WO2016145532A1 - Cmpf pour le traitement ou la prévention de la stéatose hépatique - Google Patents

Cmpf pour le traitement ou la prévention de la stéatose hépatique Download PDF

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WO2016145532A1
WO2016145532A1 PCT/CA2016/050293 CA2016050293W WO2016145532A1 WO 2016145532 A1 WO2016145532 A1 WO 2016145532A1 CA 2016050293 W CA2016050293 W CA 2016050293W WO 2016145532 A1 WO2016145532 A1 WO 2016145532A1
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cmpf
subject
liver
mice
hfd
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Judith EVERSLEY
Michael Wheeler
Kacey PRENTICE
Ying Liu
Feihan DAI
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The Governing Council Of The University Of Toronto
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/335—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin
    • A61K31/34—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin having five-membered rings with one oxygen as the only ring hetero atom, e.g. isosorbide
    • A61K31/341—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin having five-membered rings with one oxygen as the only ring hetero atom, e.g. isosorbide not condensed with another ring, e.g. ranitidine, furosemide, bufetolol, muscarine
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
    • A61P1/16—Drugs for disorders of the alimentary tract or the digestive system for liver or gallbladder disorders, e.g. hepatoprotective agents, cholagogues, litholytics
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P3/00—Drugs for disorders of the metabolism
    • A61P3/06—Antihyperlipidemics
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P5/00—Drugs for disorders of the endocrine system
    • A61P5/48—Drugs for disorders of the endocrine system of the pancreatic hormones
    • A61P5/50—Drugs for disorders of the endocrine system of the pancreatic hormones for increasing or potentiating the activity of insulin
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D307/00—Heterocyclic compounds containing five-membered rings having one oxygen atom as the only ring hetero atom
    • C07D307/02—Heterocyclic compounds containing five-membered rings having one oxygen atom as the only ring hetero atom not condensed with other rings
    • C07D307/34—Heterocyclic compounds containing five-membered rings having one oxygen atom as the only ring hetero atom not condensed with other rings having two or three double bonds between ring members or between ring members and non-ring members
    • C07D307/56—Heterocyclic compounds containing five-membered rings having one oxygen atom as the only ring hetero atom not condensed with other rings having two or three double bonds between ring members or between ring members and non-ring members with hetero atoms or with carbon atoms having three bonds to hetero atoms with at the most one bond to halogen, e.g. ester or nitrile radicals, directly attached to ring carbon atoms
    • C07D307/68—Carbon atoms having three bonds to hetero atoms with at the most one bond to halogen

Definitions

  • the present invention relates to field of metabolic disorders and more specifically to the use of 3-carboxy-4-methyl-5-propyl-2-furanpropanoic acid (CMPF) for the treatment or prevention of disorders related to lipid metabolism.
  • CMPF 3-carboxy-4-methyl-5-propyl-2-furanpropanoic acid
  • MetS The underlying pathophysiology of MetS is based in dysregulated lipid metabolism, resulting in aberrant lipid storage in the liver and muscle, increased insulin resistance, and altered circulating lipoprotein levels (Avramoglu et al., 2006; Bergman and Ader, 2000; Cao et al., 2008; Ginsberg, 2006). While this dyslipidemia is largely attributed to a ubiquitous increase in release of free fatty acids (FFAs) from the adipose tissue, systemic lipid profiling has revealed strong correlations between MetS and a specific fatty acid signature of serum lipids, rather than elevated lipids in general (Warensjo et al., 2005). Thus, individual lipids from the diet, micobiome, and de novo synthesis influenced by genetics and environment that are characteristic of the MetS plasma lipomic signature may play a more significant role in the regulation of systemic metabolic homeostasis than previously thought.
  • FFAs free fatty acids
  • Metabolomic screening has now become a widely employed method for examining the lipid signature of MetS, insulin resistance, and diabetes (Bain et al., 2009; Fiehn et al., 2010; Friedrich, 2012; Prentice et al., 2014; Wang et al., 201 1 ).
  • Several specific fatty acids, as well as some of their metabolic by-products, have been strongly implicated in directly altering metabolic homeostasis.
  • C16:0 palmitic acid
  • C18:1 n9 oleic acid
  • rodent models Joseph et al., 2004; Prentki and Nolan, 2006; Tang et al., 2007
  • metabolic by-products of palmitic acid also shown to be dysregulated in MetS, insulin resistance and diabetes, also have distinctive effects on metabolic homeostasis.
  • SREBP-1 SREBP-1 -mediated pathway in the liver results in the production of C18:0 (stearic acid), which is associated with increased hepatic insulin resistance (Chu et al., 2013).
  • Hepatic steatosis also known as fatty liver disease
  • NAFLD non-alcoholic fatty liver disease
  • NASH non-alcoholic steatohepatitis
  • alcoholic fatty liver disease is closely associated with being overweight or obese, as it is caused by excessive fat deposition in the liver. This occurs when fat intake is greater than can be processed by the adipose tissue alone, or when the liver is unable to processes fat for excretion, leading to inappropriate fat storage (Angulo, 2002).
  • Increased oxidative stress and cytokines are also thought to be contributing factors.
  • NAFLD World Gastroenterology Organization
  • NAFLD insulin resistance characteristic of obesity and diabetes
  • Angulo Angulo, 2002
  • insulin-sensitive and insulin-resistant obese subjects can be stratified based on muscle and liver lipid accumulation alone.
  • the mechanism underling NAFLD-induced insulin resistance is associated with increased diacylglycerols (DAG) accumulation, which induces activation of ⁇ and results in inhibition of insulin receptor signaling (Birkenfeld et al., 2014).
  • DAG diacylglycerols
  • NAFLD NAFLD
  • obesity is the most common risk factor (with 75-90% of obese individuals having NAFLD)
  • this line of screening is the most prevalent diagnostic tool (Torres et al, 2008).
  • Blood tests demonstrating mild to moderate elevations in serum aminotransferase levels is most commonly associated with NAFLD in addition to mild changes in serum alkaline phosphatase and g-glutamyl transpeptidase.
  • NAFLD is commonly associated with insulin resistance and metabolic syndrome, other parameters including fasting cholesterol and triglycerides, glucose and insulin are likely to also be abnormal. Further tests including liver ultrasound and more rarely liver biopsy can also be used for clinical diagnosis.
  • these advances tests are commonly reserved for the investigation of NASH, due to the elevated risk of morbidity and mortality.
  • NAFLD NAFLD
  • the primary preventative option is modification of diet to limit fat intake, and sustained weight loss. While it is widely believed that some medications prescribed for diabetes, including Metformin, may be beneficial for the treatment of NAFLD, there is no evidence that this is the case (Lavine et al., 201 1 ).
  • CMPF fu ran fatty acid metabolite 3-carboxy-4-methyl-5-propyl-2- furanpropanoic acid
  • CMPF alters whole body glucose metabolism, resulting in preferential fatty acid utilization and significantly improved insulin sensitivity in rodent models of obesity and insulin resistance.
  • Administration of CMPF also correlates with a significant reduction in fat accumulation in the liver, in terms of both eliminating existing fat deposits, as well as preventing the accumulation of new fat deposits, in mice that are fed a high fat diet.
  • CMPF treatment followed by a high fat diet increases energy expenditure and lipid metabolism in mice. This corresponds to improved insulin sensitivity, and protection against the development of hepatic steatosis.
