WO2018222701A1 - Traitement de maladies rénales avec un dérivé d'acide biliaire - Google Patents

Traitement de maladies rénales avec un dérivé d'acide biliaire Download PDF

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WO2018222701A1
WO2018222701A1 PCT/US2018/035111 US2018035111W WO2018222701A1 WO 2018222701 A1 WO2018222701 A1 WO 2018222701A1 US 2018035111 W US2018035111 W US 2018035111W WO 2018222701 A1 WO2018222701 A1 WO 2018222701A1
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compound
mice
subject
kidney
stz
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PCT/US2018/035111
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English (en)
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Moshe Levi
Luciano Adorini
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Intercept Pharmaceuticals, Inc.
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Publication of WO2018222701A1 publication Critical patent/WO2018222701A1/fr

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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
    • A61K31/56—Compounds containing cyclopenta[a]hydrophenanthrene ring systems; Derivatives thereof, e.g. steroids
    • A61K31/575—Compounds containing cyclopenta[a]hydrophenanthrene ring systems; Derivatives thereof, e.g. steroids substituted in position 17 beta by a chain of three or more carbon atoms, e.g. cholane, cholestane, ergosterol, sitosterol
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P13/00—Drugs for disorders of the urinary system
    • A61P13/12—Drugs for disorders of the urinary system of the kidneys

Definitions

  • the fastest growing group of people in the United States with impaired kidney function is the oldest age group.
  • the population older than 65 years in the United States is expected to double in the next 20 years, and that of elderly worldwide is expected to triple from 743 million in 2009 to 2 billion in 2050. This will result in a marked increase in the elderly population with chronic kidney disease (CKD).
  • CKD chronic kidney disease
  • hypertension, obesity, and insulin resistance can induce mitochondrial dysfunction, endoplasmic reticulum stress, oxidative stress, inflammation, altered lipid metabolism, and stimulation of profibrotic growth factors in the kidney, which collectively contribute to age-, diabetes-, and/or obesity-related kidney disease.
  • the rate of decline in renal function varies by gender, race, and burden of co-morbid conditions. Although greater glomerular, vascular, and interstitial sclerosis is evident on renal tissue examination of healthy kidney donors with increasing age, closer examination of processes leading to sclerosis suggests a role for metabolic and hormonal factors that can decrease the rate of sclerosis.
  • the Baltimore Longitudinal Study of Aging revealed that nearly a third of older healthy adults have little change in renal function over time. Similar findings have also been reported in rodent models of aging.
  • SREBP-1 and SREBP-2 are master regulators of fatty acid, triglyceride, and cholesterol synthesis.
  • Prevention of the age-related increases in SREBP-1 and SREBP-2 is associated with decreased renal triglyceride and cholesterol accumulation, decreased renal expression of growth factors, connective tissue growth factor (CTGF) and vascular endothelial growth factor (VEGF), matrix metalloproteinase inhibitor, and plasminogen activator inhibitor-1 (PAI-1), resulting in prevention of mesangial expansion, podocyte injury and proteinuria.
  • CTGF connective tissue growth factor
  • VEGF vascular endothelial growth factor
  • PAI-1 plasminogen activator inhibitor-1
  • the present application relates to a method of treating or preventing a renal disease, disorder, or condition in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of a compound of Formula A:
  • R1 is C1-C6 alkyl
  • R2, R3, and R5 are each independently H or OH;
  • R 4 is CO 2 H or OSO 3 H.
  • the present application also relates to a method of improving kidney function in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of a compound of Formula A, or a pharmaceutically acceptable salt or amino acid conjugate thereof.
  • the present application also relates to a method of slowing the progress of or reversing age-, diabetes-, and/or obesity-related increase in proteinuria, podocyte injury, fibronectin and/or type 4 collagen accumulation in the glomeruli of a kidney, triglyceride and/or cholesterol accumulation in the glomeruli and/or tubulointerstitium of a kidney, or TGF- ⁇ expression in a kidney, or slowing the progress of or reversing age-, diabetes-, and/or obesity- related impairments in mitochondrial biogenesis or mitochondrial function in a kidney, in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of a compound of Formula A, or a pharmaceutically acceptable salt or amino acid conjugate thereof.
  • the present application also relates to a method of increasing the level of FXR and/or TGR5 in a kidney in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of a compound of Formula A, or a pharmaceutically acceptable salt or amino acid conjugate thereof.
  • the present application also relates to a compound of Formula A, or a
  • a renal disease, disorder, or condition for treating or preventing a renal disease, disorder, or condition; improving kidney function; slowing the progress of or reversing age-, diabetes-, and/or obesity-related increase in proteinuria, podocyte injury, fibronectin and/or type 4 collagen accumulation in the glomeruli of a kidney, triglyceride and/or cholesterol accumulation in the glomeruli and/or tubulointerstitium of a kidney, or TGF- ⁇ expression in a kidney; slowing the progress of or reversing age-, diabetes-, and/or obesity-related impairments in mitochondrial biogenesis or mitochondrial function in a kidney; or increasing the level of FXR and/or TGR5 in a kidney, in a subject in need thereof.
  • the present application also relates to a compound of Formula A, or a
  • a medicament for the treatment or prevention of a renal disease, disorder, or condition; for improving kidney function; for slowing the progress of or reversing age-, diabetes-, and/or obesity-related increase in proteinuria, podocyte injury, fibronectin and/or type 4 collagen accumulation in the glomeruli of a kidney, triglyceride and/or cholesterol accumulation in the glomeruli and/or tubulointerstitium of a kidney, or TGF- ⁇ expression in a kidney; for slowing the progress of or reversing age-, diabetes-, and/or obesity-related impairments in
  • the present application also relates to use of a compound of Formula A, or a pharmaceutically acceptable salt or amino acid conjugate thereof, in the manufacture of a medicament for the treatment or prevention of a renal disease, disorder, or condition; for improving kidney function; for slowing the progress of or reversing age-, diabetes-, and/or obesity-related increase in proteinuria, podocyte injury, fibronectin and/or type 4 collagen accumulation in the glomeruli of a kidney, triglyceride and/or cholesterol accumulation in the glomeruli and/or tubulointerstitium of a kidney, or TGF- ⁇ expression in a kidney; for slowing the progress of or reversing age-, diabetes-, and/or obesity-related impairments in mitochondrial biogenesis or mitochondrial function in a kidney; or for increasing the level of FXR and/or TGR5 in a kidney, in a subject in need thereof.
  • a com ound of Formula A is Com ound 1:
  • a pharmaceutically acceptable salt of Compound 1 is the sodium salt of Compound 1 (i.e., Compound 1-Na).
  • a pharmaceutically acceptable salt of Compound 1 is the triethylammonium salt of Compound 1 (i.e., Compound 1-TEA).
  • Figure 1A is a bar graph showing relative mRNA level of FXR in 5-month-old mice fed ad lib (5mo-AL), 24-month-old mice fed ad lib (24mo-AL), or 24-month-old mice fed with caloric restriction (24mo-CR).
  • Figure 1B is a bar graph showing relative mRNA level of TGR5 in 5-month-old mice fed ad lib (5mo-AL), 24-month-old mice fed ad lib (24mo-AL), or 24-month-old mice fed with caloric restriction (24mo-CR).
  • Figure 1C is a Western blot showing relative protein level of FXR in 5-month-old mice fed ad lib (5mo-AL), 24-month-old mice fed ad lib (24mo-AL), or 24-month-old mice fed with caloric restriction (24mo-CR).
  • Figure 1D is a bar graph showing relative protein level of FXR in 5-month-old mice fed ad lib (5mo-AL), 24-month-old mice fed ad lib (24mo-AL), or 24-month-old mice fed with caloric restriction (24mo-CR).
  • Figure 2A is a bar graph showing the ratio of albumin/creatinine in 5-month-old mice fed ad lib (5mo-AL), 24-month-old mice fed ad lib (24mo-AL), 24-month-old mice fed ad lib treated with a compound of the present application (24mo-AL-INT), or 24-month-old mice fed with caloric restriction (24mo-CR).
  • Figure 2B depicts fluorescent images of synaptopodin in young mice fed ad lib (AL- young), old mice fed ad lib (AL-old), old mice fed with caloric restriction (CR-old), or old mice fed ad lib and treated with a compound of the present application (AL-old-INT).
  • Figure 2C depicts bar graphs showing relative mRNA level of TGF- ⁇ (top), fibronectin (middle), or FSP-1 (bottom) in 5-month-old mice fed ad lib (5mo-AL), 24-month- old mice fed ad lib (24mo-AL), 24-month-old mice fed ad lib and treated with a compound of the present application (24mo-AL-INT), or 24-month-old mice fed with caloric restriction (24mo-CR).
  • Figure 2D depicts immunofluorescent images of fibronectin protein in 5-month-old mice fed ad lib (5mo-AL), 24-month-old mice fed ad lib (24mo-AL), 24-month-old mice fed ad lib and treated with a compound of the present application (24mo-AL-INT), or 24-month- old mice fed with caloric restriction (24mo-CR).
  • Figure 3A is a bar graph showing mitochondria to nuclear DNA ratio in 5-month-old mice fed ad lib (5mo-AL), 24-month-old mice fed ad lib (24mo-AL), 24-month-old mice fed ad lib and treated with a compound of the present application (24mo-AL-INT), or 24-month- old mice fed with caloric restriction (24mo-CR).
  • Figure 3B is a bar graph showing relative mRNA level of Nrf-1 in 5-month-old mice fed ad lib (5mo-AL), 24-month-old mice fed ad lib (24mo-AL), 24-month-old mice fed ad lib and treated with a compound of the present application (24mo-AL-INT), or 24-month-old mice fed with caloric restriction (24mo-CR).
  • Figure 3C is a Western blot showing relative protein levels of pAMPK and AMPK in 5-month-old mice fed ad lib (5mo-AL), 24-month-old mice fed ad lib (24mo-AL), 24-month- old mice fed ad lib and treated with a compound of the present application (24mo-AL-INT), or 24-month-old mice fed with caloric restriction (24mo-CR).
  • Figure 3D is a bar graph showing pAMPK to AMPK ratio in 5-month-old mice fed ad lib (5mo-AL), 24-month-old mice fed ad lib (24mo-AL), 24-month-old mice fed ad lib and treated with a compound of the present application (24mo-AL-INT), or 24-month-old mice fed with caloric restriction (24mo-CR).
  • Figure 3E is a bar graph showing relative mRNA level of Sirt1 in 5-month-old mice fed ad lib (5mo-AL), 24-month-old mice fed ad lib (24mo-AL), 24-month-old mice fed ad lib and treated with a compound of the present application (24mo-AL-INT), or 24-month-old mice fed with caloric restriction (24mo-CR).
  • Figure 3F is a bar graph showing relative mRNA level of ERR ⁇ in 5-month-old mice fed ad lib (5mo-AL), 24-month-old mice fed ad lib (24mo-AL), 24-month-old mice fed ad lib and treated with a compound of the present application (24mo-AL-INT), or 24-month-old mice fed with caloric restriction (24mo-CR).
  • Figure 3G is a Western blot showing relative protein levels of PGC1 ⁇ , Sirt3, and MCAD in 5-month-old mice fed ad lib (5mo-AL), 24-month-old mice fed ad lib (24mo-AL), 24-month-old mice fed ad lib and treated with a compound of the present application (24mo- AL-INT), or 24-month-old mice fed with caloric restriction (24mo-CR).
  • Figure 3H is a bar graph showing Sirt3 to ⁇ -actin ratio in 5-month-old mice fed ad lib (5mo-AL), 24-month-old mice fed ad lib (24mo-AL), 24-month-old mice fed ad lib and treated with a compound of the present application (24mo-AL-INT), or 24-month-old mice fed with caloric restriction (24mo-CR).
  • Figure 3I is a bar graph showing relative mRNA level of Sirt3 in 5-month-old mice fed ad lib (5mo-AL), 24-month-old mice fed ad lib (24mo-AL), 24-month-old mice fed ad lib and treated with a compound of the present application (24mo-AL-INT), or 24-month-old mice fed with caloric restriction (24mo-CR).
  • Figure 3J is a bar graph showing PGC1 ⁇ to ⁇ -actin ratio in 5-month-old mice fed ad lib (5mo-AL), 24-month-old mice fed ad lib (24mo-AL), 24-month-old mice fed ad lib and treated with a compound of the present application (24mo-AL-INT), or 24-month-old mice fed with caloric restriction (24mo-CR).
  • Figure 3K is a bar graph showing relative mRNA level of PGC1 ⁇ in 5-month-old mice fed ad lib (5mo-AL), 24-month-old mice fed ad lib (24mo-AL), 24-month-old mice fed ad lib and treated with a compound of the present application (24mo-AL-INT), or 24-month-old mice fed with caloric restriction (24mo-CR).
  • Figure 3L is a bar graph showing MCAD to ⁇ -actin ratio in 5-month-old mice fed ad lib (5mo-AL), 24-month-old mice fed ad lib (24mo-AL), 24-month-old mice fed ad lib and treated with a compound of the present application (24mo-AL-INT), or 24-month-old mice fed with caloric restriction (24mo-CR).
  • Figure 3M is a Western blot showing the relative protein levels of acetyl-IDH2 and IDH2 in 5-month-old mice fed ad lib (5mo-AL), 24-month-old mice fed ad lib (24mo-AL), 24-month-old mice fed ad lib and treated with a compound of the present application (24mo- AL-INT), or 24-month-old mice fed with caloric restriction (24mo-CR).
