# MBX-102/JNJ39659100, a Novel Non-TZD Selective Partial PPAR-γ Agonist Lowers Triglyceride Independently of PPAR-α Activation

Authors: Apurva Chandalia; Holly J. Clarke; L. Edward Clemens; Bindu Pandey; Vic Vicena; Paul Lee; Brian E. Lavan; Francine M. Gregoire
Journal: PPAR Research
 (2009)

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## Abstract

MBX-102/JNJ-39659100 (MBX-102) is a selective, partial PPAR-γ agonist that lowers glucose in the absence of some of the side effects, such as weight gain and edema, that are observed with the TZDs. Interestingly MBX-102 also displays pronounced triglyceride lowering in preclinical rodent models and in humans. Although in vitro reporter gene studies indicated that MBX-102 acid is a highly selective PPAR-γ agonist that lacks PPAR-α activity, we sought to determine if PPAR-α activation in vivo could possibly contribute to the triglyceride lowering abilities of MBX-102. In vivo studies using ZDF and ZF rats demonstrated that MBX-102 lowered plasma triglycerides. However in ZF rats, MBX-102 had no effect on liver weight or on hepatic expression levels of PPAR-α target genes. Further in vitro studies in primary human hepatocytes supported these findings. Finally, the ability of MBX-102 to lower triglycerides was maintained in PPAR-α knockout mice, unambiguously establishing that the triglyceride lowering effect of MBX-102 is PPAR-α independent. The in vivo lipid lowering abilities of MBX-102 are therefore mediated by an alternate mechanism which is yet to be determined.


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## Body

## 1. Introduction

The peroxisome proliferator-activated receptors (PPARs) belong to the nuclear
hormone receptor superfamily of transcription factors. They are lipid sensors
known to govern numerous biological processes. The
three PPAR
subtypes (α, δ (β), and γ) regulate the expression of numerous genes
involved in a variety of metabolic pathways [1, 2]. PPAR-γ is expressed most abundantly in adipose tissue
and is a master regulator of adipogenesis and mediates the anti-diabetic activity
of the marketed insulin-sensitizing drugs that belong to the thiazolidinedione
(TZD) class-such as rosiglitazone (Avandia) and pioglitazone (Actos). PPAR-α is highly expressed in
the liver and is the molecular target
for the fibrates (e.g., fenofibrate and gemfibrozil), a class of drugs that
lower plasma triglycerides and increase HDL levels in humans [3, 4].  The function of PPAR δ(β) is still not fully
understood but recent evidence suggests that this ubiquitously expressed PPAR
isoform has pleiotropic actions that may govern diverse physiological processes,
including  the regulation of lipid and
lipoprotein metabolism [5, 6], insulin sensitivity
[7], cardiac function [8], epidermal biology [9], neuroprotection [10],  and gastrointestinal
tract function and disease [11].As
indicated above, the clinical relevance of PPAR-γ agonists is
highlighted by the currently marketed antidiabetic
blockbuster drugs, Avandia, and Actos. These drugs behave as selective PPAR-γ full agonists as they are potent and selective activators of PPAR-γ [12].  In humans, they enhance
insulin action, improve glycemic control with a significant reduction in the
level of glycohaemoglobin (HbA1C), and have variable effects on
serum triglyceride levels in patients with type 2 diabetes [13]. Despite their proven efficacy, they possess a number of
deleterious side effects, including significant weight gain and peripheral
edema [14–16],
increased risks of congestive heart failure, and increased rate of bone fracture
[15, 17, 18].The weight gain associated with the use of TZDs is observed in
preclinical species and in humans [15, 19] and
is likely due to multiple interacting factors, including increased adiposity
and fluid retention [17, 20].  Fluid retention and subsequent edema are the
most significant undesired effects of TZD treatment. Edema is a
prominent problem in patients taking TZDs particularly those who are also
taking insulin or sulfonylureas. In
susceptible patients with pre-existing conditions, fluid retention and edema
can lead to an increased incidence of congestive heart failure [21].  Moreover the inference that
TZD treatment cause a significant increase in the risk of myocardial infarction
and an increase in the risk of death from cardiovascular in type 2
diabetic patients was recently made [22, 23],
leading the FDA to request the addition of a black box warning to the label of
both Actos and Avandia.Another major side effect of glitazone use is related to their
detrimental skeletal actions as they are known to cause bone loss in rodents [24–26].
More importantly, TZDs treatment was recently shown to decrease bone
formation and accelerated bone loss in healthy and insulin resistant
individuals and/or to increase the fracture rate in diabetic women treated with
TZDs [27, 28].  Such major safety concerns have not only
restrained the clinical use of these drugs but have also led to development
failure of a large number of PPAR agonists [15, 17].During the last
decade, a major investment was made by the pharmaceutical industry to develop
safer PPAR agonists (reviewed in [20, 29]). This effort led to the description of several unique
TZD-like and non-TZD-like partial PPAR-γ agonists that display insulin-sensitizing activity associated with lower
stimulation of adipogenesis and therefore with a potential for reduced
side effects [15, 17, 20, 30–33].MBX-102/JNJ39659100 (MBX-102) is a compound in development for the
treatment of type 2 diabetes. It is a
single enantiomer of halofenate, a drug developed for lipid lowering that was
tested clinically in the 1970s as a hypolipidemic and hypouricemic agents [34, 35].
Studies with halofenate in diabetic patients also demonstrated significant
effects on plasma glucose and insulin [36, 37],
suggesting insulin sensitizing properties.
It was recently discovered that both halofenate and MBX-102 are
selective partial PPAR-γ modulators thereby
offering an explanation for their anti-diabetic properties and lack of weight
gain and edema [20, 38].The
results presented here show, in agreement with the published halofenate data,
that MBX-102 also displays significant triglyceride lowering in preclinical
rodent models. As triglycerides lowering in
preclinical species and in humans is often considered a hallmark of PPAR-α activation and because the mechanism of action by which
halofenate lowers triglycerides has not been elucidated, we performed a series of studies to assess if PPAR-α activation could possibly play a role in the hypolipidemic efficacy of
MBX-102.


