Evaluation of an Integrin α_vβ₃ Radiotracer, [¹⁸F]F-FPP-RGD₂, for Monitoring Pharmacological Effects of Integrin α_v siRNA in the NASH Liver

Abstract

Purpose

Integrin α_v is a key regulator in the pathophysiology of hepatic fibrosis. In this study, we evaluated the potential utility of an integrin α_vβ₃ positron emission tomography (PET) radiotracer, ¹⁸F-labeled cyclic arginine-glycine-aspartic acid penta-peptide ([¹⁸F]F-FPP-RGD₂), for detecting hepatic integrin α_v and function in nonalcoholic steatohepatitis (NASH) model rats using integrin α_v siRNA.

Methods

NASH model rats were produced by feeding a choline-deficient, low-methionine, high-fat diet for 8 weeks. PET/computerized tomography imaging and quantification of integrin α_v protein, serum aspartate aminotransferase, and alanine aminotransferase were performed 1 week after single intravenous injection of integrin α_v siRNA.

Results

Integrin α_v siRNA (0.1 and 0.5 mg/kg) dose-dependently decreased hepatic integrin α_v protein concentrations in control and NASH model rats. The hepatic mean standard uptake value of [¹⁸F]F-FPP-RGD₂ was decreased dose-dependently by integrin α_v siRNA. The mean standard uptake value was positively correlated with integrin α_v protein levels in control and NASH model rats. Serum aspartate aminotransferase and alanine aminotransferase concentrations were also decreased by siRNA injection and correlated with liver integrin α_v protein expression levels in NASH model rats.

Conclusion

This study suggests that [¹⁸F]F-FPP-RGD₂ PET imaging is a promising radiotracer for monitoring hepatic integrin α_v protein levels and hepatic function in NASH pathology.

Introduction

Nonalcoholic steatohepatitis (NASH) is a condition with chronic liver inflammation, which leads to the transformation of hepatic stellate cells (HSCs) to myofibroblasts. The pathology of NASH and the mechanism of how fibrosis progresses have been well documented [1]. Among these, the activation of HSCs is well known as a key event in hepatic fibrosis [2]. Activation of HSCs produces proteins, such as integrin, which play an important role in the process of transforming HSCs into fibrotic myofibroblasts.

Integrins are heterodimeric cell surface receptors, which comprise different 18 α subunits and 8 β subunits. These different pairs of integrins are closely related to the initiation, maintenance, and resolution of fibrosis [3–5]. Elevated integrin α_vβ₃ expression has been observed in activated HSC in hepatic cells [6] and in the NASH model [7–9]. These findings suggest that integrin α_vβ₃ plays a critical role in the development and progression of NASH pathology. Nonclinical and clinical reports have shown the antifibrotic effect of integrin-targeting treatments. ProAgio induces apoptosis of αHSCs in the fibrotic liver by targeting integrin α_vβ₃ [6, 10]. Integrin α_vβ₆ has also been reported to be a key player in the progression of fibrosis. In patients with liver fibrosis, integrin α_vβ₆ mRNA expression levels are increased and correlate with the stage of fibrosis [11]. These reports suggest that integrin α_v plays a pivotal role in the regulation of the fibrotic process. Additionally, therapeutic targeting of integrins may be a promising avenue for the treatment of patients with a broad range of fibrotic diseases, such as NASH.

We have previously evaluated integrin positron emission tomography (PET) imaging using ¹⁸F-labeled cyclic arginine-glycine-aspartic acid penta-peptide ([¹⁸F]F-FPP-RGD₂), integrin α_vβ₃ PET probe, in liver fibrosis animal models [12, 13]. The hepatic [¹⁸F]F-FPP-RGD₂ uptake value was increased before the onset of fibrosis, and well correlated with hepatic integrin α_v and β₃ protein expression and pathological fibrosis score in diet-induced NASH model mice [12]. Moreover, both hepatic mean standard uptake value (SUV) and volume of distribution (V_T), quantified by compartment analysis, were higher than that of control rats and positively correlated with hepatic integrin α_v and β₃ protein levels in diet-induced NASH model rats, severer than the pathological condition of mice [13]. These studies provide a potential of [¹⁸F]F-FPP-RGD₂ PET imaging for noninvasive evaluation of integrin α_v protein and pathophysiology in NASH liver.

