<title>1. INTRODUCTION</title> <p>MBX-102/JNJ39659100 is a compound
in development for the treatment of type 2 diabetes. It is a single enantiomer of halofenate, a
drug that was tested clinically in the 1970s as a hypolipidemic and
hypouricemic agent [##REF##4572799##1##–##REF##797258##6##].
Although developed for lipid lowering, studies with halofenate in diabetic
patients also demonstrated significant effects on plasma glucose and insulin
both in monotherapy [##REF##346616##7##, ##REF##323994##8##]
and in combination with other oral hypoglycemic agents [##REF##171574##9##–##REF##321288##11##]. Two decades later, it was discovered that
both halofenate and MBX-102/JNJ39659100 are selective partial PPAR-<italic>γ</italic> agonists [##REF##16936200##12##, ##UREF##3##13##]
thereby offering an explanation for its antidiabetic properties.</p> <p>Translational medicine is important
for studying the action and safety of drugs. Studies in animals allow for
interventional procedures that are not appropriate for humans. Key to interpreting
these studies is to understand the relationship of the pharmacologically active
form, (i.e., free drug) to the pharmacodynamic effects in each species studied.</p> <p>Connecting preclinical pharmacology and
safety studies in different species to the likely human experience therefore requires
an understanding of the action of the drug at the target from these different
species as well as the relationship of the free, pharmacologically active form
to total drug concentration in these species.</p> <p>For drugs with high serum protein
binding this is particularly important. High serum protein binding appears to
be a common feature of PPAR-<italic>γ</italic> agonists such as rosiglitazone,
pioglitazone, and others [##REF##7956739##14##–##REF##17012539##16##]
and previous data suggest that it may also be a feature of halofenate [##REF##1120140##17##, ##REF##935655##18##]
and therefore, also of MBX-102/JNJ39659100. Accurately determining free levels of highly
plasma protein-bound drugs is technically challenging, making comparisons
between species for these drugs extremely difficult. In the results reported herein, methods were
used that allow for comparison between mouse, rat, and human plasma protein
binding. This allowed for the appropriate interpretation of the pharmacology
and potential for human risk of 
MBX-102/JNJ39659100. This study
provides an approach that could be applied to the translational medicine and
safety assessments for other PPAR agonists.</p>
<title>2. MATERIALS AND METHODS</title> <p>[<sup>3</sup>H] MBX-102 acid (740
GBq/mmol, 20 Ci/mmol) was synthesized by Amersham Biosciences (Buckinghamshire, UK). MBX-102 acid was synthesized
at IRIX Pharmaceuticals (Florence, SC, USA).
The structure of MBX-102 acid is shown in ##FIG##0##Figure 1## in comparison to the full
agonists, rosiglitazone
and pioglitazone. For radio-labeled
binding studies, pooled frozen plasma from either Sprague Dawley rats, CD-1 mice,
or humans were purchased from Bioreclamation, Inc. (Hicksville, NY, USA). For the competitive
equilibrium dialysis experiments, fresh pooled mixed gender plasma from either CD-1
mouse, Sprague-Dawley rat, or humans obtained from Bioreclamation, Inc. (Hicksville, NY) were used. Human, mouse, and rat serum
albumins, and human alpha-1-acid glycoprotein were purchased from Sigma (St. Louis, Mo, USA). Charcoal stripped and delipidated sera from either human males, CD-1 male mice, or Sprague Dawley male rats were purchased from Biochemed (Winchester, Va, USA). FDG (Fluorescein di-<italic>β</italic>-D-galactopyranoside) was purchased from
Invitrogen (Carlsbad, Calif, USA). Lanthascreen TR-FRET PPAR-gamma Coactivator
Assay Kit and fluorescently labeled NCOR peptide
(Fluor-DPASNLGLEDIIRKALMGSFDDK) were purchased from Invitrogen (Carlsbad, Calif). Steady Glo reagent was purchased from Promega
(Madison, Wis, USA). DMEM culture media, Lipofectamine, Optimem,
and Penicillin-Streptomycin were purchased from Invitrogen (Carlsbad, Calif). Bovine Insulin, isobutylmethylxanthine, and
dexamethasone were purchased from Sigma (St.
Louis, Mo). HEK 293T cells were obtained from ATCC (Manassas, Va,
USA). Pro 293-Culture defined media was purchased
from Cambrex (East Rutherford, NJ, USA).</p> <title>2.1. Formulation of [<sup>3</sup>H] MBX-102 acid</title> <p> Radiolabeled
MBX-102 acid was prepared as a 1 mL ethanol solution at a concentration of 50 <italic>μ</italic>M (1 mCi total). Stock MBX-102 acid
dosing solutions (100-fold of final concentration) were prepared with unlabeled
MBX-102 acid in dimethyl sulfoxide (DMSO) and spiked with 1 <italic>μ</italic>L/mL (0.05 <italic>μ</italic>M) of [<sup>3</sup>H] labeled MBX-102
acid so that the final evaluated concentrations of MBX-102 acid were 400 <italic>μ</italic>M, 600 <italic>μ</italic>M, 1000 <italic>μ</italic>M, 1500 <italic>μ</italic>M, and 2000 <italic>μ</italic>M. Final solvent concentrations were 1%
of the total volume.</p> <title>2.2. Determination of plasma protein binding of MBX-102 acid by
equilibrium dialysis</title> <p>Plasma was stored at −20°C. Prior to use, it was thawed and spun at
approximately 2000 rpm for 5 minutes to remove any precipitated material. The
pH was adjusted to pH 7.4 by careful addition of NaH<sub>2</sub>PO<sub>4</sub>.
