Design, Synthesis, and Antifibrosis Activity in Liver of Nonsecosteroidal Vitamin D Receptor Agonists with Phenyl-pyrrolyl Pentane Skeleton

Article abstract of DOI:10.1021/acs.jmedchem.8b01165

Liver fibrosis is characterized by excessive deposition of extracellular matrix (ECM) components and results in impaired liver function. Vitamin D plays a critical role in the development of liver fibrosis as it inhibits transforming growth factor β1 (TGF

Full text of DOI:10.1021/acs.jmedchem.8b01165

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Article
Design, Synthesis and Antifibrosis Activity in Liver of Nonsecosteroidal
Vitamin D Receptor Agonists with Phenyl-pyrrolyl Pentane Skeleton
Cong Wang, Bin Wang, Lingjing Xue, Zisheng Kang, Siyuan Hou, Junjie Du, and Can Zhang
J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jmedchem.8b01165 • Publication Date (Web): 10 Oct 2018
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Design, Synthesis and Antifibrosis Activity in
Liver of Nonsecosteroidal Vitamin D
Receptor Agonists with Phenyl-pyrrolyl
Pentane Skeleton
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Cong Wang a,b1, Bin Wanga,1, Lingjing Xuea, Zisheng Kanga, Siyuan Houa, Junjie Dua,
Can Zhanga,*
aState Key Laboratory of Natural Medicines and Jiangsu Key Laboratory of Drug
Discovery for Metabolic Diseases, Center of New Drug Discovery, China Pharmaceutical
University, 24 Tong Jia Xiang, Nanjing, 210009, China.
bFujian Provincial Key Laboratory of Hepatic Drug Research
*Correspondence should be addressed to:
Tel/Fax: 86-25-83271171; E-mail: zhangcan@cpu.edu.cn
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ABSTRACT: Liver fibrosis is characterized by excessive deposition of extracellular
matrix (ECM) components and results to impairment of liver function. Vitamin D plays a
critical role in the development of liver fibrosis as it inhibits transforming growth factor
β1 (TGFβ1)-induced excessive deposition of ECM in activated hepatic stellate cells
(HSCs). Here, a series of novel nonsecosteroidal Vitamin D receptor (VDR) agonists
with phenyl-pyrrolyl pentane skeleton was designed and synthesized. Among them, seven
compounds including 15a exhibited more efficient inhibitory activity in collagen
deposition and fibrotic gene expression. Histological examination results displayed that
compound 15a treatment prevented the development of hepatic fibrosis that induced by
carbon tetrachloride (CCl4) injections in mice. In addition, compound 15a, unlike the
positive control calcipotriol and 1,25(OH)2D3, did not cause hypercalcemia that is toxic
to nerve, heart and many other organs. These findings provide novel insights into drug
discoveries for hepatic fibrosis using nonsecosteroidal VDR modulators.
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INTRODUCTION
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Liver fibrosis, characterized by excessive accumulation of extracellular matrix (ECM)
and loss of pliability and liver function, is the result of wound-healing responses that
triggered by either chronic or acute liver injury,1-3 such as alcohol abuse, chronic hepatitis
virus (hepatitis B virus/hepatitis C virus) infection, and increasingly, nonalcoholic
steatohepatitis (NASH), nonalcoholic fatty liver disease (NAFLD).4-6 With continuous
injury, the fibrillar collagens were progressively deposited and parenchymal nodules were
surrounded by collagen bands, eventually leading to the histological signature of hepatic
cirrhosis which represents a major global health concern. At present, the only way to treat
the end-stage cirrhosis is liver transplantation.7 However, the condition of the potential
recipients, especially the number of available donor organs, limited the applicability of
this technique even in the developed world.8 Moreover, the Food and Drug
Administration has not yet approved anti-fibrotic therapies for chronic liver disease.9
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Hepatic stellate cells (HSCs) are established as a major cellular source of ECM and
the main driver of liver fibrogenesis. In healthy liver cells, HSCs remains quiescent and
the main function is storing vitamin A.10 Once being activated following liver injury,
HSCs would entered into a α-smooth muscle actin (α-SMA) positive phenotypic
transformation and differentiate into ECM-secreting cells. The activated HSCs produce a
considerable amount of collagen I, which is the main components of ECM, resulting in
the loss of liver pliability and function.11
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Transforming growth factor β1 (TGFβ1) is one of the most potent pro-fibrotic
