WS^® 1541 contained in PRO 160/120 is a proprietary combination of 160 mg extract from saw palmetto fruits (Serenoa repens (Bartram) J.K.Small, syn. Sabal serrulata (Michx.) Schult.f), (DER 10–14.3:1), extraction solvent: ethanol 90% (m/m) (WS^® 1473) and 120 mg dry extract from stinging nettle root (Urtica dioica L.), (DER 7.6–12.5:1), extraction solvent: ethanol 60% (m/m) (WS^® 1031). The plant material was fully authenticated by morphologic and analytical means complying to the European pharmacopoeia (Ph. Eur. 11.0) by the internal pharmacognosy and quality control departments of Dr. Willmar Schwabe GmbH and Co. KG prior to extraction and specimens are stored for 10 years. Individual batches of the two extracts were taken from regular GMP production batches, with Urtica extract (batch W814698) containing 82.5 ppm scopoletin and Sabal extract (batch EXCh.952) containing 82.0% fatty acids and 0.3% sterols calculated as β-sitosterol as specified in the certificates of analyses. Furthermore, the extracts were characterized by three chemical fingerprinting methods (Supplementary Figure S1), with high performance thin layer chromatography (HPTLC) and nuclear magnetic resonance spectroscopy (NMR) used for both extracts, while gas chromatography flame ionization detector (GC-FID) was exclusively used for Sabal extract and liquid chromatography high resolution mass spectrometry (LC-HRMS) solely used for Urtica extract due to the respective phytochemical nature of the two extracts. The aforementioned characterization of the plant material, extraction process and final extract comply in most instances with the requirements of the Consensus statement on the Phytochemical Characterization of Medicinal Plant extracts (Heinrich et al., 2022).

Sulpiride was obtained from Sigma. Finasteride (Think Chemical Co., Ltd.) served as an active control in some experiments.

Data were analyzed using GraphPad Prism 10 (GraphPad Software, La Jolla, CA) and are presented as mean values ±standard deviation (SD). Experimental groups were compared using one-way analysis of variance (ANOVA) followed by Dunnett post-tests. In the in vivo study, vehicle A/vehicle B and each active treatment group were compared to the sulpiride/vehicle B group. In the in vitro study, untreated cells and those with active treatments were compared to the DMSO group unless stated otherwise. In line with the exploratory character of the experiments and recent guidelines (Michel et al., 2020a), calculated p-values should not be interpreted as hypothesis testing and only as descriptive. Relevant p-values <0.05 are presented in the figures.

Neither sulpiride nor any of the WS^® 1541 doses, nor finasteride affected body weight (Supplementary Figure S2). As reported by others (Van Coppenolle et al., 2000; Van Coppenolle et al., 2001), treatment with sulpiride for 30 days approximately doubled the weight of the lateral prostate lobes while not affecting that of the ventral and dorsal lobe or total prostate/body weight ratio (Figures 1B–E). Treatment with WS^® 1541 did not affect the weight of the lateral or any other lobe relative to vehicle B. In contrast, the active control finasteride reduced the weight of the lateral and ventral lobe and total prostate weight. Thus, our model recapitulates the known phenotype of the sulpiride model and the established effects of the active control finasteride.

We then used the validated model to explore effects of the various treatments on the expression of various genes related to oxidative stress and/or inflammation (Cox2, Nos2, Il1b, Il2, Il4, Il6, Il7, Kc, Il15, Il16, Il18, Ccl2, Ccl7, Tnfa, and Ikbkb), adhesion molecules, growth factors and transcription factors (Vegfa, Tgfb, Egr1, Icam1, Fgf2 and Egf) in lateral lobe tissue. The expression of the reference gene Gapdh was similar in all groups (Supplementary Figure S3), and all target gene expressions were presented relative to that of Gapdh. Relative to the sulpiride/vehicle B group, all target genes were expressed less in the vehicle A/vehicle B group (Figure 2), implying that induction of a lateral lobe enlargement was associated with an upregulation of all analyzed target genes. Treatment with WS^® 1541 tended to attenuate the sulpiride-induced gene expression in the majority of analyzed genes, indicative of an overall normalization of expression with a profile more similar to control animals. There was no clear evidence of a dose-response curve. However, treatment with finasteride appeared to exacerbate parameters indicative of inflammation and oxidative stress. Except for Il2 and Tnfa, finasteride increased the pro-inflammatory expression profile as seen by an upregulation of Cox2, Il1b, Il4, Il6, Kc, Il18, Ccl2 and Ccl7. Focusing on adhesion molecules, transcription and growth factors, treatment with WS^® 1541 attenuated increased expression levels of Vegfa, Tgfb, Egr1, Icam1, Fgf2 and Egf seen in sulpiride treated control animals. Finasteride treatment showed a similar expression pattern to the sulpiride/vehicle B group but elevated levels of Tgfb and Egr1 expression.