A 1 mL sample of spiked plasma was prepared by direct dilution of [<sup>3</sup>H]-MBX-102
acid stock solution into plasma and then added to one side of an equilibrium
dialysis chamber. The other chamber was filled with 1 mL of 0.01 M phosphate
buffered saline (PBS). The dialysis apparatus was placed in a water bath at 37°C
and rotated at 20 rpm. Preliminary studies indicated that equilibrium is
achieved within 5 hours (data not shown). Once equilibrium was established, the
contents of the cell chambers were removed and analyzed by liquid scintillation
counting. The chambers were sampled in triplicate. Nonspecific binding, in the
absence of plasma, was determined to be 5.3 +/− 3.9% (mean +/− SD, <italic>n</italic> = 3). The
mean recovery of [<sup>3</sup>H] MBX-102 acid was determined in triplicate by
sampling of both dialysis chambers at each concentration of MBX-102 acid. The recovery percentage was
found not to vary with MBX-102 acid concentration. The mean +/− SD % recoveries
across all MBX-102 acid concentrations for each species were 83.9 +/− 6.7%,
84.4 +/− 2.4%, and 85.8 +/− 2.6% for human, rat, and mouse plasma,
respectively.</p> <title>2.3. Determination of protein binding of MBX-102 acid to selected human
plasma proteins</title> <p>Stock solutions of human serum albumin and alpha-1-acid
glycoprotein were prepared in PBS buffer. Human serum albumin (40 mg/mL, ~600 <italic>μ</italic>M) and human alpha-1-acid glycoprotein
(22.5 <italic>μ</italic>M) were spiked with [<sup>3</sup>H]
MBX-102 acid. The spiked protein solution (175 <italic>μ</italic>L) was added to one side of an
equilibrium dialysis chamber, and an equal volume of PBS buffer was added to
the other chamber. Dialysis was allowed to reach equilibrium and the binding to
protein was determined by liquid scintillation counting of samples from both
chambers as described above. The percent recovery of [<sup>3</sup>H] MBX-102
acid with both serum proteins was between 95.7% and 98.5%.</p> <title>2.4. Determination of MBX-102 acid binding to albumin by surface plasmon resonance (SPR)</title> <p>The characterization of the binding of MBX-102 acid against human,
mouse, and rat albumin was performed using SPR-based biosensors (Biosensor
Tools, Salt Lake City, Utah, USA).
The assay methods used to assess the binding of MBX-102 acid to human, mouse,
and rat albumins have been described previously [##REF##11554715##19##]. Briefly, each albumin was
immobilized onto a CM5 sensor chip using standard amine coupling. Immobilization densities were between 10 000 and 13 000 RU. The test compound was run in a
twofold dilution series with the highest concentration of 200 <italic>μ</italic>M. Each of the 16 different
concentrations was tested in duplicate. The running buffer contained 53 mM Na<sub>2</sub>HPO<sub>4</sub>, 12.5 mM KH<sub>2</sub>PO<sub>4</sub>, 70 mM NaCl at pH 7.4, and 5% DMSO. All binding data were collected at 37°C. The binding response profile of
MBX-102 acid over the three different albumin surfaces was evaluated and the
binding constants for the high-affinity site were determined using a two-independent-site
model. Conversion from K<sub>D</sub> to %bound was performed as previously described [##REF##11554715##19##].</p> <title>2.5. Determination of species differences in protein binding of MBX-102
acid by competitive equilibrium dialysis</title> <p>A comparison of the binding to
plasma from different species was performed essentially by the method described
below. Briefly, [<sup>3</sup>H] MBX-102 acid spiked plasma samples were
formulated as described above with the exception that pH was not adjusted to
7.4 and the final DMSO concentration was 0.6%. 