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modulators. In paracrine and autocrine fashion, TGFβ1 promotes HSCs activation and
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contributes to fibrotic processes in liver.12,
Therefore, the inhibition of TGFβ1
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pathway to reduce ECM production in HSCs is recognized as an effective anti-fibrotic
strategy. While the precise mechanisms of regulating ECM synthesis via TGFβ1 pathway
have yet to be elucidated, vitamin D has been established in close relationship with
TGFβ1 and liver fibrosis development. Previous studies demonstrated a beneficial effect
for 1,25(OH)2D3 (1, Figure 1), the most active form of vitamin D, to attenuate liver
fibrosis.14-16
It is widely recognized that 1,25(OH)2D3 plays a pivotal role in the homeostasis of
calcium and phosphorus, cell proliferation and differentiation, as well as
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immunomodulation.17,
1,25(OH)2D3 exerts actions through promoting gene
transcription by binding to vitamin D receptor (VDR), which belonging to the
superfamily of nuclear receptor. VDR is robust expressed in HSCs and fully functional in
these cells.19 In 2013, Ding et al. reported that calcipotriol, an analog of 1,25(OH)2D3,
could inhibit the collagen I and α-SMA expression via reducing the occupancy of
SMAD3 at these sites and antagonizing a wide range of transcriptional responses on
profibrotic genes that dependent on TGFβ/SMAD signaling pathway.20 These findings
suggest that VDR is an checkpoint to modulate the liver wound-healing response and
VDR ligands may as a potential therapy for the treatment of liver fibrosis.
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VDR ligands have already been attractive therapeutics against psoriasis, osteoporosis,
and cancer.21-23 At present, more than 3000 VDR modulators with secosteroid skeleton
have been synthesized and biologically evaluated as drug candidates,24 such as
calcipotriol (2) and paricalcitriol (3). Almost all of the VDR ligands with high activity
have the same secosteroidal skeleton as 1-3, structurally, consisting of the A-ring that
borne two hydroxyl groups, a triene moiety or conjugated diene, a side chain, and the
CD-ring. Although many compounds exhibit efficient VDR activity in in vitro and in vivo
studies, their synthetic inconvenience, structural complexity, chemical instability, as well
as hypercalcemia limit the clinical application in the treatment of liver fibrosis. Recently,
a lot of attention has been drawn to nonsecosteroidal vitamin D mimics,25-28 such as
bisphenol derivative (4),29 tris-aromatic derivatives (5),21 carborane derivatives (6),30-33
due to their less calcium mobilization and simpler structures. Previously, we have
reported phenyl-pyrrolyl pentane skeleton as a novel nonsecosteroidal VDR ligand
skeleton which possessed the potential to inhibit proliferation of cancer cells without
inducing hypercalcemia effect.34-36 However, we found some compounds had no effect on
cancer cells but show significantly inhibitory effect on HSCs activation, indicating that
VDR agonists may affect HSCs more strongly than cancer cells. Therefore, it is
noteworthy to verify whether these nonsecosteroidal vitamin D ligands can act as
effective as 1,25(OH)2D3 or calcipotriol that with secosteroidal skeleton for preventing
the progression of liver fibrosis but have smaller side effects like hypercalcemia.
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To explore the relationship between structure and anti-fibrotic activity for these
nonsecosteroidal compounds based on the phenyl-pyrrolyl pentane skeleton, twenty-two
new compounds have been designed, synthesized, and examined with various biological
assays. Seven compounds showed much better properties than positive control
calcipotriol in the anti-collagen I synthetic activities assay. Among them, compound 15a
exhibited more potent inhibitory activity against both fibrotic gene expression and
collagen deposition by Q-PCR and western blot assays. Results of histological
examination displayed that the treatment of compound 15a prevented hepatic fibrosis
induced by carbon tetrachloride (CCl4) injection in mice. Moreover, compound 15a had
no significant change on serum calcium that can be raised by positive control calcipotriol
or 1,25(OH)2D3.
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Figure 1. Chemical structures of representative secosteroidal and nonsecosteroidal VDR
ligands.
RESULTS AND DISCUSSION
Design of target compounds.