  • CMPF also appears to act as an allosteric inhibitor of Acetyl-CoA Carboxylase (ACC) to drive lipolysis and prevent triglyceride synthesis.
  • ACC Acetyl-CoA Carboxylase
  • CMPF induces FGF21 expression and potentiates lipid uptake into hepatocytes.
  • Increased lipolysis has also been shown to be associated with induction of FGF21 expression and secretion, which acts locally on the liver to drive lipid metabolism and protect against the development of steatosis.
  • mice placed in a high fat diet prior to and concurrently with CMPF administration exhibited dramatically improved insulin sensitivity (comparable to mice fed a normal control diet), relative to control mice on the same high fat diet.
  • Insulin sensitivity in high fat diet fed mice treated with CMPF was similar to mice fed a normal control diet, and significantly improved compared to control mice fed a high fat diet.
  • Example 2 also demonstrates the effects of CMPF on reducing hepatic steatosis and improving insulin sensitivity in leptin knockout Ob/Ob mice, which is a genetic model of obesity and insulin resistance.
  • a method for the treatment or prevention of hepatic steatosis comprises administering to a subject in need thereof 3-carboxy-4-methyl-5-propyl-2-furanpropanoic acid (CMPF). Also provided is the use of CMPF for the treatment or prevention of hepatic steatosis in a subject in need thereof.
  • the subject has, or is at risk of developing non-alcoholic fatty liver disease (NAFLD) or non-alcoholic steatohepatitis (NASH).
  • NAFLD non-alcoholic fatty liver disease
  • NASH non-alcoholic steatohepatitis
  • the subject has a liver with a fat content of 5% or greater by mass.
  • the subject has a liver with a fat content of 7% or greater by mass, or a fat content between 5% and 10%.
  • the subject has metabolic syndrome, insulin resistance, is obese and/or has a high fat diet.
  • a method for the treatment or prevention of insulin resistance comprises administering CMPF to a subject in need thereof.
  • CMPF for the treatment or prevention of insulin resistance in a subject in need thereof.
  • CMPF for improving insulin sensitivity in a subject in need thereof.
  • the subject has insulin resistance and/or impaired insulin sensitivity.
  • the subject has a fasting serum insulin level greater than 25mlU/L or 174 pmol/L.
  • the subject has impaired glucose tolerance.
  • the subject has metabolic syndrome, is obese and/or has a high fat diet.
  • a method of increasing lipid metabolism comprises administering CMPF to a subject.
  • CMPF for increasing lipid metabolism in a subject in need thereof.
  • lipid metabolism is increased relative to carbohydrate metabolism in the subject.
  • CMPF increases lipolysis and/or decreases lipogenesis in the subject.
  • CMPF increases lipid metabolism in the liver.
  • CMPF increases fatty acid oxidation in the islets cells of the pancreas.
  • the cells are liver cells such as hepatocytes.
  • the methods and uses described herein include contacting a liver, or part thereof, in vivo, in vitro or ex vivo with CMPF to increase lipid metabolism and/or reduce the fat content of the liver or part thereof.
  • CMPF inhibits Acetyl-CoA Carboxylase 1 or 2 (ACC1 and ACC2). In one embodiment CMPF induces expression of FGF21 . In one embodiment, CMPF inhibits ACC in liver cells in vivo, in vitro or ex vivo. In one embodiment, CMPF induces expression of FGF21 in liver cells in vivo, in vitro or ex vivo. In one embodiment, the liver cells are hepatocytes.
  • the methods and uses described herein include the use and/or administration of a composition comprising CMPF and a pharmaceutically acceptable carrier.
  • Figure 1 shows acute treatment with CMPF induces persistent changes in whole body metabolism.
  • B) Plasma CMPF concentration following intraperitoneal injection (n 4-6).
  • C) Weekly weight gain through injection period and following placement on 60% high fat diet (n 20/group).
  • D) Food and (E) water intake over 24 hours four weeks following final injection (n 4/group).
  • F) MRI scan images and (G) quantification of fat distribution (n 4/group).
  • H) Activity over 24 hour period in x, y, and z planes (n 4/group).
  • FIG. 2 shows that CMPF treatment protects insulin sensitivity on a high fat diet.
  • A) 16hr fasting blood glucose and (B) fasting plasma insulin levels (N 8/group).
  • C) Blood glucose levels during intraperitoneal insulin tolerance test (IpITT) (n 8/group).
  • F) IpITT showing that treatment with 2,5-Furandicarboxylic acid, a control furan acid, does not have the same effect as CMPF on improving insulin sensitivity during IpITT (n 4/group) *P ⁇ 0.05, **P ⁇ 0.01 , ***P ⁇ 0.001 . All error bars SEM.
  • FIG. 3 shows CMPF treatment prevents the development of high fat diet-induced steatosis.
  • B) H&E and oil red O staining of liver sections (n 8/group).
  • C) Triglyercide content from isolated livers (n 8/group)
  • FIG. 4 shows the acute effect of CMPF on hepatocyte metabolism.
  • A) Fatty acid uptake and (B) oxidation per hour in isolated hepatocytes following 24 hour treatment (n 4/group).
  • C) Glucose oxidation per hour (n 4/group).
  • D) Demonstration of specificity of fatty acid oxidation measurements by inhibition with the CPT1 inhibitor etomoxir (N 4).
  • FIG. 5 shows the persistent effect of CMPF is associated with increased FGF21 and decreased ACC abundance.
  • D) Heat map showing significantly altered genes associated with lipogenesis and lipolysis in livers from mice isolated 4 weeks following treatment as determined by microarray (n 3/group).
  • F FGF21 levels in fasting serum at the time of sacrifice and (G) gene expression in 24 hour treated hepatocytes.
  • H Serum FGF21 levels in the days following first injection of CMPF, vehicle, or 2,5- Furandicarboxylic acid.
  • FIG. 6 shows that CMPF prevents steatosis through FGF21 A
  • FIG. 7 shows that CMPF must enter the liver through OAT transporters.
  • A) Weight gain through the injection and 6-week follow-up periods (N 2-3/group).
  • B) Blood glucose during ipITT (N 2-3/group).
  • C) Photographs and Oil red O staining of liver sections (N 2-3/group).
  • D) Liver weight as a percentage of body weight and (E) quantification of liver triglyceride (N 2-3/group). *P ⁇ 0.05. All error bars SEM.
  • FIG. 8 shows that CMPF alters whole-body metabolism in diet-induced obese mice.
  • A) Mice on a high fat diet (HFD) gain significantly more weight than chow fed mice. Injection with CMPF (HFD- CMPF) has no effect on body weight compared to HFD-Control injected mice (N 20-23/group).
  • B) Activity over a 24hour period summed in the x, y and z planes (N 3-4/group).
  • D) Representative MRI images showing adipose tissue distribution and (E) quantification of adipose area (N 4/group).
  • FIG. 10 shows that CMPF treatment ameliorates steatosis and improves hepatic insulin sensitivity in high fat diet fed mice.
  • A) Photographs and Oil red O staining of liver sections (N 8/group).
  • B) Liver weight expressed as percentage of body weight (N 4/group).
  • C) In vivo insulin signaling shown as pAKT in (C) liver and (D) muscle samples (N 4/group). *P ⁇ 0.05.
  • Figure 1 1 shows metabolomic and microarray analysis of diet- induced obese livers.
