U.S. patent number 10,233,209 [Application Number 15/371,032] was granted by the patent office on 2019-03-19 for inhibitors of the farnesoid x receptor and uses in medicine.
This patent grant is currently assigned to The Penn State Research Foundation, The United States of America, As represented by the Secretary, Department of Health and Human Services. The grantee listed for this patent is The Penn State Research Foundation, The United States of America, as represented by the Secretary, Department of Health and Human Services, The United States of America, as represented by the Secretary, Department of Health and Human Services. Invention is credited to Shantu Amin, Dhimant Desai, Frank J. Gonzalez, Changtao Jiang, Fei Li, James B. Mitchell, Andrew D. Patterson, Cen Xie.
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United States Patent |
10,233,209 |
Gonzalez , et al. |
March 19, 2019 |
Inhibitors of the farnesoid x receptor and uses in medicine
Abstract
Disclosed are inhibitors of the farnesoid X receptor, for
example of formula (I), wherein R.sup.1, R.sup.2, R.sup.4, X, Y, Z,
m, and n are as defined herein, which are useful in treating or
preventing obesity, type 2 diabetes/insulin resistance and
non-alcoholic fatty liver disease in a mammal in need thereof. Also
disclosed is a composition comprising a pharmaceutically suitable
carrier and at least one compound of the invention, a method of
method of inhibiting a farnesoid X receptor in a mammal, and a
method of treating or preventing obesity in a mammal.
##STR00001##
Inventors: |
Gonzalez; Frank J. (Bethesda,
MD), Jiang; Changtao (Beijing, CN), Xie; Cen
(Rockville, MD), Patterson; Andrew D. (State College,
PA), Li; Fei (Rockville, MD), Mitchell; James B.
(Damascus, MD), Amin; Shantu (Union City, NJ), Desai;
Dhimant (Mechanicsburg, PA) |
Applicant: |
Name |
City |
State |
Country |
Type |
The United States of America, as represented by the Secretary,
Department of Health and Human Services
The Penn State Research Foundation |
Washington
University Park |
DC
PA |
US
US |
|
|
Assignee: |
The United States of America, As
represented by the Secretary, Department of Health and Human
Services (Washington, DC)
The Penn State Research Foundation (University Park,
PA)
|
Family
ID: |
51358096 |
Appl.
No.: |
15/371,032 |
Filed: |
December 6, 2016 |
Prior Publication Data
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Document
Identifier |
Publication Date |
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US 20170152283 A1 |
Jun 1, 2017 |
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Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
Issue Date |
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14909263 |
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9540415 |
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PCT/US2014/049460 |
Aug 1, 2014 |
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62004436 |
May 29, 2014 |
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61861109 |
Aug 1, 2013 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
C07J
31/006 (20130101); C07J 41/0066 (20130101); A61P
3/10 (20180101); A61P 3/06 (20180101); A61P
5/50 (20180101); A61P 43/00 (20180101); A61P
1/16 (20180101); C07J 41/0061 (20130101); A61P
3/04 (20180101); C07J 41/0055 (20130101); C07J
9/005 (20130101) |
Current International
Class: |
C07J
9/00 (20060101); C07J 41/00 (20060101); C07J
31/00 (20060101) |
Field of
Search: |
;514/119 |
References Cited
[Referenced By]
U.S. Patent Documents
Foreign Patent Documents
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Aug 2001 |
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CA |
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101891791 |
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Nov 2010 |
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CN |
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WO 87/02367 |
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Apr 1987 |
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WO |
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WO 90/00175 |
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Jan 1994 |
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WO |
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WO 94/24147 |
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Oct 1994 |
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WO |
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WO 00/37077 |
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Jun 2000 |
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WO |
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WO 02/094865 |
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Nov 2002 |
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WO |
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WO 03/030612 |
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Apr 2003 |
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WO |
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WO 2009/136396 |
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Nov 2009 |
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WO |
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WO 2010/014836 |
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Feb 2010 |
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WO |
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WO 2011/022838 |
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Mar 2011 |
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WO |
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|
Primary Examiner: Valenrod; Yevgeny
Attorney, Agent or Firm: Leydig, Voit & Mayer, Ltd.
Government Interests
This invention was made with government support under Grant No.
ES022186 awarded by the National Institutes of Health and under
Hatch Act Project Nos. PEN04451 and PEN04607, awarded by the United
States Department of Agriculture. The Government has certain rights
in the invention.
Parent Case Text
CROSS-REFERENCE TO RELATED APPLICATIONS
This patent application is a continuation of copending U.S. patent
application Ser. No. 14/909,263, filed Feb. 1, 2016, which is a 371
of International Patent Application No. PCT/US2014/049460, filed
Aug. 1, 2014, which claims the benefit of U.S. Provisional Patent
Applications Nos. 61/861,109, filed Aug. 1, 2013, and 62/004,436,
filed May 29, 2014, which are incorporated by reference for all
purposes.
Claims
The invention claimed is:
1. A compound selected from: ##STR00030## ##STR00031## wherein
R.sup.14 is selected from .beta.-alanino, phenylalanino, tyrosino,
methionino, tryptophano, leucino, isoleucino, methyl aspartato,
asparto, valino, 2-fluoro-.beta.-alanino, 2-bromoalanino,
2-chloroalanino, 2-fluoroalanino, 2-iodoalanino, 3-bromoalanino,
3-chloroalanino, 3-fluoroalanino, 3-iodoalanino,
4-bromophenylalanino, 4-chlorophenylalanino, 4-fluorophenylalanino,
and 4-iodophenylalanino, or a pharmaceutically acceptable salt
thereof.
2. A pharmaceutical composition comprising a pharmaceutically
acceptable carrier and a compound or salt of claim 1.
3. The compound or salt of claim 1, wherein the compound is:
##STR00032##
4. The compound or salt of claim 1, wherein the compound is:
##STR00033##
5. The compound or salt of claim 1, wherein the compound is:
##STR00034##
6. The compound or salt of claim 1, wherein the compound is:
##STR00035##
7. The compound or salt of claim 1, wherein the compound is:
##STR00036##
8. The compound or salt of claim 1, wherein the compound is:
##STR00037##
9. The compound or salt of claim 1, wherein the compound is:
##STR00038##
10. The compound or salt of claim 1, wherein the compound is:
##STR00039##
11. The compound or salt of claim 1, wherein the compound is:
##STR00040##
Description
BACKGROUND OF THE INVENTION
Obesity has reached epidemic proportions worldwide and is
associated with chronic diseases such as type 2 diabetes mellitus,
cardiovascular diseases, hepatosteatosis, and cancer. Obesity
develops as a result of energy intake that exceeds energy
expenditure, leading to a net storage of excess calories in the
form of fat in adipose tissue. Obesity is metabolically linked with
type 2 diabetes (insulin resistance) and hepatosteatosis, the
latter of which can lead to steatohepatitis, hepatocarcinogenesis
and liver failure. Thus, a pharmaceutical approach that suppresses
appetite, blocks dietary fat absorption, induces fat mobilization,
or increases metabolism would be ideal in the treatment of obesity
and related metabolic disorders.
Farnesoid X Receptor (FXR) is an orphan nuclear receptor initially
identified from a rat liver cDNA library (Forman, et al., Cell
81:687-693, 1995) that is most closely related to the insect
ecdysone receptor. FXR is a member of the nuclear receptor
superfamily of transcription factors that includes receptors for
the steroid, retinoid, and thyroid hormones (Mangelsdorf, et al.,
Cell 83:841-850, 1995). Northern blotting and in situ hybridization
analysis showed that FXR is most abundantly expressed in the liver,
intestine, kidney, and adrenal (B. M. Forman, et al., Cell
81:687-693.1995; Seol, et al., Mol. Endocrinol. 9:72-85, 1995). FXR
is a ligand-activated nuclear receptor that binds to DNA as a
heterodimer with the retinoic acid receptor .alpha. (RXR.alpha.)
that is activated by the vitamin A derivative 9-cis retinoic acid.
The FXR/RXR.alpha. heterodimer preferentially binds to response
elements composed of two nuclear receptor half sites of the
consensus AG(G/T)TCA organized as an inverted repeat and separated
by a single nucleotide (IR-1 motif) (Forman, et al., Cell
81:687-693, 1995). An early report showed that rat FXR is activated
by micromolar concentrations of farnesoids such as farnesol and
juvenile hormone thus accounting for the original name (Forman, et
al., Cell 81:687-693, 1995). However, these compounds were weak
ligands and also failed to activate the corresponding mouse and
human FXR, leaving the nature of the endogenous FXR ligand in
doubt. However, several naturally-occurring bile acids were found
to bind to and activate FXR at physiological concentrations
(Makishima, et al., Science 284:1362-1365, 1999; Parks, et al.,
Science 284:1365-1368, 1999; Wang et al., Mol. Cell 3:543-553,
1999; PCT WO 00/37077, published Jun. 29, 2000). The bile acids
that serve as FXR ligands include chenodeoxycholic acid (CDCA),
deoxycholic acid (DCA), lithocholic acid (LCA), and the taurine and
glycine conjugates of these bile acids.
Bile acids are cholesterol metabolites that are formed in the liver
and secreted into the duodenum of the intestine, where they have
important roles in the solubilization and absorption of dietary
lipids and vitamins. About 95% of bile acids are subsequently
reabsorbed in the ileum and returned to the liver via the
enterohepatic circulatory system. The conversion of cholesterol to
bile acids in the liver is under feedback regulation, and bile
acids down-regulate transcription of cytochrome P450 7A1 (CYP7A1),
which encodes the enzyme that catalyzes the rate-limiting step in
bile acid biosynthesis. FXR is involved in the repression of CYP7A1
expression by bile acids through an indirect mechanism involving
the FXR target gene small heterodimer partner (SHP) and liver
receptor homolog 1 (Goodwin et al., Mol. Cell 6:517-528, 2000;
reviewed in Matsubara et al., Mol. Cell. Endocrinol. 368:17-29,
2013). In the ileum, in an FXR dependent manner, bile acids induce
the expression of the intestinal bile acid binding protein (IBABP),
a cytoplasmic protein which binds bile acids with high affinity and
may be involved in their cellular uptake and trafficking. Two
groups have now demonstrated that bile acids mediate their effects
on IBABP expression through activation of FXR, which binds to an
IR-1 type response element that is conserved in the human, rat, and
mouse IBABP gene promoters. Thus, FXR is involved in both the
stimulation (IBABP) and the repression (CYP7A1) of target genes
involved in bile acid and cholesterol homeostasis. FXR also induces
expression of the bile salt export pump (BSEP, ABC11) that
transports unconjugated and conjugated bile acids/salts from
hepatocyte into the bile (reviewed in Matsubara et al., Mol. Cell.
Endocrinol. 368:17-29, 2013).
Tempol (4-hydroxy-2,2,6,6,-tetramethylpiperidine-1-oxyl), an
antioxidant and a radiation protector, was reported to prevent
obesity in mice (Mitchell et al., Free Radic. Biol Med. 34: 93-102,
2003). A recent mass spectrometry-based investigation revealed that
tempol can affect fatty acid metabolism and the altered levels of
suspected gut microbe-generated metabolites provided initial clues
that tempol may alter the microbiome (Li et al., J. Proteome Res.,
12:1369-1376, 2013). Previous studies demonstrated that the
alteration of the gut microbiome can affect the level of bile acids
in liver, heart, and kidney (Swann et al., Proc. Natl. Acad. Sci.
USA 108:4523-4530, 2011). High fat diets can induce changes in the
expression of genes in the small intestine that are controlled by
nuclear receptors including FXR (de Wit et al., BMC Med. Genomics
1:14, 2008). Thus, there may be relationship between altered bile
acids in the intestine and FXR signaling that can alter high fat
diet-induced obesity. While there are known natural and synthetic
FXR agonist, no therapeutic agents have been disclosed which
antagonize FXR. Recent studies revealed that the secondary bile
acid tauro-.beta.-muricholic acid (T.beta.MCA) can antagonize bile
acid signaling in the intestine (Sayin et al., Cell Metab. 225-235,
2013; Li et al., Nat. Commun. 4:2384, 2013). Trisubstituted-pyrazol
carboxamide analogs have been synthesized that are FXR antagonist,
but their effect on metabolism and physiology were not investigated
(Yu et al., Bioorg. Med. Chem. 2919-2938, 2014).
Non-alcoholic fatty liver disease (NAFLD) is characterized by
massive ectopic triglyceride (TG) accumulation in the liver in the
absence of other liver disease or significant alcohol consumption
(WeiB et al., Ditsch. Arzteb.l Int. 2014; 0.447-452, 2014). NAFLD
is the most common liver disorder affecting 20-30% of the adult
population and more than 80% of obese people in the world. NAFLD
can develop into nonalcoholic steatohepatitis (NASH), fibrosis,
cirrhosis and even hepatocellular carcinoma (Browning et al., J.
Clin Invest. 114:147-152, 2004). As a component of metabolic
syndrome, NAFLD is tightly associated with obesity, insulin
resistance/type 2 diabetes, and coronary heart disease and
atherosclerosis (Bhatia et al., Eur. Heart 33:1190-1200, 2012). To
date, the underlying molecular mechanism of NAFLD development
remains largely unknown and the identification of novel targets for
NAFLD therapy is of high priority.
The foregoing shows that there is an unmet need for antagonists of
the FXR receptor and a method of treating obese patients to induce
weight loss, insulin resistance, and NAFLD.
BRIEF SUMMARY OF THE INVENTION
The present invention provides inhibitors of the nuclear receptor
farnesoid X receptor for treating or preventing obesity in mammals,
particularly humans. Compounds embodying aspects of the invention
inhibit the farnesoid X receptor and affect high fat diet-induced
obesity through signal transduction mediated by the farnesoid X
receptor. In accordance with the invention, the present invention
provides compositions comprising these compounds and methods of
using these compounds as therapeutic agents in the treatment or
prevention of obesity.
The invention also provides a pharmaceutical composition comprising
a compound or salt embodying the principles of the invention and a
pharmaceutically acceptable carrier.
The invention further provides a method of inhibiting a farnesoid X
receptor in a mammal, comprising administering to a mammal in need
thereof a compound embodying the principles of the invention or a
pharmaceutically acceptable salt thereof.
The invention additionally provides a method for treating or
preventing obesity in a mammal, comprising administering to a
mammal in need thereof a compound embodying the principles of the
invention or a pharmaceutically acceptable salt thereof.
The invention also provides a method for treating or preventing
obesity, insulin resistance and NAFLD in a mammal in need thereof,
comprising administering to the mammal a compound embodying the
principles of the invention or a pharmaceutically acceptable salt
thereof. Desirably, the compounds inhibit the farnesoid X receptor
in the intestine and affects obesity, insulin resistance and NAFLD
through signal transduction mediated only by the intestinal
farnesoid X receptor and not by the liver farnesoid X receptor.
Preferably, the compounds have minimal systemic bioavailability so
that the compounds do not inhibit the liver farnesoid X receptor
which minimizes any systemic toxicity.
The invention further provides methods of synthesizing the compound
embodiments of the invention.
BRIEF DESCRIPTION OF THE SEVERAL VIEWS OF THE DRAWING(S)
FIG. 1 depicts results of luciferase assays showing that
tauro-.beta.-muricholic acid (T.beta.MCA) antagonizes farnesoid X
receptor (FXR) activation by the FXR agonist taurocholic acid (TCA)
in cultured primary hepatocytes.
FIG. 2 depicts results of luciferase assays showing that
tauro-.beta.-muricholic acid (T.beta.MCA) antagonizes FXR
activation by the FXR agonist taurocholic acid (TCA) in Caco2
cells.
FIG. 3 illustrates ATP levels in the ileum mucosa of Fxr.sup.fl/fl
mice and Fxr.sup..DELTA.IE mice that were kept on a high fat diet
for 8 weeks.
FIG. 4 illustrates the blocking of induction of Shp mRNA with
chenodeoxycholic acid by glycine-.beta.-muricholic acid.
FIG. 5 illustrates the blocking of induction of Shp mRNA with
GW4064 by glycine-.beta.-muricholic acid.
FIG. 6 illustrates the blocking of induction of Fgf19 mRNA with
GW4064 by glycine-.beta.-muricholic acid.
FIG. 7 illustrates the reversal of Atp5g mRNA inhibition by GW4064
by glycine-.beta.-muricholic acid.
FIG. 8 depicts a synthesis of compounds in accordance with an
embodiment of the invention.
FIG. 9 depicts a synthesis of compounds in accordance with an
embodiment of the invention.
FIG. 10 depicts the structures of .beta.-muricholic acid,
tauro-.beta.-muricholic acid (T.beta.MCA),
glycine-.beta.-muricholic acid, chenodeoxycholic acid,
taurochenodeoxycholic acid (TCA), and glycine-chenodeoxycholic
acid.
FIG. 11 illustrates the body mass gain for Fxr.sup.fl/fl and
Fxr.sup..DELTA.IE mice treated with vehicle or tempol after 10
weeks of a high fat diet.
FIGS. 12A and B illustrate the fat mass in grams and as a
percentage of body mass for Fxr.sup.fl/fl and Fxr.sup..DELTA.IE
mice after 14 weeks of a high fat diet.
FIG. 13 illustrates the results of a glucose tolerance test (GTT)
for Fxr.sup.fl/fl and Fxr.sup..DELTA.IE mice after 7 weeks of a
high fat diet.
FIG. 14 illustrates the results of an insulin tolerance test (ITT)
for Fxr.sup.fl/fl and Fxr.sup..DELTA.IE mice after 13 weeks of a
high fat diet.
FIGS. 15A-C illustrate the fasted glucose, fasted serum insulin,
and HOMA index for Fxr.sup.fl/fl and Fxr.sup..DELTA.IE mice after
15 weeks of a high fat diet.
FIGS. 16A and B depict the shift from Firmicutes to Bacteroidetes
in mice being fed a normal chow diet upon treatment with
tempol.
FIGS. 17A and B depict a comparison of the ratio of Firmicutes to
Bacteroidetes and the bile salt hydrolase enzymatic activity in the
feces of mice on a normal chow diet and treated with vehicle or
tempol.
