Onvancerti for inhibiting non-adrenergic contraction of smooth muscle and prostate cell proliferation

Abstract

Methods for treating benign prostatic hyperplasia (BPH) in a patient are provided. Methods for inhibiting non-adrenergic contraction of smooth muscle are also provided. Additionally, methods for inhibiting proliferation of human prostate cells are provided.

Description

[Technical field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of U.S. Provisional Application No. 62 / 810,171, filed February 25, 2019, the entire contents of which are incorporated herein by reference.

[0002] (1) Field of the invention This application relates generally to the treatment of benign prostatic hyperplasia (BPH). More specifically, treatment of BPH with highly selective polo-like kinase 1 (PLK1) inhibitors is provided. [Background technology]

[0003] (2) Description of related fields Benign prostatic hyperplasia (BPH) is commonly associated with lower urinary tract symptoms (LUTS) due to ureteral compression resulting from prostatic enlargement and increased prostatic smooth muscle tone in BPH (Hennenberg et al., 2014). This symptomatic form of BPH, called bladder outlet obstruction (BOO), often requires medical treatment (Oelke et al., 2013). However, this has limited efficacy even for mild to moderate LUTS and is unavailable for severe LUTS (Oelke et al., 2013). This limited improvement is contrasted with the high caseload, around 6 billion patients with obstructive symptoms worldwide in 2018 (Irwin et al., 2011). Due to the age-dependent prevalence with demographic transition, the importance of LUTS suggestive of BPH is further increasing, and a high demand for new medical therapies with higher efficacy is emerging.

[0004] According to the contribution of prostatic smooth muscle tone and prostatic growth to LUTS suggestive of BPH, medical treatment in BPH aims at 1) inhibiting prostatic smooth muscle contraction for rapid symptom improvement, and 2) inhibiting prostatic growth to prevent the progression and complications of BPH (Oelke et al., 2013). Prostatic smooth muscle contraction is partially induced by activation of α1-adrenoceptors by noradrenaline released during adrenergic neurotransmission (Hennenberg et al., 2014). As a result, α1-adrenoceptor antagonists ("α1-blockers") are the first-line treatment for LUTS suggestive of BPH, as they can alleviate BOO by relaxing prostatic smooth muscle (Caine et al., 1975, 1976; Oelke et al., 2013). However, these antagonists only reduce symptoms (international prostate symptom scores, IPSS) and improve urinary flow (Q max) by at most 50%, but even placebo may produce an improvement of up to 30% (Hennenberg et al., 2014, 2017; Oelke et al., 2013; Strand et al., 2017). The phosphodiesterase-5 inhibitor tadalafil was recently approved for the treatment of obstructive LUTS, but its efficacy is not higher than that of α1-blockers (Dahm et al., 2017). 5α-reductase inhibitors (5-ARIs) are applied to reduce prostate size and stop prostate growth with the aim of preventing the progression of BPH (Oelke et al., 2013). However, the reduction in the risk of symptom progression may not exceed 35–40% with monotherapy and 66% with combination therapy (Strand et al., 2017). The unfavourable consequences of medical LUTS therapy contribute to high discontinuation rates, with up to 70% of patients discontinuing medication within 12 months after the initial prescription (Cindolo et al., 2015). The consequences are disease progression, hospitalisation and surgery for BPH (Cindolo et al., 2015). This limitation of α1-blockers may be due to the contribution of non-adrenergic mediators to prostatic smooth muscle tone, including endothelin-1, which induces prostatic smooth muscle contraction in conjunction with α1-adrenoceptors (Hennenberg et al., 2014, 2017).

[0005] Polo-like kinase (PLK) is a serine-threonine kinase that has been primarily implicated in promoting proliferation in various cell types, including airway smooth muscle cells, prostate cancer cells, and others (Jiang and Tang, 2015; Lin et al., 2019; Shao et al., 2015). In addition, recent findings have suggested a function for PLK1 in promoting airway, vascular, and human prostate smooth muscle contraction (de Carcer et al., 2017; Hennenberg et al., 2018; Li et al., 2016). However, the additive effects of PLK inhibitors and α1-blockers on prostate smooth muscle contraction and their effects on prostate stromal cell proliferation are unknown.

[0006] Onbancertive [ka] Onbancertive

[0007] Onvansertiv (also known as PCM-075, NMS-1286937, NMS-937, "compound of formula (I)" in U.S. Pat. No. 8,927,530; IUPAC name 1-(2-hydroxyethyl)-8-{[5-(4-methylpiperazin-1-yl)-2-(trifluoromethoxy)phenyl]amino}-4,5-dihydro-1H-pyrazolo[4,3-h]quinazoline-3-carboxamide) is the first PLK1 specific ATP competitive inhibitor administered by oral route to enter clinical trials with demonstrated antitumor activity in various preclinical models. Onvansertiv has favorable pharmacological parameters and good oral bioavailability. Onvansertiv has several advantages over other PLK1 inhibitors, including extremely high selectivity for PLK1 isozyme and oral bioavailability. Onvancertive has an IC of 2 nM for PLK1 and >10,000 for PLK2 and PLK3. 50 has. Summary of the Invention

[0008] A method of treating benign prostatic hyperplasia (BPH) in a patient is provided, the method comprising treating the patient with (a) onvancertive and (b) an alpha-1 blocker, a 5-alpha-reductase inhibitor, a phosphodiesterase-5 enzyme inhibitor, or a combination thereof. Also provided is a method of inhibiting non-adrenergic contraction of smooth muscle, the method comprising treating the smooth muscle with Onvancertive. Additionally, a method of inhibiting proliferation of human prostate cells is provided, the method comprising treating human prostate cells with onvancertive in a manner sufficient to inhibit proliferation of the human prostate cells. [Brief description of the drawings]