A 1 mL sample of spiked human plasma was applied to one side of the
dialysis membrane and 1 mL of spiked animal plasma was applied to the other
side. The samples were dialyzed by rotation at 20 rpm for up to 120 hours in a
37°C incubator. The ratio of free drug in plasma was calculated
according to the equation: ratio of free drug (animal versus human) = (total
cpm in human plasma)/(total cpm in animal plasma).</p> <title>2.6. Cell culture</title> <p>HEK 293T cells (ATCC) were cultured in 15-cm
dishes at subconfluence (approx. cell density was 14 000/cm<sup>2</sup>) in DMEM (high
glucose), and 10% (v/v) fetal bovine serum (FBS) supplemented with 1% (v/v) Penicillin-Streptomycin. All cells were maintained at 37°C in a
humidified atmosphere of 8% CO<sub>2</sub> in air.</p> <title>2.7. PPAR-<italic>γ</italic> reporter gene assays</title> <p>HEK-293T
cells were cultured as described above. Prior to use, the cells were trypsinized
using 0.25% trypsin/1 mM EDTA and resuspended in DMEM, 10% (v/v) FBS lacking
Penicillin-Streptomycin. For a pool
sufficient to supply 100 wells, 6 million cells were diluted into medium for a
total volume of 9 mL. The DNA-Lipofectamine 2000 mixture was prepared as per
manufacturer’s instructions. For a pool
sufficient to supply 100 wells, 5 <italic>μ</italic>g Gal 4-Mouse PPAR-<italic>γ</italic> LBD, 5 <italic>μ</italic>g pFR-Luciferase, and 500 ng
Lac-z plasmids were mixed with 40 <italic>μ</italic>L of
Lipofectamine 2000 in Optimem medium in a total volume of 1 mL. The cell
suspension was mixed with 1 mL of the DNA-Lipofectamine 2000 mixture. The
mixture was plated into a 96-well plate and incubated for 4 hours at which time
the transfection medium was removed and replaced with 100 <italic>μ</italic>L DMEM, 10% (v/v)
FBS and cultured overnight. The culture medium was then removed from the
plates and replaced with 50 <italic>μ</italic>L Pro293A medium. Compounds and charcoal stripped/delipidated
serum or serum albumin, <italic>or</italic> alpha-1
acid glycoprotein stock solutions were prepared at 2X final concentration in
Pro293A medium and mixed together prior to addition of 50 <italic>μ</italic>L to the transfected
cells and incubated for an additional 24 hours. Measurement of luciferase and
fluorescence activity was performed according to the manufacturer’s
instructions. Briefly, after removal of media, cells were incubated for 10
minutes in 100 <italic>μ</italic>L of Steady-Glo reagent. An 80 <italic>μ</italic>L lysate aliquot was transferred to
opaque white well plates and the luminescence measured. The 80 <italic>μ</italic>L aliquot was
then transferred back to the original plate. The fluorescence emission (excitation
485 nm, emission 535 nm) was measured after the addition of 100 <italic>μ</italic>L of 10 <italic>μ</italic>M fluorescein di-<italic>β</italic>-D-galactopyranoside in assay buffer (2.1 mM KH<sub>2</sub>PO<sub>4</sub>, 310.3 mM NaCl, 5.9 mM Na<sub>2</sub>HPO<sub>4</sub>-7H<sub>2</sub>O,
20 mM KCl, 2 mM MgSO<sub>4</sub>, 0.2% triton-X100). Each experimental
condition was assessed in quadruplicate. The data were normalized for each well
by dividing the luminescence measurement by the fluorescence measurement.
Dose-response curves were generated and EC<sub>50</sub> values were calculated
using Prism Graphpad version 5.1.</p> <title>2.8. Lanthascreen corepressor displacement assay</title> <p>Assays were
performed according to the manufacturer’s instructions. Briefly, GST-PPAR<italic>γ</italic>-LBD (5 nM), Tb-labeled anti-GST antibody
(5 nM), and fluorescent-peptide (125 nM) were diluted together in kit assay
buffer with 5 mM DTT and 10 <italic>μ</italic>L/well of this solution was added to 384-well
black plates (Costar, Corning Inc. Life Science, Lowell, Mass, USA). 
Ligands were prepared as stock solutions in DMSO at 100-fold their final
concentration followed by dilution to 2X concentration in kit assay buffer with
5 mM DTT containing a 2X concentration of serum albumin or charcoal
stripped/delipidated serum prior to addition of 10 <italic>μ</italic>L/well to the assay plate.
The plate was covered and incubated for 4 hours at room temperature. The time
resolved fluorescence resonance energy transfer (TR-FRET) signal was measured
using a Pherastar fluorescence counter (BMG labtech, Offenburg, Germany). The ratio of the emission intensity
of the acceptor (Fluorescein: <italic>λ</italic> =
520 nm) divided by the emission intensity of the donor (Tb: <italic>λ</italic> =
490 nm) was then calculated to determine the degree of NCOR binding. Each
measurement was performed in quadruplicate. Dose-response curves were generated
and IC<sub>50</sub> values were calculated using Prism Graphpad version 5.01.</p> <title>2.9. Statistics</title> <p>To compare logEC50 (or logIC50), ANOVA model of randomized
block design was used. If block effect (experiment effect) was not significant,
the data were reanalyzed by a reduced ANOVA model. Tukey’s test was used for
multiple comparisons (SAS). Differences were considered significant at a <italic>P</italic> value &lt;.05.</p>
<title>3. RESULTS</title> <title>3.1. Interspecies protein binding of MBX-102 acid</title> <p>MBX-102 is a
selective partial PPAR-<italic>γ</italic> modulator which is structurally
distinct from the full PPAR-<italic>γ</italic> agonists, rosiglitazone and
pioglitazone (see ##FIG##0##Figure 1##). In order to understand the relationship between