In this study, we investigated the potential utility of integrin α_vβ₃ PET radiotracer, [¹⁸F]F-FPP-RGD₂, for the detection of hepatic integrin α_v and function in NASH model rats using integrin α_v siRNA. We performed suppression of hepatic integrin α_v protein using intravenous liquid nanoparticle (LNP) formulation including siRNA, which targets liver-specific integrin α_v, developed by Shionogi & Co., Ltd (Osaka, Japan).

Materials and Methods

Animals

Twenty-three male RccHan®: WIST rats (Wistar Hannover Rcc rats, 7 weeks old) were purchased from Japan SLC, Inc. (Shizuoka, Japan). The rats were housed in a light- and temperature-controlled room (lights on at 07:00 h and off at 19:00 h). They were allowed free access to tap water and fed a normal diet (CE-2; CLEA, Tokyo, Japan) or a choline-deficient, low-methionine, high-fat diet (CDHFD; no choline, 45% fat, 0.1% methionine, and 1% cholesterol) prepared by Oriental Yeast Co., Ltd. (Tokyo, Japan) for 8 weeks. Eight weeks after feeding, control or NASH model rats received a single intravenously administration of LNP formulation including siRNA at a dosage of 0 (phosphate-buffered saline; PBS), 0.1, or 0.5 mg/kg. The experiments were approved by the Institutional Animal Care and Use Committee of Osaka University Graduate School of Medicine (approval number: 29–030-002, 21 July 2017).

Radiochemistry

[¹⁸F]F-FPP-RGD₂ was synthesized as previously reported [14] to a mean molar activity of 166.5 ± 60.5 GBq/μmol. The RGD dimeric peptide PEG₃-c[RGDyK]₂ was obtained from Peptides International (Louisville, KY, USA). The radiochemical purity of all radiosynthesis, which was analyzed by high-performance liquid chromatography on a COSMOSIL 5C18 MS-II column (Nacalai Tesque, Inc. Kyoto, Japan), was higher than 99%.

PET/Computed Tomography Scans

All dynamic PET/computed tomography (CT) scans were performed by a Triumph LabPET-12 PET/CT instrument (TriFoil Imaging, Chatsworth, CA, USA), which was designed for small animals in the laboratory, and a PET camera with an intrinsic axial resolution of 1.38 mm (full width at half maximum) [15]. The injection tube was catheterized into the tail vein for PET probe injection under 2% isoflurane anesthesia, and the rats were placed on a heated pad on the scanner bed. All PET/CT scans of the control or NASH model group were conducted 1 week after the administration of PBS or siRNA, respectively. The scans were started immediately after intravenous [¹⁸F]F-FPP-RGD₂ (8.4 ± 1.0 MBq) administration at 0.5 mL/30 s by a syringe pump (Legato 210; KD Scientific, Holliston, MA, USA) via a cannula inserted into the tail vein. PET scans were conducted with a 90-min dynamic scan mode, and then a CT scan was conducted to acquire anatomical information and correct the PET images for attenuation.

PET Image Analysis

CT images were reconstructed using the filtered back-projection method (512 slices). PET images were reconstructed into 24 frames of increasing length (6 × 10, 4 × 60, 11 × 300, and 3 × 600 s) using the three-dimensional maximum-likelihood expectation–maximization algorithm and CT-based attenuation correction. To obtain time-activity curves (TACs), volumes of interest for the liver (1280 mm³) were manually defined for each rat on their CT images using PMOD PET data analysis software (v3.905; PMOD Technologies Ltd., Zurich, Switzerland). TACs for the liver were constructed by normalizing decay-corrected measurements and are expressed as mean standardized uptake values (SUVs), where SUV = radioactivity concentration (kBq/cm³) × body mass (g)/amount of radioactivity injected (MBq)/1000.

Plasma AST and ALT Analysis

After PET/CT scanning, rats were euthanized by exsanguination under isoflurane anesthesia. Plasma samples were collected and assayed for AST and ALT concentrations by enzymatic methods using commercially available kits (Sekisui Medical, Tokyo, Japan) and a Hitachi 7170 autoanalyzer (Hitachi, Tokyo, Japan) in accordance with the manufacturer’s instructions.

