Abstract
Background/Aim: Adenosine and 4 G-protein-associated membrane receptors (A1, A2A, A2B, and A3) and their derivatives regulate the central nervous, cardiovascular, peripheral, and immune system. We developed a novel selective A3 AR antagonist, HL3501, and examined its anti-fibrotic effects across various models. Materials and Methods: The anti-fibrotic activity of HL3501 was evaluated in three cell lines (HK2, LX2, and Primary hepatic stellate cell) and a methionine–choline-deficient (MCD) model including use of mouse pharmacokinetics (PK). Results: HL3501 decreased alpha-smooth muscle actin (α-SMA) and collagen 1 in TGF-β1-induced pro-fibrotic activation in HK2 cells. HL3501 also inhibited TGF-β1-induced HSC activation, which resulted in reduction of α-SMA and fibronectin in LX2 and human primary HSCs. In the nonalcoholic fatty liver disease activity score (NAS) analysis, HL3501 showed improved anti-steatosis and anti-inflammatory activity. The mouse PK study revealed the oral bioavailability (%F) of HL3501 at 30 mg/kg and 60 mg/kg as 92.5 and 107.2%, respectively. Conclusion: HL3501 presents anti-fibrotic effects in in vitro and in vivo studies. We also demonstrated that HL3501 is orally available and has a good bioavailability (BA >90%) profile from in mouse PK. HL3501, therefore, has a therapeutic potential for various fibrotic diseases, including those of liver and kidney tissues.
Endogenous adenosine is continuously produced both at the intracellular and extracellular levels. It has a short half-life (~1.5 s) and is quickly metabolized to inosine and hypoxanthine. It may also act as a building block, an intermediate metabolite of nucleic acids and an intracellular messenger in pathological and physiological processes. Under physiological conditions, acutely increased adenosine has beneficial effects such as vasodilation and a decrease in inflammation. In contrast, chronic elevation of adenosine is responsible for pathologic condition associated with chronic inflammation, fibrosis, and organ damage. Extracellular adenosine signaling is mediated via 4 G-protein-associated membrane receptors: A1, A2A, A2B, and A3. These subtype receptors are widely distributed in almost all organs and tissues with regulating central nervous, cardiovascular, peripheral, and immune systems. With the development and generation of various adenosine receptors agonists or antagonists and four adenosine receptor knockout mouse models, adenosine signaling has been demonstrated as an essential player under pathophysiological conditions by modulation of inflammation, ischemic tissue injury, fibrosis, and tissue remodeling (1-4).
Diabetic nephropathy (DN) is a severe complication of diabetes mellitus, a metabolic disease with a high prevalence worldwide. DN is associated with progressive and irreversible renal fibrosis. Tubulointerstitial fibrosis is typically present in patients with DN, accompanied by infiltration of inflammatory cells, the activation of fibroblasts, and the expression of α-SMA and fibronectin (5, 6). Recent clinical studies have indicated that plasma adenosine and its metabolites increase significantly in patients with DN (7). Studies in diabetic rats and patients have revealed that the observed up-regulation of A3 AR expression was correlated with disease progression (8). Some reports have also noted the relationship between adenosine A3 AR antagonist and renal fibrosis (9). MRS1220, an adenosine A3 AR antagonist, inhibited α-SMA and fibronectin expression in TGF-β1-induced fibrotic HK2 cells, and significantly reduced immunostaining of α-SMA in diabetic rats (10). The A3 AR antagonist, LJ-1888, improved UUO-induced tubulointerstitial fibrosis, while another A3 AR antagonist prevented kidney fibrosis in db/db mice by decreasing the level of α-SMA (11).
Liver fibrosis and end-stage cirrhosis are common hepatopathological pathways of chronic liver disease. Myofibroblasts (derived from HSCs) and portal fibroblasts are involved in extracellular matrix production in response to liver injury and fibrosis. Caffeine, a non-selective adenosine receptor (AR) antagonist, showed protective traits on alcohol-induced liver fibrosis in in vitro cell studies (11, 12). Additionally, the A3 AR agonist CF102 showed anti-fibrotic effects in an LX2 proliferation assay and STAM and CCL4-induced liver fibrosis animal models (13).
