RXR partial agonist CBt-PMN exerts therapeutic effects on type 2 diabetes without the side effects of RXR full agonists

Article abstract of DOI:10.1021/ml300055n

Treating insulin resistance and type 2 diabetes in rodents, currently known retinoid X receptor (RXR) agonists induce significant adverse effects. Here we introduce a novel RXR partial agonist CBt-PMN (11b), which shows a potent glucose-lowering effect and improvements of insulin secretion and glucose tolerance without the serious adverse effects caused by RXR full agonists. We suggest that RXR partial agonists may be a new class of antitype 2 diabetes drug candidates.

Full text of DOI:10.1021/ml300055n

Letter
pubs.acs.org/acsmedchemlett
RXR Partial Agonist CBt-PMN Exerts Therapeutic Effects on Type 2
Diabetes without the Side Effects of RXR Full Agonists
Hiroki Kakuta,*,† Nobumasa Yakushiji,† Ryosuke Shinozaki,† Fuminori Ohsawa,† Shoya Yamada,†
Yui Ohta,† Kohei Kawata,† Mariko Nakayama,† Manabu Hagaya,† Chisa Fujiwara,† Makoto Makishima,‡
Shigeyuki Uno,‡ Akihiro Tai,§ Ami Maehara,∥ Masaru Nakayama,∥ Toshitaka Oohashi,∥ Hiroyuki Yasui,⊥
and Yutaka Yoshikawa⊥
†Division of Pharmaceutical Sciences, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences,
1-1-1 Tsushima-Naka, Okayama 700-8530, Japan
‡Division of Biochemistry, Department of Biomedical Sciences, Nihon University School of Medicine, 30-1 Oyaguchi-kamicho,
Itabashi-ku, Tokyo 173-8610, Japan
§Faculty of Life and Environmental Sciences, Prefectural University of Hiroshima, 562 Nanatsuka-Cho, Shobara, Hiroshima 727-0023,
Japan
∥Division of Medical Sciences, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, 2-5-1
Shikata-Cho, Kita-Ku, Okayama 700-8558, Japan
⊥Kyoto Pharmaceutical University, 5 Nakauchi-cho, Misasagi, Yamashina-ku, Kyoto 607-8414, Japan
S
* Supporting Information
ABSTRACT: Treating insulin resistance and type 2 diabetes
in rodents, currently known retinoid X receptor (RXR)
agonists induce significant adverse effects. Here we introduce a
novel RXR partial agonist CBt-PMN (11b), which shows a
potent glucose-lowering effect and improvements of insulin
secretion and glucose tolerance without the serious adverse
effects caused by RXR full agonists. We suggest that RXR
partial agonists may be a new class of antitype 2 diabetes drug
candidates.
KEYWORDS: Nuclear receptors, RXR, partial agonists, type 2 diabetes
heterodimers,14 we previously examined the feasibility of
etinoid X receptors (RXRs) are of interest as nuclear
Rreceptors that serve to regulate transcription of genes separating the blood glucose-lowering action of RXR agonists
from the adverse effects. For this purpose, we administered
RXR agonists NEt-TMN (5),15 NEt-3IB (6a), and NEt-3IP
(6b)16 (Figure 1), which similarly activate RXR, but differently
activate PPAR/RXR and LXR/RXR,17 to KK-Ay type 2 diabetes
model mice, and we examined the relationship of the RXR-
heterodimeric activation pattern to the antihyperglycemic effect
and adverse effects such as hepatomegaly and TG elevation.18
We found that 6a has a less potent TG-elevating activity and
hepatomegaly than the other RXR agonists examined, though
all of them showed similar blood glucose-lowering action on
repeated administration. This result suggested that it might be
possible to separate the therapeutic effects from the side effects
of RXR agonists. However, even 6a shows significant TG-
elevation and hepatomegaly. Since all RXR agonists that have
been reported to show serious side effects are RXR full agonists
that potently activate RXR, we hypothesized that there is a
relevant to diabetes. They function either as homodimers or as
heterodimers, for example with peroxisome proliferator-
activated receptors (such as PPARγ, the well-known target of
thiazolidinedione-type agents to improve insulin resistance) or
liver X receptors (LXRs, whose activation induces glucose
metabolism and improves glucose tolerance).1−5 In addition,