[0009] [Figure 1] 1A, 1B and 1C are graphs showing the effect of Onvansert on adrenergic contraction of human prostate strips. Contraction of human prostate strips was induced in the organ bath with noradrenaline (FIG. 1A), or the α1-adrenoceptor agonists phenylephrine and methoxamine (FIG. 1B) after addition of 100 nM or 1 μM concentrations of Onvansert, or DMSO (control), or with noradrenaline (FIG. 1C) after a 30-minute washout of DMSO and Onvansert (100 nM). In each experiment, DMSO and Onvansert were applied to separate samples obtained from the same prostate. To exclude heterogeneity due to individual variations, differences in the degree of BPH, or other variations in smooth muscle content, tension was expressed as % of the contraction with high molar KCl assessed before application of Onvansert or DMSO. Data are means ± SEM of tissue series from n = 5 (norepinephrine and 100 nM onvansert with no washout), n = 6 (norepinephrine and 1 μM onvansert), n = 5 (phenylephrine), n = 5 (methoxamine), and n = 5 (norepinephrine after washout) patients, with samples from each patient assigned to both control and inhibitor groups (# P < 0.05 after multivariate analysis at the concentrations indicated; P values ​​are for all groups after 2-way ANOVA as shown in the insert). [Diagram 2]FIG. 2 is a graph showing the effect of Onvansert on non-adrenergic contraction of human prostate strips. Contraction of human prostate strips was induced by endothelin-1, ATP, or the thromboxane A2 analog U46619 in the organ bath after addition of 100 nM concentration of Onvansert or DMSO (control). In each experiment, DMSO and Onvansert were applied to separate samples obtained from the same prostate. To exclude heterogeneity due to individual variations, different degrees of BPH, or other variations in smooth muscle content, tension was expressed as % of contraction with high molar KCl assessed before application of Onvansert or DMSO. Data are mean ± SEM of tissue series from n=6 (endothelin-1), n=5 (ATP), and n=5 (U46619) patients, with samples from each patient assigned to both control and inhibitor groups (P values ​​are for all groups after 2-way ANOVA as shown in the insert). [Diagram 3] 3A and 3B are graphs showing the effect of Onvansert on EFS-induced contraction of human prostate specimens. Contraction of human prostate specimens was induced by EFS in organ baths after the addition of Onvansert at a concentration of 100 nM or 1 μM, or DMSO (control) (FIG. 3A), and after a 30-minute washout of DMSO and Onvansert (100 nM) (FIG. 3B). In each experiment, DMSO and Onvansert were applied to separate samples obtained from the same prostate. To exclude heterogeneity due to individual variations, different degrees of BPH, or other variations in smooth muscle content, tension was expressed as a % of the contraction with high molar KCl assessed before application of Onvansert or DMSO. Data are means ± SEM of tissue series from n=6 (DMSO vs. 100 nM Onvancertive with no washout), n=7 (DMSO vs. 1 μM Onvancertive), and n=5 (DMSO vs. 100 nM Onvancertive after washout) patients, with samples from each patient assigned to both control and inhibitor groups (P values ​​are for all groups after 2-way ANOVA as shown in the insert). [Figure 4]Figures 4A and 4B are graphs showing the effect of alpha 1 blockers on contraction of human prostate specimens. Contraction of human prostate specimens was induced by EFS (Figure 3A) or noradrenaline (Figure 4B) in organ baths after the addition of tamsulosin (300 nM), silodosin (100 nM), or an equal volume of distilled water containing 1% DMSO (control). In each experiment, vehicle or alpha 1 blockers were applied to separate samples obtained from the same prostate. To exclude heterogeneity due to individual variations, different degrees of BPH, or other variations in smooth muscle content, tension was expressed as % of contraction with high molar KCl assessed before application of alpha 1 blockers or vehicle. Data are means ± SEM of tissue series from n = 5 (EFS / tamsulosin), n = 5 (EFS / silodosin), n = 5 (noradrenaline / tamsulosin), and n = 6 (noradrenaline / silodosin) patients, with samples from each patient assigned to both control and inhibitor groups (# P < 0.05 after multivariate analysis at the concentrations indicated; P values ​​are for all groups after 2-way ANOVA as indicated in the insert). [Diagram 5]5A and 5B are graphs showing the effect of combination of onvansert and alpha 1 blocker compared to alpha 1 blocker alone on EFS-induced human prostate specimen contraction. Contraction of human prostate specimen was induced by EFS in organ baths after addition of tamsulosin (300 nM), silodosin (100 nM), onvansert (100 nM), or combination of onvansert (100 nM) with tamsulosin or silodosin, and an equal volume of solvent (DMSO as a control for alpha 1 blocker alone without onvansert, or water / DMSO as a control for alpha 1 blocker alone without onvansert). In each experiment, alpha 1 blocker or onvansert and combination were applied to separate samples obtained from the same prostate. Each figure represents an independent experimental system using different prostate tissue (although both groups in each figure were assigned samples from the same prostate). To exclude heterogeneity due to individual variability, differences in the degree of BPH, or other variations in smooth muscle content, tension was expressed as % of the high molar KCl contraction assessed before application of Onvansert, α1-blocker, or DMSO. Data are mean ± SEM of tissue series from n = 7 (tamsulosin vs. tamsulosin and Onvansert), n = 5 (silodosin vs. silodosin and Onvansert), n = 5 (Onvansert vs. tamsulosin and Onvansert), and n = 5 (Onvansert vs. silodosin and Onvansert) patients, with samples from each patient assigned to both control and inhibitor groups (#P<0.05 after multivariate analysis at the concentrations indicated; P values ​​are for all groups after 2-way ANOVA as shown in the insert). [Figure 6]Figures 6A, 6B, 6C and 6D are photographs (Figure 6A) and graphs showing PLK1 detection in human prostate tissue and WPMY-1 cells. Figure 6A shows the results of Western blot analysis performed on human prostate tissue (n=4 patients) and WPMY-1 cells (samples from n=4 independent experiments) using an antibody raised against PLK1. A band with the expected molecular weight size of PLK1 (68 kDa) is shown. The position and size of the marker bands are shown to the right of each blot (size in kDa). Figure 6B shows the relative intensity of the estimated PLK1 band. After densitometric quantification of the 68 kDa band in (Figure 6A), the arbitrary units of all samples were normalized to the mean of the values ​​obtained for prostate tissue. Values ​​and medians of all samples from both groups are shown. Figure 6C shows the relative mRNA expression of PLK1 in human prostate tissue (n=6 patients) and WPMY-1 cells (samples from n=4 independent experiments). The Ct values ​​from RT-PCR were expressed as 2-ΔCP and finally normalized to the mean value obtained for prostate tissue. The values ​​of all samples from both groups and the median are shown. Figure 6D shows that the 2-ΔCP values ​​from the detection of PLK1 and PSA by RT-PCR in human prostate tissue were plotted against each other and Spearman's correlation analysis was performed. [Figure 7]Figures 7A, 7B, and 7C are graphs and micrographs showing the effect of onvansertiv on viability and proliferation of WPMY-1 cells. Figure 7A is a graph showing WPMY-1 cell viability assessed by CCK-8 assay after exposure to onvansertiv or vehicle at the concentrations and durations as indicated. Data are mean ± SEM of n = 5 independent experiments for each condition (# P < 0.05 vs. corresponding control). Figure 7B is a graph and micrograph showing proliferation assessed by colony formation assay. WPMY-1 cells were exposed to onvansertiv at the concentrations indicated. Representative micrographs and mean ± SEM of n = 5 independent experiments (# P < 0.05 vs. control) are shown. Figure 7C is a graph and micrograph showing proliferation assessed by EdU assay. WPMY-1 cells were exposed to onvansertiv at the concentrations indicated for 24 hours. Nuclei of proliferating cells are shown in pink and nuclei of non-proliferating cells are shown in blue. Representative images and means±SEM of n=5 independent experiments (#P<0.05 vs. control) are shown. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0010] As used herein, the singular forms "a," "an," and "the" are intended to include the plural forms as well, unless the context clearly dictates otherwise. Additionally, the use of "or" is intended to include "and / or" unless the context clearly dictates otherwise. The present invention is based, in part, on the discovery that the highly selective polo-like kinase 1 (PLK1) inhibitor onvansertiv effectively inhibits both adrenergic and non-adrenergic contractions of smooth muscle, such as human prostate tissue. This is surprising, since other PLK1 inhibitors inhibit adrenergic but not non-adrenergic contractions of smooth muscle (Hennenberg et al., 2018). Because onvansertiv inhibits non-adrenergic contractions of smooth muscle, specifically endothelin-1-induced and ATP (purinergic)-induced smooth muscle contractions (see Examples below), onvansertiv can be effectively combined with inhibitors of adrenergic smooth muscle contraction, such as alpha-1 blockers, to treat benign prostatic hyperplasia (BPH).

