeutically effective amount of the composition inhibits GO activity, LDHA activity, or a combination thereof. In embodiments, the therapeutically effective amount comprises less than about 0.
  • measuring the circulating oxalate levels of a subject comprises an enzymatic assay, mass spectrometry, or a combination thereof.
  • the subject’s circulating oxalate level is at least about 1 uM, about 1 uM, about 1.5 uM, about 2 uM, about 2.5 uM, about 3 uM, about 4 uM, about 5 uM, about 6 uM, about 7 uM, about 8 uM, about 9 uM, about 10 uM, or greater than about 10 uM.
  • FIG. 1 shows non-limiting, exemplary experimental results. Suppressed AGXT and increased oxalate in livers from humans and mi ce with NASH and in lipid-loaded hepaiocy les.
  • Panel B H&E and Picrosirius Red staining of liver samples from patients with NASH compared with disease-free and non-malignant liver samples (non-NASH).
  • Panel H Liver samples were collected from C57BL/6J mice fed a standard chow diet (control) or a high-fat, high-fructose, high-cholesterol diet (NASH diet) for 6 months, and Panel I) stained with H&E and Picrosirius Red.
  • Panel N Primary hepatocytes from mice fed a standard chow diet and HepG2 cells were treated with either BSA control or BSA-conjugated palmitic acid (200 ⁇ M) Docket No.: 2932719-000218-WO1 Date of Filing: February 28, 2024 overnight.
  • FIG. 2 shows non-limiting, exemplary experimental results. Oxalate reduction via AGXT overexpression ameliorates NASH.
  • FIG. 3 shows non-limiting, exemplary experimental results. Oxalate reduction via AGXT overexpression lowers hepatic steatosis through induction of fatty acid ⁇ -oxidation pathways. Mice were injected with AAV8-GFP or AAV8-AGXT (2x10 11 viral genomes per Docket No.: 2932719-000218-WO1 Date of Filing: February 28, 2024 mouse) and placed on the NASH diet for 6 months prior to tissue and plasma analysis.
  • Panel B Volcano plots of differentially expressed genes (DEGs) based on RNA-sequencing comparing livers from mice treated with AAV8-GFP or AAV8-AGXT (downregulated, blue and upregulated, red).
  • Panel C Pathways enriched in the upregulated DEGs based on KEGG pathway analysis comparing livers from mice treated with AAV8-GFP or AAV8-AGXT.
  • Panel G Percent-positive ORO area.
  • Panel K HepG2 cells were transfected with either GFP control (GFP) or GFP-tagged AGXT (AGXT) plasmids. Western blot analysis for AGXT protein abundance 48 h post-transfection.
  • GFP GFP control
  • AGXT GFP-tagged AGXT
  • GFP GFP control
  • AGXT AGXT
  • each point represents an individual mouse.
  • each point represents an independent experiment that included at least 2 biological repetitions. All data are expressed as mean ⁇ SEM.
  • Statistical comparisons were made using unpaired t-test (Panels B-E, L, N, O), two-way ANOVA with Bonferroni’s multiple comparisons test (Panels F, H), or one-way ANOVA with Tukey’s multiple comparisons test (G, J). Seahorse analysis and statistical comparisons for Panel I) and Panel P) are shown in Fig. 13 Panel E and Fig.14 Panel B, respectively. All individual points and Docket No.: 2932719-000218-WO1 Date of Filing: February 28, 2024 p values are shown.
  • Panel E Percent-positive F4/80 area.
  • FIG. 6 shows non-limiting, exemplary experimental results. Oxalate reduction via AGXT overexpression lowers hepatic fibrosis in NASH. Mice were injected with AAV8-GFP or AAV8-AGXT (2x10 11 viral genomes per mouse) and placed on the NASH diet for 6 months prior to tissue and plasma analysis.
  • Panel D Percent- positive Picrosirius Red area.
  • FIG. 7 shows non-limiting, exemplary experimental results. Pharmacological targeting of hepatic oxalate overproduction ameliorates established NASH.
  • Panel A Schema of glyoxylate/oxalate metabolism, chemical structure of MDMG-935P and its inhibitory effects (AGXT, alanine-glyoxylate aminotransferase; GO, glycolate oxidase; GPHPR, glyoxalate reductase/hydroxypryruvate reductase; HOGA1, 4-hydroxy-2-oxoglutarate aldolase; LDHA, lactate dehydrogenase).
  • AGXT alanine-glyoxylate aminotransferase
  • GO glycolate oxidase
  • GPHPR glyoxalate reductase/hydroxypryruvate reductase
  • HOGA1 4-hydroxy-2-oxoglutarate aldolase
  • LDHA lactate dehydrogenase
  • Panel C Liver oxalate normalized to protein concentrations in samples from mice administered vehicle, 5 mg/kg/day or 10 mg/kg/day MDMG-935P.
  • Panel D Liver-to-body weight ratios.
  • Panel E Plasma samples were analyzed for aspartate transaminase (AST), and Panel F) alanine transaminase (ALT) in mice administered vehicle, 5 mg/kg/day or 10 mg/kg/day MDMG- 935P.
  • Panel G Representative gross appearance of abdominal cavities and livers. Liver samples were collected, Panel H) stained with H&E, and Panel I) scored for steatosis, lobular inflammation, hepatocellular ballooning, and NAS. **p ⁇ 0.01, ***p ⁇ 0.001 vs. vehicle; ## p ⁇ 0.01, ### p ⁇ 0.001 vs. 5 mg/kg/day of MGMD-935P. Panel J) Liver triglycerides normalized to protein concentrations.
  • FIG. 8 shows non-limiting, exemplary experimental results.
  • Panel B Liver samples were collected and stained with F4/80 (red) and DAPI (blue) to visualize nuclei.
  • Panel C Percent-positive F4/80 area.
  • Panel D Liver samples were collected from the treated mice and fibrosis-related genes were assessed by qRT-PCR relative to Gapdh.
  • Panel E Liver sections were stained with Picrosirius Red (red) and Panel F) quantified for percent-positive picrosirius red area.
  • Panel G Hydroxyproline contents normalized to protein concentration in liver samples from mice treated with vehicle, 5 mg/kg/day or 10 mg/kg/day MDMG-935P.
  • Panel H Liver sections were scored for fibrosis.
  • Panel I Liver samples were collected and stained with ⁇ -smooth muscle actin (SMA) and DAPI (blue).
  • Panel J Percent-positive ⁇ -SMA area.
  • Panel K Schematic summary of the effects of oxalate on NASH, and inhibition of oxalate production by either AAV-AGXT overexpression or pharmacological targeting using MDMG- 935P. All data are expressed as mean ⁇ SEM. Statistical comparisons were made using One- way ANOVA with Tukey’s multiple comparisons (Panels A, C, D, F, G, H) tests or Kruskal- Wallis with Dunn’s multiple comparisons test (Panel J). All individual points and p-values are shown.
  • FIG. 9 shows a schematic of demographics of patients with and without NASH.
  • FIG. 10 shows non-limiting, exemplary experimental results. Suppression of AGXT in mice with NASH, and correlation between hepatic oxalate and NASH severity.
  • FIG. 11 shows non-limiting, exemplary experimental results.
  • mice Confirmation of hepatic-specific targeting by AAV8-TBG, body weight, liver weight and lipid peroxidation in mice overexpressing AGXT.
  • Mice were injected with AAV8-GFP or AAV8-AGXT (2x10 11 viral genomes per mouse) and placed on the NASH diet for 6 months prior to tissue and plasma analysis.
  • FIG.12 shows non-limiting, exemplary experimental results.
  • Panel B HepG2 cells were treated with 0, 125, 250, or 500 ⁇ M sodium oxalate (Oxalate) Docket No.: 2932719-000218-WO1 Date of Filing: February 28, 2024 overnight. Intracellular oxalate was normalized to protein concentrations. For primary hepatocytes, each point represents an individual mouse.
  • FIG. 13 shows non-limiting, exemplary experimental results. Effects of oxalate on genes regulating lipid metabolism, bioenergetics and mitochondrial superoxide in hepatocytes.
  • mice fed a standard chow diet and HepG2 cells were treated with or without oxalate (250 ⁇ M, primary mouse hepatocytes; 500 ⁇ M, HepG2 cells).
  • Panel F Mitochondrial superoxide was visualized with MitoSOX (red) and nuclei were labeled with Hoechst (blue) in HepG2 cells treated with or without oxalate (500 ⁇ M).
  • FIG.14 shows non-limiting, exemplary experimental results. AGXT overexpression lowers palmitic acid-induced lipid accumulation and mitochondrial superoxide formation while improving mitochondrial respiration.
  • HepG2 cells were transfected with either GFP control (GFP) or GFP-tagged AGXT (AGXT) plasmids.
  • FIG. 15 shows non-limiting, exemplary experimental results.
  • Panel A Body weight
  • Panel B liver weight at endpoint.
  • Panel D HepG2 cells were treated with either BSA control or BSA-conjugated palmitic acid (200 ⁇ M) for 12 h. The next day, cells were treated with either MDMG-935P (50 ⁇ M) or vehicle control (DMSO) for 12 h followed by analysis of neutral lipids visualized with Nile Red stain and nuclei labeled with DAPI.
  • FIG.16 shows a non-limiting, exemplary 1 H NMR spectrum of MDMG-409E.
  • FIG.17 shows a non-limiting, exemplary 1 H NMR spectrum of tert-Butyl (E)-3-(4- acetylphenyl)acrylate (FAB-564).
  • FIG. 18 shows a non-limiting, exemplary 13 C NMR spectrum of tert-Butyl (E)-3- (4-acetylphenyl)acrylate (FAB-564).
  • FIG. 19 shows a non-limiting, exemplary 1 H NMR spectrum of tert-Butyl 3-(4- acetylphenyl)propanoate (FAB-574).
  • FIG.20 shows a non-limiting, exemplary 1 H NMR spectrum of 1-(4-(6-Chlorohex- 1-yn-1-yl)phenyl)ethan-1-one (FAB-586).
  • FIG.21 shows a non-limiting, exemplary 13 C NMR spectrum of 1-(4-(6-Chlorohex- 1-yn-1-yl)phenyl)ethan-1-one (FAB-586).
  • FIG.20 shows a non-limiting, exemplary 1 H NMR spectrum of 1-(4-(6-Chlorohex- 1-yn-1-yl)phenyl)ethan-1-one (FAB-586).
  • FIG.21 shows a non-limiting, exemplary 13 C NMR spectrum of 1-(4-(6-Chlorohex- 1-yn-1-yl)phenyl)ethan-1-one (FAB-586).
  • FIG. 22 shows a non-limiting, exemplary 1 H NMR spectrum of Methyl (E)-2- hydroxy-5-(5-(3-oxo-3-(4-(trifluoromethyl)phenyl)prop-1-en-1-yl)furan-2-yl)benzoate (FAB- 542).
  • FIG. 23 shows a non-limiting, exemplary 13 C NMR spectrum of Methyl (E)-2- hydroxy-5-(5-(3-oxo-3-(4-(trifluoromethyl)phenyl)prop-1-en-1-yl)furan-2-yl)benzoate (FAB- 542).
  • FIG. 23 shows a non-limiting, exemplary 13 C NMR spectrum of Methyl (E)-2- hydroxy-5-(5-(3-oxo-3-(4-(trifluoromethyl)phenyl)prop-1-en-1-yl)furan-2-yl)benzoate (FAB- 542).
  • FIG. 23 shows
  • FIG. 24 shows a non-limiting, exemplary 1 H NMR spectrum of (E)-5-(5-(3-(4- (dimethylamino)phenyl)-3-oxoprop-1-en-1-yl)furan-2-yl)-2-hydroxybenzoic acid (FAB-544).
  • FIG. 25 shows a non-limiting, exemplary 13 C NMR spectrum of (E)-5-(5-(3-(4- (dimethylamino)phenyl)-3-oxoprop-1-en-1-yl)furan-2-yl)-2-hydroxybenzoic acid (FAB-544).
  • FIG.26 shows a non-limiting, exemplary mass spectrum of Methyl (E)-2-hydroxy- 5-(5-(3-oxo-3-(4-(trifluoromethyl)phenyl)prop-1-en-1-yl)furan-2-yl)benzoate (FAB-542).
  • FIG. 27 shows a non-limiting, exemplary 1 H NMR spectrum of Methyl (E)-2- hydroxy-5-(5-(3-(4-iodophenyl)-3-oxoprop-1-en-1-yl)furan-2-yl)benzoate (FAB-545).
  • FIG. 28 shows a non-limiting, exemplary 13 C NMR spectrum of Methyl (E)-2- hydroxy-5-(5-(3-(4-iodophenyl)-3-oxoprop-1-en-1-yl)furan-2-yl)benzoate (FAB-545).
  • FIG.29 shows a non-limiting, exemplary mass spectrum of Methyl (E)-2-hydroxy- 5-(5-(3-(4-iodophenyl)-3-oxoprop-1-en-1-yl)furan-2-yl)benzoate (FAB-545).
  • FIG. 28 shows a non-limiting, exemplary 13 C NMR spectrum of Methyl (E)-2- hydroxy-5-(5-(3-(4-iodophenyl)-3-oxoprop-1-en-1-yl)furan-2-yl)benzoate (FAB-545).
  • FIG. 30 shows a non-limiting, exemplary 1 H NMR spectrum of Methyl (E)-2- hydroxy-5-(5-(3-(4-(methylthio)phenyl)-3-oxoprop-1-en-1-yl)furan-2-yl)benzoate (FAB- 554).
  • FIG. 31 shows a non-limiting, exemplary 13 C NMR spectrum of Methyl (E)-2- hydroxy-5-(5-(3-(4-(methylthio)phenyl)-3-oxoprop-1-en-1-yl)furan-2-yl)benzoate (FAB- 554).
  • FIG.32 shows a non-limiting, exemplary mass spectrum of FAB-445.
  • FIG.33 shows a non-limiting, exemplary 1 H NMR spectrum of Methyl (E)-5-(5-(3- (4-cyanophenyl)-3-oxoprop-1-en-1-yl)furan-2-yl)-2-hydroxybenzoate (FAB-555).
  • FIG. 34 shows a non-limiting, exemplary 13 C NMR spectrum of Methyl (E)-5-(5- (3-(4-cyanophenyl)-3-oxoprop-1-en-1-yl)furan-2-yl)-2-hydroxybenzoate (FAB-555).
  • FIG.35 shows a non-limiting, exemplary mass spectrum of Methyl (E)-5-(5-(3-(4- cyanophenyl)-3-oxoprop-1-en-1-yl)furan-2-yl)-2-hydroxybenzoate (FAB-555).
  • FIG.36 shows a non-limiting, exemplary 1 H NMR spectrum of Methyl (E)-5-(5-(3- (4-butylphenyl)-3-oxoprop-1-en-1-yl)furan-2-yl)-2-hydroxybenzoate (FAB-559).
  • FIG. 37 shows a non-limiting, exemplary 13 C NMR spectrum of Methyl (E)-5-(5- (3-(4-butylphenyl)-3-oxoprop-1-en-1-yl)furan-2-yl)-2-hydroxybenzoate (FAB-559).
  • FIG.38 shows a non-limiting, exemplary mass spectrum of Methyl (E)-5-(5-(3-(4- butylphenyl)-3-oxoprop-1-en-1-yl)furan-2-yl)-2-hydroxybenzoate (FAB-559).
  • FIG. 38 shows a non-limiting, exemplary mass spectrum of Methyl (E)-5-(5-(3-(4- butylphenyl)-3-oxoprop-1-en-1-yl)furan-2-yl)-2-hydroxybenzoate (FAB-559).
  • FIG. 40 shows a non-limiting, exemplary 13 C NMR spectrum of (E)-2-Hydroxy-5- (5-(3-(4-hydroxyphenyl)-3-oxoprop-1-en-1-yl)furan-2-yl)benzoic acid (FAB-561).
  • FIG.41 shows a non-limiting, exemplary mass spectrum of (E)-2-Hydroxy-5-(5-(3- (4-hydroxyphenyl)-3-oxoprop-1-en-1-yl)furan-2-yl)benzoic acid (FAB-561).
  • FIG.42 shows a non-limiting, exemplary 1 H NMR spectrum of 5-(5-((E)-3-(4-((E)- 3-(tert-butoxy)-3-oxoprop-1-en-1-yl)phenyl)-3-oxoprop-1-en-1-yl)furan-2-yl)-2-hydroxy benzoic acid (FAB-565).
  • FIG.43 shows a non-limiting, exemplary 13 C NMR spectrum of 5-(5-((E)-3-(4-((E)- 3-(tert-butoxy)-3-oxoprop-1-en-1-yl)phenyl)-3-oxoprop-1-en-1-yl)furan-2-yl)-2-hydroxy benzoic acid (FAB-565).
  • FIG. 44 shows a non-limiting, exemplary 1 H NMR spectrum of Methyl (E)-2- hydroxy-5-(5-(3-oxo-3-(p-tolyl)prop-1-en-1-yl)furan-2-yl)benzoate (FAB-567).
  • FIG. 43 shows a non-limiting, exemplary 13 C NMR spectrum of 5-(5-((E)-3-(4-((E)- 3-(tert-butoxy)-3-oxoprop-1-en-1-yl)phenyl)-3-oxoprop-1-en-1-yl)furan-2-
  • FIG. 45 shows a non-limiting, exemplary 13 C NMR spectrum of Methyl (E)-2- hydroxy-5-(5-(3-oxo-3-(p-tolyl)prop-1-en-1-yl)furan-2-yl)benzoate (FAB-567).
  • FIG.46 shows a non-limiting, exemplary mass spectrum of FAB-567.
  • FIG. 47 shows a non-limiting, exemplary 1 H NMR spectrum of Methyl (E)-2- hydroxy-5-(5-(3-(4-(hydroxymethyl)phenyl)-3-oxoprop-1-en-1-yl)furan-2-yl)benzoate (FAB- 568).
  • FIG. 48 shows a non-limiting, exemplary 13 C NMR spectrum of Methyl (E)-2- hydroxy-5-(5-(3-(4-(hydroxymethyl)phenyl)-3-oxoprop-1-en-1-yl)furan-2-yl)benzoate (FAB- 568).
  • FIG.49 shows a non-limiting, exemplary mass spectrum of Methyl (E)-2-hydroxy- 5-(5-(3-(4-(hydroxymethyl)phenyl)-3-oxoprop-1-en-1-yl)furan-2-yl)benzoate (FAB-568).