free drug levels and the efficacy of the selective partial PPAR-<italic>γ</italic> agonist MBX-102 acid in different
species, the plasma binding properties of MBX-102 acid were determined. Pooled,
mixed sex plasma obtained from humans, Sprague Dawley rats, and CD-1 mice were
spiked with MBX-102 acid and the % MBX-102 acid bound to protein was determined
by equilibrium dialysis. The data shown in ##TAB##0##Table 1## reveal that MBX-102 acid is
99.5%–100% bound to
plasma proteins from humans, rats, and mice. The high degree of binding
observed was also independent of MBX-102 acid concentration. To identify
potential MBX-102 acid binding proteins in humans, equilibrium binding studies
were performed using purified human serum albumin and human alpha 1-acid
glycoprotein. A high level of MBX-102 acid binding (&gt;98%) to human serum
albumin was observed. In comparison, the binding to human alpha 1-acid
glycoprotein was very low (&lt;5%) (data not shown). These studies indicate
that the selective partial PPAR-<italic>γ</italic> agonist MBX-102 acid is highly protein-bound
in plasma across different species and identifies serum albumin as a protein
that binds MBX-102 acid.</p> <p>To further characterize the binding
of MBX-102 acid to albumin, we used surface plasmon resonance (SPR), a label-free
technique that can be used to provide information on the kinetics and affinity
of complex formation for drugs that are highly bound to albumin [##REF##11554715##19##, ##REF##12532383##20##]. The
binding constants (KD) and the bound percentage for
human, mouse, and rat albumin are reported in ##TAB##1##Table 2##. In full agreement with
the studies reported above, MBX-102 acid binding to albumin was &gt;98%. This
high degree of protein binding precluded any further analysis of differential
binding of MBX-102 acid to plasma proteins across species because the absolute
binding could not be determined accurately by any of the two methodologies
used. Therefore, competitive equilibrium dialysis (CED) was used to address the
question of differences in the binding of MBX-102 acid to plasma proteins among
species. CED utilizes competition dialysis between the plasma of two species to
accurately determine the ratios of the free drug fractions in these species [##REF##12211222##21##].
Using this technique, the ratio of the free fractions is inversely related to
the fold accumulation of total drug in the plasma of each species plasma at
equilibrium. The ratios of rat-to-human and mouse-to-human free fraction were
determined over several concentrations of MBX-102 acid. The data shown in ##TAB##2##Table 3## indicate that the free MBX-102 acid in rat plasma is 1.7 to 2.3 fold higher
than in human plasma and that the free MBX-102 acid concentration in mouse
plasma is 2.3 to 10.5 fold higher than in human plasma. Interestingly, both the
rat-to-human and the mouse-to-human free drug ratios were found to decrease
with total drug concentration possibly due to saturation of weak binding sites
on human binding proteins. These findings predict that at a fixed total drug
level of MBX-102 acid, the relative free drug levels across species will be in
the order mouse &gt; rat &gt; human.</p> <title>3.2. Activation of PPAR-<italic>γ</italic> by free drug in the presence of human
serum</title> <p>The finding that the partial PPAR-<italic>γ</italic> agonist MBX-102 acid is differentially
bound to plasma proteins across species suggested that the free levels,
putatively responsible for pharmacodynamic effects of MBX-102 acid, could lead
to a different dependence on total drug levels amongst the different species.
In order to fully interpret the impact of different levels of free MBX-102 acid
between species, it is essential to confirm that free drug level is responsible
for the action at the receptor and to know if there are any intrinsic
interspecies differences in PPAR-<italic>γ</italic> activity of MBX-102 acid. PPAR-<italic>γ</italic> reporter gene assays demonstrated that
there were no intrinsic differences in the ability of MBX-102 acid to activate
human, mouse, or rat PPAR-<italic>γ</italic> (data not shown). To understand the
effect of serum on the activation of PPAR-<italic>γ</italic> by MBX-102 acid, the ability of MBX-102
acid to transactivate PPAR-<italic>γ</italic> was determined in a cell-based assay in
the presence of increasing concentrations of human serum. As illustrated in
##FIG##1##Figure 2(a)##, MBX-102 acid induced PPAR-<italic>γ</italic> activity in a dose-dependent manner in
the absence of serum. In the presence of increasing concentrations of human
serum, there was a pronounced and serum concentration-dependent rightward shift
of the dose-response curve for MBX-102 acid. 
The fold changes in mean EC<sub>50</sub> values relative to no serum
were 3, 19-, and 29-fold for 2%, 10%, and 20% human serum,
respectively. At higher human serum
concentrations, there was a decrease in the window of activation precluding an
analysis of serum concentrations above 20%. Similar studies were performed for
the full PPAR-<italic>γ</italic> agonists, rosiglitazone and
pioglitazone (see Figures ##FIG##1##2(b)## and ##FIG##1##2(c)##). For both compounds, as was seen for
MBX-102 acid, a rightward shift in the dose-response curve for PPAR-<italic>γ</italic> activation was observed in the presence
of 10% human serum compared to serum free. 