  • A) Heat map showing fold change in select metabolites from the pentose phosphate pathway, pentose metabolism, and glutathione metabolism (N 4/group). Dark grey indicates significant change at P ⁇ 0.05, light grey indicates change at P ⁇ 0.10.
  • FIG. 12 shows that CMPF improves insulin sensitivity in ob/ob mice.
  • A) Body weight before and after the two week injection period (N 4- 7/group).
  • B) Representative MRI images showing adipose distribution and (C) quantification of adipose area in MRI images (N 4-7).
  • E) Random blood glucose and 16hour fasting (F) blood glucose and (F) plasma insulin levels (N 4-7).
  • FIG. 13 shows that CMPF reduces steatosis in ob/ob mice.
  • A) Photographs and (b) Oil red O staining of liver sections (N 8-14).
  • C) Liver weight expressed as percentage of body weight (N 4-7). *P ⁇ 0.05.
  • Figure 14 shows that treatment with CMPF prior to a HFD impairs glucose tolerance.
  • B) Blood glucose and (C) corresponding plasma insulin values during intraperitoneal glucose tolerance test (N 8/group). *,#P ⁇ 0.05
  • FIG 15 shows that islets from CMPF-injected mice placed on a high fat diet exhibit impaired glucose-stimulated insulin secretion (GSIS) and enhanced palmitate-stimulated insulin secretion (PSIS).
  • GSIS glucose-stimulated insulin secretion
  • PSIS palmitate-stimulated insulin secretion
  • FIG. 16 shows that HFD mice treated with CMPF have further impairment in glucose tolerance.
  • B) Blood glucose and (C) corresponding plasma insulin values during an oral glucose tolerance test (OGTT) (N 8/group).
  • D) Blood glucose and (E) corresponding plasma insulin values during intraperitoneal glucose tolerance test (IpGTT) (N 8/group).
  • FIG. 7 shows that CMPF treatment in high fat diet-fed mice causes a significant decrease in islet mass and pancreatic insulin area.
  • A) Total intracellular insulin content in islets isolated from mice treated following diet intervention (N 13-14/group).
  • B) Insulin positive area calculated using positive pixel analysis of immunohistochemically (IHC) stained whole pancreatic sections show HFD-CMPF mice have significantly reduced insulin area relative to total pancreatic area (N 5- 6/group).
  • Figure 18 shows that differences in islet size are not due to proliferation.
  • A) BrdU positive cells in pancreatic sections (N 4 mice/group).
  • B) Quantification of average cell size determined by islet area divided by number of nuclei (N 8/mice group).
  • C) Quantification of number of cells per islet (N 8/group). *P ⁇ 0.05, **P ⁇ 0.01 , ***P ⁇ 0.001 .
  • FIG 19 shows that CMPF treatment impairs glucose metabolism and promotes fatty acid metabolism in the beta cell of high fat diet-fed mice treated with CMPF.
  • B) Static secretion assays with low glucose (LG; 2m M Glucose), high glucose (HG; 20mM) and high glucose with KCI treatment (KCI; 20mM glucose plus 30mM KCI), with C) fold change in insulin secretion (high glucose/low glucose) (N 13-14/group).
  • FIG. 20 shows that CMPF treatment worsens glucose intolerance in leptin knockout Ob/Ob mice.
  • FIG. 21 shows that islets from CMPF-treated Ob/Ob Mice have enhanced palmitate-stimulated and reduced glucose-stimulated insulin secretion.
  • A) Islets isolated from Ob/Ob-CMPF mice are significantly smaller than islets from Ob/Ob-Control mice (N 4-7/group).
  • B) Fold change in glucose-stimulated insulin secretion (HG/LG) during static secretion assays show Ob/Ob-CMPF islets have reduced GSIS (N 4- 7/group).
  • hepatic steatosis also known as “fatty liver disease” refers to a condition characterized by excessive amounts of triglycerides and other fats inside liver cells.
  • subjects with hepatic steatosis exhibit elevated levels of SGOT (serum glutamic- oxaloacetic transaminase), SGPT (serum glutamic pyruvic transaminase) and/or alkaline phosphatase.
  • subjects with hepatic steatosis exhibit elevated serum levels of aspartate aminotransferase (AST) and/or serum alanine aminotransferase (ALT). In some embodiments, subjects will have an altered AST/ALT ratio.
  • non-alcoholic fatty liver disease refers to hepatic steatosis wherein at least 5% of the mass of the liver is fat.
  • NAFLD refers to hepatic steatosis wherein between 5- 10% of the mass of the liver is fat.
  • NAFLD refers to hepatic steatosis wherein at least about 5%, 6%, 7%, 8%, 9% or 10% of the mass of the liver is fat.
  • subjects with NAFLD have an aspartate aminotransferase-to-alanine aminotransferase (AST/ALT) ratio >1 .
  • non-alcoholic steatohepatitis refers to hepatic steatosis characterized by fatty liver with associated inflammation and fibrosis.
  • NASH non-alcoholic steatohepatitis
  • typically at least 5% of the mass of the liver is fat, or between 5-10% of the liver is fat.
  • insulin resistance refers to a physiological condition wherein insulin becomes less effective at lowering blood sugar levels.
  • insulin sensitivity is related to insulin resistance and refers to the effectiveness of insulin to lower blood sugar levels.
  • a subject with insulin resistance has a fasting serum insulin level greater than 25 lU/ml or 174 pmol/L.
  • subjects with insulin resistance may be identified using the homeostasis model assessment-estimated insulin resistance (HOMA-IR) index.
  • FPI fasting plasma insulin
  • FPG fasting plasma glucose
  • subjects with a HOMA-I R index greater than 2.60 are identified as having insulin resistance.
  • paired glucose tolerance refers to pre- diabetic state associated with insulin resistance. In one embodiment, “impaired glucose tolerance” refers to a subject with a fasting blood glucose level >1 10 mg/dL.
  • obese refers to a condition wherein a subject has excess body fat to the extent that it has a negative effect on health.
  • a subject is considered obese if they have a body mass index equal or greater to 30 kg/m 2
  • the term "subject" refers to any member of the animal kingdom that has a liver capable of converting acetyl-CoA to fatty acids.
  • the subject is any member of the animal kingdom that has ⁇ -cells which store and release insulin in order to control levels of glucose in the blood.
  • the subject is any member of the animal kingdom wherein CMPF induces the expression of FGF21 .
  • the subject is a human.
  • the subject is an animal such as a rat or mouse.
  • the subject has, or is suspected of having hepatic steatosis.
  • the subject has or is suspected of having a metabolic disorder.
  • the subject has insulin resistance and/or impaired glucose tolerance.
  • Treating means an approach for obtaining beneficial or desired results, including clinical results.
  • beneficial or desired clinical results can include, but are not limited to, alleviation or amelioration of one or more symptoms or conditions, diminishment of extent of disease, stabilized (i.e. not worsening) state of disease (e.g. maintaining physiologically normal levels of fat in the liver and/or physiologically normal levels of blood insulin), preventing spread of disease, delay or slowing of disease progression, amelioration or palliation of the disease state, diminishment of the reoccurrence of disease, and remission (whether partial or total), whether detectable or undetectable.
  • Treating” and “Treatment” can also mean prolonging survival as compared to expected survival if not receiving treatment.
  • “Treating” and “treatment” as used herein also include prophylactic treatment.