[0011] Thus, in some embodiments, a method of treating benign prostatic hyperplasia (BPH) in a patient is provided. The method includes treating the patient with (a) onvansertiv and (b) alpha 1 blockers, 5-alpha-reductase inhibitors, phosphodiesterase-5 enzyme inhibitors, or a combination thereof. Onvansertiv inhibits smooth muscle contraction by a different mechanism than alpha 1 blockers, 5-alpha-reductase inhibitors, or phosphodiesterase-5 enzyme inhibitors, so this combination treatment will be better than any of the individual inhibitors alone. Indeed, the combination treatment of onvansertiv with either tamsulosin or silodosin (both alpha 1 blockers) inhibits prostatic smooth muscle tissue to a greater extent than any of the individual compounds alone (see examples below). In some embodiments, the method reduces symptoms of BPH or increases urinary flow (Q max In another embodiment, the method improves urinary flow (Q max ) by more than 50%. In various embodiments, the patient is treated with onvancertive and an alpha 1 blocker. Non-limiting examples of alpha 1 blockers include silodosin, tamsulosin, alfuzosin, doxazosin, prazosin, and terazosin. Non-limiting examples of 5-alpha-reductase inhibitors include dutasteride and finasteride. Non-limiting examples of phosphodiesterase-5 inhibitors include avanafil, lodenafil, mirodenafil, sildenafil, tadalafil, vardenafil, udenafil, zaprinast, benzamidonafil, and dasantafil.

[0012] Also provided is a method of inhibiting non-adrenergic contraction of smooth muscle, the method comprising treating the smooth muscle with Onvancertive. This method can be used to inhibit any non-adrenergic smooth muscle contraction, hi some embodiments, the smooth muscle contraction is endothelin-1 induced or ATP induced (purinergic). In various embodiments of this method, the smooth muscle is prostate tissue, e.g., human prostate tissue, although the method should be effective in inhibiting non-adrenergic smooth muscle contraction of any tissue in any mammal. In some embodiments, the prostate tissue is from a patient with benign prostatic hyperplasia (BPH).

[0013] Additionally, a method of inhibiting the proliferation of prostate cells is provided, which comprises treating prostate cells with onvansert in a manner sufficient to inhibit the proliferation of human prostate cells. In various embodiments, the prostate cells are from a human BPH patient, but the method should be effective in inhibiting the proliferation of prostate cells in any mammal in vitro or in vivo. In some embodiments of these methods, the cells are also exposed to a second agent that inhibits the proliferation of human prostate cells. The second agent may be any agent known to be at least partially effective in inhibiting the proliferation of human prostate cells. A non-limiting example of such a second agent is metformin (Wang et al., 2017).

[0014] In any of the methods provided herein, onvansertiv may be administered by any means known in the art. In some embodiments, onvansertiv is administered orally in a pharma- ceutically acceptable excipient. For any particular patient, the determination of the effective dose of onvansertiv in inhibiting smooth muscle tissue contraction or prostate cell proliferation can be made without undue experimentation by methods known in the art. In some embodiments, the dose of onvansertiv administered is 6, 12, or 24 mg / m 2 In some of these embodiments, the dose of onvancertive is 50 mg / m 2 / day, e.g., 24 or 36 mg / m 2 / day. In embodiments in which onvancertive is administered with an additional agent, onvancertive can be administered before, at the same time, or after the additional agent. In addition, onvancertive and the second agent can be administered on different or the same schedule, for example, daily, 5 days on + 2 days off.

