# The Development of INT131 as a Selective PPARγ Modulator: Approach to a Safer Insulin Sensitizer

Authors: Linda S. Higgins; Christos S. Mantzoros
Journal: PPAR Research
 (2008)

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## Abstract

INT131 (formerly T0903131, T131, AMG131) is a potent
non-thiazolidinedione (TZD) selective peroxisome proliferator-activated receptorγ modulator (SPPARM) currently in Phase 2 clinical trials for treatment of type-2 diabetes mellitus (T2DM). This new chemical entity represents a second generation SPPARM approach developed after the first generation
PPARγ full agonists to address their inherent limitations. INT131 was specifically and carefully designed using preclinical models to exhibit a biological profile of strong efficacy with
de minimis side effects compared to PPARγ full agonists. As a potent PPARγ modulator, INT131 binds to PPARγ with high affinity. In pharmacology models of diabetes and in early clinical studies, it achieved a high level of efficacy in terms of antidiabetic actions such as insulin sensitization and glucose and insulin lowering, but had little activity in terms of other, undesired, effects associated with TZD PPARγ full agonists such as edema and adipogenesis. Ongoing clinical development is directed at translating these findings into establishing a novel and effective treatment for T2DM patients with an improved safety profile in relation to that currently available.


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## Body

## 1. PPARγ Full Agonists

PPARγfull
agonists are a mainstay in the treatment of insulin resistance and type-2
diabetes. While the glucose lowering action of thiazolidinediones (TZDs) was
well-known as early as 1988 [1], it was not until 1995 that the nuclear
receptor PPARγ was identified as their target [2] and
that its activation was shown to be responsible for their therapeutic benefits.
PPARγ full agonists, including the TZDs rosiglitazone
(Avandia) and pioglitazone (Actos) are powerful drugs for the treatment of
insulin resistance associated with type-2 diabetes mellitus (T2DM) [3]. Troglitazone (Rezulin) was the first TZD
approved for clinical use in the US
in 1997, but was subsequently
withdrawn from the market in 2000 due to idiosyncratic hepatotoxicity. Rosiglitazone
and pioglitazone were approved in the US
in 1999. These drugs enabled
the beneficial effect of PPARγ activating agents to be recognized in clinical
practice globally.These medications
enhance insulin sensitivity and reduce glucose and insulin levels in T2DM
patients, and have been shown to have robust and relatively durable benefit for
glucose control [4]. Insulin resistance is a key
etiologic feature in the onset and subsequent progression of the disease.
Furthermore, insulin sensitization comprises a complementary mechanism of
action to that of other commonly used therapeutic modalities such as inhibition
of gluconeogenesis by metformin, increased insulin secretion by sulfonylureas,
and administration of exogenous insulin. The potential to be used in
combination with other approaches thus further extends the clinical utility of PPARγ activating agents for glucose control and
to treat T2DM. Rosiglitazone and pioglitazone, both in the TZD class, are the
only agents currently approved for insulin sensitization as their major
mechanism of action.Realization of PPARγ maximal therapeutic potential by full
agonists is limited, however, by associated side effects. PPARγ full agonist binding to PPARγ activates a broad spectrum of PPARγmediated
effects, some of which are undesirable. Thus, use of TZDs is limited by side
effects that include weight gain, fluid retention, and decreased bone density [5]. TZD-induced peripheral edema,
which frequently occurs in patients receiving TZD monotherapy, is especially
problematic in patients receiving concomitant insulin therapy, and is of special
concern for patients who have either clinical or subclinical congestive heart
failure (CHF) and thus cannot tolerate the extra fluid volume [6, 7]. In addition, there is strong
evidence that activation of PPARγ causes adipocyte differentiation and increased