Hepatic Integrin α_v Protein Analysis

Hepatic integrin α_v subunit protein levels were determined using a JESS Automated Western Blotting system (Protein Simple, San Jose, CA, USA). Liver tissue was homogenized in RIPA Lysis and Extraction Buffer (Thermo Fisher Scientific K.K., Tokyo, Japan) which contained Halt Protease Inhibitor Cocktail (Thermo Fisher Scientific K.K., Tokyo, Japan), and 0.2 mg/mL protein in lysates was separated by a 12–230 kDa Separation Module (Protein Simple). Hepatic integrin α_v protein was detected using a mouse monoclonal antibody against CD51 (integrin α_v) (Cat. No. 611013; BD Biosciences, San Jose, CA, USA) and the Anti-Mouse Detection Module (Protein Simple) in accordance with the manufacturer’s instructions.

Statical Analysis

All statistical analyses were performed using GraphPad Prism statistical software (version 6; GraphPad Software, San Diego, CA, USA). All results are expressed as the mean ± standard deviation (SD). Statistical comparisons between the three groups were made using Dunnett’s test. Spearman’s rank correlation was used for correlation analysis between two variables (i.e., integrin α_v protein expression, SUV, AST, and ALT), and Spearman’s rank correlation coefficient was represented r_s. Statistical significance (two-tailed) was set at P < 0.05.

Results

Effects of siRNA on the Body, Liver, and Liver/Body Weight Ratio

Liver weight and the liver/body weight ratio in the NASH model group were significantly higher than those in the control group (both P < 0.01, Table 1). In contrast, there were no significant differences in body, liver, and liver/body weight ratio within the control or NASH model groups, which suggested no physiological effect of siRNA injection (Table 1).

Table 1.

Body weight, liver weight, and liver/body weight ratio

Group	Dose of siRNA (mg/kg)	Parameters
		Body weight (g)			Liver weight (g)			Liver/body weight ratio
Control	PBS	255.7	±	15.1	9.26	±	1.4	0.036	±	0.004
	0.1	278.4	±	37.7	9.94	±	1.1	0.036	±	0.001
	0.5	265.3	±	28.8	9.27	±	0.9	0.035	±	0.002
NASH model	PBS	235.0	±	21.3	16.9	±	2.5**	0.071	±	0.005**
	0.1	243.1	±	28.4	17.2	±	2.7**	0.071	±	0.008**
	0.5	238.8	±	26.1	18.9	±	2.4**	0.079	±	0.003**

Values are expressed as mean ± SD (n = 3–5). ^**P < 0.01, compared with the respective control group

NASH nonalcoholic steatohepatitis, PBS phosphate-buffered saline, siRNA small interfering RNA

Effects of siRNA on Liver Integrin α_v Protein Levels

The suppression effects of siRNA injection on integrin α_v protein levels in the liver were examined. Liver integrin α_v protein levels in the NASH model group were significantly higher than those in the control group (P < 0.01, Fig. 1). Moreover, liver integrin α_v levels in the control and NASH model groups were significantly decreased by siRNA injection (0.1 and 0.5 mg/kg) compared with PBS injection rats (P < 0.01, Fig. 1).

Fig. 1.

Effects of siRNA on hepatic integrin α_v protein expression. Hepatic integrin α_v protein expression in control (a) and NASH model groups (b). Vertical axis values indicate integrin α_v protein expression relative to mean integrin α_v protein expression in control rats with PBS injection. *P < 0.05; **P < 0.01 compared with the PBS treatment group in control or NASH model rats. Values are expressed as mean ± SD (n = 3–5). PBS phosphate-buffered saline, siRNA small interfering RNA

Effects of siRNA on Liver [¹⁸F]F-FPP-RGD₂ Uptake

[¹⁸F]F-FPP-RGD₂ uptake in the liver in the control and NASH model groups was evaluated 1 week after the injection of PBS or siRNA (0.1 and 0.5 mg/kg). Figure 2a shows that the TACs of [¹⁸F]F-FPP-RGD₂ in the NASH model groups were higher than those in the control groups, which decreased by siRNA injection. The mean standard uptake value between 60 and 90 min (SUV_60-90 min) in the control group was decreased by siRNA dose injection (0.5 mg/kg; P < 0.05, Fig. 2c). Moreover, the mean SUV_60-90 min in NASH model rats was significantly lower than that in PBS-treated rats in the control group (0.1 mg/kg, P < 0.05; 0.5 mg/kg, P < 0.01, Fig. 2d). Figure 3 shows representative color-coded SUV_60-90 min images of [¹⁸F]F-FPP-RGD₂ PET in the control and NASH model groups 1 week after the injection of PBS or siRNA (0.1 and 0.5 mg/kg).