FIGS. 18A-D illustrate a human FXR competition assay using the
synthetic agonist GW4064 and varied doses of TUDCA, T.omega.MCA,
T.beta.MCA, T.alpha.MCA. Results were normalized to Renilla
expression.
FIG. 19A shows a principal coordinates analysis plot of weighted
UniFrac distances. Circles represent cecal communities in
vehicle-treated mice and squares represent cecal communities in
tempol-treated mice. Both groups were fed a high-fat diet for 10
weeks.
FIG. 19B-G shows 16S rRNA gene sequencing analysis of genus levels
of cecum content. Data are presented as mean.+-.SD.
FIG. 20A shows a scores scatter plot of a principal components
analysis (PCA) model of urine ions in vehicle- and tempol-treated
mice fed a high-fat diet for 14 weeks.
FIG. 20B shows a loadings scatter plot of all detected urine ions
in the PCA model. The p[1] and p[2] values represent the
contributing weights of each ion to principal components 1 and 2.
The identities of two ions with the highest loading values are
annotated in the plot. All the data were obtained in electrospray
inoization negative mode (ESI.sup.-).
FIG. 20C shows urine levels of p-cresol sulfate and p-cresol
glucuronide. Values were normalized to those of vehicle-treated
mice and were expressed as relative abundance.
FIGS. 20D and E show tandem MS and chemical structures of p-cresol
sulfate (20D) and p-cresol glucuronide (20E).
FIG. 21A shows scores scatter plot of a PCA model of urine ions in
vehicle- and tempol-treated mice after 14 weeks of high-fat diet
treatment.
FIG. 21B shows loadings scatter plot of a PCA model of urine ions
in vehicle- and antibiotic-treated mice after 14 weeks of HFD. The
p[1] and p[2] values represent the contributing weights of each ion
to principal components 1 and 2. The identities of two ions with
the highest loading values are annotated in the plot. All the data
were obtained in negative mode (ESI.sup.-).
FIG. 21C shows urine levels of p-cresol sulfate and p-cresol
glucuronide in vehicle- and antibiotic-treated mice after 14 weeks
of high-fat diet treatment. Values were normalized to those of
vehicle-treated mice and were expressed as relative abundance. n=5
mice per group. All data are presented as mean.+-.SD. Analysis of
variance followed by two-tailed Student's t-test. *P<0.05,
**P<0.01 compared to vehicle-treated mice.
FIG. 22A shows representative H&E staining of liver sections
from vehicle- and tempol-treated mice fed a high-fat diet for 14
weeks.
FIG. 22B shows representative Oil Red 0 staining of lipid droplets
in liver sections from vehicle- and tempol-treated mice fed a
high-fat diet for 14 weeks.
FIG. 22C shows liver weights from vehicle- and tempol-treated mice
fed a high-fat diet for 16 weeks.
FIG. 22D shows liver weight to body weight ratios in vehicle- and
tempol-treated mice fed a high-fat diet for 16 weeks.
FIG. 22E shows liver triglyceride (TG) contents from vehicle- and
tempol-treated mice fed a high-fat diet for 16 weeks.
FIG. 23A shows representative H&E staining of liver sections
from vehicle- and tempol-treated mice fed a high-fat diet for 16
weeks.
FIG. 23B shows liver weights from vehicle- and tempol-treated mice
fed a high-fat diet for 16 weeks.
FIG. 23C shows liver weight to body weight ratios from vehicle- and
tempol-treated mice fed a high-fat diet for 16 weeks.
FIG. 23D shows liver TG contents from vehicle- and
antibiotic-treated fed a high-fat diet for 16 weeks.
FIG. 24A shows a scores scatter plot of a PCA model of ileum ions
from vehicle- and antibiotic-treated mice fed a high-fat diet for 7
weeks.
FIG. 24B shows loadings scatter plot of a PCA model of ileum ions
in vehicle- and antibiotic-treated mice fed a high-fat diet for 7
weeks. The p[1] and p[2] values represent the contributing weights
of each ion to principal components 1 and 2. The identities of two
ions with the highest loading values are annotated in the plot. All
the data were obtained in negative mode (ESI.sup.-).
FIG. 25A shows the ratio of individual taurine-conjugated bile
acids to total bile acids in the ileum from vehicle- and
antibiotic-treated mice fed a high-fat diet for 14 weeks.
FIG. 25B shows the ratio of individual taurine-conjugated bile
acids to total bile acids in the ileum from vehicle- and
tempol-treated mice fed a high-fat diet for 7 weeks.
FIG. 26A shows fecal BSH enzyme activity from vehicle- and
antibiotic-treated mice fed a high-fat diet for 7 weeks. n=4-5 mice
per group.
FIG. 26B shows western blot analysis of ileum FXR expression in
mice fed a high-fat diet for 12 weeks. Each lane represents one
mouse.
FIG. 26C shows Fxr mRNA levels and mRNA levels of the FXR target
genes Shp and Fgf15 in the ileum from mice fed a high-fat diet for
12 weeks. n=3 mice per group.
FIG. 26D shows mRNA levels of the FXR target genes Shp and Fgf15 in
the ileum from vehicle- and antibiotic-treated mice fed a high-fat
diet for 7 weeks. n=3 mice per group.
FIG. 26E shows mRNA levels of the FXR target genes Shp and Fgf15 in
the ileum after 24 hours of treatment of mice fed a high-fat diet
for 7 weeks with vehicle, taurocholic acid (TCA), and
taurine-.beta.-muricholic acid (T.beta.MCA) with TCA. n=3 mice per
group.
FIG. 27A shows a representative H&E staining of liver sections
from control-floxed (Fxr.sup.fl/fl) mice and intestine-specific
knockout mice (Fxr.sup..DELTA.IE) mice fed a high-fat diet for 14
weeks.
FIG. 27B shows a representative Oil Red 0 staining of lipid
droplets in liver sections from Fxr.sup.fl/fl and Fxr.sup..DELTA.IE
mice fed a high-fat diet for 14 weeks.
FIG. 27C shows liver weights from Fxr.sup.fl/fl and
Fxr.sup..DELTA.IE mice fed a high-fat diet for 14 weeks.
FIG. 27D shows liver triglyceride contents from Fxr.sup.fl/fl and
Fxr.sup..DELTA.IE mice fed a high-fat diet for 14 weeks.
FIG. 28A shows mRNA levels of mitochondrial oxidative
phosphorylation (OXPHOS) related enzymes from the ileum mucosa from
Fxr.sup.fl/fl and Fxr.sup..DELTA.IE mice fed a high-fat diet for 14
weeks.
FIG. 28B shows mRNA levels of mitochondrial oxidative
phosphorylation (OXPHOS)-related genes from ileum mucosa of
vehicle- and antibiotic-treated mice fed a high-fat diet for 7
weeks. n=3 mice per group.
FIG. 28C shows measured state III respiration for complex-I- and
complex-II-dependent respiration from the ileum mucosa from
Fxr.sup.fl/fl and Fxr.sup..DELTA.IE mice fed a high-fat diet for 12
weeks.
FIG. 28D shows ATP levels in the ileum mucosa of Fxr.sup.fl/fl mice
and Fxr.sup..DELTA.IE mice fed a high-fat diet for 7 weeks.
FIG. 29A shows serum free fatty acids. The bars for each fatty
acid, from left to right, are from vehicle-treated Fxr.sup.fl/fl
mice, tempol-treated Fxr.sup.fl/fl mice, vehicle-treated
Fxr.sup..DELTA.IE mice and tempol-treated Fxr.sup..DELTA.IE
mice.
FIG. 29B shows serum ceramides from vehicle- and antibiotic-treated
mice fed a high-fat diet for 7 weeks. n=3 mice per group.
FIG. 29C shows expression of mRNAs encoding ceramide synthesis- and
catabolism-related enzymes in the ileum from Fxr.sup.fl/fl and
Fxr.sup..DELTA.IE mice fed a high-fat diet for 14 weeks.
FIG. 29D shows levels of mRNAs encoding ceramide synthesis- and
catabolism-related enzymes in the ileum after 7 weeks antibiotic of
treatment of mice fed a high-fat diet for 14 weeks.
FIG. 30A shows the structure of MS fragments derived from ceramides
and
FIG. 30B-30G shows tandem MS and chemical structures of the various
ceramides.
FIG. 31A shows liver TG contents in vehicle- and antibiotic-treated
mice fed a high-fat diet for 3 days.
FIG. 31B shows Fxr, Shp and Fgf15 mRNA levels in the ileum of mice
fed a high-fat diet for 14 weeks and then treated with vehicle or
antibiotic for 3 days.
FIG. 31C shows the profile of ceramides from ileum from mice fed a
high-fat diet for 14 weeks, and then treated with vehicle or
antibiotic for 3 days.
FIG. 31D shows primary hepatocyte triglyceride (TG) content after
24 hours of incubation with vehicle and 2 .mu.M, 5 .mu.M and 10
.mu.M ceramide (n=4).
FIG. 31E shows mRNA levels of fatty acid synthesis, triglyceride
synthesis, and fatty acid catabolism related genes in primary
hepatocytes after 16 hours of incubation with vehicle and 2 .mu.M,
5 .mu.M and 10 .mu.M ceramide (left to right bar for each mRNA,
respectively, n=5).
FIG. 31F shows western blot analysis of nuclear SREBP1-N expression
in primary hepatocytes after 24 hours of incubation with vehicle,
and 2 .mu.M and 10 .mu.M ceramide (n=3).
FIG. 31G shows western blot analysis of CIDEA expression in primary
hepatocytes after 24 hours of incubation with vehicle, and 2 .mu.M
and 10 .mu.M ceramide (n=3).
FIG. 32A shows levels of mRNAs encoding fatty acid synthesis and
triglyceride synthesis related enzymes in the livers from vehicle-
and antibiotic-treated mice fed a high-fat diet for 14 weeks.
FIG. 32B shows expression of mRNAs encoding enzymes involved in
fatty acid and triglyceride synthesis in the livers of
Fxr.sup.fl/fl and Fxr.sup..DELTA.IE mice fed a high-fat diet for 14
weeks.
FIG. 32C shows mRNA levels of fatty acid oxidation-related genes in
the livers from mice fed a high-fat diet for 7 weeks.
FIG. 32D shows mRNA levels of fatty acid oxidation-related genes in
the livers from Fxr.sup.fl/fl mice and Fxr.sup..DELTA.IE mice fed a
high-fat diet for 14 weeks.
FIG. 32E shows western blot analysis of SREBP1-N protein expression
in livers from vehicle- and antibiotic-treated mice fed a high-fat
diet for 7 weeks. LAMIN A/C is used as a loading control (n=3).
FIG. 32F shows western blot analysis of CIDEA protein expression in
livers of vehicle- and antibiotic-treated mice fed a high-fat diet
for 7 weeks. 0-ACTIN is used as a loading control (n=3).
FIG. 32G shows Cyp7a1mRNA levels in the livers of vehicle- and
antibiotic-treated mice fed a high-fat diet for 7 weeks (n=3).
FIG. 32H shows Cyp7a1mRNA levels in the livers of vehicle- and
tempol-treated mice fed a high-fat diet for 7 weeks (n=3).
FIG. 32I shows mRNA levels of inflammation related genes in the
livers of vehicle- and antibiotic-treated fed a high-fat diet for 7
weeks. (n=3).
FIG. 32J shows mRNA levels of inflammation related genes in the
livers of vehicle- and tempol-treated mice fed a high-fat diet for
7 weeks (n=3).
FIG. 33A shows a representative H&E staining of liver sections
from vehicle- and antibiotic-treated Fxr.sup.fl/fl and
Fxr.sup..DELTA.IE mice fed a high-fat diet for 14 weeks.
FIG. 33B shows Oil red 0 staining of lipid droplets in liver
sections from vehicle- and antibiotic-treated Fxr.sup.fl/fl and
Fxr.sup..DELTA.IE mice fed a high-fat diet for 14 weeks.
FIG. 33C shows liver weights of vehicle- and antibiotic-treated
Fxr.sup.fl/fl and Fxr.sup..DELTA.IE mice fed a high-fat diet for 14
weeks.
FIG. 33D shows liver weight to body weight ratios of vehicle- and
antibiotic-treated Fxr.sup.fl/fl and Fxr.sup..DELTA.IE mice fed a
high-fat diet for 14 weeks.
FIG. 33E shows liver triglyceride contents of vehicle and
antibiotic-treated Fxr.sup.fl/fl and Fxr.sup..DELTA.IE mice fed a
high-fat diet for 14 weeks.
FIG. 33F shows lipidomics profile of ceramides in ileum of vehicle-
and antibiotic-treated Fxr.sup.fl/fl and Fxr.sup..DELTA.IE mice fed
a high-fat diet for 14 weeks (bars from left to right for each
ceramide, respectively).
FIG. 33G shows serum ceramides levels from vehicle- and
antibiotic-treated Fxr.sup.fl/fl and Fxr.sup..DELTA.IE mice fed a
high-fat diet for 14 weeks (bars from left to right for each
ceramide, respectively).
FIG. 34A shows representative H&E staining of liver sections
from vehicle- and tempol-treated Fxr.sup.fl/fl and
Fxr.sup..DELTA.IE mice fed a high-fat diet for 14 weeks.
FIG. 34B shows Oil red 0 staining of lipid droplets in liver
sections from vehicle and tempol-treated Fxr.sup.fl/fl mice and
Fxr.sup..DELTA.IE mice on a high-fat diet for 14 weeks.
FIG. 34C shows liver weights-of vehicle and tempol-treated
Fxr.sup.fl/fl mice and Fxr.sup..DELTA.IE mice on a high-fat diet
for 14 weeks.
FIG. 34D shows liver weight to body weight ratios from vehicle- and
tempol-treated Fxr.sup.fl/fl mice and Fxr.sup..DELTA.IE mice on a
high-fat diet for 14 weeks.
FIG. 34E shows liver triglyceride levels from vehicle- and
tempol-treated Fxr.sup.fl/fl mice and Fxr.sup..DELTA.IE mice on a
high-fat diet for 14 weeks.
FIG. 34F shows mRNA levels of fatty acid synthesis, triglyceride
synthesis, and fatty acid catabolism related genes in livers from
vehicle and tempol-treated Fxr.sup.fl/fl mice and Fxr.sup..DELTA.IE
mice on a high-fat diet for 14 weeks. The bars under each mRNA from
left to right are vehicle-treated Fxr.sup.fl/fl, tempol-treated
Fxr.sup.fl/fl, vehicle-treated Fxr.sup..DELTA.IE tempol-treated
Fxr.sup..DELTA.IE mice.
FIG. 34G shows western blot analysis of liver nuclear SREBP1-N
expression after tempol treatment of Fxr.sup.fl/fl and
Fxr.sup..DELTA.IE, mice on a high-fat diet for 16 weeks. Each lane
represents an individual mouse.
FIG. 34H shows western blot analysis of liver CIDEA expression
after tempol treatment of Fxr.sup.fl/fl and Fxr.sup..DELTA.IE, mice
on a high-fat diet for 16 weeks. Each lane represents an individual
mouse.
FIG. 35 shows the metabolism of the positive control
tauro-.beta.-muricholic acid (T.beta.MCA) and
glycine-.beta.-muricholic acid (Gly-MCA) to the product
.beta.-muricholic acid (T.beta.MCA) after incubation with fecal
protein containing intestinal bacteria.
FIG. 36 shows concentrations of Gly-MCA in mouse ileum after oral
gavage of 0, 1, 5, and 50 mg/kg of Gly-MCA.
FIG. 37A shows serum aminotransferase (ALT) levels in mice after 24
hours of treatment with Gly-MCA.
FIG. 37B shows aspartate aminotransferase (ALT) levels in mice
after 24 hours of treatment with Gly-MCA.
FIG. 38 shows luciferase activity observed in HEK293T fibroblasts
transiently co-transfected with a chimeric receptor construct as a
function of concentration of the added FXR agonist GW4064 in the
presence and absence of Gly-MCA.
FIG. 39 shows Shp mRNA expression in differentiated Caco-2 cells
treated with 100 .mu.M CDCA and vehicle (control), or 100 .mu.M and
200 .mu.M Gly-MCA with 100 .mu.M CDCA (n=3).
FIG. 40A shows levels of Shp mRNA in differentiated Caco-2 cells
after treatment with 100 .mu.M and 200 .mu.M Gly-MCA and with 2
.mu.M GW4064 or 5 .mu.M GW4064 (n=3). For each dosage of Gly-MCA
from left to right is shown further treatment with no GW4064, 2
.mu.M GW4064, and 5 .mu.M GW4064.
FIG. 40B shows levels of Fgf19 mRNA in differentiated Caco-2 cells
after treatment with 100 .mu.M and 200 .mu.M Gly-MCA and with 2
.mu.M GW4064 or 5 .mu.M GW4064 (n=3). For each dosage of Gly-MCA
from left to right is shown further treatment with no GW4064, 2
.mu.M GW4064, and 5 .mu.M GW4064.
FIG. 40C shows levels of Atp5g mRNAs in differentiated Caco-2 cells
after treatment with 100 .mu.M and 200 .mu.M Gly-MCA and with 2
.mu.M or 5 .mu.M GW4064 (n=3). For each dosage of Gly-MCA from left
to right is shown treatment with no GW4064, 2 .mu.M GW4064, and 5
.mu.M GW4064.
FIGS. 41A and 41B show growth curves of changes in body mass (A)
and % changes in initial body weight (B), over the course of 9
weeks, of vehicle- and Gly-MCA-treated mice, respectively, fed a
high-fat diet. n=5 mice per group.
FIGS. 41C and 41D show body composition as determined by NMR to
show the fat mass (C) and fat mass to lean mass ratio (D) in
vehicle and Gly-MCA-treated mice, respectively, after 9 weeks on a
high-fat diet. n=5 mice per group.
FIG. 42A shows cumulative food intake per day averaged over a
period of 1 week (from 6 to 7 weeks) in vehicle- and
Gly-MCA-treated mice fed a high-fat diet.
FIG. 42B shows 24 h energy expenditure using an indirect energy
balance (TEE.sub.bal) for an average period of 1 week (from 6 to 7
weeks) in vehicle- and Gly-MCA-treated mice fed a high-fat diet.
n=5 mice per group.