  • treatment methods comprise administering to a subject a therapeutically effective amount of CMPF as described herein and optionally consists of a single administration, or alternatively comprises a series of administrations.
  • an effective amount or “therapeutically effective amount” means an amount effective, at dosages and for periods of time necessary to achieve the desired result.
  • an effective amount is an amount that helps reduce the level of fat in in the liver compared to the response obtained without administration of CMPF.
  • an effective amount is an amount that helps reduce the level of fat in the liver to less than about 5% by mass.
  • an effective amount is an amount that changes the presence of biomarkers for hepatic steatosis in the subject.
  • an effective amount of CMPF is an amount that measurably reduces levels of serum aminotransferase and/or g-glutamyl transpeptidase in the subject.
  • an effective amount of CMPF is an amount that measurably reduces serum triglyceride levels in the subject.
  • an effective amount is an amount that helps improve glucose homeostasis, reduces the fasting amount of insulin in the blood and/or improves HOMA-I R in the subject. Effective amounts may vary according to factors such as the disease state, age, sex and weight of the subject.
  • the amount of a given compound that will correspond to such an amount will vary depending upon various factors, such as the given drug or compound, the pharmaceutical formulation, the route of administration, the type of disease or disorder, the identity of the subject or host being treated, and the like, but can nevertheless be routinely determined by one skilled in the art.
  • CMPF for the Treatment of Hepatic Steatosis and/or Insulin Resistance
  • CMPF 3-carboxy-4-methyl-5-propyl-2-furanpropanoic acid
  • CMPF-HFD mice exhibited dramatically improved insulin sensitivity compared to Control-HFD mice ( Figure 2, part C).
  • CMPF alters whole body glucose metabolism in rodent models of obesity and insulin resistance, resulting in preferential fatty acid utilization and significantly improved insulin sensitivity. In one embodiment, this correlates with a significant reduction in fat accumulation in the liver, in terms of both eliminating existing fat deposits, as well as preventing the accumulation of new fat deposits, while mice are fed a high fat diet.
  • CMPF has also been demonstrated to alter lipid metabolism and improve insulin sensitivity in ob/ob mice, which represents a genetic model of obesity and insulin resistance. CMPF therefore appears to act on cells in the liver and beta cells in the pancreas but may also have actions beyond these cells.
  • CMPF is for use or administered to a subject with hepatic steatosis or a subject at risk of developing hepatic steatosis.
  • the subject has, or is at risk of developing, non-alcoholic fatty liver disease (NAFLD).
  • NAFLD non-alcoholic fatty liver disease
  • the methods and uses described herein can also be for the treatment or prevention of more severe forms of hepatic steatosis such as non-alcoholic steatohepatitis (NASH).
  • NASH non-alcoholic steatohepatitis
  • subjects at risk of developing hepatic steatosis or who may benefit from the use or administration of CMPF include subjects with metabolic syndrome, insulin resistance, and/or a high fat diet.
  • CMPF is for use or administered to a subject with insulin resistance with a fasting serum insulin level greater than 25 IU/L or 174 pmol/L.
  • the subject has impaired glucose tolerance.
  • subjects who may benefit from improving insulin sensitivity or from the use or administration of CMPF include subjects with metabolic syndrome, insulin resistance, obesity and/or a high fat diet.
  • CMPF has also been demonstrated to alter lipid metabolism resulting in preferential fatty acid utilization. Accordingly, in one embodiment the present description provides uses and methods for increasing lipid metabolism with CMPF.
  • the method comprises administering CMPF to a subject.
  • the use or administration of CMPF increases the metabolism of lipids relative to that of carbohydrates. For example, in one embodiment CMPF increases beta oxidation and inhibits glucose oxidation.
  • CMPF increases lipolysis and/or decreases lipogenesis.
  • CMPF increases lipolysis and/or decreases lipogenesis in the liver or pancreas. Also provided are uses and methods for increasing lipid metabolism with CMPF in vivo, in vitro or ex vivo.
  • CMPF increases lipid metabolism in liver cells (hepatocytes). In one embodiment, CMPF increases lipid metabolism in the pancreas. For example, in one embodiment, CMPF increases fatty acid oxidation in the islets cells of the pancreas. In one embodiment, subjects who may benefit from the use or administration of CMPF for increasing lipid metabolism include subjects with metabolic syndrome, insulin resistance, obesity and/or a high fat diet. In one embodiment, CMPF may be used to alter lipid metabolism and/or reduce fat content in a liver, or part thereof, in vitro or ex vivo.
  • CMPF has also been demonstrated to have an effect on the expression or activity of proteins associated with lipid metabolism.
  • CMPF inhibits Acetyl-CoA Carboxylase (ACC).
  • ACC expression and/or activity is reduced in a subject even after CMPF has been removed from the circulation.
  • CMPF induces the expression of Fibroblast Growth Factor-21 (FGF21 ).
  • FGF21 Fibroblast Growth Factor-21
  • CMPF increases the expression of one or more genes selected from leptin receptor (Lepr), PCK1 and CPT1 a.
  • CMPF decreases the expression or protein levels of one or more genes selected from ACC1 , ACC2, Gck and Scd1 .
  • CMPF may be formulated for use and/or prepared for administration to a subject in need thereof as known in the art. Conventional procedures and ingredients for the selection and preparation of suitable formulations are described, for example, in Remington's Pharmaceutical Sciences (2003 - 20th edition) and in The United States Pharmacopeia: The National Formulary (USP 24 NF19) published in 1999.
  • Administration can be effected in one dose, continuously or intermittently (e.g., in divided doses at appropriate intervals) throughout the course of treatment. Methods of determining the most effective means and dosage of administration are well known to those of skill in the art and will vary with the formulation used for therapy, the purpose of the therapy, the target cell(s) being treated, and the subject being treated. Single or multiple administrations can be carried out with the dose level and pattern being selected by the treating physician, veterinarian, or clinician.
  • Routes of administration include, but are not limited to, oral (e.g., by ingestion); buccal; sublingual; transdermal (including, e.g., by a patch, plaster, etc.); transmucosal (including, e.g., by a patch, plaster, etc.); intranasal (e.g., by nasal spray, drops or from an atomiser or dry powder delivery device); ocular (e.g., by eyedrops); pulmonary (e.g., by inhalation or insufflation therapy using, e.g., an aerosol, e.g., through the mouth or nose); rectal (e.g., by suppository or enema); vaginal (e.g., by pessary); parenteral, for example, by injection, including subcutaneous, intradermal, intramuscular, intravenous, intraarterial, intracardiac, intrathecal, intraspinal, intracapsular, subcapsular, intraorbital
  • the route of administration is oral (e.g., by ingestion). In another embodiment, the route of administration is parenteral (e.g., by injection).
  • compositions comprising CMPF and a pharmaceutically acceptable carrier.
  • the composition comprises CMPF and a pharmaceutically acceptable carrier for the treatment or prevention of hepatic steatosis.
  • the composition comprises CMPF and a pharmaceutically acceptable carrier for improving insulin sensitivity.
  • the composition comprises CMPF and a pharmaceutically acceptable carrier for increasing lipid metabolism. Also provided is the use of CMPF for the manufacture of a medicament for the treatment or prevention of one or more conditions as described herein.
  • compositions include, albeit not exclusively, solutions of CMPF in association with one or more pharmaceutically acceptable vehicles or diluents, and optionally contained in buffered solutions with a suitable pH.