adipose tissue mass, contributing to weight gain [3]. The dose response curve for the therapeutic
effects of TZDs overlaps with the dose response for side effects, such that
increasing doses produce both greater benefits for glucose control as well as
greater incidence and higher degrees of side effects [8].
Thus, doses which would produce the maximal clinical benefit of PPARγ full agonists may not be tolerated by a
significant number of patients and the full potential of PPARγ activation for insulin sensitization and
glucose control may not be realized at approved clinical doses of rosiglitazone
or pioglitazone.As a consequence of
the known safety issues, TZDs are not recommended for patients with New York
Heart Association Class 3 and 4 CHF, and the potential clinical impact of cardiovascular
side effects prompted the American Heart and the American Diabetes Associations
to issue a joint consensus statement advising against the use of TZDs in
patients with advanced heart failure [9]. Awareness of the safety issues
associated with TZDs was dramatically increased following the publication of a
meta-analysis in May of 2007 showing a nonstatistically significant trend
towards an increase in macrovascular events in patients taking rosiglitazone [10]. As a result of a detailed
examination of the safety record for the TZD class, both rosiglitazone and
pioglitazone received black box safety warnings for the increased risk of CHF
due to fluid retention. Only rosiglitazone was further implicated for a
“possible” risk of increased ischemic cardiovascular events [11] and obtained
an additional black box warning, but data suggesting this risk have not been
replicated by all studies. Finally, a series of scientific papers has
demonstrated an association between TZD use and bone fracture, especially in women [12]. Despite these well-known limitations,
Actos and Avandia represent a
combined annual global market of more than $5 billion even following a rapid decrease
and then stabilization of total sales and a switch from rosiglitazone to
pioglitazone or other antidiabetic medications following heightened awareness
of safety concerns in 2007. The continued use of the TZDs is a strong testament to the utility
of insulin sensitization as a mode of action for treatment of T2DM, but also
underscores the need for a safer treatment for insulin resistance.Historically, the
proven therapeutic utility of activating the PPARγ nuclear receptor to reduce glucose and
HbA1c led the pharmaceutical industry to focus on a search for greater and
broader efficacy through more potent PPARγ full agonists as well as through the
development of dual αand γ (“α/γ”) PPAR agonists. The
latter were intended to combine the insulin sensitizing effects of PPARγ activation with the lipid lowering effects
of PPARα activation. Unfortunately, no new agents
deriving from these programs have been approved for clinical use. In the case
of full PPARγ agonists, efficacy and side effects have been
shown to be intrinsically linked, with higher efficacy compounds associated
with greater propensity for side effects. Similarly, PPARα/γ dual agonists have been plagued with side
effects. For example, muraglitazar, a dual PPARα/γ agonist, was taken through a comprehensive
development program and demonstrated remarkable efficacy in lowering HbA1c as
well as improving lipid profile in T2DM patients. However, preclinical and
clinical safety signals associated with edema, weight gain, and increased
cardiovascular events led to a request in 2005 by FDA for outcome studies prior
to approval and resulted in abandonment of the program by the sponsor in 2006. In
summary, accumulated experience with PPARγ and PPARα/γ ligands has led to an understanding of a
spectrum of desirable and undesirable activities, as graphically depicted in
Figure 1.Figure 1
Spectrum
of PPARγ effects. The range of biological activities, both desired antidiabetic therapeutic
effects and undesired effects related to tolerability and safety issues,
increases for ligands characterized as antagonists, selective agonists, full
agonists, and broad selectivity full agonists.