FIG. 43A shows the glucose tolerance test (GTT) in vehicle- and
Gly-MCA-treated mice after 6 to 7 weeks of feeding a high-fat diet.
n=5 mice per group.
FIG. 43B shows the area under the curve (AUC) of the glucose
tolerance test depicted in FIG. 45A.
FIG. 43C shows the insulin tolerance test (ITT) in vehicle- and
Gly-MCA-treated mice after 6 to 7 weeks of feeding a high-fat diet.
n=5 mice per group.
FIG. 44A shows representative H&E staining of liver sections in
vehicle- and Gly-MCA-treated mice fed a high-fat diet for 7
weeks.
FIG. 44B shows liver weights in vehicle- and Gly-MCA-treated mice
fed a high-fat diet for 7 weeks. n=5 mice per group.
FIG. 44C shows liver weight to body weight ratios in vehicle- and
Gly-MCA-treated mice fed a high-fat diet for 7 weeks. n=5 mice per
group.
FIG. 44D shows liver triglyceride content of vehicle- and
Gly-MCA-treated mice fed a high-fat diet for 9 weeks. n=5 mice per
group.
FIGS. 45A and 45B show serum alanine aminotransferase (ALT) and
aspartate aminotransferase (AST) levels of vehicle- and
Gly-MCA-treated mice, respectively, fed a high-fat diet for 9
weeks. n=5 mice per group.
FIG. 46A shows a scores scatter plot of a PCA model of feces ions
from vehicle- and Gly-MCA-treated mice fed a high-fat diet for 9
weeks.
FIG. 46B shows a scatter plot of partial least squares discriminant
analysis (PLS-DA) of feces ions from vehicle and Gly-MCA-treated
mice fed a high-fat diet for 9 weeks. Each point represents an
individual mouse feces ion. The labeled ions are identified as
.beta.-MCA, T.beta.MCA, taurocholic acid (TCA) and Gly-MCA, which
are affected by Gly-MCA treatment. The p(corr)[1]P values represent
the interclass difference and p[1]P values represent the relative
abundance of the ions. Data were obtained in negative ionization
mode (ESI.sup.-).
FIG. 46C shows individual bile acid compositions in feces ions from
vehicle- and Gly-MCA-treated mice fed a high-fat diet for 9
weeks.
FIG. 46D shows Gly-MCA levels in feces of vehicle- and
Gly-MCA-treated mice fed a high-fat diet for 9 weeks. n=5 mice per
group. All data are presented as mean.+-.SD.
FIG. 47A shows a scores scatter plot of a PCA model of ileum ions
in vehicle- and Gly-MCA-treated mice fed a high-fat diet for 9
weeks.
FIG. 47B shows a scatter plot of PLS-DA of ileum ions from vehicle
and Gly-MCA-treated mice fed a high-fat diet for 9 weeks. Each
point represents an individual mouse feces ion. The labeled ions
are identified as T-.alpha.-MCA, T.beta.MCA, taurocholic acid
(TCA), tauroursodeoxycholic acid (TCDCA), taurodeoxycholic acid
(TDCA) and taurochenodeoxycholic acid (TCDCA) and Gly-MCA, which
are induced by Gly-MCA treatment. The p(corr)[1]P values represent
the interclass difference and p[1]P values represent the relative
abundance of the ions. All the data are obtained in negative mode
(ESI-).
FIG. 47C shows the bile acid composition in ileum from vehicle and
Gly-MCA-treated mice fed a high-fat diet for 9 weeks.
FIG. 47D shows Gly-MCA levels in ileum of vehicle- and
Gly-MCA-treated mice fed a high-fat diet for 9 weeks. n=5 mice per
group. All data are presented as mean.+-.SD.
FIG. 48A shows serum total triglyceride levels of vehicle- and
Gly-MCA-treated mice fed a high-fat diet for 9 weeks. n=5 mice per
group.
FIG. 48B shows the profile of serum triglyceride species from
vehicle- and Gly-MCA-treated mice fed a high-fat diet for 9 weeks.
n=5 mice per group. All data are presented as mean.+-.SD.
FIG. 49A shows profiles of serum ceramides from vehicle- and
Gly-MCA-treated mice fed a high-fat diet for 9 weeks. n=5 mice per
group.
FIG. 49B shows profiles of ileum ceramides from vehicle- and
Gly-MCA-treated mice fed a high-fat diet for 9 weeks. n=5 mice per
group. All data are presented as mean.+-.SD.
FIG. 50A shows mRNA levels of FXR target genes Shp and Fgf15 in the
ileum of vehicle- and Gly-MCA-treated mice fed a high-fat diet for
9 weeks. n=5 mice per group.
FIG. 50B shows levels of mRNAs encoding genes involved in ceramide
metabolism in ileum from vehicle- and Gly-MCA-treated mice fed a
high-fat diet for 9 weeks. n=5 mice per group. All data are
presented as mean.+-.SD.
FIG. 51A shows mRNA levels of the FXR target gene Shp in the liver
of vehicle- and Gly-MCA-treated mice fed a high-fat diet for 9
weeks. n=5 mice per group.
FIG. 51B shows mRNA levels of Cyp7a1 in the liver of vehicle- and
Gly-MCA-treated mice fed a high-fat diet for 9 weeks. n=5 mice per
group. All data are presented as mean.+-.SD.
FIG. 52 shows growth curves of genetically obese leptin
receptor-deficient (db/db) treated with vehicle and Gly-MCA for 6
weeks. n=5 mice per group. All data are presented as
mean.+-.SD.
FIGS. 53A and 53B show the body composition, as determined by NMR,
of the fat mass, and fat mass to lean mass ratio in db/db mice
treated with vehicle and Gly-MCA for 6 weeks. n=5 mice per group.
All data are presented as mean.+-.SD.
FIG. 54A shows representative H&E staining of liver sections in
db/db mice treated with vehicle and Gly-MCA for 6 weeks. n=5 mice
per group.
FIGS. 54B and 54C shows liver weights, and liver weight to body
weight ratios in db/db mice treated with vehicle and Gly-MCA for 6
weeks. n=5 mice per group.
FIG. 54D shows liver triglyceride content of db/db mice treated
with vehicle and Gly-MCA for 6 weeks. n=5 mice per group. All data
are presented as mean.+-.SD.
FIGS. 55A and 55B show serum ALT and AST levels in db/db mice
treated with vehicle and Gly-MCA for 6 weeks. n=5 mice per group.
All data are presented as mean.+-.SD.
FIGS. 56A and 56B shows the bile acid composition in feces and
ileum from vehicle and Gly-MCA-treated db/db mice for 6 weeks. n=5
mice per group.
FIG. 56C shows relative levels of Gly-MCA in ileum, feces, liver
and serum of vehicle and Gly-MCA-treated db/db mice for 6 weeks.
n=5 mice per group. All data are presented as mean.+-.SD.
FIG. 57A shows the profile of serum triglyceride species in db/db
mice treated with vehicle and Gly-MCA for 6 weeks. n=5 mice per
group.
FIGS. 57B and 57C shows profiles of serum and ileum ceramides
species from vehicle and Gly-MCA-treated mice fed a high-fat diet
for 9 weeks. n=5 mice per group. All data are presented as
mean.+-.SD.
FIG. 58 show the curves of body mass of HFD-induced obese mice
treated with vehicle- and Gly-MCA for 6 weeks. n=5 mice per
group.
FIG. 59 show body composition as determined by NMR in high-fat
diet-induced obese mice treated with vehicle- and Gly-MCA for 6
weeks. n=5 mice per group.
FIG. 60A shows representative H&E staining of liver sections in
high-fat diet-induced obese mice treated with vehicle- and Gly-MCA
for 6 weeks. n=5 mice per group.
FIGS. 60B and 60C shows liver weights and liver weight to body
weight ratios in high-fat diet-induced obese mice treated with
vehicle- and Gly-MCA for 6 weeks. n=5 mice per group.
FIGS. 61A and 61B shows the bile acid composition in feces and
ileum from high-fat diet-induced obese mice treated with vehicle-
and Gly-MCA for 6 weeks. n=5 mice per group.
FIG. 61C shows relative levels of Gly-MCA in the ileum, feces,
liver and serum of vehicle and Gly-MCA-treated high-fat
diet-induced obese mice for 6 weeks. n=5 mice per group. All data
are presented as mean.+-.SD.
DETAILED DESCRIPTION OF THE INVENTION
In an embodiment, the invention provides a compound of formula (I)
or (II):
##STR00002## wherein R.sup.1 and R.sup.2 are independently selected
from hydrogen, alkyl, and C(.dbd.O)R.sup.3, R.sup.4 is selected
from hydrogen, alkyl, and C(.dbd.O)R.sup.3, X is selected from
C.dbd.O and CH.sub.2, Y is selected from CH.sub.2, NR.sup.5, O, S,
SO, SO.sub.2, and Se, or X and Y taken together form C.dbd.C, Z is
selected from COOR.sup.6, SO.sub.3R.sup.7, and
P(.dbd.O)(OR.sup.8).sub.2, R.sup.3, R.sup.5, R.sup.6, R.sup.7, and
R.sup.8 are independently selected from hydrogen, alkyl, and aryl,
R.sup.4 is selected from hydrogen, alkyl, and C(.dbd.O)R.sup.3, m
is an integer of 1 to 6, and n is an integer of 1 to 6, or a
pharmaceutically acceptable salt thereof, with the proviso that,
when the compound is of formula (I), m is 1, n is 2, X is C.dbd.O,
Y is NH, le and R.sup.2 are both hydrogen, and R.sup.4 is hydrogen,
then Z is not SO.sub.3H.
In accordance with certain embodiments, when the compound is of
formula (I), m is 1, X is C.dbd.O, Y is NH, R.sup.1 and R.sup.2 are
both hydrogen, R.sup.4 is hydrogen, and n is 1, then Z is not
COOH.
In accordance with certain embodiments, the compound is of formula
(I).
In accordance with any of the above embodiments, R.sup.4 is
hydrogen.
In accordance with certain embodiments, R.sup.1 and R.sup.2 are
hydrogen.
In accordance with certain embodiments, X is C.dbd.O.
In accordance with certain embodiments, m is 2.
In accordance with certain embodiments, Y is NH.
In accordance with certain embodiments, n is an integer of 1 to
6.
In accordance with certain preferred embodiments, the compound is
selected from:
##STR00003##
In accordance with certain preferred embodiments, the compound is
selected from:
##STR00004##
In accordance with certain embodiments, X is CH.sub.2.
In accordance with certain embodiments, Y is selected from NH, O,
S, and Se.
In accordance with certain embodiments, m is 2
In accordance with certain embodiments, n is 2.
In accordance with certain embodiments, Z is SO.sub.3H.
In accordance with certain preferred embodiments, the compound is
selected from:
##STR00005##
In accordance with certain embodiments, the compound is of formula
(II).
In accordance with certain embodiments, R.sup.1 and R.sup.2 are
hydrogen.
In accordance with certain embodiments, X is C.dbd.O.
In accordance with certain embodiments, m is 2.
In accordance with certain embodiments, Y is NH.
In accordance with certain embodiments, n is an integer of 1 to
6.
In accordance with certain preferred embodiments, the compound is
selected from:
##STR00006##
In accordance with certain preferred embodiments, the compound is
selected from:
##STR00007##
In certain embodiments, X is CH.sub.2.
In accordance with certain embodiments, Y is selected from NH, O,
S, and Se.
In certain embodiments, m is 2.
In certain embodiments, n is 2.
In certain embodiments, Z is SO.sub.3H.
In accordance with certain preferred embodiments, the compound is
selected from:
##STR00008##
In accordance with certain more preferred embodiments, the compound
is selected from:
##STR00009## ##STR00010## ##STR00011## ##STR00012##
The invention also provides a compound of formula (III):
##STR00013##
wherein R.sup.1 and R.sup.2 are independently selected from
hydrogen, alkyl, and C(.dbd.O)R.sup.3,
R.sup.11 is hydrogen, halo, alkyl, OR.sup.2, and
C(.dbd.O)R.sup.3,
R.sup.12 is hydrogen, halo, alkyl, OR.sup.4, or
C(.dbd.O)R.sup.3,
R.sup.13 is hydrogen, alkyl, OR.sup.14, or C(.dbd.O)R.sup.3,
R.sup.4 is selected from hydrogen, alkyl, and C(.dbd.O)R.sup.3,
R.sup.3 is hydrogen, alkyl, or aryl,
R.sup.15, R.sup.16, R.sup.17, and R.sup.18 are independently
selected from hydrogen and halo, and R.sup.14 is selected from
glycino, alanino, .beta.-alanino, phenylalanino, tyrosino,
methionino, tryptophano, leucino, isoleucino, methyl aspartato,
asparto, methyl glutamo, glutamo, methyl prolino, prolino, valino,
2-fluoro-.beta.-alanino, 2-bromoalanino, 2-chloroalanino,
2-fluoroalanino, 2-iodoalanino, 3-bromoalanino, 3-chloroalanino,
3-fluoroalanino, 3-iodoalanino, 4-bromophenylalanino,
4-chlorophenylalanino, 4-fluorophenylalanino, taurino, and
4-iodophenylalanino,
or a pharmaceutically acceptable salt thereof,
with the provisos that, when R.sup.1 is hydrogen, R.sup.11 and
R.sup.12 are both .beta.-hydroxyl, R.sup.13, R.sup.15, R.sup.16,
R.sup.17, and R.sup.18 are all hydrogen, then R.sup.14 is not
glycine or taurine, and,
when R.sup.1 is hydrogen, R.sup.11 is .alpha.-hydroxyl, R.sup.12,
R.sup.13, R.sup.15, R.sup.16, R.sup.17, and R.sup.18 are all
hydrogen, then R.sup.14 is not glycine or taurine.
In certain embodiments, the compound is selected from:
##STR00014## ##STR00015##
In certain embodiments, R.sup.11 is halo, R.sup.12 and R.sup.13 is
hydroxyl, and R.sup.15, R.sup.16, R.sup.17, and R.sup.18 are all
hydrogen.
In certain embodiments, and R.sup.12 are both halo, and R.sup.15,
R.sup.16, R.sup.17, and R.sup.18 are all hydrogen.
In certain embodiments, R.sup.18 is halo and R.sup.15, R.sup.16,
and R.sup.17 are all hydrogen.
Referring now to terminology used generically herein, the term
"alkyl" means a straight-chain or branched alkyl substituent
containing from, for example, 1 to about 6 carbon atoms, preferably
from 1 to about 4 carbon atoms, more preferably from 1 to 2 carbon
atoms. Examples of such substituents include methyl, ethyl, propyl,
isopropyl, n-butyl, sec-butyl, isobutyl, tert-butyl, pentyl,
isoamyl, hexyl, and the like.
The term "aryl" refers to an unsubstituted or substituted aromatic
carbocyclic substituent, as commonly understood in the art, and the
term "C.sub.6-C.sub.10 aryl" includes phenyl and naphthyl. It is
understood that the term aryl applies to cyclic substituents that
are planar and comprise 4n+2.pi. electrons, according to Huckel's
Rule.
In any of the above embodiments, the C-20 carbon atom of the
compound or salt of Formula (I) or (II) can have an R
configuration, an S configuration, or can be a mixture of R and S
isomers.
In any of the above embodiments, when the stereochemistry at a
chiral carbon atom is not specified, the chiral carbon atom can
have an R configuration, an S configuration, or can be a mixture of
R and S isomers.
The phrase "pharmaceutically acceptable salt" is intended to
include nontoxic salts synthesized from the parent compound which
contains a basic or acidic moiety by conventional chemical methods.
Generally, such salts can be prepared by reacting the free acid or
base forms of these compounds with a stoichiometric amount of the
appropriate base or acid in water or in an organic solvent, or in a
mixture of the two. Generally, nonaqueous media such as ether,
ethyl acetate, ethanol, isopropanol, or acetonitrile are preferred.
Lists of suitable salts are found in Remington's Pharmaceutical
Sciences, 18th ed., Mack Publishing Company, Easton, Pa., 1990, p.
1445, and Journal of Pharmaceutical Science, 66, 2-19 (1977).
Suitable bases include inorganic bases such as alkali and alkaline
earth metal bases, e.g., those containing metallic cations such as
sodium, potassium, magnesium, calcium and the like. Non-limiting
examples of suitable bases include sodium hydroxide, potassium
hydroxide, sodium carbonate, and potassium carbonate. Suitable
acids include inorganic acids such as hydrochloric acid,
hydrobromic acid, hydroiodic acid, sulfuric acid, phosphoric acid,
and the like, and organic acids such as p-toluenesulfonic,
methanesulfonic acid, benzenesulfonic acid, oxalic acid,
p-bromophenylsulfonic acid, carbonic acid, succinic acid, citric
acid, benzoic acid, acetic acid, maleic acid, tartaric acid, fatty
acids, long chain fatty acids, and the like. Preferred
pharmaceutically acceptable salts of inventive compounds having an
acidic moiety include sodium and potassium salts. Preferred
pharmaceutically acceptable salts of inventive compounds having a
basic moiety (e.g., a dimethylaminoalkyl group) include
hydrochloride and hydrobromide salts. The compounds of the present
invention containing an acidic or basic moiety are useful in the
form of the free base or acid or in the form of a pharmaceutically
acceptable salt thereof.
It should be recognized that the particular counterion forming a
part of any salt of this invention is usually not of a critical
nature, so long as the salt as a whole is pharmacologically
acceptable and as long as the counterion does not contribute
undesired qualities to the salt as a whole.
It is further understood that the above compounds and salts may
form solvates, or exist in a substantially uncomplexed form, such
as the anhydrous form. As used herein, the term "solvate" refers to
a molecular complex wherein the solvent molecule, such as the
crystallizing solvent, is incorporated into the crystal lattice.
When the solvent incorporated in the solvate is water, the
molecular complex is called a hydrate. Pharmaceutically acceptable
solvates include hydrates, alcoholates such as methanolates and
ethanolates, acetonitrilates and the like. These compounds can also
exist in polymorphic forms.
The present invention is further directed to a pharmaceutical
composition comprising a pharmaceutically acceptable carrier and at
least one compound or salt described herein.