  • the compositions include described herein one or more pharmaceutically acceptable carriers, diluents, excipients, adjuvants, fillers, buffers, preservatives, anti-oxidants, lubricants, stabilisers, solubilisers, surfactants (e.g., wetting agents), masking agents, colouring agents, flavouring agents, and sweetening agents.
  • the formulation may further comprise other active agents, for example, other therapeutic or prophylactic agents.
  • the present disclosure also includes a kit comprising CMPF, or a composition comprising CMPF, e.g., preferably provided in a suitable container and/or with suitable packaging; and (b) instructions for use, e.g., written instructions on the use or administration of CMPF for e.g. the treatment or prevention of hepatic steatosis, the treatment or prevention of insulin resistance and/or for increasing lipid metabolism as described herein.
  • EXAMPLE 1 CMPF enhances lipid metabolism, protects insulin sensitivity and induces FGF21 to prevent steatosis
  • mice were injected intraperitoneally (IP) with 6mg/kg CMPF or vehicle control at 24h intervals for seven days and assessed for body weight, blood glucose and plasma insulin. All experiments were approved by the Animal Care Committee (University of Toronto) and animals handled according to the Canadian Council of Animal Care guidelines.
  • CMPF was purchased from Cayman Chemical (product number 10007133) and dissolved in 70% ethanol to a stock concentration of 100mM. CMPF was stored at 4C. For injections, CMPF was dissolved in 10Oul sterile saline within an insulin syringe for injection.
  • mice Seven-week-old male CD1 mice were purchased from Charles River and allowed to acclimate for one week prior to the beginning of experiments. Mice were maintained on standard chow diet while being injected intraperitoneally (i.p.) with 6mg/kg CMPF or vehicle control at 24h intervals for seven days, as previously described (Prentice et al., 2014). 24 hours following the final injection, mice were switched to either a 60%kcal from fat HFD (HFD; OpenSource diets D12492, Research Diets Inc, USA) or a sucrose-matched control (OpenSource diets D12450J, Research Diets Inc, USA) for 4 weeks. Mice were also monitored weekly for individual body weights and whole-cage food consumption.
  • HFD fat HFD
  • OpenSource diets D12492, Research Diets Inc, USA a sucrose-matched control
  • mice were fasted overnight for 14h before an intraperitoneal glucose tolerance test (ipGTT).
  • Mice were injected ip with 1 g/kg sucrose. IpGTTs, and measurement of plasma insulin were performed as previously described (Allister et al., 2013).
  • Blood ( ⁇ 25ul) was collected from the tail vein at 0 (fasting), 10, 20 and 30 minutes from quantification of plasma insulin and blood glucose. Blood glucose was also measured at 60 and 120 minutes post injection.
  • IP insulin tolerance tests ipITT were performed following a 4h fast. 1 .5IU/kg insulin was injected and blood glucose was measured at 0, 10, 20, 30, 45, 60 and 120 minutes.
  • Plasma insulin was quantified by ELISA (Alpco Ultrasensitive Insulin ELISA). All experiments were approved by the Animal Care Committee at the University of Toronto and animals handled according to the Canadian Council of Animal Care guidelines.
  • mice were anesthetized using isofluorance and imaged by a 7 tesla pre-clinical MRI at the STTARR facility at the University Health Network, Toronto, ON. A heating pad through the imaging process maintained body temperature. 26 images were captured from each mouse, and fat distribution was assessed using TissueStudio where area of visceral and subcutaneous fat depots was quantified in each section for each animal. Total areas were summed and averaged within groups.
  • mice were housed individually in CLAMs with monitoring of food and water consumption, activity in x,y,and z planes, oxygen consumption and C02 production were measured every 10 minutes over 48 hours.
  • the cages were housed in a room with a standard 12 hour light-dark cycle. Mice were provided with the same diet they were maintained on before entering the cages. The first 24 hours of data was removed from analysis while the mice were acclimated to the cages. Analysis was performed on the final 24 hours within the cages. Quantification of Circulating Factors
  • Circulating leptin, adiponectin, FGF21 , and triglyceride were measured in 14hr fasting samples obtained at the time of sacrifice. Total free fatty acids were quantified from 14hr fasting samples and following a 1 hr reefed with ad libitum access to food. Assays were performed according to manufacturer protocols (BioVision, USA).
  • mice were given a 1 1 U/kg insulin bolus by tail vein injection. Mice were sacrificed 10 minutes following injection, tissues isolated and flash frozen in liquid nitrogen. Liver and muscle tissues for analysis were ground in liquid nitrogen and lysed in RIPA buffer (Cell Signaling, USA) containing protease inhibitor cocktail (Roche, Canada) and stored at -20C prior to use. Lysates were spun at 12,000rpm and supernatant was evaluated for protein content by Bradford assay (BioRad, Canada).
  • Equal amount of protein were then combined with sample buffer containing DTT and loaded onto a 4-15% SDS-PAGE gradient gel (BioRad, Canada) and run at increasing voltages of 50V for 30minutes, 75V for 30 minutes, and then 100V for 30 minutes. Proteins were then transferred onto PVDF membrane using a Turbo Blotter (BioRad, Canada). The membrane was probed with antibodies and imaged using Kodak Imager 4000pro (Carestream, USA).
  • Livers were isolated, embedded into TissueTek and frozen. Staining of sections was performed as previously described. For H&E staining, livers were weighed and fixed in 10% neutral buffered formalin at the time of sacrifice. Tissue processing and immunostaining for insulin have been described previously (Luu et al., 2013). Briefly, pancreata were dehydrated, cleared and embedded in paraffin. Paraffin sections were cut at 4um from the middle of the pancreas. Sections were then dried and deparaffinized for staining. Images of each section were acquired using Aperio Imagescope at 40x magnification (Aperio Image scope).
  • Fatty acid and glucose uptake and oxidation experiments were performed as previously described. Briefly, primary hepatocytes were isolated from 7-9 week old wild type mice and plated at a density of 400,000 cells per well in 12-well plates. Cells were allowed to attach in DMEM media supplemented with 10% FBS for 4 hours. Cells were then washed and cultured in Williams Solution without FBS. Cell were treated with vehicle or 200uM for CMPF starting at this time point for 18 hours. Compound C and AICAR were incubated for 4 hours prior to oxidation and uptake. CMPF was conjugated to fatty acid free BSA for 4 hours prior to starting treatment.
  • FGF21KO mice Six to nine week old male FGF21 KO mice were obtained from Taconic along with age-matched c57 controls. Mice were allowed to acclimate for one week prior to the beginning of the treatment period and randomized to chow and HFD control and CMPF treatment groups. Mice were injected with 6mg/kg/day CMPF for seven days and changed to HFD and matched sucrose control as described above. Mice were maintained on the diet for six weeks prior to evaluation. FGF21 deletion was confirmed by PCR.
  • CMPF furan fatty acid metabolite
  • Acute treatment with the furan fatty acid metabolite CMPF has been shown to alter whole-body glucose utilization, including a reduction in both glucose appearance and disappearance rates in a hyperinsulinemic, euglycemic clamp condition (Prentice et al., 2014).
  • Reduced glucose utilization may be consistent with increased reliance on alternate energy sources, including fatty acids. This is consistent with an elevation of CMPF in gestational diabetes, as during pregnancy maternal metabolism switches to preferential fat oxidation to preserve glucose for the developing fetus.