## 2. Material and Methods

### 2.1. Chemicals

MBX-102,
pioglitazone, and rosiglitazone maleate were synthesized at Metabolex (Metabolex
Inc, Hayward, CA). Fenofibrate
and GW7647 were obtained from Sigma-Aldrich (Saint-Louis, MO).
WY-14643 was obtained from Eagle Picher Pharmaceutical Services (Lenexa, KS).


### 2.2. Cell-Based Reporter Assays

The determination of mouse PPAR-α, δ, and γactivation was performed as
previously described [38]. Briefly, HEK-293T cells were transfected with Gal4 chimeras and
reporter gene plasmids using Lipofectamine 2000 (InVitrogen, Carlsbad, CA)
and incubated for 4 hours before treatment with compound for 20–24 hours.  Expression was assayed using the Steady-Glo
assay system (Promega, Madison, WI).


#### 2.3. Human Primary Hepatocytes

Cryo-preserved
primary human hepatocytes were obtained from Celsis (Baltimore, MD).
Cells were quickly thawed in a 37C° water bath and placed into 5 mL of warm
InvitroGRO CP medium (Celsis Baltimore, MD) with 2.2% Torpedo antibiotic (Celsis
Baltimore, MD).  A total of 350 000 cells/well were plated in
24-well collagen-coated plates (Becton Dickinson, San Jose, CA)
and incubated overnight.  The following
day the media was replaced with fresh InvitroGRO HI medium (Celsis Baltimore, MD)
containing either DMSO (0.5%) or the test compounds, and the cells were incubated
for 24 hours.  Cells were then harvested
and processed for gene expression analysis. Total RNA
was isolated using Trizol (Invitrogen, Carlsbad,
CA), and cDNA was prepared by reverse
transcription using the High Capacity cDNA Reverse Transcription Kit (Applied
Biosystems, Foster City, CA).
RT-PCR (Taqman) was performed in 96-well plates containing Taqman fast
universal PCR master mix (Applied Biosystems, Foster City, CA) and the
appropriate gene expression assay mixes for human HADHB,
HMGCS2, CYP4a11, and RPLP0 (Applied Biosystems, Foster City, CA).  The “fold change versus vehicle” in gene
expression was calculated using the comparative Ct method for
relative quantification.  For each
compound, two to five independent experiments were performed, and in each
experiment the compounds were tested in at least 2 replicate wells.  The “fold change versus vehicle” data for
replicate experiments were pooled prior to statistical analysis.


##### 2.4. In Vivo Studies

The Metabolex Institutional Animal Care and Use Committee approved
all animal care and experimental procedures described below.  All
animals were housed in temperature (22  ±  3C°)
and humidity (55  ±  4%) controlled rooms, with 12 hour light (6AM-6PM)/dark
cycle.  Unless specified otherwise, mice
were housed 4 to 5 mice/cage, and rats were housed 2 rats/cage and were allowed ad libitum access to tap water and Purina
Rodent Chow (Laboratory
Rodent Diet 5001, St. Louis, Mo., USA).


##### 2.4.1. Reagents and Assays

Plasma glucose
levels were measured using the method of Trinder [39] (Glucose Oxidase G7016, Peroxidase P8125, Sigma Chemical Co.,
St.  Louis, MO).  Plasma
triglycerides were measured using a triglyceride Diagnostic Kit (Sigma Chemical
Co., MO). Plasma-free fatty acid (FFA) levels
were measured using the HR Series NEFA-HR [2] (Wako, Richmond, VA).
Plasma insulin
levels were determined using either a rat or a mouse insulin EIA kit (ALPCO
Chem. Windham, NH).


#### 2.4.2. Zucker Diabetic Fatty Rat Study

9 week-old
Zucker diabetic fatty (ZDF) rats were obtained from Charles River (Boston, MA).
Vehicle and drug suspensions were administered to the rats daily by oral gavage
for 11 days. Six rats were assigned to each of the following groups: Vehicle
(10 mL/kg), rosiglitazone maleate (4 mg/kg), and MBX-102 (100 mg/kg). Body weight
and food intake were recorded weekly. On day 11, rats were fasted for 6 hours
and blood samples (~500μL) were collected via cardiac puncture at the time of
necropsy.


#### 2.4.3. Zucker Fatty Rat Study

10 week-old male
Zucker Fatty (ZF) rats were obtained from Harlan (Indianapolis, IN).
Vehicle and drug suspensions were administered to the rats daily by oral gavage
for 32 days. Eight rats were assigned to each of the
following groups:  ZF Vehicle (5 mL/kg),
ZF  +  fenofibrate (450 mg/kg), and ZF  +  MBX-102 (100 mg/kg). Body weight and food
intake were recorded every 2 or 3 days in the fed state until day 28 of the
study.  At day 33 (24 to 28 hours post-last
dose), blood samples were collected following a 6
hour fast from each rat via cardiac puncture for total triglyceride and insulin
determinations. Liver weights were also
recorded. Following necropsy, a
small (~100–200 mg) section of liver was excised, placed into a cryovial and
immediately frozen in liquid nitrogen. Tissue homogenates for gene expression
analysis were prepared as follows: frozen liver samples were placed into a 2 mL
homogenization vial containing HTG tissue lysis buffer (1 mL/100 mg of tissue,
High Throughput Genomics, Tucson, AZ) and a 5 mm steel bead. Tissues
were homogenized for 5 minutes (25 pulses/second) in a Qiagen Tissue Lyser.  Homogenates were heated at 95C° for 10
minutes, frozen at −80C°, and shipped to high throughput genomics (HTG, Inc.,
Tuscson, AZ) for mRNA measurement using a custom qNPA multiplex array.  The HTG quantitative nuclease protection
(qNPA) technology was used to analyze changes in mRNA expression levels.  All raw values were obtained by imaging with
a high-resolution imager and were normalized against two endogenous house
keeping genes, RPL10a (rat ribosomal protein L10A) and Arbp (rat acidic
ribosomal phosphoprotein P0).  For the
treatment groups, the fold changes (FC) were calculated using the
Vehicle-treated values as 100% (FC  =  1).