  • Figure 3N is a bar graph showing acetyl-IDH2 to IDH2 ratio in 5-month-old mice fed ad lib (5mo-AL), 24-month-old mice fed ad lib (24mo-AL), 24-month-old mice fed ad lib and treated with a compound of the present application (24mo-AL-INT), or 24-month-old mice fed with caloric restriction (24mo-CR).
  • Figure 3O is a bar graph showing Complex I activity in 5-month-old mice fed ad lib (5mo-AL), 24-month-old mice fed ad lib (24mo-AL), 24-month-old mice fed ad lib and treated with a compound of the present application (24mo-AL-INT), or 24-month-old mice fed with caloric restriction (24mo-CR).
  • Figure 3P is a bar graph showing Complex IV activity in 5-month-old mice fed ad lib (5mo-AL), 24-month-old mice fed ad lib (24mo-AL), 24-month-old mice fed ad lib and treated with a compound of the present application (24mo-AL-INT), or 24-month-old mice fed with caloric restriction (24mo-CR).
  • Figure 4 depicts bar graphs showing relative mRNA levels of TNF- ⁇ (top), TLR2 (middle), and TLR4 (bottom) in human podocytes treated with serum from 4-month-old mice (Young), 28-month-old mice (Old), or 28-month-old mice treated with a compound of the present application (Old-INT).
  • Figures 5A-5I are bar graphs showing the relative mRNA levels of FXR (Figure 5A), TGR5 (Figure 5B), Nrf1 (Figure 5C), Sirt1 (Figure 5D), PGC1 ⁇ (Figure 5E), ERR ⁇ (Figure 5F), Sirt3 (Figure 5G), Cox4 (Figure 5H), and LCAD ( Figure 5I) in Ames dwarf mice.
  • Figure 6A is a bar graph showing glomerular and tubular FXR mRNA levels in kidney biopsy samples obtained from human subjects with diabetic- and obesity-related kidney disease, with glomerular and tubular cells obtained by laser capture microdissection.
  • Figure 6B are immunohistochemical staining images of kidney biopsy samples obtained from healthy human subjects (left) and human subjects with diabetic kidney disease (right).
  • Figure 6C is a graph displaying FXR protein expression in kidney biopsy samples obtained from human subjects with diabetic kidney disease as determined by
  • Figure 6D is a graph displaying TGR5 protein expression in kidney biopsy samples obtained from human subjects with diabetic kidney disease as determined by
  • Figure 7A is a Venn diagram displaying transcript numbers regulated by Compound 1- Na compared with Compound 2 or Compound 3 as determined by RNA-Seq analysis.
  • Figure 7B is a graph displaying activated pathways enriched in DBA/2J mice with STZ-induced hyperglycemia treated with Compound 2 but not enriched in DBA/2J mice with STZ-induced hyperglycemia treated with Compound 3 as determined by RNA-Seq analysis.
  • Figure 7C is a graph displaying activated pathways enriched in DBA/2J mice with STZ-induced hyperglycemia treated with Compound 3 but not enriched in DBA/2J mice with STZ-induced hyperglycemia treated with Compound 2 as determined by RNA-Seq analysis.
  • Figure 7D is a graph displaying additional activated pathways enriched in DBA/2J mice with STZ-induced hyperglycemia treated with Compound 1-Na.
  • Figure 7E is a Western blot showing relative protein level of nSREBP-1 in nondiabetic DBA/2J mice (CON), DBA/2J mice with STZ-induced hyperglycemia (STZ), and DBA/2J mice with STZ-induced hyperglycemia treated with Compound 2, Compound 1-Na, or Compound 3.
  • Figure 7F is a bar graph showing relative protein level of the ratio of active SREBP- 1/ ⁇ -actin in nondiabetic DBA/2J mice (CON), DBA/2J mice with STZ-induced
  • STZ hyperglycemia
  • DBA/2J mice with STZ-induced hyperglycemia treated with Compound 2, Compound 1-Na, or Compound 3.
  • Figure 7G is a bar graph showing relative mRNA level of SCD-1 in nondiabetic DBA/2J mice (CON), DBA/2J mice with STZ-induced hyperglycemia (STZ), and DBA/2J mice with STZ-induced hyperglycemia treated with Compound 2, Compound 1-Na, or Compound 3.
  • CON nondiabetic DBA/2J mice
  • STZ DBA/2J mice with STZ-induced hyperglycemia
  • STZ STZ-induced hyperglycemia treated with Compound 2, Compound 1-Na, or Compound 3.
  • ⁇ P ⁇ 0.05 versus STZ *P ⁇ 0.05 versus CON.
  • Figure 7H is a bar graph showing relative mRNA level of SCD-2 in nondiabetic DBA/2J mice (CON), DBA/2J mice with STZ-induced hyperglycemia (STZ), and DBA/2J mice with STZ-induced hyperglycemia treated with Compound 2, Compound 1-Na, or Compound 3.
  • CON nondiabetic DBA/2J mice
  • STZ DBA/2J mice with STZ-induced hyperglycemia
  • STZ STZ-induced hyperglycemia treated with Compound 2, Compound 1-Na, or Compound 3.
  • ⁇ P ⁇ 0.05 versus STZ *P ⁇ 0.05 versus CON.
  • Figure 7I is a bar graph showing relative mRNA level of Fit-1 in nondiabetic DBA/2J mice (CON), DBA/2J mice with STZ-induced hyperglycemia (STZ), and DBA/2J mice with STZ-induced hyperglycemia treated with Compound 2, Compound 1-Na, or Compound 3.
  • CON nondiabetic DBA/2J mice
  • STZ DBA/2J mice with STZ-induced hyperglycemia
  • STZ STZ-induced hyperglycemia treated with Compound 2, Compound 1-Na, or Compound 3.
  • ⁇ P ⁇ 0.05 versus STZ *P ⁇ 0.05 versus CON.
  • Figure 7J is a Western blot showing relative protein level of Sirt1 in non-diabetic mature mice (db/m), diabetic mice (db/db) and diabetic mice (db/db) treated with Compound 2, Compound 1-Na, or Compound 3.
  • Figure 7K is a bar graph showing relative protein level of the ratio of Sirt1/ ⁇ -actin in non-diabetic mature mice (db/m), diabetic mice (db/db) and diabetic mice (db/db) treated with Compound 2, Compound 1-Na, or Compound 3.
  • db/m non-diabetic mature mice
  • db/db diabetic mice
  • db/db diabetic mice
  • db/db diabetic mice
  • db/db diabetic mice treated with Compound 2, Compound 1-Na, or Compound 3.
  • ⁇ P ⁇ 0.05 versus db/db *P ⁇ 0.05 versus db/m.
  • Figure 7L is a Western blot showing relative protein level of PGC-1 ⁇ in non-diabetic mature mice (db/m), diabetic mice (db/db) and diabetic mice (db/db) treated with Compound 2, Compound 1-Na, or Compound 3.
  • Figure 7M is a bar graph showing relative protein level of the ratio of PGC-1 ⁇ / ⁇ -actin in non-diabetic mature mice (db/m), diabetic mice (db/db) and diabetic mice (db/db) treated with Compound 2, Compound 1-Na, or Compound 3.
  • Figure 7N is a Western blot showing relative protein level of ERR ⁇ in non-diabetic mature mice (db/m), diabetic mice (db/db) and diabetic mice (db/db) treated with Compound 2, Compound 1-Na, or Compound 3.
  • Figure 7O is a bar graph showing relative protein level of the ratio of ERR ⁇ / ⁇ -actin in non-diabetic mature mice (db/m), diabetic mice (db/db) and diabetic mice (db/db) treated with Compound 2, Compound 1-Na, or Compound 3.
  • db/m non-diabetic mature mice
  • db/db diabetic mice
  • db/db diabetic mice
  • db/db diabetic mice
  • db/db diabetic mice
  • db/db diabetic mice treated with Compound 2, Compound 1-Na, or Compound 3.
  • ⁇ P ⁇ 0.05 versus db/db *P ⁇ 0.05 versus db/m.
  • Figure 7P is a schematic diagram displaying that Compound 1-Na can simultaneously activate both FXR and TGR5 signaling, and their nonoverlapping pathways, with potential additive effects.
  • Figure 8A is a bar graph showing relative albuminuria level defined by the ratio of albumin/creatinine (ACR) in nondiabetic DBA/2J mice (CON), DBA/2J mice with STZ- induced hyperglycemia (STZ), and DBA/2J mice with STZ-induced hyperglycemia treated with Compound 1-Na.
  • ACR albumin/creatinine
  • Figure 8B are representative periodic acid-Schiff (PAS) staining images of kidney sections from nondiabetic DBA/2J mice (CON, top), DBA/2J mice with STZ-induced hyperglycemia (STZ, middle), and DBA/2J mice with STZ-induced hyperglycemia treated with Compound 1-Na (bottom). Scale bar, 50 ⁇ m.
  • PAS periodic acid-Schiff
  • Figure 8C is a bar graph showing glomerular area ( ⁇ m 2 ) as determined by periodic acid-Schiff (PAS) staining of kidney sections from nondiabetic DBA/2J mice (CON), DBA/2J mice with STZ-induced hyperglycemia (STZ), and DBA/2J mice with STZ-induced hyperglycemia treated with Compound 1-Na.
  • PAS periodic acid-Schiff
  • Figure 8D is a bar graph showing mesangial expansion index as determined by periodic acid-Schiff (PAS) staining of kidney sections from nondiabetic DBA/2J mice (CON), DBA/2J mice with STZ-induced hyperglycemia (STZ), and DBA/2J mice with STZ-induced hyperglycemia treated with Compound 1-Na.
  • Mesangial expansion index was defined by the percentage of mesangial area in glomerular tuft area, and the mesangial area was determined by assessment of PAS-positive and nucleus-free areas in the mesangium. ⁇ P ⁇ 0.05 versus STZ, *P ⁇ 0.05 versus CON.
  • Figure 8E are representative Masson trichrome staining images of kidney sections showing tubulointerstitial fibrosis (blue) from nondiabetic DBA/2J mice (CON, top), DBA/2J mice with STZ-induced hyperglycemia (STZ, middle), and DBA/2J mice with STZ-induced hyperglycemia treated with Compound 1-Na (bottom).
  • Figure 8F are representative merged two-photon excitation (green)-SHG (red) images of kidney sections showing tubulointerstitial fibrosis (red) from nondiabetic DBA/2J mice (CON, top), DBA/2J mice with STZ-induced hyperglycemia (STZ, middle), and DBA/2J mice with STZ-induced hyperglycemia treated with Compound 1-Na (bottom). Scale bar, 50 ⁇ m.
  • Figure 8G are immunofluorescence staining images for fibronectin of kidney sections from nondiabetic DBA/2J mice (CON, top), DBA/2J mice with STZ-induced hyperglycemia (STZ, middle), and DBA/2J mice with STZ-induced hyperglycemia treated with Compound 1-Na (bottom). Scale bar, 20 ⁇ m.
  • Figure 8H are immunofluorescence staining images for collagen IV of kidney sections from nondiabetic DBA/2J mice (CON, top), DBA/2J mice with STZ-induced hyperglycemia (STZ, middle), and DBA/2J mice with STZ-induced hyperglycemia treated with Compound 1-Na (bottom). Scale bar, 20 ⁇ m.
  • Figure 8I is a bar graph showing relative protein level of fibronectin in nondiabetic DBA/2J mice (CON), DBA/2J mice with STZ-induced hyperglycemia (STZ), and DBA/2J mice with STZ-induced hyperglycemia treated with Compound 1-Na. ⁇ P ⁇ 0.05 versus STZ, *P ⁇ 0.05 versus CON.
  • Figure 8J is a bar graph showing relative protein level of collagen IV in nondiabetic DBA/2J mice (CON), DBA/2J mice with STZ-induced hyperglycemia (STZ), and DBA/2J mice with STZ-induced hyperglycemia treated with Compound 1-Na. ⁇ P ⁇ 0.05 versus STZ, *P ⁇ 0.05 versus CON.
  • Figure 8K are immunohistochemical staining images for ⁇ -SMA of kidney sections from nondiabetic DBA/2J mice (CON, top), DBA/2J mice with STZ-induced hyperglycemia (STZ, middle), and DBA/2J mice with STZ-induced hyperglycemia treated with Compound 1-Na (bottom).
  • Figure 8L are immunohistochemical staining images for WT-1 of kidney sections from nondiabetic DBA/2J mice (CON, top), DBA/2J mice with STZ-induced hyperglycemia (STZ, middle), and DBA/2J mice with STZ-induced hyperglycemia treated with Compound 1-Na (bottom). Scale bar, 20 ⁇ m.
  • Figure 8M is a bar graph showing relative podocyte density as numbers of podocytes per glomerular area in nondiabetic DBA/2J mice (CON), DBA/2J mice with STZ-induced hyperglycemia (STZ), and DBA/2J mice with STZ-induced hyperglycemia treated with Compound 1-Na. ⁇ P ⁇ 0.05 versus STZ, *P ⁇ 0.05 versus CON.
  • Figure 8N are immunofluorescence staining images for the podocyte marker nephrin of kidney sections from nondiabetic DBA/2J mice (CON, top), DBA/2J mice with STZ- induced hyperglycemia (STZ, middle), and DBA/2J mice with STZ-induced hyperglycemia treated with Compound 1-Na (bottom).