  • FIG.50 shows a non-limiting, exemplary 1 H NMR spectrum of Methyl (E)-5-(5-(3- (4-ethylphenyl)-3-oxoprop-1-en-1-yl)furan-2-yl)-2-hydroxybenzoic acid (FAB-571e). Docket No.: 2932719-000218-WO1 Date of Filing: February 28, 2024 [0061]
  • FIG. 51 shows a non-limiting, exemplary 13 C NMR spectrum of Methyl (E)-5-(5- (3-(4-ethylphenyl)-3-oxoprop-1-en-1-yl)furan-2-yl)-2-hydroxybenzoic acid (FAB-571e).
  • FIG.52 shows a non-limiting, exemplary mass spectrum of Methyl (E)-5-(5-(3-(4- ethylphenyl)-3-oxoprop-1-en-1-yl)furan-2-yl)-2-hydroxybenzoic acid (FAB-571e).
  • FIG. 53 shows a non-limiting, exemplary 1 H NMR spectrum of (E)-5-(5-(3-(4- Ethylphenyl)-3-oxoprop-1-en-1-yl)furan-2-yl)-2-hydroxybenzoate (FAB-571a).
  • FIG. 53 shows a non-limiting, exemplary 1 H NMR spectrum of (E)-5-(5-(3-(4- Ethylphenyl)-3-oxoprop-1-en-1-yl)furan-2-yl)-2-hydroxybenzoate (FAB-571a).
  • FIG. 54 shows a non-limiting, exemplary 13 C NMR spectrum of (E)-5-(5-(3-(4- Ethylphenyl)-3-oxoprop-1-en-1-yl)furan-2-yl)-2-hydroxybenzoate (FAB-571a).
  • FIG. 55 shows a non-limiting, exemplary mass spectrum of(E)-5-(5-(3-(4- Ethylphenyl)-3-oxoprop-1-en-1-yl)furan-2-yl)-2-hydroxybenzoate (FAB-571a).
  • FIG. 55 shows a non-limiting, exemplary mass spectrum of(E)-5-(5-(3-(4- Ethylphenyl)-3-oxoprop-1-en-1-yl)furan-2-yl)-2-hydroxybenzoate (FAB-571a).
  • FIG. 56 shows a non-limiting, exemplary 1 H NMR spectrum of Methyl (E)-2- hydroxy-5-(5-(3-(4-isopropylphenyl)-3-oxoprop-1-en-1-yl)furan-2-yl)benzoate (FAB-572).
  • FIG. 57 shows a non-limiting, exemplary 13 C NMR spectrum of Methyl (E)-2- hydroxy-5-(5-(3-(4-isopropylphenyl)-3-oxoprop-1-en-1-yl)furan-2-yl)benzoate (FAB-572).
  • FIG.58 shows a non-limiting, exemplary mass spectrum of Methyl (E)-2-hydroxy- 5-(5-(3-(4-isopropylphenyl)-3-oxoprop-1-en-1-yl)furan-2-yl)benzoate (FAB-572).
  • FIG. 59 shows a non-limiting, exemplary 1 H NMR spectrum of (E)-5-(5-(3-(3- Ethyl-4-(hydroxymethyl)phenyl)-3-oxoprop-1-en-1-yl)furan-2-yl)-2-hydroxybenzoic acid (FAB-578a).
  • FIG. 59 shows a non-limiting, exemplary 1 H NMR spectrum of (E)-5-(5-(3-(3- Ethyl-4-(hydroxymethyl)phenyl)-3-oxoprop-1-en-1-yl)furan-2-yl)-2-hydroxybenzoic acid (FAB-578a).
  • FIG. 60 shows a non-limiting, exemplary 13 C NMR spectrum of (E)-5-(5-(3-(3- Ethyl-4-(hydroxymethyl)phenyl)-3-oxoprop-1-en-1-yl)furan-2-yl)-2-hydroxybenzoic acid (FAB-578a).
  • FIG.61 shows a non-limiting, exemplary mass spectrum of (E)-5-(5-(3-(3-Ethyl-4- (hydroxymethyl)phenyl)-3-oxoprop-1-en-1-yl)furan-2-yl)-2-hydroxybenzoic acid (FAB- 578a).
  • FIG. 62 shows a non-limiting, exemplary 1 H NMR spectrum of (E)-5-(5-(3-(4-(3- (tert-Butoxy)-3-oxopropyl)phenyl)-3-oxoprop-1-en-1-yl)furan-2-yl)-2-hydroxybenzoic acid (FAB-581atBu).
  • FIG. 62 shows a non-limiting, exemplary 1 H NMR spectrum of (E)-5-(5-(3-(4-(3- (tert-Butoxy)-3-oxopropyl)phenyl)-3-oxoprop-1-en-1-yl)furan-2-yl)-2-hydroxybenzoic acid (FAB-581atBu).
  • FIG. 63 shows a non-limiting, exemplary 13 C NMR spectrum of (E)-5-(5-(3-(4-(3- (tert-Butoxy)-3-oxopropyl)phenyl)-3-oxoprop-1-en-1-yl)furan-2-yl)-2-hydroxybenzoic acid (FAB-581atBu).
  • FIG.64 shows a non-limiting, exemplary mass spectrum of (E)-5-(5-(3-(4-(3-(tert- Butoxy)-3-oxopropyl)phenyl)-3-oxoprop-1-en-1-yl)furan-2-yl)-2-hydroxybenzoic acid (FAB- 581atBu).
  • FIG. 64 shows a non-limiting, exemplary mass spectrum of (E)-5-(5-(3-(4-(3-(tert- Butoxy)-3-oxopropyl)phenyl)-3-oxoprop-1-en-1-yl)furan-2
  • FIG. 65 shows a non-limiting, exemplary 1 H NMR spectrum of (E)-5-(5-(3-(4-(6- Chlorohex-1-yn-1-yl)phenyl)-3-oxoprop-1-en-1-yl)furan-2-yl)-2-hydroxybenzoic acid (FAB- 583a).
  • FIG. 66 shows a non-limiting, exemplary 13 C NMR spectrum of (E)-5-(5-(3-(4-(6- Chlorohex-1-yn-1-yl)phenyl)-3-oxoprop-1-en-1-yl)furan-2-yl)-2-hydroxybenzoic acid (FAB- 583a).
  • FIG. 66 shows a non-limiting, exemplary 13 C NMR spectrum of (E)-5-(5-(3-(4-(6- Chlorohex-1-yn-1-yl)phenyl)-3-oxoprop-1-en-1-yl)furan-2
  • FIG. 67 shows a non-limiting, exemplary mass spectrum of (E)-5-(5-(3-(4-(6- Chlorohex-1-yn-1-yl)phenyl)-3-oxoprop-1-en-1-yl)furan-2-yl)-2-hydroxybenzoic acid (FAB- 583a).
  • FIG.68 shows a non-limiting, exemplary 1 H NMR spectrum of Methyl (E)-5-(5-(3- (4-hexylphenyl)-3-oxoprop-1-en-1-yl)furan-2-yl)-2-hydroxybenzoate (FAB-590e).
  • FIG. 70 shows non-limiting, exemplary mass spectra of Methyl (E)-5-(5-(3-(4- hexylphenyl)-3-oxoprop-1-en-1-yl)furan-2-yl)-2-hydroxybenzoate (FAB-590e).
  • FIG. 70 shows non-limiting, exemplary mass spectra of Methyl (E)-5-(5-(3-(4- hexylphenyl)-3-oxoprop-1-en-1-yl)furan-2-yl)-2-hydroxybenzoate (FAB-590e).
  • FIG. 70 shows non-limiting, exemplary mass spectra of Methyl (E)-5-(5-(3-(4- hexylphenyl)-3-oxoprop-1-en-1-yl)furan-2-yl)-2-hydroxybenzoate (FAB-590e).
  • FIG. 70 shows non-limiting, exemplary mass spectra of Methyl (
  • FIG. 71 shows a non-limiting, exemplary 1 H NMR spectrum of (E)-5-(5-(3-(4- Hexylphenyl)-3-oxoprop-1-en-1-yl)furan-2-yl)-2-hydroxybenzoic acid (FAB-590a). Docket No.: 2932719-000218-WO1 Date of Filing: February 28, 2024 [0082]
  • FIG. 72 shows a non-limiting, exemplary 13 C NMR spectrum of (E)-5-(5-(3-(4- Hexylphenyl)-3-oxoprop-1-en-1-yl)furan-2-yl)-2-hydroxybenzoic acid (FAB-590a). [0083] FIG.
  • FIG. 73 shows non-limiting, exemplary spectra of (E)-5-(5-(3-(4-Hexylphenyl)-3- oxoprop-1-en-1-yl)furan-2-yl)-2-hydroxybenzoic acid (FAB-590a).
  • FIG. 74 shows a non-limiting, exemplary 1 H NMR spectrum of (E)-2-Hydroxy-5- (5-(3-(4-octylphenyl)-3-oxoprop-1-en-1-yl)furan-2-yl)benzoic acid (FAB-594a).
  • FIG. 74 shows a non-limiting, exemplary 1 H NMR spectrum of (E)-2-Hydroxy-5- (5-(3-(4-octylphenyl)-3-oxoprop-1-en-1-yl)furan-2-yl)benzoic acid (FAB-594a).
  • FIG. 75 shows a non-limiting, exemplary 13 C NMR spectrum of (E)-2-Hydroxy-5- (5-(3-(4-octylphenyl)-3-oxoprop-1-en-1-yl)furan-2-yl)benzoic acid (FAB-594a).
  • FIG. 76 shows non-limiting, exemplary spectra of (E)-2-Hydroxy-5-(5-(3-(4- octylphenyl)-3-oxoprop-1-en-1-yl)furan-2-yl)benzoic acid (FAB-594a).
  • FIG.77 shows a non-limiting, exemplary 1 H NMR spectrum of Methyl 2-hydroxy- 5-(5-(3-oxo-3-(4-(trifluoromethyl)phenyl)propyl)furan-2-yl)benzoate (FAB-546).
  • FIG.78 shows a non-limiting, exemplary 13 C NMR spectrum of Methyl 2-hydroxy- 5-(5-(3-oxo-3-(4-(trifluoromethyl)phenyl)propyl)furan-2-yl)benzoate (FAB-546).
  • FIG.79 shows a non-limiting, exemplary 1 H NMR spectrum of Methyl 5-(5-(3-(4- butylphenyl)-3-oxopropyl)furan-2-yl)-2-hydroxybenzoate (FAB-582).
  • FIG.80 shows a non-limiting, exemplary 13 C NMR spectrum of FAB-582.
  • FIG.81 shows a non-limiting, exemplary 1 H NMR spectrum of Methyl 5-(5-(3-(4- butylphenyl)-3-hydroxypropyl)furan-2-yl)-2-hydroxybenzoate (FAB-584).
  • FIG.82 shows a non-limiting, exemplary 13 C NMR spectrum of Methyl 5-(5-(3-(4- butylphenyl)-3-hydroxypropyl)furan-2-yl)-2-hydroxybenzoate (FAB-584).
  • FIG. 83 shows a non-limiting, exemplary 1 H NMR spectrum of (E)-2-Hydroxy-5- (5-(3-(4-iodophenyl)-3-oxoprop-1-en-1-yl)furan-2-yl)benzoic acid (FAB-548).
  • FIG. 85 shows a non-limiting, exemplary 13 C NMR spectrum of (E)-2-Hydroxy-5- (5-(3-(4-iodophenyl)-3-oxoprop-1-en-1-yl)furan-2-yl)benzoic acid (FAB-548). Docket No.: 2932719-000218-WO1 Date of Filing: February 28, 2024 [0095]
  • FIG.85 shows a non-limiting, exemplary mass spectrum of (E)-2-Hydroxy-5-(5-(3- (4-iodophenyl)-3-oxoprop-1-en-1-yl)furan-2-yl)benzoic acid (FAB-548).
  • FIG.86 shows a non-limiting, exemplary 1 H NMR spectrum of 2-Hydroxy-5-(5-(3- oxo-3-(4-(trifluoromethyl)phenyl)propyl)furan-2-yl)benzoic acid (FAB-549).
  • FIG.87 shows a non-limiting, exemplary mass spectrum of 2-Hydroxy-5-(5-(3-oxo- 3-(4-(trifluoromethyl)phenyl)propyl)furan-2-yl)benzoic acid (FAB-549).
  • FIG.86 shows a non-limiting, exemplary 1 H NMR spectrum of 2-Hydroxy-5-(5-(3- oxo-3-(4-(trifluoromethyl)phenyl)propyl)furan-2-yl)benzoic acid (FAB-549).
  • FIG. 88 shows a non-limiting, exemplary 1 H NMR spectrum of (E)-5-(5-(3-(4- Cyanophenyl)-3-oxoprop-1-en-1-yl)furan-2-yl)-2-hydroxybenzoic acid (FAB-558).
  • FIG. 89 shows a non-limiting, exemplary 13 C NMR spectrum of (E)-5-(5-(3-(4- Cyanophenyl)-3-oxoprop-1-en-1-yl)furan-2-yl)-2-hydroxybenzoic acid (FAB-558).
  • FIG. 89 shows a non-limiting, exemplary 13 C NMR spectrum of (E)-5-(5-(3-(4- Cyanophenyl)-3-oxoprop-1-en-1-yl)furan-2-yl)-2-hydroxybenzoic acid (FAB-558).
  • FIG. 89 shows a non-limiting, exemplary 13 C NMR spectrum of (E)-5-(5
  • FIG. 90 shows a non-limiting, exemplary mass spectrum of (E)-5-(5-(3-(4- Cyanophenyl)-3-oxoprop-1-en-1-yl)furan-2-yl)-2-hydroxybenzoic acid (FAB-558).
  • FIG. 91 shows a non-limiting, exemplary 1 H NMR spectrum of (E)-5-(5-(3-(4- Butylphenyl)-3-oxoprop-1-en-1-yl)furan-2-yl)-2-hydroxybenzoic acid (FAB-563).
  • FIG. 91 shows a non-limiting, exemplary 1 H NMR spectrum of (E)-5-(5-(3-(4- Butylphenyl)-3-oxoprop-1-en-1-yl)furan-2-yl)-2-hydroxybenzoic acid (FAB-563).
  • FIG. 92 shows a non-limiting, exemplary 13 C NMR spectrum of (E)-5-(5-(3-(4- Butylphenyl)-3-oxoprop-1-en-1-yl)furan-2-yl)-2-hydroxybenzoic acid (FAB-563).
  • FIG. 93 shows a non-limiting, exemplary mass spectrum of (E)-5-(5-(3-(4- Butylphenyl)-3-oxoprop-1-en-1-yl)furan-2-yl)-2-hydroxybenzoic acid (FAB-563).
  • FIG. 93 shows a non-limiting, exemplary mass spectrum of (E)-5-(5-(3-(4- Butylphenyl)-3-oxoprop-1-en-1-yl)furan-2-yl)-2-hydroxybenzoic acid (FAB-563).
  • FIG. 95 shows a non-limiting, exemplary 13 C NMR spectrum of (E)-2-Hydroxy-5- (5-(3-(4-(methylthio)phenyl)-3-oxoprop-1-en-1-yl)furan-2-yl)benzoic acid (FAB-566).
  • FIG.96 shows a non-limiting, exemplary mass spectrum of (E)-2-Hydroxy-5-(5-(3- (4-(methylthio)phenyl)-3-oxoprop-1-en-1-yl)furan-2-yl)benzoic acid (FAB-566). Docket No.: 2932719-000218-WO1 Date of Filing: February 28, 2024 [00107]
  • FIG. 97 shows a non-limiting, exemplary 1 H NMR spectrum of (E)-2-Hydroxy-5- (5-(3-oxo-3-(p-tolyl)prop-1-en-1-yl)furan-2-yl)benzoic acid (FAB-573).
  • FIG. 97 shows a non-limiting, exemplary 1 H NMR spectrum of (E)-2-Hydroxy-5- (5-(3-oxo-3-(p-tolyl)prop-1-en-1-yl)furan-2-yl)benzoic acid (FAB
  • FIG. 98 shows a non-limiting, exemplary 13 C NMR spectrum of (E)-2-Hydroxy-5- (5-(3-oxo-3-(p-tolyl)prop-1-en-1-yl)furan-2-yl)benzoic acid (FAB-573).
  • FIG.99 shows a non-limiting, exemplary mass spectrum of (E)-2-Hydroxy-5-(5-(3- oxo-3-(p-tolyl)prop-1-en-1-yl)furan-2-yl)benzoic acid (FAB-573).
  • FIG.100 shows a non-limiting, exemplary 1 H NMR spectrum of (E)-2-Hydroxy-5- (5-(3-(4-(hydroxymethyl)phenyl)-3-oxoprop-1-en-1-yl)furan-2-yl)benzoic acid (FAB-575).
  • FIG.101 shows a non-limiting, exemplary 13 C NMR spectrum of (E)-2-Hydroxy-5- (5-(3-(4-(hydroxymethyl)phenyl)-3-oxoprop-1-en-1-yl)furan-2-yl)benzoic acid (FAB-575).
  • FIG.100 shows a non-limiting, exemplary 1 H NMR spectrum of (E)-2-Hydroxy-5- (5-(3-(4-(hydroxymethyl)phenyl)-3-oxoprop-1-en-1-yl)furan-2-yl)benzoic acid (FAB-575).
  • FIG.100 shows a non-limiting, exemplary 1 H NMR spectrum
  • FIG. 102 shows a non-limiting, exemplary mass spectrum of (E)-2-Hydroxy-5-(5- (3-(4-(hydroxymethyl)phenyl)-3-oxoprop-1-en-1-yl)furan-2-yl)benzoic acid (FAB-575).
  • FIG.103 shows a non-limiting, exemplary 1 H NMR spectrum of (E)-2-Hydroxy-5- (5-(3-(4-isopropylphenyl)-3-oxoprop-1-en-1-yl)furan-2-yl)benzoic acid (FAB-576).
  • FIG.104 shows a non-limiting, exemplary 13 C NMR spectrum of (E)-2-Hydroxy-5- (5-(3-(4-isopropylphenyl)-3-oxoprop-1-en-1-yl)furan-2-yl)benzoic acid (FAB-576).
  • FIG. 105 shows a non-limiting, exemplary mass spectrum of (E)-2-Hydroxy-5-(5- (3-(4-isopropylphenyl)-3-oxoprop-1-en-1-yl)furan-2-yl)benzoic acid (FAB-576).
  • FIG. 105 shows a non-limiting, exemplary mass spectrum of (E)-2-Hydroxy-5-(5- (3-(4-isopropylphenyl)-3-oxoprop-1-en-1-yl)furan-2-yl)benzoic acid (FAB-576).
  • FIG. 106 shows a non-limiting, exemplary 1 H NMR spectrum of 5-(5-(3-(4- Butylphenyl)-3-oxopropyl)furan-2-yl)-2-hydroxybenzoic acid (FAB-585).