## 2. Selective PPARγ Modulation Separates Efficacy and Side Effect Dose Reponse Curves

A very different
approach to leveraging PPARγ antidiabetic therapeutic benefits would
focus on minimizing side effects (Figure 1, left) by limiting the spectrum of activation. This approach would require selective PPARγ modulation which by design would minimize
side effects while maintaining desired therapeutic benefit.After the
identification of PPARγ as the target for TZDs, the crystal structure
of the PPARγ binding pocket as well as its activity
relationships were probed, providing an important tool for pursuing selective
modulation of the receptor. For example, in the case of the TZD PPARγfull
agonists, a key interaction occurs between the ligand and the activation helix
(helix 12) of PPARγ [13, 14]. Binding of activating ligands to the
nuclear receptor PPARγ leads to conformational changes favoring
binding of PPARγ to the RXR nuclear receptor, which is
required for PPARγ driven gene transcription, as well as to
altered association with cofactors (Figure 2). Different types of PPARγ ligands lead to sufficiently different
conformations of the bound receptor heterodimer complex that different combinations
and patterns of coactivators and corepressors are recruited for differential
transcriptional control [15]. That is, the composition of the protein complex
of PPARγ, RXR, and specific cofactors determines
the pattern of the ensuing gene transcription and hence the cellular response to
the PPARγ ligand. Since the repertoire of cofactors
available for recruitment to the PPARγ-RXR complex varies among cell types, PPARγ responses are context-dependent. Thus, full
agonists such as TZDs would be expected to lead to a different pattern of
cofactor recruitment, gene transcription, and cellular response than a
SPPARM.PPARγ activation. Upon ligand
binding, the nuclear receptor PPARγ associates with nuclear receptor RXR as
well as with coactivators and corepressors which are present in a cell type and
state specific pattern. This complex binds to PPAR response elements to enhance
or repress gene transcription.
(a)(b)Theoretically, SPPARMs
can be identified or designed which would produce a pattern of cofactor
recruitment, gene transcription, and cellular response whereby the dose
response curves for desired and undesired effects seen in patients could
potentially be sufficiently separated to establish a broad therapeutic window
(Figure3). Is there precedence for the success of a modulator approach for another
nuclear receptor? Both tamoxifen and its successor raloxifene are selective
estrogen receptor modulators (SERMs) which are designed to optimize the
therapeutic actions of estrogen receptor activation while minimizing the side
effects [16]. A number of SPPARMs have to date been identified by in vitro and
preclinical studies and some have entered early clinical studies [11, 14] but no reports have been published on any of
these molecules reaching advanced stages of clinical development.Figure 3
Selective PPARγ modulation
separates dose response curves of different PPARγ effects. Left: PPARγ full
agonists activate the range of
receptor responses in a linked fashion. Hence, increasing concentration (or
dose) increases responses in concert. In the clinical setting, higher doses of
TZDs produce greater efficacy as well as greater side effects.
Right: selective PPAR modulation is
response and context-dependent. Depending on the cellular setting and the
response being measured, SPPARM activity may have different potency (top) or
different maximal activity (efficacy, bottom) compared to a full agonist.
Hence, increasing concentration (or dose) may lead to increases in some
responses without linked increases in others. This offers the potential in the
clinical setting for separation of antidiabetic efficacy from side effects such
as edema and weight gain.