### 2.4.4. PPAR-α KO Study

Male wild-type (C57BL/6N) and PPAR-α knockout mice (B6.129S4-Pparatm1Gonz, on C57BL/6N background, N12) were received from Taconic (Germantown, New-York) at 4–6 weeks of age. Animals were allowed access ad libitum to tap water and Rodent Chow (RD D12450B, New Brunswick, NJ). Ten wild-type (WT) and 10 knockout (KO) mice were assigned to each of the following groups: vehicle (5 mL/kg), WY-14643 (130 mg/kg), and MBX-102 (200 mg/kg). Compounds or vehicle were delivered by oral gavage once daily for 7 days.  At the end of the drug treatment, blood samples from each mouse were collected, following a 6 hour fast, via cardiac puncture for total triglyceride and free fatty acid determinations. Three independent studies were performed to evaluate the ability of MBX-102 to lower triglycerides in WT and KO mice. Datasets obtained from the 3 studies were pooled prior to statistical analysis.


### 2.5. Statistical Analysis

Data are expressed as mean± SEM.  Prism software (GraphPad v 5.01, San Diego, CA)
was used for all statistical analyses.
Unless specified otherwise in the figure legends, 1-way ANOVA followed by either Tukey's multiple comparison test or
Newman-Keul multiple comparison test or 2-way ANOVA followed by Bonferroni post
test was used to assess statistical differences between groups.  All P-values of less than .05 were
considered statistically significant.


## 2.1. Chemicals

MBX-102,
pioglitazone, and rosiglitazone maleate were synthesized at Metabolex (Metabolex
Inc, Hayward, CA). Fenofibrate
and GW7647 were obtained from Sigma-Aldrich (Saint-Louis, MO).
WY-14643 was obtained from Eagle Picher Pharmaceutical Services (Lenexa, KS).


## 2.2. Cell-Based Reporter Assays

The determination of mouse PPAR-α, δ, and γactivation was performed as
previously described [38]. Briefly, HEK-293T cells were transfected with Gal4 chimeras and
reporter gene plasmids using Lipofectamine 2000 (InVitrogen, Carlsbad, CA)
and incubated for 4 hours before treatment with compound for 20–24 hours.  Expression was assayed using the Steady-Glo
assay system (Promega, Madison, WI).


### 2.3. Human Primary Hepatocytes

Cryo-preserved
primary human hepatocytes were obtained from Celsis (Baltimore, MD).
Cells were quickly thawed in a 37C° water bath and placed into 5 mL of warm
InvitroGRO CP medium (Celsis Baltimore, MD) with 2.2% Torpedo antibiotic (Celsis
Baltimore, MD).  A total of 350 000 cells/well were plated in
24-well collagen-coated plates (Becton Dickinson, San Jose, CA)
and incubated overnight.  The following
day the media was replaced with fresh InvitroGRO HI medium (Celsis Baltimore, MD)
containing either DMSO (0.5%) or the test compounds, and the cells were incubated
for 24 hours.  Cells were then harvested
and processed for gene expression analysis. Total RNA
was isolated using Trizol (Invitrogen, Carlsbad,
CA), and cDNA was prepared by reverse
transcription using the High Capacity cDNA Reverse Transcription Kit (Applied
Biosystems, Foster City, CA).
RT-PCR (Taqman) was performed in 96-well plates containing Taqman fast
universal PCR master mix (Applied Biosystems, Foster City, CA) and the
appropriate gene expression assay mixes for human HADHB,
HMGCS2, CYP4a11, and RPLP0 (Applied Biosystems, Foster City, CA).  The “fold change versus vehicle” in gene
expression was calculated using the comparative Ct method for
relative quantification.  For each
compound, two to five independent experiments were performed, and in each
experiment the compounds were tested in at least 2 replicate wells.  The “fold change versus vehicle” data for
replicate experiments were pooled prior to statistical analysis.


#### 2.4. In Vivo Studies

The Metabolex Institutional Animal Care and Use Committee approved
all animal care and experimental procedures described below.  All
animals were housed in temperature (22  ±  3C°)
and humidity (55  ±  4%) controlled rooms, with 12 hour light (6AM-6PM)/dark
cycle.  Unless specified otherwise, mice
were housed 4 to 5 mice/cage, and rats were housed 2 rats/cage and were allowed ad libitum access to tap water and Purina
Rodent Chow (Laboratory
Rodent Diet 5001, St. Louis, Mo., USA).


#### 2.4.1. Reagents and Assays

Plasma glucose
levels were measured using the method of Trinder [39] (Glucose Oxidase G7016, Peroxidase P8125, Sigma Chemical Co.,
St.  Louis, MO).  Plasma
triglycerides were measured using a triglyceride Diagnostic Kit (Sigma Chemical
Co., MO). Plasma-free fatty acid (FFA) levels
were measured using the HR Series NEFA-HR [2] (Wako, Richmond, VA).
Plasma insulin
levels were determined using either a rat or a mouse insulin EIA kit (ALPCO
Chem. Windham, NH).