[0015] Preferred embodiments are described in the following examples. Other embodiments within the scope of the claims herein will be apparent to those skilled in the art from consideration of the specification or practice of the invention as disclosed herein. The specification, together with the examples, are intended to be exemplary only, with the scope and spirit of the invention being indicated by the claims which follow the examples. EXAMPLES

[0016] Example: Effect of Onvancertive on prostatic smooth muscle function in human benign prostatic hyperplasia Prostatic smooth muscle contraction and prostatic dilation contribute to lower urinary tract symptoms suggestive of benign prostatic hyperplasia. Recent evidence has demonstrated that inhibitors of polo-like kinase (PLK) inhibit smooth muscle contraction in human prostatic tissue. However, their additive effects with α1-blockers and effects on prostatic growth are unknown. Here, we investigated the effects of onvancertive, a novel and highly selective PLK1 inhibitor, on prostatic smooth muscle contraction, alone and in combination with α1-blockers, and on the proliferation and viability of prostatic stromal cells (WPMY-1). Prostatic tissue was obtained from radical prostatectomy. Contraction was studied in organ baths. Proliferation and viability were assessed by plate colony, EdU, and CCK-8 assays. Electrical field stimulation (EFS)-induced contractions of human prostate tissue were inhibited by 34% by 100 nM and 1 μM onvancertive at 32 Hz and by 48% and 47% by the alpha-1 blockers tamsulosin and silodosin. Combinations of onvancertive with tamsulosin or silodosin further reduced EFS-induced contractions compared to alpha-1 blockers alone (59% and 61%, respectively) and onvancertive alone (both 68%). Contractions induced by noradrenaline, phenylephrine, methoxamine, endothelin-1, and ATP were inhibited to a similar extent by onvancertive (100 nM). Viability and proliferation of WPMY-1 cells were reduced in a concentration- and time-dependent manner (24-72 h, 10-100 nM). Onvancertive inhibits neurogenic, adrenergic, and endothelin-1 and ATP-induced contraction of human prostatic smooth muscle and proliferation of stromal cells. Contraction is reduced by up to 50% by alpha 1 blockers. The combination of alpha 1 blockers and onvancertive provides additive inhibition of prostatic contraction.

[0017] Materials and Methods Human Prostate Tissue Human prostate tissues were obtained from patients undergoing radical prostatectomy for prostate cancer (n=107). Patients who had previously undergone transurethral resection of the prostate (TURP) were excluded. The study was performed in accordance with the Declaration of Helsinki of the World Medical Association and approved by the Ethics Committee of the Ludwig Maximilian University, Munich, Germany (Ethikkommission bei der LMU Munchen, approval number 19-737). Informed consent was obtained from all patients. In accordance with the ethical approval, samples and data were collected anonymously so that no data regarding patient characteristics were stored. Samples were taken immediately after radical prostatectomy after gross examination by a uropathologist. Considering that most prostate cancers arise in the peripheral region (Pradidarcheep et al., 2011; Shaikhibrahim et al., 2012), all tissues were taken from the periurethral region. Only tissue samples that showed no histological signs of neoplasia, cancer, or inflammation upon pathological evaluation were collected. BPH is present in 80–83% of patients with prostate cancer (Alcaraz et al., 2009; Orsted and Bojesen, 2013). In general, such tissues show variations in prostate-specific antigen (PSA) content, as well as differences in smooth muscle and glandular epithelium content, reflecting the presence of BPH to different degrees (Wang et al., 2016a, 2016c). For gross examination and sampling, the prostate was dissected with a single longitudinal incision from the capsule to the urethra. Both intersections were then visually inspected for obvious tumor invasion. As tumors were usually located in the peripheral region, tumor invasion in the periurethral region (where three samples were taken) was extremely rare (seen in less than 1% of prostates). Prostates showing tumor in the periurethral region on gross examination were not sampled and were not included in the study. Organ bath studies were performed immediately after sample collection, whereas samples for molecular analysis were shock frozen in liquid nitrogen and stored at −80°C.

[0018] tension measurement Prostate specimens (6 x 3 x 3 mm) were inserted into 10 ml aerated (95% O2 and 5% CO2) tissue baths (Danish Myotechnology, Aarhus, Denmark) containing Krebs-Henseleit solution (37 °C, pH 7.4) with the following composition: 118 mM NaCl, 4.7 mM KCl, 2.55 mM CaCl2, 1.2 mM KH2PO4, 1.2 mM MgSO4, 25 mM NaHCO3, 7.5 mM glucose. In each single test, two of the four specimens from the same prostate were assigned to the control group (no inhibitor or antagonist) and the other two to the inhibitor / antagonist group (resulting in two replicates for each group in each single test). All four samples from one experiment were examined in the four chambers of the same organ bath. As a result, the control and inhibitor curves in each series / figure were obtained from the same prostate, but for the different series / figures different prostates were examined. The amount of solvent differed between series due to the difference in inhibitor concentration (100 nM, 1 μM) and the combination of onvansertib and α1 blocker, which precluded comparison between contraction levels of different series. Therefore, taking into account that prostate tissue from radical prostatectomy can show significant heterogeneity, statistical comparisons were only made between groups within the same series (i.e., containing tissue from the same prostate for inhibitor and control groups) and not between series obtained from different prostates (i.e., not across different series of organ bath experiments). Only one curve was recorded for each sample, i.e., for one type of agonist or electrical field stimulation (EFS).

[0019] After insertion into the organ bath chamber, the preparation was stretched to 4.9 mN and equilibrated for 45 min. During the initial phase of the equilibration period, a spontaneous decrease in tension is usually observed. Therefore, tension was adjusted three times during the equilibration period until a stable resting tension of 4.9 mN was achieved. After the equilibration period, the maximal contraction induced by 80 mM KCl was evaluated. The chamber was then washed three times with Krebs-Henseleit solution for a total of 30 min. Cumulative concentration-response curves for noradrenaline, phenylephrine, methoxamine, endothelin-1, and U46619, or frequency-response curves induced by EFS were generated 30 min after the addition of onvansertib, tamsulosin, silodosin, and / or dimethyl sulfoxide (DMSO) as a control. The reversibility of the Onvansert effect was evaluated in another series in which DMSO and Onvansert were washed out by replacing them with Krebs-Henseleit solution three times within 30 min starting 30 min after application of DMSO or Onvansert, after which concentration-response curves for noradrenaline or frequency-response curves with EFS were performed.