  • Figure 9A are oil red O staining images for neutral lipid accumulation of kidney sections from nondiabetic DBA/2J mice (CON, top), DBA/2J mice with STZ-induced hyperglycemia (STZ, middle), and DBA/2J mice with STZ-induced hyperglycemia treated with Compound 1-Na (bottom). Scale bar, 50 ⁇ m.
  • Figure 9B is a bar graph showing relative kidney triglyceride level ( ⁇ mol/g) as determined by biochemical content analysis of kidney lipid extracts in nondiabetic DBA/2J mice (CON), DBA/2J mice with STZ-induced hyperglycemia (STZ), and DBA/2J mice with STZ-induced hyperglycemia treated with Compound 1-Na. ⁇ P ⁇ 0.05 versus STZ, *P ⁇ 0.05 versus CON.
  • Figure 9C is a bar graph showing relative kidney cholesterol level ( ⁇ gl/g) as determined by biochemical content analysis of kidney lipid extracts in nondiabetic DBA/2J mice (CON), DBA/2J mice with STZ-induced hyperglycemia (STZ), and DBA/2J mice with STZ-induced hyperglycemia treated with Compound 1-Na.
  • Figure 10A are immunofluorescence staining images for CD68 (red) and wheat-germ agglutinin (staining whole nephron; green) of kidney sections from nondiabetic DBA/2J mice (CON, top), DBA/2J mice with STZ-induced hyperglycemia (STZ, middle), and DBA/2J mice with STZ-induced hyperglycemia treated with Compound 1-Na (bottom).
  • Figure 10B is a bar graph showing relative mRNA level of p65 in nondiabetic DBA/2J mice (CON), DBA/2J mice with STZ-induced hyperglycemia (STZ), and DBA/2J mice with STZ-induced hyperglycemia treated with Compound 1-Na. ⁇ P ⁇ 0.05 versus STZ, *P ⁇ 0.05 versus CON.
  • Figure 10C is a bar graph showing relative mRNA level of p50 in nondiabetic DBA/2J mice (CON), DBA/2J mice with STZ-induced hyperglycemia (STZ), and DBA/2J mice with STZ-induced hyperglycemia treated with Compound 1-Na. ⁇ P ⁇ 0.05 versus STZ, *P ⁇ 0.05 versus CON.
  • Figure 10D is a bar graph showing relative renal NF- ⁇ B transcriptional activation as determined by DNA binding assay in nondiabetic DBA/2J mice (CON), DBA/2J mice with STZ-induced hyperglycemia (STZ), and DBA/2J mice with STZ-induced hyperglycemia treated with Compound 1-Na. ⁇ P ⁇ 0.05 versus STZ, *P ⁇ 0.05 versus CON.
  • Figure 10E is a bar graph showing relative oxidative carbonylation of proteins in kidney homogenate as measured by ELISA in nondiabetic DBA/2J mice (CON), DBA/2J mice with STZ-induced hyperglycemia (STZ), and DBA/2J mice with STZ-induced hyperglycemia treated with Compound 1-Na. ⁇ P ⁇ 0.05 versus STZ, *P ⁇ 0.05 versus CON.
  • Figure 10F is a bar graph showing relative mRNA level of Nox-2 in nondiabetic DBA/2J mice (CON), DBA/2J mice with STZ-induced hyperglycemia (STZ), and DBA/2J mice with STZ-induced hyperglycemia treated with Compound 1-Na. ⁇ P ⁇ 0.05 versus STZ, *P ⁇ 0.05 versus CON.
  • Figure 10G is a bar graph showing relative mRNA level of p22-phox in nondiabetic DBA/2J mice (CON), DBA/2J mice with STZ-induced hyperglycemia (STZ), and DBA/2J mice with STZ-induced hyperglycemia treated with Compound 1-Na. ⁇ P ⁇ 0.05 versus STZ, *P ⁇ 0.05 versus CON.
  • Figure 10H is a bar graph showing relative mRNA level of Nox-4 in nondiabetic DBA/2J mice (CON), DBA/2J mice with STZ-induced hyperglycemia (STZ), and DBA/2J mice with STZ-induced hyperglycemia treated with Compound 1-Na. ⁇ P ⁇ 0.05 versus STZ, *P ⁇ 0.05 versus CON.
  • Figure 11A is a bar graph showing relative mRNA levels of HIF-1 ⁇ in nondiabetic DBA/2J mice (CON), DBA/2J mice with STZ-induced hyperglycemia (STZ), and DBA/2J mice with STZ-induced hyperglycemia treated with Compound 1-Na. ⁇ P ⁇ 0.05 versus STZ, *P ⁇ 0.05 versus CON.
  • Figure 11B is a bar graph showing relative mRNA level of HIF-2 ⁇ in nondiabetic DBA/2J mice (CON), DBA/2J mice with STZ-induced hyperglycemia (STZ), and DBA/2J mice with STZ-induced hyperglycemia treated with Compound 1-Na. ⁇ P ⁇ 0.05 versus STZ, *P ⁇ 0.05 versus CON.
  • Figure 11C is a Western blot showing relative protein level of Glut1 in nondiabetic DBA/2J mice (CON), DBA/2J mice with STZ-induced hyperglycemia (STZ), and DBA/2J mice with STZ-induced hyperglycemia treated with Compound 1-Na.
  • Figure 11D is a bar graph showing relative protein level of Glut1 in nondiabetic DBA/2J mice (CON), DBA/2J mice with STZ-induced hyperglycemia (STZ), and DBA/2J mice with STZ-induced hyperglycemia treated with Compound 1-Na. ⁇ P ⁇ 0.05 versus STZ, *P ⁇ 0.05 versus CON.
  • Figure 11E is a Western blot showing relative protein level of phospho-EIF-2 ⁇ and total EIF-2 ⁇ in nondiabetic DBA/2J mice (CON), DBA/2J mice with STZ-induced hyperglycemia (STZ), and DBA/2J mice with STZ-induced hyperglycemia treated with Compound 1-Na
  • Figure 11F is a bar graph showing relative protein level of the ratio of phospho-EIF- 2 ⁇ /EIF-2 ⁇ in nondiabetic DBA/2J mice (CON), DBA/2J mice with STZ-induced
  • STZ hyperglycemia
  • DBA/2J mice with STZ-induced hyperglycemia treated with Compound 1-Na.
  • Figure 12A is a bar graph showing relative protein level of FXR in non-diabetic mature mice (db/m), diabetic mice (db/db), non-diabetic mature mice (db/m) treated with Compound 1-Na and diabetic mice (db/db) treated with Compound 1-Na.
  • Figure 12B is a bar graph showing relative protein level of TGR5 in non-diabetic mature mice (db/m), diabetic mice (db/db), non-diabetic mature mice (db/m) treated with Compound 1-Na and diabetic mice (db/db) treated with Compound 1-Na.
  • Figure 12C are immunohistochemical staining images for FXR of kidney sections from nondiabetic mature mice (db/m, top), diabetic mice (db/db, middle), and diabetic mice treated with Compound 1-Na (bottom).
  • Figure 12D is a bar graph showing relative albuminuria protein level defined by the ratio of albumin/creatinine (ACR) in non-diabetic mature mice (db/m), diabetic mice (db/db), and diabetic mice (db/db) treated with Compound 1-Na.
  • ACR albumin/creatinine
  • Figure 12E are periodic acid-Schiff (PAS) staining images of kidney sections from non-diabetic mature mice (db/m, top), diabetic mice (db/db, middle), and diabetic mice (db/db) treated with Compound 1-Na (bottom).
  • PAS periodic acid-Schiff
  • Figure 12F is a bar graph showing relative mesangial matrix index as determined by periodic acid-Schiff (pas) staining of kidney sections in non-diabetic mature mice (db/m), diabetic mice (db/db), and diabetic mice (db/db) treated with Compound 1-Na. ⁇ P ⁇ 0.05 db/db + Compound 1-Na versus db/db, *P ⁇ 0.05 db/db versus db/m.
  • Figure 12G are immunofluorescence microscopy staining images for podocyte marker synaptopodin of kidney sections from non-diabetic mature mice (db/m, top), diabetic mice (db/db, middle), and diabetic mice (db/db) treated with Compound 1-Na (bottom).
  • Figure 12H is a bar graph showing relative protein level of podocyte marker synaptopodin in non-diabetic mature mice (db/m), diabetic mice (db/db), and diabetic mice (db/db) treated with Compound 1-Na. ⁇ P ⁇ 0.05 db/db + Compound 1-Na versus db/db, *P ⁇ 0.05 db/db versus db/m.
  • Figure 12I are immunohistochemical staining images for type 1 collagen (collagen I) of kidney sections from nondiabetic mature mice (db/m, top), diabetic mice (db/db, middle), and diabetic mice treated with Compound 1-Na (bottom).
  • collagen I type 1 collagen
  • Figure 12J is a bar graph showing relative protein level of collagen I in non-diabetic mature mice (db/m), diabetic mice (db/db), and diabetic mice (db/db) treated with Compound 1-Na. ⁇ P ⁇ 0.05 db/db + Compound 1-Na versus db/db, *P ⁇ 0.05 db/db versus db/m.
  • Figure 12K are immunohistochemical staining images for type 3 collagen (collagen III) of kidney sections from nondiabetic mature mice (db/m, top), diabetic mice (db/db, middle), and diabetic mice treated with Compound 1-Na (bottom).
  • collagen III type 3 collagen
  • Figure 12L is a bar graph showing relative protein level of collagen III in non-diabetic mature mice (db/m), diabetic mice (db/db), and diabetic mice (db/db) treated with Compound 1-Na. ⁇ P ⁇ 0.05 db/db + Compound 1-Na versus db/db, *P ⁇ 0.05 db/db versus db/m.
  • Figure 12M are immunofluorescence microscopy staining images for fibronectin of kidney sections from non-diabetic mature mice (db/m, top), diabetic mice (db/db, middle), and diabetic mice (db/db) treated with Compound 1-Na (bottom).
  • Figure 12N is a bar graph showing relative protein level of fibronectin in non-diabetic mature mice (db/m), diabetic mice (db/db), and diabetic mice (db/db) treated with Compound 1-Na.
  • Figure 12O are immunofluorescence microscopy staining images for collagen IV of kidney sections from non-diabetic mature mice (db/m, top), diabetic mice (db/db, middle), and diabetic mice (db/db) treated with Compound 1-Na (bottom).
  • Figure 12P is a bar graph showing relative protein level of collagen IV in non-diabetic mature mice (db/m), diabetic mice (db/db), and diabetic mice (db/db) treated with Compound 1-Na. ⁇ P ⁇ 0.05 db/db + Compound 1-Na versus db/db, *P ⁇ 0.05 db/db versus db/m.
  • Figure 12Q is a bar graph showing relative urinary H 2 O 2 level in non-diabetic mature mice (db/m), diabetic mice (db/db), and diabetic mice (db/db) treated with Compound 1-Na. ⁇ P ⁇ 0.05 db/db + Compound 1-Na versus db/db, *P ⁇ 0.05 db/db versus db/m.
  • Figure 12R is a bar graph showing relative urinary thiobarbituric acid-reacting substances (TBARS) level in non-diabetic mature mice (db/m), diabetic mice (db/db), and diabetic mice (db/db) treated with Compound 1-Na.
  • TBARS urinary thiobarbituric acid-reacting substances
  • Figure 13A is a Western blot showing relative protein level of phospho-AMPK and total AMPK in non-diabetic mature mice (db/m), diabetic mice (db/db), non-diabetic mature mice (db/m) treated with Compound 1-Na, and diabetic mice (db/db) treated with Compound 1-Na.
  • Figure 13B is a bar graph showing relative protein level of the ratio of phospho- AMPK/AMPK in non-diabetic mature mice (db/m), diabetic mice (db/db), non-diabetic mature mice (db/m) treated with Compound 1-Na, and diabetic mice (db/db) treated with Compound 1-Na.
  • Figure 13C is a Western blot showing relative protein level of SIRT-1 in non-diabetic mature mice (db/m), diabetic mice (db/db), non-diabetic mature mice (db/m) treated with Compound 1-Na, and diabetic mice (db/db) treated with Compound 1-Na.
  • Figure 13D is a bar graph showing relative protein level of the ratio of SIRT1/ ⁇ -actin in non-diabetic mature mice (db/m), diabetic mice (db/db), non-diabetic mature mice (db/m) treated with Compound 1-Na, and diabetic mice (db/db) treated with Compound 1-Na.
  • Figure 13E is a Western blot showing relative protein level of PGC-1 ⁇ in non-diabetic mature mice (db/m), diabetic mice (db/db), non-diabetic mature mice (db/m) treated with Compound 1-Na, and diabetic mice (db/db) treated with Compound 1-Na.
  • Figure 13F is a bar graph showing relative protein level of the ratio of PGC-1 ⁇ / ⁇ -actin in non-diabetic mature mice (db/m), diabetic mice (db/db), non-diabetic mature mice (db/m) treated with Compound 1-Na, and diabetic mice (db/db) treated with Compound 1-Na.
  • Figure 14A is a graph showing relative free NADH level in control low fat diet-fed mice (LF), high fat diet-fed mice (HF), and high fat (HF) diet-fed mice treated with
  • Compound 1-Na as determined by label-free imaging with fluorescence lifetime imaging microscopy (FLIM).
  • Figure 14B is an exemplary phasor plot of free NADH (green cursor) and bound NADH (red cursor) with the line joining the two cursors as a metabolic trajectory.