  • FIG. 107 shows a non-limiting, exemplary 13 C NMR spectrum of 5-(5-(3-(4- Butylphenyl)-3-oxopropyl)furan-2-yl)-2-hydroxybenzoic acid (FAB-585).
  • FIG. 107 shows a non-limiting, exemplary 13 C NMR spectrum of 5-(5-(3-(4- Butylphenyl)-3-oxopropyl)furan-2-yl)-2-hydroxybenzoic acid (FAB-585).
  • FIG. 109 shows a non-limiting, exemplary mass spectrum of 5-(5-(3-(4- Butylphenyl)-3-oxopropyl)furan-2-yl)-2-hydroxybenzoic acid (FAB-585). Docket No.: 2932719-000218-WO1 Date of Filing: February 28, 2024 [00119]
  • FIG. 109 shows a non-limiting, exemplary 1 H NMR spectrum of 5-(5-(3-(4- Butylphenyl)-3-hydroxypropyl)furan-2-yl)-2-hydroxybenzoic acid (FAB-587). [00120] FIG.
  • FIG. 110 shows a non-limiting, exemplary 13 C NMR spectrum of 5-(5-(3-(4- Butylphenyl)-3-hydroxypropyl)furan-2-yl)-2-hydroxybenzoic acid (FAB-587).
  • FIG. 111 shows a non-limiting, exemplary mass spectrum of 5-(5-(3-(4- Butylphenyl)-3-hydroxypropyl)furan-2-yl)-2-hydroxybenzoic acid (FAB-587).
  • FIG. 111 shows a non-limiting, exemplary mass spectrum of 5-(5-(3-(4- Butylphenyl)-3-hydroxypropyl)furan-2-yl)-2-hydroxybenzoic acid (FAB-587).
  • FIG. 112 shows a non-limiting, exemplary 1 H NMR spectrum of 5-(5-((E)-3-(4- ((E)-2-Carboxyvinyl)phenyl)-3-oxoprop-1-en-1-yl)furan-2-yl)-2-hydroxybenzoic acid (FAB- 570).
  • FIG. 113 shows a non-limiting, exemplary 13 C NMR spectrum of 5-(5-((E)-3-(4- ((E)-2-Carboxyvinyl)phenyl)-3-oxoprop-1-en-1-yl)furan-2-yl)-2-hydroxybenzoic acid (FAB- 570).
  • FIG.114 shows a non-limiting, exemplary mass spectrum of 5-(5-((E)-3-(4-((E)-2- Carboxyvinyl)phenyl)-3-oxoprop-1-en-1-yl)furan-2-yl)-2-hydroxybenzoic acid (FAB-570).
  • FIG.115 shows a non-limiting, exemplary 1 H NMR spectrum of (E)-5-(5-(3-(4-(2- Carboxyethyl)phenyl)-3-oxoprop-1-en-1-yl)furan-2-yl)-2-hydroxybenzoic acid (FAB-581aa).
  • FIG.116 shows a non-limiting, exemplary 13 C NMR spectrum of (E)-5-(5-(3-(4-(2- Carboxyethyl)phenyl)-3-oxoprop-1-en-1-yl)furan-2-yl)-2-hydroxybenzoic acid (FAB-581aa).
  • FIG. 117 shows a non-limiting, exemplary mass spectrum of (E)-5-(5-(3-(4-(2- Carboxyethyl)phenyl)-3-oxoprop-1-en-1-yl)furan-2-yl)-2-hydroxybenzoic acid (FAB-581aa).
  • FIG. 117 shows a non-limiting, exemplary mass spectrum of (E)-5-(5-(3-(4-(2- Carboxyethyl)phenyl)-3-oxoprop-1-en-1-yl)furan-2-yl)-2-hydroxybenzoic acid (FAB-581aa).
  • FIG. 118 shows a non-limiting, exemplary 1 H NMR spectrum of 5-(4-hydroxy-3- nitrophenyl)-2-furaldehyde (FAB-536).
  • FIG. 119 shows a non-limiting, exemplary 13 C NMR spectrum of 5-(4-hydroxy-3- nitrophenyl)-2-furaldehyde (FAB-536). Docket No.: 2932719-000218-WO1 Date of Filing: February 28, 2024 [00130] FIG.
  • FIG. 120 shows a non-limiting, exemplary 1 H NMR spectrum of (E)-1-(4- Bromophenyl)-3-(5-(4-hydroxy-3-nitrophenyl)furan-2-yl)prop-2-en-1-one (FAB-541).
  • FIG. 121 shows a non-limiting, exemplary 13 C NMR spectrum of (E)-1-(4- Bromophenyl)-3-(5-(4-hydroxy-3-nitrophenyl)furan-2-yl)prop-2-en-1-one (FAB-541).
  • FIG. 121 shows a non-limiting, exemplary 13 C NMR spectrum of (E)-1-(4- Bromophenyl)-3-(5-(4-hydroxy-3-nitrophenyl)furan-2-yl)prop-2-en-1-one (FAB-541).
  • FIG. 122 shows a non-limiting, exemplary mass spectrum of (E)-1-(4- Bromophenyl)-3-(5-(4-hydroxy-3-nitrophenyl)furan-2-yl)prop-2-en-1-one (FAB-541).
  • FIG. 123 shows activities of selected salicylate derivatives against recombinant human glycolate oxidase and lactate dehydrogenase A.
  • FIG. 124 shows a non-limiting, exemplary l list of compounds for evaluation of intracellular oxalate in hepatocytes of Primary Hyperoxaluria mice (AGXT-/-).
  • Compounds named FAB are salicylate derivatives
  • compounds named FL are flavonoids.
  • FIG.125 shows a non-limiting, exemplary schematic of a study design.
  • FIG. 126 shows a graph of non-limiting, exemplary results of DMSO with and without glycolate.
  • FIG. 127 shows graphs of non-limiting, exemplary results of MDMG-935P, FAB- 541, FAB-544, and FAB-548.
  • FIG. 128 shows graphs of non-limiting, exemplary results of FAB-549, FAB-563, FAB-566, and FAB-570.
  • FIG.129 shows graphs of non-limiting, exemplary results of FAB-571a, FAB-573, FAB-576, and FAB-578a.
  • FIG. 140 shows graphs of non-limiting, exemplary results of FAB-571a, FAB-573, FAB-576, and FAB-578a.
  • FIG. 130 shows graphs of non-limiting, exemplary results of FAB-581aa, FAB- 581atBu, FAB-583a, and FAB-585.
  • FIG. 131 shows graphs of non-limiting, exemplary results of FAB-587, FL-6, FL- 19, and FL-22.
  • FIG.132 shows a non-limiting, exemplary heat map of compounds described herein. Docket No.: 2932719-000218-WO1 Date of Filing: February 28, 2024
  • FIG.133 shows a non-limiting schema of the proposal.
  • FIG. 134 shows a non-limiting schema of glyoxylate/oxalate metabolism. Orange boxes: enzymes known to promote glyoxylate or oxalate production.
  • FIG. 135 shows non-limiting data indicating increased oxalate and suppressed AGXT in NASH.
  • Panel B Confirmation of NASH by H&E (scale bar: 50 ⁇ m).
  • Panel C AGXT protein normalized to ⁇ -Actin.
  • Panel D Liver oxalate normalized to protein concentrations. Data are means ⁇ SEM. Unpaired t test.
  • FIG. 137 shows non-limiting data indicating suppressed AGXT in atherosclerosis.
  • Panel B Confirmation of atherosclerosis using oil red O (ORO) staining of whole aortas.
  • Panel C AGXT protein normalized to ⁇ -Actin, as we reported. 14 Data are means ⁇ SEM. Unpaired t test.
  • FIG. 137 shows non-limiting, exemplary data indicating increased oxalate and suppressed AGXT in steatotic hepatocytes.
  • FIG. 138 shows non-limiting, exemplary data indicating oxalate exacerbates lipid accumulation and mitochondrial dysfunction in vitro.
  • Patent C MitoSOX (scale bar: 100 ⁇ m). Data are means ⁇ SEM. Two-way ANOVA followed by Holm-Sidak’s test.
  • FIG.139 shows non-limiting, exemplary data indicating enhanced NASH in Agxt -/- mice.
  • FIG.140 shows non-limiting, exemplary data indicating oxalate suppresses PPAR ⁇ targets and induces CCL5 in vitro.
  • FIG. 141 shows non-limiting, exemplary data indicating enhanced atherosclerosis in Agxt -/- / Apoe -/- mice.
  • FIG. 142 shows non-limiting, exemplary data indicating WD +HLP increases oxalate and atherosclerosis.
  • Panel B Liver oxalate.
  • Panel C Atherosclerosis in whole aortas using ORO. Data are means ⁇ SEM. Unpaired t test.
  • FIG. 143 shows non-limiting, exemplary data indicating liver-specific loss of PPAR ⁇ confirmed by (Panel A) Western blot. (Panel B) Morphology of male Ppara LKO and Ppara flox mice on NASH diet (12 weeks). [00154] FIG. 144 shows non-limiting, exemplary data indicating concurrent (Panel A) NASH, and (Panel B) atherosclerosis confirmed by H&E histology in male Ldlr-/- mice fed NASH diet for 16 weeks (scale bar: 50 um). Docket No.: 2932719-000218-WO1 Date of Filing: February 28, 2024 [00155] FIG.
  • FIG.146 shows non-limiting, exemplary data indicating oxalate reduction via AGXT overexpression lowers NASH.
  • Panel B AGXT overexpression was confirmed by Western blot.
  • Panel C Liver oxalate.
  • Panel D Plasma AST.
  • Panel E Gross morphology.
  • Panels F-G H&E histology (scale bar: 50 ⁇ m) used for NAS.
  • FIG. 147 shows non-limiting, exemplary data indicating unbiased transcriptomics following AGXT overexpression in NASH. RNA-seq was performed on livers from mice described in Fig.14.
  • (Panel A) PCA. Pathways enriched in the (Panel B) upregulated (red) or (Panel C) downregulated (blue) DEGs. Fisher’s exact test followed by Benjamini-Hochberg multiple testing adjustment (n 4).
  • FIG. 148 shows non-limiting, exemplary data indicating oxalate reduction via AGXT overexpression attenuates atherosclerosis.
  • Panel B Western blot confirming hepatic AGXT overexpression with (Panel C) reduced oxalate.
  • Panel D Plasma CCL5.
  • FIG. 150 shows non-limiting, exemplary data indicating dysregulated glyoxylate/oxalate metabolic genes in NAFLD/NASH. (Panel A) DEGs assessed by RNA-seq in humans and mice with NASH/ controls as described in Fig.147. Scale bar: log2fold-change.
  • Panel A Liver oxalate.
  • Panel B AGXT protein normalized to GAPDH. Data are means ⁇ SEM. Unpaired t test.
  • the term “about” can refer to approximately, roughly, around, or in the region of. When the term “about” is used in conjunction with a numerical range, it modifies that range by extending the boundaries above and below the numerical values set forth. In general, the term “about” is used herein to modify a numerical value above and below the stated value by a variance of 20 percent up or down (higher or lower). In embodiments, the term “about” can be denoted by “ ⁇ ”.
  • the term “substantially the same” or “substantially” can refer to variability typical for a particular method is taken into account.
  • the terms “sufficient” and “effective”, as used interchangeably herein, can refer to an amount (e.g., mass, volume, dosage, concentration, and/or time period) needed to achieve one or more desired result(s).
  • alkyl refers to the radical of saturated aliphatic groups, including straight- chain alkyl groups, branched-chain alkyl groups, cycloalkyl (alicyclic) groups, alkyl- substituted cycloalkyl groups, and cycloalkyl-substituted alkyl groups.
  • a straight chain or branched chain alkyl has 30 or fewer carbon atoms in its backbone (e.g., C1-C30 for straight chains, C3-C30 for branched chains), 20 or fewer, 12 or fewer, or 7 or fewer.
  • cycloalkyls have from 3-10 carbon atoms in their ring structure, e.g., have 5, 6 or 7 carbons in the ring structure.
  • alkyl (or “lower alkyl) as used throughout the specification, examples, and claims can include both “unsubstituted alkyls” and “substituted alkyls”, the latter of which refers to alkyl moieties having one or more substituents replacing a hydrogen on one or more carbons of the hydrocarbon backbone.
  • substituents include, but are not limited to, halogen, hydroxyl, carbonyl (such as a carboxyl, alkoxycarbonyl, formyl, or an acyl), thiocarbonyl (such as a thioester, a thioacetate, or a thioformate), alkoxyl, phosphoryl, phosphate, phosphonate, a hosphinate, amino, amido, amidine, imine, cyano, nitro, azido, sulfhydryl, alkylthio, sulfate, sulfonate, sulfamoyl, sulfonamido, sulfonyl, heterocyclyl, aralkyl, or an aromatic or heteroaromatic moiety.
  • carbonyl such as a carboxyl, alkoxycarbonyl, formyl, or an acyl
  • thiocarbonyl such as a thioester, a
  • lower alkyl as used herein can refer to an alkyl group, as defined herein, but having from one to ten carbons, or from one to six carbon atoms in its backbone structure. Likewise, “lower alkenyl” and “lower alkynyl” have similar chain lengths. In some embodiments, alkyl groups are lower alkyls. In some embodiments, a substituent described herein as alkyl can be a lower alkyl. [00176] It will be understood by those skilled in the art that the moieties substituted on the hydrocarbon chain can themselves be substituted, if appropriate.
  • the substituents of a substituted alkyl can include halogen, hydroxy, nitro, thiols, amino, azido, imino, amido, phosphoryl (including phosphonate and phosphinate), sulfonyl (including sulfate, sulfonamido, sulfamoyl and sulfonate), and silyl groups, as well as ethers, alkylthios, carbonyls (including ketones, aldehydes, carboxylates, and esters), -CF3, -CN and the like. Cycloalkyls can be substituted in the same manner.
  • heteroalkyl refers to straight or branched chain, or cyclic carbon-containing radicals, or combinations thereof, containing at least one heteroatom. Suitable heteroatoms include, but are not limited to, O, N, Si, P, Se, B, and S, wherein the phosphorous and sulfur atoms are optionally oxidized, and the nitrogen heteroatom is optionally quaternized. Heteroalkyls can be substituted as defined herein for alkyl groups.
  • alkylthio refers to an alkyl group, as defined herein, having a sulfur radical attached thereto.
  • the "alkylthio" moiety is represented by one of -S-alkyl, -S-alkenyl, and -S-alkynyl.
  • Representative alkylthio groups include methylthio, and ethylthio.
  • alkylthio also encompasses cycloalkyl groups, alkene and cycloalkene groups, and alkyne groups.
  • Arylthio refers to aryl or heteroaryl groups. Alkylthio groups can be substituted as defined herein for alkyl groups.
  • alkenyl and alkynyl refer to unsaturated aliphatic groups analogous in length and possible substitution to the alkyls described herein, but that contain at least one double or triple bond respectively.
  • alkoxyl or alkoxy refers to an alkyl group, as defined herein, having an oxygen radical attached thereto. Representative alkoxyl groups include methoxy, ethoxy, propyloxy, and tert-butoxy.
  • An "ether,” for example, can be two hydrocarbons covalently linked by an oxygen.
  • an alkyl that renders that alkyl an ether is or resembles an alkoxyl, such as can be represented by one of -O- alkyl, -O-alkenyl, and -O-alkynyl.
  • Aroxy can be represented by -O-aryl or O-heteroaryl, wherein aryl and heteroaryl are as defined herein.
  • the alkoxy and aroxy groups can be substituted as described herein for alkyl.
  • amine and “amino” are art-recognized and refer to both unsubstituted and substituted amines, e.g., a moiety that can be represented by the general formula: wherein R9, R10, and R gen, an alkyl, an alkenyl, - (CH 2 ) m - Rs or R 9 and R 10 taken together with the N atom to which they are attached complete a heterocycle having from 4 to 8 atoms in the ring structure; Rs represents an aryl, a cycloalkyl, a cycloalkenyl, a heterocycle or a polycycle; and m is zero or an integer in the range of 1 to 8.
  • R9 or R10 can be a carbonyl, e.g., R9, R10 and the nitrogen Docket No.: 2932719-000218-WO1 Date of Filing: February 28, 2024 together do not form an imide.
  • the term “amine” does not encompass amides, e.g., wherein one of R 9 and R 10 represents a carbonyl.
  • R9 and R10 each independently represent a hydrogen, an alkyl or cycloalkyl, an alkenyl or cycloalkenyl, or alkynyl.
  • alkylamine can refer to an amine group, as defined herein, having a substituted (as described hereinfor alkyl) or unsubstituted alkyl attached thereto, i.e., at least one of R 9 and R 10 is an alkyl group.
  • the term “imide” can refer to -C(O)NR’R’’, wherein R’ and R’’ are each independently hydrogen, or a substituted or unsubstituted alkyl, cycloalkyl, alkenyl, alkynyl, aryl aralkyl, heterocyclyl or heterocyclylalkyl group as defined herein.
  • halogen can refer to -F, -Cl, -Br or -I; the term “sulfhydryl” can refer to -SH; the term “hydroxyl” can refer to -OH; and the term “sulfonyl” can refer to -SO2-.
  • substituted refers to permissible substituents of the compounds described herein. In the broadest sense, the permissible substituents include acyclic and cyclic, branched and unbranched, carbocyclic and heterocyclic, aromatic and nonaromatic substituents of organic compounds.
  • Illustrative substituents include, but are not limited to, halogens, hydroxyl groups, or any other organic groupings containing any number of carbon atoms, for example 1-14 carbon atoms, and optionally include one or more heteroatoms such as oxygen, sulfur, or nitrogen grouping in linear, branched, or cyclic structural formats.
  • substituents include alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, phenyl, substituted phenyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, halo, hydroxyl, alkoxy, substituted alkoxy, phenoxy, substituted phenoxy, aroxy, substituted aroxy, alkylthio, substituted alkylthio, phenylthio, substituted phenylthio, arylthio, substituted arylthio, cyano, isocyano, substituted isocyano, carbonyl, substituted carbonyl, carboxyl, substituted carboxyl, amino, substituted amino, amido, substituted amido, sulfonyl, substituted sulfonyl, sulfonic acid, phosphoryl, substituted phosphoryl, phosphonyl, substituted phosphonyl, polyaryl
  • R As used herein in reference to an “R” group, the name used to describe said “R” group can be the chemical name prior to the removal of a hydrogen. For example, wherein “R” is described as an “alkane” can refer to an “alkyl” group.
  • Heteroatoms such as nitrogen can have hydrogen substituents and/or any permissible substituents of organic compounds described herein which satisfy the valences of the heteroatoms.
  • substitution or “substituted” includes the implicit proviso that such substitution is in accordance with permitted valence of the substituted atom and the substituent, and that the substitution results in a stable compound, i.e., a compound that does not spontaneously undergo transformation such as by rearrangement, cyclization, elimination, etc.