### 2.4.2. Zucker Diabetic Fatty Rat Study

9 week-old
Zucker diabetic fatty (ZDF) rats were obtained from Charles River (Boston, MA).
Vehicle and drug suspensions were administered to the rats daily by oral gavage
for 11 days. Six rats were assigned to each of the following groups: Vehicle
(10 mL/kg), rosiglitazone maleate (4 mg/kg), and MBX-102 (100 mg/kg). Body weight
and food intake were recorded weekly. On day 11, rats were fasted for 6 hours
and blood samples (~500μL) were collected via cardiac puncture at the time of
necropsy.


### 2.4.3. Zucker Fatty Rat Study

10 week-old male
Zucker Fatty (ZF) rats were obtained from Harlan (Indianapolis, IN).
Vehicle and drug suspensions were administered to the rats daily by oral gavage
for 32 days. Eight rats were assigned to each of the
following groups:  ZF Vehicle (5 mL/kg),
ZF  +  fenofibrate (450 mg/kg), and ZF  +  MBX-102 (100 mg/kg). Body weight and food
intake were recorded every 2 or 3 days in the fed state until day 28 of the
study.  At day 33 (24 to 28 hours post-last
dose), blood samples were collected following a 6
hour fast from each rat via cardiac puncture for total triglyceride and insulin
determinations. Liver weights were also
recorded. Following necropsy, a
small (~100–200 mg) section of liver was excised, placed into a cryovial and
immediately frozen in liquid nitrogen. Tissue homogenates for gene expression
analysis were prepared as follows: frozen liver samples were placed into a 2 mL
homogenization vial containing HTG tissue lysis buffer (1 mL/100 mg of tissue,
High Throughput Genomics, Tucson, AZ) and a 5 mm steel bead. Tissues
were homogenized for 5 minutes (25 pulses/second) in a Qiagen Tissue Lyser.  Homogenates were heated at 95C° for 10
minutes, frozen at −80C°, and shipped to high throughput genomics (HTG, Inc.,
Tuscson, AZ) for mRNA measurement using a custom qNPA multiplex array.  The HTG quantitative nuclease protection
(qNPA) technology was used to analyze changes in mRNA expression levels.  All raw values were obtained by imaging with
a high-resolution imager and were normalized against two endogenous house
keeping genes, RPL10a (rat ribosomal protein L10A) and Arbp (rat acidic
ribosomal phosphoprotein P0).  For the
treatment groups, the fold changes (FC) were calculated using the
Vehicle-treated values as 100% (FC  =  1).


## 2.3. Human Primary Hepatocytes

Cryo-preserved
primary human hepatocytes were obtained from Celsis (Baltimore, MD).
Cells were quickly thawed in a 37C° water bath and placed into 5 mL of warm
InvitroGRO CP medium (Celsis Baltimore, MD) with 2.2% Torpedo antibiotic (Celsis
Baltimore, MD).  A total of 350 000 cells/well were plated in
24-well collagen-coated plates (Becton Dickinson, San Jose, CA)
and incubated overnight.  The following
day the media was replaced with fresh InvitroGRO HI medium (Celsis Baltimore, MD)
containing either DMSO (0.5%) or the test compounds, and the cells were incubated
for 24 hours.  Cells were then harvested
and processed for gene expression analysis. Total RNA
was isolated using Trizol (Invitrogen, Carlsbad,
CA), and cDNA was prepared by reverse
transcription using the High Capacity cDNA Reverse Transcription Kit (Applied
Biosystems, Foster City, CA).
RT-PCR (Taqman) was performed in 96-well plates containing Taqman fast
universal PCR master mix (Applied Biosystems, Foster City, CA) and the
appropriate gene expression assay mixes for human HADHB,
HMGCS2, CYP4a11, and RPLP0 (Applied Biosystems, Foster City, CA).  The “fold change versus vehicle” in gene
expression was calculated using the comparative Ct method for
relative quantification.  For each
compound, two to five independent experiments were performed, and in each
experiment the compounds were tested in at least 2 replicate wells.  The “fold change versus vehicle” data for
replicate experiments were pooled prior to statistical analysis.


### 2.4. In Vivo Studies

The Metabolex Institutional Animal Care and Use Committee approved
all animal care and experimental procedures described below.  All
animals were housed in temperature (22  ±  3C°)
and humidity (55  ±  4%) controlled rooms, with 12 hour light (6AM-6PM)/dark
cycle.  Unless specified otherwise, mice
were housed 4 to 5 mice/cage, and rats were housed 2 rats/cage and were allowed ad libitum access to tap water and Purina
Rodent Chow (Laboratory
Rodent Diet 5001, St. Louis, Mo., USA).


### 2.4.1. Reagents and Assays

Plasma glucose
levels were measured using the method of Trinder [39] (Glucose Oxidase G7016, Peroxidase P8125, Sigma Chemical Co.,
St.  Louis, MO).  Plasma
triglycerides were measured using a triglyceride Diagnostic Kit (Sigma Chemical
Co., MO). Plasma-free fatty acid (FFA) levels
were measured using the HR Series NEFA-HR [2] (Wako, Richmond, VA).
Plasma insulin
levels were determined using either a rat or a mouse insulin EIA kit (ALPCO
Chem. Windham, NH).


## 2.4. In Vivo Studies

The Metabolex Institutional Animal Care and Use Committee approved
all animal care and experimental procedures described below.  All
animals were housed in temperature (22  ±  3C°)
and humidity (55  ±  4%) controlled rooms, with 12 hour light (6AM-6PM)/dark
cycle.  Unless specified otherwise, mice
were housed 4 to 5 mice/cage, and rats were housed 2 rats/cage and were allowed ad libitum access to tap water and Purina
Rodent Chow (Laboratory
Rodent Diet 5001, St. Louis, Mo., USA).