  • Figure 14C is a bar graph showing relative mRNA level of proinflammatory TLR4 in control low fat diet-fed mice (LF), high fat diet-fed mice (HF), and high fat (HF) diet-fed mice treated with Compound 1-Na.
  • LF low fat diet-fed mice
  • HF high fat diet-fed mice
  • HF high fat diet-fed mice
  • Figure 14D are label-free imaging (SHG-FLIM) images of kidney sections from low fat diet-fed mice (LF, top), high fat diet-fed mice (HF, middle), and high fat (HF) diet-fed mice treated with Compound 1-Na (bottom).
  • Figure 14E is a bar graph showing relative fibrosis level as determined by SHG and FLIM in control low fat diet-fed mice (LF), high fat diet-fed mice (HF), and high fat (HF) diet-fed mice treated with Compound 1-Na.
  • LF low fat diet-fed mice
  • HF high fat diet-fed mice
  • HF high fat diet-fed mice
  • Figure 14F is a bar graph showing relative kidney ceramide level of total and major ceramide species accumulation in control low fat diet-fed mice (LF), high fat diet-fed mice (HF), and high fat (HF) diet-fed mice treated with Compound 1-Na as determined by liquid chromatography-tandem mass spectrometry (LC-MS/MS).
  • LF low fat diet-fed mice
  • HF high fat diet-fed mice
  • HF high fat diet-fed mice treated with Compound 1-Na as determined by liquid chromatography-tandem mass spectrometry
  • Figure 14G is a bar graph showing relative kidney triglyceride level of total triglyceride species accumulation in control low fat diet-fed mice (LF), high fat diet-fed mice (HF), and high fat (HF) diet-fed mice treated with Compound 1-Na as determined by liquid chromatography-tandem mass spectrometry (LC-MS/MS).
  • LF low fat diet-fed mice
  • HF high fat diet-fed mice
  • HF high fat
  • HF high fat
  • Figure 14H is a bar graph showing relative kidney triglyceride level of major triglyceride species accumulation in control low fat diet-fed mice (LF), high fat diet-fed mice (HF), and high fat (HF) diet-fed mice treated with Compound 1-Na as determined by liquid chromatography-tandem mass spectrometry (LC-MS/MS).
  • LF low fat diet-fed mice
  • HF high fat diet-fed mice
  • HF high fat
  • HF high fat
  • Figure 15A is a bar graph showing relative bile acid level of total and major bile acid species composition in control low fat diet-fed mice (LF), high fat diet-fed mice (HF), and high fat (HF) diet-fed mice treated with Compound 1-Na.
  • LF low fat diet-fed mice
  • HF high fat diet-fed mice
  • HF high fat
  • Figure 15B is a graph showing kidney bile acid composition in control low fat diet-fed mice (LF).
  • Figure 15C is a graph showing kidney bile acid composition in high fat diet-fed mice (HF).
  • Figure 15D is a graph showing kidney bile acid composition in high fat (HF) diet-fed mice treated with Compound 1-Na.
  • Figure 15E is a bar graph showing relative mRNA level of Cyp7b1 in control low fat diet-fed mice (LF), high fat Western diet-fed mice (WD), and high fat Western diet-fed mice (WD) treated with Compound 1-Na.
  • LF low fat diet-fed mice
  • WD high fat Western diet-fed mice
  • WD high fat Western diet-fed mice
  • Figure 15F is a bar graph showing relative mRNA level of Cyp27a1 in control low fat diet-fed mice (LF), high fat Western diet-fed mice (WD), and high fat Western diet-fed mice (WD) treated with Compound 1-Na.
  • LF low fat diet-fed mice
  • WD high fat Western diet-fed mice
  • WD high fat Western diet-fed mice
  • Figure 15G is a bar graph showing relative mRNA level of ASBT in control low fat diet-fed mice (LF), high fat Western diet-fed mice (WD), and high fat Western diet-fed mice (WD) treated with Compound 1-Na.
  • LF low fat diet-fed mice
  • WD high fat Western diet-fed mice
  • WD high fat Western diet-fed mice
  • Figure 15H is a bar graph showing relative mRNA level of OST ⁇ in control low fat diet-fed mice (LF), high fat Western diet-fed mice (WD), and high fat Western diet-fed mice (WD) treated with Compound 1-Na.
  • LF low fat diet-fed mice
  • WD high fat Western diet-fed mice
  • WD high fat Western diet-fed mice
  • Figure 15I is a bar graph showing relative mRNA level of OST ⁇ in control low fat diet-fed mice (LF), high fat Western diet-fed mice (WD), and high fat Western diet-fed mice (WD) treated with Compound 1-Na.
  • LF low fat diet-fed mice
  • WD high fat Western diet-fed mice
  • WD high fat Western diet-fed mice
  • Figure 15J is a bar graph showing relative mRNA level of MRP3 in control low fat diet-fed mice (LF), high fat Western diet-fed mice (WD), and high fat Western diet-fed mice (WD) treated with Compound 1-Na.
  • LF low fat diet-fed mice
  • WD high fat Western diet-fed mice
  • WD high fat Western diet-fed mice
  • Figure 15K is a schematic diagram illustrating bile acid synthesis mediated by Cyp27a1 and Cyp7b1 and bile transport from the urine mediated by bile acid transporter genes ASBT, OST ⁇ , OST ⁇ , and MRP3.
  • DETAILED DESCRIPTION is a schematic diagram illustrating bile acid synthesis mediated by Cyp27a1 and Cyp7b1 and bile transport from the urine mediated by bile acid transporter genes ASBT, OST ⁇ , OST ⁇ , and MRP3.
  • the present application is based at least in part on the discovery that a compound of Formula A or a pharmaceutically acceptable salt or amino acid conjugate thereof is effective in reversing age-, diabetes-, and/or obesity-related increase in proteinuria, podocyte injury, fibronectin and/or type 4 collagen accumulation in the glomeruli of a kidney, triglyceride and/or cholesterol accumulation in the glomeruli and/or tubulointerstitium of a kidney, or TGF- ⁇ expression, and reversing age-, diabetes-, and/or obesity-related impairments in mitochondrial biogenesis and mitochondrial function in a kidney.
  • the present application relates to a method of treating or preventing a renal disease, disorder, or condition in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of a compound of Formula A:
  • R 1 is C 1 -C 6 alkyl
  • R2, R3, and R5 are each independently H or OH;
  • the renal disease, disorder, or condition is a renal disease, disorder, or condition that is modulated by FXR (e.g., where the expression of FXR plays a role in the initiation and/or development of the renal disease, disorder, or condition).
  • the renal disease, disorder, or condition is a renal disease, disorder, or condition that is modulated by TGR5 (e.g., where the expression of TGR5 plays a role in the initiation and/or development of the renal disease, disorder, or condition).
  • the renal disease, disorder, or condition is a renal disease, disorder, or condition that is modulated by FXR and TGR5.
  • the renal disease, disorder, or condition is an age-, diabetes-, and/or obesity-related renal disease, disorder, or condition.
  • the age-, diabetes-, and/or obesity-related renal disease, disorder, or condition is modulated by FXR.
  • the age-, diabetes-, and/or obesity-related renal disease, disorder, or condition is modulated by TGR5.
  • the age-, diabetes-, and/or obesity- related renal disease, disorder, or condition is modulated by FXR and TGR5.
  • the renal disease, disorder, or condition or age-, diabetes-, and/or obesity-related renal disease, disorder, or condition is a renal disease, disorder, or condition that is associated with decreased expression of FXR.
  • the renal disease, disorder, or condition or age-, diabetes-, and/or obesity-related renal disease, disorder, or condition is a renal disease, disorder, or condition that is associated with decreased expression of TGR5.
  • the renal disease, disorder, or condition or age-, diabetes-, and/or obesity- related renal disease, disorder, or condition is a renal disease, disorder, or condition that is associated with decreased expression of FXR and TGR5.
  • the renal disease, disorder, or condition is a diabetes- and/or obesity-related renal disease, disorder, or condition.
  • the diabetes- and/or obesity-related renal disease, disorder, or condition is modulated by FXR.
  • the diabetes- and/or obesity-related renal disease, disorder, or condition is modulated by TGR5. In one embodiment, the diabetes- and/or obesity-related renal disease, disorder, or condition is modulated by FXR and TGR5. In one embodiment, the renal disease, disorder, or condition or diabetes- and/or obesity-related renal disease, disorder, or condition is a renal disease, disorder, or condition that is associated with decreased expression of FXR. In one embodiment, the renal disease, disorder, or condition or diabetes- and/or obesity- related renal disease, disorder, or condition is a renal disease, disorder, or condition that is associated with decreased expression of TGR5. In one embodiment, the renal disease, disorder, or condition or diabetes- and/or obesity-related renal disease, disorder, or condition is a renal disease, disorder, or condition that is associated with decreased expression of FXR and TGR5.
  • an age-related renal disease, disorder, or condition is a renal disease, disorder, or condition that starts to develop in a subject in a particular age group.
  • the subject is between 45 and 95 years of age, between 50 and 95 years of age, between 55 and 95 years of age, between 60 and 95 years of age, between 65 and 95 years of age, between 45 and 85 years of age, between 50 and 85 years of age, between 55 and 85 years of age, between 60 and 85 years of age, between 65 and 85 years of age, between 45 and 75 years of age, between 50 and 75 years of age, between 55 and 75 years of age, between 60 and 75 years of age, or between 65 and 75 years of age.
  • the renal disease, disorder, or condition is selected from renal inflammation, renal oxidative stress, renal lipid accumulation, renal fibrosis, renovascular diseases (e.g., narrowing or blockage of the renal artery caused by deposits of fatty acids, cholesterol, calcium, and/or other substances in the renal arteries), diabetic nephropathy, and chronic kidney disease.
  • renal inflammation e.g., renal oxidative stress, renal lipid accumulation, renal fibrosis, renovascular diseases (e.g., narrowing or blockage of the renal artery caused by deposits of fatty acids, cholesterol, calcium, and/or other substances in the renal arteries), diabetic nephropathy, and chronic kidney disease.
  • the present application also relates to a method of improving one or more kidney functions in a subject in need thereof, comprising administering to the subject a
  • the kidney functions include regulation of extracellular fluid volume, for example, to ensure an adequate quantity of plasma to keep blood flowing to vital organs, regulation of the osmolarity of extracellular fluid, maintaining ion concentration (e.g., sodium ions, potassium ions, and calcium ions), maintaining the pH of the blood plasma, excretion of wastes and toxins into urine, producing hormones or red blood cells, maintaining bone health, and controlling blood pressure.
  • ion concentration e.g., sodium ions, potassium ions, and calcium ions
  • the present application also relates to a method of regulating one or more
  • the regulating includes, but is not limited to, improving proteinuria, preventing podocyte injury, preventing mesangial expansion, preventing tubulointerstitial fibrosis, inhibiting endoplasmic reticulum stress, inhibiting enhanced renal fatty acid and cholesterol metabolism, preventing mitochondrial dysfunction, preventing oxidative stress, preventing kidney fibrosis, and/or stimulating a signaling cascade involving, for example, AMP-activated protein kinase, sirtuin 1, PGC-1 ⁇ , sirtuin 3, estrogen-related receptor- ⁇ , and/or Nrf-1.
  • one or more kidney functions are improved by at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100% after the administration of a compound of the present application.
  • one or more kidney functions in the subject are impaired (e.g., to 90%, 80%, 70%, 60%, 50%, 40%, or 30% of the normal level of the one or more kidney functions), and improving one or more kidney functions by administration of a compound of the present application restores the one or more kidney functions to 50%, 60%, 70%, 80%, 90%, or 100% of the normal level of the one or more kidney functions before the impairment.
  • one or more kidney functions in the subject are decreased as compared to a control subject (e.g., a control subject as described herein). In one embodiment, one or more kidney functions in the subject are decreased (e.g., to 90%, 80%, 70%, 60%, 50%, 40%, or 30% of the level of the one or more kidney functions in a control subject), and improving one or more kidney functions by administration of a compound of the present application restores the one or more kidney functions to 50%, 60%, 70%, 80%, 90%, or 100% of the level of the one or more kidney functions in the control subject.
  • the present application also relates to a method of slowing the progress of or reversing age-, diabetes-, and/or obesity-related increase in proteinuria, podocyte injury, fibronectin and/or type 4 collagen accumulation in the glomeruli of a kidney, triglyceride and/or cholesterol accumulation in the glomeruli and/or tubulointerstitium of a kidney, or TGF- ⁇ expression in a kidney, or slowing the progress of or reversing age-, diabetes-, and/or obesity- related impairments in mitochondrial biogenesis or mitochondrial function in a kidney, in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of a compound of Formula A, or a pharmaceutically acceptable salt or amino acid conjugate thereof.
  • proteinuria, podocyte injury, fibronectin and/or type 4 collagen accumulation in the glomeruli of a kidney, triglyceride and/or cholesterol accumulation in the glomeruli and/or tubulointerstitium of a kidney, or TGF- ⁇ expression in a kidney is decreased by at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100% after the
  • proteinuria, podocyte injury, fibronectin and/or type 4 collagen accumulation in the glomeruli of a kidney, triglyceride and/or cholesterol accumulation in the glomeruli and/or tubulointerstitium of a kidney, or TGF- ⁇ expression in a kidney is increased in the subject (e.g., to 120%, 150%, 160%, 170%, 180%, 190%, 200%, 250%, 300%, or 400% of the normal level), and by administration of a compound of the present application, proteinuria, podocyte injury, fibronectin and/or type 4 collagen accumulation in the glomeruli of a kidney, triglyceride and/or cholesterol accumulation in the glomeruli and/or tubulointerstitium of a kidney, or TGF- ⁇ expression is decreased to 150%, 140%, 130%, 120%, 100%, or 90% of the normal level.
