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Vactosertib, a Novel, Orally Bioavailable Activin Receptor-Like Kinase 5 Inhibitor, Promotes Regression of Fibrotic Plaques in a Rat Model of Peyronie's Disease

Purpose To examine the therapeutic effect of Vactosertib, a small molecule inhibitor of transforming growth factor-β (TGF-β) type I receptor (activin receptor-like kinase-5, ALK5), in an experimental model of Peyronie's disease (PD) and determining anti-fibrotic mechanisms of Vactosertib in primary fibroblasts derived from human PD plaques. Materials and Methods Male rats were randomly divided into three groups (n=6 per group); control rats without treatment; PD rats receiving vehicle; and PD rats receiving Vactosertib (10 mg/kg). PD-like plaques were induced by administering 100 µL of each of human fibrin and thrombin solutions into the tunica albuginea on days 0 and 5. Vactosertib was given orally five times a week for 2 weeks. On day 30, we performed electrical stimulation of the cavernous nerve to measure erectile function, and the penis was obtained for histological examination. Fibroblasts isolated from human PD plaques were used to determine the anti-fibrotic effects of Vactosertib in vitro. Results Vactosertib induced significant regression of fibrotic plaques in PD rats in vivo through reduced infiltration of inflammatory cells and reduced expression of phospho-Smad2, which recovered erectile function. Vactosertib also abrogated TGF-β1-induced enhancement of extracellular matrix protein production and hydroxyproline content in PD fibroblasts in vitro by hindering the TGF-β1-induced Smad2/3 phosphorylation and nuclear translocation, and fibroblast-to-myofibroblast transdifferentiation. Conclusions In view of the critical role of TGF-β and the Smad pathway in the pathogenesis of PD, inhibition of this pathway with an ALK5 inhibitor may represent a novel, targeted therapy for PD.


INTRODUCTION
Peyronie's disease (PD) is characterized by excessive fibrosis and plaque formation in the tunica albuginea (TA), resulting penile deformities and pain on erectile status [1,2]. Inflammatory process and dysregulated wound healing following repeated trauma to the penis resulting from sexual intercourse are known to be responsible for fibrotic processes [3,4]. Despite promising clinical results with intralesional injection of collagenase clostridium histolyticum [5], surgical intervention is still the mainstay of treatment for PD [6,7]. Therefore, novel medical treatments are needed, and they may emerge from our increased knowledge of this disorder at the cellular and molecular levels.
Transforming growth factor-β1 (TGF-β1), a member of TGF-β superfamily, is known as a potent pro-fibrotic factor and is upregulated in human PD plaques [8][9][10]. Previous studies have shown that the expression and activity of the Smad transcriptional factors are increased in primary cultured fibroblast [9] and in fibrotic plaque tissues from PD patients [10]. TGF-β1 has also been regarded as a key component related to tissue fibrosis in a variety of organs [11]. The profibrotic effects of TGF-β1 include increased synthesis of extracellular matrix proteins and the induction of myofibrotic differentiation [11]. The desire to block TGF-β1 signaling has led to the development of several small molecule inhibitors that block the binding of TGF-β1 to its receptors. We recently observed that antagonizing TGF-β signaling through the use of a small molecule inhibitor of activin receptor-like kinase 5 (ALK5) (IN-1130), a TGF-β type I receptor, induced the regression of fibrotic plaques in PD rats in vivo [12]. However, the oral bioavailability of IN-1130 and its kinase selectivity against p38α, one of the most homologous kinase domains to that of ALK5, is relatively low in mice and rats [13], which greatly limits study for its clinical application.
In the present study, the anti-fibrotic effects of Vactosertib were determined in a rat model of PD induced by multiple intratunical administration of fibrin and in primary cultured fibroblasts derived from human PD plaques.