## 2.4.1. Reagents and Assays

Plasma glucose
levels were measured using the method of Trinder [39] (Glucose Oxidase G7016, Peroxidase P8125, Sigma Chemical Co.,
St.  Louis, MO).  Plasma
triglycerides were measured using a triglyceride Diagnostic Kit (Sigma Chemical
Co., MO). Plasma-free fatty acid (FFA) levels
were measured using the HR Series NEFA-HR [2] (Wako, Richmond, VA).
Plasma insulin
levels were determined using either a rat or a mouse insulin EIA kit (ALPCO
Chem. Windham, NH).


## 2.4.2. Zucker Diabetic Fatty Rat Study

9 week-old
Zucker diabetic fatty (ZDF) rats were obtained from Charles River (Boston, MA).
Vehicle and drug suspensions were administered to the rats daily by oral gavage
for 11 days. Six rats were assigned to each of the following groups: Vehicle
(10 mL/kg), rosiglitazone maleate (4 mg/kg), and MBX-102 (100 mg/kg). Body weight
and food intake were recorded weekly. On day 11, rats were fasted for 6 hours
and blood samples (~500μL) were collected via cardiac puncture at the time of
necropsy.


## 2.4.3. Zucker Fatty Rat Study

10 week-old male
Zucker Fatty (ZF) rats were obtained from Harlan (Indianapolis, IN).
Vehicle and drug suspensions were administered to the rats daily by oral gavage
for 32 days. Eight rats were assigned to each of the
following groups:  ZF Vehicle (5 mL/kg),
ZF  +  fenofibrate (450 mg/kg), and ZF  +  MBX-102 (100 mg/kg). Body weight and food
intake were recorded every 2 or 3 days in the fed state until day 28 of the
study.  At day 33 (24 to 28 hours post-last
dose), blood samples were collected following a 6
hour fast from each rat via cardiac puncture for total triglyceride and insulin
determinations. Liver weights were also
recorded. Following necropsy, a
small (~100–200 mg) section of liver was excised, placed into a cryovial and
immediately frozen in liquid nitrogen. Tissue homogenates for gene expression
analysis were prepared as follows: frozen liver samples were placed into a 2 mL
homogenization vial containing HTG tissue lysis buffer (1 mL/100 mg of tissue,
High Throughput Genomics, Tucson, AZ) and a 5 mm steel bead. Tissues
were homogenized for 5 minutes (25 pulses/second) in a Qiagen Tissue Lyser.  Homogenates were heated at 95C° for 10
minutes, frozen at −80C°, and shipped to high throughput genomics (HTG, Inc.,
Tuscson, AZ) for mRNA measurement using a custom qNPA multiplex array.  The HTG quantitative nuclease protection
(qNPA) technology was used to analyze changes in mRNA expression levels.  All raw values were obtained by imaging with
a high-resolution imager and were normalized against two endogenous house
keeping genes, RPL10a (rat ribosomal protein L10A) and Arbp (rat acidic
ribosomal phosphoprotein P0).  For the
treatment groups, the fold changes (FC) were calculated using the
Vehicle-treated values as 100% (FC  =  1).


## 2.4.4. PPAR-α KO Study

Male wild-type (C57BL/6N) and PPAR-α knockout mice (B6.129S4-Pparatm1Gonz, on C57BL/6N background, N12) were received from Taconic (Germantown, New-York) at 4–6 weeks of age. Animals were allowed access ad libitum to tap water and Rodent Chow (RD D12450B, New Brunswick, NJ). Ten wild-type (WT) and 10 knockout (KO) mice were assigned to each of the following groups: vehicle (5 mL/kg), WY-14643 (130 mg/kg), and MBX-102 (200 mg/kg). Compounds or vehicle were delivered by oral gavage once daily for 7 days.  At the end of the drug treatment, blood samples from each mouse were collected, following a 6 hour fast, via cardiac puncture for total triglyceride and free fatty acid determinations. Three independent studies were performed to evaluate the ability of MBX-102 to lower triglycerides in WT and KO mice. Datasets obtained from the 3 studies were pooled prior to statistical analysis.


## 2.5. Statistical Analysis

Data are expressed as mean± SEM.  Prism software (GraphPad v 5.01, San Diego, CA)
was used for all statistical analyses.
Unless specified otherwise in the figure legends, 1-way ANOVA followed by either Tukey's multiple comparison test or
Newman-Keul multiple comparison test or 2-way ANOVA followed by Bonferroni post
test was used to assess statistical differences between groups.  All P-values of less than .05 were
considered statistically significant.