  • proteinuria, podocyte injury, fibronectin and/or type 4 collagen accumulation in the glomeruli of a kidney, triglyceride and/or cholesterol accumulation in the glomeruli and/or tubulointerstitium of a kidney, or TGF- ⁇ expression in the subject is increased as compared to a control subject (e.g., a control subject as described herein).
  • proteinuria, podocyte injury, fibronectin and/or type 4 collagen accumulation in the glomeruli of a kidney, triglyceride and/or cholesterol accumulation in the glomeruli and/or tubulointerstitium of a kidney, or TGF- ⁇ expression in the subject is increased (e.g., to 120%, 150%, 160%, 170%, 180%, 190%, 200%, 250%, 300%, or 400% of the level in a control subject), and administration of a compound of the present application decreases proteinuria, podocyte injury, fibronectin and/or type 4 collagen accumulation in the glomeruli of a kidney, triglyceride and/or cholesterol accumulation in the glomeruli and/or tubulointerstitium of a kidney, or TGF- ⁇ expression to 150%, 140%, 130%, 120%, 100%, or 90% of the level in the control subject.
  • the level of proteinuria, podocyte injury, fibronectin and/or type 4 collagen accumulation in the glomeruli of a kidney, triglyceride and/or cholesterol accumulation in the glomeruli and/or tubulointerstitium of a kidney, or TGF- ⁇ expression can be determined by methods and materials known in the art.
  • the level of proteinuria can be determined by measuring the amount of albuminuria or the ratio of albuminuria to creatinine.
  • the level of fibronectin accumulation or TGF- ⁇ expression can be determined either by measuring the amount of mRNA or protein of fibronectin or TGF- ⁇ .
  • Podocyte injury can be manifested in many ways. Abnormal podocyte death (e.g., through programmed cell death, also known as apoptosis), detachment of podocytes from glomerular basement membrane (GBM), lack of podocyte proliferation, and/or abnormal podocyte mitosis are several indications of injuries to podocytes in a kidney. These abnormalities can be identified by various methods known in the art. For example, apoptotic or detached podocytes can be detected in urine through immunostaining with podocyte- specific antibodies, such as nephrin, podocin, and Glepp-1. In addition, irregular proliferation and mitosis of podocytes can be assessed with cultured podocyte in vitro.
  • apoptotic or detached podocytes can be detected in urine through immunostaining with podocyte- specific antibodies, such as nephrin, podocin, and Glepp-1.
  • irregular proliferation and mitosis of podocytes can be assessed with cultured podocyte in vitro.
  • the level of mitochondrial biogenesis or mitochondrial function is increased by at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100% after the administration of a compound of the present application.
  • the level of mitochondrial biogenesis or mitochondrial function is decreased in a kidney in the subject (e.g., to 90%, 80%, 70%, 60%, 50%, 40%, or 30% of the normal level of mitochondrial biogenesis or mitochondrial function), and increasing the level of mitochondrial biogenesis or mitochondrial function by administration of a compound of the present application restores the level of mitochondrial biogenesis or mitochondrial function to 50%, 60%, 70%, 80%, 90%, or 100% of the normal level of mitochondrial biogenesis or mitochondrial function before the decrease.
  • the level of mitochondrial biogenesis or mitochondrial function is decreased in a kidney in the subject (e.g., to 90%, 80%, 70%, 60%, 50%, 40%, or 30% of the normal level of mitochondrial biogenesis or mitochondrial function), and increasing the level of mitochondrial biogenesis or mitochondrial function by administration of a compound of the present
  • mitochondrial function in the subject is decreased as compared to a control subject (e.g., a control subject as described herein).
  • the level of mitochondrial biogenesis or mitochondrial function in the subject is decreased (e.g., to 90%, 80%, 70%, 60%, 50%, 40%, or 30% of the level of mitochondrial biogenesis or mitochondrial function in a control subject), and increasing the level of mitochondrial biogenesis or mitochondrial function by administration of a compound of the present application restores the level of mitochondrial biogenesis or mitochondrial function to 50%, 60%, 70%, 80%, 90%, or 100% of the level of mitochondrial biogenesis or mitochondrial function in the control subject.
  • mitochondrial biogenesis or mitochondrial function are known in the art.
  • the activity of mitochondrial complex I or complex IV can be determined through measurement of NADH dehydrogenase or cytochrome C oxidase activity in a cell.
  • mitochondrial biogenesis can be assessed by measuring the ratio of the amount of mitochondria DNA to the amount of nuclear DNA in a cell.
  • the present application also relates to a method of increasing the level of FXR and/or TGR5 in a kidney in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of a compound of Formula A, or a pharmaceutically acceptable salt or amino acid conjugate thereof.
  • the level of FXR and/or TGR5 is increased by at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100% after the administration of a compound of the present application.
  • the level of FXR and/or TGR5 is decreased in a kidney in the subject (e.g., to 90%, 80%, 70%, 60%, 50%, 40%, or 30% of the normal level of FXR and/or TGR5), and increasing the level of FXR and/or TGR5 by administration of a compound of the present application restores the level of FXR and/or TGR5 to 50%, 60%, 70%, 80%, 90%, or 100% of the normal level of FXR and/or TGR5 before the decrease.
  • the level of FXR and/or TGR5 in the subject is decreased as compared to a control subject (e.g., a control subject as described herein). In one embodiment, the level of FXR and/or TGR5 in the subject is decreased (e.g., to 90%, 80%, 70%, 60%, 50%, 40%, or 30% of the level of FXR and/or TGR5 in a control subject), and increasing the level of FXR and/or TGR5 by administration of a compound of the present application restores the level of FXR and/or TGR5 to 50%, 60%, 70%, 80%, 90%, or 100% of the level of FXR and/or TGR5 in the control subject.
  • a control subject e.g., a control subject as described herein.
  • the level of FXR and/or TGR5 in the subject is decreased (e.g., to 90%, 80%, 70%, 60%, 50%, 40%, or 30% of the level of FXR and/or TGR5 in a control subject), and
  • the methods of the present application further comprise measuring the level of one or more targets in the subject or a cell from a kidney of the subject, wherein the target is selected from FXR, TGR5, synaptopodin, Nrf-1, pAMPK, Sirt1, Sirt3, ERR ⁇ , PGC1 ⁇ , MCAD, Cox4, LCAD, TGF- ⁇ , fibronectin, FSP-1, TNF- ⁇ , TLR2, TLR4, and acetyl- IDH2.
  • the target is selected from FXR, TGR5, synaptopodin, Nrf-1, pAMPK, Sirt1, Sirt3, ERR ⁇ , PGC1 ⁇ , MCAD, Cox4, LCAD, TGF- ⁇ , fibronectin, FSP-1, TNF- ⁇ , TLR2, TLR4, and acetyl- IDH2.
  • the methods of present application comprise administering to a subject in need thereof a therapeutically effective amount of a compound of the present application, when the level of one or more targets is decreased in the subject as compared to the normal level of the one or more targets in the subject or as compared to a control subject.
  • the target is selected from CD36, LPL, FXR, TGR5, synaptopodin, Nrf-1, pAMPK, Sirt1, Sirt3, ERR ⁇ , PGC1 ⁇ , MCAD, Cox4, and LCAD.
  • the target is involved in mitochondrial biogenesis, including Nampt (nicotinamide phosphoribosyl transferase), Sirt1, Sirt3, PGC-1 ⁇ , ERR- ⁇ , Nrf1, and LCAD (long-chain acyl CoA
  • the target is a M2 macrophage marker, including CD163 and CD206.
  • the methods of present application comprise administering to a subject in need thereof a therapeutically effective amount of a compound of the present application, when the level of one or more targets is increased in the subject as compared to the normal level of the one or more targets in the subject or as compared to a control subject.
  • the target is selected from SREBP-1, SCD-1, SCD-2, Fit-1, kidney triglyceride, kidney cholesterol, p65, p50, NF ⁇ B, Nox-2, Nox-4, Hif1a, Hif2a, Glut1, p- EIF2 ⁇ , collagen I, collagen III, collagen IV, p22-phox, CD68, ICAM-1, Cox2, CTGF, FSP-1, Snail, ZEB1, TGF- ⁇ , fibronectin, FSP-1, acetyl-IDH2, TNF- ⁇ , TLR2, and TLR4.
  • the methods of the present application further comprise measuring the ratio of the amount of mitochondria DNA to the amount of nuclear DNA in a cell from a kidney of the subject. In one embodiment, the methods of the present application further comprise measuring the ratio of the amount of pAMPK to the amount of AMPK in a cell from a kidney of the subject. In one embodiment, the methods of the present application further comprise measuring the ratio of the amount of acetyl-IDH2 to the amount of IDH2 in a cell from a kidney of the subject.
  • the methods of present application comprise administering to a subject in need thereof a therapeutically effective amount of a compound of the present application, when the ratio of the amount of mitochondria DNA to the amount of nuclear DNA is decreased in the subject as compared to the normal ratio in the subject or as compared to a control subject. In one embodiment, the methods of present application comprise administering to a subject in need thereof a therapeutically effective amount of a compound of the present application, when the ratio of the amount of pAMPK to the amount of nuclear AMPK is decreased in the subject as compared to the normal ratio in the subject or as compared to a control subject.
  • the methods of present application comprise administering to a subject in need thereof a therapeutically effective amount of a compound of the present application, when the ratio of the amount of acetyl-IDH2 to the amount of IDH2 is increased in the subject as compared to the normal ratio in the subject or as compared to a control subject.
  • the methods of the present application further comprise measuring the amount of albuminuria or the ratio of the amount of albuminuria to the amount of creatinine in the subject. In one embodiment, the methods of present application comprise administering to a subject in need thereof a therapeutically effective amount of a compound of the present application, when the amount of albuminuria or the ratio of the amount of albuminuria to the amount of creatinine is increased in the subject as compared to the normal amount of albuminuria or normal ratio of the amount of albuminuria to the amount of creatinine in the subject, or as compared to a control subject.
  • the methods of the present application further comprise measuring the activity of mitochondrial complex I and/or complex IV in the subject. In one embodiment, the methods of present application comprise administering to a subject in need thereof a therapeutically effective amount of a compound of the present application, when the activity of mitochondrial complex I and/or complex IV is decreased as compared to the normal activity of mitochondrial complex I and/or complex IV in the subject or as compared to a control subject.
  • the methods of the present application comprise administering to a subject in need thereof a therapeutically effective amount of a compound of the present application, in combination with reducing the calorie uptake in the subject.
  • reducing calorie uptake comprises administering to the subject a diet that has a reduced amount of calorie.
  • the amount of calorie is reduced by 10%, by 20%, by 30%, by 40%, by 50%, by 60%, or by 70% as compared to the normal calorie uptake by the subject.
  • the subject is a human. In one embodiment, the subject is a human over 45 years of age, over 50 years of age, over 55 years of age, over 60 years of age, over 65 years of age, or over 70 years of age. In one embodiment, the subject is a human over 65 years of age.
  • the subject is a human between 45 and 95 years of age, between 50 and 95 years of age, between 55 and 95 years of age, between 60 and 95 years of age, between 65 and 95 years of age, between 45 and 85 years of age, between 50 and 85 years of age, between 55 and 85 years of age, between 60 and 85 years of age, between 65 and 85 years of age, between 45 and 75 years of age, between 50 and 75 years of age, between 55 and 75 years of age, between 60 and 75 years of age, or between 65 and 75 years of age.
  • control subject is a human. In one embodiment, the control subject is a human less than 70 years of age, less than 65 years of age, less than 60 years of age, less than 55 years of age, less than 50 years of age, or less than 45 years of age. In one embodiment, the control subject is a human less than 65 years of age.
  • the subject has one or more diseases, disorders, or conditions in addition to the renal disease, disorder, or condition.
  • the additional disease, disorder, or condition is selected from a cardiovascular disease, hypertension, a metabolic syndrome, diabetes, obesity, and insulin resistance.
  • dosages of the co-administered compounds will of course vary depending on the type of co-drug employed, on the specific drug employed, on the condition being treated and so forth. For example, synergistic effects can occur with substances.
  • Combination therapy includes the administration of the subject compounds in further combination with one or more other biologically active ingredients (such as, but not limited to, a FXR agonist, a TGR5 agonist, a second compound of Formula A, a second and different compound of Formula A) and non-drug therapies (such as, but not limited to, surgery or dietary treatment).
  • the compounds of the application can be used in combination with other pharmaceutically active compounds, preferably compounds that are able to enhance the effect of the compounds of the application.
  • the compounds of the application can be administered simultaneously (as a single preparation or separate preparation) or sequentially to the other drug therapy or treatment modality.
  • a combination therapy envisions administration of two or more drugs during a single cycle or course of therapy.
  • the compounds may be administered in combination with one or more separate pharmaceutical agents, e.g., a chemotherapeutic agent, an immunotherapeutic agent, or an adjunctive therapeutic agent.
  • R 1 is C 1 -C 6 alkyl selected from methyl, ethyl, n-propyl, i-propyl, n-butyl, i-butyl, t-butyl, pentyl, and hexyl.