To date, there have been only a few studies on liver fibrosis with the A3 AR antagonist. This study aimed to investigate the antifibrotic effect of a novel selective A3 AR antagonist, HL3501(14) in kidney cell and in the liver cell or tissue using preclinical studies.
Materials and Methods
Reagents. HL3501 was synthesized at the Handok R&D Center (Seoul, Korea). MRS1220 (#1217) was purchased from Tocris (Park Ellisville, MI, USA). IB-MECA(I146) and obeticholic acid (SML3096) were purchased from Sigma-Aldrich (St. Louis, MO, USA).
Cell culture. Immortalized human hepatic stellate cell lines, LX-2, were provided kindly by Prof. Fang. S (Yonsei Univ, Seoul, Republic of Korea) and maintained in Dulbecco’s Modified Eagle’s Medium (Gibco, Waltham, MA, USA) supplemented with 10% fetal bovine serum (Gibco), penicillin (100 U/ml), and streptomycin (100 μg/ml). Primary human hepatic stellate cells (INNOPROT, Bizkaia, Spain) were cultured with a stellate cell medium kit (INNOPROT) as instructed by the manufacturer. HK-2, an immortalized proximal tubule epithelial cell line, was obtained from the American Type Culture Collection (ATCC, Manassas, VA, USA) and grown in keratinocyte serum-free medium (K-SFM) supplemented with bovine pituitary extract and epidermal growth factor. The cells were incubated in 37°C, 5% CO2 humidified atmosphere.
Comparative Quantitative Real-Time PCR (qPCR). Mouse liver tissue was homogenized in TRIzol reagent (Life Technologies, Grand Island, NY, USA), and total RNA was isolated according to the protocol provided by the manufacturer. The concentration of RNA was measured using BioDrop Duo (Biodrop, Cambridge, UK), and cDNA was synthesized by a High-Capacity cDNA Reverse Transcription System (Life Technologies). qPCR was performed in duplicate for each sample using SYBR® Premix Ex TaqTM (Life Technologies) and CFX96 Real-Time PCR System (Bio-Rad, Hercules, CA, USA). The used primers are shown in Table I.
The qPCR primer list for fibrosis marker expression in mouse liver tissue.
Western blot. Each cell was seeded onto a six-well plate at a density of 10×104 cells/well (LX-2), 7×104 cells/well (primary HSC), and 10×104 cells/well (HK-2) and incubated for 24 h. After TGF-β1(10 ng/ml) (R&D system, Minneapolis, MN, USA) treatment with or without glucose (5.5 or 60 mM) for 24 h, chemicals were treated for 48 h. Cells were washed twice with PBS and lysed with radioimmunoprecipitation assay buffer (RIPA buffer, Sigma-Aldrich, St Louis, MO, USA) with protease inhibitor cocktail (ThermoScientific, Rochester, MN, USA), phosphate inhibitor (ThermoScientific), and 0.1% SDS. The cell lysates were then incubated on ice for 30 min and subsequently centrifuged at 13,000 × g for 15 min at 4°C. PierceTM BCA Protein Assay Kit (Pierce, Rockford, IL, USA) was used to quantify the protein concentration. Samples were denatured with a buffer containing 10% SDS, 0.5M DTT, 50% glycerol, 0.25% bromophenol blue, and 0.25 M Tris–HCl at 90-100°C for 6 min, then cooled at room temperature for 5 min. A sample of 15 μg of each protein was resolved in 8-16% gradient SDS-PAGE gel (Bio-Rad) and transferred to polyvinylidene difluoride (PVDF) membrane (Bio-Rad). The membrane was blocked with 5% skim milk in TBS-T at room temperature for 1 h and incubated with primary antibodies overnight at 4°C. Next, the membranes were washed with TBS-T and incubated with secondary antibodies conjugated to horseradish peroxidase for 1 h. The protein bands were photographed with enhanced chemiluminescence (ECL) reagents (Bio-Rad) using a chemidoc gel imaging system (Bio-Rad). The densities of each band were normalized to those of the GAPDH band. Anti-fibronectin (ab2413, Abcam, Cambridge, MA, USA), anti-collagen I (ab138492, Abcam), anti-alpha-SMA (ab5694, Abcam), and anti-GAPDH (015-25473, Wako Pure Chemical Industries, Osaka, Japan) were used in the western blot analysis.