  • the permissible substituents include acyclic and cyclic, branched and unbranched, carbocyclic and heterocyclic, aromatic and nonaromatic substituents of organic compounds.
  • Illustrative substituents include, for example, those described herein.
  • the permissible substituents can be one or more and the same or different for appropriate organic compounds.
  • the heteroatoms such as nitrogen can have hydrogen substituents and/or any permissible substituents of organic compounds described herein which satisfy the valencies of the heteroatoms.
  • the substituent is selected from alkoxy, aryloxy, alkyl, alkenyl, alkynyl, amide, amino, aryl, arylalkyl, carbamate, carboxy, cyano, cycloalkyl, ester, ether, formyl, halogen, haloalkyl. heteroaryl, heterocyclyl, hydroxyl, ketone, nitro, phosphate, sulfide, sulfinyl, sulfonyl, sulfonic acid, sulfonamide, and thioketone, each of which optionally is substituted with one or more suitable substituents.
  • the substituent is selected from alkoxy, aryloxy, alkyl, alkenyl, alkynyl, amide, amino, aryl, arylalkyl, carbamate, carboxy, cycloalkyl, ester, ether, formyl, haloalkyl.
  • heteroaryl, heterocyclyl, ketone, phosphate, sulfide, sulfinyl, sulfonyl, sulfonic acid, sulfonamide, and thioketone wherein each of the alkoxy, aryloxy, alkyl, alkenyl, alkynyl, amide, amino, aryl, arylalky l, carbamate, carboxy, cycloalkyl, ester, ether, formyl, haloalkyl, heteroaryl, heterocyclyl, ketone, phosphate, sulfide, sulfinyl, sulfonyl, sulfonic acid, sulfonamide, and thioketone can be further substituted with one or more suitable substituents.
  • substituents include, but are not limited to, halogen, azide, alkyd, aralkyl, alkenyl, alkynyl, cycloalky l, hydroxyl, alkoxy 1, amino, nitro, sulfhydryl, imino, amido, phosphonate, phosphinate, carbonyl, carboxyl, silyl, ether, alkylthio, sulfonyl, sulfonamido, ketone, aldehyde, thioketone, ester, heterocyclyl, -CN, ary l, aryloxy, perhaloalkoxy, aralkoxy, heteroaryl, heteroar loxy, heteroarylalkyl, heteroaralkoxy, azido, alkylthio, oxo, acylalkyl, carboxy esters, carboxamido, acyloxy, aminoalkyl, alkylaminoaryl,
  • carboxamidoalkylaryl carboxamidoaryl, hydroxyalkyl, haloalkyl, alkylaminoalkylcarboxy, aminocarboxamidoalkyl, cyano, alkoxyalkyl, perhaloalkyl, arylalkyloxyalkyl, and the like.
  • the substituent is selected from cyano, halogen, hydroxyl, and nitro.
  • Pharmaceutically acceptable salts can include, but are not limited to, amine salts, such as but not limited to N,N'-dibenzylethylenediamine, chloroprocaine, choline, ammonia, diethanolamine and other hydroxyalkylamines, ethylenediamine, N-methylglucamine, procaine, N-benzylphenethylamine. 1 -para-chlorobenzy 1-2-pyrrolidin- F- ylmethylbenzimidazole, diethylamineand other alkylamines.
  • amine salts such as but not limited to N,N'-dibenzylethylenediamine, chloroprocaine, choline, ammonia, diethanolamine and other hydroxyalkylamines, ethylenediamine, N-methylglucamine, procaine, N-benzylphenethylamine. 1 -para-chlorobenzy 1-2-pyrrolidin- F- ylmethylbenzimidazole, dieth
  • alkali metal salts such as but not limited to lithium, potassium and sodium
  • alkali earth metal salts such as but not limited to barium, calcium and magnesium
  • transition metal salts such as but not limited to zinc
  • other metal salts such as but not limited to sodium hydrogen phosphate and disodium phosphate
  • salts of mineral acids such as but not limited to hydrochlorides and sulfates
  • salts of organic acids such as but not limited to acetates, lactates, malates, tartrates, citrates, ascorbates, succinates, butyrates, valerates and fumarates.
  • salts of the disclosure can be synthesized from the parent compound that contains a basic or acidic moiety by conventional chemical methods such as methods described in Pharmaceutical Salts: Properties. Selection, and Use, P. Heinrich Stahl (ed), Camille G. Wermuth (ed), ISBN:3-90639-026-8, Hardcover, 388 pages, August 2002.
  • such salts can be prepared by reacting the free acid or base forms of these compounds with the appropriate base or acid in water or in an organic solvent, or in a mixture of the two; for example, non-aqueous media such as ether, ethyl acetate, ethanol, isopropanol, or acetonitrile are used.
  • esters can include, but are not limited to, alkyl, alkenyl, alkynyl, aryl, heteroaryl, aralkyl, heteroaralkyl, cycloalkyl and heterocyclyl esters of acidic groups, including, but not limited to, carboxylic acids, phosphoric acids, phosphinic acids, sulfonic acids, sulfinic acids and boronic acids.
  • solvates and hydrates are complexes of a compound with one or more solvent or water molecules, or 1 to about 100, or 1 to about 10, or one to about 2, 3 or 4, solvent or water molecules.
  • a reference to a compound of the disclosure and sub-groups thereof also includes ionic forms, salts, solvates, isomers, tautomers, esters, prodrugs, isotopes and protected forms thereof; such as, the salts or tautomers or isomers or solvates thereof; and more advantageously, the salts or tautomers or solvates thereof.
  • the term “isomer” can refer to molecules or polyamtoic ions with identical molecular formulas, but distinct arrangements of atoms in space.
  • constitutional isomers and stereoisomers of compounds described herein are also embodiments of the invention.
  • the enantiomers and diastereomers of compounds described herein can be aspects of the invention.
  • (R .S) of a compound is described herein, (R, R), (S, R), and (S, S) can also be aspects of the invention.
  • the term “enantiomer” can refer to molecules which are nonsuperimposable mirror images of each other.
  • the term “diastereomer” can refer to a stereoisomer of a compound having two or more chiral centers that is not a mirror image of another stereoisomer of the same compound.
  • R1 comprises a hydrogen, -Ph, a furan, a thiophene, a pyridine, a halogen, -CF 3 , -CN, -N(R 4 )(R 5 ), -OR 4 , -SR 4 , -CH 2 -NH-(CH 2 )n-N(R 4 )(R 5 ), -COOH, -COOR 4 , i Pr, t Bu, -(CH2)nCH3, -(CH2)n i Pr, -(CH2)n t Bu, -(CH2)nOR4, -(CH2)nN(R4)(R5), -(CH2)nCOOR4, -(CH 2 ) n CON(R 4 )(R 5 ), -(CH 2 ) n SR 4 , -(CH 2 ) n
  • R4 is hydrogen, alkyl, aryl and R 5 is hydrogen, alkyl, aryl.
  • R 3 comprises -COOH, -COOR 6 , or -NO 2 .
  • R4 comprises hydrogen, acyclic alkyl, C1-C9 alkyl, or aryl.
  • R 5 is hydrogen, acyclic alkyl, C 1 -C 9 alkyl, or aryl.
  • R6 is -CH3 or -CH2CH3.
  • A-B is -CO- or -(CHOH)- .
  • the halogen is fluorine, chlorine, bromine, or iodine.
  • A-B is
  • the compound comprises:
  • the compound is:
  • compositions comprising a compound of any one of the compounds described herein, or a combination thereof, and a pharmaceutically acceptable carrier, excipient, or diluent.
  • the formulations or pharmaceutical composition can also be included, or packaged, with other non-toxic compounds, such as pharmaceutically acceptable carriers, excipients, binders and fillers including, but not limited to, glucose, lactose, gum acacia, gelatin, mannitol, xanthan gum, locust bean gum, galactose, oligosaccharides and/or polysaccharides, starch paste, magnesium trisilicate, talc, com starch, starch fragments, keratin, colloidal silica, potato starch, urea, dextrans, dextrins, and the like.
  • pharmaceutically acceptable carriers including, but not limited to, glucose, lactose, gum acacia, gelatin, mannitol, xanthan gum, locust bean gum, galactose, oligosaccharides and/or polysaccharides, starch paste, magnesium trisilicate, talc, com starch, starch fragments, ker
  • the pharmaceutically acceptable carriers, excipients, binders, and fillers for use in the practice of the present invention are those which render the compounds of the invention amenable to intranasal delivery’, oral delivery, parenteral delivery', intravitreal delivery, intraocular delivery', ocular delivery, subretinal delivery, intrathecal delivery, intravenous delivery, subcutaneous delivery, transcutaneous delivery, intracutaneous delivery, intracranial delivery, topical delivery and the like.
  • the packaging material can be biologically inert or lack bioactivity, such as plastic polymers or silicone, and can be processed internally by the subject without affecting the effectiveness of the composition/formulation packaged and/or delivered therewith.
  • compositions described herein can be administered via oral administration.
  • the disclosed compositions are formulated in a pharmaceutically acceptable oral dosage form.
  • Oral dosage forms can comprise oral liquid dosage forms (such as tinctures, drops, emulsions, syrups, elixirs, suspensions, and solutions, and the like) and oral solid dosage forms.
  • the pharmaceutical compositions can also be prepared as formulations suitable for parenteral administration, intramuscular, subcutaneous, intraperitoneal, or intravenous injection, comprising physiologically acceptable sterile aqueous or non-aqueous solutions, dispersions, suspensions or emulsions, liposomes, and sterile powders for reconstitution into sterile injectable solutions or dispersions.
  • Parenteral administration can refer to administration via injection or infusion.
  • Parenteral administration includes, but is not limited to, subcutaneous administration, intravenous administration, intramuscular administration.
  • compounds and compositions described herein can be administered via intraperitoneal injection (I.P.), intravenous injection (I.V.), and intramuscular injection (I.M.).
  • the composition or pharmaceutical composition can be used alone or in combination with appropriate additives to make tablets, powders, granules or capsules, for example, with conventional additives, such as lactose, mannitol, com starch or potato starch; with binders, such as crystalline cellulose, cellulose derivatives, acacia, com starch or gelatins; with disintegrators, such as com starch, potato starch or sodium carboxymethylcellulose; with lubricants, such as talc or magnesium stearate; and if desired, with diluents, buffering agents, moistening agents, preservatives and flavoring agents.
  • conventional additives such as lactose, mannitol, com starch or potato starch
  • binders such as crystalline cellulose, cellulose derivatives, acacia, com starch or gelatins
  • disintegrators such as com starch, potato starch or sodium carboxymethylcellulose
  • lubricants such as talc or magnesium
  • Oral solid dosage can comprise lozenges, troches, tablets, capsules, caplets, powders, pellets, multiparticulates, beads, spheres, and/or any combinations thereof.
  • Oral solid dosage forms can be formulated as immediate release, controlled release, sustained release, extended release, or modified release formulations.
  • the disclosed oral solid dosage forms can be in the form of a tablet (including a suspension tablet, a fast-melt tablet, a bite-disintegration tablet, a rapid-disintegration tablet, an effervescent tablet, or a caplet), a pill, a powder (including a sterile packaged powder, a dispensable powder, or an effervescent powder), a capsule (including both soft or hard capsules, e.g., capsules made from animal-derived gelatin or plant-derived HPMC, or “sprinkle capsules”), solid dispersion, solid solution, bioerodible dosage form, controlled release formulations, pulsatile release dosage forms, multiparticulate dosage forms, pellets, granules, or an aerosol.
  • a tablet including a suspension tablet, a fast-melt tablet, a bite-disintegration tablet, a rapid-disintegration tablet, an effervescent tablet, or a caplet
  • a pill including a sterile packaged powder
  • the pharmaceutical formulation can be in the form of a powder. In still other embodiments, the pharmaceutical formulation can be in the form of a tablet, including a fast-melt tablet. Additionally, pharmaceutical formulations can be administered as a single capsule or in multiple capsule dosage form. In some embodiments, the pharmaceutical formulation can be administered in two, three, four, or more capsules or tablets.
  • Oral solid dosage forms can contain pharmaceutically acceptable excipients such as fillers, diluents, lubricants, surfactants, glidants, binders, dispersing agents, suspending agents, disintegrants, viscosity-increasing agents, film-forming agents, granulation aid, flavoring agents, sweetener, coating agents, solubilizing agents, and combinations thereof.
  • pharmaceutically acceptable excipients such as fillers, diluents, lubricants, surfactants, glidants, binders, dispersing agents, suspending agents, disintegrants, viscosity-increasing agents, film-forming agents, granulation aid, flavoring agents, sweetener, coating agents, solubilizing agents, and combinations thereof.
  • Oral solid dosage forms also can comprise one or more pharmaceutically acceptable additives such as a compatible carrier, complexing agent, ionic dispersion modulator, disintegrating agent, surfactant, lubricant, colorant, moistening agent, plasticizer, stabilizer, penetration enhancer, wetting agent, anti-foaming agent, alone or in combination, as well as supplementary active compound(s).
  • a compatible carrier complexing agent, ionic dispersion modulator, disintegrating agent, surfactant, lubricant, colorant, moistening agent, plasticizer, stabilizer, penetration enhancer, wetting agent, anti-foaming agent, alone or in combination, as well as supplementary active compound(s).
  • Oral liquid dosage forms include tinctures, drops, emulsions, syrups, elixirs, suspensions, and solutions, and the like. These oral liquid dosage forms may be formulated with any pharmaceutically acceptable excipient known to those of skill in the art for the preparation of liquid dosage forms, and with solvents, diluents, carriers, excipients, and the like chosen as appropriate to the solubility and other properties of the active agents and other ingredients. Solvents may be. for example, water, glycerin, simple syrup, alcohol, medium chain triglycerides (MCT), and combinations thereof.
  • MCT medium chain triglycerides
  • Liquid dosage forms for oral administration may be in the form of pharmaceutically acceptable emulsions, syrups, elixirs, suspensions, and solutions, which may contain an inactive diluent, such as water.
  • Pharmaceutical formulations can be prepared as liquid suspensions or solutions using a sterile liquid, such as but not limited to, an oil, water, an alcohol, and combinations of these pharmaceutically suitable surfactants, suspending agents, emulsifying agents, can be added for oral or parenteral administration.
  • Liquid formulations also may be prepared as single dose or multi-dose beverages.
  • Suspensions may include oils. Such oils include peanut oil, sesame oil, cottonseed oil, com oil, and olive oil.
  • Suitable oils also include carrier oils such as MCT and long chain triglyceride (LCT) oils.
  • Suspension preparation may also contain esters of fatty acids such as ethyl oleate, isopropyl myristate, fatty acid glycerides, and acetylated fatty acid glycerides.
  • Suspension formulations may include alcohols, (such as ethanol, isopropyl alcohol, hexadecyl alcohol), glycerol, and propylene glycol.
  • Ethers such as poly(ethylene glycol), petroleum hydrocarbons such as mineral oil and petrolatum, and water may also be used in suspension formulations.
  • Suspension can thus include an aqueous liquid or a non-aqueous liquid, an oil-in-w ater liquid emulsion, or a w ater- in-oil emulsion.
  • formulations comprising the disclosed compositions and at least one dispersing agent or suspending agent for oral administration to a subject.
  • the formulation may be a powder and/or granules for suspension, and upon admixture with water, a substantially uniform suspension is obtained.
  • the aqueous dispersion can comprise amorphous and non-amorphous particles consisting of multiple effective particle sizes such that a drug is absorbed in a controlled manner over time.
  • Supplementary active compounds include preservatives, antioxidants, antimicrobial agents including biocides and biostats such as antibacterial, antiviral and antifungal agents.
  • Preservatives can be used to inhibit microbial growth or increase stability of the active ingredient thereby prolonging the shelf life of the formulation.
  • Suitable preservatives are known in the art and include EDTA, EGTA, benzalkonium chloride or benzoic acid or benzoates, such as sodium benzoate.
  • Antioxidants include vitamin A, vitamin C (ascorbic acid), vitamin E, tocopherols, other vitamins or provitamins, and compounds such as alpha lipoic acid.
  • Unit dosage forms for oral administration such as syrups, elixirs, and suspensions
  • each dosage unit for example, teaspoonful, tablespoonful, tablet or suppository'
  • unit dosage forms for injection or intravenous administration can comprise the composition or pharmaceutical composition in a composition as a solution in sterile water, normal saline or another pharmaceutically acceptable carrier.
  • Embodiments of the composition or pharmaceutical composition can be formulated into preparations for injection by dissolving, suspending, or emulsifying them in an aqueous or non-aqueous solvent, such as vegetable or other similar oils, synthetic aliphatic acid glycerides, esters of higher aliphatic acids or propylene glycol; and if desired, with conventional additives such as solubilizers, isotonic agents, suspending agents, emulsifying agents, stabilizers and preservatives.
  • Embodiments of the composition or pharmaceutical composition can be utilized in aerosol formulation to be administered via inhalation.
  • Embodiments of the composition or pharmaceutical composition can be formulated into pressurized acceptable propellants such as dichlorodifluoromethane, propane, nitrogen and the like.
  • Embodiments of the composition or pharmaceutical composition can be formulated in an injectable composition in accordance with the disclosure.
  • injectable compositions are prepared as liquid solutions or suspensions; solid forms suitable for solution in, or suspension in, liquid vehicles prior to injection can also be prepared.
  • the preparation can also be emulsified or the active ingredient (triamino-pyridine derivative and/or the labeled triamino-pyridine derivative) encapsulated in liposome vehicles in accordance with the disclosure.
  • composition or pharmaceutical composition can be formulated for delivery by a continuous delivery system.
  • continuous delivery system is used interchangeably herein with “controlled delivery system” and encompasses continuous (e.g., controlled) delivery' devices (e.g., pumps) in combination with catheters, injection devices, and the like, a wide variety of which are known in the art.
  • Embodiments of the composition or pharmaceutical composition can be administered to a subject in one or more doses.
  • dose levels can vary as a function of the specific composition or pharmaceutical composition administered, the severity of the symptoms and the susceptibility of the subject to side effects. Dosages for a given compound are readily determinable by those of skill in the art by a variety of means.
  • compositions or pharmaceutical composition are administered.
  • the frequency of administration of the composition or pharmaceutical composition can vary depending on any of a variety of factors, e.g.. severity of the symptoms, and the like.
  • the composition or pharmaceutical composition can be administered once per month, twice per month, three times per month, every other week (qow), once per week (qw), twice per week (biw), three times per week (tiw), four times per week, five times per week, six times per week, every other day (qod), daily (ad), twice a day (qid). three times a day (tid), or four times a day.
  • the composition or pharmaceutical composition is administered 1 to 4 times a day over a 1 to 10- day time period.