## 3. Result

MBX-102/JNJ-39659100 (Figure1(a)) is the (–) enantiomer
of halofenate, a drug previously described as a partial PPAR-γ agonist [38]. MBX-102 is a prodrug ester (Figure 1(a)), that is rapidly and
completely modified in vivo by
non-specific serum esterases to the mature free acid form MBX-102 acid (Figure 1(b)), which is the circulating
form of the drug.  For these reasons
MBX-102 was utilized for in vivo studies, whereas the acid form was utilized for all in vitro studies.Chemical
structures of the prodrug ester (a) and active-free acid form (b).
(a)(b)As
previously described for halofenate, cell-based in vitro studies revealed that MBX-102 acid also behaves as a
selective, weak partial PPAR-γ agonist. As shown in Figure 2(a), a dose-dependent
activation of mouse GAL4-PPAR-γ was observed in
response to MBX-102 acid and rosiglitazone, with EC50s of ~12 μM for MBX-102 acid and ~1.5 μM for rosiglitazone. Compared to the
full agonist rosiglitazone, MBX-102 acid was
a much weaker transactivator of PPAR-γ,   as indicated by its lower transactivation activity (~10% of that observed with
rosiglitazone).  MBX-102 acid selectivity
toward PPAR-γ was confirmed by the
lack of transactivation of mouse GAL4-PPAR-α or δ(Figures
2(b) and 2(c)).  A similar PPAR
activation profile of MBX-102 acid was also observed for human and rat PPARs,
including selectivity for PPAR-γ, partial agonism, and
similar EC50s for PPAR-γ activation (data not shown).Gal4 Reporter assay data for mouse PPAR-γ
(a), mouse PPAR-α
(b), and mouse PPAR-δ(c). Values are plotted as mean
±
SEM and are representative of at least 2
independent experiments.
(a)(b)(c)Halofenate was initially developed as a hypolipidemic agent, and
MBX-102 is reported to share this ability. In order to assess MBX-102
efficacy we evaluated the lipid lowering properties of MBX-102 as well as its
antidiabetic effects, using the male Zucker Diabetic Fatty (ZDF) rat model. ZDF
rats were treated with MBX-102 (100 mg/kg) or rosiglitazone (4 mg/kg) for 11
days. As shown in Figure3, after a 6 hours fast, MBX-102 significantly decreased triglyceride
(Figure 3(b)), free fatty acid (Figure 3(c)), and cholesterol (Figure 3(d)) levels. The magnitude of reduction
in these lipid parameters was significantly higher than what was observed for
rosiglitazone (TG 89% versus 57%; FFA 86% versus 49% and Cholesterol 57% versus 10%,
for MBX-102 and rosiglitazone, resp.), suggesting superior hypolipidemic
activity of MBX-102 compared to rosiglitazone.  Moreover, both MBX-102 and rosiglitazone significantly
reduced fasting blood glucose (Figures 3(a)
and 3(e)), confirming that MBX-102 is an efficacious antidiabetic agent. This effect was anticipated as antidiabetic properties including
glucose lowering, and insulin sensitization in preclinical models is a hallmark
of full PPAR-γ agonists and has also
been reported for partial agonists [20].  In addition, significant increases in body
weight (Figure 4(a)) and adipose
tissue weight (Figure 4(b)) were observed
with rosiglitazone treatment only, indicating that MBX-102 does not display the
classical weight gain effects of the full PPAR-γ
agonists.Effect of
MBX-102 (100 mg/kg) and rosiglitazone (4 mg/kg) on fasting plasma glucose (a), triglycerides (b), FFA (c), and cholesterol (d) levels during the course of treatment of male ZDF rats.
Values are plotted as mean±  SEM
(*: P<.05, **: P<.01, ***: P<.001 versus ZDF vehicle; #: P<.05,
##: P<.01, ###: P<.001 versus MBX-102-treated group, 2-way ANOVA followed
by Bonferroni post tests). (e) Fasting plasma glucose, insulin, triglycerides, and FFA
levels on day 11. Values are plotted as mean percentage of vehicle  ±  SEM
(NS: P>.05, *: P<.05, **: P<.01, ***: P<.001 versus ZDF
vehicle, ##: P<.01 versus MBX-102-treated group, 1-way ANOVA and Tukey's
multiple comparison test).
(a)(b)(c)(d)(e)Effect of MBX-102 (100 mg/kg) and
rosiglitazone (4 mg/kg) on body weight (BW) (a) and white adipose tissue
weights (b) after 11 days of
treatment of male ZDF rats. For the adipose tissue weight, the values are
plotted as mean percentage of vehicle±  SEM (*: P<.05, **: P<.01, ***: P<.001 versus ZDF Vehicle; #:
P<.05, ##: P<.01 versus MBX-102, (a)
2-way ANOVA and Bonferroni post tests or (b)
1-way ANOVA and Tukey's multiple comparison test).
(a)(b)In order to evaluate further the lipid lowering ability of
MBX-102, male Zucker Fatty (ZF) rats, a well-established model
for hypertriglyceridemia and obesity, were
used.  The PPAR-α agonist fenofibrate,
a known triglyceride lowering agent, was included in the study as a comparator.
As ZF rats are hyperinsulinemic and
insulin resistant, the insulin sensitizing effect of MBX-102 was also assessed.
ZF male rats were treated with either vehicle, fenofibrate (450 mg/kg) or
MBX-102 (100 mg/kg) for 32 days.  In this
study, no significant differences in body weight or food intake were observed
upon drug treatment (data not shown). As shown in Figure 5(a), both MBX-102 and fenofibrate treatment significantly
lowered fasting plasma insulin after 32 days of treatment.  However, the reduction observed for
MBX-102-treated ZF rats was significantly greater when compared to the
reduction observed for the fenofibrate-treated animals. In this rat model, MBX-102