Animal models. Six-week-old C57 BL/6 male mice were purchased from OrientBio (Seongnam, Republic of Korea) and housed at the laboratory animal facility (temperature, 22°C±2°C; relative humidity, 55%±5%; 12/12-hour light/dark cycle) at the Asan Institute for Life Sciences under specific pathogen-free conditions according to ICLAS (International Council for Laboratory Animal Science). After 1 week of acclimation (7 weeks old), mice were randomly assigned to the control group (n=9/group), MCD only group (n=13/group), MCD+OCA (20 mg/kg, po) group (n=13/group), or the MCD+ HL3501 (30 mg/kg, po) group (n=13/group). Mice in MCD intake groups were fed an MCD (methionine–choline-deficient) diet for 7 weeks to induce nonalcoholic steatohepatitis. Meanwhile, the vehicle control groups were fed a MCS (methionine–choline-supplement) diet for the same period. After the first 3 weeks of induction, test article with oral dosing (HL3501, 30 mg/kg, po and OCA, 20 mg/kg, po) was initiated while the animals were retained on an MCD diet for 4 weeks. Individual body weight was measured once a week during the disease-induction period and every day before dosing during the treatment period. Food consumption was measured once a week. Mice were euthanized using CO2 at the end of the experiments, and blood and liver samples were collected. The levels of plasma alanine aminotransferase (ALT) and aspartate aminotransferase (AST) were measured. Each median and left lobe was fixed with 10% neutral buffered formalin for 24-48 h and processed to produce formalin-fixed paraffin-embedded tissue blocks. The liver slides were stained with hematoxylin and eosin (H&E) and then scored blindly according to NAFLD (nonalcoholic fatty liver diseases) activity score (NAS). Histopathological lesions of the NAFLD evaluation were analyzed semi-quantitatively according to the NASH Pathology Committee system for NAFLD Activity Score (NAS) (15, 16). This study was conducted according to the regulations of the Institutional Animal Care and Use Committee at Asan Medical Center (IACUC No.2019-11-132).
Mouse PK analysis. Six to eight weeks old male ICR mice were used for PK analysis of HL3501. HL3501 was administered orally (30 and 60 mg/kg) and through intravenous injection (3 mg/kg) to mice (n=3). Blood samples were collected via mandibular vein puncture at the set time points (0.083, 0.167, 0.5, 1, 2, 4, 6, 8, 12 h; 9 points for IV group and 0.25, 0.5, 1, 2, 4, 6, 8, 12, 24 h; 9 points for the po group). The concentration of HL3501 plasma was analyzed using a validated LC-MS/MS assay. Data obtained from the concentration–time profile were calculated using noncompartmental analysis with Phoenix-WinNonlin 7.0 (Pharsight, Sunnyvale, CA, USA).