  • the duration of administration of the composition or pharmaceutical composition analogue can vary, depending on any of a variety of factors, including patient response.
  • the composition or pharmaceutical composition in combination or separately can be administered over a period of time of about one day to one week, about one day to two weeks.
  • Embodiments of the composition or pharmaceutical composition can be administered to a subject using available conventional methods and routes suitable for delivery of conventional drugs, including systemic or localized routes. Routes of administration can include, but are not limited to, enteral administration, parenteral administration, or inhalation.
  • Routes of administration can include, but are not limited to, enteral administration, parenteral administration, or inhalation.
  • Other compositions, compounds, methods, features, and advantages of the disclosure will be or become apparent to one having ordinary skill in the art upon examination of the following drawings, detailed description, and examples. It is intended that all such additional compositions, compounds, methods, features, and advantages be included within this description, and be within the scope of the disclosure.
  • aspects of the invention are drawn towards method of treating a subject afflicted with an oxalate production-related disease or disorder, the method comprising: administering to the subject a therapeutically effective amount of a compound described herein.
  • the disease comprises a cardiometabolic disease, a cardiovascular disease, a metabolic disease, a liver disease, a renal disease, or a combination thereof.
  • a metabolic disease can refer to to a group of diseases and/or disorders in which errors of metabolism, imbalances in metabolism, or sub-optimal metabolism occur.
  • the metabolic diseases as described herein can comprise diseases that can be treated through the modulation of metabolism, although the disease itself can by a specific metabolic defect. Such metabolic diseases can involve, for example, glucose and fatty acid oxidation pathways.
  • metabolic disorder or “metabolic disease” refers to a condition characterized by an alteration or disturbance in metabolic function.
  • Metabolic and “metabolism” are terms known in the art and can comprise a range of biochemical processes that occur within a living organism. Metabolic and cardiovascular disease includes, but is not limited to.
  • the dyslipidemia can be hyperlipidemia.
  • the hyperlipidemia can be hypercholesterolemia, hypertriglyceridemia, or both hypercholesterolemia and hypertriglyceridemia.
  • the NAFLD can be hepatic steatosis or steatohepatitis.
  • the diabetes can be type 2 diabetes or type 2 diabetes with dyslipidemia.
  • methods are presented herein that are applicable to metabolic diseases related to glucose dysregulation and/or accumulation of lipids in the body, circulation or various organs, for example, the liver, and the pathological sequelae resulting therefrom, for example, (non-alcoholic fatty liver disease (NAFLD), non-alcoholic steatohepatitis (NASH), hyperglycemia, prediabetes, diabetes (type I and ty pe II), obesity, insulin resistance, metabolic syndrome and diabetic dyslipidemia.
  • Certain such metabolic diseases, disorders or conditions include, but are not limited to, hyperglycemia, prediabetes, diabetes (type I and type II), obesity 7 , insulin resistance, metabolic syndrome and diabetic dyslipidemia.
  • the metabolic disease, disorder or condition can be characterized by numerous physical symptoms. Any symptom known to one of skill in the art to be associated with the metabolic disease, disorder or condition can be prevented, treated, ameliorated or otherwise modulated with the compounds and methods described herein.
  • the symptom can be any of, but not limited to, excessive urine production (polyuria), excessive thirst and increased fluid intake (polydipsia), blurred vision, unexplained weight loss and lethargy.
  • cardiovascular diseases, disorders or conditions can comprise aortic stenosis, aneurysm (e.g., abdominal aortic aneurysm), angina, arrhythmia, atherosclerosis, cerebrovascular disease, coronary artery disease, carotid artery' disease, coronary' heart disease, dyslipidemia, heart failure, hypercholesterolemia, hyperlipidemia, hypertension, hypertriglyceridemia, myocardial infarction, peripheral vascular disease (e.g. peripheral artery disease, peripheral artery occlusive disease), thromboembolic diseases, retinal vascular occlusion, or stroke.
  • aneurysm e.g., abdominal aortic aneurysm
  • angina e.g., arrhythmia, atherosclerosis
  • cerebrovascular disease CAD
  • coronary artery disease e.g., carotid artery' disease
  • coronary' heart disease e.g., dyslipidemia, heart failure
  • nonalcoholic steatohepatitis NASH
  • metabolic dysfunction-associated steatohepatitis MASH
  • NASH nonalcoholic steatohepatitis
  • NAFLD nonalcoholic fatty liver disease
  • MASLD metabolic dysfunction-associated steatotic liver disease
  • liver disease and “hepatic disease” can be used interchangeably and can refer to damage to or a disease of the liver.
  • Non-limiting examples of liver disease include intrahepatic cholestasis (e.g..).
  • liver abscess e.g., amebic liver abscess
  • liver cirrhosis e.g., alcoholic, biliary, and experimental liver cirrhosis
  • alcoholic liver diseases e.g., fatty liver, hepatitis, cirrhosis
  • parasitic liver disease e.g., hepatic echinococcosis, fascioliasis, amebic liver abscess
  • jaundice e.g., hemolytic, hepatocellular, cholestatic jaundice
  • cholestasis portal hypertension, liver enlargement, ascites, hepatitis (e.g., alcoholic hepatitis,
  • hepatitis C hepatitis D, hepatitis E
  • granulomatous hepatitis secondary biliary cirrhosis, hepatic encephalopathy, varices, primary biliary cirrhosis, primary sclerosing cholangitis, hepatic steatosis or steatohepatitis, hepatocellular adenoma, hemangiomas, bile stones, liver failure (e.g., hepatic encephalopathy, acute liver failure), angiomyolipoma. calcified liver metastases, cystic liver metastases, fibrolamellar hepatocarcinoma.
  • hepatic adenoma hepatoma
  • hepatic cysts e.g.. Simple cysts. Polycystic liver disease, hepatobiliary cystadenoma, choledochal cyst
  • mesenchymal tumors meenchymal hamartoma, infantile hemangioendothelioma, hemangioma, peliosis hepatis, lipomas, inflammatory pseudotumor
  • epithelial tumors e.g., bile duct hamartoma, bile duct adenoma
  • focal nodular hyperplasia nodular regenerative hyperplasia
  • hepatoblastoma hepatocellular carcinoma
  • cholangiocarcinoma, cystadenocarcinoma tumors of blood vessels, angiosarcoma, Karposi's sarcoma, hemangioendothelioma, embry onal sar
  • the liver disease can comprise fibrosis, a liver disease characterized in that the liver has excess cholesterol, triglycerides or other lipids that is illustrative of liver diseases such as NAFLD, NASH or alcoholic related steatosis of the liver, liver cirrhosis or liver inflammation or hepatocellular carcinoma.
  • liver diseases such as NAFLD, NASH or alcoholic related steatosis of the liver, liver cirrhosis or liver inflammation or hepatocellular carcinoma.
  • renal disease and “kidney disease” can be used interchangeably and can refer to a disease or condition in which the function of a subject’s kidney is impaired.
  • the renal disease can comprise Abderhalden- Kaufmann- Lignac syndrome (Nephropathic Cystinosis), Nephrotoxicity.
  • Acute Kidney Failure/ Acute Kidney Injury Acute Lobar Nephroma, Acute Phosphate Nephropathy, Acute Tubular Necrosis, Adenine Phosphoribosyltransferase Deficiency, Adenovirus Nephritis, Alagille Syndrome, Alport Syndrome, Amyloidosis, ANCA Vasculitis Related to Endocarditis and Other Infections, Angiomyolipoma, Analgesic Nephropathy, Antiphospholipid Syndrome, Anti-TNF-a Therapy-related Glomerulonephritis, APOL1 Mutations, Apparent
  • Collapsing Glomerulopathy Collapsing Glomerulopathy Related to CMV, Combination Antiretroviral (cART) Related-Nephropathy, Congenital Anomalies of the Kidney and Urinary Tract (CAKUT), Congenital Nephrotic Syndrome, Congestive Renal Failure, Conorenal syndrome (Mainzer-Saldino Syndrome or Saldino-Mainzer Disease), Contrast Nephropathy, Cortical Necrosis.
  • Cryocrystalglobulinemia Cryoglobuinemia, Cystic Kidney Disease, Acquired, Cystinuria, Dasatinib-Induced Nephrotic-Range Proteinuria, Dense Deposit Disease (MPGN Type 2), Dent Disease (X-linked Recessive Nephrolithiasis), DHA Cry stalline Nephropathy, Dialysis Disequilibrium Syndrome, Diabetes and Diabetic Kidney Disease, Diabetes Insipidus, Diffuse Mesangial Sclerosis, Diuresis, Duplicated Ureter.
  • EAST syndrome Ebola and the Kidney, Ectopic Kidney, Ectopic Ureter, Edema, Swelling, Erdheim-Chester Disease, Fabry 's Disease, Familial Hypocalciuric Hypercalcemia, Fanconi Syndrome, Fraser syndrome, Fibronectin Glomerulopathy, Fibrillary Glomerulonephritis and Immunotactoid Glomerulopathy , Fraley syndrome, Fluid Overload, Hypervolemia, Focal Segmental Glomerulosclerosis, Focal Sclerosis.
  • Focal Glomerulosclerosis Galloway Mowat syndrome, Giant Cell (Temporal) Arteritis with Kidney Involvement, Gestational Hypertension, Gitelman Syndrome, Glomerular Diseases, Glomerular Tubular Reflux, Glycosuria, Goodpasture Syndrome, HANAC Syndrome, Heat Stress Nephropathy, Hematuria (Blood in Urine), Hemolytic Uremic Syndrome (HUS), Atypical Hemolytic Uremic Syndrome (aHUS).
  • Hemophagocytic Syndrome Hemorrhagic Cystitis
  • Hemorrhagic Fever with Renal Syndrome HFRS, Hantavirus Renal Disease, Korean Hemorrhagic Fever, Epidemic Hemorrhagic Fever, Nephropathis Epidemica
  • Hemosiderinuria Hemosiderosis related to Paroxysmal Nocturnal Hemoglobinuria and Hemolytic Anemia.
  • Hepatic Glomerulopathy Hepatic Veno- Occlusive Disease, Sinusoidal Obstruction Syndrome, Hepatitis C-Associated Renal Disease, Hepatocy te Nuclear Factor 10- Associated Kidney Disease, Hepatorenal Syndrome, Herbal Supplements and Kidney Disease, High Altitude Renal Syndrome, High Blood Pressure and Kidney Disease, HIV- Associated Immune Complex Kidney Disease (HIVICK), HIV- Associated Nephropathy (HIVAN), HNFIB-related Autosomal Dominant Tubulointerstitial Kidney Disease.
  • HAVICK HIV- Associated Immune Complex Kidney Disease
  • HNFIB HIV- Associated Nephropathy
  • Renal Artery Dissection Spontaneous, Renal Artery Stenosis, Renal Cell Cancer, Renal Cyst, Renal Hypouricemia with Exercise-induced Acute Renal Failure, Renal Infarction, Renal Osteodystrophy, Renal Tubular Acidosis, Renin Mutations and Autosomal Dominant Tubulointerstitial Kidney Disease, Renin Secreting Tumors (Juxtaglomerular Cell Tumor), Reset Osmostat, Retroperitoneal Fibrosis, Rhabdomyolysis, Rheumatoid Arthritis-Associated Renal Disease, Sarcoidosis Renal Disease, Salt Wasting, Renal and Cerebral, Schistosomiasis and Glomerular Disease, Schimke immuno-osseous dysplasia, Scleroderma Renal Crisis, Serpentine Fibula- Polycystic Kidney Syndrome, Exner Syndrome, Sickle Cell Nephropathy, Silica Exposure and Chronic Kidney Disease, Sri Lankan farmers' Kidney Disease, Sjogren's
  • the term hyperoxaluria can refer to a high concentration of oxalate in urine.
  • the hyperoxaluria can comprise primary hyperoxalurias (PH-1, PH- 2 and PH-3), secondary hyperoxaluria, idiopathic calcium oxalate urolithiasis.
  • the disease can comprise primary hyperoxaluria, for example PH-1.
  • hyperoxaluria refers to a high concentration of oxalate in urine. This situation may have a number of different causes. Hyperoxalurias can be classified as primary and secondary. Primary hyperoxalurias (PH) are a group of autosomal recessive genetic disorders involving enzyme failures that lead to an endogenous surplus production of oxalate. PH can comprise PHI, PH2, and PH3, with PHI being the most common and the most aggressive. [(1) Bhasin, B. Primary and Secondary Hyperoxaluria: Understanding the Enigma.
  • Secondary hyperoxalurias can be due to an excessive absorption of oxalate or its precursors in the bowel. This is linked to a diet rich in said precursors, or in the case of enteric hyperoxaluria, to an absorption disorder after bowel resection.
  • PH-1 Primary hyperoxaluria type 1 (PH-1) is a serious hereditary disease due to a deficiency of the AGT enzyme (encoded by the Agxtl gene) in hepatocytes [Zhang, X ; Roe,
  • This enzy me, AGT is in charge of metabolizing glyoxylate in hepatic peroxisomes by transamination into glycine.
  • PH-1 where AGT activity is absent or the enzyme is erroneously located in the mitochondria, glyoxylate accumulation occurs as a result.
  • Glyoxylate comes to be metabolized by oxidation to oxalate, a process that is catalyzed by glycolate oxidase (GO) enzymes in peroxisomes and lactate dehydrogenase (LDH) in the cytoplasm.
  • GO glycolate oxidase
  • LDH lactate dehydrogenase
  • PH-1 is a rare disease with an estimated incidence in Europe of 1 : 100000 births per year [Cochat, P.; Hulton, S.A.; Acquaviva, C.; Danpure, C. J.; Daudon, M.; Marchi, M. D.; Fargue, S.; Groothoff, J.; Harambat, J.; Hoppe, B.; et al. Nephrol. Dial. Transplant.
  • the disease comprises a GO-associated disease and/or lactate dehydrogenase (LDHA)-associated disease.
  • LDHA lactate dehydrogenase
  • the compound described herein comprises:
  • subject or “patient” can refer to any organism to which aspects of the invention can be administered, e.g., for experimental, diagnostic, prophylactic, and/or therapeutic purposes.
  • subjects to which compounds of the disclosure can be administered include animals, such as mammals.
  • mammals include primates, such as humans.
  • veterinary applications a wide variety of subjects will be suitable, e.g., livestock such as cattle, sheep, goats, cows, swine, and the like; poultry such as chickens, ducks, geese, turkeys, and the like; and domesticated animals for example pets such as dogs and cats.
  • living subject can refer to a subject noted above or another organism that is alive.
  • living subject can refer to the entire subject or organism and not just a part excised (e.g., a liver or other organ) from the living subject.
  • pharmaceutically acceptable derivatives of a compound can include salts, esters, enol ethers, enol esters, acetals, ketals, orthoesters, hemiacetals, hemiketals, acids, bases, solvates, hydrates or prodrugs thereof.
  • Such derivatives can be readily prepared by those of skill in this art using known methods for such derivatization.
  • the compounds produced can be administered to animals or humans without substantial toxic effects and either are pharmaceutically active or are prodrugs.
  • the method can comprise measuring the circulating oxalate levels of a subject.
  • therapeutic levels of the compounds described herein can be administered.
  • a subject’s oxalate levels can be measured by enzymatic assay, mass spectrometry, or a combination thereof.
  • the mass spectrometry can comprise LC-MS/MS.
  • a clinician of ordinary skill in the art can determine if the subject’s oxalate levels are elevated.
  • elevated circulating oxalate levels can comprise less than about 1 uM, about 1 uM, about 1.5 uM, about 2 uM, about 2.5 uM, about 3 uM, about 4 uM, about 5 uM, about 6 uM, about 7 uM, about 8 uM, about 9 uM, about 10 uM, or greater than about 10 uM.
  • administering can refer to introducing a substance, such as the compounds described herein, or derivatives thereof, isomers thereof, or a combination thereof into a subject. Any route of administration can be utilized including, for example, intranasal, topical, oral, parenteral, intravitreal, intraocular, ocular, subretinal, intrathecal, intravenous, subcutaneous, transcutaneous, intracutaneous, intracranial and the like administration. In embodiments, "administering” can also refer to providing a therapeutically effective amount of a formulation or pharmaceutical composition to a subject.
  • the formulation or pharmaceutical compound can be administered alone, but can be administered with other compounds, excipients, fillers, binders, carriers or other vehicles selected based upon the chosen route of administration and standard pharmaceutical practice. Administration can be by way of carriers or vehicles, such as injectable solutions, including sterile aqueous or nonaqueous solutions, or saline solutions; creams; lotions; capsules; tablets; granules; pellets; powders; suspensions, emulsions, or microemulsions; patches; micelles; liposomes; vesicles; implants, including microimplants; eye drops; other proteins and peptides; synthetic polymers; microspheres; nanoparticles; and the like.
  • carriers or vehicles such as injectable solutions, including sterile aqueous or nonaqueous solutions, or saline solutions; creams; lotions; capsules; tablets; granules; pellets; powders; suspensions, emulsions, or microemulsions; patches; micelles; lip
  • the therapeutically effective amount of the composition inhibits GO activity, LDHA activity, or a combination thereof.
  • a therapeutically effective amount of a composition described herein can comprise less than about 0.1 mg/kg, about 0.1 mg/kg, about 0.5 mg/kg, about 1.0 mg/kg, about 2.5 mg/kg, about 5 mg/kg, about 7.5 mg/kg, about 10 mg/kg, about 15 mg/kg, about 20 mg/kg, about 25 mg/kg, about 30 mg/kg, about 35 mg/kg, about 40 mg/kg, about 45 mg/kg, about 50 mg/kg, about 55 mg/kg, about 60 mg/kg, about 70 mg/kg, about 80 mg/kg, about 90 mg/kg, about 100 mg/kg, about 120 mg/kg, about 135 mg/kg, about 150 mg/kg.
  • a therapeutically effective amount of a composition described herein can comprise a concentration of less than about 1 nM, about 1 nM, about 5 nM, about 10 nM, about 20 nM, about 25 nM, about 30 nM, about 40 nM, about 50 nM, about 60 nM, about 75 nM, about 80 nM, about 90 nM, about 100 nM, about 200 nM, about 250 nM, about 300 nM, about 400 nM. about 500 nM, about 600 nM, about 700 nM, about 800 nM, about 900 nM, about 1000 nM. and greater than about 1000 nM.
  • the concentration can be about 500 nM.
  • the concentration can be about 700 nM.
  • Example 1 Genetic and pharmacological targetin of hepatic oxalate overproduction ameliorates nonalcoholic steatohepatitis
  • oxalate promotes lipid accumulation in hepatocytes by impairing the transcription of peroxisome proliferator activated receptor-alpha (PPARa) and inhibiting fatty acid P-oxidation (FAO).