It is preferred that the pharmaceutically acceptable carrier be one
that is chemically inert to the active compounds and one that has
no detrimental side effects or toxicity under the conditions of
use.
The choice of carrier will be determined in part by the particular
compound of the present invention chosen, as well as by the
particular method used to administer the composition. Accordingly,
there is a wide variety of suitable formulations of the
pharmaceutical composition of the present invention. In certain
embodiments, the formulation is suitable for administration to the
alimentary tract, and in particular, to the small intestine.
Formulations suitable for oral administration can consist of (a)
liquid solutions, such as a therapeutically effective amount of the
inventive compound dissolved in diluents, such as water, saline, or
orange juice, (b) capsules, sachets, tablets, lozenges, and
troches, each containing a predetermined amount of the active
ingredient, as solids or granules, (c) powders, (d) suspensions in
an appropriate liquid, and (e) suitable emulsions. Liquid
formulations may include diluents, such as water and alcohols, for
example, ethanol, benzyl alcohol, and the polyethylene alcohols,
either with or without the addition of a pharmaceutically
acceptable surfactant, suspending agent, or emulsifying agent.
Capsule forms can be of the ordinary hard- or soft-shelled gelatin
type containing, for example, surfactants, lubricants, and inert
fillers, such as lactose, sucrose, calcium phosphate, and corn
starch. Tablet forms can include one or more of lactose, sucrose,
mannitol, corn starch, potato starch, alginic acid,
microcrystalline cellulose, acacia, gelatin, guar gum, colloidal
silicon dioxide, croscarmellose sodium, talc, magnesium stearate,
calcium stearate, zinc stearate, stearic acid, and other
excipients, colorants, diluents, buffering agents, disintegrating
agents, moistening agents, preservatives, flavoring agents, and
pharmacologically compatible excipients. Lozenge forms can comprise
the active ingredient in a flavor, usually sucrose and acacia or
tragacanth, as well as pastilles comprising the active ingredient
in an inert base, such as gelatin and glycerin, or sucrose and
acacia, emulsions, gels, and the like containing, in addition to
the active ingredient, such excipients as are known in the art.
In some embodiments, the formulation can be suitable to prolonging
the amount of time that the compound of the present invention is
contacted with the alimentary tract of the mammal, and in
particular with the small intestine of the mammal. In this regard,
various formulations such as extended release formulation and
formulations designed to prolong the amount of time that the
compound is retained in the stomach before release into the small
intestine can be utilized. A number of suitable formulations are
presented in Remington: The Science and Practice of Pharmacy,
Gennaro, A. R., ed., pp. 858-929, Lippincott Williams and Wilkins
(2000).
In some embodiments, the compound or salt of the present invention
can be administered in the form of a food additive, that is, in
admixture with foodstuffs or beverages. For use as a food additive,
the compound or salt can be mixed with a foodstuff or beverage per
se, or can be formulated as a composition comprising one or more
suitable excipients prior to mixing with a foodstuff or beverage.
The foodstuff or beverage can be any suitable foodstuff or
beverage. In some embodiments, the foodstuff or beverage has a
relatively high fat content.
It will be appreciated by one of ordinary skill in the art that, in
addition to the aforedescribed pharmaceutical compositions, the
compound or salt of the present invention may be formulated as
inclusion complexes, such as cyclodextrin inclusion complexes, or
liposomes. Liposomes serve to target the compounds to a particular
tissue, such as lymphoid tissue or cancerous hepatic cells.
Liposomes can also be used to increase the half-life of the
inventive compound. Liposomes useful in the present invention
include emulsions, foams, micelles, insoluble monolayers, liquid
crystals, phospholipid dispersions, lamellar layers and the like.
In these preparations, the active agent to be delivered is
incorporated as part of a liposome, alone or in conjunction with a
suitable chemotherapeutic agent. Thus, liposomes filled with a
desired inventive compound or salt thereof, can be directed to the
site of a specific tissue type, hepatic cells, for example, where
the liposomes then deliver the selected compositions. Liposomes for
use in the invention are formed from standard vesicle-forming
lipids, which generally include neutral and negatively charged
phospholipids and a sterol, such as cholesterol. The selection of
lipids is generally guided by consideration of, for example,
liposome size and stability of the liposomes in the blood stream. A
variety of methods are available for preparing liposomes, as
described in, for example, Szoka et al., Ann. Rev. Biophys.
Bioeng., 9, 467 (1980), and U.S. Pat. Nos. 4,235,871, 4,501,728,
4,837,028, and 5,019,369. For targeting to the cells of a
particular tissue type, a ligand to be incorporated into the
liposome can include, for example, antibodies or fragments thereof
specific for cell surface determinants of the targeted tissue type.
A liposome suspension containing a compound or salt of the present
invention may be administered intravenously, locally, topically,
etc. in a dose that varies according to the mode of administration,
the agent being delivered, and the stage of disease being
treated.
In certain embodiments, the pharmaceutical composition can be
administered parenterally, e.g., intravenously, subcutaneously,
intradermally, or intramuscularly. Thus, the invention provides
compositions for parenteral administration that comprise a solution
or suspension of the inventive compound or salt dissolved or
suspended in an acceptable carrier suitable for parenteral
administration, including aqueous and non-aqueous isotonic sterile
injection solutions. Many such compositions are known in the
art.
In accordance with an embodiment, the invention provides a method
of inhibiting a farnesoid X receptor in a mammal in need thereof,
which method comprises administering to the mammal an effective
amount of a compound of the invention.
Preferably, the animal is a mammal. More preferably, the mammal is
a human.
The term "mammal" includes, but is not limited to, the order
Rodentia, such as mice, and the order Logomorpha, such as rabbits.
It is preferred that the mammals are from the order Carnivora,
including Felines (cats) and Canines (dogs). It is more preferred
that the mammals are from the order Artiodactyla, including Bovines
(cows) and Swines (pigs) or of the order Perssodactyla, including
Equines (horses). It is most preferred that the mammals are of the
order Primates, Ceboids, or Simioids (monkeys) or of the order
Anthropoids (humans and apes). An especially preferred mammal is
the human.
In certain embodiments, the FXR-mediated disease or condition is
cardiovascular disease, atherosclerosis, arteriosclerosis,
hypercholesteremia, or hyperlipidemiachronic liver disease,
gastrointestinal disease, renal disease, cardiovascular disease,
metabolic disease, cancer (i.e., colorectal cancer), or
neurological indications such as stroke. In certain embodiments,
the chronic liver disease is primary biliary cirrhosis (PBC),
cerebrotendinous xanthomatosis (CTX), primary sclerosing
cholangitis (PSC), drug induced cholestasis, intrahepatic
cholestasis of pregnancy, parenteral nutrition associated
cholestasis (PNAC), bacterial overgrowth or sepsis associated
cholestasis, autoimmune hepatitis, chronic viral hepatitis,
alcoholic liver disease, nonalcoholic fatty liver disease (NAFLD),
nonalcoholic steatohepatitis (NASH), liver transplant associated
graft versus host disease, living donor transplant liver
regeneration, congenital hepatic fibrosis, choledocholithiasis,
granulomatous liver disease, intra- or extrahepatic malignancy,
Sjogren's syndrome, Sarcoidosis, Wilson's disease, Gaucher's
disease, hemochromatosis, or alpha 1-antitrypsin deficiency. In
certain embodiments, the gastrointestinal disease is inflammatory
bowel disease (IBD) (including Crohn's disease and ulcerative
colitis), irritable bowel syndrome (IBS), bacterial overgrowth,
malabsorption, post-radiation colitis, or microscopic colitis. In
certain embodiments, the renal disease is diabetic nephropathy,
focal segmental glomerulosclerosis (FSGS), hypertensive
nephrosclerosis, chronic glomerulonephritis, chronic transplant
glomerulopathy, chronic interstitial nephritis, or polycystic
kidney disease. In certain embodiments, the cardiovascular disease
is atherosclerosis, arteriosclerosis, dyslipidemia,
hypercholesterolemia, or hypertriglyceridemia.
In accordance with a preferred embodiment, the invention provides a
method of treating or preventing obesity in a mammal in need
thereof, comprising administering to the mammal an effective amount
of a compound or salt of the invention.
As used herein, obesity can be considered as a condition in which
excess body fat may put a person at health risk (see Barlow and
Dietz, Pediatrics 102: E29, 1998; National Institutes of Health,
Obes. Res. 6 (suppl. 2):51S-209S, 1998). Excess body fat is a
result of an imbalance of energy intake and energy expenditure. In
one embodiment in humans, the Body Mass Index (BMI) is used to
assess obesity. In one embodiment, a BMI of 25.0 kg/m.sup.2 to 29.9
kg/m.sup.2 is overweight (also called grade I obesity), while a BMI
of 30 kg/m.sup.2 is truly obese (also called grade II obesity).
In another embodiment in humans, waist circumference is used to
assess obesity. In this embodiment, in men a waist circumference of
102 cm or more is considered obese, while in women a waist
circumference of 89 cm or more is considered obese. Strong evidence
shows that obesity affects both the morbidity and mortality of
individuals. For example, an obese individual is at increased risk
for heart disease, non-insulin dependent (type 2) diabetes,
hypertension, stroke, cancer (e.g. endometrial, breast, prostate,
and colon cancer), dyslipidemia, gall bladder disease, sleep apnea,
reduced fertility, and osteoarthritis, amongst others (see Lyznicki
et al., Am. Fam. Phys. 63:2185, 2001).
The dose administered to a mammal, particularly, a human, in
accordance with the present invention should be sufficient to
effect the desired response. Such responses include reversal or
prevention of the undesirable effects of the disease or disorder
mediated by the farnesoid X receptor expressed in the intestine for
which treatment is desired or to elicit the desired benefit. In
certain embodiments, the disorder is non-alcoholic fatty liver
disease, obesity and type 2 diabetes (insulin resistance). One
skilled in the art will recognize that dosage will depend upon a
variety of factors, including the age, condition, and body weight
of the human, as well as the extent of the non-alcoholic fatty
liver disease in the human. The size of the dose will also be
determined by the route, timing and frequency of administration as
well as the existence, nature, and extent of any adverse
side-effects that might accompany the administration of a
particular compound and the desired physiological effect. It will
be appreciated by one of skill in the art that successful treatment
of non-alcoholic fatty liver disease, obesity, type 2 diabetes
(insulin resistance) or other disease or disorder may require
prolonged treatment involving multiple administrations.
In this regard, treatment of NAFLD via inhibition of the intestinal
farnesoid X receptor can be regarded as a reduction in the clinical
manifestations of hepatic steatosis in a mammal. While in many
cases NAFLD does not cause signs or symptoms, NAFLD may cause
fatigue, pain, particularly in the upper right abdomen, and weight
loss. In some instances, NAFLD may progress to nonalcoholic
steatohepatitis, an inflammation in the liver. NAFLD may progress
to nonalcoholic fatty liver disease-associated cirrhosis which is a
scarring of the liver accompanied by markedly decreased liver
function. Over time, scarring can become so severe that the liver
no longer functions adequately.
NAFLD can be assessed, for example, by ultrasound, computed
tomography, magnetic resonance studies, or by liver biopsy. In
certain embodiments, the mammal is consuming a high fat diet. A
high fat diet can be considered as one that provides more than 30%
of energy as fat (see, for example, Churchill Livingstone's
Dictionary of Sport and Exercise Science and Medicine, S. Jennett,
Elsevier Limited, 2008). In other embodiments, the invention
provides a method of preventing non-alcoholic fatty liver disease
in a mammal. Preventing non-alcoholic fatty liver disease can be
regarded as reducing an expected manifestation of hepatic steatosis
in a mammal that is subjected to a dietary change from a low fat or
intermediate fat diet to a high fat diet.
Currently, no standard treatment for NAFLD exists. Physicians
typically treat the risk factors that contribute to the disease.
For example, physicians assist afflicted patients with weight loss
programs and choice of a healthy diet, control of diabetes, and
lowering of cholesterol.
In this regard, treatment of obesity via inhibition of the
farnesoid X receptor can be regarded as a reduction in the rate of
weight gain in a mammal. In certain embodiments, the mammal is
consuming a high fat diet. A high fat diet can be consider as one
which provides more than 30% of energy as fat (see, for example,
Churchill Livingstone's Dictionary of Sport and Exercise Science
and Medicine, S. Jennett, Elsevier Limited, 2008). In other
embodiments, the invention provides a method of preventing obesity
in a mammal. Preventing obesity can be regarded as reducing an
expected weight gain in a normal weight mammal that is subjected to
a dietary change from a low fat or intermediate fat diet to a high
fat diet.
In this regard, treatment of diabetes via inhibition of the
farnesoid X receptor can be regarded as a reduction of insulin
resistance in a patient afflicted therewith. Insulin resistance can
result in hyperglycemia, and a reduction in insulin resistance can
result in a lowering of blood glucose levels. Non-limiting examples
of symptoms that be treated via inhibition of the farnesoid X
receptor include brain fogginess and inability to focus, high blood
sugar, intestinal bloating, sleepiness, weight gain, fat storage,
difficulty losing weight, increased blood triglyceride levels,
increased blood pressure, increased pro-inflammatory cytokines
associated with cardiovascular disease, depression, acanthosis
nigricans, and increased hunger.
The dose administered to a mammal, particularly, a human, in
accordance with the present invention should be sufficient to
effect the desired response. Such responses include reversal or
prevention of the bad effects of the disease or disorder mediated
by the farnesoid X receptor for which treatment is desired or to
elicit the desired benefit. In certain embodiments, the disorder is
obesity. One skilled in the art will recognize that dosage will
depend upon a variety of factors, including the age, condition, and
body weight of the human, as well as the extent of the obesity in
the human. The size of the dose will also be determined by the
route, timing and frequency of administration as well as the
existence, nature, and extent of any adverse side-effects that
might accompany the administration of a particular compound and the
desired physiological effect. It will be appreciated by one of
skill in the art that successful treatment of obesity or other
disease or disorder may require prolonged treatment involving
multiple administrations.
In this regard, treatment of obesity via inhibition of the
farnesoid X receptor can be regarded as a reduction in the rate of
weight gain in a mammal. In certain embodiments, the mammal is
consuming a high fat diet. A high fat diet can be consider as one
which provides more than 30% of energy as fat (see, for example,
Churchill Livingstone's Dictionary of Sport and Exercise Science
and Medicine, S. Jennett, Elsevier Limited (2008)). In other
embodiments, the invention provides a method of preventing obesity
in a mammal. Preventing obesity can be regarded as reducing an
expected weight gain in a normal weight mammal that is subjected to
a dietary change from a low fat or intermediate fat diet to a high
fat diet.
Suitable doses and dosage regimens can be determined by
conventional range-finding techniques known to those of ordinary
skill in the art. Generally, treatment is initiated with smaller
dosages that are less than the optimum dose of the compound.
Thereafter, the dosage is increased by small increments until the
optimum effect under the circumstances is reached. The present
inventive method typically will involve the administration of about
0.1 to about 300 mg (e.g., about 0.1 to about 150 mg, about 0.1 to
about 100 mg, or about 0.1 to about 50 mg) of one or more of the
compounds described above per kg body weight of the mammal.
The therapeutically effective amount of the compound or compounds
administered can vary depending upon the desired effects and the
factors noted above. Typically, dosages will be between 0.01 mg/kg
and 250 mg/kg of the subject's body weight, and more typically
between about 0.05 mg/kg and 100 mg/kg, such as from about 0.2 to
about 80 mg/kg, from about 5 to about 40 mg/kg or from about 10 to
about 30 mg/kg of the subject's body weight. Thus, unit dosage
forms can be formulated based upon the suitable ranges recited
above and the subject's body weight. The term "unit dosage form" as
used herein refers to a physically discrete unit of therapeutic
agent appropriate for the subject to be treated.
Alternatively, dosages are calculated based on body surface area
and from about 1 mg/m.sup.2 to about 200 mg/m.sup.2, such as from
about 5 mg/m.sup.2 to about 100 mg/m.sup.2 will be administered to
the subject per day. In particular embodiments, administration of
the therapeutically effective amount of the compound or compounds
involves administering to the subject from about 5 mg/m.sup.2 to
about 50 mg/m.sup.2, such as from about 10 mg/m.sup.2 to about 40
mg/m.sup.2 per day. It is currently believed that a single dosage
of the compound or compounds is suitable, however a therapeutically
effective dosage can be supplied over an extended period of time or
in multiple doses per day. Thus, unit dosage forms also can be
calculated using a subject's body surface area based on the
suitable ranges recited above and the desired dosing schedule.
As demonstrated herein, farnesoid X receptor is implicated in the
development of obesity. Thus, administration of inhibitors of
farnesoid X receptor is expected to treat or prevent the
development of obesity, particularly in a mammal consuming a high
fat diet.
Here, it has also been shown that intestinal farnesoid X receptor
plays an essential role in the progression of NAFLD. Inhibition of
intestinal farnesoid X receptor in embodiments of the invention has
been shown to ameliorate NAFLD induced by a high fat diet.
Through studies on tempol and antibiotics that remodel and kill gut
bacteria, respectively, a novel pathway was uncovered in which
these agents alter the population of the gut microbiota resulting
in loss of bacteria that express the enzyme bile salt hydrolase
(BSH). Lower BSH results in increased levels of conjugated bile
acids in the intestine, such as T-.beta.-MCA. T-.beta.-MCA in turn
is an antagonist of intestinal FXR. Lower FXR signaling in the
intestine results in decreased obesity, insulin resistance and
NAFLD in mice fed a high-fat diet, and in genetically obese mice.
These studies led to the hypothesis that inhibiting FXR would be a
promising approach for treating patients with obesity, insulin
resistance and NAFLD. Oral administration of a new chemical entity
glycine .beta.-muricholic acid (Gly-MCA) decreases obesity, insulin
resistance and NAFLD in high-fat diet-treated mice and in
genetically obese mice. It is proposed that any compound that is
orally administered and retained in the intestine and that inhibits
intestinal FXR and has no effect on FXR expressed in liver, would
have utility in the treatment of patients with obesity, insulin
resistance and NAFLD.