LC-MS/MS analysis method. The LC-MS/MS system consisted of a CTC Autosampler, Agilent™ 1200 series (Santa Clara, CA, USA) and API4000 (applied Biosystem/MDS SCIEX, Toronto, Canada). The separation was performed using a Poroshell 120 EC-C18 analytical column (4.1 μm 2.1×50 mm) (Agilent, Santa Clara, CA, USA). The mobile phase consisted of 0.1 % formic acid in H2O (A) and 0.1% Formic Acid in Acetonitrile (B); the gradients were 5% B (0 to 1.2 min), 95% B (3.2 to 3.7 min), and 5% B (3.8 to 4.5 min); the flow rate was 0.4 mL/min; and the injection volume was 2 μl. The optimized MS/MS parameters were as follows: ion spray voltage (IS), +5500 V; ion source gas 1 (GS1) pressure, 50 psi; ion source gas 2 (GS2) pressure, 60 psi; curtain gas (CUR) pressure, 20 psi; collision gas (CAD), medium. The compounds parameters, i.e., declustering potential, collision energy and collision exit potential for HL3501 and IS (internal standard, Tolbutamide) were 111, 41, 12 V and 64, 25, 12 V, respectively. The dwell time was 150 ms. Detection of the ions was performed in the multiple reaction monitoring (MRM) mode, monitoring the transition of the m/z 376 precursor ion to the m/z 123 product ion for HL3501 and m/z 271 precursor ion to the m/z 155 product ion for IS. Quadrupole Q1 and Q3 were set on unit resolution. The analytical data were processed by Analyst software (version 1.5.3).
Statistical analyses. All in vitro data from LX2, primary HSC, and HK2 cells are presented as mean±SD. Data from MCD animal models are presented as the mean±SEM, and the statistical analysis was carried out by one-way ANOVA followed by Tukey’s post-hoc tests. Comparisons between multiple groups were determined using one-way ANOVA, followed by Fisher’s least significant difference test. p-Values <0.05 were considered statistically significant.
Results
Anti-fibrotic effects of HL3501 in HK2 cells. TGF- β1 and high-glucose treatment increased the α-SMA, fibronectin and collagen 1 protein levels of control group in HK2 cells (Figure 1). MRS1220 is an A3 AR antagonist (8) that we used as a comparative compound. We evaluated MRS1220 and HL3501 at the same concentration of 1 μM. HL3501 caused a decrease in α-SMA and collagen 1 expression in TGF-β1-induced pro-fibrotic activation HK2 cells at doses of 0.1 and 1 μM. Comparatively, MRS1220 appeared to reduce only α-SMA at a dose of 1 μM. These anti-fibrotic results suggest that HL3501 may more efficiently improve renal fibrosis than MRS1220.
HL3501 exhibits anti-fibrotic activity in HK2 cells. (a) HK-2 cells were pre-incubated for 24 h under low glucose (5.5 mM) or high glucose (60 mM) conditions with the presence or absence of TGF-β1 followed by treatment with HL3501 (0.01, 0.1, 1 μM) or MRS1220. The protein levels of α-SMA, fibronectin, and collagen 1 were measured by western blotting and normalized to the expression of GAPDH. ##p<0.01 vs. control; *p<0.05, **p<0.01 vs. TGF-β1 alone (n=4). The original western blot images for cropped images are provided as supplementary file.
Anti-fibrotic effects of HL3501 in LX2 and HSCs. We evaluated the effects of HL3501 on TGF-β1-induced liver fibrosis by the LX2 cell assay. LX2 cells were activated by TGF-β1, and HL3501 was added into the culture media at doses of 0.1, 1, 10 μM. α-SMA and fibronectin levels were evaluated with Western blots. HL3501 significantly decreased α-SMA expression at 1 and 10 μM compared to TGF-β1 alone (Figure 2a). HL3501 had a reduced inhibitory effect on the up-regulation of fibronectin compared to α-SMA levels. In addition, the human primary HSCs (INNOPROT) were cultured to evaluate anti-fibrotic activity. TGF-β1 treatment increased the levels of pro-fibrotic markers, α-SMA and fibronectin, in primary HSCs. Whereas HL3501 significantly decreased α-SMA and fibronectin at dosages of 1, 10, 10 μM respectively, compared to TFG-β1 alone (Figure 2b). These results indicate that HL3501 inhibits TGF-β1-induced liver fibrosis in LX-2 and human primary HSCs.