## 3. Int131 Specific Design and Development for Molecular and in Vitro SPPARM Activity

INT131 (formerly T0903131,
T131, AMG131) was developed focusing on a strategy to design a SPPARM which would bind to PPARγwith high affinity
but could potentially activate only a subset of the full spectrum of
activities. Such a specifically designed molecule would thereby retain the
antidiabetic actions of full PPARγ agonists such as rosiglitazone and
pioglitazone but would have minimal, if any, side effects (including weight
gain and fluid retention) caused by these TZDs. In fact, a primary screening assay assed only moieties which antagonized roziglitazone induced activity associated with side effects INT131 was thus designed and
developed as a non-TZD PPARγ modulator which represents a new chemical
class of PPARγ ligands. INT131 binds to PPARγ within the same binding pocket as the TZDs,
but occupies a unique space in the pocket and contacts the receptor at distinct
points from the TZDs [17]. Importantly, the interaction with
the activation helix of PPARγ by INT131 and by TZDs differs. The net
result of the different binding by the two types of ligands is alternative
conformational change of PPARγ, leading to distinct patterns of association
with cofactors by this nuclear receptor, and thus ultimately to unique patterns
of gene transcription [15, 17].INT131 binds to
PPARγ and displaces rosiglitazone with a Ki of ~10 nM [17], demonstrating ~20-fold higher affinity than either rosiglitazone or
pioglitazone [18], and with greater than 1000-fold
selectivity for PPARγ over PPARα, PPARδ, or a set of other nuclear receptors [17].
Characterization beyond binding reveals that selected PPARγ receptor activities are induced by INT131. In
a cell-based reporter assay designed to detect full agonist activity, INT131
activates PPARγ with an efficacy of only about 10% of that of
rosiglitazone (Figure 4(a)). Similarly, in fluorescence
resonance energy transfer assays, INT131 causes recruitment of coactivator
DRIP205, which is important for adipocyte differentiation, with an efficacy of
about 20–25% of that of a
set of full agonists including rosiglitazone, pioglitazone, and troglitazone
(Figure 4(b)). Consistent with its high potency, selective activity profile in
the full agonist cell-based reporter and FRET assays, INT131 causes little
adipocyte differentiation or triglyceride accumulation in cultured mouse (Figure
4(c)) or human preadipocytes [17, 19]. Moreover, INT131 blocks most of the
potent effects of rosiglitazone to promote fat cell differentiation [17]. Thus,
INT131 shows selectivity among the full spectrum of PPARγ effects and has the desired, nonadipogenic
profile.PPARγ full agonists, but
not INT131, activate expression of a full agonist reporter gene, induce recruitment
of DRIP205 coactivator peptide to PAPRγ, and
cause lipid accumulation. (a) An
expression construct bearing a PPAR response element designed to be activated
by PPARγ full agonists was used to detect reporter
gene expression. Transfected HEK cells were exposed to a range of
concentrations of the indicated PPAR ligands, and expression measured. The
maximal expression stimulated by INT131 was about 10% that promoted by
rosiglitazone, pioglitazone, troglitazone, farglitazar, and BPx. (b) A homogenous time-resolved
fluorescence energy transfer (FRET) assay was used to measure association of a
DRIP205 coactivator peptide to PPARγ upon exposure to a range of concentrations
of the indicated PPAR ligands. The maximal association stimulated by INT131 was
about 20–25% that was promoted
by rosiglitazone, pioglitazone, troglitazone, farglitazar, and netoglitazone.
(c) Lipid accumulation was measured in
murine preadipocytes exposed to a range of concentrations of the indicated PPAR
ligands. The maximal lipid accumulation stimulated by INT131 was about 10% that
was promoted by rosiglitazone, pioglitazone, troglitazone, farglitazar, and BPx,
Data on file.
(a)(b)(c)PPARγ activation by a SPPARM is predicted to be
context-dependent. Maximal activity of INT131 is sensitive to cellular
environment of PPARγ. That is, using the same reporter
construct and assay designed to detect PPARγ full agonist activity, INT131 potency and
efficacy may be less than, equal to, or greater than the comparator full
agonists rosiglitazone depending on the host-cell type
(Figure 5).PPARγ activation by
INT131 is cell-type-dependent. Cell-based reporter assays were performed
by transfecting three different cell types (HEK293, CV-1, CHO) with the same
reporter construct and stimulating with increasing concentrations of
rosiglitazone (black) or INT131 (red). Adapted from [17].
(a)(b)(c)


## 4. Pharmacology of Int131 is Consistent with SPPARM Activity

INT131 is potent
and highly efficacious in animal models of diabetes, but causes much less
weight gain and volume expansion than marketed TZDs. For example, in Zucker
fatty rats, a standard rodent model of T2DM, INT131 was more potent than
rosiglitazone in reducing serum glucose
(Figure6), insulin, triglyceride, and
NEFA concentrations and in improving glucose tolerance [17]. Notably, INT131 increased levels
of the adipokine adiponectin in the Zucker fatty rat model and in normal rats
with equal or greater potency than does rosiglitazone
(Figure 7). Adiponectin levels are suppressed in obesity
and in T2DM, and increased adiponectin production is thought to be a key
mediator for the insulin sensitizing and anti-inflammatory effects of PPARγ [20].Figure 6
Glucose level in
Zuker fatty rat is reduced
in response to either INT131 or rosiglitazone treatment. Fourteen-day treatment with the indicated
daily oral dose of INT131 or rosiglitazone
in increasing doses reduce glucose levels. Adapted from
[17, 19].Figure 7
Adiponectin
levels increase in a dose responsive manner following either INT131 or
rosiglitazone treatment. Zucker fatty rats were treated orally with the
indicated dose of INT131 or rosiglitazone once daily for 15 days, and plasma
adiponectin was measured.P<0.05 compared to vehicle group adapted from [17, 19].In a variety of
animal models, full agonists cause fluid retention and increased heart weight,
probably as a result of the increased cardiac load caused by plasma volume
expansion. As expected, administration of rosiglitazone to Zucker diabetic
fatty rats for two weeks caused a significant decrease in hematocrit, a marker
for increased plasma volume expansion (Figure8(a)); increase in heart weight
(Figure 8(b)); and increased lung weight
(Figure 8(c)) consistent with a
secondary effect to cardiac hypertrophy and developing CHF. INT131 at the same supratherapeutic dose did not cause these effects. Thus, SPPARM activity
is observed in this rodent model of T2DM, and the antidiabetic effects of PPAR
activation have been separated from fluid retention and adverse cardiac
effects.INT131 does not increase plasma volume,
heart weight, or lung weight. Zucker
fatty rats were treated orally once daily for 14 days with 80 mg/kg/day of
INT131 or rosiglitazone (n=6/group). (a) Hematocrit, (b) heart weight,
and (c) lung weight were measured,
and organ weights normalized to body weight. Adapted from [17, 19].
(a)(b)(c)


