A novel intestinal-restricted FXR agonist

Article abstract of DOI:10.1016/j.bmcl.2017.06.003

In this study, a new intestinal-restricted FXR agonist named fexaramine-3 (Fex-3) was developed and investigated both in vitro and in vivo. Fex-3 could selectively activate intestinal FXR and promote the expression of BSEP and SHP while suppressing CYP7A1 which is involved in bile acids syntheses better than the reported intestinal-restricted FXR agonist fexaramine (Fex). We demonstrated that Fex-3 targeted on FXR in ileum and has better selectivity than Fex. And the study of utilizing Fex-3 to reduce obesity was undergoing.

Full text of DOI:10.1016/j.bmcl.2017.06.003

Accepted Manuscript
A novel intestinal-restricted FXR agonist
Hong Wang, Zhou Zhao, Jiyu Zhou, Yitong Guo, Guangji Wang, Haiping Hao,
Xiaowei Xu
PII:
S0960-894X(17)30603-0
DOI:
http://dx.doi.org/10.1016/j.bmcl.2017.06.003
BMCL 25037
Reference:
To appear in:
Bioorganic & Medicinal Chemistry Letters
Received Date:
Revised Date:
Accepted Date:
3 March 2017
12 May 2017
2 June 2017
A novel intestinal-restricted FXR
Please cite this article as: Wang, H., Zhao, Z., Zhou, J., Guo, Y., Wang, G., Hao, H., Xu, X.,
agonist, Bioorganic & Medicinal Chemistry Letters (2017), doi: http://dx.doi.org/10.1016/j.bmcl.2017.06.003
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Bioorganic & Medicinal Chemistry Letters
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A novel intestinal-restricted FXR agonist
a,b,‡
Hong Wang , Zhou Zhao , Jiyu Zhou , Yitong Guo , Guangji Wang , Haiping Hao
a,b,‡
a,b
a,b
a,b
a,c,*
a,b,*
and Xiaowei Xu
a
State Key Laboratory of Natural Medicines, China Pharmaceutical University, Tongjiaxiang#24, Nanjing 210009, China
b
c
Key Lab of Drug Metabolism and Pharmacokinetics, China Pharmaceutical University, Tongjiaxiang#24, Nanjing 210009, China
School of Pharmacy, China Pharmaceutical University, Tongjiaxiang#24, Nanjing 210009, China
ARTICLE INFO
ABSTRACT
Article history:
Received
In this study, a new intestinal-restricted FXR agonist named fexaramine-3 (Fex-3) was
developed and investigated both in vitro and in vivo. Fex-3 could selectively activate
intestinal FXR and promote the expression of BSEP and SHP while suppressing CYP7A1
which is involved in bile acids syntheses better than the reported intestinal-restrcted FXR
agonist fexaramine (Fex). We demonstrated that Fex-3 targeted on FXR in ileum and has
better selectivity than Fex. And the study of utilizing Fex-3 to reduce obesity was
undergoing.
Revised
Accepted
Available online
Keywords:
Farnesoid X Receptor
Agonist
Intestinal-restricted
fexaramine
2009 Elsevier Ltd. All rights reserved.
Farnesoid X Receptor (FXR), as a member of nuclear receptor superfamily, is a vital transcriptional factor encoded by
1
2
Nr1h4 in mouse and NR1H4 in human . In 1995, farnesol metabolites were identified as activators of FXR . Endogenous
3,4,5
ligands of FXR were bile acids which were identified by three groups independently in 1999 . FXR is abundantly
expressed in organs involved in the enterohepatic circulation of bile acids including liver and intestine. In addition to liver
2
and intestine, FXR is also expressed in other tissues, such as the adrenal gland, kidney, stomach and adipocytes . FXR
subfamily includes two members, FXRα and FXRβ. FXRβ, also known as NR1H5,
*
Corresponding author. E-mail address: xw@cpu.edu.cn (Xiaowei Xu);haipinghao@cpu.edu.cn (Haiping Hao).
‡
These authors contribute equally to this work.
6
encoded by NR1H5P which was pseudogene in humans, is not a bile acid-activated receptor .
As the most dedicated bile acids receptor, FXR plays a pivotal role in the homeostasis of bile acids. Activation of FXR
7,8,9
in the liver suppresses the synthesis of bile acids and reduces the bile acids pool size . FXR controls the synthesis of bile
1
acids via transcriptional induction of the expression of fibroblast growth factor 19 (FGF19) and small heterodimer
0
11
partner (SHP) . In addition to the maintenance of bile acids homeostasis, FXR also takes control of lipids, glucose and
cholesterol metabolism in the liver and intestine. In wild type but not in FXR-null mice, fasting-induced FXR expression
1
led to decreased plasma triglyceride levels . In diabetic mice, activation of FXR by synthetic agonist improves
2
1