  • PPARa peroxisome proliferator activated receptor-alpha
  • FAO fatty acid P-oxidation
  • blocking oxalate overproduction through hepatocyte-specific AGXT overexpression or pharmacological inhibition of glycolate oxidase and lactate dehydrogenase potently lowers hepatic oxalate, steatosis, inflammation, and fibrosis by inducing PPARa- driven FAO, and suppressing leukocyte chemotaxis, and nuclear factor-kappaB and transforming growth factor-beta targets.
  • Nonalcoholic fatty liver disease has become the most common chronic liver disease worldwide, affecting an alarming one-third of the global population 1 .
  • NAFLD encompasses a spectrum of liver pathologies, ranging from simple steatosis, nonalcoholic steatohepatitis (NASH) that can or cannot be accompanied by hepatic fibrosis, and ultimately cirrhosis, which is associated with late-stage liver disease and can lead to hepatocellular carcinoma and liver failure 2,3 .
  • NASH nonalcoholic steatohepatitis
  • hepatic lipid overload occurs with excessive uptake of fatty acids released from the adipose tissue 4 , enhanced de novo lipogenesis from excessive carbohydrate intake 5 , or inhibition of fatty acid ⁇ -oxidation (FAO).
  • FEO fatty acid ⁇ -oxidation
  • ER endoplasmic reticulum
  • inflammasome activation leads to the release and accumulation of proinflammatory mediators, which facilitate leukocyte infiltration and hepatic stellate cell activation, promoting NASH and hepatic fibrosis 2,3,4, .
  • AGXT loss-of-function results in the accumulation of glyoxylate 15 .
  • Glyoxylate can also be produced from glycolate by glycolate oxidase (GO) and is rapidly converted by lactate dehydrogenase (LDHA) to the terminal endproduct oxalate 16 .
  • GO glycolate oxidase
  • LDHA lactate dehydrogenase
  • Fig. 1 Panel H We fed male C57BL/6J mice a standard chow diet (control) or a high-fat, high-fructose, high-cholesterol diet (NASH diet) for 6 months (Fig.1 Panel H).
  • This dietary model is known to induce steatohepatitis and hepatic fibrosis that mimic the human disease 10,25,26,27 , as we confirmed using H&E and Picrosirius Red staining (Fig. 1 Panel I).
  • livers from mice with NASH demonstrated a significant reduction in Agxt mRNA expression (Fig. 1 Panel J) and AGXT protein abundance (Fig. 1 Panel K and Panel L).
  • HepG2 human hepatoma cell line which largely retain the biochemical pathways of oxalate metabolism 29 .
  • HepG2 cells also demonstrated a significant reduction in AGXT following palmitic acid treatment (Fig. 1 Panel N and Panel O). Accordingly, a significant accumulation of intracellular oxalate was found in both primary' hepatocytes (Fig. 1 Panel P) and HepG2 ceils (Fig. 1 Panel Q).
  • hepatic AGXT is suppressed leading to oxalate overproduction in both humans and mice with NASH as well as in lipid- loaded hepatocytes.
  • PCA Principal component analysis revealed that the gene expression pattern of livers from mice treated with AAV8-GFP was distinct from those treated with AAV8-AGXT (Fig.3 Panel A).
  • Volcano plot showed over 700 differentially expressed genes (DEGs), with 508 significantly reduced and 199 significantly elevated in livers from mice overexpressing AGXT compared to mice treated with AAV8-GFP (Fig.3 Panel B).
  • KEGG pathway analysis showed a most significant enrichment in the peroxisome pathway together with other key pathways related to FAO, including fatty acid degradation and PPAR signaling pathways (Fig.3 Panel C).
  • RNA-sequencing analysis (Fig.3 Panel D), validated by qRT-PCR analyses (Fig.3 Panel E), revealed that key genes driving hepatic FAO, including peroxisome proliferator activated receptor alpha (Ppara), the master regulator of FAO 30, , PPAR ⁇ coactivator-1 ⁇ (Ppargc1a), and numerous of PPAR ⁇ target genes (carnitine palmitoyltransferase 1A, [Cpt1a], acyl-CoA dehydrogenase, medium chain [Acadm], acyl-CoA dehydrogenase, long Chain [Acadl], acyl- CoA dehydrogenase very long Chain [Acadvl], acyl-CoA oxidase 1 [Acox1], hydroxyacyl-CoA dehydrogenase subunit alpha [Hadha], Hadhb, acetyl-CoA acyltransferase 2 [Acaa2], and acy
  • livers from mice overexpressing AGXT had significantly reduced neutral lipid accumulation (Fig. 3 Panels F and G). Furthermore, biochemical analysis of liver lysates confirmed a significant reduction in hepatic triglycerides with AGXT overexpression (Fig. 3 Panel H).
  • livers from mice overexpressing AGXT had significantly reduced levels of the lipid peroxidation marker, malondialdehyde (MDA), assessed by the thiobarbituric acid reactive substances (TBARS) assay (Fig. 11 Panel E).
  • MDA malondialdehyde
  • TBARS thiobarbituric acid reactive substances
  • Oxalate induces lipid accumulation in hepatocytes through suppression of PPARa- regulated fatty acid 0-oxidation
  • Oxalate induced lipid accumulation as assessed by Nile Red staining in both mouse primary hepatocytes and HepG2 cells Fig. 4 Panels A and B.
  • oxalate promotes lipid accumulation in hepatocytes
  • oxalate did not significantly alter the expression of key genes regulating fatty' acid uptake and transport including CD36, solute carrier family 27 member 2 (SLC27A2), SI.C27A4. and SLC27A5.
  • fatty acid and lipid biosynthesis In regards to fatty acid and lipid biosynthesis, primary hepatocytes treated with oxalate showed a significant increase in the key lipogenic genes, fatty acid synthase (Fasn) and acetyl-CoA carboxylase alpha (Acaca). with no significant differences in the expression of stearoyl-Coenzyme A desaturase 1 (ScdP) or sterol regulatory element binding transcription factor 1 (SrebfT). the master regulator of fatty acid and lipid biosynthesis 31 (Fig. 13 Panel C). Furthermore, HepG2 cells treated with oxalate showed no significant alterations in genes regulating fatty' acid and lipid biosynthesis (Fig. 13 Panel D).
  • oxalate significantly reduced the protein abundance of CPTl ⁇ , which is regulated by PPARa and catalyzes the rate-limiting step of FAO by converting acyl- CoAs into acylcamitines allowing their subsequent mitochondrial ⁇ -oxidation 30 (Fig. 4 Panel E).
  • oxalate directly regulates PPARa transcriptional activity
  • HepG2 cells were transfected with a PPAR response element (PPRE) reporter (PPREx3-TK-luciferase), human PPARa and Renilla constructs, and treated with or without oxalate or with the PPARa agonist Wy 14,643 32 as a positive control.
  • PPRE PPAR response element
  • PPREx3-TK-luciferase PPAR response element
  • Oxalate reduction via AGXT overexpression lowers leukocyte infiltration, hepatic inflammation and fibrosis in NASH
  • RNA-sequencing analysis revealed a significant downregulation of multiple pathways involved in inflammation including chemokine signaling, cytokine-cytokine receptor interaction, as well as nuclear factor-kappaB (NF-KB) and tumor necrosis factor (TNF) signaling pathways (Fig. 5 Panel A).
  • HepG2 cells were plated in the lower well of a transwell, and the transmigration of fluorescently-labeled human primary blood monocytes (hPBMs) was measured in cells treated with or without oxalate (Fig.5 Panel F). Consistent with our in vivo findings, there was a significant elevation of monocytes transmigrating toward the bottom well containing oxalate-treated HepG2 cells compared with control cells (Fig. 5 Panels G and H).
  • Hepatic fibrosis is the main determinant of liver-related events and mortality in NASH 35 .
  • the KEGG pathway analysis revealed a significant downregulation of pathways associated with hepatic fibrosis including focal adhesion signaling, regulation of actin cytoskeleton and extracellular matrix (ECM)-receptor interactions (Fig. 5 Panel A).
  • RNA-sequencing analysis Fig. 6 Panel A
  • Fig. 6 Panel A validated by qRT- PCR analyses
  • Oxalate is produced from glyoxylate in the liver by multiple enzymatic pathways 16 (Fig. 7A). Because AGXT overexpression lowers hepatic oxalate overproduction and prevents NASH, we evaluated 1) whether these protective effects are mediated by AGXT or through an oxalate-lowering effect and 2) to assess the therapeutic value of pharmacologically targeting hepatic oxalate overproduction in established NASH.
  • MDMG-935P a salicylic acid derivative we recently developed that potently decreases oxalate production by inhibiting GO and LDHA 37 .
  • mice While GO generates glyoxylate, which is cleared by AGXT, LDHA catalyzes the oxidation of glyoxylate to form oxalate. 16
  • mice To assess the therapeutic potential of oxalate lowering, we devised an experimental approach to test MDMG-935P in mice with established NASH. Mice w ere fed the NASH diet for 3 months, then orally administered either vehicle, 5 mg/kg or 10 mg/kg MDMG-935P daily for an additional 3 months on the NASH diet (Fig. 7 Panel B).
  • Fig. 7 Panel E markers of liver injury in plasma
  • ALT Fig. 7 Panel F
  • mice treated with 10 mg/kg/day of MDMG-935P were significantly decreased in mice treated with 10 mg/kg/day of MDMG-935P.
  • gross morphology of the abdominal body cavity show ed no obvious differences in adiposity in all groups (Fig. 7 Panel G)
  • histopathological analysis of the livers revealed significant reductions in steatosis, lobular inflammation, hepatocellular ballooning, and overall NAS in mice treated with 10 mg/kg/day of MDMG-935P (Fig. 7 Panel T).
  • liver lysates confirmed a significant reduction in hepatic triglycerides in mice treated with 10 mg/kg/day of MDMG-935P (Fig. 7 Panel J).
  • genes regulating FAO were significantly upregulated in livers from mice treated with 10 mg/kg/day of MDMG-935P including Cptla, Acadl, Acadvl, Haclha. and Hadhb (Fig. 7 Panel K).
  • mice administered 10 mg/kg/day of MDMG-935P exhibited significant reductions in the expression of key chemokines and cytokines driving the proinflammatory response in NASH including Ccl2. Ccl5. and Tnf (Fig. 8 Panel A).
  • hepatic oxalate suppresses transcription of PPAR ⁇ and the expression of its target genes controlling FAO (CPT1 ⁇ ), leading to impaired mitochondrial fatty acid utilization, lipotoxicity, leukocyte chemotaxis, hepatic inflammation, and fibrosis.
  • CPT1 ⁇ FAO
  • lowering hepatic oxalate in NASH by genetic (hepatocyte-specific AGXT overexpression by AAV8-AGXT) and pharmacological (GO and LDHA inhibition by MDMG-935P) targeting of oxalate overproduction ameliorates hepatic steatosis, inflammation, and fibrosis through induction of PPAR ⁇ -driven FAO, and suppression of NF- ⁇ B and TGF ⁇ targets (Fig.8 Panel K).
  • dysregulated oxalate metabolism can cause renal and cardiovascular diseases 17-21 , studies have focused on the deleterious effects of oxalate on the kidneys and renal cells as well as monocytes and macrophages 17-20,38 .
  • liver fibrosis markers previously found after 6 months on this diet include a significant upregulation of pathways/genes related to fibrogenesis and ECM remodeling (Col1a1, Col1a2, Col3a1, Col4a1, Col4a2, Timp1, and Serpine1), TGF ⁇ signaling (Tgfb1, Tgfb2, Tgfb3, Tgfbr1, and Tgfbr2), SMAD signaling (SMAD2 Ser465/467 phosphorylation), as well as perisinusoidal and portal fibrosis10,25,26,27.
  • pathways/genes related to fibrogenesis and ECM remodeling Cold1a1, Col1a2, Col3a1, Col4a1, Col4a2, Timp1, and Serpine1
  • TGF ⁇ signaling Tgfb1, Tgfb2, Tgfb3, Tgfbr1, and Tgfbr2
  • SMAD2 Ser465/467 phosphorylation as well as perisinusoidal and portal fibrosis10,25,
  • Exogenous oxalate not only induces chronic kidney disease, but also causes cardiac fibrosis in C57BL/6 mice 18 , and accelerates atherosclerosis development in apolipoprotein E-deficient (Apoe -/- ) mice 19 .
  • hepatic oxalate overproduction due to the loss of AGXT enhances atherosclerosis, while overexpression of AGXT in hepatocytes lowers oxalate and ameliorates atherosclerosis in Apoe -/- mice 19 .
  • Plasma samples were obtained from peripheral blood specimens following centrifugation and separation and stored at -80°C until analysis.
  • Disease-free liver tissue was obtained during laparoscopic, robotic-assisted liver resection (partial lobectomy) in patients with no existing or prior history of liver disease (2015. 101. C).
  • NASH tissue was obtained during orthotopic liver transplantation for end-stage liver disease (2010.179, 2020.039).
  • Liver specimens were placed into formalin and further processed to formalin-fixed, paraffin-embedded tissue blocks or flash-frozen in liquid nitrogen and stored at -80°C until analysis.
  • Patient demographics and laboratory values were exported from the electronic medical record and are described in Fig 9 Panels A and B.
  • mice were fed the NASH diet (Research Diets D17010103) ad libitum for 6 months.
  • MDMG-935P is a salicylic acid derivative we recently developed that potently decreases oxalate production by inhibiting GO and LDHA.
  • the therapeutic potential of MDMG-935P was evaluated in mice with NASH using established protocols 10,25 ’ 27 ’ 37 . Eight-week-old C57BL/6J male mice were fed the NASH diet (Research Diets D17010103) ad libitum for 3 months. Mice were then randomized to receive MDMG-935P solubilized in 0.5% methylcellulose by oral gavage at a concentration of 0 mg/kg/day (vehicle), 5 mg/kg/day, or 10 mg/kg/day for an additional 3 months on the NASH diet until euthanasia and tissue harvest.
  • hepatocytes were isolated from 8-10-week-old male C57BL/6J mice fed a standard diet. Following euthanasia by isoflurane. the portal vein was cannulated using a 24G IV catheter (Terumo #SR-OX2419CA). The catheter was held within the portal vein by applying a surgical knot. The inferior vena cava was cut and 50 mL of warm liver perfusion media (Gibco #17701038 supplemented with 1% penicillin/streptomycin [GenClone #25-512]) was perfused through the liver at 5 mL/min.
  • warm liver perfusion media Gibco #17701038 supplemented with 1% penicillin/streptomycin [GenClone #25-512]
  • Cells were dissociated from the liver by gentle mechanical separation using forceps in ice-cold plating and thawing media (William’s E Media [Gibco #A1217601] supplemented with thawing and plating supplements [Gibco #CM3000]) and passed through a 100 pm filter. Cells were rinsed twice by centrifugation at 50 g for 3 min and purified by percoll gradient (20% percoll [Cytiva #45- 001-748]), 80% thawing and plating media). Percoll was removed by centrifugation at 150 g for 3 minutes, and cells were rinsed twice by centrifugation at 50 g for 3 min.
  • Hepatocytes were maintained in maintenance media and utilized for experiments no longer than 24 hours post-isolation. Hepatocytes were plated at approximately 80% confluence and allowed to adhere for approximately 6 hours prior to treatment. HepG2 cells were maintained in DMEM (Gibco #1059-010) supplemented with 10% FBS (Gibco #10438-026) and 1% penicillin/ streptomycin (GenClone #25-512) and plated at approximately 5 x 10 4 cells per cm 2 .
  • IxlO 4 HepG2 cells per well were plated in a 96-well plate and transfected with 80 ng/well of PPREx3-TK-luciferase (pGL3/PPREx3, Addgene), 10 ng/well recombinant human PPARa (pcDNA3.1/hPPARa, NM_001001930), and 10 ng/well Renilla (pRL-TK, Promega) constructs using Lipofectamine 3000 (Invitrogen #L3000-015). Approximately 18 hours following transfection, cells were treated with or without sodium oxalate (500 ⁇ M) or Wy 14,643 (25 ⁇ M) for 24 hours.
  • PPREx3-TK-luciferase pGL3/PPREx3, Addgene
  • 10 ng/well recombinant human PPARa pcDNA3.1/hPPARa, NM_001001930
  • pRL-TK Promega
  • HepG2 cells were lysed and luminescence was measured (Promega #E1980) as per manufacturer’s instructions on a CLARIOstar Plus High-Performance Multimode Microplate Reader.
  • HepG2 cells were plated at 2.5 x 10 5 cells/well in a 12-well plate with Lipofectamine 3000 according to manufacturer's instructions with pcDNA3.1-GFP control (1 pg DNA per 2 x 10 5 cells) or pcDNA3.1-AGXT-GFP (1 pg DNA per 2 x 10 5 cells, OriGene. NM_000030).
  • hPBMs Human peripheral blood monocytes
  • H99-064 Human peripheral blood monocytes
  • blood was drawn by median cubital vein venipuncture from healthy volunteers and centrifuged through a Ficoll Histopaque 1077 gradient (Sigma) to isolate mononuclear cells. Cells were then washed with saline and monocytes were isolated by centrifugation through a Percoll (Pharmacia) gradient. Cells were washed once in serum-free RPMI medium and re-suspended in serum-free RPMI medium. hPBMs were used within 24 hours.
  • hPBMs were suspended in warm HBSS (Gibco #14025-076) at a concentration of approximately 1 x 10 6 cells/mL and 5 ⁇ L/mL of Vybrant DiO cell-labeling solution (Invitrogen #V22886) was added as per manufacturer’s instructions. Cells were incubated at 37°C for 20 min, then rinsed twice with fresh HBSS before a final resuspension of 5 x 10 6 cells/mL prior to transwell experiments. [00289] Cloning and production of AAV 8-GFP and AAV 8-AGXT
  • AAV 8-AGXT expressing the human AGXT and AAV 8-GFP control were prepared as we previously described 19 . Briefly, plasmids for AAV8 package (pAdDeltaF6, pAAV2/8, pAAV-TBG-GFP, pAAV-TBG-MCS) were kindly provided by Dr. Jiandie Lin (University of Michigan). The human AGXT was cloned from plasmid #RG212899 (Origene) into the backbone plasmid pAAV-TBG-MCS using the Gibson assembly kit (New England Biolabs). The AGXT sequence and proper insertion were confirmed by Sanger sequencing.
  • pAAV-TBG- GFP with the same backbone but expressing GFP was used as a control.