Boehm et al. reported the first nonsecosteroidal analogs of vitamin D3, LG190178 (4)
and found that propane-1.2-diol of 4 are important for the binding affinity. Based on the
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phenyl-pyrrolyl pentane skeleton and the structure feature of 4, we designed derivatives
13 using a scaffold hopping strategy and introducing different R1 substituents to identify
anti-fibrotic VDR ligands. Then, phenyl-pentane group on pyrrole ring C-4 position
instead of the C-5 position was designed to investigate the influence of the substitution
positions of the pyrrole ring, and we obtained compounds 15. Finally, we further
investigated the A ring part of target compounds and designed compounds 17, 19, and 20
by substitution of the A ring with other hydrophilic groups, such as the butanoic acid,
pentanoic acid, alanine, β-alanine, and succinic acid.
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Figure 2. Design of the novel nonsecosteroidal VDR ligands.
Synthetic procedures of target compounds.
The synthetic pathway of target compounds 13a-h is outlined in Scheme 1. The
intermediate 7 was prepared using previously reported approach,34 then it reacted with
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ethyl pyrrole-2-carboxylate at 0°C in the presence of lewis acid BF3·Et2O to produce
intermediate 8a. After the management with iodoethane in DMF, intermediate 9a was
obtained. The reduction reaction of 9a give the intermediate 10a, which was hydrolyzed
by KOH to produce 11a in high yield. By reaction with the corresponding amines, 11a
gave intermediates 12a-i, respectively. Finally, target compounds 13a-h were obtained by
electrophilic substitution of glycidol with intermediates 12a-h in the presence of NaH.
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The synthetic pathway of compounds 15a-i is outlined in Scheme 2. Intermediate 8b,
the regioselectivity isomer of 8a, was obtained by reacting with ethyl
pyrrole-2-carboxylate at 20°C , instead of at 0°C, in the presence of lewis acid BF3·Et2O
in moderate yield. By the same method as described in the synthesis of 9a-13, target
compounds 15a-i were obtained.
The synthetic pathway of compounds 17a-b, 19a-b, and 20 is outlined in Scheme 3.
Intermediate 14a was subjected to nucleophilic substitution with the corresponding
halohydrocarbon to give intermediates 16a-b, which were further treated by hydrolysis of
ester groups to afford target compounds 17a-b in high yield. On the other hand,
intermediates 18a-b were synthesized by treatment of intermediate 14a with
corresponding amino acids. Deprotection of 18a-b using CF3COOH yielded target
compounds 19a-b. Compound 20 was obtained by the same method as described in the
synthesis of intermediates 16a-b.
Scheme 1. Synthesis of compounds 13a-ha
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aReagents and conditions: (a) Ethyl 1H-pyrrole-2-carboxylate, BF3·Et2O, 0°C , 1 h, 73%;
(b) C2H5I, NaH, DMF, 0-25°C , 2 h, 82.4%; (c) Pd/C, HCOONH4, CH3OH/EtOAc (10:1),
25°C , 1 h, 98%; (d) 2 mol/L KOH, EtOH, rt, 1 h, 94%; (e) EDCI, HOBt, TEA, RNH2,
DCM, rt, overnight, 35-96%; (f) Glycidol, NaH, DMF, 80°C , 5 h, 54-82%.
Scheme 2. Synthesis of compounds 15a-ia
aReagents and conditions: (a) Ethyl 1H-pyrrole-2-carboxylate, BF3·E t2O, 25°C , 1 h, 44%;
(b) C2H5I, NaH, DMF, 0-25°C , 2 h, 85%; (c) Pd/C, HCOONH4, CH3OH/EtOAc (10:1),
25°C , 1 h, 97%; (d) 2 mol/L KOH, EtOH, rt, 1 h, 95%; (e) EDCI, HOBt, TEA, RNH2,
DCM, rt, overnight, 32-89%; (f) Glycidol, NaH, DMF, 80°C , 5 h, 47-78%.
Scheme 3. Synthesis of compounds 17a-b, 19a-b, and 20a
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aReagents and conditions: (a) Ethyl 4-bromobutyrate or ethyl 5-bromopentyrate, NaH,
DMF, 80°C , 5 h, 58-67%; (b) 2 mol/L KOH, EtOH, rt, 1 h, 95-97%; (c) Boc-AA, EDCI,
DMAP, DCM, rt, overnight, 43-52%; (d) TFA, DCM, rt, 1 h, 87-92%; (e) Succinic
anhydride, NaH, DMF, 80°C , 2 h, 43%.