LXR activation is reported to induce glucokinase expression,
which is coregulated with insulin,6 and to promote insulin
secretion via stimulation of pancreatic islets.7 So-called
permissive RXR-heterodimers, such as PPAR/RXR and LXR/
RXR, can be activated by RXR agonists alone.8 Therefore, RXR
agonists seem to be promising candidates for improving both
insulin resistance and glucose tolerance. Several RXR agonists
1−4 (Figure 1) have been evaluated for the treatment of insulin
resistance and type 2 diabetes in rodents.9 However, all of them
induce significant adverse effects, such as blood triglyceride
(TG) elevation,10 weight gain,11 hepatomegaly,12 and hypo-
thyroidism.13
Received: March 2, 2012
Accepted: April 8, 2012
Published: April 9, 2012
On the basis of a report that structurally different RXR
agonists show different patterns of activation of RXR-
© 2012 American Chemical Society
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ACS Medicinal Chemistry Letters
Letter
In general, representative RXR agonists consist of a
hydrophobic domain including 1,1,4,4-tetramethyltetralin, an
acidic domain bearing benzoic acid, nicotinic acid, or
pyrimidinecarboxylic acid, and a linking domain connecting
these domains, as shown in Figure 2. Our design strategy to
Figure 3. Results of reporter gene assays of 5, 11a, and 11b. COS-1
cells were transfected with three kinds of vectors consisting of a RXR
receptor subtype, a luciferase reporter gene under the control of the
appropriate RXR response element (CRBPII-tk-Luc), and secreted
alkaline phosphatase (SEAP) gene as a background. (a) RXRα, (b)
RXRβ, and (c) RXRγ, based on the luciferase activity of 1 μM 1 (RXR
full agonist) taken as 1.0. Circles, triangles, and squares indicate 5, 11a,
and 11b, respectively. The data (n = 3) represent the mean sem.
Data for NEt-TMN were taken from ref 18, because these experiments
were performed at the same time.
Figure 1. Chemical structures of known RXR ligands.
threshold difference between the therapeutic effects and
adverse effects of RXR activation. Therefore, we decided to
focus on RXR partial agonists, which would activate RXR only
moderately or whose transcriptional efficacy would be limited.
We further considered that this idea might be consistent with
reports that RXR antagonists HX531 (7)19 and danthron (8)20
(Figure 1) show blood glucose-lowering effects in type 2
diabetes model mice, because partial antagonists are also
thought to behave as partial agonists.
To test our hypothesis, we aimed to synthesize RXR partial
agonists by modifying the representative RXR agonist structure,
focusing on restriction of the molecular flexibility by linking the
hydrophobic or acidic domain and the linking domains to form
a new ring moiety (Figure 2). Screening of the synthesized
Figure 4. Relative transactivation activities of 5, 11a, and 11b toward
PPARγ, PPARγ/RXRα, LXRα, and LXRα/RXRα. COS-1 cells were
transfected with four kinds of vectors, consisting of RXRα, a partner
receptor (PPARγ or LXRα), the partner response element (tk-
PPREx3-Luc for PPARγ or tk-rBARx3-Luc for LXRα), and secreted
alkaline phosphatase (SEAP) gene as a background. (a) Chemical
structures of TIPP70321 (PPAR pan-agonist) and carba-T090131722
(LXR pan-agonist). (b) Relative transactivation data for PPARγ, based
on the luciferase activity of 1 μM TIPP703 taken as 1.0. (c) Relative
transactivation data for PPARγ/RXRα, based on the luciferase activity
of 1 μM TIPP703 taken as 1.0. (d) Relative transactivation data for
LXRα, based on the luciferase activity of 1 μM carba-T0901317 (LXR
pan-agonist) taken as 1.0. (e) Relative transactivation data for LXRα/
RXRα, based on the luciferase activity of 1 μM carba-T0901317 taken
as 1.0. TIPP703 or carba-T0901317 at 1 μM give each Emax value.
Circles, triangles, and squares indicate 5, 11a, and 11b, respectively.
The data (n = 3−6) represent the mean sem.