Sulpiride-treated rats exhibited an approximate doubling of oxidized protein as assessed by immunohistochemistry (Figures 3A, B). While this parameter was not affected by finasteride, all three doses of WS^® 1541 inhibited the increase in oxidized protein to levels similar to the double control animals.

Treatment with sulpiride induced epithelial hypertrophy as evidenced by an increase in epithelial thickness by approximately 50%. This increase was completely prevented by all treatments (Figures 3C, D).

For further evaluation of potential mechanisms of action underlying the observed treatment effect of WS^® 1541 against oxidative, inflammatory and hyperproliferative changes, the combination product WS^® 1541 and its individual extract components WS^® 1473 and WS^® 1031, were tested in a set of in vitro experiments using BPH-1 cells. In this immortalized luminal epithelial cell line isolated from a 68-year-old patient with benign prostrate hyperplasia, both single extracts concentration-dependently reduced basal production of reactive oxygen species (Figure 4A). To reflect the relative abundance of the individual extracts in the clinically used combination product, a separate experiment compared 25 μg/mL WS^® 1473, 21.9 μg/mL WS^® 1031 and 46.9 μg/mL WS^® 1541 as well as 100 µM finasteride (Figure 4B). This experiment confirmed the lack of effect of finasteride on ROS production. In contrast, both WS^® 1473 and WS^® 1031 reduced ROS abundance and the extract combination WS^® 1541 caused a more prominent inhibition than either extract alone.

We next examined the effects of the extracts and of finasteride on apoptosis and cell cycle progression. While neither finasteride nor WS^® 1031 affected the amount of annexin V⁺ cells, WS^® 1473 and WS^® 1541 stimulated apoptosis (Figures 4C, D). Using the EdU assay to assess possible cell cycle effects, we found that treatment with finasteride resulted in increased cells numbers in G₁-phase. The single extracts WS^® 1031 and WS^® 1473 showed little effect on cell cycle progression. However, their combination in WS^® 1541 increased the amount of cells in G₁-phase as seen with finasteride, indicating possible synergism of both extracts. Consistent with these results, finasteride and WS^® 1541 decreased the cell number in S-phase, thus inhibiting cell cycle progression by holding the cells in G₁-phase. No change was observed for any of the treatments on the G₂ phase (Figures 4E,F).

To obtain insight into the effect of the treatments on cell migration as a marker for epithelial-mesenchymal-transition (EMT), a gap-closing assay was performed. Concentration-response experiments showed that WS^® 1473 was ineffective whereas WS^® 1031 concentration-dependently inhibited migration (Figure 5A). In another experimental setting with constant concentrations, we confirmed that WS^® 1473 was ineffective in this assay, while WS^® 1031, WS^® 1541 and finasteride decreased migration retaining more of the initial cell free area (Figures 5B, C).

As TGF-β is a key driver of EMT, we examined the effect of WS^® 1473 and WS^® 1031 on mRNA expression of the three TGF-β isoforms and its two receptors. We observed no changes due to different treatments in mRNA expression of HPRT verifying HPRT as suitable reference gene (Supplementary Figure S4). All three TGF-β isoforms and their receptors were expressed in BPH-1 cells. To compare expression levels of the different isoforms, we normalized all expression levels to TGFB1 expression, which showed the highest expression level. Expression levels of the other TGF-β isoforms normalized to TGFB1 were much lower with 0.20 and 0.02 for isoforms 2 and 3, respectively. Concerning TGF-β receptors, a 2-fold higher expression level was found for TGFBR2 vs. TGFBR1 with normalized expression levels of 0.99 and 0.48, respectively (Figure 6A). WS^® 1031 moderately decreased expression of TGFB1, TGFB2, TGFB3 and TGFBR1 without a clear concentration-response effect. WS^® 1473 had no effect on expression of either TGFB1 or TGFB3 at concentrations up to 30 μg/mL, but concentration-dependently reduced the mRNA expression of TGFB2. Both WS^® 1031 and WS^® 1473 had a minor effect on the expression of TGFBR2 (Figures 6B–F). F-Actin-Polymerization was not affected by WS^® 1473 and WS^® 1031 treatment (Supplementary Figure S5).