Table 1. The structures of all compounds.
Compd
X
N
Y
C
R1
R2
R3
H
R4
13a
C2H5
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13b
13c
13d
13e
13f
N
N
N
N
N
N
N
C
C
C
C
C
C
C
C
C
C
N
N
N
C2H5
C2H5
C2H5
C2H5
C2H5
C2H5
C2H5
H
H
H
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H
H
H
13g
13h
15a
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15c
H
H
C2H5
C2H5
C2H5
H
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15d
15e
15f
C
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C
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N
N
N
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H
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H
C2H5
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C2H5
C2H5
C2H5
C2H5
C2H5
C2H5
C2H5
C2H5
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15g
15h
15i
17a
17b
19a
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C
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C2H5
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VDR binding affinity.
The VDR binding affinity of synthesized compounds was tested with VDR competitor
assay and 1,25(OH)2D3 was applied as the positive control. All compounds were
evaluated for their binding affinity in triplicates at 100 nM. The binding affinity of
compounds was exhibited by a reference value to 1,25(OH)2D3, which is assigned as
100%. Table 1 and Table 2 showed the structure-activity relationships (SARs) for these
compounds. Firstly, we focused on the important pharmacophore side chain with C-5
position of pyrrole ring that bearing phenyl-pentane group, the results showed that
compounds with the terminal hydrophobic groups in side chain section, such as
tert-butoxide (13a), tert-butyl (13b), trifluoromethyl (13e) displayed significant binding
affinities. However, introducing flexible hydrophobic substitution n-pentyl group to give
compound 13h resulted in decreased affinity, which suggests that flexible substitution is
not preferred in the VDR ligand binding pocket (LBP). Moreover, substitutions of
hydrophilic groups, such as 1-hydroxy (13d), aminos (13f-g), dramatically decreased the
binding affinities. By removing the substitution on the pyrrole group C-5 position to C-4
position, compounds 15a-i were synthesized to explore the influence of substitution
position on the binding affinity. Although most compounds displayed decreased binding
affinities compared to the pyrrole group C-5 position, compound 15a bearing
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tert-butoxide group showed better activity than the corresponding compound 13a and
turned out to be the most potent molecule. Subsequently, we further investigated the A
ring part of target compounds by substitution of the A ring with other hydrophilic groups,
such as butanoic acid (17a), pentanoic acid (17b), alanine (19a), β-alanine (19b),
butanedioic acid (20). As a result, no improvement of the binding affinities was detected
compared with that of compound 15a. Meanwhile, carboxylic acid as the terminal
hydrophilic group, such as compounds 17a-b and 20 displayed better binding affinities
than that of anime groups (19a-b).
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Table 2. The affinities of VDR binding and activities of anti-collagen I synthetic at 100
nM.
Relative VDR
Relative VDR
Anti-collagen I
at 100 nM (%)b
Anti-collagen I
at 100 nM (%)b
Compd
Compd
binding
binding
ability(%)a
ability(%)a
13a
13b
13c
13d
43±3.2
45±5.3
27±4.1
—
150±1 3.6*
159±9.3*
71±7.2
—
15e
15f
39±4.8
—
133±1 4.5*
—
15g
15h
—
—
16±1.3
51±5.3
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13e
13f
37±2.5
—
135±1 5.9*
—
15i
17a
17b
19a
19b
20
—
—
32±3.9
30±3.2
20±2.9
22±4.8
30±3.1
93±8.5
100
112±7.8
90±9.2
73±4.8
66±5.2
80±6.1
95±9.2
100
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13g
13h
15a
15b
15c
15d
—
—
21±3.5
62±4.5
31±2.3
—
70±7.9
210±20.1*
119±7.3*
—
2
—
—
1
aThe values represent the mean ± SD of three independent experiments. 1,25(OH)2D3 (1)
is assigned as 100%. 1,25(OH)2D3 (1) and Calcipotriol (2) are as the positive control.
bThe values represent the mean ± SD of three independent experiments. 1,25(OH)2D3 (1)
is assigned as 100%. 1,25(OH)2D3 (1) and Calcipotriol (2) are as the positive control. *P<
0.05 vs. 1,25(OH)2D3 (1).
Transactivation.