Figure 2. Molecular design strategy for creating RXR partial agonists.
compounds identified CBt-PMN (11b) as a novel RXR partial
agonist. Studies in mice and rats showed that 11b did not
induce the side effects typically caused by RXR full agonists,
while oral administration to KK-Ay mice, a widely used model
of type 2 diabetes, resulted in a potent glucose-lowering effect
and improved insulin secretion and glucose tolerance. These
results indicate that RXR partial agonists may be a new class of
antitype 2 diabetes drug candidates without the serious adverse
effects shown by RXR full agonists. In this article, we describe
the molecular design strategy and the results of the in vitro and
in vivo experiments, and we discuss the mechanism of action of
11b.
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ACS Medicinal Chemistry Letters
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Figure 5. Evaluation of antitype 2 diabetes effects of repeated oral administration of 5 or 11b at 10 mg/kg/day to male KK-Ay mice for 14
consecutive days. (a) Time course of blood glucose levels. (b) Time course of body weight change. (c) Results of oral glucose tolerance tests
(OGTT) in KK-Ay mice treated with vehicle and compounds. (d) Effects of compounds on serum insulin levels. (e) Effects of compounds on serum
HbA1c levels. (f) Effects of compounds on adiponectin levels. (g−i) Effects of compounds on liver weight, serum triglyceride, and total cholesterol,
respectively. The white, gray, and black bars indicate vehicle, 5, and 11b treatment, respectively. The data (n = 3−7) represent the mean sem. Data
for vehicle control and 5 were taken from ref 18, because these experiments were performed at the same time. Statistical analysis was performed by
analysis of variance (ANOVA). Significant differences: * p < 0.05 vs vehicle. ** p < 0.01 vs vehicle.
obtain RXR partial agonistic activity was to form a ring between
the hydrophobic or acidic domain and the linking domain to
decrease the molecular flexibility.
presence of increasing concentrations of 11b, indicating that
11b acts as a partial antagonist (Supporting Information Figure
S1). In addition, 11b showed moderate RAR activation
(Supporting Information Figure S2). These results support
the idea that 11b acts as a RXR partial agonist.
The activities of 11a and 11b toward PPARγ/RXRα and
LXRα/RXRα were next examined, with RXR full agonist 5 as a
positive control.18 None of the compounds showed PPARγ
activity, but compounds 11a and 11b showed similar PPARγ/
RXRα activation, though they were both less potent than 5
(Figure 4b,c). Compounds 11a and 11b also activated both
LXRα and LXRα/RXRα less potently than 5 (Figure 4d,e).
Since these compounds showed both PPARγ/RXRα and
LXRα/RXRα agonistic activities in vitro, we next examined
their in vivo activities.
Compounds were synthesized as illustrated in Schemes S1
and S2 (see Supporting Information) and evaluated by
measuring their RXR-agonistic activities in reporter-gene assays.
Although the compounds possessing ring structures between
the hydrophobic and the linking domains did not show RXR
agonistic activities, compounds 11a and 11b, in which the ring
was formed between the acidic and the linking domains, did
show agonistic activity (Supporting Information Table S2).
Interestingly, while 11a, possessing a 2-methylimidazole
structure, showed full agonistic activity, 11b, possessing a
triazole structure, showed only partial agonistic activity toward
RXRα (EC50 = 143 nM, Emax = 75%) (Figure 3a and
Supporting Information Table S2). The RXR partial agonist
activity of 11b is thought to be due to not only the closed ring
structure but also other factors, including the polarity/positions
of the nitrogen atoms. Moreover, 11b behaved similarly toward
other RXR subtypes (Figure 3b,c). The RXR agonistic activity
of the full agonist LGD1069 (1) at 1 μM was reduced in the
First, it was confirmed that oral single administration of each
compound at 30 mg/kg resulted in a serum concentration over
1 μM in ICR male mice (Supporting Information Figure S3).
Then, changes in body weight, hepatomegaly, and serum
triglyceride (TG) elevation were assessed when each
compound was administered orally to ICR mice at 30 mg/
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Letter
kg/day for 7 days (Supporting Information Figure S4). The
mice given the RXR full agonists 5 and 11a showed greater
body weight gain than did the vehicle control group, and the
increase was significant in the case of 5. In contrast, the group
treated with 11b showed similar body weight change to the
control group (Supporting Information Figure S4a). Liver
weight was increased significantly by both 5 and 11a, but not by
11b, compared to the control group (Supporting Information
Figure S4b). Compound 11b also did not increase serum TG
and total cholesterol values compared to the control group,
whereas 5 significantly increased the TG level (Supporting
Information Figure S4c,d). Further, when 11b was adminis-
tered orally to male and female SD rats at 30 mg/kg/day for 28
days, no significant difference in body weight change, water
intake, or food intake was observed, compared with the vehicle
control (Supporting Information Figure S5). The testes of
animals treated with 11b were slightly enlarged, but the other
agents had no such effect (Supporting Information Table S4).