Treatment with the dopamine D₂ receptor antagonist sulpiride leads to hyperprolactinemia. While this is an adverse effect in clinical use, it can be leveraged to induce BPH in rats (Van Coppenolle et al., 2000; Van Coppenolle et al., 2001). The design of the present in vivo study in rats follows that of a previous similar study in mice in which hyperprolactinemia was induced by transgenic expression of prolactin (Pigat et al., 2019). Of note, the sulpiride model induces an enlargement of the lateral lobe whereas the ventral and dorsal lobes are insensitive to sulpiride-induced hyperprolactinemia (Van Coppenolle et al., 2000; Van Coppenolle et al., 2001). This phenotype with preferential enlargement of the lateral lobes was recapitulated in the present study, thereby validating the sulpiride treatment.

Our in vivo study had a preventive design (i.e., treatment starting concomitant with sulpiride), which may be more sensitive to treatment effects than designs in which medication was administered after prostatic enlargement had manifested.

Several measures were implemented to increase data robustness of our experiments. First, we used finasteride as a positive control in most of our experiments in high doses or concentrations (5 mg/kg/day or 100 µM) to exclude false negative effects due to underdosing. The 5α-reductase inhibitor finasteride is an established and guideline-recommended medical treatment to delay progression of BPH (Gravas et al., 2022). Second, we applied three extract doses in the in vivo study and several extract concentrations in the in vitro experiments to obtain more robust data. As high concentrations or doses of plant extracts can have non-specific effects due to the presence of toxoflavins, enones, quinones or catechols (Baell and Walters, 2014), it is important to demonstrate that the observed effects have specificity and are not due to non-specific effects. Therefore, we specifically compared how the single extracts WS^® 1473 and WS^® 1031 contribute to the effects of the clinically used WS^® 1541 in vitro. Third, while it is usually assumed that reference gene expression used for normalization of mRNA data is stable, examples of regulation have been reported for all commonly used reference genes (Hendriks-Balk et al., 2007; Bollmann et al., 2012; Michel-Reher and Michel, 2015). Therefore, we explicitly tested the reference gene used for normalization purposes in our studies; Gapdh was stably expressed within the in vivo experimental setting and in vitro HPRT expression in BPH-1 cells was irrespective of treatment. Fourth, based on various guidelines on data robustness (Vollert et al., 2020; Vollert et al., 2022), we classify our experiments as exploratory, i.e., not designed to test a specific statistical null hypothesis. Rather, we applied multiple complementary approaches to reach robust conclusion. Finally, in line with the exploratory character of our experiments and with recommendation from leading statisticians (Amrhein et al., 2019; Michel et al., 2020b), we did not perform hypothesis-testing statistical analysis. Rather, all calculated p-values should be interpreted as descriptive only.

These aspects should be considered in the interpretation of our data. In the following we will discuss our findings based on mechanistic pathways by integrating those from the in vivo and in vitro studies, followed by a discussion of the individual contributions of WS^® 1473 and WS^® 1031 to the overall effects of the clinically used WS^® 1541.

As male LUTS are assumed to be caused by bladder outlet obstruction due to benign prostatic enlargement, reducing prostate growth or even shrinking prostate size is a mechanism of action useful for delaying BPH progression and reducing the risk of complications, but not for symptomatic improvement. Among the chemically defined medicines for the treatment of BPH, 5α-reductase inhibitors reduce prostate size whereas α₁-adrenoceptor antagonists and PDE-5 inhibitors do not. By contrast, α1-adrenoceptor antagonists and PDE-5 inhibitors have a rapid and robust effect on LUTS, while the improvement of LUTS is delayed and limited with 5α-reductase inhibitors (Bschleipfer et al., 2023; Michel et al., 2023). Obviously, pharmacological mechanisms unrelated to prostate size are most relevant for reduction of symptoms.

One of the suggested mechanisms of action of Sabal serrulata fruit extracts is inhibition of 5α-reductase (Koch, 2001; Buck, 2004; Pigat et al., 2019; Csikós et al., 2021). Multiple studies have reported a concentration-dependent and non-competitive inhibition of 5α-reductase activity by hexane, ethanol and supercritical CO₂ Sabal serrulata extracts (European Medicines Agency, 2015). Of note, the potency of Sabal serrulata fruit extracts to inhibit 5α-reductase differs markedly between them (Scaglione et al., 2008). However, the more relevant question is whether and to which extent such inhibition of 5α-reductase activity contributes to their clinical effects. Our data argue against a relevant contribution of this mechanism of action in the sulpiride BPH model because the effects of the Sabal serrulata extracts tested here differed from those of finasteride for most outcome parameters.