For rosiglitazone, there was a 14-fold increase in EC<sub>50</sub>, and
for pioglitazone, there was an 8-fold increase in EC<sub>50</sub>. Serum protein binding therefore affects the degree to which PPAR-<italic>γ</italic> can be activated by agonists in a
cellular environment. Similar studies were performed for all three PPAR-<italic>γ</italic> agonists in the presence of human serum
albumin. As expected, the EC<sub>50</sub>s for activation of PPAR-<italic>γ</italic> were rightward shifted in the presence of
human serum albumin for all three PPAR-<italic>γ</italic> agonists (see Figures ##FIG##2##3(a)##, ##FIG##2##3(b)##, and ##FIG##2##3(c)##). Concentrations of serum albumin greater than
0.08% caused interference in the reporter assay precluding an analysis of the
effect of higher and more physiologically relevant albumin concentrations. To
further confirm the selectivity of the albumin effect, the EC<sub>50</sub> for
activation of PPAR-<italic>γ</italic> was also evaluated in the presence of alpha
1-acid glycoprotein. As anticipated, no shift in EC<sub>50</sub> was detected
even in the presence of the highest concentration of alpha 1-acid
glycoprotein tested (0.14%, data not shown).</p> <title>3.3. Differential activation of PPAR-<italic>γ</italic> across species</title> <p>On the basis of the
finding that MBX-102 acid is differentially bound to serum proteins from human,
mouse, and rat, and the confirmation that free drug levels determine the
ability of MBX-102 acid to activate PPAR-<italic>γ</italic>, it is predicted that MBX-102 acid
should differentially activate PPAR-<italic>γ</italic> in the presence of serum from different
species. As illustrated in ##FIG##3##Figure 4##, this was found to be the case. In the presence of 10% human, rat, or mouse
serum, MBX-102 acid activated PPAR-<italic>γ</italic> with EC<sub>50</sub>s of 260 <italic>μ</italic>M, 196 <italic>μ</italic>M, and 170 <italic>μ</italic>M, respectively. These differences in EC<sub>50</sub> were
found to be highly statistically significant. Similar studies were also
performed with the full PPAR-<italic>γ</italic> agonists, rosiglitazone and
pioglitazone. As summarized in ##TAB##3##Table 4##,
MBX-102 acid activation of PPAR-<italic>γ</italic> was affected differently in the
presence of 10% serum from different species compared to the effects seen with
rosiglitazone and pioglitazone. For MBX-102
acid, the EC<sub>50</sub> in the presence of mouse and rat serum occurred at
lower concentrations than in human serum, whereas for both rosiglitazone and
pioglitazone the opposite effect was observed, namely, that higher
concentrations were needed in the presence of rat and mouse serum. These data
suggest that the differential effect of serum on PPAR-<italic>γ</italic> activation observed with MBX-102 acid
is a property of MBX-102 acid and not of the serum proteins.</p> <title>3.4. Differential corepressor displacement from PPAR-<italic>γ</italic> across species</title> <p>The cell-based
PPAR-<italic>γ</italic> reporter assay is adversely affected by
mouse serum concentrations greater than 10% precluding analysis of
cross-species differential serum binding at serum concentrations closer to
physiological levels. An alternate in
vitro assay was developed that allowed the assessment of the effect of
much higher and more physiologically relevant serum concentrations on MBX-102
acid action. The data shown in ##FIG##4##Figure 5## demonstrate that a peptide derived from
the corepressor NCOR is constitutively bound to the ligand-binding domain of
PPAR-<italic>γ</italic> and can be fully displaced by MBX-102
acid with an IC<sub>50</sub> of 11 <italic>μ</italic>M. Increasing concentrations of human
serum caused a rightward shift of the dose-response curve resulting in up to a
19-fold shift in the IC<sub>50</sub> at 40% human serum. Differential
displacement of NCOR by MBX-102 acid was assessed at 40% serum for human, rat,
and mouse (see ##FIG##5##Figure 6##). The fold changes in IC<sub>50</sub> for human-to-rat
serum and human-to-mouse serum were 4 and 7, respectively. These data are very
consistent with the relative free drug ratios predicted by the competitive
equilibrium dialysis studies.</p>
<title>4. DISCUSSION</title> <p>The data presented here demonstrate
that MBX-102/JNJ39659100 is highly protein-bound, as had been suggested by
previous studies with halofenate, and that at least one of the MBX-102 acid
binding proteins is serum albumin. Our goal was to understand the serum binding
properties of MBX-102 acid across species and to use this information in
interpreting the pharmacodynamic and toxicological effects across species. The
use of competitive equilibrium dialysis studies successfully demonstrated that
MBX-102 acid is indeed differentially bound to plasma with the order of
tightness of binding being human &gt; rat &gt; mouse. The studies performed using
the cell-based PPAR-<italic>γ</italic> reporter assay confirmed, at least
qualitatively, our hypothesis that the pharmacodynamic effects of MBX-102 acid
are dictated by free drug levels and, further, that the differential binding of
MBX-102 acid to serum proteins across species also results in a predictable and
highly reproducible effect on pharmacodynamics. From these studies, the order
of binding of MBX-102 acid to serum across species is predicted to be
human &gt; rat &gt; mouse, which is in agreement with the data from the CED
studies. Although we observed good
qualitative correlations with the reporter assay and the CED assay, the
magnitude of shifts in EC<sub>50</sub> in the reporter assay was much smaller than those seen with the CED
assay. One limitation of these reporter assay studies was the inability to
investigate the effect of serum concentrations higher than 10% which could
possibly explain the quantitative differences observed between these two
assays. For this reason, we developed a new assay for measuring PPAR-<italic>γ</italic> activity in vitro that was able to tolerate serum concentrations as high