1
hyperglycemia and hyperlipidemia . Obesity and insulin resistance are emerging as the major contributors of dysfunction
3
or diseases of cardiac, vascular, liver and other system. There is an urgent demand for drug discovery to reduce obesity and
1
insulin resistance .
4
Over the past decade, FXR agonists have emerged in large numbers. Some endogenous bile acids including CDCA,
3
DCA, LCA and CA are FXR agonists . In addition, several semi-synthetic bile acids were determined to be potent FXR
1
5
16
agonists, such as 6-ECDCA and TC-100 . Among the semi-synthetic FXR agonists, 6-ECDCA is the most exemplary
1
7,18
one which has entered into a number of clinical trials
and has been approved for treatment of Primary Biliary
1
Cholangitis . A number of synthetic FXR agonists have been reported, including fexaramine, GW4064, PX-102, XL335
9
2
0,21,22,23,24
and GSK2324
.
2
Solution and solid-phase synthesis led to the discovery of Fex . Fex is poorly absorbed into circulation when delivered
1
2
orally . In contrast to systemic agonism of FXR, Fex did not activate FXR downstream genes in the liver. Orally delivered
5
Fex improved metabolic profile of high-fat diet induced obesity, including weight gain, increased insulin sensitization,
2
6
decreased inflammation and browning of white adipose tissue . Gut-restricted FXR agonism led to whole-body benefits.
These results suggested that intestinal FXR activation would be a promising and safer treatment for obesity, insulin
2
6
resistance and metabolic syndrome . To further improve the intestinal-restricted property, a series of Fex derivatives were
designed and synthesized by our group. Herein, we reported the Fex derivatives for the treatment of diet-induced obesity in
mice with more potent gut-restricted FXR activation.
Scheme 1 The Structures of Fex-1, Fex-2, Fex-3, Fex-4 and Fexaramine.
The synthesis routes of these compounds were showed in scheme S1 and scheme S2. Firstly, we investigated the agonist
effect of these compounds in vitro. We performed the experiments on Caco-2 and HepG-2 cells but the results showed no
significant agonist effect on FXR (data not shown). We speculated that the two cell lines may not be suitable for detection
of agonist effect on FXR. Then rat primary hepatocytes were utilized for in vitro study. We found that Fex-1 and Fex-2 had
no agonist effect on rat primary hepatocytes. However, from Figure 1, Fex-3 and Fex-4 could significantly promote the
mRNA expression of BSEP and SHP and suppress CYP7A1, all of which are the target genes of FXR. From this results we
2

supposed that the biphenyl group was very important as Fex-3 and Fex-4 kept this group but Fex-1 and Fex-2 had this
group replaced by naphthoyl-group and this was consistence with the reported literature (Scheme 1). Compared with Fex,
the results elucidated that Fex-3 and Fex-4 could also act as FXR agonists. As Fex-3 was a little better than Fex-4, we
focus on Fex-3 in the following experiments. Through MTT assay, Fex-3 has no cell cytotoxicity even at 100 µM (Figure
S14).
Figure 1 The mRNA level changes of BSEP, CYP7A1, FXR and SHP after treating on the rat primary hepatocytes with Fex-3, Fex-4 and
Fex (50 µM). This results were repeated three times with n = 3 per group. ** P<0.01.
Since our new compound Fex-3 could be a new FXR ligand, we try to demonstrate that Fex-3 was an intestinal-
restricted FXR agonist which only activated FXR in the intestine not in the liver or other organs. Initially, we investigated
this in Caco-2 cells with transwell experiments. From figure 2 and table 1, we found that the accumulative amount of Fex-
3
and Fex in cell was nearly the same but the penetrating velocity was different and Fex was faster than Fex-3 obviously.
In addition, Fex uptaking amount increased during the time while Fex-3 went to platform at about 3 hours (Figure S17).
The concentration curves also show that Fex-3 could be absorbed into Caco-2 cells more easily than Fex (Figure S18).
These results indicated that Fex-3 was more intestinal-restricted than Fex, inferred to that Fex-3 could not enter into
circulation.
Figure 2 Uptake of Fex and Fex-3 in Caco-2 cells.
Papp
Papp
Efflux Ratio
0.87
-
AP-BL(×10 cm/s)
6
-6
BL-AP(×10 cm/s)
Con(µM)
Fex
20
27.67±5.08
23.99±5.02
3