As reported by others (Van Coppenolle et al., 2000; Van Coppenolle et al., 2001), treatment with sulpiride for 30 days approximately doubled the weight of the lateral prostate lobes while not affecting that of the ventral and dorsal lobe or total prostate/body weight ratio.

In contrast to finasteride, WS^® 1541 did not inhibit the sulpiride-induced enlargement of the lateral prostate lobe (Figure 1). On the other hand, WS^® 1541 at doses of 600 and 900 mg/kg/day has previously been reported to reduce prostate size in the mouse model of transgenic overexpression of prolactin (Pigat et al., 2019). While these data appear contradictory, this may be related to anatomical differences between mouse and rat prostate. Recently, Dos Santos et al. published that cell plasticity in the above-mentioned mouse model drives amplification of an androgen-independent epithelial cell population which is sensitive to antioxidant therapy indicating different states of disease progression (Dos Santos et al., 2023). This indicates different stages of disease development and progression which might be targeted by different treatment options or the combination of different treatments. This may be another explanation why prostate size, particularly the enlargement of the lateral lobes, was not affected in our sulpiride study in contrast to the study published by Pigat et al. (Pigat et al., 2019). More importantly, Sabal serrulata fruit extracts did not reduce prostate volume or prostate specific antigen levels in serum in clinical studies in BPH patients (Carraro et al., 1996), which sharply contrast the consistently observed reduction of prostate size and prostate specific antigen upon treatment with finasteride (Rittmaster, 1994).

The lack of effect on prostate size notwithstanding, WS^® 1541 had several growth-related effects in vivo and in vitro. Thus, WS^® 1541 reduced the mRNA expression of several growth factors in the sulpiride model, including Vegfa, Egf, Egr1, Icam1, Tgfb and Fgf2 (Figure 2), and diminished epithelial thickness in lateral lobes of sulpiride treated rats as does finasteride. This is in line with the data from the transgenic mouse model (Pigat et al., 2019). However, finasteride had minor effects on these factors but tended to decrease Egf1 expression and to increase Tgfb and Egr1 expression. On a cellular level, the extract combination WS^® 1541 promoted apoptosis of BPH-1 cells in vitro. This activity was apparently contributed by the Urtica extract WS^® 1031 whereas the Sabal extract WS^® 1473 had no effect on apoptosis (Figures 4C, D). Interestingly, in our experimental setting, we saw no induction of apoptosis by finasteride. Cell cycle assessment revealed that finasteride prevented cell cycle progression by holding the cells in G₁-phase. A similar effect was observed by treating BPH-1 cells with WS^® 1541, possibly reflecting synergistic effects of WS^® 1031 and WS^® 1473 (Figures 4E, F).

Inflammation and oxidative stress are tightly intertwined and are considered to be important pathophysiological factors in human BPH (Ficarra et al., 2014; Minciullo et al., 2015; De Nunzio et al., 2020). The aging prostate stroma is characterized by upregulation of several chemokines and cytokines as IL-1β, IL-2, IL-6 IL-8, IL-15 and IL-17 (Chughtai et al., 2011; De Nunzio et al., 2016). Tissue microarrays from prostatic specimen obtained from 282 BPH patients showed that the majority had infiltration of both T and B lymphocytes as well as macrophages (Robert et al., 2009). Inflammatory mediators secreted by prostatic stroma can promote slow but constant proliferation of both epithelial and stromal fibroblasts. More importantly, the International Prostate Symptom Score (IPSS) and prostate volume were higher in patients with high-grade prostatic inflammation (Robert et al., 2009).

Accordingly, several previous studies have reported anti-inflammatory effects of Sabal serrulata fruit extracts (Latil et al., 2015) and specifically of the combined WS^® 1541 extract (Pigat et al., 2019) and its WS^® 1473 component (Saponaro et al., 2020). Our present data confirm and extend those observations in several ways. WS^® 1541 reduced the mRNA expression of various inflammatory mediators including Il1b, Il2, Il4, Il6, Kc, Il15, TNFa and Ikbkb as well as Cox2 and Nos2 in the sulpiride model (Figure 2). Thus, WS^® 1541 exhibited an anti-inflammatory effect. Interestingly, finasteride increased expression of some inflammatory marker, especially Il4, Il6, Il18, Ccl2 and Ccl7 (Figure 2). This is consistent with an increased prevalence of intraprostatic inflammation especially in benign areas found in cancer patients treated with finasteride (Murtola et al., 2016).