Chemistry
Compounds of formula 1, wherein W is OR.sup.4, R.sup.4 is hydrogen,
R.sup.1 and R.sup.2 are hydrogen, X is C.dbd.O, m is 1 and Y is NH,
such as .beta.-Muricholic acid 9 and conjugates thereof, such as
the representative embodiments of tauro-.beta.-Muricholic acid 10
and glycine-.beta.-Muricholic acid 16 can be prepared as
illustrated in the scheme set forth in FIG. 8. Esterification of
the dihydroxy acid 1 with, for example, methanol under acid
catalysis provides ester 2. Protection of the A-ring hydroxyl group
with ethyl chloroformate provides carbonate 3. Oxidation of the
7-hydroxyl group with, for example potassium chromate gives ketone
4. Bromination with, for example, bromine in HBr gives bromo ketone
5. Reduction of the ketone with, for example, gives bromo alcohol
6. Reductive elimination of bromine using, for example, zinc metal
provides olefin 7. Cis-dihydroxylation with, for example, osmium
tetroxide gives cis diol 8. Hydrolysis of both esters provides
.beta.-muricholic acid 9. .beta.-muricholic acid 9 can be
conjugated with taurine using a suitable coupling agent provides
tauro-.beta.-muricholic acid 10. Glycine can be substituted for
taurine to provide the glycine conjugate of .beta.-muricholic acid
2-aminoethylphosphonic acid can be substituted for taurine to
provide the phosphonic acid analog of tauro-.beta.-muricholic acid.
The chemistry is as described in Iida, T., et al., Lipid, 16: 863-5
(1981), Lida T., et al., Journal of Lipid Research, 30: 1267-1279
(1989), and Tserng K-Y., et al., J Lipid Research, 18: 404-407
(1977).
Compounds of formula I, wherein X is CH.sub.2, wherein m is 2, can
be prepared for example by the route illustrated in FIG. 9,
starting with the illustrative embodiment of .beta.-muricholic acid
9. The carboxylic acid is protected via acid-catalyzed
esterification to provide compound 11. The hydroxyl groups in
compound H can be protected using any suitable protecting group
such as benzyl (Bzl) to give compound 12. Reduction of the carboxyl
group using any suitable reducing agent, for example, lithium
aluminum hydride provides alcohol 13. Conversion of the hydroxyl
group to a suitable leaving group, for example, bromo, using any
suitable reagents such as triphenylphosphine and carbon
tetrabromide gives compound 14. Displacement of the leaving group
in 13 using, for example, a nucleophilic reagent of the formula:
HYCH.sub.2CH.sub.2SO.sub.3H wherein Y is NH, O, S, or Se followed
by deprotection gives taurine conjugated analog 15.
In an embodiment, the invention provides a method of synthesizing
the compound of formula (I):
##STR00016##
wherein R.sup.1, and R.sup.2, and R.sup.4 are hydrogen,
X is CH.sub.2,
Y is selected from CH.sub.2, NR.sup.5, O, S, SO, SO.sub.2, and
Se,
Z is selected from COOR.sup.6, SO.sub.3R.sup.7,
P(.dbd.O)(OR.sup.8).sub.2 and NR.sup.9R.sup.10,
R.sup.3, R.sup.5, R.sup.6, R.sup.7, R.sup.8 R.sup.9, and R.sup.10
are independently selected from hydrogen, alkyl, and aryl,
R.sup.4 is selected from hydrogen, alkyl, and C(.dbd.O)R.sup.3,
m is an integer of 1 to 6, and
n is an integer of 1 to 6,
comprising the steps of:
(i) providing a compound of formula (IV):
##STR00017##
(ii) treating the compound of formula (IV) with an alcohol to
provide a compound of formula (V):
##STR00018##
(iii) protecting the hydroxyl groups in the compound of formula (V)
to provide a compound of formula (VI):
##STR00019##
(iv) treating the compound of formula (VI) with a reducing agent to
provide a compound of formula (VII):
##STR00020##
(v) converting the compound of formula (VII) to a compound of
formula (VIII), wherein LG is a leaving group:
##STR00021##
(vi) treating the compound of formula (VIII) with a compound of the
formula: HY(CH.sub.2).sub.nZ wherein Y is NH, S, O, or Se to
provide a compound of formula (IX):
##STR00022## and
(vii) converting the compound of formula (IX) into the compound of
formula (I).
In an embodiment, the invention provides a method of synthesizing
the compound of formula (II):
##STR00023##
wherein R.sup.1 and R.sup.2 are hydrogen,
X is CH.sub.2,
Y is selected from CH.sub.2, NR.sup.5, O, S, SO, SO.sub.2, and
Se,
Z is selected from COOR.sup.6, SO.sub.3R.sup.7,
P(.dbd.O)(OR.sup.8).sub.2 and NR.sup.9R.sup.10,
R.sup.3, R.sup.5, R.sup.6, R.sup.7, R.sup.8 R.sup.9, and R.sup.10
are independently selected from hydrogen, alkyl, and aryl,
R.sup.4 is selected from hydrogen, alkyl, and C(.dbd.O)R.sup.3,
m is an integer of 1 to 6, and
n is an integer of 1 to 6,
comprising the steps of:
(i) providing a compound of formula (X):
##STR00024##
(ii) treating the compound of formula (X) with an alcohol to
provide a compound of formula (XI):
##STR00025##
(iii) protecting the hydroxyl groups in the compound of formula
(XI) to provide a compound of formula (XII):
##STR00026##
(iv) treating the compound of formula (XII) with a reducing agent
to provide a compound of formula (XIII):
##STR00027##
(v) converting the compound of formula (XIII) to a compound of
formula (XIV), wherein LG is a leaving group:
##STR00028##
(vi) treating the compound of formula (XIV) with a compound of the
formula: HY(CH.sub.2).sub.nZ wherein Y is NH, S, O, or Se to
provide a compound of formula (XV):
##STR00029## and
(vii) converting the compound of formula (XV) into the compound of
formula (II).
The following examples further illustrate the invention but, of
course, should not be construed as in any way limiting its
scope.
Luciferase Assay
The PGL4-Shp-TK firefly luciferase construct and human Fxr
expression plasmid were provided by Grace L. Guo of Rutgers
University. The human Asbt expression plasmid was provided by Paul
A. Dawson of Wake Forest University School of Medicine. The
plasmids were transfected into cells using X-TREMEGENE.TM. HP DNA
Transfection Reagent (Roche). The cells were lysed, and luciferase
activities measured with a DUAL-LUCIFERASE.TM. assay kit (Promega).
Firefly luciferase activity was normalized to Renilla luciferase
activity.
ATP Assay
ATP detection was performed using the following protocol. For
extraction of ATP, 10 mg of ileum mucosa were homogenized with 1.0
mL of ice-cold TE saturated phenol (Sigma-Aldrich). A mixture of
200 .mu.L of chloroform and 150 .mu.L of deionized water were added
and the homogenate thoroughly shaken for 20 s and centrifuged at
10,000 g for 5 min at 4.degree. C. The aliquot from the supernatant
was diluted 100-fold with deionized water, and 10 .mu.L of the
diluted extract was measured by ATP determination kit (Invitrogen
Corp.) (Chida et al., Analytica Chimica Acta 727: 8-12 (2012).
Tempol, bacitracin, neomycin, and streptomycin were purchased from
Sigma-Aldrich (St. Louis, Mo.). Bile acids were ordered from
Steraloids, Inc. (Newport, R.I.) and Sigma (St. Louis, Mo.), and
taurocholic acid-d5 sodium salt was from Toronto Research Chemicals
Inc. (Toronto, Ontario). Ceramides were obtained from Avanti Polar
Lipids. HFD (60 kcal % fat) were purchased from Bio-Sery
(Frenchtown, N.J.). T-.beta.-MCA and Gly-MCA were synthesized as
according to the scheme shown in FIG. 41 and described in Example
1. All solvents and organic reagents were of the highest grade
available.
Animal Studies
High-fat diet (HFD) (60% kcal consisting of fat) was purchased from
Bioserv. Inc. Intestine-specific Fxr-null (Fxr.sup..DELTA.IE) mice
and wild-type (Fxr.sup.fl/fl) mice had a C57BL/6N genetic
background. Fxr.sup.fl/fl and Fxr.sup..DELTA.IE (Kim et al., J.
Lipid Res. 48:2664-2672, 2007) mice were backcrossed with C57BL/6N
mice for over 10 generations. For the antibiotic (the combination
of bacitracin, neomycin, and streptomycin) study, male C57BL/6N
mice from 6 weeks of age were fed a high-fat diet ("HFD) and
administered 0.1% (w/v) of each compound (the combination of
bacitracin, neomycin, and streptomycin) in the drinking water. For
the tempol study, male C57BL/6N mice from 6 weeks of age were fed a
HFD and administered 0.064% (w/v) tempol in the drinking water. For
T.beta.MCA in vivo, male C57BL/6N mice from 6 weeks of age were fed
a HFD and treated with the antibiotics (0.1% of each compound of
bacitracin, neomycin, and streptomycin combination) for 3 days.
Vehicle (saline), TCA (400 mg/kg body weight, dissolved in saline)
or a combination of TCA and T.beta.MCA (400 mg/kg body weight of
each compound, dissolved in saline) were orally administered to the
mice and followed by a second dose 12 h later. The mice were killed
2 h later for tissue collection. For the Gly-MCA study, Gly-MCA was
custom synthesized. Bacon-flavored dough pills were produced as
described (Walker et al., Toxicol. Appl. Pharmacol. 260:65-69,
2012) for oral administration of Gly-MCA (0.25 mg Gly-MCA/pill,
dose of 10 mg/kg). Mice were trained to eat the dough pills prior
to the study. For the prevention of obesity, insulin resistance and
NAFLD, male wild-type (WT) C57BL/6N mice, 6- to 8-weeks-old, were
fed a high-fat diet (Bio-Serv, Frenchtown, N.J.; 60 kcal % fat)
from the age of 6 weeks and were orally administered with vehicle
(control pills) or Gly-MCA (0.25 mg/pill/day, dose 10 mg/kg).
C57BL/6N mice fed a high-fat diet for 12 weeks were administered
(0.25 mg Gly-MCA/pill, dose of 5 mg/kg). Leptin-deficient db/db
mice, 6- to 8-weeks-old, fed a chow diet, were administered Gly-MCA
(0.25 mg/pill/day, 10 mg/kg). Mice were housed individually in
their home cages. Cumulative food intake and TEE.sub.bal were
measured for 1 week in vehicle and Gly-MCA-treated mice from 6 to 7
weeks of HFD. TEE.sub.bal was measured as previously described
(Ravussin et al., Int. J. Obesity 37:399-403, 2013).
All animal studies were performed in accordance with the Institute
of Laboratory Animal Resources guidelines and approved by the NCI
Animal Care and Use Committee.
Preparation and Culture of Primary Hepatocytes
Primary hepatocytes from 6-week-old C57BL/6N mice were obtained by
collagenase 1 (Invitrogen, Carlsbad, Calif.) perfusion. The cells
were purified by 45% Percoll (Sigma, St. Louis, Mo.) density
centrifugation and cultured in DMEM (Invitrogen, Carlsbad, Calif.)
with 10% fetal bovine serum and 1%
Insulin-Transferrin-Selenium-Ethanolamine (ITS-X) (Invitrogen,
Carlsbad, Calif.). The viability of hepatocytes was determined
using trypan blue dye exclusion, and those with higher than 85%
viability were used. The medium was changed to DMEM with 1% fetal
bovine serum after culturing for 4 hours. After starvation for 4
hours, the cells were exposed to ceramide. At the prescribed time
points, cells were harvested and subjected to qPCR analysis and TG
content detection.
RNA Analysis
The mucosa of intestine was gently scraped and liver was taken and
both were flash frozen in liquid nitrogen and stored at -80.degree.
C. until RNA was prepared. RNA was extracted from frozen intestine
and liver using TRIzol reagent (Invitrogen, Carlsbad, Calif.). cDNA
was synthesized from 1 .mu.g total RNA using Superscript II reverse
transcriptase (Invitrogen, Carlsbad, Calif.). qPCR primers were
designed with qPrimerDepot. Measured mRNA levels were normalized to
those of 18S ribosomal RNA and expressed as fold change relative to
those of control group.
Western Blot Analysis
Liver whole-cell or nuclear extracts were prepared. Membranes were
incubated with antibodies against FXR (Santa Cruz Biotechnology,
Inc., Santa Cruz, Calif.), SREBP1 (BD Biosciences, San Jose,
Calif.), and CIDEA (Abcam, Cambridge, Mass.). The signals obtained
were normalized to .beta.-ACTIN (Abcam) for whole cell extract and
LAMIN A/C (Santa Cruz) for nuclear extracts.
16S rRNA Gene Sequencing of the Intestinal Microbiome
The bacteria in feces and cecum content were extracted using
PowerSoil DNA Isolation Kit (Mo Bio laboratory, Inc., Carlsbad,
Calif.). The PCR products (approximately 1000 bps) were purified
using the Agencourt AMPure technology (Beckman Coulter, Brea,
Calif.) as described in 454 Technical Bulletin #2011-002, Short
Fragment Removal Procedure. After purification, the products were
quantified by both Qubit (Lifetech, Carlsbad, Calif.) and qPCR,
using the KAPA Biosystems Library Quantification Kit
(KapaBiosystems, Woburn, Mass.), pooled based on molar amounts, run
on a 1% agarose gel and extracted. After purification with a
QIAquick PCR Purification kit (Qiagen, Valencia, Calif.), the
quality and quantity were assessed using a DNA 7500LabChip on the
Agilent 2100 Bioanalyzer (Agilent Technologies, Santa Clara,
Calif.) and Qubit quantification. The sequencing was performed
using a quarter PTP plate on a 454 Life Sciences Genome Sequencer
FLX+ (Roche Diagnostics, Indianapolis, Ind.). 16S rRNA gene
sequencing analysis was performed as previously described (Lozupone
and Knight, Appl. Environ. Microbiol. 71:8228-8235, 2005). Weighted
UniFrac analysis to assess changes in the bacterial abundance was
performed on the Galaxy web-based platform (Blankenberg et al.,
Bioinformatics 26:1783-1785, 2010; Goecks et al., Genome Biol. 11:
126, 2010; Giardine et al., Genome Res. 15:1451-1455, 2005).
Metagenomic Data Analysis
After quality filtering and deduplication, each sample contained on
average 11 thousand reads. The Mothur software package was used to
preprocess the sequencing data and the RDP multi-classifier to
assign each sequence to a taxonomic rank. Preprocessing consisted
of filtering reads for an average quality of 20, removing
duplicated sequences and splitting into samples by barcodes while
allowing for one mismatch in the barcode. To account for
differences in total reads per sample, classifications were
converted to percent of total reads. This approach then permitted
accurate comparisons within and between groups.
Metabolomics Analysis
Lipidomics analysis: For serum lipidomics analysis 25 .mu.l serum
were extracted by 4-fold cold chloroform:methanol (2:1) solution
containing 2 .mu.M LPC (17:0), PC (17:0), SM (17:0) and CER (17:0)
(Avanti Polar Lipids, Alabaster, Ala.) as internal standards. The
samples were vortexed for 30 s and then allowed to stand for 5 min
at room temperature. The mixture was centrifuged at 13,000 rpm for
5 min and then the lower organic phase was collected and evaporated
at room temperature under vacuum. The residue was dissolved in
chloroform:methanol (1:1), followed by diluting with
isopropanol:acetonitrile:H.sub.2O (2:1:1) containing 2 .mu.M PC
(17:0) prior to UPLC-MS analysis. For tissue lipidomics analysis,
about 50 mg of accurately weighted tissues were homogenized with
700 .mu.L methanol: H.sub.2O (4:3) solution and then extracted
using 800 .mu.L chloroform containing 2 .mu.M LPC (17:0), SM (17:0)
and CER (17:0) as internal standards. The homogenate was incubated
at 37.degree. C. for 20 min followed by centrifuged for 20 min at
13,000 rpm. The lower organic phase was transferred to a new tube
and dried under vacuum. The residue was suspended with 100
chloroform:methanol (1:1) solution and then diluted with
isopropanol:acetonitrile:H.sub.2O (2:1:1) solution containing 2
.mu.M PC (17:0) before injection. For lipidomics discovery, samples
were analyzed by UPLC-ESI-QTOF MS using a Water Acquity CSH 1.7 um
C18 column (2.1.times.100 mm) under the following conditions: UPLC:
A-acetonitrile/water (60/40), B-isopropanol/acetonitrile (90/10).
Both A and B contained 10 mM Ammonium acetate and 0.1% formic acid.
Gradient: initial 60% A to 57% A at 2 min, to 50% A at 2.1 min*, to
46% A at 12 min, to 30% A at 12.1 min*, to 1% A at 18 min before
returning to initial conditions at 18.5 min with equilibration for
2 additional minutes (an *indicates ballistic gradient). Flow rate
was 0.4 ml/min. Column temperature was maintained at 55.degree. C.
MS, same conditions as above, except run time was 18 min.
Global metabolomics analysis: urine samples were prepared by adding
20 .mu.L of urine to 180 .mu.L 50% aqueous acetonitrile (50:50
water/acetonitrile). Samples were vortexed for 5 min and
centrifuged at 18000.times.g for 20 min at 4.degree. C. to remove
particulates and precipitated protein. The supernatant was
transferred to an autosampler vial for analysis. 50 mg tissue
samples were homogenized in 500 mL 50% aqueous acetonitrile
containing 5 .mu.M of chlorpropamide (internal standard). The
samples were vortexed and centrifuged at 13,000 rpm for 20 min at
4.degree. C. to remove particulates and precipitate protein. The
supernatant was transferred to an autosampler vial for analysis.