  • R1 is C1-C4 alkyl selected from methyl, ethyl, n-propyl, i-propyl, n-butyl, i-butyl, and t-butyl.
  • R1 is methyl, ethyl, n-propyl, or i-propyl.
  • R 1 is methyl or ethyl.
  • R 1 is methyl.
  • R 1 is ethyl.
  • R2 is H and R3 is OH. In one embodiment, R3 is H and R2 is OH. In one embodiment, R 5 is H. In one embodiment, R 5 is OH.
  • R 2 is H, R 3 is OH, and R 5 is H. In one embodiment, R 2 is H, R 3 is OH, and R5 is OH.
  • R4 is CO2H. In one embodiment, R4 is OSO3H.
  • R 2 is H
  • R 3 is OH
  • R 4 is CO 2 H
  • R 5 is H
  • R2 is H
  • R3 is OH
  • R4 is OSO3H
  • R5 is OH
  • R1 is ethyl
  • R2 is H
  • R3 is OH
  • R4 is CO2H
  • R5 is H
  • R 1 is ethyl
  • R 2 is H
  • R 3 is OH
  • R 4 is OSO 3 H
  • R 5 is H.
  • a compound of Formula A is Compound 1:
  • a com ound of Formula A is Com ound 2:
  • a pharmaceutically acceptable salt of Compound 1 is the sodium salt of Compound 1 (i.e., Compound 1-Na).
  • a pharmaceutically acceptable salt of Compound 1 is the triethylammonium salt of Compound 1 (i.e., Compound 1-TEA).
  • a compound of the present application is administered once daily, twice daily, three times daily, once every 6 hours, or once every 4 hours. In one embodiment, a compound of the present application is administered for one day, two days, three days, four days, five days, six days, or seven days a week. In one embodiment, a compound of the present application is not administered every day of the week. In one embodiment, a compound of the present application is administered every other day, once every three days, once every four days, once every five days, once every six days, or once every seven days.
  • a compound of the present application is administered for a period of one week, two weeks, three weeks, four weeks, six weeks, two months, three months, four months, six months, or more.
  • the period in which a compound of the present application is administered comprises one or more segments (e.g., one or more days, one or more weeks, or one or more months) during which the compound is not administered.
  • a“compound of the application” or“compound of the present application” as used herein encompasses Compound 1, 1-Na, 1-TEA, or a pharmaceutically acceptable salt or amino acid conjugate thereof.
  • amino acid conjugate refers to a conjugate of the compound of the present application with any suitable amino acid.
  • suitable amino acid conjugate of a compound of the present application will have the added advantage of enhanced integrity in bile or intestinal fluids.
  • Suitable amino acids include but are not limited to glycine and taurine.
  • the present invention encompasses the glycine and taurine conjugates of the compound of the present application (e.g., Compound 1).
  • FXR refers to Farnesoid X Receptor, which is a member of the nuclear receptor family of ligand-activated transcription factors that includes receptors for the steroid, retinoid, and thyroid hormones. FXR binds to DNA as a heterodimer with the 9-cis retinoic acid receptor (RXR).
  • RXR 9-cis retinoic acid receptor
  • TGR5 refers to a G-protein-coupled receptor that is responsive to bile acids (BAs).
  • a“subject in need thereof” is a subject having a disease, disorder, or condition against which a compound of the application is effective, or a subject having an increased risk of developing a disease, disorder, or condition against which a compound of the application is effective relative to the population at large.
  • A“subject” includes a mammal.
  • the mammal can be any mammal, e.g., a human, primate, bird, mouse, rat, fowl, dog, cat, cow, horse, goat, camel, sheep or a pig. Particularly, the mammal is a human.
  • treating refers to any indicia of success in the treatment or amelioration of any of the diseases, disorders, or conditions described herein. Treating can include, for example, reducing or alleviating the severity of one or more symptoms of any of the diseases, disorders, or conditions described herein, or it can include reducing the frequency with which symptoms of any of the diseases, disorders, or conditions described herein are experienced by a patient.“Treating” can also refer to reducing or eliminating any of the diseases, disorders, or conditions described herein of a part of the body, such as a cell, tissue or bodily fluid, e.g., podocytes.
  • the term“preventing” refers to the partial or complete prevention of any of the diseases, disorders, or conditions described herein in an individual or in a population, or in a part of the body, such as a cell, tissue or bodily fluid (e.g., podocytes).
  • the term“prevention” does not establish a requirement for complete prevention of a disease, disorder, or condition in the entirety of the treated population of individuals or cells, tissues, or fluids of individuals.
  • treat or prevent is used herein to refer to a method that results in some level of treatment or amelioration of any of the diseases, disorders, or conditions described herein, and contemplates a range of results directed to that end, including but not restricted to prevention of any of the diseases, disorders, or conditions described herein entirely.
  • “pharmaceutically acceptable” refers to a material that is not biologically or otherwise undesirable, e.g., the material may be incorporated into a pharmaceutical composition administered to a patient without causing any significant undesirable biological effects or interacting in a deleterious manner with any of the other components of the composition in which it is contained.
  • Pharmaceutically acceptable carriers or excipients have met the required standards of toxicological and manufacturing testing and/or are included on the Inactive Ingredient Guide prepared by the U.S. Food and Drug administration.
  • A“pharmaceutically acceptable excipient” means an excipient that is useful in preparing a pharmaceutical composition that is generally safe, non-toxic and neither biologically nor otherwise undesirable, and includes excipient that is acceptable for veterinary use as well as human pharmaceutical use.
  • A“pharmaceutically acceptable excipient” as used in the specification and claims includes both one and more than one such excipient.
  • therapeutically effective amount refers to an effective amount comprising an amount sufficient to treat a disease, disorder, or condition described herein or to prevent or delay a disease, disorder, or condition described herein. In some embodiments, an effective amount is an amount sufficient to delay the development of the disease, disorder, or condition. In some embodiments, an effective amount is an amount sufficient to prevent or delay recurrence. An effective amount can be administered in one or more administrations.
  • a therapeutically effective amount can be estimated initially either in cell culture assays, e.g., in a cell from the kidney, such as podocytes, or animal models, usually rats, mice, rabbits, dogs, or pigs.
  • the animal model may also be used to determine the appropriate concentration range and route of administration. Such information can then be used to determine useful doses and routes for administration in humans.
  • Therapeutic/prophylactic efficacy and toxicity may be determined by standard pharmaceutical procedures in cell cultures or experimental animals, e.g., ED50 (the dose therapeutically effective in 50% of the population) and LD50 (the dose lethal to 50% of the population).
  • the dose ratio between toxic and therapeutic effects is the therapeutic index, and it can be expressed as the ratio,
  • compositions that exhibit large therapeutic indices are preferred.
  • a regimen can include periods of active administration and periods of rest as known in the art. Active administration periods include administration of a compound of the application in a defined course of time, including, for example, the number of and timing of dosages of the compositions. In some regimens, one or more rest periods can be included where no compound is actively administered, and in certain instances, includes time periods where the efficacy of such compounds can be minimal.
  • “combination therapy” means that a compound of the application can be administered in conjunction with another therapeutic agent.“In conjunction with” refers to administration of one treatment modality in addition to another treatment modality, such as administration of a compound of the application as described herein in addition to
  • administration of another therapeutic agent to the same subject refers to administration of one treatment modality before, during, or after delivery of a second treatment modality to the subject.
  • a“pharmaceutical composition” or “pharmaceutical formulation” refers to a formulation containing a compound of the present application in a form suitable for administration to a subject.
  • the pharmaceutical composition is in bulk or in unit dosage form. It can be advantageous to formulate compositions in dosage unit form for ease of administration and uniformity of dosage.
  • the specification for the dosage unit forms is dictated by and directly dependent on the unique characteristics of the active reagent and the particular therapeutic effect to be achieved.
  • the unit dosage form is any of a variety of forms, including, for example, a capsule, an IV bag, a tablet, a single pump on an aerosol inhaler, or a vial.
  • Possible formulations include those suitable for oral, sublingual, buccal, parenteral (e.g., subcutaneous, intramuscular, or intravenous), rectal, topical including transdermal, intranasal, and inhalation administration. Most suitable means of administration for a particular patient will depend on the nature and severity of the disease being treated, the nature of the therapy being used, and the nature of the active compound.
  • Formulations suitable for oral administration may be provided as discrete units, such as tablets, capsules, cachets, lozenges, each containing a predetermined amount of the active compound; as powders or granules; as solutions or suspensions in aqueous or non-aqueous liquids; or as oil-in-water or water-in-oil emulsions.
  • Formulations suitable for sublingual or buccal administration include lozenges comprising a compound of the application and typically a flavored base, such as sugar and acacia or tragacanth and pastilles comprising the active compound in an inert base, such as gelatin and glycerin or sucrose acacia.
  • a flavored base such as sugar and acacia or tragacanth
  • pastilles comprising the active compound in an inert base, such as gelatin and glycerin or sucrose acacia.
  • Formulations suitable for parenteral administration typically comprise sterile aqueous solutions containing a predetermined concentration of the active compound; the solution may be isotonic with the blood of the intended recipient. Additional formulations suitable for parenteral administration include formulations containing physiologically suitable co-solvents and/or complexing agents such as surfactants and cyclodextrins. Oil-in-water emulsions are also suitable formulations for parenteral formulations. Although such solutions may be administered intravenously, they may also be administered by subcutaneous or intramuscular injection.
  • Formulations suitable for rectal administration may be provided as unit-dose suppositories comprising a compound of the application in one or more solid carriers forming the suppository base, for example, cocoa butter.
  • Formulations suitable for topical or intranasal application include ointments, creams, lotions, pastes, gels, sprays, aerosols, and oils.
  • Suitable carriers for such formulations include petroleum jelly, lanolin, polyethyleneglycols, alcohols, and combinations thereof.
  • Oral formulations generally include an inert diluent or an edible pharmaceutically acceptable carrier. They can be enclosed in gelatin capsules or compressed into tablets. For the purpose of oral administration, the active ingredient can be incorporated with excipients and used in the form of tablets, troches, or capsules. Oral formulations can also be prepared using a fluid carrier for use as a mouthwash, wherein the active ingredient in the fluid carrier is applied orally and swished and expectorated or swallowed. Pharmaceutically compatible binding agents, and/or adjuvant materials can be included as part of the composition.
  • the tablets, pills, capsules, troches and the like can contain any of the following ingredients, or compounds of a similar nature: a binder such as microcrystalline cellulose, gum tragacanth or gelatin; an excipient such as starch or lactose, a disintegrating agent such as alginic acid, Primogel, or corn starch; a lubricant such as magnesium stearate or Sterotes®; a glidant such as colloidal silicon dioxide; a sweetening agent such as sucrose or saccharin; or a flavoring agent such as peppermint, methyl salicylate, or orange flavoring.
  • a binder such as microcrystalline cellulose, gum tragacanth or gelatin
  • an excipient such as starch or lactose, a disintegrating agent such as alginic acid, Primogel, or corn starch
  • a lubricant such as magnesium stearate or Sterotes®
  • a glidant such as colloidal silicon dioxide
  • Pharmaceutically compatible diluents may also include starch, dextrin, sucrose, glucose, lactose, mannitol, sorbitol, xylitol, microcrystalline cellulose, calcium sulfate, calcium hydrogen phosphate, calcium carbonate, and the like.
  • Pharmaceutically compatible wetting agents included water, ethanol, isopropanol, and the like.
  • Pharmaceutically compatible binders may also include starch pulp, dextrin, syrup, honey, glucose solution, microcrystalline cellulose, mucilage of arabic gum, gelatin mucilage, sodium hydroxymethylcellulose, methylcellulose,
  • Pharmaceutically compatible disintegrants may also include dry starch, microcrystalline cellulose, low-substituted hydroxypropylcellulose, cross-linked polyvinylpyrrolidone, croscarmellose sodium, sodium carboxymethyl starch, sodium bicarbonate and citric acid, polyoxyethylene sorbitol fatty acid esters, sodium dodecyl sulfonate and the like.
  • Pharmaceutically compatible lubricants and glidants may also include talc powder, silica, stearate, tartaric acid, liquid paraffin, polyethylene glycol, and the like.
  • compositions suitable for injectable use e.g., intravenous,
  • intramuscular include sterile aqueous solutions (where water soluble),
  • Suitable carriers include physiological saline,
  • the carriers can also be a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyethylene glycol, and the like), and suitable mixtures thereof.
  • the composition must be sterile and should be fluid to the extent that easy syringeability exists. It must be stable under the conditions of manufacture and storage and must be preserved against contaminating by microorganisms such as bacteria and fungi. The proper fluidity can be maintained, for example, by the use of agents such as lecithin, by the maintenance of the required particle size in the case of dispersion and by the use of surfactants.
  • microorganisms can be achieved by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, ascorbic acid, thimerosal, and the like. In many cases, it will include isotonic agents, for example, sugars, polyalcohols such as mannitol, sorbitol, sodium chloride in the composition.
  • isotonic agents for example, sugars, polyalcohols such as mannitol, sorbitol, sodium chloride in the composition.
  • Other excipients include, but are not limited to, antioxidants such as ascorbic acid or sodium bisulfite; chelating agents such as ethylenediaminetetraacetic acid; buffers such as acetates, citrates or phosphates; and agents for the adjustment of tonicity such as sodium chloride or dextrose.
  • the pH can be adjusted with acids or bases, such as hydrochloric acid or sodium hydroxide.