  • mice were treated with CMPF acutely, as described previously (Prentice et al., 2014), and then placed on a high fat diet (HFD; 60% kcal from fat) for four-six weeks to induce insulin resistance (Figure 1 , part A).
  • HFD high fat diet
  • plasma concentrations peaked within one hour of injection to concentrations observed in diabetic populations (Prentice et al. , 2014), and declined rapidly thereafter, returning to baseline within 24 hours of injection ( Figure 1 , part B).
  • CMPF was not elevated in the plasma for the duration of dietary intervention.
  • CMPF-HFD mice were significantly more active than Control- HFD mice, particularly during the light phase ( Figure 1 , part H). Consistent with a decrease in whole-body glucose utilization, CMPF-HFD had a significantly reduced respiratory exchange ratio (RER) compared to Control- HFD and Control-Chow mice, suggesting preferential fatty acid utilization ( Figure 1 , parts I and J). In spite of reduced subcutaneous fat area, there were no differences in circulating leptin, adiponectin, or free fatty acid levels ( Figure 1 , parts K-M). CMPF-HFD mice had significantly reduced circulating TG than Control-HFD, with no difference compared to chow-fed animals (Fig. 1 N).
  • Insulin Sensitivity is Protected with Previous CMPF Treatment
  • CMPF-HFD mice exhibited significantly elevated fasting blood glucose and no change in plasma insulin levels ( Figure 2, parts A, B). Interestingly, in spite impaired glucose tolerance, CMPF-HFD mice exhibited dramatically improved insulin sensitivity compared to Control-HFD mice during intraperitoneal insulin tolerance test (IpITT) ( Figure 2, part C). The rate of glucose clearance in response to insulin was equal in the Control-Chow and CMPF-HFD groups.
  • CMPF 2,5-Furandicarboxylic acid
  • CMPF Acutely Inhibits Acetyl-CoA Carboxylase
  • CMPF Direct inhibition of ACC by CMPF may be responsible for the increase in fat utilization during the 7 day injection period, however, CMPF is eliminated both from circulation ( Figure 1 , part B) and the liver while the mice are maintained on a high fat diet ( Figure 5, part A).
  • Figure 1 , part B the AMPK- ACC pathway was investigated in liver isolated from CMPF-HFD mice and controls at the end of the 4 week diet period. Activation of AMPK leads to downstream inhibition of ACC transcription, which may potentiate a feedback loop to promote beta-oxidation.
  • CMPF-HFD mice had both increased levels of phosphorylated AMPK (Figure 5, part B), as well as lower total ACC protein, with a higher ratio of phosphorylated ACC to total ACC ( Figure 5, part C).
  • the AMPK-ACC pathway is therefore likely critical for the prevention of steatosis development by CMPF.
  • CMPF may act to directly modulate the pyruvate dehydrogenase complex, regulating the switch between glucose and lipid oxidation.
  • FGF21 is known to attenuate hepatic steatosis through regulation of lipolysis in addition to an increase in beta-oxidation (Fisher et al., 201 1 ). Closer examination of FGF21 target genes indicated that FGF21 acts locally on the liver in CMPF-HFD mice to regulate lipolysis (Fig. 5D and E). This includes potent changes in key FGF21 target genes including leptin receptor (Lepr) and Sterol-CoA Desaturase 1 (Scd1 ), which were significantly increased and decreased, respectively.
  • Lepr leptin receptor
  • Scd1 Sterol-CoA Desaturase 1
  • CMPF Three days following the first CMPF injection, plasma levels of FGF21 are significantly increased (2.57-fold) in the CMPF treated group compared to vehicle and FDCA controls, and remain elevated at least one week into the HFD feeding period (Fig 5H).ln addition, expression of key genes regulating lipolysis and glucose metabolism were significantly altered in 24 hour treated hepatocytes, including ACC1 and ACC2, and Gck ( Figure 5, part I). Together this suggests that CMPF induces the expression of FGF21 acutely following the start of treatment, and therefore, FGF21 is likely essential for the long-term prevention of development of steatosis when CMPF is cleared from circulation.
  • FGF21 expression is normally elevated under fasting or nutrient restrictive conditions. Treatment with CMPF may mimic this status in the liver of chow-fed mice, as ACC inhibition drives beta-oxidation, the primary fuel source during fasting, as well as inhibits glucose utilization. This "fasting-like state” may drive FGF21 expression, activating a feedback loop that continues after CMPF is eliminated.
  • FGF21KO Mice are Resistant to CMPF Treatment
  • CMPF (6mg/kg) was administered to mice with global deletion of FGF21 (FGF21 KO) and age-matched c57 controls (WT) for 7 days, followed by 6 weeks of 60% kcal from fat HFD feeding (Fig. 6a).
  • FGF21 KO global deletion of FGF21
  • WT age-matched c57 controls
  • Fig. 6a 60% kcal from fat HFD feeding
  • both WT and FGF21 KO mice treated with CMPF gained slightly less weight than HFD-controls (Fig. 6b).
  • insulin sensitivity was evaluated by IplTT.
  • CMPF treatment had significantly improved insulin sensitivity compared to both HFD controls, as observed previously ( Figure 6C), however, CMPF treatment had the opposite effect of worsening insulin sensitivity in FGF21 KO mice when compared to FGF21 KO-HFD controls (Fig. 6C).
  • the lack of improvement in insulin sensitivity corresponded to both worsening of fasting hyperinsulinemia (Fig. 6D) and no difference in hepatic triglyceride content in FGF21 KO-CMPF mice (Fig. 6E-G).
  • CMPF treatment significantly reduced triglyceride content in HFD-fed WT control mice, as observed in CD1 mice (Fig. 6E-G).
  • CMPF prevents the development of steatosis through induction of FGF21 .
  • CMPF is known to be a substrate for the Organic Anion Transporter (OAT) family of transporters, particularly through OAT1 (SLC22A6) and OAT3 (SLC22A8) (outlined in Prentice et al., 2014).
  • OAT Organic Anion Transporter
  • Probenecid an FDA-approved drug for the treatment of gout, is a nonspecific OAT inhibitor.
  • mice 8-week-old male c57bl6 mice were treated twice daily with 30mg/kg probenecid for 3 days, prior to initiating treatment for 7 days with once daily 6mg/kg CMPF simultaneously with continued Probenecid treatment. 24 hours following the final injection, mice were placed on a 60% kcal from fat HFD for 6 weeks to induce insulin resistance. Probenecid-treated control (PBN-CON) and CMPF-treated (PBN- CMPF) mice had no significant difference in weight gain over the HFD period (Fig. 7A).
  • mice treated with PBN had no significant difference between control and CMPF-treated groups (Fig. 7B).
  • Evaluation of hepatic lipid accumulation by Oil red O staining revealed no difference in triglyceride accumulation between PBN-groups, while there was a significant improvement in CMPF-treated controls compared to HFD-controls (Fig. 7C).
  • Fig. 7 D, E there was no difference in liver weight or liver triglyceride between PBN-CON and PBN-CMPF mice.
  • CMPF must enter the liver through OAT transporters in order to mediate its effect on reducing lipid accumulation and improving insulin sensitivity. Discussion
  • CMPF may play dual roles in the context of metabolic disorders, with a beneficial effect on hepatic insulin sensitivity and lipid metabolism, and a conversely detrimental role for beta cell function (Prentice et al., 2014).