For metabolomics discovery, a 5 .mu.l aliquot of supernatant
samples was injected into the UPLC-ESI-QTOFMS system (Waters,
Milford, Mass.) with a Waters Acquity BEH 1.7 um C18 (2.1.times.50
mm) column. The gradient mobile phase comprises 0.1% formic acid in
water (A) and 0.1% formic acid in acetonitrile (B). The gradient
was maintained at initial 95% A for 0.5 min, to 40% A at 4 min, and
then to 1% A at 8 min. The column was flushed for 1 min, then
equilibrated at initial conditions for 1.5 min. Flow rate was 0.5
ml/min. Column temperature was maintained at 60.degree. C. Waters
Synapt HDMS Q-TOF was operated in both positive and negative modes,
scanning 50-850 amu, at a rate of 0.3 scans/sec. The following
instrument conditions were used: capillary 3 kV, source temperature
120.degree. C., sampling cove 30V, desolvation gas flow 850 L/h at
400.degree. C. Biomarker identification and quantitation:
Biomarkers were screened by analyzing ions in the loading scatter
plot, and metabolomics databases (METLIN and Madison Metabolomics
Consortium Database) were searched to find potential candidates. To
confirm the identities of the putative markers, the authentic
standards were compared with the metabolites based on MS/MS
fragmentation pattern and retention time. Concentrations of the
metabolites were determined by multiple reaction-monitoring mass
spectrometry based on standard curves using authentic
standards.
Data Processing and Multivariate Data Analysis
Chromatographic and spectral data were deconvoluted by MarkerLynx
software (Waters). A multivariate data matrix containing
information on sample identity, ion identity (retention time and
m/z), and ion abundance was generated through centroiding,
deisotoping, filtering, peak recognition, and integration. The
intensity of each ion was calculated by normalizing the single ion
counts vs. the total ion counts in the whole chromatogram. The data
matrix was further exported into SIMCA-P software (Umetrics,
Kinnelon, N.J.) and transformed by mean-centering and pareto
scaling, a technique that increases the importance of low abundance
ions without significant amplification of noise. Statistical models
including principal components analysis (PCA), partial least
squares-discriminant analysis (PLS-DA), and orthogonal projections
to latent structures-discriminant analysis (OPLS-DA) were
established to represent the major latent variables in the data
matrix.
NMR-Based Metabolomics Experiments
Methanol, K.sub.2HPO.sub.4, NaH.sub.2PO.sub.4 (all in analytical
grade), sodium 3-trimethylsilyl [2,2,3,3-d4] propionate (TSP-d4)
and D.sub.2O (99.9% in D) were purchased from Sigma-Aldrich (St.
Louis, Mo.). Phosphate buffer (0.1 M
K.sub.2HPO.sub.4/NaH.sub.2PO.sub.4 and PH 7.4) was prepared with
K.sub.2HPO and NaH.sub.2PO.sub.4 for their good solubility and
low-temperature stability. Liver samples (.about.50 mg) were
extracted three times with 0.6 mL 600 .mu.L of precooled
methanol-water mixture (2/1, v/v) using the PreCellys Tissue
Homogenizer (Bertin Technologies, Rockville, Md.). After
centrifugation at 11180.times.g for 10 min at 4.degree. C., the
combined supernatants were dried. Each of the aqueous extracts was
separately reconstituted into 600 .mu.L phosphate buffer containing
50% D.sub.2O and 0.005% TSP-d4 (chemical shift reference).
Following centrifugation, 550 .mu.L of each extract was transferred
into a 5 mm NMR tube. Cecal content samples were directly extracted
using an optimized procedure described previously (Wu et al.,
2010). Briefly, samples (.about.50 mg) were mixed with 600 .mu.L
precooled phosphate buffer, vortexed for 30 s and subjected to
three consecutive freeze-thaws followed by homogenization using the
Precellys.TM. Tissue Homogenizer. After centrifugation
(11,180.times.g, 4.degree. C.) for 10 min, the supernatants (550
.mu.L) were transferred into 5 mm NMR tubes for NMR analysis.
.sup.1H NMR Spectroscopy
.sup.1H NMR spectra of aqueous liver and fecal extracts were
recorded at 298 K on a Bruker Avance III 850 MHz spectrometer
(operating at 850.23 MHz for 1H) equipped with a Bruker inverse
cryogenic probe (Bruker Biospin, Germany). A typical
one-dimensional NMR spectrum was acquired for each of all samples
employing the first increment of NOESY pulse sequence (NOESYPR1D).
To suppress the water signal, a weak continuous wave irradiation
was applied to the water peak during recycle delay (2 s) and mixing
time (100 ms). The 90.degree. pulse length was adjusted to
approximately 10 .mu.s for each sample and 64 transients were
collected into 32 k data points for each spectrum with spectral
width of 20 ppm. To facilitate NMR signal assignments, a range of
2D NMR spectra were acquired and processed as described previously
(Dai et al., 2010; Ding et al., 2009) for selected samples
including .sup.1H-.sup.1H correlation spectroscopy (COSY),
.sup.1H-.sup.1H total correlation spectroscopy (TOCSY),
.sup.1H-.sup.13C heteronuclear single quantum correlation (HSQC),
and .sup.1H-.sup.13C heteronuclear multiple bond correlation
spectra (HMBC).
Spectral Data Processing and Multivariate Data Analysis
All free induction decays (FID) were multiplied by an exponential
function with a 1 Hz line broadening factor prior to Fourier
transformation. .sup.1H NMR spectra were corrected manually for
phase and baseline distortions and spectral region .delta. 0.5-9.5
was integrated into regions with equal width of 0.004 ppm (2.4 Hz)
using AMIX software package (V3.8, Bruker-Biospin, Germany). Region
.delta. 4.45-5.20 was discarded by imperfect water saturation.
Regions .delta. 1.15-1.23 and .delta. 3.62-3.69 were also removed
for ethanol contaminations in the cecal contents during mice
dissection process. Each bucketed region was then normalized to the
total sum of the spectral integrals to compensate for the overall
concentration differences prior to statistical data analysis.
Multivariate data analysis was carried out with SIMCAP+ software
(version 13.0, Umetrics, Sweden). Principal Component Analysis
(PCA) was initially carried out on the NMR data to generate an
overview and to assess data quality. Orthogonal projection to
latent structures with discriminant analysis (OPLS-DA) was
subsequently conducted on the NMR data. The OPLS-DA models were
validated using a 7-fold cross validation method and the quality of
the model was described by the parameters R2X and Q2 values. To
facilitate interpretation of the results, back-transformation
(Cloarec et al., Anal. Chem. 77:517-526, 2005) of the loadings
generated from the OPLS-DA was performed prior to generating the
loadings plots, which were color-coded with the Pearson linear
correlation coefficients of variables (or metabolites) using an
in-house developed script for MATLAB (The Mathworks Inc.; Natwick,
Mass.). In this study, a cutoff value of |r|>0.811 (r>0.755
and r<-0.755) was chosen for correlation coefficient as
significant based on the discrimination significance
(p<0.05).
Bile Salt Hydrolase Activity
Fecal proteins were prepared from feces samples (0.5 g) in pH 7.4
phosphate buffered saline (PBS, 5.0 mL) using sonication. Bile salt
hydrolase (BSH) activity was measured based on the generation of
CDCA from TCDCA in the feces. Briefly, incubation was carried out
in 3 mM sodium acetate buffer, pH 5.2, containing 0.1 mg/ml fecal
protein and 50 .mu.M TCDCA-d5 in a final volume of 200 .mu.L. After
a 20 min incubation at 37.degree. C., the reaction was stopped by
plunging the samples into dry ice. 100 .mu.L of acetonitrile was
directly added to the reaction mix. After centrifuging at
14,000.times.g for 20 min, 5 .mu.L of the supernatant was
transferred to an auto sampler vial subjected to analysis by a UPLC
system coupled with a XEVO triple quadrupole tandem mass
spectrometer (Waters Corp., Milford, Mass.).
Mitochondrial Isolation and Functional Studies
For intestinal mitochondria, the mucosa of ileum was gently
scraped, washed 2.times. with PBS, minced in ice-cold mitochondrial
homogenization buffer (225 mM mannitol, 75 mM sucrose, 5 mM MOPS,
0.5 mM EGTA and 2 mM taurine (pH 7.25)) containing 0.2% BSA, and
homogenized in a loose fitting homogenizer. Homogenates were
centrifuged at 500.times.g for 10 min at 4.degree. C. The
supernatant was then centrifuged at 10,000.times.g for 10 min at
4.degree. C. The final mitochondrial pellet was resuspended in
mitochondrial isolation buffer containing 0.2% BSA at a
concentration of 0.5 mg/ml before functional assessment.
The oxygen consumption of isolated mitochondria was measured in a
chamber connected to a Clark-type O.sub.2 electrode (Instech) and
O.sub.2 monitor (Model 5300, YSI Inc) at 25.degree. C. Mitochondria
were incubated in respiration buffer (120 mM KCl, 5 mM MOPS, 0.1 mM
EGTA, 5 mM KH.sub.2PO.sub.4 and 0.2% BSA) with substrates for
either complex I (5 mM glutamate and 5 mM malate), or complex II (5
mM succinate and 1 .mu.M rotenone). State 3 (maximal) respiration
activity was measured after addition of 1 mM ADP. ADP independent
respiration activity (State 4) was monitored after addition of 2
.mu.M oligomycin. The respiratory control ratio was determined by
the state 3/state 4 respiration rates.
Histological Analysis
Hematoxylin and eosin (H&E) staining were performed on formalin
fixed paraffin embedded sections using a standard protocol. Oil red
0 staining was performed on frozen liver sections using a standard
protocol. At least three discontinuous liver sections were
evaluated for each mouse.
Triglycerides Content Quantification
Hepatic lipids were extracted using a 2:1 chloroform-methanol
solution. Liver triglycerides were measured with a triglyceride
colorimetric assay kit, according to the manufacturer's
recommendation (Bioassay Systems, Hayward, Calif.).
Cell Culture
Caco-2 (ATCC.TM. HTB-37.TM.) cells were induced to differentiate
following the method as described previously (Ferraretto et al.,
Anticancer Res. 27:3919-3925, 2007). The differentiated Caco-2
cells were incubated for 8 hours with DMEM media with 1% fetal
bovine serum, and then exposed to Gly-MCA/CDCA/GW4064 for 24 hours.
RNA was extracted from frozen intestine using TRIzol reagent
(Invitrogen). cDNA was synthesized from 1 .mu.g total RNA using
Superscript II reverse transcriptase (Invitrogen).
Gly-MCA Hydroxylation by Gut Bacterial
Fecal proteins were prepared from the fecal sample (0.5 g) in pH
7.4 PBS (5.0 ml) using sonication. Incubation was carried out in 3
mM sodium acetate buffer, pH 5.2, containing 0.1 mg/ml fecal
protein and 50 .mu.M Gly-MCA or T-.beta.-MCA in a final volume of
200 ml. After a 20-min incubation at 37.degree. C., the samples
were plunged into dry ice to stop the reaction. 100 of .mu.L
methanol was directly added to the 100 ml reaction mixture. After
centrifuging at 14,000 g for 20 min, 5 ml of the supernatant was
transferred to an autosampler vial subjected to analysis by a UPLC
system coupled with a XEVO triple quadrupole tandem mass
spectrometer (Waters Corp., Milford, Mass.).
Animal Studies
High fat diet (HFD) (60% kcal consisting of fat) was purchased from
Bioserv. Inc. Gly-MCA was custom synthesized.
Bacon-flavored dough pills were produced as described (Walker et
al., Toxicol. Appl. Pharmacol. 260:65-69, 2012) for oral
administration of Gly-MCA (0.25 mg Gly-MCA/pill). Mice were trained
to eat the dough pills prior to the study.
Male wild-type (WT) C57BL/6N mice, 6- to 8-weeks-old, were fed a
HFD (Bio-Serv, Frenchtown, N.J.; 60 kcal % fat) from the age of 6
weeks and were orally administered with vehicle (control pills) or
Gly-MCA (0.25 mg/pill/day, 10 mg/kg). Mice were housed individually
in their home cages. Cumulative food intake and TEE.sub.bal were
measured for 1 week in vehicle and Gly-MCA-treated mice from 6 to 7
weeks of HFD. TEE.sub.bal was measured as previously described
(Ravussin et al., Int. J. Obesity 37:399-403, 2013). All animal
studies were performed in accordance with the Institute of
Laboratory Animal Resources guidelines and approved by the NCI
Animal Care and Use Committee.
Metabolic Assays
For the glucose tolerance test (GTT), mice were fasted for 16 h,
blood was drawn, and mice were injected intraperitoneally (i.p.)
with 1 g/kg glucose. For the insulin tolerance test (ITT), mice
were fasted 4 h, blood was drawn, and then were injected with
insulin (Eli Lilly, Washington, D.C.), by i.p. at a dose of 1 U/kg
body weight. Blood samples were taken from the tail at 15, 30, 60,
and 90 min after injection, and glucose measured using a Glucometer
(Bayer, Pittsburgh, Pa.).
Example 1
This example demonstrates that tauro-.beta.-muricholic (T.beta.MCA)
acid antagonized FXR activation by taurocholic acid (TCA) in
primary mouse hepatocytes.
Primary hepatocytes from Fxr.sup.+/+ and Fxr.sup.-/- mice were
transfected with PGL4-Shp-TK firefly luciferase construct and the
control plasmid phRL-SV40. After 24 h, the cells were treated with
100 .mu.M taurocholic acid (TCA), T.beta.MCA, or T.beta.MCA with
TCA. The cells were lysed, and luciferase activities measured as
describe herein. The results are depicted in FIG. 1.
As is apparent from the results depicted in FIG. 1, T.beta.MCA
antagonized FXR activation by TCA in primary hepatocytes from
Fxr.sup.+/+ mice, but not from Fxr.sup.-/- mice.
Example 2
This example demonstrates that T.beta.MCA antagonized FXR
activation by TCA in Caco-2 cells.
Caco-2 cells were transfected with PGL4-Shp-TK firefly luciferase
construct, the control plasmid phRL-SV40, and human FXR and human
ASBT expression plasmids. After 24 h, the cells were treated with
100 .mu.M TCA, T.beta.MCA, or T.beta.MCA with 100 .mu.L 100 .mu.M
TCA. The cells were lysed, and luciferase activities measured as
describe herein. The results are depicted in FIG. 2.
As is apparent from the results depicted in FIG. 2, T.beta.MCA
antagonized FXR activation by TCA in Caco-2 cells.
Example 3
This example demonstrates that ATP levels in mouse ileum mucosa
were markedly elevated in Fxr.sup..DELTA.IE mice as compared to
Fxr.sup.fl/fl mice after 14 weeks on a high fat diet.
Two separate groups of Fxr.sup.fl/fl mice and Fxr.sup..DELTA.IE
mice were kept on a high fat diet for 14 weeks. ATP levels in the
ileum mucosa of both groups of mice were determined as described
herein. The results are depicted in FIG. 3.
As is apparent from the results depicted in FIG. 3, ATP levels in
the ileum mucosa of Fxr.sup..DELTA.IE mice, which do not express
farnesoid X receptor (FXR) in the intestine, were markedly elevated
as compared with ATP levels in the ileum mucosa of control
Fxr.sup.fl/fl mice that express intestinal FXR. These results
indicate increased energy expenditure occurred in the small
intestine in the absence of the nuclear receptor FXR.
Example 4
This example demonstrates that glycine-.beta.-muricholic acid
(Gly-MCA) is an FXR antagonist.
Mice make T.beta.MCA in the liver while humans preferentially make
Gly-MCA. Thus, it was of interest to determine whether Gly-MCA was
also an FXR antagonist. Chenodeoxycholic acid (CDCA), an FXR
agonist at a dose of 100 .mu.M, increased expression of the Fxr
target gene Shp mRNA four-fold and the induction of Shp mRNA with
CDCA was inhibited by Gly-MCA in a dose dependent manner (FIG. 4).
Gw4064, a synthetic FXR agonist, induced expression of the FXR
target genes Shp and Fgf19 at both 2 .mu.M and 5 .mu.M
concentrations, and induction of both genes was blocked by Gly-MCA
.quadrature. in a dose dependent manner (FIGS. 5 and 6). In
addition, Gw4064 treatment inhibited Atp5g mRNA expression and
Gly-MCA reversed this inhibition (FIG. 7). These data indicate that
Gly-MCA, produced in humans, is an FXR antagonist similar to
T.beta.MCA.
Example 5
This example demonstrates the effect of tempol on body mass of
high-fat diet-treated Fxr.sup.fl/fl and Fxr.sup..DELTA.IE mice.
Vehicle and tempol-treated Fxr.sup.fl/fl and Fxr.sup..DELTA.IE mice
were maintained on a high-fat diet for 10 weeks. FIG. 11 depicts
the body mass gain in grams for vehicle and tempol-treated
Fxr.sup.fl/fl and Fxr.sup..DELTA.IE mice after 10 weeks of a
high-fat diet feeding.
As is apparent from the results depicted in FIG. 11, tempol
treatment of Fxr.sup.fl/fl mice resulted in a weight gain that was
approximately 65% less of the weight gain exhibited by vehicle
treated mice. Tempol treatment of Fxr.sup..DELTA.IE mice, which are
intestinal-specific Fxr-null mice, resulted in an insignificant
difference in weight gain, thereby implicating intestinal FXR in
mediating the lower weight gain by tempol of mice fed a high-fat
diet.
Example 6
This example demonstrates the role of intestinal FXR in lipid and
glucose metabolism.
Male Fxr.sup.fl/fl and Fxr.sup..DELTA.IE mice were fed a high fat
diet revealing that Fxr.sup..DELTA.IE mice were resistant to high
fat diet-induced obesity. The fat mass in grams and as a percentage
of body mass was measured in non-anesthetized mice using an Echo
3-in-1 NMR analyzer (Echo Medical Systems, Houston, Tex.), and the
results depicted in FIGS. 12A and B. The results show that fat mass
and the ratio of fat and body mass of Fxr.sup.fl/fl mice were
higher than for Fxr.sup..DELTA.IE mice. The glucose tolerance test
(GTT) revealed that Fxr.sup..DELTA.IE mice had improved glucose
intolerance compared to Fxr.sup.fl/fl mice, which is depicted in
FIG. 13, which shows the area under the curve for blood glucose (in
mg/dL) as a function of time. The insulin tolerance test (ITT),
which is depicted in FIG. 14, demonstrated that the insulin
sensitivity in Fxr.sup..DELTA.IE mice was significantly increased
as compared to Fxr.sup.fl/fl mice. In addition, fasted serum
insulin levels and the HOMA in Fxr.sup..DELTA.IE mice was
significantly increased as compared to Fxr.sup.fl/fl mice, while
fasted glucose was approximately the same in both groups of mice,
as depicted in FIGS. 15A-C.