To estimate agonistic abilities of the nonsecosteroidal ligands bearing phenyl-pyrrolyl
pentane skeleton, a transactivation assay was performed in HEK293 cells using
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pGL4.27-SPP×3 -Luci reporter plasmid. Compounds 13a-b, 15a and 15e with strong
binding affinities were selected, calcipotriol and 1,25(OH)2D3 were used as positive
control. As shown in Figure 3, all four compounds acted as potent agonists with
concentration-dependent transcriptional activity. Among them, compound 15a was the
most potent compound and displayed transcriptional activity at 100 nM, while
compounds 13a-b and 15e did not reach the optimal transcription level even at 1000 nM.
This may due to the interaction of heterodimer partners with VDR, such as nuclear
receptor corepressor 1 and steroid receptor coactivator 1, which has an effect on the
biological activity of VDR. In addition, a significantly higher increase of transcripts
encoding 25-hydroxyvitamin D-24-hydroxylase (CYP24A1) by compounds 15a was
showed (Figure S1).
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Figure 3. Transcriptional activities of the compounds were examined. HEK293 cells
were co-transfected with TK-SPP × 3-Luci reporter plasmid, pCMX-Renilla,
pENTER-CMV-hRXRα and pCMX−VDR. Eight hours after transfection, test compounds,
calcipotriol and 1,25(OH)2D3 were added. 24 hours later, luciferase activity assay was
performed using the Dual-Luciferase Assay System. Renilla luciferase activity was as the
reference to normalize the firefly luciferase activity. All the experiments were performed
three times. *P< 0.05 vs. DMSO.
Anti-collagen I synthetic activities in vitro.
Liver fibrosis is characterized by the replacement of functional hepatic tissue with
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highly cross-linked collagen I-rich ECM and TGFβ1 is recognized as one of the most
potent pro-fibrotic modulator responsible for collagen I synthesis. Consequently,
inhibiting the production of collagen I induced by TGFβ1 is an effective strategy to
against fibrotic progress. To examined the anti-fibrotic effects of all target compounds, an
stable and unlimited source of human HSCs, LX-2 cells, were employed as a valuable
cell model to study human hepatic fibrosis.43 Calcipotriol and 1,25(OH)2D3 were applied
as positive control (Table 2). Compared with calcipotriol and 1,25(OH)2D3, seven of the
synthesized analogues (13a-b, 13e, 15a-b, 15e, and 17a) at the concentration of 100 nM,
which was little cytotoxic to LX-2 (Table S1), demonstrated more effective inhibitory
properties against collagen I synthesis, with the values at the range of 112-210%, and six
compounds (13c, 13h, 17b, 19a-b, and 20) displayed an equivalent inhibitory potency.
This discrepancy between binding affinity and agonistic activity could be interpreted by
the interactions between the cofactors and VDR ligand complex. It is required AF-2
transactivation motif of VDR to interacts with cofactors such as VDR interacting proteins
(DRIPs) for the VDR transcriptional activation.37-39 Table 2 highlights the important
SARs features of inhibitory potencies. Similar to the SARs of binding affinities, 13a-b
and 13e bearing terminal hydrophobic groups in the side chain section also displayed
significant inhibitory activities. In addition, compound 13h, which showed decreased
binding affinity compared with other compounds bearing terminal hydrophobic groups as
described above, was also only moderately active. Replacement of hydrophobic groups
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with hydrophilic groups, such as 1-hydroxy (13d), aminos (13f-g), dramatically
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weakened the inhibitory activities. On this point, it could be proved that the inhibitory
activities of synthesized compounds are positively correlative with VDR binding
affinities. Varying the position of the substitution from pyrrole group C-5 position to C-4
had dramatically effects on inhibitory activities. As likely as the binding affinities,
compound 15a showed better activity than the corresponding compound 13a and turned
out to be the most potent molecule. In addition, all compounds varying propane-1.2-diol
to other hydrophilic groups displayed moderate inhibitor activities against collagen I
synthesis and had no better results than 15a. Meanwhile, carboxylic acid as the terminal
hydrophilic group, such as compounds 17a-b and 20 displayed better inhibitory activities
than that of anime groups (19a-b).
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Effects on the expression of collagen I and α-SMA in LX-2 cells.