## 5. Toxicology of Int131 Demonstrates a Safety Profile Distinct from Tzds and Consistent with a SPPARM

Preclinical
safety experience with PPARγ full agonists has produced a consistent
profile of target mediated effects. Prominent among these are: fluid retention
as manifested by a drop in hematocrit and related hematological measures of
increased plasma volume as well as in edema; weight gain due to increased
adipose tissue together with fluid retention; cardiac hypertrophy and heart
failure; and fatty infiltration and replacement of bone marrow. Appearance of
these adverse effects follows a predictable steep time and dose relationship in
multiple species (Figure 9, [21]), and has been predictive of
clinical experience. Therefore, preclinical results from subchronic and chronic
safety studies take on heightened importance for PPAR ligands in clinical
development. Based on experience with many PPAR full agonist programs, the 2008
FDA draft guidance for development of diabetes drugs [22] includes specific recommendations
for preclinical studies with PPAR ligands. These include detailed measures to
detect cardiac changes, fatty infiltration of organs, and fluid retention.
According to the draft guidance, appearance of safety signals in preclinical
programs which have been predictive of clinical safety issues for other PPAR
ligands could lead to a requirement for more detailed clinical safety studies or
outcome studies prior to approval.Figure 9
Preclinical
LOAEL for PPARγ full agonists follows a steep time dependence. The lowest observable adverse effect level
(LOAEL) at various exposure times in mouse, rat, and monkey is depicted for
aggregate data for the class and expressed as multiple of clinical exposure level.
Data from [21].INT131 is well
tolerated in rats treated for 6 months with doses resulting in up to two to
three orders of magnitude greater exposure than exposure attained at efficacious
clinical doses in humans. Of particular note was the lack of the toxicities
characteristic of PPARγ full agonists, including signs of fluid
accumulation or increased heart weight at doses representing these high safety
multiples. These adverse effects are typically observed at or near efficacious
exposure levels for potent PPARγ full agonists. Thus, the therapeutic
window for INT131 is predicted to be significantly greater than it is for the
older classes of compounds.Safety testing of
INT131 in cynomolgus monkeys for one and six months at exposures up to >70-fold
(highest dose and duration tested) over the exposures expected at the highest
dose in the ongoing clinical development program showed that all doses were
well tolerated. Confirming the rat safety study results, typical PPAR full agonist
effects such as fluid retention, increased adiposity, fatty replacement of
marrow, or cardiac changes detected by echocardiography, pathology, or
histology were not observed in INT131 treated monkeys.An additional area
of concern for the general PPARligand class of compounds is carcinogenicity. In
July 2004, FDA provided guidance regarding preclinical and clinical safety
assessments for any molecules in clinical development affecting PPAR superfamily
members. Cumulative rodent data reviewed by the agency for a number of PPARγ dual α/γ agonists in development had shown an increased
incidence of carcinogenicity. Based on these data, the FDA mandated that
clinical dosing could not exceed six months with any PPAR ligand (α, γ, δ, α/γ dual, or α/γ/δ pan agonist) unless two-year rodent
carcinogenicity studies were completed and satisfactorily reviewed by the
agency.SPPARMs such as INT131
would appear to be at lower risk for demonstrating carcinogenic activity than
PPARγfull
agonists and dual
PPARα/γ agonists (Figure 10) for several reasons.
First, many of the PPAR binding molecules that caused tumors in the rodent
studies were PPARα/γ dual agonist with which multispecies,
multitissue, and both-sex tumor incidence occurred [23]. INT131 is highly selective for PPARγ, with no binding to PPARα or δat
10 μM, 1000 fold over the Ki for PPARγ [19].Figure 10
Carcinogenicity
risk in relation to spectrum of PPAR activation. Propensity for demonstrating
carcinogenic activity is predicted to increase with broader PPAR activating
activity.While
carcinogenicity is less of a concern for PPARγ agonists than for PPARα or α/γ dual agonists, the two most prevalent
types of tumors associated with PPARγ full agonist molecules which do occur are lipomas
and hemangiosarcomas. These cancers derive from adipose tissue and vascular
endothelium, respectively. Since INT131 shows little propensity to promote
adipocyte differentiation in vitro or adipose proliferation in vivo, it would
be reasonable to expect that INT131 would convey minimal, if any, risk for
these malignancies. Similarly, the lack of edema in preclinical models suggests
a weak activity in the vascular endothelium and thus would be unlikely to invoke
the activation associated with hemangiosarcomas at very high doses of full PPARγ agonists. Taken together, it is likely that
selectivity of a SPPARM such as INT131 will reduce the potential for
carcinogenicity that plague PPAR full agonists, but this remains to be conclusively
shown by ongoing studies.