as 40%. The data from this new assay confirmed the predicted order of binding
for MBX-102 acid to serum across species as human &gt; rat &gt; mouse and also
provided quantitatively very similar fold changes to the CED assay. The basis
of the differential binding of MBX-102 to serum albumin from different species
is unknown. Although at the protein level, mouse and rat albumins are highly
conserved (~90% homology), the degree of conservation is much lower between
human and mouse (~72%) and human and rat (~73%). Such differences may, at least
in part, be responsible for the differential binding observed between species.</p> <p>The approaches described here will
be generally useful for interpreting preclinical pharmacology data in different
species as well as toxicology studies and how these will relate to the human
experience. Whilst confined initially to PPAR-<italic>γ</italic>, the approaches could easily be adapted
for PPAR-<italic>α</italic> and PPAR-<italic>δ</italic> and indeed to virtually any other
ligand-modulated receptor.</p>
<p>Recommended by Anne Miller</p> <p>Drug binding to plasma proteins restricts their free and active concentrations, thereby affecting their pharmacokinetic properties. Species differences in plasma protein levels complicate the understanding of interspecies pharmacodynamic and toxicological effects. MBX-102 acid/JNJ39659100 is a novel PPAR-<italic>γ</italic> agonist in development for the treatment of type 2 diabetes. Studies were performed to evaluate plasma protein binding to MBX-102 acid and evaluate species differences in free drug levels. Equilibrium dialysis studies demonstrated that MBX-102 acid is highly bound (&gt;98%) to human, rat and mouse albumin and that free MBX-102 acid levels are higher in rodent than in human plasma. Interspecies differences in free drug levels were further studied using PPAR-<italic>γ</italic> transactivation assays and a newly developed PPAR-<italic>γ</italic> corepressor displacement (biochemical) assay. PPAR-<italic>γ</italic> transactivation and corepressor displacement by MBX-102 acid was higher in rat and mouse serum than human serum. These results confirm the relevance of interspecies differences in free MBX-102 acid levels.</p>
<fig id="fig1" position="float"><label>Figure 1</label><caption><p>Structure of rosiglitazone, pioglitazone, and MBX-102 acid
(active form). </p></caption></fig> <fig id="fig2" position="float"><label>Figure 2</label><caption><p>
<italic>PPAR-<italic>γ</italic> activation by (a) MBX-102 acid, (b)
rosiglitazone, and (c) pioglitazone in the presence of increasing human
serum.</italic> Normalized reporter assay data were
calculated as the percentage of maximum signal by expressing each data point as
a percentage of the mean for the maximum signal. The percentage of maximum
signal for the curves representing 0%, 2%, 10%, and 20% (v/v) serum was
calculated independently. The dose-response curves shown are from a
representative experiment. Values are EC<sub>50</sub> (<italic>μ</italic>M) determined from 3 experiments
and shown as the mean ± SD.</p></caption></fig> <fig id="fig3" position="float"><label>Figure 3</label><caption><p>
<italic>PPAR-<italic>γ</italic> activation by (a) MBX-102 acid, (b)
rosiglitazone, and (c) pioglitazone in the presence of increasing human serum
albumin.</italic> Normalized reporter assay data were
calculated as the percentage of maximum signal as described in ##FIG##1##Figure 2##. The
percentage of maximum signal for the curves representing 0 and 0.08% serum
albumin was calculated independently. The dose-response curves shown are from a
representative experiment. Values are EC<sub>50</sub> (<italic>μ</italic>M) determined from 2–6 experiments and
shown as the mean ± SD.</p></caption></fig> <fig id="fig4" position="float"><label>Figure 4</label><caption><p>
<italic>Activation of PPAR-<italic>γ</italic> by MBX-102 acid in the presence of
human serum compared to mouse and rat serum.</italic> Normalized reporter assay data
are expressed as the percentage of maximum signal as described in ##FIG##1##Figure 2##. The
dose-response curves shown are from representative experiments. MBX-102 acid
activation of PPAR-<italic>γ</italic> in the presence of 10% (v/v) human (H),
mouse (M), or rat (R) serum. The dose-response curves shown are from a
representative experiment. Values are EC<sub>50</sub> (<italic>μ</italic>M) determined from 3
experiments and shown as the mean ± SD. FC is the ratio of EC<sub>50</sub>s for human: rat or human: mouse (∗ = <italic>P</italic> &lt; .05, ∗∗; = <italic>P</italic> &lt; .01, ∗∗∗ = <italic>P</italic> &lt; .001 by ANOVA with
Tukey post hoc test).</p></caption></fig> <fig id="fig5" position="float"><label>Figure 5</label><caption><p>
<italic>Displacement of NCOR corepressor peptide from PPAR-<italic>γ</italic> by MBX-102 acid in the presence of
human serum.</italic> MBX-102 acid induced displacement of NCOR corepressor peptide
from the human PPAR-<italic>γ</italic> ligand-binding domain in the presence
of human serum at 0, 10%, or 40% (v/v). Normalized FRET assay data are
expressed as the percentage of maximum signal (as described in ##FIG##1##Figure 2##). The
dose-response curves shown are from a representative experiment. Values are IC<sub>50</sub> (<italic>μ</italic>M) determined from 3 experiments and shown as the mean ± SD.</p></caption></fig> <fig id="fig6" position="float"><label>Figure 6</label><caption><p>
<italic>
Displacement of NCOR corepressor peptide from PPAR-<italic>γ</italic> ligand-binding domain by MBX-102 acid
in the presence of human serum compared to mouse and rat serum.</italic> MBX-102
acid induced displacement of NCOR corepressor peptide from human PPAR-<italic>γ</italic> ligand-binding domain in the presence
of 40% (v/v) human (H), mouse (M), or rat (R) serum. Normalized FRET assay data
are expressed as the percentage of maximum signal (“percentage of maximal signal,” as described
in ##FIG##1##Figure 2(a)##).