  • Prolonged absorption of the injectable compositions can be brought about by including in the composition an agent which delays absorption, for example, aluminum monostearate and gelatin.
  • the preparation can be enclosed in ampoules, disposable syringes or multiple dose vials made of glass or plastic.
  • Sterile injectable solutions can be prepared by incorporating the active ingredient in the required amount in an appropriate solvent with one or a combination of ingredients enumerated above, as required, followed by filtered sterilization.
  • dispersions are prepared by incorporating the active ingredient into a sterile vehicle that contains a basic dispersion medium and the required other ingredients from those enumerated above.
  • methods of preparation are vacuum drying and freeze-drying that yields a powder of the active ingredient plus any additional desired ingredient from a previously sterile-filtered solution thereof.
  • Formulations of the application may be prepared by any suitable method, typically by uniformly and intimately admixing a compound of the application with liquids or finely divided solid carriers or both, in the required proportions and then, if necessary, shaping the resulting mixture into the desired shape.
  • a tablet may be prepared by compressing an intimate mixture comprising a powder or granules of the active ingredient and one or more optional ingredients, such as a binder, lubricant, inert diluent, or surface active dispersing agent, or by molding an intimate mixture of powdered active ingredient and inert liquid diluent.
  • Suitable formulations for administration by inhalation include fine particle dusts or mists which may be generated by means of various types of metered dose pressurized aerosols, nebulizers, or insufflators.
  • formulations of the present application may include other agents known to those skilled in the art of pharmacy, having regard for the type of formulation in issue.
  • formulations suitable for oral administration may include flavoring agents and formulations suitable for intranasal administration may include perfumes.
  • Dosage and administration may be adjusted to provide sufficient levels of the active agent(s) (e.g., a compound of the present application) to maintain a desired effect.
  • Factors which may be taken into account include the severity of the disease state, general health of the subject, age, weight, and gender of the subject, diet, time and frequency of administration, drug combination(s), reaction sensitivities, and tolerance/response to therapy.
  • compositions may be administered every 1-96 hours, preferably every 2-72 hours, preferably every 3-48 hours, preferably every 4-24 hours, preferably every 6-12 hours.
  • Long-acting pharmaceutical compositions may be administered every 3 to 4 days, every week, or once every two weeks depending on half-life and clearance rate of the particular formulation.
  • a pharmaceutical composition is administered in a dosage form which comprises a compound of the application in a daily total amount of from about 0.1- 1500 mg, 0.2-1200 mg, 0.3-1000 mg, 0.4-800 mg, 0.5-600 mg, or 0.6-500 mg.
  • a pharmaceutical composition is administered in a dosage form which comprises a compound of the application in a daily total amount of from 0.5-20 mg, preferably 1-15 mg, preferably 2-14 mg, preferably 4-12 mg, preferably 5-10 mg.
  • a pharmaceutical composition is administered in a dosage form which comprises a compound of the application in a daily total amount of about 50 mg. In one embodiment, a pharmaceutical composition is administered in a dosage form which comprises a compound of the application in a daily total amount of about 40 mg. In one embodiment, a pharmaceutical composition is administered in a dosage form which comprises a compound of the application in a daily total amount of about 30 mg. In one embodiment, a pharmaceutical composition is administered in a dosage form which comprises a compound of the application in a daily total amount of about 20 mg. In one embodiment, a pharmaceutical composition is administered in a dosage form which comprises a compound of the application in a daily total amount of about 15 mg.
  • a pharmaceutical composition is administered in a dosage form which comprises a compound of the application in a daily total amount of about 10 mg. In one embodiment, a pharmaceutical composition is administered in a dosage form which comprises a compound of the application in a daily total amount of about 5 mg. In one embodiment, a pharmaceutical composition is administered in a dosage form which comprises a compound of the application in a daily total amount of about 2.5 mg. In one embodiment, a pharmaceutical composition is administered in a dosage form which comprises a compound of the application in a daily total amount of about 1 mg.
  • a pharmaceutical composition is administered in a dosage form which comprises a compound of the application in a daily total amount of less than 10 mg/kg, preferably less than 5 mg/kg, such as, for example 0.1-5.0 mg/kg, preferably 0.5-4.5 mg/kg, preferably 1.0-4.0 mg/kg, preferably 1.2-3.5 mg/kg, preferably 1.4-3.0 mg/kg, preferably 1.5- 2.5 mg/kg, preferably 1.6-2.4 mg/kg.
  • Example 1 Synthesis of a compound of the present application
  • a compound of the present application can be prepared by methods known in the art (e.g., those described in U.S. Patent No.7,932,244).
  • a compound of the present application can be prepared by a process as shown in Scheme 1 and disclosed in WO
  • Step 1 is the esterification of Compound 2 to obtain Compound 4.
  • Step 2 is a reaction to form Compound 5 from Compound 4.
  • Step 3 is the protection of the hydroxy group at the C3 position of Compound 5 to afford Compound 6.
  • Step 4 is the oxidative cleavage of Compound 6 to afford Compound 7.
  • Step 5 is the reduction of Compound 7 to afford Compound 8.
  • Step 6 is the sulfonation of Compound 8 to afford the sodium salt of
  • Compound 1 (1-Na) The sodium salt of Compound 1 can be converted to its free acid form (i.e., Compound 1) or other salt forms (e.g., Compound 1-TEA or the triethylammonium salt of Compound 1) according to procedures known in the art.
  • Example 2 Methods of the present application
  • mice 3-month-old and 22-month-old C57BL/6 mice fed ad lib, and 22- month-old C57BL/6 mice with caloric restriction were obtained through the NIA aging colony. The mice were continued to be fed ad lib with NIH31 diet or caloric restricted NIH31 diet as per the NIA instructions. A group of 22-month-old ad lib fed mice were also fed with diet containing Compound 1-Na (6 ⁇ -ethyl-3 ⁇ , 7 ⁇ , 23-trihydroxy-24-nor-5- ⁇ -cholan-23 sulfate sodium salt) at 30 mg/kg body weight/day.
  • Compound 1-Na (6 ⁇ -ethyl-3 ⁇ , 7 ⁇ , 23-trihydroxy-24-nor-5- ⁇ -cholan-23 sulfate sodium salt
  • Ames Mice Ames dwarf mice and their controls were acquired from Jackson Laboratories (Bar Harbor, ME) and studied at 21-month-old.
  • DBA/2J mice Eight-week-old male DBA/2J mice were obtained from the Jackson Laboratories (Bar Harbor, ME). They were maintained on a 12-hour light/12-hour dark cycle. Mice were injected with streptozotocin (STZ) (Sigma-Aldrich, St. Louis, MO)
  • mice were treated for 8 weeks with (i) Western diet only; (ii) Compound 1-Na (6 ⁇ -ethyl-3 ⁇ , 7 ⁇ , 23-trihydroxy-24-nor-5- ⁇ -cholan-23 sulfate sodium salt): 30 mg/kg body weight/day admixed with Western diet; (iii) Compound 2 (obeticholic acid): 20 mg/kg body wt/d admixed with Western diet; or (iv) Compound 3: 30 mg/kg body wt/d admixed with Western diet.
  • Compound 1-Na (6 ⁇ -ethyl-3 ⁇ , 7 ⁇ , 23-trihydroxy-24-nor-5- ⁇ -cholan-23 sulfate sodium salt): 30 mg/kg body weight/day admixed with Western diet
  • Compound 2 obeticholic acid
  • Compound 3 30 mg/kg body wt/d admixed with Western diet.
  • db/db Mice Six-week-old male db/m and db/db mice (BLKS/J) were obtained from the Jackson Laboratories. They were maintained on a 12-hour light/12-hour dark cycle. They were on (i) a regular chow diet, (ii) Compound 1-Na (6 ⁇ -ethyl-3 ⁇ , 7 ⁇ , 23-trihydroxy-24- nor-5- ⁇ -cholan-23 sulfate sodium salt) (30 mg/kg body wt/d), (iii) Compound 2 (obeticholic acid) (20 mg/kg body wt/d), or (iv) Compound 3 (30 mg/kg body wt/d) admixed with chow for 2 weeks.
  • Compound 1-Na (6 ⁇ -ethyl-3 ⁇ , 7 ⁇ , 23-trihydroxy-24- nor-5- ⁇ -cholan-23 sulfate sodium salt) (30 mg/kg body wt/d)
  • Compound 2 obeticholic acid
  • Plasma lipid levels were measured with kits (Wako Chemical, Richmond, VA).
  • Urine albumin and creatinine concentrations were determined with kits (Exocell, Philadelphia, PA) according to the manufacturer’s manual.
  • RNA seq kit modified per the manufacturer’s
  • Quantitative real-time PCR was performed according to methods known in the art, for example, as in Jiang T. et al., Diabetes 56, 2485 (2007), Wang XX. et al., Diabetes 59, 2916 (2010), Wang XX. et al., Am. J. Physiol. Renal Physiol.297, F1587 (2009), or Wang XX. et al., J. Am. Soc. Nephrol.27, 1362 (2016).
  • Cortical homogenate protein content was measured by BCA assay (Thermo Fisher Scientific, Waltham, MA). Equal amount of total protein was separated by SDS-PAGE gels and transferred onto PVDF membranes.
  • SREBP-1 catalog no. H-160; Santa Cruz Biotechnology, Dallas, TX
  • Glut1 catalog no.07-1401; Cell Signaling, Danvers, MA
  • p-AMPK/AMPK catalog nos.4184 and 2795; Cell Signaling
  • SIRT1 catalog no.07- 131; Millipore, Billerica, MA
  • PGC-1 ⁇ catalog no.
  • ⁇ -actin (catalogue number A5316, Sigma, St Louis, MO) was used as a loading control and all signals were normalized to ⁇ -actin signal.
  • Lipids from the kidneys were extracted by the method of Bligh and Dyer. Triglyceride and cholesterol composition were measured by gas chromatography (Agilent Technologies, Wilmington, DE).
  • Mitochondrial fraction was isolated from the kidney and used for the measurement of complex I (NADH dehydrogenase) and complex IV (cytochrome c oxidase) activity with kits from Abcam (Cambridge, UK).
  • Podocyte cell culture Human podocytes obtained were maintained in RPMI-1640, 1% Insulin-Transferrin- Selenium, 10% fetal bovine serum, 100 U/ml penicillin, and 100 ⁇ g/ml streptomycin at 33 °C. Podocyte differentiation is induced by thermo-shifting the cells from 33°C to 37°C for 7 days. The differentiated podocytes were then cultured in the presence of 5% of the 4-month old C57/BL6 mouse serum or 5% of the 28-month old mouse serum obtained from NIA to replace fetal bovine serum for 72 hours. In the last 24 hours, 10 ⁇ M Compound I-Na was added to the treatment group.
  • Kidney tissue was homogenized with 500 ⁇ L methanol:H2O (4:3, vol/vol) solution and then extracted using 700 ⁇ L chloroform containing SM (17:0), PC (17:0), and CER (17:0) at 1 ⁇ M as internal standards. The homogenate was shaken and incubated at 37 °C for 20 minutes followed by centrifugation at 15,000 ⁇ g for another 15 minutes. The lower organic phase was collected and evaporated to dryness under vacuum. The residue was then suspended with 100 ⁇ L chloroform:methanol (1:1, vol/vol) solution and then diluted with isopropanol:acetonitrile: H 2 O (2:1:1, vol/vol/vol) solution before injection.
  • Lipidomics analysis was performed on an Acquity UPLC/Synapt G2 Si HDMS QTOFMS system (Waters Corp., Milford, MA) equipped with electrospray ionization (ESI) source. Separation was achieved on an Acquity UPLC CSH C18 column (100 ⁇ 2.1-mm internal diameter, 1.7 mm; Waters Corp.).
  • the mobile phase was a mixture of acetonitrile/water (60/40, vol/vol; A) and isopropanol/acetonitrile (90/10, vol/vol; B), and both A and B contained 10 mM ammonium acetate and 0.1% formic acid.
  • the gradient elution program consisted of a 2-minute linear gradient of 60% A to 57% A to 50% A at 2.1 minutes, a linear decrease to 46% A at 12 minutes to 30% A at 12.1 minutes, and a linear decrease to 1% A at 18 minutes before returning to initial conditions at 18.5 minutes to equilibrate the column.
  • the column temperature was maintained at 55 °C, and the flow rate was 0.4 mL/min.
  • Mass spectrometry data was acquired in both the positive and negative ESI modes at a range of m/z 100-1200.
  • Kidney tissue was homogenized with 200 ⁇ L acetonitrile containing 1 ⁇ M
  • the gradient elution was started from 80% A for 4 minutes, decreased linearly to 60% A over 11 minutes, to 40% A over the next 5 minutes, and to 10% A for the succeeding 1 minute, and finally, increased to 80% A for 4 minutes to re-equilibrate the column.
  • Column temperature was maintained at 45 °C, and the flow rate was 0.4 mL/min.
  • Mass spectrometry detection was operated in negative mode. A mass range of m/z 50-1000 was acquired.
  • Nuclear protein extracts were prepared from kidney tissue. The nuclear extracts were used for the measurement of NF- ⁇ B transcriptional activity with a kit from Marligen
  • the amount of oxidized proteins in kidney homogenates was determined by using an OxyElisa Oxidized Protein Quantitation Kit (Millipore) according to the manufacturer’s instructions.
  • the index of the mesangial expansion was defined as the ratio of mesangial area to glomerular tuft area.