  • Seventy pg of AAV shuttle vector, 200 pg Delta F6 helper plasmid and 70 ⁇ g AAV2/8- Rep/Cap plasmid were prepared with EndoFree Plasmid Maxi Kit (QIAGEN) and transfected into 15-cm plates of HEK293T cells using PEI transfection reagent (Sigma-Aldrich). After 96 h. the cells were lysed (20 mM Tris, pH 8.0, 150 mM NaCl) and 1 M MgCl 2 and 25 KU/ml benzonase were added after 3 freeze-thaw cycles between liquid nitrogen and 37°C.
  • AAVs in the supernatant were purified by ultracentrifugation in a density gradient iodixanol solution with a T865 rotor for 160 min at 60,000 rpm and 14°C.
  • AAVs were concentrated in PBS with 0.01% Poloxamer 188 (Sigma-Aldrich) using a lOOkDa filter tube (Millipore, Cat# 910096) and the titer was quantified by qPCR.
  • cDNA synthesis was performed in a Mastercycler nexus gradient thermocycler (Eppendorf). Primers were purchased from Integrated DNA Technologies (Tables 1 and 2) and qRT-PCR was performed using SSoAdvanced Uniersal SYBR Green Supermix (Bio-Rad #175271) with a CFX96 Touch Real-Time PCR Detection System (BioRad) according to manufacturer’s instructions. Results were normalized to housekeeping genes (GAPDH, ACTB, HPRT1) and expressed as a fold change from control treatments using the AACt threshold cycle method of normalization.
  • Table 1 Human primers used for qRT-PCR analyses.
  • RNA was isolated from liver samples of mice treated with AAV8-GFP or AAV8-AGXT (n 4 per group, randomly selected). Samples were quantitated with a Qubit RNA assay (ThermoFisher Scientific) and RNA quality was evaluated with the Agilent TapeStation RNA assay (Agilent Technologies). All samples had RNA integrity numbers (RINs) of at least 8.3. Libraries were prepared with the Stranded mRNA Prep, Ligation Kit (Illumina). One pg of RNA was processed for each sample and mRNA was purified and fragmented. cDNA was synthesized, and 3’ ends were adenylated.
  • Anchor sequences were ligated to each sample and a limited-cycle PCR was performed to amplify and index the libraries.
  • the average library' size was evaluated using an Agilent TapeStation D1000 assay (Agilent Technologies) and libraries were quantitated with qPCR (Bio-rad CFX96 Touch Real-Time PCR. NEB Library Quant Kit for Illumina). Libraries were normalized to 0.5 nM and pooled. The library' pool was denatured and diluted to approximately 100pM. A 1% library of 2.5pM PhiX was spiked in as an internal control. Paired-end 76 x 76 base pair sequencing was performed on an Illumina NovaSeq 6000.
  • Trimmomatic v.0.35 was used to trim the low-quality reads with the parameters: SLIDINGWINDOW:4:20 MINLEN:25.
  • the resulted high-quality reads were then mapped to the mouse reference genome (GRCm38.90) usingHISAT2 v.2.1.0.13.
  • Gene level quantification was performed using HTSeq-counts v0.6.0 based on the GRCm38.90 genome annotations.
  • the R package DESeq2 was then used to identify significant differentially expressed genes (DEGs). Genes with adjusted p value ⁇ 0.05 were considered as significant DEGs.
  • RNA-sequencing data have been deposited in NCBIs Gene Expression Omnibus (GEO) database (accession number: GSE224097).
  • liver tissues samples protein was isolated from approximately 50 mg of liver tissue. Tissue was lysed in RIPA lysis and extraction buffer (G Biosciences #786-489) supplemented with 1% Halt protease inhibitor cocktail (Thermo Scientific #78429) and 1% phosphatase inhibitor cocktail A (Alfa Aesar #J65354.LQ) and Precellys soft tissue homogenizing ceramic beads (Cayman Chemical Company #10011152) in a Precellys Evolution homogenizer (Bertin Technologies).
  • Membranes were labeled with rabbit anti-AGXT (1: 1000, Sigma #HPA035370), mouse anti-AGXT (1:500, Santa Cruz Biotechnology #SC-517388), rabbit anti-CPTla (1 : 1000, Abeam #ab234111), mouse anti-(3-actin (1 : 1000, Cell Signaling Technology #3700S), mouse anti-GAPDH (1:5000, Santa Cruz Biotechnology #sc365062).
  • Primary antibodies were visualized by fluorescent secondary donkey anti-rabbit antibody (1 :20,000, Li-Cor #926-68073) or donkey anti-mouse antibody (1:20,000 Li-Cor #926-32212) on a Li-Cor Odyssey XF Imager. Densitometry' was performed using Image Studio Lite v5.2 software and normalized to (3-actin or GAPDH.
  • hepatocytes or HepG2 cells were trypsinized, pelleted by centrifugation, and lysed in 150 ⁇ L ice-cold PBS by sonication. Intracellular oxalate was measured using the Oxalate Assay Kit (Abeam #abl96990) and normalized to total protein using Quick Start Bradford lx Dye Reagent. For triglyceride analysis, approximately 50 mg of frozen liver samples was homogenized in PBS, and the soluble fraction was removed by centrifugation. Lipids were extracted as described previously 10,25,49 Briefly, lipids were extracted using 3:2 hexane:isopropanol.
  • Triglycerides were evaluated using the LabAssay Triglyceride measurement kit (Fuji Film, #632-50991) as per manufacturer’s instructions. Liver hydroxyproline was measured using a hydroxy proline assay kit (Abeam #ab222941) as per manufacturer’s instructions. MDA was measured using the TBARS assay kit (Cayman Chemical Company# 10009055) according to manufacturer’s instructions.
  • Hepatocyte ballooning was scored from 0-2 (0: normal hepatocytes, 1 : normal-sized with pale cytoplasm, 2: pale and enlarged hepatocytes, at least 2-fold).
  • Lobular inflammation was scored from 0-3 based on foci of inflammation counted at 20X (0: none, 1 : ⁇ 2 foci; 2: 2-4 foci; 3: >4 foci).
  • NAS was calculated as the sum of steatosis, hepatocyte ballooning and lobular inflammation scores.
  • Picrosirius Red staining slides were treated with 0.2 N phosphomoly bdic acid for 3 min and transferred to 0.1% Sirius Red saturated in picric acid (Rowley Biochemical Inc.) for 90 min, then transferred to 0.01 N hydrochloric acid for 3 min.
  • Picrosirius Red staining was used to score hepatic fibrosis from 0-4 (0: no fibrosis; 1 : perisinusoidal or portal fibrosis; 2: perisinusoidal and portal fibrosis; 3: bridging fibrosis; 4: cirrhosis).
  • the NAS and fibrosis score 50 were evaluated by two independent investigators blinded to experimental groups, and the average scores are presented.
  • Frozen section processing was used for Oil Red O staining.
  • Formalin-fixed liver samples were cryoprotected in 20% sucrose at 4°C overnight, blotted, then liquid nitrogen-snap frozen in OCT compound (Tissue-Tek) and stored at -80°C.
  • OCT compound Tissue-Tek
  • frozen blocks Prior to sectioning, frozen blocks were brought up to about -20°C, then sectioned at 5 pm on a Cryotome SME (Thermo-Shandon).
  • slides Prior to staining, slides were thawed to room temperature for 30 min and then fixed in 10% neutral buffered formalin for 20 min, rinsed in DDW, followed by rinsing in 60% isopropanol before being placed in working ORO-isopropanol stain (Rowley Biochemical Inc., H-503-1B) for 5 min. Slides were then rinsed in 60% isopropanol followed by three changes of DDW and nuclear counterstained with Harris Hematoxylin.
  • Immunofluorescence was performed using rat anti-F4/80 (Bio-Rad ABD Serotec MCA497R, 1 :400), mouse anti-smooth muscle actin-Cy3 (1:400, Sigma #C6198), or rabbit anti-arginasel (Argl, Sigma #HPA024006. 1 :200) and nuclei were visualized with DAPI.
  • rat anti-F4/80 Bio-Rad ABD Serotec MCA497R, 1 :400
  • mouse anti-smooth muscle actin-Cy3 (1:400, Sigma #C6198
  • rabbit anti-arginasel Argl, Sigma #HPA024006. 1 :200
  • F4/80 goat-ant-rat secondary antibody conjugated to Cy5 fluorophore was used (1:200, Invitrogen # A-21247).
  • Nile Red in hepatocytes cells were fixed with 3.7% neutral buffered formalin and stained with Nile Red (1 :2,000 TCI Chemicals N0659) and DAPI (1 :50,000 MP Biometicals #0215757410) for 30 min.
  • mitochondrial superoxide analysis cells were treated with MitoSOX superoxide fluorescent dye (4 ⁇ M, Invitrogen #M36008) and Hoechst nuclear stain (2 ⁇ M, ThermoScientific #62249) for 30 minutes. Liver sections and cells were imaged on a Keyence BZ-X810 all-in-one fluorescence microscope. Images were analyzed using the Keyence BZ-X800 analyzer software.
  • Oxygen consumption rates and dependency on fatty acid -oxidation (FAO) were assessed using an Agilent Seahorse XFe24 Analyzer at the Cellular Metabolism Core, Louisiana State University Health Sciences Center-Shreveport. As we previously described 10 , HepG2 cells were seeded at 2.5 x 10 4 per well in XF24 cell culture microplates (Agilent # 103015-100). The next day, cells were treated with or without 500 ⁇ M NaOX for approximately 18 hours. XFe24 sensor cartridges were hydrated in accordance with the manufacturer’s protocol.
  • Oligomycin, FCCP, rotenone+antimycin A (R/A) (Agilent #103015- 100), and etomoxir (Cayman Chemical Company #11969) were used at final concentrations of 2.5 ⁇ M, 1 ⁇ M, 0.5 ⁇ M, and 20 ⁇ M, respectively.
  • Transwell chemotaxis assay HepG2 cells were plated at approximately 1 x 10 5 cells per well of a 24-well plate and allowed to adhere overnight in 500 pL DMEM supplemented with 10% FBS and 1% penicillin/streptomycin. HepG2 cells were then treated with or without 500 ⁇ M oxalate for approximately 18 hours. hPBMs were labeled with Vybrant DiO cell-labeling solution and suspended at approximately 5 x 10 6 cells per mL of warmed HBSS.
  • Millicell cell culture hanging inserts with 8 pm pores were inserted into each well, and 100 pL of hPBMs were added to the top well of each insert. hPBMs were permitted to incubate for approximately 18 hours in the insert and pass through the pores into the bottom well. Following hPBM transmigration, inserts were carefully removed and the bottom well containing both HepG2 cells and hPBMs were fixed in 3.7% neutral buffered formalin for at least 20 min. Cells were visualized on a Keyence BZ-X810 all-in-one fluorescence microscope, and the total number of hPBMs that passed into the bottom w ell were quantified using Keyence BZ-X800 analyzer software. Representative images of the fluorescent hPBMs (shown in green) were taken with a brightfield overlay to visualize equal numbers of HepG2 cells for each treatment.
  • Example 2 Hepatic oxalate overproduction as a driver and therapeutic target in NASH and atherosclerosis
  • NASH nonalcoholic fatty liver disease
  • CVD atherosclerotic cardiovascular disease
  • oxalate metabolism commonly dysregulated in NAFLD and CVD was uncovered.
  • Alanine-glyoxylate aminotransferase (AGXT) a liver-specific enzyme that detoxifies glyoxylate, the oxalate precursor, was reduced and oxalate was markedly increased in correlation with NASH severity.
  • oxalate was also increased both in patients and mice with atherosclerosis.
  • MDMG-935P is included in the family of compounds with general structure aminomethylfurylsalicylic acids.
  • Structure of the salicylic acid derivative MDMG-935P (Moya-Garzon et al., New Salicylic Acid Derivatives, Double Inhibitors of Glycolate Oxidase and Lactate Dehydrogenase, as Effective Agents Decreasing Oxalate Production. European Journal of Medicinal Chemistry 2022, 237. 114396 and EP 3593803)
  • the compound FAB-541 has been texted testing primary hyperoxaluria (in vitro). Without wishing to be bound by theory, it can be tested in lipid loaded hepatocytes (in vitro) and NASH mouse models (in vivo).
  • Flavonoid compounds have been tested against recombinant enzymes GO and
  • LDHA LDHA. They have undergone biological testing in hyperoxaluric hepatocytes (in vitro) and, without wishing to be bound by theory, in lipid loaded hepatocytes (in vitro) or in NASH mouse models (in vivo). These compounds are natural products and are not structurally related to MDMG-935P or FAB compounds. They are not salicylic acid derivatives.
  • Example 4 Salicylate derivatives: Double GO/LDHA inhibitors lowering oxalate production
  • Salicylate derivatives Double GO/LDHA inhibitors lowering oxalate production
  • Structure [00543] The compounds are derivatives of salicylic acid bearing lateral chains that contain aromatic and aliphatic moieties.
  • Synthesis [00545] The compounds can be synthesized in 3-5 steps.
  • Activity on recombinant enzymes [00547] The compounds are dual hGO/hLDHA inhibitors. The activities of a selection of compounds are depicted in the table. [00548] Fig.
  • Example 5 shows activities of selected salicylate derivatives against recombinant human glycolate oxidase and lactate dehydrogenase A.
  • Example 5 New results using salicylic acid derivatives as glycolate oxidase and lactate dehydrogenase inhibitors.
  • Diversely substituted furylsalicylic acids are efficient scaffolds inhibiting glycolate oxidase and lactate dehydrogenase enzymes. 1 Their dual inhibitory activity raises their potential to become therapeutic agents against primary hyperoxaluria type 1 (PH1). Their efficiency to reduce oxalate production has been proved in vitro (AGXT-KO mouse hepatocytes) as well as in vivo (AGXT-KO mice).
  • Example 6 New compounds for evaluation of intracellular oxalate in hepatocytes of Primary Hyperoxaluria mice (AGXT-/-) [00554] Fig. 124 shows a non-limiting, exemplary list of compounds for evaluation of intracellular oxalate in hepatocytes of Primary Hyperoxaluria mice (AGXT-/-). Compounds named FAB can be salicylate derivatives, compounds named FL are flavonoids.
  • EXAMPLE 7 [00555]
  • Example 7 [00556]
  • NAFD Non-alcoholic fatty liver disease
  • Non- alcoholic steatohepatitis (NASH), the more severe form of NAFLD, is characterized by hepatocyte injury and lobular inflammation associated with liver fibrosis. 2,3
  • NASH Non- alcoholic steatohepatitis
  • CVD cardiovascular disease
  • 5-11 there is an urgent need to identify new pathways that can be targeted for concurrent treatment of NASH and associated atherosclerosis. Such efforts have been hampered by limited understanding of the pathophysiology and metabolic pathways linking NASH with atherosclerosis.
  • Oxalate is an end product of glyoxylate metabolism in the liver, accounting for 85-90% of total circulating oxalate. 12,17,18 While the deleterious effects of oxalate in the kidneys are well known, 17-28 the effects of oxalate in hepatocytes, the primary cells responsible for its formation, 17 have not been systematically studied yet.
  • Alanine-glyoxylate aminotransferase (AGXT), a liver-specific enzyme that detoxify glyoxylate, was reduced in livers from both humans and mice with NASH while oxalate was increased in correlation with disease severity. Remarkably, oxalate was also increased both in patients and mice with atherosclerosis.
  • AGXT Alanine-glyoxylate aminotransferase
  • 13-14 hepatic oxalate overproduction (Agxt -/- ) increased both NASH and atherosclerosis with suppressed hepatic fatty acid ⁇ - oxidation (FAO) and induced proinflammatory pathways. Exogenous oxalate also enhanced atherosclerosis development.
  • oxalate induced mitochondrial dysfunction and lipid accumulation while downregulating peroxisome proliferator-activated receptor ⁇ (PPAR ⁇ ) targets and upregulating C-C motif chemokine ligand 5 (CCL5).
  • PPAR ⁇ peroxisome proliferator-activated receptor ⁇
  • CCL5 C-C motif chemokine ligand 5
  • mice with hepatic oxalate overproduction and PPAR ⁇ deficiency (Agxt -/- / Ppara LKO / Ldlr -/- )
  • new dietary models, genetic (Ccl5 -/- ) and Docket No.: 2932719-000218-WO1 Date of Filing: February 28, 2024 pharmacological (MitoTEMPO, Wy-14643) approaches, combined with in vitro models, we evaluated the mechanisms by which oxalate 1) causes mitochondrial dysfunction, and 2) suppresses PPAR ⁇ /FAO to induce CCL5 in hepatocytes.
  • Oxalate reduction can be therapy for NASH and associated atherosclerosis. Without wishing to be bound by theory, oxalate-reducing treatments ameliorate established NASH and atherosclerosis by inducing PPAR ⁇ /FAO and suppressing CCL5.
  • Non-limiting aspects described herein comprise 1) identify metabolic pathway linking NASH and atherosclerosis and 2) advance translation of oxalate reduction as a concurrent treatment for these diseases, a significant unmet clinical need.
  • NAFLD has reached epidemic proportions with no pharmacological therapy available.
  • NAFLD a spectrum of liver pathologies, ranges from simple steatosis, NASH, to cirrhosis that can lead to hepatocellular carcinoma and liver failure.
  • 29 Lipid overload is central to the pathogenesis of NAFLD and NASH.
  • 3,30,31 Fatty acids are supplied in excess to the liver via 1) enhanced flow from adipose tissue, and 2) increased synthesis from carbohydrates, primarily fructose, via de novo lipogenesis.
  • NAFLD is associated with increased risk of liver-related mortality; however, the most common cause of death in patients with NAFLD, for example those with NASH, is CVD, 5-11
  • the main driver of most CVDs is atherosclerosis arising from imbalanced lipid metabolism and a maladaptive immune response. 44,45
  • Underlying risk factors of atherosclerosis, including obesity, dyslipidemia and type 2 diabetes, 46 are common in patients with NAFLD/NASH. 2
  • NASH is associated with increased atherosclerosis, independent of those risk factors.
  • HLP lactate dehydrogenase A
  • glycine and glycolate are the precursors for glyoxylate, which is readily oxidized to oxalate by lactate dehydrogenase A (LDHA). 17 Secreted oxalate can complex with calcium in the kidneys to form calcium oxalate stones, the most common nephrolithiasis, which is associated with NAFLD and CVD. 68-73 Notably, humans have no enzymes capable of degrading oxalate. 74 However, specific hepatic enzymes can prevent oxalate overproduction via glyoxylate detoxification.
  • LDHA lactate dehydrogenase A
  • Alanine-glyoxylate aminotransferase is expressed only in the liver and plays a central role by converting glyoxylate to glycine.17,75 Glyoxylate can also be converted to glycolate via glycolate reductase/hydroxypyruvate reductase (GRHPR).
  • Glyoxylate can be oxidized to glyoxylate by glycolate oxidase (GO, encoded by HAO1).