  • the underlying mechanism of CMPF in inducing fatty acid metabolism while reducing glucose metabolism appears to be constant between these two tissues.
  • the driving force allowing for long-term protection against the development of steatosis is dependent on induction of FGF21 expression and activity. FGF21 expression is normally elevated under fasting or nutrient restrictive conditions (Galman et al., 2008).
  • Treatment with CMPF may mimic this status in the liver of chow- fed mice through increasing beta-oxidation, the primary fuel source during fasting, as well as inhibition of glucose utilization. Importantly, this "fasting-like state" is induced directly, rapidly, and independently of FGF21 , as increases in fatty acid uptake and oxidation are observed during in vitro treatment of isolated hepatocytes from FGF21 KO animals (data not shown). This potent stimulation of beta-oxidation is likely to stimulate FGF21 expression and secretion. Increased FGF21 , combined with a persistent drive toward beta- oxidation induced by CMPF over the chronic 7-day treatment period likely activates a feedback loop that continues to perpetuate after CMPF is eliminated.
  • FGF21 activates AMPK, resulting in inhibition of SREBPI c, and reduced expression of ACC 1 and 2 (Potthoff et al., 2009). These enzymes are rate-limiting for the production of malonyl-CoA, which is required for both triglyceride synthesis, as well as inhibition of beta-oxidation (Abu-Elheiga et al., 2001 ). Livers isolated from mice treated with CMPF have a significant reduction in total ACC levels, while those from FGF21 KO livers have no significant difference, supporting this hypothesis. Loss of ACC likely explains the inability of CMPF-treated mice to accumulate hepatic triglyceride, even when maintained on a 60% kcal from fat HFD. EXAMPLE 2: CMPF for the Treatment of Steatosis, Insulin Resistance and Increasing Fatty Acid Metabolism
  • CMPF As CMPF is found to be elevated in conditions of Gestational and Type 2 Diabetes, two conditions characterized by insulin insufficiency in the face of insulin resistance, the relationship between CMPF and insulin resistance was investigated.
  • CMPF-injected mice developed an increased reliance on non-glucose energy source, suggesting a switch in metabolism from primarily carbohydrates to fats or amino acids. This correlates with the switch in metabolism observed in gestational diabetes, which is established in order to preserve glucose for the developing fetus.
  • HFD high-fat diet
  • mice fed HFD were significantly heavier than chow- fed controls ( Figure 8, part A).
  • HFD-CMPF and HFD-Control were significantly heavier than chow-fed mice.
  • mice were placed in metabolic cages. Mice were observed to have significantly reduced activity (in the x,y,z planes) in the light phase as compared to HFD-Control mice (Fig. 8b). This reduction in activity did not correspond to any changes in food consumption (Fig. 8c). Consistent with reduced activity, HFD-CMPF mice had greater total adipose tissue area by MRI imaging, primarily attributed to the subcutaneous depot (Fig. 8d,e). This increase in adipose area did not correspond to changes in adipocyte size (Fig. 8f), or circulating levels of leptin or adiponectin (Fig. 8g,h).
  • NAFLD is the prominent source of insulin resistance in the DIO murine models of diabetes, thus we examined whether the liver was the source of the observed improvement in insulin sensitivity.
  • HFD-Control mice had significantly greater lipid accumulation in the hepatocytes compared to Chow-Control livers, as determined by gross anatomy, as well as histological sections and Oil Red-0 staining of whole liver sections ( Figure 10a). Quite unexpectedly, the livers of the HFD-CMPF treated mice were remarkably similar to Chow-Controls, both in visual appearance, as well as with Oil Red- O Staining of lipid droplets.
  • mice were injected intravenously with a bolus of insulin and sacrificed 10 minutes later.
  • Harvested tissues were evaluated for pAkt as a marker of insulin signalling.
  • Levels of pAkt were significantly increased in the livers of the HFD-CMPF mice compared to HFD-Controls, consistent with improved insulin sensitivity (Fig. 10c).
  • Fig. 10d there was no difference in insulin signalling in the muscle, suggesting that the improved phenotype is due to the liver and not other peripheral insulin-sensitive tissues (Fig 10d). Therefore, CMPF rescues diet-induced NAFLD to improve insulin sensitivity.
  • CMPF Treatment Alters the Liver Metabolomic Profile Indicating Alternate Glucose Utilization
  • CMPF Alterations in the glutathione pathway, including lower levels of several gamma glutamyl amino acids and the glutathione catabolite 5- oxoproline suggests that CMPF induces alterations in glutathione synthesis. Glutathione is essential for maintaining the redox status of cells, thus alterations in this pathway are consistent with CMPF inducing a state of oxidative stress. Increases in flux through the PPP may be occurring in response to the decreased level of glutathione synthesis to increase NADPH levels (pathway illustrated in Figure 1 1 , part B). Overall, CMPF induces an altered metabolic state in the liver of obese animals.
  • Microarray analysis was performed on the livers of Chow- and HFD-controls, as well as HFD-CMPF mice at the end of the 6 week diet period. Consistent with alterations in glucose and amino acid metabolism, several genes involved in metabolic processes were significantly altered. Of primary interest, the mTORcl target gene Lipinl was significantly increased in HFD-CMPF livers (Fig.1 1 c). Furthermore, increased activity of this gene, as determined by increased nuclear localization, was confirmed by western blot in livers HFD-CMPF mice compared to controls (Fig. 1 1 d). Lipinl is a regulator of numerous downstream transcription factors that regulate hepatic lipid accumulation. Thus, CMPF may alter mTOR signaling to reduce hepatic lipid accumulation and increase insulin sensitivity in obese mice.
  • CMPF Treatment Improves Insulin Sensitivity in a Genetic Model of Insulin Resistance
  • Leptin is a hormone produced by the adipose tissue that acts to regulate fat storage through influencing satiety signals. Deletion of the leptin gene (ob/ob mouse strain) induces a loss of satiety signals and results in massive obesity due to overeating on a standard chow diet.
  • the ob/ob mice are a common model of obesity, diabetes, and NAFLD 11 . Therefore, the effect of CMPF was examined in the ob/ob mouse strain to determine if the 'switch' from carbohydrate to fat metabolism induced by CMPF is sufficient to overcome genetic-induced obesity and NAFLD.
  • CMPF Treatment Reduces Hepatic Liver Content and Alters Whole-Body Fat Distribution in a Genetic Model of Insulin Resistance
  • CMPF is able to improve insulin sensitivity and reduce hepatic lipid accumulation in ob/ob mice. Livers from ob/ob-control and -CMPF mice were isolated and a reduction in visible lipid accumulation in the livers of ob/ob- CMPF mice was observed ( Figure
  • CMPF appears to cause a shift in whole-body metabolism from being glucose to fat-driven. In the beta cell this corresponds to impaired glucose sensing, and enhanced insulin secretion with palmitate stimulation. In the peripheral tissues, this shifted metabolism enhances fat metabolism in the liver, resulting in either elimination of fat deposits, or prevention from fat accumulation. Importantly, this reduction in hepatic lipid accumulation can occur in the presence of excess fat intake associated with a HFD, as well as under conditions of genetic-induced obesity.
  • EXAMPLE 3 CMPF alters Islet Metabolism to Induce Preferential Fatty Acid Oxidation and Reduce Glucose Metabolism
  • CMPF was demonstrated to impair pancreatic beta cell function in chow-fed mice, leading to impaired insulin biosynthesis and secretion, ultimately resulting in the development of diabetes.