5
2
5
0
0
0
761.7±137.39**
18.72±10.38
97.60±32.51
873.2±187.87**
6.62±0.26
1.14
0.35
0.77
Fex-3
75.00±9.61
Table 1 Transwell results of Fex and Fex-3 in Caco-2 cells within two hours. AP-BL means the velocity of compounds penetrating into the
cells, BL-AP is the velocity of compounds going out of the cells ** P < 0.01, n = 3.
With the results obtained from the above in vitro experiments we tried to explore the ability of Fex-3 in vivo. After one
week accommodating, the mice were treated with Fex or Fex-3 (control with vehicle) for 5 days (the doses of Fex and Fex-
-
1
are both 100 mg kg ) and sacrificed 1 hour after the final treatment with tissues (liver, kidney, duodenum, jejunum and
3
ileum) and serum collected. We measured the concentration of Fex and Fex-3 in jejunum, ileum, liver and plasma by LC-
MS/MS. From figure 3, we found that Fex accumulated mostly in ileum, then in jejunum, but nearly in liver and serum
which was consistent with the reported literature. Interestingly, Fex-3 only accumulated in the ileum and the detectable
drug concentration Fex-3 was obviously higher than Fex. This result might demonstrate that Fex-3 could be a better
intestinal-restricted FXR agonist than Fex.
Figure 3 Fex and Fex-3 concentration in jejunum, ileum, liver and plasma from p.o. treated mice.
Next, the mRNA expression of FXR in duodenum, jejunum, ileum, liver and kidney were detected by PCR. The FXR
mRNA level in Fex-3 treated mouse ileum was significantly higher than vehicle (Fig 4). Fex-3 had no influence on the
FXR mRNA expression in other organs, including the liver. This was consistent with the results shown in Fig 3, which
means that Fex-3 mainly activated FXR of the ileum.
Figure 4 FXR mRNA expression in different organs after treated with Fex-3 for 5 days.
In order to explain that why Fex-3 exhibits better intestinal selectivity, we calculated the LogP of Fex and Fex-3 out as
4

7
.01 and 7.42. Since Fex-3 has the higher LogP value, we supposed that the solubility of Fex-3 in aqueous solution was
poorer than Fex. The calculation results implied that Fex-3 would accumulate in the intestinal epithelial cells and does not
enter into the blood. However, we found that Fex-3 could not be dissolved in DMSO/mineral oil medium and the drug
dissolved out and was adhered to the intestine in i.p. treated mice. Hence, the results from i.p. treated mice could not be
used for elucidating our hypothesis.
Figure 5 Induction of FXR target genes after p.o treated with Fex-3 and Fex.* P < 0.05, ** P < 0.01, n = 6-8 per group.
-1
Then in exploring the in vivo effects, we discovered that Fex-3 p.o. treated (100mg kg for 5 d) produced robust
induction of the FXR target gene Shp in the ileum. However, Fex-3 cannot induce the expression of Shp in the liver, kidney
and other part of intestine. Additionally, we found that Fex-3 induced FXR downstream genes Shp and Bsep significantly
than Fex while suppressed the expression of Cyp7a1 (Figure 5). We could make a conclusion that Fex-3 works only in the
ileum and exhibits better tissue-selectivity than Fex.
Figure 6 Molecular docking by Discovery Studio v4.0. Fex-3(purple, A) and superimposition with Fex (green, B).
To investigate the binding mode between Fex-3 and FXR, molecular docking was conducted based on the previously
reported co-crystal structure of Fex and FXR (PDB ID: 1OSH). The green (Fex) and purple (Fex-3) structures occupy
5

nearly the same cavity of FXR (Figure 6). However, Fex-3 could stick into the protein inner than Fex as shown in the
figure. For further investigation into the binding affinity of Fex and Fex-3 with FXR, the K value was measured by
d
fortebio. As shown in Figure S18, the binding affinity of Fex-3 with FXR was significantly higher than Fex with nearly
two order of magnitude.
In summary, we have successfully designed and synthesized a novel intestinal-restricted FXR agonist Fex-3. In
comparison with Fex, Fex-3 exhibits slightly higher FXR agonism activity and much better selectivity in the ileum.
Intestinally restricted not systemic FXR activation leads to decrease in obesity and insulin resistance and other whole-body
benefits. To further investigation into in vivo efficacy of Fex-3, animal experiments are being conceived and designed.
Acknowledgments
This work was supported by the Natural Science Foundation of China (Nos. 21602254, 81603194), the Natural Science
Foundation of Jiangsu province, China (BK20160767) and National Found for Fostering Talents of Basic Science
(
NFFTBS, J1310032).
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1
2
3
. A new FXR agonist Fex-3 was developed.
. Fex-3 could selectively activate intestinal FXR and promote the expression of BSEP and SHP while suppressing CYP7A1.
. We demonstrated that Fex-3 targeted on FXR in ileum and has better selectivity than Fex.
9

Graphical Abstract
A novel intestinal-restricted FXR agonist
Hong Wang , Zhou Zhao , Jiyu Zhou , Yitong Guo ,
Leave this area blank for abstract info.
a,b,‡
a,b,‡
a,b
a,b
a,b a,c,*
Guangji Wang , Haiping Hao and Xiaowei Xu
a,b,*
a State Key Laboratory of Natural Medicines, China Pharmaceutical University, Tongjiaxiang#24, Nanjing 210009, China
b Key Lab of Drug Metabolism and Pharmacokinetics, China Pharmaceutical University, Tongjiaxiang#24, Nanjing 210009, China
c School of Pharmacy, China Pharmaceutical University, Tongjiaxiang#24, Nanjing 210009, China
1
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