Animals and study design
Four-month-old male Sprague-Dawley (SD) rats were used in this study. To establish an optimal condition to generate PD-like plaques, rats received repeated injections of fibrin (Greenplast; Green Cross Co., Yongin, Korea) into the TA at 100 μL of each of human fibrin and thrombin solutions on days 0 and 5 as we previously described [17]. Then, we determined the efficacy of Vactosertib (molecular weight, 399.42) in a rat model of PD. The chemical structure of Vactosertib and the experimental design are illustrated in Fig. 1.
Age-matched SD rats were randomly divided into the following three groups (n=6 per group): control without treatment; PD rats receiving vehicle; and PD rats receiving Vactosertib (10 mg/kg). Vactosertib was dissolved in an artificial gastric fluid formulation consisting of 7 mL of 37% hydrochloric acid, 2.0 g of NaCl, and 3.2 g of pepsin in 1,000 mL of water. The artificial gastric fluid was used as a vehicle. Vactosertib or vehicle was given orally five times a week for 2 weeks. Evaluation of erectile function was done at day 30 by cavernous nerve electrical stimulation. The penis was then harvested for histologic examination.

Measurement of erectile function
Rats were anesthetized with ketamine (100 mg/kg) and xylazine (5 mg/kg) intramuscularly. Erectile function was measured as we previously described [18]. The platinum wire electrodes were placed around the cavernous nerve under the dissecting microscope (Zeiss, Göttingen, Germany). Electrical stimulation was performed with 3 V current, 12 Hz pulse frequency, and 1 ms pulse width for 1 minute duration. During tumescence, the maximal intracavernous pressure (ICP) and total ICP were recorded. The total ICP of tumescence, expressed by area under the curve, was measured from the beginning of cavernous nerve stimulation to 20 seconds after stimulus termination. Noninvasive computerized tail-cuff system was used for measuring systemic blood pressure (BP-2000; Visitech Systems, Apex, NC, USA). The ratios of maximum ICP to mean systolic blood pressure (MSBP) and total ICP to MSBP were calculated to normalize the variations in systemic blood pressure.

Histological examinations
A midportion of penile segment was collected and fixed in 10% formalin phosphate-buffered solution (PBS) immediately before paraffin embedding. The sections were stained with H&E. The collagen deposition and disorganization in the fibrotic plaque were determined with Masson trichrome staining. Quantitative image analysis of plaque area was performed using image processing program (NIH ImageJ 1.34; http:// rsbweb.nih.gov/ij/), and we analyzed two sections from each animal. The plaque area of the vehicle-treated PD group was arbitrarily set to 100%, and the area of each group was expressed as a percentage of the vehicletreated PD group.

Primary fibroblast culture
The plaque tissue samples were collected from patients with PD who underwent surgical correction after obtaining informed consent from all the subjects. All of procedures were performed in accordance with relevant approved guidelines by the Inha University Institutional Review Board (IRB No. 2007-730). Fibroblasts were isolated from human PD plaques as previously described [10].

Hydroxyproline assay
Collagen protein levels were estimated by hydroxyproline determinations as previously described [19]. Briefly, aliquots of standard hydroxyproline or fibroblast samples were hydrolyzed in an alkali solution and mixed with a buffered chloramine-T reagent for 25 minutes at room temperature. Ehrlich's reagent was added to produce the chromophore. Using a spectrophotometer, the reddish-purple complex of absorbance was measured at 550 nm. Absorbance measurement was recorded and plotted against the concentration of standard hydroxyproline. The concentration of hydroxyproline in fibroblast lysates was calculated based on the standard curve created for each microplate.

Statistical analysis
The results of each measurement are represented by the means±standard errors. Comparisons of parametric data between groups were performed by one-way analysis of variance followed by Newman-Keuls post hoc tests for the multiple comparisons and Kruskal-Wallis tests were used for nonparametric data. Statistical analysis was done with Sigmastat v3.5 software (Systat Software Inc., Richmond, CA, USA). The data were tested for its normality and homogeneity variance. The p-values less than 5% were considered as statistically significant.