  • GDM gestational
  • T2D type 2 diabetes
  • mice Eight week old male CD1 mice were treated once daily for 7 days with either 6mg/kg CMPF (Prentice et al., 2014) or vehicle while maintained on a chow diet. At the end of the treatment period, mice were placed on a 60%kcal from fat HFD for 4-5 weeks to induce insulin resistance (outlined in Figure 14, part A). As anticipated, control-HFD mice exhibited significant glucose intolerance during IpGTT ( Figure 14, part B). Interestingly, despite improved insulin sensitivity (outlined in Example 2), CMPF-HFD mice had worsened glucose tolerance compared to Control-HFD mice ( Figure 14, part B).
  • CMPF-HFD mice had an absence of glucose-stimulated insulin secretion (GSIS) during IpGTT ( Figure 14, part C).
  • GSIS glucose-stimulated insulin secretion
  • islets isolated from CMPF- HFD mice exhibited significantly reduced GSIS compared to islets from Control-HFD mice ( Figure 15, part A).
  • PSIS palmitate-stimulated insulin secretion
  • CMPF-HFD islets did not exhibit a significant difference in ROS (Figure 15, part D). This may be due to beta cell compensation for increased ROS in the form of increase antioxidant expression, as observed in the 7-day treated chow-fed mice (Prentice et al., 2014). As anticipated, islets from the Control-HFD mice were significantly larger than those from the Control-Chow mice. However, interestingly, islets from the CMPF-HFD mice were significantly smaller than both those from the Control-HFD and Control-Chow mice, ( Figure 15, part E). This observation may be due to the improvement in insulin sensitivity.
  • CMPF-HFD islets had an impaired hyperpolarization of the mitochondrial membrane potential (MMP) in response to the addition of high glucose as compared to both Control-Chow and Control-HFD groups ( Figure 15, part F).
  • MMP mitochondrial membrane potential
  • pancreatic islets Under conditions of insulin resistance, as observed in diet- induced obesity (DIO), the pancreatic islets exhibit compensatory increases in mass and function to maintain euglycemia.
  • DIO diet- induced obesity
  • mice fed a HFD for 6 weeks with either vehicle or 6mg/kg CMPF once daily for 2 weeks outlined in Figure 16, part A. Mice were maintained on the HFD throughout the injection period. As anticipated, HFD-control mice exhibited worsened glucose tolerance compared to Chow-control mice during both OGTT and IpGTT ( Figure 16, parts B,D).
  • pancreatic islets are known to increase in size and secretory capacity in DIO (HFD) models.
  • HFD DIO
  • islets from 7-day CMPF- treated chow-fed mice exhibited impaired glucose-stimulated insulin secretion, which was attributed to decreased insulin biosynthesis, as well as impaired glucose sensing.
  • islets were isolated from the mice and examined ex vivo. Interestingly, no correspondence to any significant difference in total insulin content in the isolated islets (Figure 17, part A) was observed.
  • Islets isolated from the HFD-CMPF mice exhibited significantly impaired glucose-stimulated insulin secretion (GSIS), as well as impaired KCI-induced insulin secretion compared to Chow-Control and HFD-Control islets ( Figure 19, parts B, C).
  • GSIS glucose-stimulated insulin secretion
  • KCI-induced insulin secretion compared to Chow-Control and HFD-Control islets ( Figure 19, parts B, C).
  • CMPF may be inducing a 'switch' in substrate utilization from carbohydrate-driven metabolism to preferential utilization of other substrates such as fats or amino acids.
  • PSIS static palmitate- stimulated insulin secretion assays
  • islets HFD-CMPF mice had greater PSIS as compared to both Chow- and HFD-Controls ( Figure 19, parts D, E).
  • mitochondrial membrane potential measurements MMP
  • islets from HFD-CMPF mice had a greater depolarization of the MMP in response to the addition of palmitate than in response to high glucose.
  • both the Chow- and HFD-controls had a greater response to the addition of high glucose ( Figure 19, parts F, G).
  • islets isolated from HFD-CMPF mice had greater levels of reactive oxygen species (ROS) than either Chow- or HFD-Controls.
  • ROS reactive oxygen species
  • CMPF Treatment Enhances Palmitate-Stimulated Insulin Secretion and Decreases Islet Mass in a Genetic Model of Insulin Resistance
  • the ob/ob mouse model is a genetic induction of insulin resistance and obesity.
  • the effect of CMPF on the islets of ob/ob mice closely mirrors the phenotype observed in islets isolated from the HFD- CMPF mice.
  • islets isolated from the ob/ob-CMPF mice were significantly smaller compared to controls ( Figure 21 , part A).
  • Static GSIS assays demonstrated a significantly lower fold change in insulin secretion under high glucose conditions in the ob/ob-CMPF islets compared to the ob/ob-Controls ( Figure 21 , part B), due to significantly greater insulin secretion in the basal, low glucose condition.
  • CMPF appears to cause a shift in whole-body metabolism from being glucose to fat-driven. In the beta cell this corresponds to impaired glucose sensing, and enhanced insulin secretion with palmitate stimulation. In the peripheral tissues, this shifted metabolism enhances fat metabolism in the liver, resulting in either elimination of fat deposits, or prevention from fat accumulation. Importantly, this reduction in hepatic lipid accumulation can occur in the presence of excess fat intake associated with a HFD, as well as under conditions of genetic-induced obesity.
  • CMPF prevents and improves insulin resistance and prevents and improves fatty liver.
  • CMPF likely has a direct effect on the liver but may act by reducing insulin secretion to improve liver function and IR.
  • Galman, C, Lundasen, T. Kharitonenkov, A., Bina, H.A., Eriksson, M. , Hafstrom, I., Dahlin, M., Amark, P., Angelin, B., and Rudling, M.
  • the circulating metabolic regulator FGF21 is induced by prolonged fasting and PPARalpha activation in man. Cell metabolism 8, 169-174
  • FGF21 induces PGC-1 alpha and regulates carbohydrate and fatty acid metabolism during the adaptive starvation response. Proceedings of the National Academy of Sciences of the United States of America 106, 10853- 10858 Prentice, K. J. et al. The Furan Fatty Acid Metabolite CMPF Is Elevated in Diabetes and Induces beta Cell Dysfunction. Cell metabolism 19, 653- 666, doi: 10.1016/j.cmet.2014.03.008 (2014).

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Abstract

L'invention concerne des méthodes et des utilisations permettant le traitement ou la prévention de la stéatose hépatique. L'invention concerne également des méthodes et des utilisations permettant le traitement ou la prévention de l'insulino-résistance. L'acide carboxy-4-méthyl-5-propyl-2-furane propanoïque (CMPF) a montré sa capacité à prévenir la stéatose hépatique, améliorer le métabolisme lipidique et améliorer la sensibilité à l'insuline.
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EP4385508A1 (fr) * 2022-12-16 2024-06-19 Centre de Coopération Internationale en Recherche Agronomique pour le Développement (CIRAD) Acides gras furaniques pour augmenter la masse musculaire
WO2024126642A1 (fr) * 2022-12-16 2024-06-20 Centre De Cooperation Internationale En Recherche Agronomique Pour Le Developpement (Cirad) Acides gras de furane pour améliorer la masse musculaire

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