Fig. 2.

Time-activity curves of the liver and mean SUV_60-90 min. Hepatic time-activity curves of [¹⁸F]F-FPP-RGD₂ in the control group (a) and NASH model group (b) after siRNA (0, 0.1, and 0.5 mg/kg intravenously) administration. Hepatic mean SUV_60-90 min in the control group (c) and NASH model group (d). *P < 0.05; **P < 0.01 compared with the PBS treatment group in control or NASH model rats. Values are expressed as mean ± SD (n = 3–5). PBS phosphate-buffered saline, siRNA small interfering RNA

Fig. 3.

Representative color-coded PET/CT images. Representative PET/CT fusion images at 60–90 min after [¹⁸F]F-FPP-RGD₂ administration. PBS phosphate-buffered saline, siRNA small interfering RNA

Correlation Analysis Between Integrin α_v Protein Levels and the SUV of [¹⁸F]F-FPP-RGD₂ in the Liver

We determined whether liver integrin α_v protein levels are correlated with the SUV of [¹⁸F]F-FPP-RGD₂ in the liver after the injection of PBS or siRNA (0.1 and 0.5 mg/kg). We performed a correlation analysis between liver integrin α_v protein levels and the mean SUV_60-90 min of [¹⁸F]F-FPP-RGD₂ in the liver in the same rats. Liver integrin α_v protein levels were strongly correlated with the mean SUV_60-90 min of [¹⁸F]F-FPP-RGD₂ in the liver in the control and NASH model groups with or without siRNA injection (r_s = 0.901, P < 0.01, Fig. 4).

Fig. 4.

Correlation analysis of hepatic integrin α_v protein expression levels and mean SUV_60-90 min. Correlation between hepatic integrin α_v protein expression levels and the mean SUV_60-90 min in the control and NASH model groups after siRNA injection. Open circles, control rats with PBS; open triangles, control rats with 0.1 mg/kg siRNA; open squares, control rats with 0.5 mg/kg siRNA; filled circles, NASH model rats with PBS; filled triangles, NASH model rats with 0.1 mg/kg siRNA; filled squares, NASH model rats with 0.5 mg/kg siRNA. PBS phosphate-buffered saline, siRNA small interfering RNA

Correlation Analysis Between Integrin α_v Protein Levels and Serum AST and ALT Concentrations

We also conducted a correlation analysis between liver integrin α_v protein levels and serum AST and ALT concentrations. Hepatic integrin α_v protein expression levels were strongly correlated with serum AST (r_s = 0.875, P < 0.01) and ALT (r_s = 0.862, P < 0.01) concentrations in the control and NASH model groups with or without siRNA injection (Fig. 5).

Fig. 5.

Correlation analysis of hepatic integrin α_v protein expression levels and AST or ALT concentrations. Correlation between hepatic integrin α_v protein expression levels and AST (a) or ALT (b) in the control group and NASH model group after siRNA injection. Open circles, control rats with PBS; open triangles, control rats with 0.1 mg/kg siRNA; open squares, control rats with 0.5 mg/kg siRNA; filled circles, NASH model rats with PBS; filled triangles, NASH model rats with 0.1 mg/kg siRNA; filled squares, NASH model rats with 0.5 mg/kg siRNA. PBS phosphate-buffered saline, siRNA small interfering RNA, AST aspartate aminotransferase, ALT alanine aminotransferase

Correlation Analysis Between the SUV of [¹⁸F]F-FPP-RGD₂ and Serum AST and ALT Concentrations

The SUV of [¹⁸F]F-FPP-RGD₂ were also correlated with serum AST (r_s = 0.680, P < 0.01) and ALT (r_s = 0.741, P < 0.01) concentrations in the control and NASH model groups with or without siRNA injection (Fig. 6).

Fig. 6.