The anti-fibrotic activities of selected compounds 13a, 13b, 15a, and 15e, which
displayed optimal property on binding affinities and anti-collagen I synthetic activities,
were analyzed using western blot and Q-PCR assays. The increased expression of
collagen I and α-SMA is the markers of activated HSCs3. As described above, liver
fibrosis, regardless of its cause, is featured by progressive accumulation of ECM
proteins and the mainly component is collagen I. Moreover, the α-SMA-positive
myofibroblasts is considered as the key promoter in the progression of liver fibrosis.
Collagen I alpha 1 (COL1A1) is the direct target of VDR20, in addition, a-SMA and
collagen I are both upregulated by TGFβ1 in HSCs. Therefore, the expression of α-SMA
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and collagen I were selected to determine the anti-fibrotic activities of selected
compounds. As shown in Figure 4C and D, the activity of these molecules was
significantly affected by the pyrrole group substitution position, compounds 15a and 15e
(C-4 substitution) at the concentration of 100 nM significantly reduced α-SMA and
collagen I protein levels in TGFβ1-treated LX-2 cells, while compounds 13a and 13b
(C-4 substitution) exhibited no significant influence on ACTA2 expression. Compared
with the hydroxyl group contained analog 15a, the molecules 15e showed less activity,
which suggest that hydroxyl group in the side chain terminal is essential. Q-PCR results
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(Figure 4A and B) showed that compound 15a significantly down-regulated COL1A1 and
ACTA2 mRNA expression. Moreover, compared with positive control calcipotriol and
1,25(OH)2D3, compound 15a showed more effective inhibitory potency against COL1A1
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mRNA expression. The results impel us continuously to test the anti-fibrotic effect of
these compounds.
Figure 4. Effects of compounds on activation of LX-2 cells. LX-2 cells were cultured
with compounds, calcipotriol or 1,25(OH)2D3 for 24 hours at 100 nM. (A and B) The
expression levels of ACTA2 and COL1A1 were measured by Q-PCR. (C and D)
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Expression of α-SMA and collagen I on LX-2 cells was determined by western blot. The
representative gel electrophoresis bands are shown (C), and expression levels of proteins
were normalized to the expression of β-actin (D). Densitometry data are shown as mean ±
SD. *P< 0.05 vs. TGFβ1, #P< 0.05 vs. TGFβ1+Calcipotriol.
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15a inhibited activation of LX-2 cells through VDR.
VDR is recognized the potential therapy target for liver fibrosis and the
above-mentioned results suggested the compounds may be VDR agonists. To confirm
that compound 15a repressed fibrotic gene expression via VDR, RNA interference (RNAi)
was used in LX-2 cells. Loss of VDR abolished 15a-mediated repression of collagen I
and α-SMA expression was shown in Figure 5. These data demonstrated that compound
15a exerts its repressing effect on HSCs activation through interaction with VDR.
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Figure 5. 15a inhibited activation of LX-2 cells via VDR. (A) VDR-specific (siVDR) or
negative control (siNC) siRNA-transfected LX-2 cells were treated with 15a (100 nM),
TGFβ1 (1 ng/mL), or TGFβ1 plus 15a for 24 hours. The expression of VDR, α-SMA and
collagen I on LX-2 cells was tested by western blot. The representative gel
electrophoresis bands are shown. (B, C and D) Expression levels of VDR, collagen I and
α-SMA were normalized to the expression of β-actin. The quantified densitometry data
are shown as mean ± SD. *P< 0.05.
Effects on suppressing the expression of α-SMA in CCl4-induced hepatic fibrosis
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mice.
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Based on in vitro results, compound 15a was selected for further studies in vivo. To
explore whether compound 15a could repress the expression of fibrotic gene and inhibit
hepatic fibrogenesis in vivo, CCl4 was used to induce liver fibrosis by intraperitoneal (IP)
injection in C57BL/6J mice. The anti-fibrotic property of compound 15a was determined
by histological examination. As shown in Figure 6, consistent with the above studies in
vitro, oral administration of compound 15a to CCl4-treated mice reduced α-SMA levels
in the liver tissues according to Q-PCR and IHC staining. In addition, compound 15a
also increased the mRNA levels of Cyp24a1, suggesting 15a inhibits fibrotic progress
through agitating VDR (Figure S2).