The dose-response curves shown are from a representative experiment. Values are
IC<sub>50</sub> (<italic>μ</italic>M) determined from 3 experiments and shown as the mean ± SD.
FC is the IC<sub>50</sub> fold change of mouse or rat compared to human (∗∗∗ = <italic>P</italic> &lt; .001 by ANOVA with
Tukey post hoc test).</p></caption></fig>
<table-wrap id="tab1" position="float"><label>Table 1</label><caption><p>
<italic>Binding of MBX-102 acid to rat, mouse, and human plasma
determined by equilibrium dialysis.</italic> Binding of [<sup>3</sup>H] MBX-102 acid
to plasma was conducted by equilibrium dialysis against PBS buffer at 37°C
and the percentage
of total radiolabel bound to plasma was determined by dividing the amount of
sample in the plasma compartment by the combined total amounts in the plasma
and PBS buffer compartments. Values represent the result of a representative
experiment and are the mean ± SD of triplicate determinations.</p></caption><table frame="hsides" rules="groups"><thead><tr><th align="left" rowspan="2" colspan="1">
MBX-102
acid (<italic>μ</italic>M) </th><th align="center" colspan="3" rowspan="1">
%Protein Binding ± SD </th></tr><tr><th align="center" rowspan="1" colspan="1"> Human </th><th align="center" rowspan="1" colspan="1"> Mouse </th><th align="center" rowspan="1" colspan="1"> Rat </th></tr></thead><tbody><tr><td align="left" rowspan="1" colspan="1"> 400 </td><td align="center" rowspan="1" colspan="1">99.8 ± 0.1</td><td align="center" rowspan="1" colspan="1">99.8 ± 0.0</td><td align="center" rowspan="1" colspan="1">99.8 ± 0.1</td></tr><tr><td align="left" rowspan="1" colspan="1"> 600 </td><td align="center" rowspan="1" colspan="1">99.8 ± 0.1</td><td align="center" rowspan="1" colspan="1">99.7 ± 0.0</td><td align="center" rowspan="1" colspan="1">99.8 ± 0.1</td></tr><tr><td align="left" rowspan="1" colspan="1"> 1000 </td><td align="center" rowspan="1" colspan="1">99.7 ± 0.1</td><td align="center" rowspan="1" colspan="1">99.5 ± 0.1</td><td align="center" rowspan="1" colspan="1">99.7 ± 0.0</td></tr><tr><td align="left" rowspan="1" colspan="1"> 1500 </td><td align="center" rowspan="1" colspan="1"> 100 ± 0.1 </td><td align="center" rowspan="1" colspan="1">99.8 ± 0.1</td><td align="center" rowspan="1" colspan="1">99.8 ± 0.2</td></tr><tr><td align="left" rowspan="1" colspan="1"> 2000 </td><td align="center" rowspan="1" colspan="1">99.8 ± 0.1</td><td align="center" rowspan="1" colspan="1">99.5 ± 0.1</td><td align="center" rowspan="1" colspan="1">99.5 ± 0.1</td></tr></tbody></table></table-wrap> <table-wrap id="tab2" position="float"><label>Table 2</label><caption><p>
<italic>Binding of MBX-102 acid to rat, mouse, and human albumin
determined by plasmon resonance-based biosensors.</italic> The binding constants for
the high-affinity site were determined at 37°C. Values represent
the mean of duplicate determinations (HSA: human serum albumin, MSA: mouse
serum albumin, RSA: rat serum albumin).</p></caption><table frame="hsides" rules="groups"><thead><tr><th align="left" rowspan="1" colspan="1"> Interaction </th><th align="center" rowspan="1" colspan="1">K<sub>D</sub>
(<italic>μ</italic>M) </th><th align="center" rowspan="1" colspan="1"> %Bound </th></tr></thead><tbody><tr><td align="left" rowspan="1" colspan="1"> HSA:MBX-102 </td><td align="center" rowspan="1" colspan="1"> 5.8 </td><td align="center" rowspan="1" colspan="1"> 99.1 </td></tr><tr><td align="left" rowspan="1" colspan="1"> MSA:MBX-102 </td><td align="center" rowspan="1" colspan="1"> 5.5 </td><td align="center" rowspan="1" colspan="1"> 99.2 </td></tr><tr><td align="left" rowspan="1" colspan="1"> RSA:MBX-102 </td><td align="center" rowspan="1" colspan="1"> 12.8 </td><td align="center" rowspan="1" colspan="1"> 98.1 </td></tr></tbody></table></table-wrap> <table-wrap id="tab3" position="float"><label>Table 3</label><caption><p>
<italic>Interspecies free MBX-102 acid ratios determined by
competitive equilibrium dialysis</italic>. [<sup>3</sup>H] MBX-102 acid distribution