## 6. Early Clinical Results with Int131 Show Separation of Efficacy from Side Effects

Four Phase 1 studies
have demonstrated that INT131 besylate is well tolerated and has highly
desirable pharmacokinetic and pharmacodynamic properties. The rapid and robust
stimulation of adiponectin levels (Figure11) provides evidence of activation
of PPARγ pathways associated with therapeutic efficacy,
confirming preclinical pharmacology results [15].Figure 11
Adiponectin level is stimulated in human subjects exposed to INT131. Circulating
adiponectin was measured in healthy volunteers exposed to 8 consecutive daily
fixed oral doses of INT131. Adapted from [24].A 4-week Phase 2a
multicenter, randomized, double blind, placebo controlled study was conducted
to establish the glucose lowering activity of INT131 besylate in subjects with
T2DM. INT131 was well tolerated, with no significant safety signals [19]. A reduction in fasting plasma glucose (the primary endpoint
of the study) was observed at week 1 and week 4, unusually early for this mechanism of
action, and was statistically significant despite the short duration of
treatment. Stimulation of adiponectin
levels, seen in healthy volunteers in Phase I, was confirmed in the T2DM
population in the Phase 2a study. Most notably, the SPPARM activity of INT131
was supported by separation of the observed antidiabetic effects from edema and
weight gain, differentiating INT131 from TZD PPARγ full agonists. These results provided the foundation
for an ongoing multicenter double blind placebo controlled Phase 2b study of 4
doses of INT131 and pioglitazone comparator in T2DM patients, which is designed
to rigorously test the SPPARM activity of INT131 for separation of PPARγ mediated efficacy in treating insulin
resistance from TZD side effects.


## 7. Conclusion

The non-TZD
selective PPARγ modulator INT131 is the culmination of a
molecular target-based strategy to develop an improved insulin-sensitizing drug
that does not cause the weight gain and edema that plague the PPAR full agonists.
As predicted by its unique PPARγ profile, INT131 shows potential as a potent
and efficacious insulin-sensitizing molecule in T2DM patients that causes
little if any weight gain at therapeutically efficacious doses. This emerging clinical
profile of efficacy/side-effect separation is consistent with the
underlying molecular biology design, the in vitro study data and the robust
preclinical data. It thus represents the final part of an accordant continuum testing
the hypothesis that selective modulation of PPARγ can create a clinically relevant
therapeutic window which is hoped to eventually provide tangible benefits to patients.



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