  • the mesangial area was determined by assessment of the periodic acid-Schiff-positive and nucleus-free area in the mesangium using ScanScope image analyzer (Aperio Technologies, Vista, CA).
  • the samples were excited with a 40X, 0.8 NA water objective (Olympus) for harmonic and FLIM measurements.
  • the sample is placed directly on top of the detector assembly input window below the objective, and two photon-induced fluorescence, SHG, and THG are detected by a large area photomultiplier (PMT; R7600P-300; Hammatsu).
  • the detector assembly consists of a sealed chamber with the filter wheel/shutter inside and the housing with PMT.
  • the refractive index matching liquid fills the inside of the housing, and removes loss of photons due to internal reflections and thus, achieves efficient collection of photons.
  • Two BG39 filters serve as input and output windows of the chamber and block NIR excitation light from entering PMT and transmitting UV and visible fluorescence as well harmonic signals.
  • the only optical elements in the detector assembly are BG39 filters and the glss filter of the filter wheel, allowing detection of emitted photons from 320- to 650-nm-wavelenth range.
  • the transmission geometry of detection system allows more efficient detection of SHG and THG signals due to forward propagating nature of the harmonic signals.
  • the signal from the PMT is collected using an FLIMBox and directly transferred to the phasor plot. Briefly, this method of lifetime analysis involves transferring the
  • Phasor approach toward FLIM is a fit-free approach, and increases the speed of the analysis and decreases the computational difficulty associated with FLIM technique.
  • Free NADH and bound NADH have lifetimes of 0.4 and approximately 3.4 ns, respectively, and their individual fluorescence intensity decay seems close to the universal circle.
  • the phasor positions of free and bound NADH are represented by the blue and the red cursors, respectively and the line joining the two cursors is called the metabolic trajectory.
  • the distribution of the points along the metabolic trajectory can be then transformed to a distribution depicting the number of pixels belonging to a certain fraction of free NADH using the law of linear addition in phasor space.
  • the distribution of free NADH can then be compared with the difference in metabolism of different samples.
  • the state of fibrosis was analyzed by a ratiometric method.
  • a ratio of the area covered by SHG to the area covered by FLIM is calculated.
  • This analysis takes into account of both collagen accumulation due to fibrosis and the changes in tissue architecture. The higher the value of this ratio, the higher the fibrosis.
  • the green and red colors in Figure 14B were used to show the SHG and FLIM signals, respectively, and thus, the ratio has been defined as f green /f red .
  • the data collection and analysis were carried out by using SimFCS developed by the Laboratory for Fluorescence Dynamics, University of California, Irvine.
  • SHG imaging was carried out with a 710-nm pulsed laser for excitation and a combination of UG-11 and BG39 filters for the efficient detection of 355-nm SHG photons.
  • the combination of BG39 and UG-11 creates a spectral window of observation with maxima around 355 nm.
  • the SHG signal (green) when excited at 710 nm can be very efficiently collected using these two filters.
  • Results were presented as the means ⁇ SE for at least three independent experiments. Data were analyzed by ANOVA and Student-Newman-Keuls tests for multiple comparisons or by Student's t test for unpaired data between two groups. Statistical significance was accepted at the P ⁇ 0.05 level.
  • a compound of the present application e.g., Compound 1-Na
  • the treatment induced a significant decrease in urinary albumin, similar to beneficial effects achieved with life-long caloric restriction (Figure 2A).
  • the compound of the present application e.g., Compound 1- Na
  • the beneficial effects were associated with prevention of the age-related increases in the level of transforming growth factor- ⁇ (TGF- ⁇ ), fibroblast specific protein-1 (FSP-1), and fibronectin ( Figures 2C and 2D).
  • TGF- ⁇ transforming growth factor- ⁇
  • FSP-1 fibroblast specific protein-1
  • Example 5 A compound of the present application reverses age-related decrease in mitochondrial biogenesis and function.
  • the compound of the present application e.g., Compound 1-Na
  • acetyl-IDH2/IDH2 another target of SIRT3 activity
  • This is associated with the reversal of the decreased mitochondrial complex I and complex IV activity in aged kidneys by the 2-month treatment, at levels identical to life-long caloric restriction ( Figures 3O and 3P).
  • Example 6 A compound of the present application decreases inflammation in human podocytes conditioned with aging serum.
  • the Ames dwarf mice that exhibit delayed aging and extended longevity were studied to determine if FXR and TGR5 expression were regulated similar to caloric restriction (see, e.g., Wang (2017)). Similar to caloric restriction, the FXR and TGR5 mRNA levels increased in the kidneys of Ames dwarf mice ( Figures 5A and 5B). In addition, the genes involved in the mitochondrial biogenesis and function, including NRF1, SIRT1, PGC1 ⁇ , ERR ⁇ , SIRT3, COX4, and LCAD, also increased in the Ames kidneys, consistent with the findings with the treatment of aging C57BL/6 mice with a compound of the present application (e.g.,
  • FXR mRNA is markedly reduced in both glomeruli and tubules in kidney biopsies obtained from human subjects with nephropathy associated with diabetes and obesity, after laser capture microdissection, RNA extraction, and quantitative RT-PCR ( Figure 6A) (see, e.g., Wang (2016)). Immunohistochemistry for FXR in kidney biopsies obtained from human subjects with diabetic nephropathy was also performed. FXR staining in control subjects was concordant with the expression reported from rat tubular segments with predominant expression in the S1 segment of the proximal tubule and cortical thick ascending limb of the loop of Henle, with less expression in the S2 and S3 segment and distal convoluted tubule.
  • both Compound 2 and Compound 1-Na, but not Compound 3 can regulate lipogenesis pathway mediated by SREBP-1 and targets SCD-1, SCD-2, and FIT-1 mRNA (Figure 7E, Figure 7F, Figure 7G, Figure 7H, Figure 7I).
  • Compound 3 and Compound 1-Na, but not Compound 2 can both induce mitochondrial biogenesis pathway as shown by increases in SIRT1, PGC-1 ⁇ , and ERR- ⁇ protein expression ( Figure 7J, Figure 7K, Figure 7L, Figure 7M, Figure 7N, and Figure 7O).
  • Compound 1-Na can simultaneously activate both FXR and TGR5 signaling and their nonoverlapping pathways, with potential additive effects (Figure 7P).
  • Example 10 Compound 1-Na decreases albuminuria and prevents renal histopathologic alterations and renal fibrosis in diabetic DBA/2J mice.
  • a Western diet 42 kcal% milkfat, 34% sucrose, 0.20% cholesterol; approximating the human Western diet
  • Treatment with Compound 1-Na also decreased the glomerular area and mesangial matrix expansion ( Figure 8B, Figure 8C, and Figure 8D).
  • Immunofluorescence microscopy showed increased fibronectin and type 4 collagen in the glomeruli of the diabetic kidney which were prevented by Compound 1-Na treatment ( Figure 8G, Figure 8H, Figure 8I, and Figure 8J).
  • immunohistochemistry showed increased staining with ⁇ -smooth muscle actin ( ⁇ -SMA), which was prevented by Compound 1-Na as well (Figure 8K).
  • Example 12 Compound 1-Na modulates renal lipid metabolism and prevents renal triglyceride and cholesterol accumulation in diabetic DBA/2J mice.
  • Diabetic DBA/2J mice were shown to have increased kidney neutral lipid accumulation in both glomeruli and tubulointerstitium by oil red O staining (Figure 9A) (see, e.g., Wang (2016)).
  • the lipid accumulation was mediated by increases in SCD-2 as well as ChREBP- ⁇ and liver pyruvate kinase (Table 1).
  • Biochemical analysis of kidney lipid extracts showed increased kidney triglyceride and cholesterol accumulation, which was significantly decreased by Compound 1-Na treatment (Figure 9B and Figure 9C). The effects of
  • Compound 1-Na in decreasing renal triglyceride and cholesterol content were mediated by coordinated effects inducing (i) decreased expression of SREBP-1c and its target genes SCD- 1 and SCD-2, which mediate fatty acid and triglyceride synthesis (Table 1); (ii) decreased expression of liver pyruvate kinase, which also mediates fatty acid and triglyceride synthesis (Table 1); (iii) decreased expression of SREBP-2, which mediates cholesterol synthesis (Table 1); (iv) increased expression of lipolysis gene LPL (Table 1); and (v) decreased expression of lipid droplet formation gene FIT-1 (Table 1).
  • Compound 1-Na also decreases serum triglycerides and LDL cholesterol in diabetic DBA/2J mice.
  • STX treatment of DBA/2J mice fed a Western diet resulted in marked increases in serum glucose, triglyceride, and cholesterol levels, with most of the cholesterol derived from LDL (14.0 ⁇ 2.1 mg/dl in control versus 654 ⁇ 79 mg/dl in diabetic mice) (Table 1).
  • Treatment with Compound 1-Na did not decrease serum glucose levels in diabetic DBA/2J mice but significantly decreased plasma triglyceride, total cholesterol, and LDL cholesterol levels (654 ⁇ 79 mg/dl in diabetic mice versus 40.5 ⁇ 2.5 mg/dl in diabetic mice treated with Compound 1-Na) (Table 1).
  • Example 13 Compound 1-Na prevents inflammation, oxidative stress, and endoplasmic reticulum stress in diabetic DBA/2J mice.
  • Compound 1-Na markedly decreased the expression of macrophage marker CD68 in diabetic kidneys (Figure 10A) (see, e.g., Wang (2016)). This was consistent with the inhibition by Compound 1-Na treatment of NF- ⁇ B p65 and p50 heterodimeric complexes expression (Figure 10B and Figure 10C) and NF- ⁇ B activity, the master transcription factor regulating inflammation ( Figure 10D).
  • the expression of NF- ⁇ B-dependent proinflammatory mediators like intercellular adhesion molecule-1 and cyclooxygenase-2, was also
  • HIF-1 ⁇ and HIF-2 ⁇ were significantly increased in diabetic DBA/2J mice (see, e.g., Wang (2016)).
  • Treatment with Compound 1-Na prevented the increased expression of HIF-1 ⁇ and HIF-2 ⁇ in diabetic kidneys ( Figure 11A and Figure 11B).
  • Glut1 expression was also increased in diabetic kidneys but reversed by Compound 1-Na treatment ( Figure 11C and Figure 11D).
  • Example 15 Renal effects of Compound 1-Na in the db/db mouse model of type 2 diabetes mellitus and obesity.
  • Compound 1-Na stimulated FXR and TGR5 mRNA in both db/m and db/db mice ( Figure 12A and Figure 12B).
  • FXR mRNA was increased in db/db mice
  • FXR protein abundance as determined by FXR immunohistochemistry was decreased in db/db mice (- 35.2% relative to db/m)
  • Compound 1-Na treatment activated FXR protein expression to levels seen in nondiabetic db/m mice ( Figure 12C).
  • Compound 1-Na treatment of db/db mice did not alter blood glucose levels, but significantly decreased plasma total cholesterol and triglyceride levels (Table 2).
  • treatment of db/db mice with Compound 1-Na resulted in significantly decreased albuminuria (Figure 12D).
  • Treatment with Compound 1-Na also decreased mesangial matrix expansion (Figure 12E and Figure 12F), podocyte loss as shown by synaptopodin immunofluorescence microscopy (Figure 12G), renal fibrosis indicated by the decreased collagen 1 ( Figure 12I and Figure 12J), and collagen 3 ( Figure 12K and Figure 12L) protein abundance as determined by
  • Example 16 Compound 1-Na induces mitochondrial biogenesis and metabolism pathways in db/db mouse mode of type 2 diabetes mellitus and obesity.
  • Compound 1-Na treatment also increased PGC-1 ⁇ mRNA (table 2) and protein ( Figure 13E and Figure 13F), ERR- ⁇ , and Nrf1 mRNA (Table 2), the transcriptional regulators of mitochondrial biogenesis and activity, as well as several enzymes that mediated fatty acid and glucose oxidation, including carnitine palmitoyltransferase-1A, pyruvate dehydrogenase kinase 4, long-chain acyl CoA dehydrogenase, and acetyl CoA synthetase 2 (Table 2).
  • Example 17 Compound 1-Na prevents mitochondrial dysfunction, oxidative stress, inflammation, and fibrosis in mice with diet-induced obesity.

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Abstract

La présente invention concerne des méthodes de traitement ou de prévention d'une maladie , d'un trouble ou d'une affection rénale (par exemple, l'âge, le diabète et/ou une maladie, un trouble ou une affection rénale liée au diabète et/ou à l'obésité) chez un sujet en ayant besoin, comprenant l'administration d'une quantité thérapeutiquement efficace d'un composé de la demande.
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Publication number Priority date Publication date Assignee Title
WO2020163201A1 (fr) * 2019-02-04 2020-08-13 Intercept Pharmaceuticals, Inc. Traitement et prévention de maladies inflamatoires de l'intestin avec un dérivé d'acide biliaire
EP3920937A4 (fr) * 2019-02-04 2022-11-02 Intercept Pharmaceuticals, Inc. Traitement et prévention de maladies inflamatoires de l'intestin avec un dérivé d'acide biliaire
US11512065B2 (en) 2019-10-07 2022-11-29 Kallyope, Inc. GPR119 agonists
US11279702B2 (en) 2020-05-19 2022-03-22 Kallyope, Inc. AMPK activators
US11851429B2 (en) 2020-05-19 2023-12-26 Kallyope, Inc. AMPK activators
US11407768B2 (en) 2020-06-26 2022-08-09 Kallyope, Inc. AMPK activators

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