between either mouse and human plasma or rat and human plasma was conducted by
competitive equilibrium dialysis at 37°C. Values represent mean ± SD
for 5 independent experiments.</p></caption><table frame="hsides" rules="groups"><thead><tr><th align="left" rowspan="2" colspan="1"> MBX-102 Acid (<italic>μ</italic>M) </th><th align="center" colspan="2" rowspan="1"> Free Fraction Ratio
(<italic>n</italic> = 5
± SD)</th></tr><tr><th align="center" rowspan="1" colspan="1">Rat:Human </th><th align="center" rowspan="1" colspan="1">Mouse:Human </th></tr></thead><tbody><tr><td align="left" rowspan="1" colspan="1"> 100 </td><td align="center" rowspan="1" colspan="1">2.3 ± 0.6</td><td align="center" rowspan="1" colspan="1">10.5 ± 5.5</td></tr><tr><td align="left" rowspan="1" colspan="1"> 300 </td><td align="center" rowspan="1" colspan="1">2.3 ± 0.6</td><td align="center" rowspan="1" colspan="1">5.9 ± 3.6</td></tr><tr><td align="left" rowspan="1" colspan="1"> 700 </td><td align="center" rowspan="1" colspan="1">2.0 ± 0.3</td><td align="center" rowspan="1" colspan="1">3.7 ± 1.9</td></tr><tr><td align="left" rowspan="1" colspan="1"> 1000 </td><td align="center" rowspan="1" colspan="1">1.8 ± 0.2</td><td align="center" rowspan="1" colspan="1">2.6 ± 1.1</td></tr><tr><td align="left" rowspan="1" colspan="1"> 1300 </td><td align="center" rowspan="1" colspan="1">1.7 ± 0.2</td><td align="center" rowspan="1" colspan="1">2.3 ± 0.7</td></tr></tbody></table></table-wrap> <table-wrap id="tab4" position="float"><label>Table 4</label><caption><p>
<italic>Differential activation of PPAR-<italic>γ</italic> by PPAR-<italic>γ</italic> agonists in the presence of 10% of
human, rat, and mouse serum.</italic> Values are EC<sub>50</sub> (<italic>μ</italic>M) determined
from 3 experiments and shown as the mean ± SD. FC is the ratio of EC<sub>50</sub>s for human: rat or human: mouse (∗ = <italic>P</italic> &lt; .05, ∗∗ = <italic>P</italic> &lt; .01, ∗∗∗ = <italic>P</italic> &lt; .001
by ANOVA with Tukey post hoc test).</p></caption><table frame="hsides" rules="groups"><thead><tr><th align="left" rowspan="2" colspan="1"> PPAR
agonist </th><th align="center" colspan="3" rowspan="1"> Mean
EC<sub>50</sub> (<italic>μ</italic>M) ± SD </th><th align="center" colspan="2" rowspan="1"> Fold
Change in EC<sub>50</sub>
</th></tr><tr><th align="center" rowspan="1" colspan="1">Human </th><th align="center" rowspan="1" colspan="1">Rat </th><th align="center" rowspan="1" colspan="1">Mouse </th><th align="center" rowspan="1" colspan="1">Human:Rat </th><th align="center" rowspan="1" colspan="1">Human:Mouse </th></tr></thead><tbody><tr><td align="left" rowspan="1" colspan="1"> MBX-102
acid </td><td align="center" rowspan="1" colspan="1"> 260
± 16.9 </td><td align="center" rowspan="1" colspan="1"> 196
± 18 </td><td align="center" rowspan="1" colspan="1"> 169
± 5.2 </td><td align="center" rowspan="1" colspan="1"> 1.33**</td><td align="center" rowspan="1" colspan="1"> 1.53***</td></tr><tr><td align="left" rowspan="1" colspan="1"> Rosiglitazone </td><td align="center" rowspan="1" colspan="1">2.0 ± 0.1</td><td align="center" rowspan="1" colspan="1">5.2 ± 0.3</td><td align="center" rowspan="1" colspan="1">4.5 ± 0.3</td><td align="center" rowspan="1" colspan="1"> 0.39***</td><td align="center" rowspan="1" colspan="1"> 0.45***</td></tr><tr><td align="left" rowspan="1" colspan="1"> Pioglitazone </td><td align="center" rowspan="1" colspan="1">8.3 ± 0.4</td><td align="center" rowspan="1" colspan="1">11.4 ± 1.2</td><td align="center" rowspan="1" colspan="1">9.7 ± 1.4</td><td align="center" rowspan="1" colspan="1"> 0.73<sup>NS</sup>
</td><td align="center" rowspan="1" colspan="1"> 0.86<sup>NS</sup>
</td></tr></tbody></table></table-wrap>
<graphic xlink:href="PPAR2008-465715.001"/> <graphic xlink:href="PPAR2008-465715.002"/> <graphic xlink:href="PPAR2008-465715.003"/> <graphic xlink:href="PPAR2008-465715.004"/> <graphic xlink:href="PPAR2008-465715.005"/> <graphic xlink:href="PPAR2008-465715.006"/>
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PPAR Res. 2008 Sep 14; 2008:465715