[0020] Application of EFS mimics action potentials and results in the release of endogenous neurotransmitters, including noradrenaline. The inhibitor of neurotransmitter release, tetrodotoxin, was used and has previously been shown to explain two-thirds of EFS-induced contractions in the human prostate (Angulo et al., 2012). For EFS, tissue specimens were placed between two parallel platinum electrodes connected to a CS4 stimulator (Danish Myotechnology). Square waves were applied at a voltage of 50 V, 1 ms interval, with a pulse train duration of 10 s and a delay of 1 ms between single pulses. EFS-induced contractile responses were examined at frequencies of 2, 4, 8, 16, and 32 Hz, with a pulse train interval of 30 s between stimuli. Data analysis and calculations were based on the maximum peak height of contraction. For calculation of agonist or EFS induced contractions, tension is expressed as % of KCl induced contraction, as this can correct for differences in stromal / epithelial ratios, differences in smooth muscle content, variations in the degree of BPH, or any other heterogeneity between prostate samples and patients.

[0021] Western blot analysis Frozen prostate tissue was homogenized using the FastPrep®-24 system (MP Biomedicals, Illkirch, France) with matrix A in a buffer containing 25 mM Tris / HCl, 10 μM phenylmethanesulfonyl fluoride, 1 mM benzamidine, and 10 μg / ml leupeptin hemisulfate. After centrifugation (20,000 g, 4 min), the protein concentration of the supernatant was assayed using a Dc assay kit (Biorad, Munich, Germany) and boiled for 10 min in sodium dodecyl sulfate (SDS) sample buffer (Roth, Karlsruhe, Germany). Cultured stromal cell samples were prepared as described below. Samples (20 μg / lane) were subjected to SDS-polyacrylamide gel electrophoresis and proteins were blotted onto Protran® nitrocellulose membranes (Schleicher & Schuell, Dassel, Germany). Membranes were blocked overnight in phosphate-buffered saline (PBS) containing 5% milk powder (Roth, Karlsruhe, Germany) and incubated with rabbit anti-PLK1 (208G4) (#4513) (Cell Signalling Technology, Danvers, MA, USA) and mouse monoclonal anti-β-actin antibody (sc-47778) (Santa Cruz Biotechnology, Santa Cruz, CA, USA).

[0022] Primary antibodies were diluted in PBS containing 0.1% Tween 20 (PBS-T) and 5% milk powder. Detection was then continued with biotinylated goat anti-rabbit or horse anti-mouse secondary IgG (BA-1000, BA-2000) (Vector Laboratories, Burlingame, CA, USA) followed by incubation with avidin and biotinylated horseradish peroxidase (HRP) (both diluted 1:200 in PBS) from the "Vectastain ABC kit" (Vector Laboratories, Burlingame, CA, USA). After incubation with primary or secondary antibodies or biotin-HRP, the membranes were washed with PBS-T. Finally, the blots were developed with enhanced chemiluminescence (ECL) using ECL Hyperfilm (GE Healthcare, Freiburg, Germany). The intensity of the bands obtained for PLK1 was quantified by densitometry using "Image J" (National Institutes of Health, Bethesda, MD, USA).

[0023] RT-PCR RNA from frozen prostate tissue or cells was isolated using the RNeasy Mini Kit (Qiagen, Hilden, Germany). For tissue isolation, 30 mg of tissue was homogenized using the FastPrep®-24 system with matrix A (MP Biomedicals, Illkirch, France). RNA concentration was measured spectrophotometrically. Reverse transcription to cDNA was performed with 1 μg of isolated RNA using the Reverse Transcription System (Promega, Madison, WI, USA). RT-PCR for PLK1, PSA, and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was performed in a Roche Light Cycler (Roche, Basel, Switzerland) using primers provided by Qiagen (Hilden, Germany) as ready-to-use mixes based on Ref-Seq accession numbers snM_005030 for PLK1, NM_001030047 for KLK3 (synonym PSA), and nM_002046 for GAPDH. PCR reactions were performed in a volume of 25 μl containing 5 μl of LightCycler® FastStart DNA MasterPlus SYBR Green I (Roche, Basel, Switzerland), 1 μl of template, 1 μl of primer, and 18 μl of water. Denaturation was performed at 95° C. for 10 min, and amplification was performed for 45 cycles of 95° C. for 15 s followed by 60° C. for 60 s. The specificity of the primers and the amplification was demonstrated by subsequent melting point analysis, which revealed a single peak for each target. Results were expressed using the ΔΔCP method, where the cycle number (Ct) at which the fluorescent signal crossed a defined threshold for GAPDH was subtracted from the Ct value for PLK (Ct target -Ct GAPDH = ΔCP), value of 2 -ΔCP and normalized to each other or to the mean value of the prostate tissue.

[0024] cell culture WPMY-1 cells are an immortalized cell line derived from human prostatic stroma free of prostate cancer (Webber et al., 1999). Cells were obtained from the American Type Culture Collection (ATCC; Manassas, VA, USA) and maintained at 37°C and 5% CO2 in RPMI 1640 (Gibco, Carlsbad, CA, USA) supplemented with 10% fetal calf serum (FCS) and 1% penicillin / streptomycin. Before addition of Onvansertiv or DMSO, the medium was changed to FCS-free medium. For Western blot analysis, cells were lysed using radioimmunoprecipitation assay (RIPA) buffer (Sigma-Aldrich, St. Louis, MO, USA) and removed from the flasks after 15 min of incubation on ice. Cell debris was removed by centrifugation (10,000 g, 10 min, 4° C.) and different aliquots of the supernatant were subjected to protein determination or boiled with SDS sample buffer.

[0025] Cytotoxicity assay Cell viability was assessed using Cell Counting Kit-8 (CCK-8) (Sigma-Aldrich, St. Louis, MO, USA). Cells were grown in 96-well plates (5000 cells / well) for 24 h, after which onvancertive or vehicle was added at the indicated concentrations. Cells were then grown for different periods (24, 48, 72 h). Separate controls were performed for each period. At the end of this period, 10 μl of [2-(2-methoxy-4-nitrophenyl)-3-(4-nitrophenyl)-5-(2,4-disulfophenyl)-2H-tetr azolium monosodium salt (WST-8) from CCK-8 was added and after 2 h of incubation at 37°C, the absorbance of each well was measured at 450 nM.

[0026] Colony formation assay Approximately 100 cells were placed in each well of a 6-well culture plate and treated with onvansert (final concentrations 10, 50, 100 nM) or vehicle. Cells were incubated at 37°C for 14 days, then washed twice with PBS and fixed overnight (4°C) with 2 ml of 10% trichloroacetic acid. All plates were then washed five times with cold water and stained with 0.4% sulforhodamine B solution (diluted in 1% acetic acid) for 30 minutes at room temperature. All plates were labeled and washed five times with 1% acetic acid before being photographed. The number of colonies containing 50 cells or more was counted under a microscope.