HL3501 inhibited TGF-β1-induced pro-fibrotic markers activation in vitro. (a) LX-2 cells were treated with HL3501 (0.1, 1, 10 μM) for 48 h after TGF-β1 activation for 24 h (10 ng/ml). These α-SMA and fibronectin protein levels were measured by western blotting and normalized to the expression of GAPDH. ##p<0.01 vs. control; *p<0.05, **p<0.01 vs. TGF-β1 alone (n=5). (b) Primary human hepatic stellate cells were treated with HL3501 (0.1, 1, 10 μM) for 48 h after TGF-β1 activation for 24 h (10 ng/mL). The protein levels of α-SMA and fibronectin were measured by Western blots and normalized to the expression of GAPDH. ##p<0.01 vs. control; *p<0.05, **p<0.01 vs. TGF-β1 alone (n=4).
Effects of HL3501 in the MCD-diet NASH mouse model. To further evaluate the in vivo effect of HL3501, we used the NASH model with mice fed the MCD (methionine-choline-deficient) diet. A schematic of the experimental procedure using the MCD model is depicted in Figure 3a. NAS scores (ballooning degeneration, lobular inflammation, steatosis) were considered the primary endpoint for testing liver damage. We also performed qRT-PCR analysis for pro-fibrotic biomarkers (α-SMA, fibronectin, collagen 3A1) change in liver tissue. Obeticholic acid (OCA), a FXR agonist, is a clinical candidate for NASH indication; we thus used it as a comparative control in this study (15). The scores of each NAS component and ALT/AST levels across all groups are presented in Table II. The NAS score was significantly higher in the MSC-only group in the NASH model compared to the control group. Additionally, the HL3501 treatment group exhibited significantly reduced lobular inflammation and a lower steatosis score compared to the MCD-only group. HL3501 also improved serum ALT and AST levels. For the OCA treatment group, a weak reduction in ballooning and steatosis was detected, but there was no effect on inflammation (Table II). H&E staining revealed that the MCD-only group had hepatocellular ballooning, inflammatory infiltration, and steatosis. However, HL3501 showed reduced inflammatory infiltration and steatosis (Figure 3b). qRT-PCR of mouse liver revealed that HL3501 tended to decrease pro-fibrotic markers (α-SMA, fibronectin, collagen 3A1) compared to the MCD-only group. HL3501 significantly decreased α-SMA levels in the MCD-only group. The comparative control, OCA, caused a decrease in α-SMA levels but did not affect fibronectin or Col3A1 (Figure 3c). These results suggest that HL3501 ameliorated liver fibrosis in the MCD mice model.
HL3501 inhibited pro-fibrotic markers in the methionine-choline-deficient diet-fed (MCD) mouse model. (a) The flow chart of mouse MCD model study. (b) Representative images of H&E staining from each treatment group. HL3501 (30 mg/kg, po); obeticholic acid (OCA, 20 mg/kg, po). The scale bar represents 50 μm. (c) The mRNA levels of α-SMA, fibronectin, collagen 3A1, (n=5 per group) were analyzed by RT-PCR. Data are expressed as the mean±SE. #p<0.05 vs. control. *p<0.05 vs. MCD alone group.
The NAS (inflammation, ballooning, steatosis) score was analyzed, and serum alanine transaminase (ALT) and aspartate transaminase (AST) levels were assessed in each group. Data represent the mean±SEM.
PK study of HL3501in mice. The time–concentration curve of HL3501 after single oral and intravenous administration in ICR male mice is shown in Figure 4. The PK parameters are presented in Table III. The oral dose regimens of HL3501 showed dose-dependent PK relationships, such as increasing the oral dose from 30 mg/kg to 60 mg/kg increased the AUC from 23 to 55 μg*h/ml. The mean elimination half-life of HL3501 following the i.v. route was a moderate 2 h. The estimated oral bioavailability (%F) of HL3501 at 30 mg/kg to 60 mg/kg was high at 92.5 and 107.2%, respectively. HL3501 has a high oral bioavailability and good PK profiles in mouse PK.