Ethics statement
All experiments were conducted in accordance with laboratory animal and institutional animal care guidelines. Protocols used in this study were approved by the Institutional Animal Care and Use Subcommittee

Vactosertib promotes the regression of fibrotic plaques in Peyronie's disease rats through reduced inflammatory cell infiltration
Repeated intratunical injections of fibrin induced fibrotic scarring in the TA. PD rats treated with Vactosertib showed a remarkable regression of fibrotic plaques and improved the disorganization of collagen distribution (Fig. 2). H&E and immunohistochemical staining for vimentin revealed infiltration of lymphocytes and fibroblasts in fibrotic plaques of the vehicletreated PD rats. Oral administration of Vactosertib significantly decreased inflammatory cell infiltration (Fig. 3A).

Vactosertib suppresses the transnuclear expression and phosphorylation of Smad2/3 in Peyronie's disease rats in vivo and in human Peyronie's disease fibroblasts in vitro
To examine the effect of Vactosertib on the TGF-β signaling pathway, we performed immunohistochemical staining for the phosphorylated form of Smad2. P-Smad2 immunoreactivity was weak in control rats. Strong expression of p-Smad2 was noted in fibrotic plaques in the vehicle-treated PD rats, whereas the expression was reduced significantly in PD rats treated with Vactosertib (Fig. 3B).

Vactosertib inhibits myofibroblastic differentiation induced by transforming growth factor-β β1 in human Peyronie's disease fibroblasts
The expression of smooth muscle α-actin, a marker for myofibroblasts, at the protein level was determined by Western blot and immunocytochemistry. Treatment of PD fibroblasts with TGF-β1 resulted in an increase in α-actin fiber formation, which was attenuated by treatment with Vactosertib (Fig. 5).

Vactosertib decreases the production of hydroxyproline and extracellular matrix proteins induced by transforming growth factor-β β1 in human Peyronie's disease fibroblasts
The amount of hydroxyproline was measured to determine total collagen content. The hydroxyproline content in TGF-β1-treated fibroblasts was significantly increased compared with that in untreated fibroblasts. Collagen production induced by TGF-β1 was significantly reduced by Vactosertib treatment (Fig. 6A).
Both Western blot analysis and fluorescent immunocytochemistry showed that Vactosertib significantly inhibited TGF-β1-induced production of extracellular matrix proteins, such as PAI-1, fibronectin, collagen I, and collagen IV in PD fibroblasts (Fig. 6B-6D). However, no significant difference was noted in collagen III expression among three experimental groups (Fig. 6B,  6C).

Vactosertib restores erectile function in Peyronie's disease rats
Representative ICP traces during stimulation of the cavernous nerve with 3 V current, 12 Hz pulse frequency, and 1 ms pulse width in age-matched control and PD rats treated with vehicle or Vactosertib are shown in Fig. 7A. The ratios of maximal ICP and total ICP to MSBP were significantly lower in the vehicletreated PD rats than in age-matched controls. Oral administration of Vactosertib completely restored erectile function in PD rats (Fig. 7B, 7C).