Correlation analysis of hepatic mean SUV_60-90 min and AST or ALT concentrations. Correlation between hepatic mean SUV_60-90 min and AST (a) or ALT (b) in the control group and NASH model group after siRNA injection. Open circles, control rats with PBS; open triangles, control rats with 0.1 mg/kg siRNA; open squares, control rats with 0.5 mg/kg siRNA; filled circles, NASH model rats with PBS; filled triangles, NASH model rats with 0.1 mg/kg siRNA; filled squares, NASH model rats with 0.5 mg/kg siRNA. PBS phosphate-buffered saline, siRNA small interfering RNA, AST aspartate aminotransferase, ALT alanine aminotransferase

Discussion

In this study, we investigated whether [¹⁸F]F-FPP-RGD₂, integrin α_vβ₃ PET imaging can assess the suppression of hepatic integrin α_v protein by siRNA injection in NASH model rats. PET imaging with [¹⁸F]F-FPP-RGD₂, integrin α_vβ₃ PET radiotracer could detect a decrease in integrin α_v protein expression levels induced by siRNA in the control and NASH model groups. Additionally, there was a strong correlation between the hepatic PET quantitative value (mean SUV_60-90 min) and hepatic integrin α_v protein levels in the control and NASH model groups. These findings suggested that [¹⁸F]F-FPP-RGD₂ PET imaging may be a noninvasive method for monitoring the levels of integrin α_v protein in the liver.

The diet-induced NASH animal model is suitable for reflecting the clinically relevant onset and progression of hepatic fibrosis [16, 17]. In our previous study, we observed the progression of NASH pathology by histological and biochemical analyses in CDHFD-induced NASH model rats [13]. We found that hepatic integrin α_v protein expression levels increased with the severity of the pathological score. These previous findings are similar to other previous studies that were performed in similar animal models [12, 16, 18]. Furthermore, we validated that [¹⁸F]F-FPP-RGD₂ PET uptake correlated well with pathological change of hepatic fibrosis and expression of integrin α_v protein [12, 13]. In the present study, we performed PET imaging in the NASH model induced by feeding CDHFD for 8 weeks. The NASH pathological stage of rats fed CDHFD for 8 weeks is similar to that in rats fed for 9–10 weeks in our previous study [13]. Kleiner et al. [19] used the NASH model with CDHFD feeding for 8 weeks and found borderline NASH and frank NASH according to the nonalcoholic fatty liver disease activity score. In the present study, integrin α_v protein expression levels were higher in the NASH model group than in the control group, with an eightfold difference (Fig. 1). We performed a single intravenous injection of integrin α_v siRNA to rats in these pathological stages. Our compound (including siRNA) was designed to elicit the maximum suppression effect a week after administration in rats. Therefore, integrin α_v protein expression levels were evaluated 1 week after administration of the compound. As shown in Fig. 1, integrin α_v protein levels in siRNA-treated rats (0.1 or 0.5 mg/kg) were significantly lower (< 50%) than those in PBS-treated rats in the control and NASH model groups. This finding indicated that the increased hepatic integrin α_v in NASH pathology can be adequately suppressed by siRNA injection.

A ninety-minute dynamic PET scan was conducted to determine the hepatic TACs of [¹⁸F]F-FPP-RGD₂ in the control and NASH model groups with integrin α_v siRNA injection. In both groups, the hepatic uptake of [¹⁸F]F-FPP-RGD₂ rapidly peaked within several minutes after PET probe administration and gradually decreased until the end of scanning at 90 min (Fig. 2a and b). This phenomenon was the same in both groups, regardless of the presence or absence of siRNA injection. Moreover, we found that the TACs in the NASH model group showed a higher overall trend than that in the control group (Fig. 2a and b). Subsequently, we evaluated the change in the mean SUV_60-90 min by siRNA injection in both groups. The mean SUV_60-90 min of rats with siRNA injection was significantly lower than that in PBS-treated rats in both groups (Fig. 2c and d), which was also visually obvious (Fig. 3). The decrease of SUV_60-90 min in control rats by siRNA could be due to a decrease in basal expression of integrin α_v. The reduction of basal levels had no effect on the physiological parameters (body and liver weight, AST, and ALT). To the best of our knowledge, no reports have shown that PET imaging can noninvasively and visually detect a suppression effect of integrin α_v siRNA in the NASH animal model. In this study, we clearly showed that the [¹⁸F]F-FPP-RGD₂ PET probe, which binds to integrin α_vβ₃, could detect the suppression of hepatic integrin α_v expression by siRNA in NASH model rats.