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Figure 6. 15a suppressed the expression of α-SMA in CCl4-induced hepatic fibrosis
lesions and protected the liver from impairment. Mice (n=5 in each group) received either
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DMSO or CCl4 (0.5 mL/kg body weight) intraperitoneally for three weeks before
intragastric administration of 15a, calcipotriol, 1,25(OH)2D3 (20 μg/kg body weight) or
DMSO. (A) α-SMA expression in the injured liver was tested by immunohistochemistry
(×200). (B) The expression levels of α-SMA were quantified using Image-Pro Plus 6.0.
Data are shown as mean ± SD. *P< 0.05 vs. CCl4. (C) Expression of Acta2 in the injured
liver was examined by Q-PCR (mean ± SD. *P＜0.05).
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Effects on suppressing the expression of collagen in CCl4-induced hepatic fibrosis
mice.
In addition, we measured collagen content to further examine the anti-fibrotic effect
of compound 15a. Histopathologically, compound 15a treatment resulted in the
inhibition of collagen accumulation in CCl4 mice liver based on H&E and Masson’s
trichrome staining (Figure 7A). The amounts of hepatic hydroxyproline in liver tissue
were estimated, which was a major component of the collagen. As shown in Figure 7B,
treatment of compound 15a had a significant reduction in hydroxyproline content, with
slightly stronger potency than positive control calcipotriol and 1,25(OH)2D3. Moreover,
mRNA levels of Col1α1 was elevated in the liver fibrosis models, the results showed
that the expression of Col1α1 was also reduced by compound 15a treatment (Figure 7C).
These results suggest that compound 15a treatment prevents CCl4-induced liver injury
and hepatic fibrosis.
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Figure 7. 15a inhibited the CCl4-induced hepatic lesions and collagen deposition. Mice
(n=5 in each group) received either DMSO or CCl4 (0.5 mL/kg body weight)
intraperitoneally for three weeks before intragastric administration of 15a, calcipotriol,
1,25(OH)2D3 (20 μg/kg body weight) or DMSO. (A and B) CCl4-induced hepatic fibrosis
lesions were examined by H&E staining (×100), the collagen deposition was determined
by Masson’s trichrome staining (×200) and hydroxyl proline measurement (mean ± SD.
*P＜0.05 vs. CCl4). (C) Expression of Col1a1 in the injured liver was examined by
Q-PCR (mean ± SD. *P＜0.05).
Effects on liver function and serum calcium of fibrotic models.
Serum alanine transaminase (ALT), aspartate transaminase (AST), and total bile acid
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(TBA) levels are commonly measured clinically as biomarkers for liver health.40
Significantly elevated levels of AST, ALT and TBA often suggest the existence of liver
damage. As shown in Figure 8A, B and C, the levels of AST, ALT and TBA were
significantly decreased in compound 15a-treated mice as compared with control animals.
Moreover, compound 15a displayed better results than positive control calcipotriol and
1,25(OH)2D3, which are very promising for the reduction of liver damage. To determine
the effect of novel designed nonsecosteroidal analogs on inducing hypercalcemic,
calcium concentration was measured by calcemic activity assay in vivo. Ma et al. reported
that nonsecosteroidal VDR modulators showed poor activity in intestinal cells.41
Moreover, the nonsecosteroidal compound has the ability to activate VDR and is weak in
binding to vitamin D-binding proteins, so that it does not accumulate excessively in the
intestinal, resulting in no excessive calcium absorption.29 In our study, compound 15a
displayed small impact on the expression of intestinal Trpv6, which is a VDR target gene
involved in calcium metabolism (Figure S3). As shown in Figure 8D, compared with
calcipotriol and 1,25(OH)2D3, there was no significant effect on serum calcium when
treated with compound 15a in mice, which suggests that nonsecosteroidal analog 15a
results in a high dissociation of anti-fibrotic potency from calcemic effects.
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Figure 8. 15a protected the liver from impairment. Mice (n=5 in each group) received
either DMSO or CCl4 (0.5 mL/kg body weight) intraperitoneally for three weeks before
intragastric administration of 15a, calcipotriol, 1,25(OH)2D3 (20 μg/kg body weight) or
DMSO. (A, B, and C) The serum levels of ALT, AST and TBA were determined. (D) The
serum calcium concentration was determined by calcium assay kit (mean ± SD. *P＜0.05,
**P＜0.01).
In Vivo Pharmacokinetics Study.
Pharmacokinetic studies of compound 15a and 1,25(OH)2D3 were performed in rats.
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