HL3501 revealed an orally available PK profile in ICR mice. Mean blood concentration vs. time profiles of HL3501 following oral administration of 30 and 60 mg/kg and IV administration of 3 mg/kg, respectively. PO: Orally dosed; IV: intravenous injected.
The pharmacokinetic parameters of HL3501 in mice.
Discussion
In recent clinical studies, diabetic patients have revealed that the observed up-regulation of A3 AR expression was correlated with disease progression (8). It was reported that selective A3 AR antagonists such as MRS1220 and LJ-1888 showed an antifibrotic effect in the kidney using in vitro and in vivo models (10-12).
In addition, non-alcoholic steatohepatitis (NASH) is a form of fibrosis in the liver among the various fibrotic diseases. To date, there have been only a few studies on liver fibrosis with the A3 AR antagonist.
HL3501, A3 AR antagonist is a new compound and structurally different from those compounds (14). We demonstrated that HL3501 may exert anti-fibrotic effects on HK2 kidney cells, human HSCs (LX2 cells and primary HSCs), and the MCD animal model.
HL3501 showed anti-fibrotic efficacy in TGF-β1-induced activation in the HK2 cell assay and also effectively reduced α-SMA and collagen1. The anti-renal fibrosis efficacy of HL3501 is consistent with other A3 AR antagonists (9, 10). We therefore suggest that HL3501 would be protective against renal fibrosis.
Moreover, HL3501 inhibited TGF-β1-induced HSC activation, which resulted in a reduction of α-SMA and fibronectin in human HSCs. To confirm the anti-hepatic fibrotic efficacy of HL3501, we used the MCD animal model and compared it with the comparative control OCA, a clinical candidate of the NASH indication. In the mouse MCD model, HL3501 tended to cause a reduction in profibrotic markers observed in qRT-PCR compared to the vehicle. MCD diet-induced steatosis and liver inflammatory score significantly improved in the HL3501 treatment group in the NAS analysis. Moreover, HL3501 has a different primary target from OCA; however, HL3501 showed improved NAS score and reduction of pro-fibrotic markers (α-SMA, fibronectin, col3A1) than OCA in qRT-PCR.
Another study by Fishman et al., reported that the A3 AR agonist, CF-102 exhibited anti-inflammatory and antifibrotic effects through the NF-B and the Wnt/β-catenin pathways (13). HL3501, A3 AR antagonist may have a different mechanism from CF-102 on the regulation of hepatic fibrosis. In summary, our study indicates that HL3501, an A3 AR antagonist, shows anti-fibrotic effects in preclinical NASH models by inhibiting the expression of pro-fibrotic markers (α-SMA, fibronectin, Col3A1). Moreover, HL3501 reduced pro-fibrotic protein expression in renal fibrosis cell assays, which correlates with the A3 AR antagonists. HL3501 is orally available and has a wide bioavailability (BA: >90%) profile in mouse PK experiments.
Conclusion
HL3501 could be used as a therapeutic option for various fibrotic diseases, including disease of liver and kidney tissues. Further studies are warranted to reveal the detailed mechanism of HL3501 in fibrosis across all tissues.
Footnotes
Authors’ Contributions
YK, WCS, and BGM planned the experiments. YK, DMK carried out the experimental part. YK, DMK and WCS analyzed the data, carried out the statistical analysis, and prepared the tables and figures. YK wrote the final manuscript. All Authors read and approved the final manuscript.
Conflicts of Interest
The Authors declare that they have no commercial or associative conflicts of interest.
- Received July 4, 2022.
- Revision received July 20, 2022.
- Accepted July 28, 2022.
- Copyright © 2022, International Institute of Anticancer Research (Dr. George J. Delinasios), All rights reserved
This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY-NC-ND) 4.0 international license (https://creativecommons.org/licenses/by-nc-nd/4.0).