Example 7
This example demonstrates that tempol affects bile acid homeostasis
via inhibition of the genus Lactobacillus.
Significant phylum-level shifts from Firmicutes to Bacteroidetes in
the gut microbiome composition were observed in mouse cecum
following 5 days of tempol treatment by gavage (250 mg/kg) of mice
on normal chow diet. Heat map diagrams of 16S rRNA sequencing
indicated that tempol treatment dramatically decreased the family
Lactobacillacieae. It was found that tempol treatment robustly
reduced the genus Lactobacillus. Similar to the results of acute
treatment via gavage, qPCR analysis of suspected fecal microbes
obtained from mice on a high fat diet revealed total bacteria
remain unchanged between vehicle and tempol treated mice, while
tempol treatment cause a shift from Firmicutes to Bacteroidetes, as
depicted in FIGS. 16A and B. These results indicate that the
effects of tempol on the gut microbiome are independent of diet and
obesity conditions. Furthermore, the genus Lactobacillus of the
Lactobacillaceae was decreased, coincident with significant
downregulation of bile salt hydrolase (BSH) enzymatic activity in
the feces, as depicted in FIGS. 17A and B. Bile salt hydrolase
(BSH) deconjugates taurine-conjugated bile acids produced in the
liver to free bile acids.
These results indicate that tempol affects bile acid homeostasis
via inhibition of the genus Lactobacillus.
Example 8
This example demonstrates the results of a human FXR competition
assay using the synthetic agonist Gw4064 and varied doses of TUDCA,
TWMCA, T.beta.MCA, T.alpha.MCA. Results were normalized to Renilla
expression.
HEK293T cells were co-transfected with: 1) a chimeric receptor
construct in which the carboxy terminal portions of human FXR
(containing the native ligand-binding domain and AF2
transactivation domain) was fused to an amino terminal GAL4
DNA-binding domain under regulatory control of the constitutively
active SV40 promoter; 2) a firefly luciferase reporter plasmid
driven by the UAS GAL4 DNA response element; and, 3) a Renilla
luciferase reporter gene (pRL-luciferase; Promega; Madison, Wis.)
as a transfection efficiency control. Luciferase detection was
conducted using the Dual Luciferase Reporter Assay kit (Promega
Corp., Madison, Wis.) and a Tecan GeniosPro luminescent plate
reader (Research Triangle Park, N.C.). The results are illustrated
in FIGS. 18A-D.
As is apparent from the results illustrated in FIG. 18, all of the
bile acid conjugates TUDCA, TWMCA, T.beta.MCA, and T.alpha.MCA
inhibited FXR in the presence of the synthetic agonist Gw4064.
Example 9
This example demonstrates that changes in the gut microbiota
brought about by tempol are correlated with NAFLD.
High-fat diet (HFD) is extensively used as a mouse model for NAFLD.
The antioxidant tempol selectively modulates the gut microbiota
composition and metabolism under normal diet conditions (Li et al.,
Nat. Commun. 4: 2384, 2013). In an effort to determine whether
tempol modifies the gut microbiome in the HFD-induced NAFLD model,
16S rRNA gene sequencing analysis was carried out. Weighted
UniFrac.TM. analysis showed distinct clustering of cecal
communities isolated from vehicle and tempol-treated groups on a
HFD for 12 weeks. Principal coordinate 1 (PC1) explains 56.08% of
the variation, indicating that tempol had a stronger effect on
microbiota composition than vehicle in mice on a HFD for 12 weeks
(FIG. 19A). The separation of samples in the principal components
analysis plot reflects abundance differences in significantly
decreased Firmicutes and markedly increased Proteobacteria. The
genus Desulfovibrio was identified as a major contributor of the
increased Proteobacteria (FIG. 19B), which was found to be
significantly lower in obese subjects (Karlsson et al., Obesity
20:2257-2261, 2012). A dramatic increase in the genus Roseburia was
observed (FIG. 19C), which is negatively correlated with body
weight in dogs (Handi et al., FEMS Microbiol. Ecol. 84332-343,
2013). The genus Clostridium sensu stricto and Lactobacillus levels
were also significantly decreased in tempol-treated mice, whereas
the levels of genus Bacteroides and Streptococcus remained similar
(FIG. 19D-G).
To identify gut microbiota related markers in urine,
ultra-performance liquid chromatography coupled with electrospray
ionization quadrupole time-of-flight mass spectrometry
(UPLC-ESI-QTOFMS)-based metabolomics analysis was employed. PCA
modeling of UPLC-ESI-QTOFMS negative mode data from mouse urine
demonstrated clear discrimination between the tempol and the
control group (FIG. 20A). Loadings scatter plot analysis revealed
that two compounds, p-cresol sulfate (m/s 187.0060 with retention
time 2.61 min) and p-cresol glucuronide (m/s 283.0812 with
retention time 3.04 min) were significantly reduced in urine of the
tempol-treated group (FIGS. 20B and C). The identities of these
compounds were confirmed by MS/MS analysis (FIGS. 20D and E). These
results indicated that tempol remodeled the gut microbiota
composition and altered gut microbiota-related metabolism markers
in mice on HFD for 14 weeks. Similar to the results of the tempol
treatment model to specifically modulate the gut flora,
metabolomics analysis revealed that the urinary levels of p-cresol
sulfate and p-cresol glucuronide were almost absent in
antibiotic-treated mice on a HFD for 14 weeks (FIGS. 21A-C).
Following the change of the gut microbiota composition and related
metabolites, liver histology indicated a significant reduction in
hepatic lipid droplets in tempol-treated mice on a HFD for 16 weeks
and antibiotic-treated mice on a HFD for 7 weeks (FIGS. 22A and B,
and FIG. 23A). Tempol treatment and antibiotic treatment, which
also changes the gut microbiota composition, decreased liver
weights and liver/body mass ratios, respectively (FIGS. 22C and D,
FIGS. 23A and B). Hepatic triglyceride (TG) contents were decreased
to approximately 50% and 35% in mice treated with antibiotic and
tempol, respectively (FIG. 22E and FIG. 23D).
Example 10
This example demonstrates that gut microbiota modifies bile acid
metabolism and affects FXR signaling.
The gut microbiota is tightly associated with bile acid metabolism.
UPLC-ESI-QTOFMS-based metabolomics analysis was adopted to
determine bile acid composition and levels of bile acid metabolites
in the intestine. Scores scatter plot of a PCA model of the
UPLC-ESI-QTOFMS negative mode data from mouse ileum indicated
distinct metabolic profiles between the vehicle and antibiotic
groups (FIG. 24A). The top enriched metabolite, T.beta.MCA (m/z
514.2871, retention time=6.64 min), was increased in the
antibiotic-treated mice on a HFD for 7 weeks as revealed in the
loading scatter plot (FIG. 24B) according to previous methods; this
increase was similar to what was observed with tempol treatment (Li
et al., J. Proteome Res., 12:1369-1376, 2013). Analysis of ileum
bile acid composition revealed that the levels of
taurine-conjugated bile acid T.beta.MCA were significantly
increased after antibiotic treatment (FIG. 25A). Similar results
were obtained from tempol-treated mice on a HFD for 16 weeks (FIG.
25B). The gut microbiota can modify bile acid composition by
microbial enzymatic activities. The activity of bile salt hydrolase
(BSH), a bacterial enzyme that hydrolyzes taurine-conjugated bile
acids to free bile acids, was greatly reduced in the
antibiotic-treated mice on a HFD for 7 weeks (FIG. 26A). This
likely accounts for the most significantly enriched bile acid in
the ileum of antibiotic- and tempol-treated mice on a HFD that was
T.beta.MCA, an FXR antagonist (Li et al., J. Proteome Res.,
12:1369-1376, 2013; Sayin et al., Cell Metab. 225-235, 2013).
Western blot and qPCR analysis indicated that 12 weeks of HFD
treatment significantly induced FXR protein levels (FIG. 26B) and
FXR signaling in the ileum as revealed by increases in mRNAs from
the FXR target genes, small heterodimer partner (Shp) and
fibroblast growth factor 15 (Fgf15) mRNAs (FIG. 26 C). Conversely,
antibiotic treatment decreased Shp and Fgf15 mRNAs indicating that
FXR signaling was inhibited in the ileum (FIG. 26D). The question
arose as to whether T.beta.MCA inhibited FXR signaling in mice on
HFD treatment in vivo. T.beta.MCA treatment significantly blunted
the Shp and Fgf15 induction by the FXR agonist TCA in the ileum of
mice treated with antibiotic on a HFD for three days (FIG. 26E).
These results indicated that both antibiotic and tempol treatments
regulated bile acid composition, mainly by increasing T.beta.MCA as
a result of lower bacterial BSH activity, which inhibited FXR
signaling in the ileum of HFD-fed mice.
Example 11
This example demonstrates that intestine-specific Fxr disruption
reduces hepatic lipid accumulation in high-fat diet fed mice.
To further clarify the role of intestinal FXR in the development of
NAFLD, intestine-specific Fxr-null (Fxr.sup..DELTA.IE) mice were
treated with HFD for 14 weeks. H&E staining and Oil red 0
staining of liver sections showed a significant decrease in lipid
accumulation in livers of Fxr.sup..DELTA.IE mice compared to
wild-type (Fxr.sup.fl/fl) mice (FIGS. 27A and B). Fxr.sup..DELTA.IE
mice displayed significantly reduced liver weight and ratio of
liver weight (FIG. 27C). This change in liver weight was largely
due to hepatic triglyceride (TG) levels that were 50% lower in
Fxr.sup..DELTA.IE mice compared to Fxr.sup.fl/fl mice on a HFD for
14 weeks (FIG. 27D). Mechanistic studies revealed that the
expression of mitochondrial electron transport chain (ETC) complex
II related genes such as succinate dehydrogenase complex, subunit
D, integral membrane protein (Sdhd), complex III related gene such
as cytochrome c1 (Cyc1), complex IV related gene such as
mitochondrially-encoded cytochrome c oxidase II (mt-Co2),
cytochrome c oxidase subunit IV isoform 1 (Cox4i1), cytochrome c
oxidase subunit Va (Cox5a), ATP synthase, H+ transporting,
mitochondrial F0 complex, subunit C1 (subunit 9) (Atp5g) and ATP
synthase, H+ transporting, mitochondrial F0 complex, subunit D
(Atp5h), were elevated in the ileum epithelium of Fxr.sup..DELTA.IE
mice (FIG. 28A). Similar results were obtained from
antibiotic-treated mice (FIG. 28B). Subsequently, there was an
approximately 70% increased activity of complex II and no
significant elevation in activity of complex I in the ileum
mitochondria of Fxr.sup..DELTA.IE mice compared to Fxr.sup.fl/fl
mice (FIG. 28C). Ileum ATP levels in Fxr.sup..DELTA.IE mice were
also significantly higher than in Fxr.sup.fl/fl mice (FIG. 28D).
Free fatty acids are closely associated with the development of
hepatic steatosis (Donnelly et al., J. Clin. Invest. 115:1343-1351,
20052005). However, serum lipidomics revealed that a subset of
species of free fatty acids were at similar levels in vehicle- and
tempol-treated Fxr.sup..DELTA.IE mice and Fxr.sup.fl/fl mice (FIG.
29A). LC-MS/MS quantitation confirmed that ileum C16:0, C18:0,
C20:0, C22:0, C24:0 and C24:1 ceramide levels were significantly
reduced in antibiotic-treated mice on a HFD for 7 weeks (FIG. 29B).
Accordingly, serum C16:0, C18:0, C20:0, C24:0 and C24:1 ceramide
levels in antibiotic-treated mice were also significantly lower
than in vehicle-treated mice (FIG. 29C). The identity of each
ceramide was confirmed by LC-MS fragmentography (FIG. 30A-G).
Further, intestinal mRNAs encoding de novo ceramide
synthesis-related genes, such as serine palmitoyltransferase, long
chain base subunit 3 (Sptic3), ceramide synthase 4 (Cers4),
degenerative spermatocyte homolog 1 (Degs1), and sphingomyelin
phosphodiesterase 3 (Smpd3) waned significantly in
Fxr.sup..DELTA.IE mice and antibiotic-treated mice (FIGS. 29C and
D). Ceramide synthase 2 (Cers2) mRNA levels were significantly
decreased in antibiotic-treated mice, and have a reduced trend
(P=0.06) in Fxr.sup..DELTA.IE mice. The expression of genes
involved in ceramide catabolism such as sphingomyelin synthase 1
and 2 (Sgms1 and Sgms2), and alkaline ceramidase 1 and 3 (Acer1 and
Acer3) remained similar in Fxr.sup..DELTA.IE mice and
antibiotic-treated mice (FIGS. 29C and D)
Example 12
This example demonstrates that ceramide regulates the SREBP1c-CIDEA
pathway in the liver.
To establish a causal relationship between the decrease in ceramide
levels and improvement of NAFLD, mice on a HFD were treated with
antibiotics for a short duration. Three days of antibiotic
treatment did not decrease triglyceride content in the liver (FIG.
31A). Subsequently, the FXR signaling pathway was inhibited as
revealed by decreased expression of the FXR target gene Shp and
Fgf15 mRNAs (FIG. 31B). As early as 3 days after antibiotic
treatment, ceramide levels in the ileum of antibiotic-treated mice
were significantly decreased (FIG. 31C). These results indicated
ceramide might be the cause rather than the result of the
development of NAFLD and a a biomarker to monitor NAFLD. The
contribution of ceramide to NAFLD was further evaluated in cultured
primary mouse hepatocytes. Ceramide treatment induced a
significantly increased triglyceride contents in primary
hepatocytes in a dose-dependent manner (FIG. 31D). To elucidate the
mechanisms by which ceramide leads to hepatic steatosis, the
expression of the genes involved in hepatic lipogenesis and fatty
acid oxidation were measured. Fatty acid synthesis-related genes
such as sterol response element-binding protein 1c (Srebp1c), DNA
fragmentation factor-alpha-like effector a (Cidea), elongation of
very-long-chain fatty acids protein 6 (Elovl6) and TG formulation
related genes such as diacylglycerol O-acyltransferase 2 (Dgat2)
were significantly upregulated by ceramide in primary hepatocytes
(FIG. 31E). In contrast, the expression of genes involved in fatty
acid .beta.-oxidation such as carnitine palmitoyltransferase 1
(Cpt1), acyl-coenzyme A oxidase 1 (Acox1), enoyl-coenzyme A,
hydratase/3-hydroxyacyl coenzyme A dehydrogenase (Ehhadh), and
acetyl-coenzyme A acyltransferase 1A (Acaa1a) were not affected by
ceramide treatment (FIG. 31E). In agreement with the mRNA results,
ceramide exposure at 2 .mu.M and 10 .mu.M significantly induced the
protein levels of the mature nuclear form of SREBP1-N and the
SREBP1-N target gene protein CIDEA (FIGS. 31F and G). In vivo,
mRNAs encoded by the hepatic fatty acid synthesis related genes
Srebp1c, Cidea, fatty acid synthase (Fasn), and Elovl6 were
decreased in antibiotic-treated mice compared to vehicle-treated
mice, and Fxr.sup..DELTA.IE compared to Fxr.sup.fl/fl mice (FIGS.
32A and B). The expression of genes involved in fatty acid remained
at similar levels in antibiotic-treated mice compared to
vehicle-treated mice, and Fxr.sup..DELTA.IE compared to
Fxr.sup.fl/fl mice (FIGS. 32C and D). Western blot analysis further
revealed that the protein levels of the mature nuclear form of
SREBP1-N and CIDEA were significantly downregulated in livers of
antibiotic-treated mice on a HFD for 7 weeks (FIGS. 32E and F). The
rate limiting enzyme cholesterol 7.alpha.-hydroxylase (CYP7A1)
initiates the classic pathway for bile acid synthesis and plays an
important role in regulating lipid metabolism. Cyp7a1 mRNA levels
were marginally induced in antibiotic-treated mice, but not in
tempol-treated mice (FIGS. 32G and H). In addition,
inflammation-related genes such as toll-like receptor 2 (Tlr2),
toll-like receptor 4 (Tlr4), toll-like receptor 9 (Tlr9) and tumor
necrosis factor .alpha. (Tnf.alpha.), were comparable in
antibiotic- and tempol-mice (FIGS. 32I and J). The present findings
revealed that inhibition of ceramide metabolism might be a major
contributing factor to improve HFD-induced NAFLD development in
antibiotic-treated mice.
Example 13
This example demonstrates that inhibition of intestinal FXR is
required for gut microbiome-mediated progression of NAFLD.
Fxr.sup..DELTA.IE mice were employed to determine the role of
intestinal FXR in the progression of the NAFLD. Liver histology
revealed that antibiotic and tempol treatment decreased hepatic
lipid droplets in Fxr.sup.fl/fl mice on a HFD for 14 and 16 weeks,
respectively; no changes in hepatic lipid were observed in
Fxr.sup..DELTA.IE mice with these treatments (FIGS. 33A and B and
FIGS. 34A and B). The liver weights and liver/body mass ratios of
antibiotic- and tempol-treated Fxr.sup.fl/fl mice were
significantly reduced, whereas the liver weights and liver/body
mass ratios were similar in Fxr.sup..DELTA.IE and Fxr.sup.fl/fl
mice (FIGS. 33C and D, FIGS. 34C and D). Hepatic triglyceride
content analysis confirmed that antibiotic and tempol treatment did
not alleviate hepatic steatosis in Fxr.sup..DELTA.IE mice (FIG. 33E
and FIG. 34E). Ileum and serum C16:0, C18:0, C20:0, C22:0, C24:0
and C24:1 ceramide levels were significantly decreased in
Fxr.sup..DELTA.IE mice and tempol-treated Fxr.sup.fl/fl mice, but
not in Fxr.sup..DELTA.IE mice (FIGS. 33F and G). In
Fxr.sup..DELTA.IE mice, hepatic fatty acid synthesis related genes
such as Srebp1c, Cidea, Fasn, and Elovl6 remained unchanged between
vehicle-treated and antibiotic-treated mice (FIG. 34F). Further,
the protein levels of the mature nuclear form of SREBP1 and CIDEA
proteins were significantly reduced in the liver of tempol-treated
mice, whereas no decrease was noted in Fxr.sup..DELTA.IE mice
treated with tempol (FIGS. 34G and H). The present findings
revealed that inhibition of intestinal FXR mediates the
amelioration of NAFLD caused by antibiotic and tempol
treatments.