[0027] EdU proliferation assay WPMY-1 cells were seeded at a cell line specific density (50,000 / well) on 16-well chambered coverglass (Thermo Scientific, Waltham, MA, USA). After 24 h, cells were treated with onvansert (final concentrations 10, 50, 100 nM) or vehicle. After another 24 h, the medium was replaced with 10 mM 5-ethynyl-2'-deoxyuridine (EdU) solution in FCS-free medium containing onvansert or vehicle. After 20 h, cells were fixed with 3.7% formaldehyde. EdU incorporation was determined using the "EdU-Click 555" cell proliferation assay (Baseck, Tutzing, Germany) according to the manufacturer's instructions. In this assay, EdU incorporation into DNA is assessed by detection with fluorescent 5-carboxytetramethylrhodamine (5-TAMRA). All nuclei were counterstained with DAPI. Cells were analyzed by fluorescence microscopy (excitation: 546 nM; emission: 479 nM).

[0028] Drugs and Nomenclature Onvancertive (PCM-075) (1-(2-hydroxyethyl)-8-[5-(4-methylpiperazin-1-yl)-2-(trifluoromethoxy)anilino]-4,5-dihydropyrazolo[4,3-h]quinazoline-3-carboxamide) is a PLK inhibitor with high selectivity for PLK1 (Beria et al., 2011). Phenylephrine ((R)-3-[-1-hydroxy-2-(methylamino)ethyl]phenol) and methoxamine (α-(1-aminoethyl)-2,5-dimethoxybenzyl alcohol) are selective agonists of the α1-adrenoceptor. U46619 ((Z)-7-[(1S,4R,5R,6S)-5-[(E,3S)-3-hydroxyoct-1-enyl]-3-oxabicyclo[2.2.1]heptan-6-yl]hept-5-enoic acid) is an analogue of thromboxane A2 (TXA2) that specifically activates the thromboxane A2 receptor and is often used as an agonist of the thromboxane A2 receptor (Malmsten, 1976; Shen and Tai, 1998). Tamsulosin (5-[(2R)-2-[2-(2-ethoxyphenoxy)ethylamino]propyl]-2-methoxybenzenesulfonamide) and silodosin (1-(3-hydroxypropyl)-5-[(2R)-2-[2-[2-(2,2,2-trifluoroethoxy)phenoxy]ethylamino]propyl]-2,3-dihydroindole-7-carboxamide) are alpha- 1AIt is an α1-adrenoceptor antagonist with high subtype selectivity. Both are available and commonly used to treat LUTS suggestive of BPH (Oelke et al., 2013). Stock solutions of onvancertive (10 mM) and appropriate dilutions were prepared in DMSO and stored at -20°C until use. Stock solutions of noradrenaline, phenylephrine, and methoxamine (all at 10 mM), and adenosine 5'-triphosphate (ATP) (500 mM suspension) were prepared fresh before each experiment. Stock solutions of U46619 (10 mM) were prepared in ethanol and stored at -80°C until use. Stock solutions of endothelin-1 (0.4 mM) were stored at -20°C until use. Stock solutions of tamsulosin and silodosin (10 mM) were prepared in DMSO, further diluted (1:100, to give 100 μM) in distilled water, and stored at −20° C. until use. Onvancertive was supplied by Trovagene Inc. (San Diego, CA, USA). Noradrenaline, phenylephrine, methoxamine, and ATP were obtained from Sigma (Munich, Germany), U46619, tamsulosin, and silodosin from Tocris (Bristol, UK), and endothelin-1 from Enzo Life Sciences (Rehala, Germany).

[0029] statistical analysis Data are presented as mean ± standard error of the mean (SEM) with indication of the number of experiments (n). Multivariate analysis of variance (ANOVA) and two-way ANOVA were used for unpaired observations and were performed using SPSS® version 20 (IBM SPSS Statistics, IBM Corporation, Armonk, NY, USA). P values ​​< 0.05 were considered statistically significant. All groups included in the statistical analysis were based on ≥ 5 independent experiments and, in the case of experiments performed on human tissues, each group contained tissues from ≥ 5 patients. Thus, the minimum group size subjected to statistical tests was n = 5. Furthermore, all groups compared to each other by statistical tests showed the same group size, and as a result, no statistical comparisons were made between groups with different sample sizes or between groups consisting of tissues from different patients. Spearman's correlation analysis and calculation of pEC50 values ​​by curve fitting were performed using GraphPadPrism 6 (Statcon, Witzenhausen, Germany).

[0030] result Effect of Onvancertive on adrenergic contraction To evaluate the effects of Onvansertiv and α1-blockers on prostatic smooth muscle contraction, contraction of human prostatic tissue was induced in organ baths. The effect of Onvansertiv on noradrenaline-induced contraction of human prostatic tissue was tested using two different concentrations of Onvansertiv. Both concentrations, i.e., 100 nM and 1 μM, caused significant inhibition of noradrenaline-induced contraction (FIG. 1A). The degree of inhibition was comparable at both concentrations, and thus increasing the Onvansertiv concentration from 100 nM to 1 μM did not increase the degree of inhibition (100 μM noradrenaline-induced contraction was inhibited by 45±9.7% by 100 nM Onvansertiv and 41±6.2% by 1 μM Onvansertiv). In contrast to these contractile forces, Onvancertive did not alter the pEC50 of noradrenaline (5.9±0.2 M after 100 nM Onvancertive and 5.5±0.2 M in matched controls; 5.7±0.3 M after 1 μM Onvancertive and 6±0.3 M in matched controls).