DISCUSSION
We showed here that oral administration of Vactosertib decreased infiltration of inflammatory cells, suppressed the expression of p-Smad2, and ameliorated tunica fibrosis in PD rats in vivo. Furthermore, Vactosertib also diminished TGF-β1-induced extracellular matrix production and hydroxyproline content in hu- man PD fibroblasts in vitro by blocking TGF-β1-induced nuclear translocation of Smad2 and Smad3, and by inhibiting TGF-β1-induced differentiation of fibroblasts into myofibroblasts. Finally, Vactosertib induced a significant recovery of erectile function in PD rats.
In the present study, Vactosertib significantly reduced infiltration of inflammatory cells in PD rats. Local delivery of fibrin glue into the TA increases the expression of TGF-β1 in the fibrotic area [21]. TGF-β is mitogenic for inflammatory cells and promotes the accumulation of extracellular matrix proteins [22]. Moreover, TGF-β is produced by multiple cell types, including fibroblasts, myofibroblasts, and lymphocytes [23,24]. Vactosertib also attenuated TGF-β1-induced Smad2/3 phosphorylation and nuclear translocation in human PD fibroblasts in vitro, and decreased p-Smad2 expression in PD rats in vivo, which results in a decrease in the production of extracellular matrix proteins. There-fore, reduced infiltration of inflammatory cells and blockage of the TGF-β signaling pathway may play a major role in the Vactosertib-mediated regression of fibrosis.
A previous study in renal epithelial cells reported that TGF-β1 can regulate extracellular matrix genes by several mechanisms, including the Smad pathway, the p38 MAP kinase pathway, and a combination of the two, as well as other kinase pathways [25]. In the present study, Vactosertib also abrogated TGF-β1-induced phosphorylation of p38. From this finding, we believe that antifibrotic therapies aimed at inhibiting both the Smad and p38 MAP kinase pathways would be more effective than blocking a single pathway.
TGF-β activates Smad2 and Smad3 upon ligand binding, which in turn induces myofibroblastic differentiation and promotes the synthesis of extracellular matrix proteins [26]. In the present study, treating PD fibro- www.wjmh.org blasts with TGF-β1 induced myofibroblastic differentiation, as evidenced by both Western blot analysis and fluorescent immunocytochemistry for smooth muscle α-actin. Vactosertib suppressed the TGF-β1-induced fibroblast-to-myofibroblast transition. Because myofibroblasts are the main cell type that responsible for the production of extracellular matrix proteins, blocking myofibroblastic differentiation is another important mechanism responsible for the Vactosertib-mediated amelioration of fibrotic processes. Previous study reported that erectile function was impaired in elastin haploinsufficient mice [27], indicating importance of elastin component in normal penile erection. We previously reported that a small molecule inhibitor of ALK5 (IN-1130) restored elastin fibers in PD rats in vivo [12]. However, we did not measure the effects of Vactosertib on elastin fibers and this is a limitation of our study.
Small molecule inhibitors of ALK5, such as SB-505124, SB-525334, GW6604, SD-208, and LY-2157299, were devised to directly block the catalytic activity of ALK5 [13]. Among them, LY-2157299 has progressed to phase II clinical trials for various cancers [28]. A preclinical study in 4-week-old rats with Vactosertib at doses up to 120 mg/kg revealed little toxicity. The maximum tolerated dosage of Vactosertib in rats appeared to be 50 mg/kg/d for males and 20 mg/kg/d for females. Notably, a daily dose of 2.5 mg/kg of Vactosertib was more efficacious than a daily dose of 150 mg/kg of LY-2157299 against melanoma [29]. Additionally, Vactosertib prolonged the lifespan of the hepatic fibrosis model induced by carbon tetrachloride or bile duct ligation as well as the bleomycin-induced pulmonary fibrosis model [13,16]. Currently, phase I clinical trials for patients with advanced solid tumors (NCT02160106, NIH) and phase 1/2 clinical trials in myelodysplastic syndrome (NCT03074006, NIH) have been initiated to investigate the safety, tolerability, and efficacy of Vactosertib. However, no anti-TGF-β therapy is currently available for fibrotic diseases. Therefore, further clinical trials are necessary to validate the therapeutic potential of Vactosertib in patients with PD. Moreover, additional studies are needed to address whether Vactosertib induces the regression of fibrotic plaques when administered at a chronic or stable phase of disease.

CONCLUSIONS
Vactosertib promoted the regression of tunica fibrosis by blocking the TGF-β pathway and inhibiting infiltration of inflammatory cell in PD rats in vivo. Vactosertib also abrogated TGF-β1-induced extracellular matrix production and differentiation of fibroblasts into myofibroblasts by blocking the TGF-β1-induced activation of the Smad2/3 pathway in human PD fibroblasts in vitro. Inhibition of the TGF-β pathway via a novel ALK5 inhibitor, especially as an orally bioavailable formulation, may represent a viable therapeutic strategy for the treatment of PD.