Clinical studies have shown that integrins α_vβ₃ and α_vβ₆ are upregulated in the liver tissue of patients with NASH [6, 11], with integrin α_vβ₃ playing an important role in the development and progression of NASH pathology. Specifically, integrin α_vβ₃ is a key molecule in the transformation of hepatic stellate cells into myofibroblasts. Furthermore, several integrin inhibitors have been shown to reduce fibrosis in animal models. The integrin α_vβ₃ inhibitor ProAgio induces apoptosis of αHSC in the fibrotic liver [6, 10]. Additionally, the integrin α_vβ₁, α_vβ₃, and α_vβ₆ antagonist IDL-2965 shows strong antifibrotic effects in liver fibrosis animal models. Prophylactic treatment of IDL-2965 significantly reduced histopathological fibrosis score in CCl₄-induced liver fibrosis mice. And therapeutic treatment of IDL-2965 significantly reduced liver α-SMA and collagen levels measured by immunohistochemistry, in diet-induced liver fibrosis mice. These effects are correlated with a decrease in relevant plasma biomarkers [20]. Unfortunately, none of the low-molecular-weight drugs targeting integrins have been launched into the market. Although the reason for this is unclear, our development candidate, LNP formulation including integrin α_v siRNA, has at least two features that its predecessor does not. The first is that our drug candidate suppresses the production of integrin α_v protein regardless of the type of β subunit, which is a feature that no other compound has. In this study, we confirmed that hepatic integrin α_v protein expression levels were strongly correlated with serum AST and ALT concentrations (Fig. 5), indicating that integrin α_v siRNA may deliver therapeutic effects. The second feature is the specificity of the integrin α_v siRNA in the LNP formulation for the liver, thereby reducing concerns about side effects on other organs. Although further research is needed, these characteristics indicated that our candidate, by specificity suppressing the mRNA expression level of integrin α_v siRNA in the liver, will be safer and more therapeutically effective than previous small molecules.

A previous report [21] showed that F-FPP-RGD₂ has a high affinity for integrin α_vβ₃, with a half-maximal inhibitory concentration (IC₅₀) of 3.8 ± 0.42 nM, and a moderate affinity for integrin α_vβ₆, with an IC₅₀ of 86 ± 7 nM. In contrast, its affinities for two other integrins, α_vβ₅ and α_vβ₈, are 503 ± 55 nM and > 10,000 nM, respectively. These data indicate that F-FPP-RGD₂ has a higher selectivity for integrin α_vβ₃ than for other integrins. In our study, hepatic integrin α_v expression levels in both groups were strongly positively correlated with the hepatic mean SUV_60-90 min of [¹⁸F]F-FPP-RGD₂ (Fig. 4). Moreover, hepatic integrin α_v expression levels were positively correlated with serum AST and ALT concentrations (Fig. 5). Further studies will be needed to clarify the effects of siRNA on pathologic fibrosis in NASH. Our findings suggest that the hepatic mean SUV_60-90 min of [¹⁸F]F-FPP-RGD₂, which is obtained noninvasively in a clinical static PET scan, may be useful for monitoring the hepatic integrin α_v expression and pathophysiology of NASH.

In the future, we will examine whether this decrease in integrin α_v expression, including the regression of fibrosis and recovery of liver pathology, is correlated with PET data in rodents and primates.

Conclusion

This study shows that [¹⁸F]F-FPP-RGD₂ PET imaging can detect integrin α_v protein suppression by siRNA in a diet-induced NASH animal model. Moreover, the mean SUV_60-90 min of [¹⁸F]F-FPP-RGD₂ is strongly correlated with hepatic integrin α_v protein expression levels and hepatic function (ALT and AST). These findings indicate that [¹⁸F]F-FPP-RGD₂ PET imaging may be useful for the noninvasive detection of liver integrin α_v protein and hepatic function in NASH pathology.

Author Contribution

SH performed the PET experiments and data analysis and drafted the manuscript. MI performed the PET experiments. HI synthesized the PET probe. IM performed the protein and AST/ALT analysis. JN performed the design and synthesization of LNP formulation. KM and ES were the supervisors of this study. KA coordinated and designed the study and drafted the manuscript. All authors read and approved the final version of the manuscript.

Funding

This study was funded by Shionogi & Co., Ltd.

Data Availability

All data generated or analyzed during this study are included in this published article and its supplementary information files.

Declarations

Conflict of Interest

Shuichi Hiroyama, Keiko Matsunaga, Miwa Ito, Hitoshi Iimori, Ippei Morita, Jun Nakamura, Eku Shimosegawa, and Kohji Abe declare that they have no competing interests.

Ethics Approval

The experimental protocols were reviewed and approved by the Institutional Animal Care and Use Committee of Shionogi & Co. Ltd. and Osaka University Graduate School of Medicine.

Consent to Participate

Not applicable.

Consent for Publication

Not applicable.