  • HYPDH hydroxyproline dehydrogenase
  • PRODH2 4-hydroxy- 2-oxoglutarate
  • oxalate was increased in correlation with NASH severity.
  • sCAD coronary artery disease
  • the role of oxalate in NASH and associated atherosclerosis, its mechanisms of action and ability for targeting oxalate overproduction as a therapeutic approach is evaluated.
  • Dysregulated oxalate metabolism in human NASH and atherosclerosis is understudied.
  • hepatic oxalate overproduction drives NASH and atherosclerosis via mitochondrial dysfunction, impaired PPAR ⁇ /FAO, and CCL5 induction, while suppression of oxalate formation reduces established NASH and atherosclerosis.
  • Non-limiting objectives are 1) to evaluate the mechanisms by which oxalate drives NASH and associated atherosclerosis, 2) to evaluate oxalate reduction as a potential therapy for NASH and associated atherosclerosis, and 3) to evaluate oxalate overproduction and its genetic regulation in human NASH and atherosclerosis.
  • NAFLD affects nearly one third of the global population; 1 yet, despite considerable efforts in drug development, no pharmacological therapy currently exists to treat this disease. 4
  • the major cause of death in patients with NAFLD/NASH is CVD driven by accelerated atherosclerosis, independent of traditional risk factors.
  • mice with liver- specific deficiency of PPAR ⁇ (Ppara LKO ).
  • Ppara LKO liver- specific deficiency of PPAR ⁇
  • 1314 allowed us to generate new mouse strains for this project that will be made available to the scientific community: Agxt -/- / Ppara LKO / Ldlr -/- ; Agxt -/- / Ccl5 -/- / Ldlr -/- ; and Agxt -/- / Ppara LKO / Ccl5 -/- / Ldlr -/- mice.
  • Non-limiting therapeutic innovation Supported by data showing that liver-specific AGXT overexpression reduces oxalate, NASH and atherosclerosis in mice, we will explore genetic and dietary approaches to lower oxalate as treatments for established NASH and associated atherosclerosis and define their underlying mechanisms of action, which can change current clinical paradigms.
  • Species, sex and age as biological variables Sex- and age-specific prevalence of NAFLD, NASH and atherosclerosis in humans have been clearly defined, 93-95 which is considered in the human data in Fig.
  • mice/sex/genotype/treatment can be used to reach statistical significance (p ⁇ 0.05, power 0.80, 25% difference).
  • mice can be required.
  • 13,14,63,81,92 we will use primary mouse hepatocytes as well as HepG2 and AML-12 cell lines purchased from the ATCC (please, see Authentication of Key Biological and/or Chemical Resources).
  • NASH diet Research Diets D17010103, 40% fat, 22% fructose, 2% cholesterol) 13,63 supplemented with sodium oxalate (NaOx, 50 ⁇ moles/g) 101,102 or 0.1% Wy-14643.
  • RNA-seq indicated suppressed hepatic PPAR ⁇ /FAO, activation of proinflammatory pathways, and increased CCL5.
  • oxalate drives NASH and associated atherosclerosis through hepatic mitochondrial dysfunction, suppression of PPAR ⁇ -mediated FAO and induction of CCL5.
  • RNA- seq and pathway analysis revealed suppression of oxidative phosphorylation (OXPHOS) and fatty acid degradation with downregulated PPAR ⁇ targets in livers from Agxt -/- mice. In contrast, proinflammatory pathways were upregulated (Fig.
  • RNA-seq indicated a significant enrichment of proinflammatory pathways in livers from Agxt -/- / Apoe -/- mice with upregulated Ccl5 observed by qPCR (Fig. 141 panels D-E).
  • Non-limiting exemplary experimental strategy Evaluate the mechanisms by which oxalate induces mitochondrial dysfunction in hepatocytes.
  • Cells at 70-80% confluence will be switched from 10%- to 1%-FBS medium and treated with BSA- conjugated PA (200 ⁇ M) 13 or BSA (Cayman Chemicals) with or without NaOX (500 ⁇ M, Sigma-Aldrich) 20 for 18 h.
  • ETC electron transport chain
  • BN ⁇ PAGE blue native polyacrylamide gel electrophoresis
  • 109-112 mitochondrial ETC complex activity using spectrophotometric analysis of NADH oxidation (340 nm, Complex I), DCIP reduction (600 nm, Complex II), and Cytochrome c reduction (550 nm, (Complex III), 113 3) mitochondrial transmembrane potential ( ⁇ m) using JC-1 assay (Sigma-Aldrich), 114 4) high- resolution respirometry and oxygen fluxes using Oroboros Oxygraph-2k (available at LSUHS) 115 with various substrates to assess electron flow at complex I (pyruvate, 4 mM, glutamate, 10 mM, and malate, 4 mM), complex I linked OXPHOS (ADP, 2 mM), mitochondrial membrane integrity (Cytochrome C, 10 ⁇ M), complex I and II
  • luciferase assays all reagents are available at the Rom lab. HepG2 and AML-12 cells will be co- transfected with PPREx3-TK- luciferase, PPAR ⁇ and Renilla plasmids (80, 10 and 10 ng, respectively). After 24 h, the cells will be treated with vehicle (EtOH), PPAR ⁇ agonist or antagonist (Wy-14643 or GW6471, 10 ⁇ M, 117,118 Cayman Chemicals), or NaOX (500 ⁇ M).
  • vehicle EtOH
  • PPAR ⁇ agonist or antagonist Wy-14643 or GW6471, 10 ⁇ M, 117,118 Cayman Chemicals
  • NaOX 500 ⁇ M
  • PPAR ⁇ target genes Hadha, Acaa2, Acsl1, Acadl, Acadvl, and Acot3
  • PPAR ⁇ target genes Hadha, Acaa2, Acsl1, Acadl, Acadvl, and Acot3
  • PPAR ⁇ /coactivators PLC1 ⁇ , CBP1, TRAP1, NCOR2, SRC1/2/3, and SHP1/2
  • To study endogenous oxalate overproduction we will feed Agxt +/+ and Agxt -/- mice our NASH diet for 12 weeks. 13 Docket No.: 2932719-000218-WO1 Date of Filing: February 28, 2024
  • To study exogenous oxalate we will feed C57BL6/J mice the NASH diet with or without NaOX (50 ⁇ moles/g 101,102 ).
  • mice will be treated with MitoTEMPO (0.7 mg/kg/d) 126 or vehicle (EtOH/saline) via osmotic minipumps (ALZET) while on the NASH diets.
  • MitoTEMPO 0.7 mg/kg/d
  • vehicle EtOH/saline
  • AZET osmotic minipumps
  • liver and plasma oxalate Abcam
  • liver steatosis, inflammation and fibrosis using lipid extraction, H&E, ORO, F4/80, and Sirius Red staining
  • biochemical plasma AST, alanine aminotransferase [ALT], alkaline phosphatase [ALP], and lipid profile
  • inflammatory plasma CCL5
  • hepatic expression of PPAR ⁇ target genes and Ccl5, as described. 1314,63 Mitochondrial function will be assessed in livers and isolated hepatocytes as described herein.
  • Oxalate overload will increase CCL5 luciferase activity in a PPAR ⁇ - dependent manner via NF- ⁇ B and will enhance recruitment of p65 to the endogenous promoter. These effects will be attenuated by MitoTEMPO. Oxalate-induced PPAR ⁇ suppression can change nuclear localization of p65 which can be addressed by subcellular fractionation and Western blot. Without wishing to be bound by theory, transcription factors other than NF- ⁇ B mediate CCL5 induction can be addressed by systematic serial deletions of the CCL5 promoter region in luciferase assays followed by ChIP.
  • exogenous and endogenous oxalate will accelerate NASH in mice via mitochondrial dysfunction, suppressed PPAR ⁇ /FAO and CCL5 induction, which will be attenuated by MitoTEMPO administration.
  • oxalate overload will increase NASH, which will be attenuated by treatment with Wy-14643.
  • Ccl5 -/- mice oxalate overload will increase hepatic steatosis by suppressing PPAR ⁇ -mediated FAO, but hepatic inflammation and fibrosis will be attenuated compared to Ccl5 +/+ mice.
  • oxalate overload will result in comparable hepatic steatosis, but inflammation and fibrosis will be reduced compared to Docket No.: 2932719-000218-WO1 Date of Filing: February 28, 2024 Ppara flox / Ccl5 +/+ mice.
  • oxalate overload will increase NASH-associated atherosclerosis, which will be attenuated by MitoTEMPO treatment.
  • FAO-related pathways e.g., Peroxisome, Fatty acid degradation and PPAR signaling
  • pathways related to a proinflammatory response were downregulated (e.g., Chemokine signaling, Cytokine-cytokine receptor interaction, and NF- ⁇ B signaling)
  • Fig. 147 panels A-C We validated the RNA-seq data by qPCR and found that Ppara target genes (Hadha [shown], Acaa2, Acsl1, Acadl, Acadvl, and Acot3 [not shown]) were upregulated, while Ccl5 was down- regulated (Fig. 147 panel D).
  • Oxalate reduction via AGXT overexpression attenuates atherosclerosis.
  • AGXT over- expression in Apoe -/- mice fed a WD for 12 weeks lowered liver oxalate (Fig.148 panels A-C), liver (not shown) and plasma CCL5, and atherosclerosis (Fig.148 panels D-E), independent of plasma lipids, as we reported. 14
  • oxalate reduction via AGXT over- expression attenuates athero-sclerosis and NASH, implying the potential of this strategy for simultaneous treatment.
  • Oxalate-reducing agents will be tested in mouse AML-12 hepatocytes, human HepG2 cells, and primary hepatocytes from wild-type and Ppara -/- mice loaded with PA (200 ⁇ M, 18 h). 1) After PA loading, the cells will be transfected with GFP-tagged AGXT or GFP plasmid.
  • the cells will be treated with increasing pyridoxine concentrations (Sigma- Aldrich, 0-250 ⁇ M, 24 h), 133 followed by assessment of cellular oxalate, lipids, mitochondrial function, FAO, and CCL5. 3) GO (HAO1) or 4) LDHA inhibition lower oxalate by preventing oxidation of glycolate to glyoxylate or glyoxylate to oxalate, respectively.
  • AAV8-TBG-AGXT or AAV8-TBG-GFP I.P., 2x10 11 vg/mouse
  • mice will be given orally (gavage) pyridoxine (50 mg/kg/d) 135 or water (control).
  • mice will be injected with AAV8-H1-shHao1 or AAV8-H1-shLdha vs.
  • AAV8-H1- shGfp I.P., 2x10 11 vg/mouse.
  • the above oxalate-reducing agents will be given for 8 weeks followed by endpoint analyses of liver/plasma oxalate, steatohepatitis, fibrosis, PPAR ⁇ /FAO and CCL5 as described herein.
  • oxalate-reducing treatments will lower established NASH by preventing PPAR ⁇ suppression, enhancing hepatic FAO and reducing CCL5.
  • oxalate-reducing treatments will lower hepatic steatosis by preserving PPAR ⁇ /FAO while hepatic inflammation and fibrosis will be comparable to the treated Ccl5 +/+ mice.
  • Ccl5 -/- / Ldlr -/- mice oxalate-reducing treatments will lower hepatic steatosis while atherosclerosis will be comparable to the treated Ccl5 +/+ / Ldlr -/- mice.
  • Ppara LKO / Ccl5 -/- / Ldlr -/- mice treated with oxalate-reducing agents hepatic steatosis will be higher while atherosclerosis will be comparable to the treated Ppara flox / Ccl5 +/+ / Ldlr -/- mice.
  • oxalate production is enhanced in human NASH and atherosclerosis due to dysregulation of glyoxylate/oxalate metabolic genes.
  • Glyoxylate/oxalate metabolic genes are dysregulated in human NASH. First, we analyzed RNA-seq of livers from humans with NASH, 140 and our mice with diet-induced NASH. 13 Pathway analysis revealed clear similarities between human NASH and our mouse model.
  • the glyoxylate and dicarboxylate pathway as well as the glycine, serine and threonine metabolic pathway were suppressed in NASH in both species, while proinflammatory/fibrotic pathways (e.g., Chemokine signaling and Extracellular matrix- receptor interaction) were upregulated (Fig 149).
  • proinflammatory/fibrotic pathways e.g., Chemokine signaling and Extracellular matrix- receptor interaction
  • AGXT, GRHPR and HOGA1 as well as PPARA, were downregulated in human NASH, while the proinflammatory/ fibrotic genes, CCL5 and TGFB1, were upregulated (Fig. 150 panel A).
  • the samples are being systematically characterized for genomic, transcriptomic, lipidomic, proteomic, and histological indices (H&E, ORO, Sirius red, and NAS). Current histological characterization indicates 35% NAFLD and 12% NASH.
  • Reads will be mapped to GRCh38 and methylation analysis will be done using differential methylation analysis package (DMAP). 149 Resulting regions will be annotated using HOMER package. 150 Differentially modified genomic regions (FDR ⁇ 0.2) will be identified by t test based on the difference of methylation ratio. 151,152 The hepatic expression of glyoxylate/oxalate metabolic enzymes and promoter methylation patterns will be analyzed relative to the diagnosis of NAFLD, NASH and cardiometabolic comorbidities, the above biochemical/histological indices, expression of PPAR ⁇ targets and CCL5, with or without adjusting for confounding factors.
  • DMAP differential methylation analysis package
  • FDR ⁇ 0.2 Differentially modified genomic regions
  • liver-related blood traits of relevance to NAFLD/NASH and CVD including transaminases (ALT, AST. ALP, and gamma- glutamyltransferase (GGT), lipid profile (total cholesterol, triglycerides, LDL-C, and HDL-C), and inflammatory markers (C-reactive protein).
  • ALT transaminases
  • AST. ALP gamma- glutamyltransferase
  • GTT gamma- glutamyltransferase
  • lipid profile total cholesterol, triglycerides, LDL-C, and HDL-C
  • C-reactive protein inflammatory markers
  • hepatic and plasma oxalate will be increased in samples from donors with NAFLD and further increase in those with NASH.
  • the transcript and protein levels of AGXT, GRHPR, and H0GA1 will be reduced in samples from donors with NAFLD due to promoter hypermethylation, which will be enhanced in those with NASH.
  • Ldha was significantly upregulated in NASH, which can also increase oxalate formation.
  • Our analyses of human liver samples from large cohorts can reveal enhanced LDHA in NASH that can be pursued mechanistically by promoter methylation, 153 or alternatively, by HIF1 regulation.
  • our GWAS is not limited to known PH mutations in AGXT, GRHPR, and HOGA1 and will include much larger cohorts than previously studied, 77 it is possible that the number of carriers in our human liver samples (over 600 in total) will be too small to establish associations with hepatic oxalate, NAFLD/NASH or cardiometabolic comorbidities.
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  • the splicing regulator SLU7 is required to preserve DNMT1 protein stability 7 and DNA methylation. Nucleic Acids Res. 2021 Sep 7;49(15): 8592-8609.
  • Valvona CJ Fillmore HL, Nunn PB, Pilkington GJ.
  • the Regulation and Function of Lactate Dehydrogenase A Therapeutic Potential in Brain Tumor. Brain Pathol. 2016 Jan;26(l):3-17. Semenza GL, Jiang BH, Leung SW, Passantino R, Concorde! JP, Maire P, Giallongo A. Hypoxia response elements in the aldolase A, enolase 1. and lactate dehydrogenase A gene promoters contain essential binding sites for hypoxia-inducible factor 1. J Biol Chem. 1996 Dec 20;271(51):32529-37. Liu DJ, et al., Exome-wide association study of plasma lipids in >300,000 individuals.
  • Magdy T Jouni M, Kuo HH, Weddle CJ, Lyra-Leite D, Fonoudi H, Romero-Tejeda M, Gharib M, Javed H, Fajardo G, Ross CJD, Carleton BC, Bernstein D, Burridge PW. Identification of Drug Transporter Genomic Variants and Inhibitors That Protect Against Doxorubicin-Induced Cardiotoxicity. Circulation. 2022 Jan 25;145(4):279-294.
  • Magdy T Jiang Z. Jouni M. Fonoudi H. Lyra-Leite D, Jung G.
  • Example 8 Genetic and pharmacological targeting of hepatic oxalate overproduction ameliorates metabolic dysfunction-associated steatohepatitis (MASH)
  • oxalate promotes lipid accumulation in hepatocytes by impairing the transcription of peroxisome proliferator activated receptor-alpha (PPARa) and inhibiting fatty acid 0-oxidation (FAO).
  • PPARa peroxisome proliferator activated receptor-alpha
  • FAO fatty acid 0-oxidation
  • blocking oxalate overproduction through hepatocyte-specific overexpression of AGXT or pharmacological inhibition of glycolate oxidase (GO) and lactate dehydrogenase A (LDHA) potently lowers hepatic oxalate, steatosis, inflammation, and fibrosis by inducing PPARa-driven FAO, and suppressing leukocyte chemotaxis, and nuclear factor-kappa B and transforming growth factor-beta targets.

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Abstract

La présente invention concerne des compositions et leurs procédés d'utilisation.
PCT/US2024/017626 2023-02-28 2024-02-28 Compositions et leurs procédés d'utilisation Ceased WO2024182489A1 (fr)

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US20200197418A1 (en) * 2017-03-10 2020-06-25 Universidad De Granada Compounds for the treatment of diseases caused by oxalate accumulation

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US20200197418A1 (en) * 2017-03-10 2020-06-25 Universidad De Granada Compounds for the treatment of diseases caused by oxalate accumulation

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Title
DAS SREEPARNA; MITRA INDRANI; BATUTA SHAIKH; NIHARUL ALAM MD.; ROY KUNAL; BEGUM NAZNIN ARA: "Design, synthesis and exploring the quantitative structure–activity relationship of some antioxidant flavonoid analo", BIOORGANIC & MEDICINAL CHEMISTRY LETTERS, ELSEVIER, AMSTERDAM NL, vol. 24, no. 21, 17 September 2014 (2014-09-17), Amsterdam NL , pages 5050 - 5054, XP029077486, ISSN: 0960-894X, DOI: 10.1016/j.bmcl.2014.09.028 *
DATABASE PUBCHEM SUBSTANCE 15 June 2022 (2022-06-15), ANONYMOUS: "304896-59-1", XP093209998, Database accession no. 464490893 *
MOYA-GARZON ET AL.: "New Salicylic Acid .Derivatives, double inhibitors of glycolate oxidase and", LACTATE DEHYDROGENASE, AS EFFECTIVE AGENTS DECREASING OXALATE PRODUCTION IN EUROPEAN JOURNAL OF MEDICINAL CHEMISTRY, vol. 237, 2022, pages 1 - 16, XP087063745, DOI: 10.1016/j.ejmech.2022.114396 *

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