robustly decreased fasting plasma triglycerides after 32 days of treatment (Figure 5(b)). Although fenofibrate also
led to a reduction in plasma triglyceride levels, the reduction was less pronounced
when compared to MBX-102 (31% versus 60%, Figure
5(b)).Effect of
MBX-102 (100 mg/kg) and fenofibrate (450 mg/kg) on fasting plasma insulin (a), triglycerides (b), and liver weights (c) after 32 days of treatment of male ZF rats. Values are
plotted as mean±  SEM (*: P<.05, ***: P<.001 versus ZF vehicle,
#: P<.05, ###: P<.001, MBX-102 versus fenofibrate, 1-way ANOVA, and
Newman-Keuls multiple comparison test).
(a)(b)(c)To
determine if PPAR-α activation
might be responsible for the triglyceride lowering ability of MBX-102, liver
weight and liver gene expression levels of several PPAR-α
responsive genes were assessed in this study.
As shown in Figure 5(c), fenofibrate
treatment markedly increased liver weight while MBX-102 treatment caused
minimal change in this parameter. In addition, a
slight but not statistically significant upregulation of ACO (Figure 6(a)), significant
upregulation of HADHB (Figure 6(b)), and significant downregulation of apoC-III (Figure 6(c)) mRNA levels were also detected upon treatment with fenofibrate.
In
contrast, MBX-102 treatment had no effect on the mRNA expression levels of
these three PPAR-α responsive genes, suggesting that MBX-102 lowered
triglycerides independently of PPAR-α activation.Gene expression levels of
PPAR-α responsive genes in
livers derived from male ZF rats treated for 32 days with either MBX-102
(100 mg/kg) or fenofibrate (450 mg/kg). Expression levels of ACO (a), HADHB (b), and apoC-III (c) mRNA.
Values
represent mean  ±  SEM (NS: P>.05, ***: P<.001 versus Vehicle-treated, 1-way ANOVA, and Newman-Keuls multiple comparison
test).
(a)(b)(c)In order to further explore
the PPAR selectivity of MBX-102 in a physiologically relevant cell-based
system, primary human hepatocytes were used to evaluate the expression levels
of several PPAR-α responsive genes. Primary human hepatocytes were
treated with known PPAR-α agonists including GW7647, WY-14643, and fenofibric
acid as well as with the PPAR-γ agonists rosiglitazone, pioglitazone, and
MBX-102 acid. As shown in Figure 7, HADHB (a), HMGCS2 (b), and CYP4a11 (c) mRNA levels were significantly
upregulated by treatment with all PPAR-α agonists. The extent of upregulation
was similar for all three PPAR-α agonists.  Interestingly, these three genes were also
significantly up-regulated by pioglitazone although the magnitude of this
effect was less than for the three PPAR-α agonists.  In contrast,
although MBX-102 acid treatment was able to induce mRNA levels of the PPAR-γ responsive genes CD36 and FABP4 in these cells (data
not shown), it had no effect on any of the PPAR-α responsive gene tested supporting
the in vivo results observed in the
ZF rats.Effect
of PPAR-α agonists, rosiglitazone, pioglitazone, and MBX-102 acid on HADHB (a), HMGCS2 (b), and CYP4a11 (c) mRNA
levels in primary human hepatocytes. Values represent
mean  ±  SEM
(*: P<.05, ***: P<.001 versus Vehicle-treated, 1-way ANOVA and Tukey's
multiple comparison test).
(a)(b)(c)Based on these
results, we speculated that MBX-102 would be able to lower triglycerides in
mice lacking PPAR-α.  Therefore,
the effect of MBX-102 on triglyceride levels was evaluated in wild-type (WT)
and PPAR-α knockout (KO) mice. WT and KO mice were treated with either vehicle,
the PPAR-α selective agonist WY-14643 (130 mg/kg), or MBX-102 (200 mg/kg) for 7
days.  Prior to
evaluating triglyceride lowering, single, and repeated doses, pharmacokinetic
analyses were performed with both compounds in both WT and KO mice, and no
difference in plasma drug exposure was observed (data not shown).  As shown in Figure 8(a), treatment with
WY-14643 significantly
reduced plasma triglycerides in WT mice. This effect was totally abolished in
the PPAR-α KO mice, confirming that PPAR-α was required for this
effect.  In contrast, a significant
reduction in plasma triglycerides was observed upon treatment with MBX-102 both
in WT and PPAR-α KO mice, demonstrating this effect was independent of PPAR-α activation. Plasma FFA levels in WT and KO mice are depicted in Figure 8(b).  Compared to vehicle-treated WT mice, plasma
FFA levels were markedly elevated in vehicle-treated KO mice.  Treatment with WY-14643
had little (WT) to no effect (KO) on plasma FFA levels. In contrast, although
MBX-102 had no impact on FFA levels in WT mice, it led to significant FFA
lowering in the KO animals (Figure 8(b)). At the end of the study changes in liver weight upon compound
treatment were evaluated. As expected, treatment with WY-14643 increased liver
weight by 52% in WT mice, and the effect was totally abolished in the PPAR-α KO
mice (Figure 8(c)). MBX-102 treatment
mildly increased liver weight to a similar extent in both WT and KO mice, indicating
this effect occurred independently of PPAR-α activation.Effect of MBX-102 (200 mg/kg) and WY-14643
(130 mg/kg) on plasma triglyceride
levels (a), FFA levels (b), and liver weights (c) in WT and KO mice after 7 days
treatment of PPAR-α KO mice. Values
represent mean ± SEM (*: P<.05, ***: P<.001 versus Vehicle, 2-way
ANOVA, and Bonferroni post-tests).
(a)(b)(c)