As for serum constituents, although some significant differences
from the vehicle were observed, the data were within the ranges
considered normal by the suppliers (Charles River, Ltd.)
(Supporting Information Table S5). Thus, it seems unlikely
that the RXR partial agonist 11b would cause the side effects
associated with RXR full agonists.
Next, we examined the antitype 2 diabetes activity in male
KK-Ay mice. Compound 5 or 11b was orally administered at 10
mg/kg/day for 14 days. The average blood glucose level in
vehicle-treated mice was about 500 mg/dL, while the level in 5-
treated mice was reduced to about 300 mg/dL from day 3 after
the start of administration, showing a significant blood glucose-
lowering effect. Compound 11b also showed a significant blood
glucose-lowering effect, although the lag time was longer than
that in the case of 5 (Figure 5a). Moreover, 11b significantly
reduced serum insulin concentration (Figure 5d) and HbA1c
(hemoglobin A1c, which is correlated with blood glucose levels
over a period of time) (Figure 5e) and produced an
improvement in the oral glucose tolerance test (OGTT)
(Figure 5c), showing significant antitype 2 diabetes effects.
Since 11b improved insulin resistance in KK-Ay mice, we
quantitated adiponectin, which is reported to be associated with
insulin resistance, and found that 5 increased adiponectin
significantly, whereas 11b did so only moderately (Figure 5f).
Since a low adiponectin level is related to insulin resistance,
adiponetin elevation by RXR agonists may be correlated with
their antitype 2 diabetes effects.
Figure 6. Fold changes in mRNA expression of Irs1 (a), Irs2 (b),
Slc2a1 (c), Slc2a2 (d), G6pc (e), Pck (f), Gck (g), Scd1 (h), Fasn (i),
and Srebp1c (j) in the liver tissue of male KK-Ay mice treated with
vehicle, 5, or 11b at 10 mg/kg/day for 14 consecutive days. These
measurements were performed using the same mice as in the case of
Figure 5. The white, gray, and black bars indicate vehicle, 5, and 11b
treatment, respectively. The data (n = 3−7) represent the mean
sem. Statistical analysis was performed by analysis of variance
(ANOVA). Significant differences: * p < 0.05 vs vehicle. ** p <
0.01 vs vehicle.
agonists increase Slc2a1 (GLUT1) and Slc2a2 (GLUT2),
thereby inducing an increase of liver glucose intake.23 Gck
expression is also increased by RXR agonists.23 Other changes
include suppression of G6p and Pck expression, and increase in
expression of Gck (related to glycolysis), Scd1, Fasn, and
Srebp1c, which are associated with increased lipid synthesis
induced by LXR agonists.6 We also examined changes in
expression of Irs1 and Irs2. Neither 5 nor 11b influenced gene
expression of Irs1, Irs2, Slc2a1, Slc2a2, G6pc, and Pck. However,
11b increased Gck expression significantly, indicating that one
of the mechanisms of the glucose-lowering effect of 11b is
induction of glycolysis via Gck. While 5 markedly increased the
gene expression of Scd1 and Fasn, 11b did not. Since Scd1 and
Fasn are associated with lipid synthesis, this result may explain
the lack of serum TG elevation by 11b.
Expression of Gck, Scd1, and Fasn is regulated by
Srebp1c,24−26 but 5 had no effect on the expression of Srebp1c.
It has been reported that Sreb1c expression in liver is
suppressed by adiponectin.27 Therefore, the reason why 5
had no effect on Srebp1c expression may be that it induced a
significant increase of adiponectin, as shown in Figure 6. On the
other hand, the partial agonist 11b activates RXR only
moderately, and this may be sufficient to induce expression
of Srebp1c, which lowers blood glucose, without causing
overexpression of Scd1 and Fasn, which induce lipid synthesis.
These results are consistent with our hypothesis that there is a
threshold difference between the therapeutic and adverse
effects of RXR activation. Although an appropriate low dosage
of a RXR full agonist may show similar beneficial effects to a
RXR partial agonist 11b, in the case of the overdose medical
malpractice, RXR full agonists can cause several adverse effects.