[0031] The effect of Onvansertib on α1-adrenergic prostatic smooth muscle contraction was confirmed using two α1-adrenoceptor agonists, phenylephrine and methoxamine. Onvansertib (100 nM) caused significant inhibition of phenylephrine- and methoxamine-induced contractions (Figure 1B). The degree of inhibition was comparable for both agonists and was similar to the inhibition of noradrenaline-induced contractions by Onvansertib (100 nM Onvansertib inhibited 30 μM phenylephrine-induced contractions by 47 ± 16.2% and 100 nM Onvansertib inhibited 100 μM methoxamine-induced contractions by 43 ± 19.4%). In contrast to these contractile forces, Onvansertib inhibited the pEC 50 values ​​were unchanged (7.8±1.3 M for phenylephrine after 100 nM Onvancertive and 6.7±0.8 M for matched controls; 5.2±0.2 for methoxamine after 100 nM Onvancertive and 5±0.1 for matched controls). To assess the reversibility of this effect of onvansertib on adrenergic contractions, noradrenaline-induced contractions were assessed in a separate experimental system after washout of DMSO and onvansertib (100 nM). Under these conditions, noradrenaline-induced contractions were not different, i.e., were comparable after washout of DMSO and onvansertib (Figure 1C).

[0032] Effect of Onvancertive on non-adrenergic contractions The effect of Onvancertive on prostatic smooth muscle contraction induced by endothelin-1, ATP, or the thromboxane A2 analog U46619 was examined. Onvancertive (100 nM) caused significant inhibition of endothelin-1-induced contraction (45±6.6% inhibition of 3 μM endothelin-1-induced contraction with 100 nM Onvancertive) and significant inhibition of ATP-induced contraction (51±18.6% inhibition of 10 mM ATP-induced contraction with 100 nM Onvancertive) (Figure 2). Onvancertive (100 nM) did not alter U46619-induced contraction (Figure 2).

[0033] Effect of Onvancertive on EFS-induced contractions The effect of Onvancertive on EFS-induced neurogenic contractions of human prostate tissue was tested using two different concentrations of Onvancertive. Both concentrations, i.e., 100 nM and 1 μM, caused significant inhibition of EFS-induced contractions (Figure 3A). The degree of inhibition was comparable at both concentrations, and thus increasing the Onvancertive concentration from 100 nM to 1 μM did not increase the degree of inhibition (100 nM Onvancertive inhibited 32 Hz-induced contractions by 34 ± 11.3%, and 1 μM Onvancertive inhibited 32 Hz-induced contractions by 34 ± 14%). To assess the reversibility of this effect of onvansertib on EFS-induced contractions, EFS-induced contractions were assessed in a separate experimental system after washout of DMSO and onvansertib (100 nM). Under these conditions, EFS-induced contractions were not different, i.e., were comparable after washout of DMSO and onvansertib (Figure 3B).

[0034] Effects of α1-blockers on EFS-induced and noradrenaline-induced contractions The α1 blockers tamsulosin and silodosin significantly and to the same extent inhibited EFS-induced contractions of human prostate tissue (FIG. 4A). The degree of inhibition was comparable for both compounds (300 nM tamsulosin inhibited 32 Hz-induced contractions by 48±18.3%, and 100 nM silodosin inhibited 32 Hz-induced contractions by 47±16.4%). In contrast to this incomplete inhibition of EFS-induced contractions, the same concentrations of tamsulosin and silodosin almost completely inhibited noradrenaline-induced contractions (300 nM tamsulosin inhibited 100 μM noradrenaline-induced contractions by 93±3.5%, and 100 nM silodosin inhibited 100 μM noradrenaline-induced contractions by 84±4.99%) (FIG. 4B).

[0035] Effect of Onvancertive and α1-blocker combination on EFS-induced contractions EFS-induced contractions were compared between human prostate tissues after addition of α1-blockers alone or in combination with 100 nM Onvancertive (Figure 5a). EFS-induced contractions after addition of the combination of α1-blockers and Onvancertive were significantly lower than EFS-induced contractions after addition of α1-blockers alone (Figure 5a). This additive effect of Onvancertive was comparable with that of tamsulosin and silodosin (59 ± 11.5% inhibition of 32 Hz-induced contractions with Onvancertive compared to tamsulosin alone, and 61 ± 9.8% inhibition of 32 Hz-induced contractions with Onvancertive compared to silodosin alone). In two separate experimental systems, the effect of the combination of onvancertive and an α1-blocker on EFS-induced contractions was compared to the effect of onvancertive (100 nM) alone (Figure 5b). EFS-induced contractions after the addition of the combination of onvancertive and an α1-blocker were significantly lower than EFS-induced contractions after the addition of onvancertive alone (Figure 5b). This additive effect of the α1-blocker was comparable when tamsulosin and silodosin were used (68 ± 12.2% inhibition of 32 Hz-induced contractions with tamsulosin compared to onvancertive alone, and 68 ± 12.7% inhibition of 32 Hz-induced contractions with silodosin compared to onvancertive alone).

[0036] Detection of PLK1 in WPMY-1 cells To compare the potential expression level of PLK1 in the human prostate stromal cell line WPMY-1 with human prostate tissue, we performed Western blot analysis using an antibody raised against PLK1. Detection revealed bands with the size of the expected molecular weight of PLK1 (Figure 6). In agreement with previous findings (Hennenberg et al., 2018), the intensity of these bands was highly variable between samples of prostate tissue from different patients (Figure 6A). In contrast, this intensity was constant in four different samples of WPMY-1 cells and was in the range of the highest expression levels found in human prostate tissue (Figure 6B). A similar pattern was seen after detection of PLK1 mRNA by RT-PCR (Figure 6C). Thus, PLK1 mRNA content showed high variability in human prostate tissue (Figure 6C). In WPMY-1 cells, the content of PLK1 mRNA was constant and in the range of levels comparable to the highest content found in prostate tissue (Figure 6C). In human prostate tissues, a correlation was found between PLK1 and PSA mRNA levels (R = -0.543, P = 0.297) (Fig. 6D).