## 4. Discussion

Type
2 diabetes mellitus is a chronic disease characterized by glucose intolerance,
hyperinsulinemia, and dyslipidemia, [40]. PPAR-γ agonists such as rosiglitazone
and pioglitazone belong to the thiazolidinedione (TZD) class and are currently
in clinical use for lowering glucose levels in diabetes [41, 42].Our
results show that MBX-102 acid, a non-TZD PPAR agonist, is a partial, selective PPAR-γ agonist which has
the potential to offer antidiabetic efficacy comparable to rosiglitazone. More
importantly, compared to rosiglitazone, treatment of ZDF rats with MBX-102 did
not significantly affect body weight and white adipose tissue mass, suggesting
that in humans, MBX-102 will not display the classical adverse effects of the
full PPAR-γ
agonists [15, 20]. These
data are in agreement with a previously published report that established that
halofenate, the racemic mixture from which MBX-102 is derived,  had comparable insulin
sensitization to rosiglitazone in the absence of body weight gain [38].Among
the efficacy parameters measured in our studies, the most differentiating
feature of MBX-102 was its impressive lipid lowering abilities.  MBX-102 was much
more efficacious than rosiglitazone and fenofibrate at lowering plasma
triglycerides in the diabetic, insulin-resistant rat models tested. In rodents,
differences in feeding behavior can induce significant fluctuation in plasma
triglycerides and free fatty acid levels. Such an artifact can be excluded in
the present studies as all measurements were performed on 6 hour post-fasting
plasma samples.In the clinical setting, fibrate therapy is known to achieve significant
triglyceride lowering, an expected feature of PPAR-α agonists [43, 44].
In contrast, the lipid effects of the marketed PPAR-γ agonists are
not as clear, as pioglitazone displays beneficial effects on lipid profile in diabetic patients while rosiglitazone
does not [13, 45].
Our data suggest that MBX-102 will display beneficial effects on lipid profile in humans, and this was recently confirmed in a phase 2a clinical trial [46].
Overall, these results are not unexpected based on the history of halofenate,
the parent molecule from which MBX-102 was derived.  Halofenate was tested
clinically in the 1970s as a hypolipidemicand hypouricemic agent
and was shown to lower serum triglycerides and uric acid in patients
with a variety of hyperlipidemias [36, 37, 47–49].Although the mechanism
by which halofenate and MBX-102 reduce triglycerides in preclinical rodent
models and in humans remains unclear, a major concern was that MBX-102 may exert
its hypolipidemic action through PPAR-α activation. As
mentioned above, triglyceride lowering is a well-known feature of PPAR-α agonists. Although
the classical in vitro reporter gene
assays we used to assess MBX-102 selectivity toward PPAR-γ clearly show their
inability to transactivate human, mouse, or rat PPAR-α, the biological
relevance of these assays remains unclear as they do not truly represent the
interaction between the ligand and its receptor in a physiologically relevant
setting [17]. The discontinuation of several
dual α/γ PPAR agonists at mid
to late stage of development due to major safety concerns including
dose-limiting toxicities and carcinogenicity-related issues clearly highlights
the potential for increased risk of safety liabilities for dual agonists
compared to selective agonists [15, 17]. The carcinogenic
risk is of particular interest as duals agonists appear to have enhanced rodent
carcinogenicity potential compared to selective gamma agonists (http://www.fda.gov/cder/present/DIA2004/Elhage.ppt), increasing the burden of developing such agents for use in humans.  Therefore in order to demonstrate that
MBX-102 can lower triglycerides independently of PPAR-α activation, we
undertook a series of studies in which we used physiologically relevant
readouts of PPAR-α activation.Although both fenofibrate and MBX-102 had the ability to modulate
triglyceride levels in ZF rats, MBX-102 only had a small effect on rat liver,
which is unlikely mediated by PPAR-α
activation as MBX-102 treatment led to a similar liver weight increase in PPAR-α KO mice. Moreover, MBX-102 was
unable to regulate the hepatic expression levels of the 3 known PPAR-α target genes tested, suggesting
its inability to transactivate rat PPAR-α in vivo. In contrast, the anticipated regulation of these genes
(i.e., upregulation of 2 key genes involved in fatty acid oxidation and
downregulation of apoC-III) was observed with fenofibrate [50, 51].Primary
human hepatocytes represent a biologically relevant cell line to model clinical
effects of PPAR-α agonism and therefore
were used to further explore the PPAR
selectivity of MBX-102 acid.  In this
cell-based system, we were unable to detect any induction of PPAR-α responsive
genes upon MBX-102 acid treatment, further confirming its lack of PPAR-α activity.
Moreover, the finding that MBX-102 still
lowers triglycerides in PPAR-α deficient mice unambiguously demonstrates
that MBX-102 can lower triglycerides effectively in the absence of PPAR-α.Although
these results corroborate that MBX-102 is a selective
PPAR-γ
agonist, the mechanism by which it lowers triglycerides
in preclinical species and in the clinic still needs to be addressed.  Pioglitazone also possesses triglyceride
lowering effects in humans but in this case partial contribution of PPAR-α activation cannot be
ruled out. Our hepatocyte data indeed
show that pioglitazone  upregulates
PPAR-α responsive genes, in
agreement with published reports showing that pioglitazone binds to and
activates the human PPAR-α receptor [52].
Moreover,  pioglitazone has recently been
shown to raise hepatic apoA-I and HDL through a PPAR-α-dependent pathway [53].Studies
performed in the 1970s with halofenate may provide a potential clue as to how
MBX-102 lowers triglycerides. In normal rats, sustained reduction of serum
triglyceride levels upon treatment with halofenate was suggested to be mediated
through the inhibition of hepatic triglyceride formation. Although the
mechanism of action  mediating this
effect is not yet elucidated, it was also suggested that the inhibition of
hepatic triglyceride formation might be related to drug-induced decreases in
the availability of fatty acids for triglyceride synthesis [54].
Our results in ZDF rats are in agreement with this hypothesis as a marked
lowering of circulating free fatty acids was indeed observed upon MBX-102
treatment.Taken as a whole, the
data from these studies provide definitive evidence that MBX-102 acid does not
activate PPAR-α.  As such,
the lowering of triglycerides in vivo by MBX-102 is not a PPAR-α mediated effect, but is rather mediated by an
alternate mechanism which has yet to be determined. Additional studies are
required to determine if MBX-102, like halofenate, is capable of inhibiting
liver triglyceride formation.  More
importantly, studies designed to understand how such inhibition may occur will
be required. Among these, measurement of serum and hepatic triglyceride
formation and turnover will be necessary.



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*Source: 706852-2009-04-23.xml*