Therefore, RXR partial agonists will be more attractive antitype
2 diabetes drug candidates than RXR full agonists.
We also evaluated adverse effects in male KK-Ay mice treated
with RXR full agonist 5 or RXR partial agonist 11b.
Examination of body weight change, liver weight, serum TG,
and total cholesterol revealed that while 5 induced significant
increases similar to those seen in ICR mice, 11b did not alter
these parameters in comparison with the vehicle (Figure 5b,g−
i). Thus, 11b appears to have a favorable profile of therapeutic
and side effects. The reason why 11b showed antitype 2
diabetes effects at 10 mg/kg p.o. but did not produce significant
side effects even when orally administered at 30 mg/kg/day,
which provides a serum concentration sufficient to produce the
In summary, we hypothesized that there are different
thresholds for the therapeutic effects and side effects of RXR
activation. Therefore, we aimed to synthesize RXR partial
agonists by reducing the molecular flexibility of the
representative RXR agonist structure. As we had hoped, ring
formation between the acidic and linking domains with a
triazole structure afforded a RXR partial agonist 11b. This
compound showed a significant antitype 2 diabetes effect in
KK-Ay diabetic model mice but did not induce the side effects
associated with RXR full agonists in ICR mice or SD rats.
Compound 11b did not induce expression of genes associated
with lipid synthesis, whereas the full agonist 5 did induce
expression of these genes. These results support our hypothesis
Emax, is considered to be its RXR partial agonist character,
though other factors such as differences in timing or
mechanism of action may also be involved.
To address the mechanism of the antitype 2 diabetes activity
of 11b, we examined changes in the expression levels of genes
associated with glucose/lipid metabolism in the liver of KK-Ay
mice by means of RT-PCR (Figure 6). It is reported that RXR
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ASSOCIATED CONTENT
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of selective nuclear receptor modulators: RAR and RXR as a case
study. Nat. Rev. Drug Discovery 2007, 6, 811−820.
General information, synthetic procedures, combustion analysis
data, HPLC charts, luciferase reporter gene assay, and in vivo
experimental procedures. This material is available free of
charge via the Internet at http://pubs.acs.org.
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AUTHOR INFORMATION
■
Corresponding Author
*Tel & Fax: +81-(0)86-251-7963. E-mail: kakuta@pharm.
okayama-u.ac.jp.
Author Contributions
H.K. conceived and designed the project. N.Y., R.S., and M.H.
synthesized compounds. F.O., S.Y., and Y.O. performed
reporter gene assays. M.M. prepared plasmids. K.K., M.N.,
and C.F. performed in vivo experiments with ICR mice and SD
rats. A.T. performed HPLC analysis. Y.Y. and H.Y. performed
in vivo experiments with KK-Ay mice. C.F., S.U., A.M., M.N.,
and T.O. performed PCR analysis. The manuscript was written
by H.K. and F.O.
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Funding
This work was supported by Health and Labour Science
research grants for Research on seeds for Publicly Essential
Drugs and Medical Devices (23080401) from the Ministry of
Health, Labour, and Welfare of Japan. We also thank the
Ministry of Education, Science, Culture and Sports of Japan,
and Takeda Science Foundation for financial support.
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
We thank Dr. Miyachi (Okayama University) for kindly
providing TIPP703 and carba-T0901317. We also thank Dr.
Kagechika (School of Biomedical Science, Tokyo Medical and
Dental University) for kindly providing PA452. The authors are
also grateful to Professor Yoshio Naomoto and Dr. Takuya
Fukazawa (Department of General Surgery, Kawasaki Medical
School) for preparing plasmides. We also thank Dr. Aiba
(Okayama University) for assistance with dissection.
ABBREVIATIONS
■
RXR, retinoid X receptor; PPAR, peroxisome proliferator-
activated receptors; LXR, liver X receptor; RAR, retinoic acid
receptor; TG, triglyceride; RT-PCR, reverse transcriptase
polymerase chain reaction; Irs, insulin receptor substrate;
GLUT, glucose transporter; G6p, glucose-6-phosphatase; Pck,
phosphoenolpyruvate carboxykinase; Gck, glucokinase; Scd1,
stearoyl-CoA desaturase 1; Fasn, fatty acid synthase; Srebp1c,
sterol regulatory element-binding protein 1c
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