Inflammation processes during BPH pathogenesis can cause the generation of free radicals and an impaired efficiency of antioxidant defense mechanisms. The balance between ROS and the antioxidant component also has a role in developing prostate disease (Minciullo et al., 2015). WS^® 1541 and its components WS^® 1031 and WS^® 1473 concentration-dependently reduced ROS in BPH-1 cells (Figure 4). Accordingly, we found diminished abundance of oxidized proteins in sections of WS^® 1541 treated BPH rats showing a relevant antioxidative potential of WS^® 1541 in vivo (Figure 3). An earlier study using BPH-1 cells reported that WS^® 1473 if anything increased ROS production whereas WS^® 1541 decreased it in the presence and absence of H₂O₂; however, these effects were not seen at all concentrations (1, 10 and 20 μg/mL) or all time points (3 and 24 h) (Saponaro et al., 2020).

On a cellular level, stromal cell proliferation and pro-fibrotic tissue remodeling besides epithelial proliferation result in prostate hyperplasia accompanied by LUTS. During BPH pathogenesis, the process of EMT might be a source of pathophysiological relevant stromal cells (Rodriguez-Nieves and Macoska, 2013; De Nunzio et al., 2016). In sulpiride treated rats Tgfb mRNA expression is induced compared to healthy animals and could be diminished by simultaneous treatment with WS^® 1541 but not with finasteride. Members of the TGF-β family are key players in EMT (Katsuno and Derynck, 2021). During EMT, epithelial cells adapt the pro-migratory phenotype of mesenchymal cells, which we mimicked in the gap-closing assay using BPH patient-derived luminal epithelial cell line BPH-1. We did not see any effect on phalloidin staining in treated BPH-1 cells ruling out possible inhibitory effects of the extracts on f-actin polymerization that could have affected migratory potential of BPH-1 cells (Supplementary Figure S5). WS^® 1031 was able to reduce migration activity of BPH-1 cells assumingly suppressing EMT. As WS^® 1473 had no effect in this assay, the activity to inhibit cell migration seen for the combination of these extracts in WS^® 1541 appears to be contributed by WS^® 1031 (Figure 5). We investigated the mRNA expression of the TGF-β isoform 1, 2 and 3 and TGF-β receptor 1 and 2 in dependence of WS^® 1031 and WS^® 1473 treatment, showing a moderate decrease in expression of TGFB1, TGFB2, TGFB3 and TGFBR1 upon WS^® 1031 treatment but without a clear concentration-response relationship. WS^® 1473 did not affect TGFB1, TGFB3 or TGFBR1 but concentration-dependently reduced TGFB2 expression. Both WS^® 1031 and WS^® 1473 had a minor effect on the expression of TGFBR2 (Figures 6B–E). Taking into account that the TGF-β1 isoform is majorly expressed in BPH-1 cells (Figure 6A), inhibitory effects on migration might be accounted for by its attenuated expression due to WS^® 1031. Interestingly, ROS are able to activate TGF-β from latent TGF-β binding proteins triggering downstream signaling and thus expression of ECM proteins and EMT supporting genes (Morry et al., 2017; Katsuno and Derynck, 2021).

Our combined in vivo and in vitro data demonstrate growth inhibiting effects of WS^® 1541 on prostate epithelial cells albeit this did not translate into a diminished prostate lobe size in the rat sulpiride model. Moreover, we found anti-inflammatory effects and reduction of markers of oxidative stress. Importantly, our in vitro experiments demonstrated that each extract has a specific effect profile. Thus, Sabal serrulata extract WS^® 1031 reduced ROS and attenuated cell migration without affecting f-actin polymerization. On the other hand, Urtica dioica extract WS^® 1473 also reduced ROS but in addition stimulated apoptosis. The combination of WS^® 1031 and WS^® 1473 in WS^® 1541 synergistically hindered cell cycle progression by impeding the entry in the S-phase. This data supports a scientific rationale of combining WS^® 1473 and WS^® 1031 in the clinically used WS^® 1541.