Example 14
This example demonstrates the systemic responses of mice on a
high-fat diet, to tempol and antibiotic treatment.
A total of 53 metabolites involved in the metabolism of amino
acids, carbohydrates and nucleotides were identified by .sup.1H
NMR. 1D .sup.1H NMR spectra of the cecal contents are dominated by
short chain fatty acids (SCFAs), nucleotides, oligosaccharides and
some amino acids. Glycogen, glucose, amino acids and nucleotides
are the dominant metabolites observed in the .sup.1H NMR spectra of
liver.
In order to obtain the metabolic variations associated with
different biological sample groups, pair-wise OPLS-DA was performed
between data obtained from cecal contents or liver of mice after
tempol or antibiotic treatment. The quality of these models was
further validated by evaluation with CV-ANOVA (p<0.05) and
permutation test (200 tests) for the OPLS-DA and PLS-DA models.
Compared with the vehicle-treated wild-type mice, tempol treatment
significantly decreased the levels of SCFAs (acetate, propionate,
and butyrate) but significantly elevated the levels of
oligosaccharides and glucose in the cecal contents. Similar changes
in SCFAs and oligosaccharides were also observed from the cecal
contents of the antibiotic-treated wild type mice compared to those
from the respective controls. However, no significant differences
in the levels of SCFAs and oligosaccharides were observed in the
cecal contents between tempol-treated and vehicle-treated
Fxr.sup..DELTA.IE mice.
Tempol treatment significantly decreased the levels of lipid and
unsaturated fatty acid (UFA) in the livers, whereas tempol
treatment significantly elevated the levels of glucose, glycogen,
bile acids and a range of nucleotide metabolites (e.g., uridine,
hypoxanthine and 5'-IMP), nicotinurate, and choline in comparison
with the vehicle-treated wild-type mice. These observations are
consistent with reduced lipogenesis in the liver due to tempol
treatment. However, no significant change in lipid and glucose
metabolism was observed in the liver of Fxr.sup..DELTA.IE mice
after tempol-treatment. In addition, antibiotic treatment
significantly elevated the levels of bile acids, trimethylamine
oxide (TMAO, choline, fumarate, formate, amino acids including
branched chain amino acids (leucine, isoleucine and valine),
alanine, glycine, tyrosine and phenylalanine, and some nucleic
acids such as hypoxanthine, uridine and 5'-IMP in the liver.
Compared with the vehicle-treated Fxr.sup.fl/fl mice,
Fxr.sup..DELTA.IE mice exhibit lower lipid and UFA levels but
higher taurine and glycogen levels in the livers.
Example 15
This example demonstrates a synthesis of .beta.-muricholic acid 9,
glycine-.beta.-muricholic acid (Gly-MCA) 16, and
tauro-.beta.-muricholic acid (T-.beta.-MCA) 10 in accordance with
an embodiment of the invention.
.beta.-Muricholic acid (.beta.-MCA) 9 was prepared as illustrated
in FIG. 41 by following the literature procedure (Lida T, Momose T,
et al., Journal of Lipid Research, 30: 1267-1279 (1989)). In
general, esterification of the dihydroxy acid 1 with methanol under
acid catalysis provided ester 2 in quantitative yield. Protection
of the hydroxyl group in the 3 position with ethyl chloroformate
provided carbonate 3. Oxidation of the 6-hydroxyl group with
potassium chromate gave ketone 4 in quantitative yield. Bromination
with 47% HBr solution gave bromo ketone 5, which on reduction with
NaBH.sub.4 gave bromohydrin 6 in moderate yield. Reductive
dehydrobromination with zinc metal provided olefin 7 in about 80%
yield. Cis-dihydroxylation with osmium tetroxide to give cis diol 8
followed by hydrolysis provided s-muricholic acid 9 in quantitative
yield. r-muricholic acid 9 was conjugated with glycine to provide
glycine-.beta.-muricholic acid (Gly-MCA) 16. A suspension of ethyl
glycinate was reacted with .beta.-MCA 9 and EEDQ by refluxing
overnight. The residue obtained after workup was dissolved in
boiling ethanol and hydrolyzed with 10% K.sub.2CO.sub.3. The
aqueous solution was acidified to give Gly-MCA 16 as a white powder
in 68% yield. .sup.1H NMR (CDCl.sub.3) 0.75 (s, 3H, 18-Me), 1.01
(d, 3H, J=6.5 Hz, 21-Me), 1.14 (s, 3H, 19-Me), 3.44-3.56 (m, 2H),
3.58-3.61 (m, 1H), 3.91 (s, 2H).
T.beta.MCA 11 was similarly prepared from 9 by conjugation with
taurine instead of glycine.
Example 16
This example demonstrates that Gly-MCA is stable in the
intestine.
Fecal extracts were prepared as described above. Gly-MCA (50 .mu.M)
was incubated with fecal extract (0.1 mg/mL). The negative control
was fecal extract alone. The positive control was fecal extract
(0.1 mg/mL) and T.beta.MCA acid (50 .mu.M). The samples were
analyzed by UPLC to determine the amount of RR-MCA (hydrolysis
product) and the results shown in FIG. 35.
Gly-MCA was given to mice via oral gavage at dosages of 0, 1, 5,
and 50 mg/kg of Gly-MCA, with the Gly-MCA dosed in corn oil.
Gly-MCA was detected using ultra performance liquid
chromatography-electrospray ionization-quadrupole time-of-flight
mass spectrometry (UPLC-ESI-QTOFMS). The results are shown in FIG.
36.
As is apparent from the results shown in FIGS. 35 and 36, Gly-MCA
is stable in the intestine.
Example 17
This example demonstrates that mice treated with Gly-MCA do not
develop significant liver toxicity.
Mice were dosed with vehicle or Gly-MCA at 1 mg/kg, 5 mg/kg, and 50
mg/kg. After 24 h, serum aminotransferase (ALT) and aspirate
aminotransferase (AST) levels were determined and the results shown
in FIG. 37.
As is apparent from the results shown in FIG. 37, Gly-MCA did not
exhibit significant liver toxicity at each of the doses as compared
with vehicle.
Example 18
This example demonstrates that Gly-MCA significantly inhibited the
FXR activity induced by the synthetic FXR agonist GW4064.
HEK293T fibroblasts were transiently co-transfected with (1) a
chimeric receptor construct in which the carboxy terminal portions
of human FXR (containing the native ligand-binding domain and AF2
transactivation domain) was fused to an amino terminal GAL4
DNA-binding domain under regulatory control of the constitutively
active SV40 promoter, (2) a firefly luciferase reporter plasmid
driven by the UAS GAL4 DNA response element, and (3) a Renilla
luciferase reporter gene (pRL-luciferase; Promega; Madison, Wis.)
as a transfection efficiency control. GW4064 or GW4064 and Gly-MCA
were added to the media for 24 h, the cells were harvested, and
cell extracts prepared. Luciferase detection was conducted using
the Dual Luciferase Reporter Assay kit (Promega; Madison, Wis.) and
a Tecan GeniosPro.TM. luminescent plate reader (Research Triangle
Park, N.C.). The results are shown in FIG. 38.
As is apparent from the results shown in FIG. 38, Gly-MCA
significantly inhibited the FXR activity induced by GW4064.
Example 19
This example demonstrates that Gly-MCA is a potent antagonist of
FXR.
Differentiated Caco-2 cells were treated with 100 .mu.M of the FXR
agonist chenodeoxycholic acid (CDCA) and with 0, 100 .mu.M, or 200
.mu.M Gly-MCA, and expression of the FXR target gene Shp mRNA
measured. As is apparent from the results shown in FIG. 39, CDCA
caused a 4-fold increase in expression of Shp mRNA. Gly-MCA
inhibited the induction of Shp mRNA with CDCA in a dose-dependent
manner.
Differentiated Caco-2 cells were treated with 0.2 .mu.M or 5 .mu.M
GW4064 and with 100 .mu.M or 200 .mu.M Gly-MCA. Control cells were
not treated with either agent. Relative expression of the FXR
target gene mRNAs, Shp mRNA, Fgf19 mRNA, and Atp5g mRNA were
determined and the results shown in FIGS. 40A-C, respectively.
Expression of Shp mRNA and Fgf19 mRNA induced by GW4064 was blocked
by Gly-MCA in a dose-dependent manner (FIGS. 40A and B). GW4064
treatment inhibited expression of the FXR target gene Atp5g mRNA
and Gly-MCA reversed the inhibition (FIG. 40C).
Example 20
This example demonstrates that inhibition of FXR signaling by
Gly-MCA is a potent therapeutic strategy for treatment of obesity,
insulin resistance and NAFLD.
To determine whether inhibition of intestinal FXR could be a
therapeutic target for high-fat diet (HFD)-induced obesity, insulin
resistance and NAFLD, and confirm that this transcription factor is
a suitable drug target, HFD-treated mice were orally administered
Gly-MCA. Gly-MCA treatment reduced body weight gain after one week
of treatment with a HFD (FIGS. 41A and B). The absolute fat mass
and the fat/lean mass ratio, measured by NMR, were significantly
decreased in Gly-MCA-treated mice after 7 weeks of treatment
compared with vehicle-treated mice (FIGS. 41C and D). To explore
the mechanism of reduced adiposity in Gly-MCA-treated mice,
cumulative food intake, energy expenditure (EE) using an energy
balance technique (TEE.sub.bal: food energy intake and body
composition change) were measured. Food intake was comparable
between the two groups (FIG. 44A). Gly-MCA treatment increased the
energy expenditure significantly, which could contribute to the
decreased body weight gain of mice on a HFD compared with
vehicle-treated mice (FIG. 42B). To clarify the role of Gly-MCA in
obesity-related glucose homeostasis, glucose and insulin tolerance
tests (GTT and ITT, respectively) were performed. The GTT revealed
that after 6 weeks of HFD challenge, Gly-MCA-treated mice displayed
significantly reduced blood glucose levels after glucose loading
compared with vehicle-treated mice (FIGS. 43A and B). The ITT
demonstrated that the insulin sensitivity was significantly
increased after Gly-MCA treatment (FIG. 43C). These results
indicated that Gly-MCA improved HFD-induced obesity and insulin
resistance. Liver histology indicated a marked reduction in hepatic
lipid droplets after Gly-MCA treatment of mice that were fed a HFD
for 7 weeks (FIG. 44A). Gly-MCA treatment decreased liver weights
and liver/body mass ratios (FIG. 44B). Hepatic triglyceride
contents were decreased to approximately 51% in mice treated with
Gly-MCA (FIG. 44D). These results indicated that Gly-MCA treatment
protected mice from HFD-induced non-alcoholic fatty liver disease
(NAFLD). To exclude the possibility that the effect of Gly-MCA on
body weight and NAFLD were due to a non-specific toxicological
effects, serum aminotransferase (ALT) and aspartate
aminotransferase (AST) biomarkers of liver toxicity were
determined. ALT and AST were significantly higher on a HFD and
GlyMCA treatment significantly decreased serum ALT and AST levels
(FIGS. 45A and B), thus indicating that the dose of Gly-MCA
employed was not toxic, but actually decreased HFD-induced hepatic
toxicity. NAFLD is tightly associated with bile acid metabolism.
UPLC-ESI-QTOFMS-based metabolomics analysis was adopted to
determine bile acid composition and levels of bile acid metabolites
in the feces and intestine. A Scores scatter plot of a PCA model of
the UPLC-ESI-QTOFMS negative mode data from mouse feces and ileum
indicated distinct metabolic profiles between the vehicle- and
Gly-MCA-treated groups (FIGS. 46A and B). The top enriched
metabolite, T.beta.MCA (m/z 514.2871, retention time=6.64 min), was
increased in the Gly-MCA-treated mice on a HFD for 9 weeks as
revealed in the loading scatters plot (FIGS. 46B and 47B). Levels
of T-.beta.-MCA were significantly increased whereas TCA levels
were significantly decreased in feces after Gly-MCA treatment (FIG.
46C). The levels of taurine-conjugated bile acids were increased in
the ileum of Gly-MCA-treated mice, notably, levels of T.beta.MCA
were significantly increased (FIG. 46C). Gly-MCA levels were
markedly increased in the feces and ileum after Gly-MCA treatment
for 9 weeks (FIGS. 46D and 47D, respectively). Serum triglyceride
levels remained similar between the two groups on a HFD for 9 weeks
(FIGS. 48A and B). Serum C16:0, C20:0, C22:0, and C24:1 ceramides
levels, and ileum C16:0, C18:1, and C24:0 ceramides levels were
reduced in Gly-MCA treated mice on a HFD for 9 weeks (FIGS. 49A and
B). Gly-MCA treatment decreased Shp and Fgf15 mRNAs indicating that
FXR signaling was inhibited in the ileum (FIG. 50A). Intestinal
mRNAs encoding ceramide de novo synthesis-related genes, such as
serine Sptic3, Cers4, Degs1, and Smpd3 were significantly lower in
Gly-MCA-treated mice (FIG. 50B). The expression of Shp mRNA was
similar between two groups indicating that FXR signaling wasn't
affected in the liver (FIG. 51A). Cyp7a1 mRNA levels were induced
in Gly-MCA-treated mice (FIG. 51B). Since Fgf15 mRNA levels were
lower, this might contribute to the increase of Cyp7a1 mRNA levels
in Gly-MCA-treated mice. In a model of genetically-induced obesity,
leptin receptor-deficient (db/db) mice treated with Gly-MCA for 6
weeks had reduced body weight as compared to vehicle-treated mice;
weight loss was significant after just one week of treatment (FIG.
52). The absolute fat mass and the fat/lean mass ratio, as measured
by NMR, were significantly decreased in Gly-MCA-treated db/db mice
after 6 weeks of Gly-MCA treatment compared with vehicle-treated
mice (FIGS. 53A and B). Liver histology indicated a significant
decrease in hepatic lipid droplets after Gly-MCA treatment (FIG.
54A). Gly-MCA treatment decreased liver weights and liver/body mass
ratios (FIGS. 54B and C). Liver TG contents were dramatically
improved in mice treated with Gly-MCA (FIG. 54D). Gly-MCA treatment
significantly decreased serum ALT and AST levels (FIGS. 55A and
55B), thus indicating that the dose of Gly-MCA employed was not
toxic to the db/db mice and reduced liver toxicity in this mouse
model. Levels of T-.alpha.-MCA and T.beta.MCA were significantly
increased in feces and ileum after Gly-MCA treatment (FIGS. 56A and
56B). The accumulation of Gly-MCA in the ileum is far much more
than liver, feces, and serum (FIG. 56C). Serum triglyceride levels
remained similar after 6 weeks of Gly-MCA treatment (FIG. 57A).
Serum C16:0, C20:0, C22:0, and C24:1 ceramides levels, and ileum
C16:0, C18:0, C18:1, C20:0, C22:0, C24:0 and C24:1 ceramides levels
were reduced in Gly-MCA treated mice compare to vehicle treatment
(FIGS. 57B and C). In another model of HFD-induced obesity,
C57BL/6N mice made obese by 12 weeks of feeding a high-fat diet,
were treated with Gly-MCA. Due to limited amounts of Gly-MCA, these
mice were treated with only 5 mg/kg GMCA. Despite the lower dosing,
they had reduced body weight gain as compared to vehicle-treated
mice from two weeks of treatment (FIG. 58). The absolute fat mass,
as measured by NMR, were significantly decreased in Gly-MCA-treated
obese mice after 6 weeks of treatment compared with vehicle-treated
mice (FIG. 59). Liver histology indicated a marked amelioration in
hepatic lipid droplets after Gly-MCA treatment (FIG. 60A). Gly-MCA
treatment reduced liver weights and liver/body mass ratios (FIGS.
60B and C). Levels of T.alpha.MCA and T.beta.MCA were significantly
enhanced in feces and ileum after Gly-MCA treatment (FIGS. 61A and
61B). The accumulation of Gly-MCA in the ileum is far greater than
liver, feces, and serum (FIG. 61C).
All references, including publications, patent applications, and
patents, cited herein are hereby incorporated by reference to the
same extent as if each reference were individually and specifically
indicated to be incorporated by reference and were set forth in its
entirety herein.
The use of the terms "a" and "an" and "the" and "at least one" and
similar referents in the context of describing the invention
(especially in the context of the following claims) are to be
construed to cover both the singular and the plural, unless
otherwise indicated herein or clearly contradicted by context. The
use of the term "at least one" followed by a list of one or more
items (for example, "at least one of A and B") is to be construed
to mean one item selected from the listed items (A or B) or any
combination of two or more of the listed items (A and B), unless
otherwise indicated herein or clearly contradicted by context. The
terms "comprising," "having," "including," and "containing" are to
be construed as open-ended terms (i.e., meaning "including, but not
limited to,") unless otherwise noted. Recitation of ranges of
values herein are merely intended to serve as a shorthand method of
referring individually to each separate value falling within the
range, unless otherwise indicated herein, and each separate value
is incorporated into the specification as if it were individually
recited herein. All methods described herein can be performed in
any suitable order unless otherwise indicated herein or otherwise
clearly contradicted by context. The use of any and all examples,
or exemplary language (e.g., "such as") provided herein, is
intended merely to better illuminate the invention and does not
pose a limitation on the scope of the invention unless otherwise
claimed. No language in the specification should be construed as
indicating any non-claimed element as essential to the practice of
the invention.
Preferred embodiments of this invention are described herein,
including the best mode known to the inventors for carrying out the
invention. Variations of those preferred embodiments may become
apparent to those of ordinary skill in the art upon reading the
foregoing description. The inventors expect skilled artisans to
employ such variations as appropriate, and the inventors intend for
the invention to be practiced otherwise than as specifically
described herein. Accordingly, this invention includes all
modifications and equivalents of the subject matter recited in the
claims appended hereto as permitted by applicable law. Moreover,
any combination of the above-described elements in all possible
variations thereof is encompassed by the invention unless otherwise
indicated herein or otherwise clearly contradicted by context.
* * * * *