[0037] Effect of Onvancertive on the viability and proliferation of WPMY-1 cells The effect of onvancertive on the viability of WPMY-1 cells was evaluated by CCK-8 assay. Onvancertive induced a concentration-dependent (10 nM, 50 nM, 100 nM) and time-dependent (24-72 h) decrease in viability (Figure 7A). The decrease ranged from 15 ± 8.8% at 10 nM to 63 ± 2.2% at 100 nM after 24 h, 28 ± 8.3% at 10 nM to 86 ± 1.9% at 100 nM after 48 h, and 40 ± 4.4% at 10 nM to 89 ± 1.4% at 100 nM after 72 h (Figure 7A). The effect of Onvansertiv on the proliferation of WPMY-1 cells was assessed by two different readouts, the results of which confirmed each other (Figures 7B and C). In the plate colony assay, colony formation was reduced by Onvansertiv in a concentration-dependent manner, reaching a reduction of 39±4.5% by 10 nM, 89±1.9% by 50 nM, and 97±0.4% by 100 nM (Figure 7B). In the EdU assay, the percentage of proliferating cells was reduced by Onvansertiv in a concentration-dependent manner, reaching a reduction of 18±0.1% by 10 nM, 24±2.1% by 50 nM, and 35±2.0% by 100 nM after 24 hours (Figure 7C).

[0038] Consideration Our findings suggest that the PLK1 inhibitor onvansertib inhibits 1) neurogenic contraction, α1-adrenergic contraction, and endothelin-induced contraction in human prostates, and 2) proliferation of prostatic stromal cells. Notably, the combination of onvansertib with α1-blockers caused greater inhibition of neurogenic contraction than α1-blockers alone. This additive effect of onvansertib with α1-blockers, together with their simultaneous effects on growth, may be of particular interest from a clinical perspective. Both prostatic smooth muscle contraction and prostatic growth may contribute to BOO and LUTS in BPH (Hennenberg et al., 2014). As a result, although both are medical targets, they are still affected by poor efficacy, common use of combination therapy, and low adherence, ultimately leading to complications, hospitalization, and surgery (Hennenberg et al., 2014; Oelke et al., 2013). Identification of novel targets is a prerequisite to overcome these limitations in the future. Based on our findings, onvancertive may be an attractive compound to be tested for LUTS suggestive of BPH in vivo.

[0039] We tested the effect of onvansertiv on EFS-induced and noradrenaline-induced contractions using two different concentrations. Both 100 nM and 1 μM concentrations of onvansertiv inhibited contractions to the same extent. Thus, together with the reported IC50 values, we conclude that the inhibition of contractions is largely dependent on PLK1 inhibition, and no other contraction-mediating kinases were involved in this range up to 1 μM. Indeed, onvansertiv is reported to be highly specific for PLK1, with IC50 values ​​of 2 nM for PLK1, 10 μM for PLK2, and 10 μM for PLK3 in biochemical assays (Beria et al., 2011). In another screen with biochemical assays, onvansertiv was tested against 296 kinases and showed reduced activation (<50%) of 11 kinases (including PLK1) when tested at 1 μM, When tested at 100 nM, it showed inhibition of only PLK1 (Valsasina et al., 2012). In a separate series of biochemical assays from the same study conducted with 63 kinases, it showed IC50 values ​​of 510 nM for FLT3, 744 nM for MELK, and 826 nM for CK2. 50 The value of onvansertiv was revealed (Valsasina et al., 2012). Thus, the concentration of 100 nM used in most of our experiments may be highly specific to PLK1, and most of their effects may be due to PLK1 inhibition. Since the concentration of 1 μM did not provide greater inhibition than that obtained from 100 nM, we hypothesize that 1) 1 μM does not result in off-target inhibition of contraction-mediating kinases, and 2) 100 nM is sufficient to obtain maximal PLK1-dependent effects. Consistent with previous studies reporting that onvansertiv binds to the ATP pocket of PLK1 by hydrogen bonds and thus acts as an ATP-competitive inhibitor with reversible dissociation (Beria et al., 2011; Valsasina et al., 2012), other studies also found that the effect of onvansertiv was reversible. Thus, after washout of onvansertiv, contraction returned to the level of the corresponding control.

[0040] In our organ bath experiments, inhibition of smooth muscle contraction was observed, without taking into account the medical history of the patients included. Thus, tissue samples were anonymous, and no patient data was collected, stored, or analyzed for this study. To eliminate individual variability or heterogeneity from previous treatments, we referred to the contraction as high molar KCl-induced contraction and confirmed our findings in different contexts, i.e., with different α1-adrenoceptor agonists or different combination conditions. Nevertheless, the lack of reference to patient characteristics, especially previous medications that may affect contractility, needs to be considered as a potential limitation. The inhibition of neurogenic contraction and α1-adrenergic prostatic smooth muscle contraction is consistent with recent findings obtained with four other inhibitors with assumed selectivity for PLK1 (Hennenberg et al., 2018). Similar to our current results, inhibition of EFS-induced contraction by other PLK1 inhibitors was in the range of around 50% (Hennenberg et al., 2018). Here, we tested the effect of two alpha 1 blockers on EFS-induced contraction, comparing the combination of onvansert and alpha 1 blocker with alpha 1 blocker and onvansert alone. Both alpha 1 blockers, tamsulosin and silodosin, showed very similar results, inhibiting EFS-induced contraction by less than 50%. This incomplete inhibition by alpha 1 blockers is consistent with previous results that reported comparable or even smaller effects of alpha 1 blockers on EFS-induced contraction of human prostate tissue (Angulo et al., 2012; Buono et al., 2014; Chueh et al., 1996; Oger et al., 2009, 2010). We exclude that this incomplete inhibition of EFS-induced contraction by tamsulosin and silodosin was due to too low alpha 1 blocker concentration. Indeed, the same concentrations used in the EFS experiments completely reduced noradrenaline-induced prostate contraction (at least in the range of noradrenaline concentrations applied here).We found that the EFS-induced contraction after application of the combination of onvansertib and α1 blocker was significantly lower than the tension after α1 blocker or onvansertib alone, which may be considered as an important finding of our study. It can be speculated that the addition of onvansertib or other PLK1 inhibitors to α1 blockers may result in higher LUTS improvement in BPH patients than α1 blockers alone. Consistent with previous results obtained at the protein level, we here confirm that PLK1 mRNA expression does not correlate with PSA expression, suggesting that it acts independently of the degree of BPH (Hennenberg et al., 2018).

[0041] In contrast to recent findings that the PLK1 inhibitors SBE 13 and cyclaporin did not alter endothelin-1-induced contraction of human prostate tissue, we found here an inhibition of endothelin-1-induced contraction by onvansertib (Hennenberg et al., 2018). The reason for this discrepancy remains speculative at this stage but may reflect divergent pharmacological profiles. However, it is noteworthy that even the inhibition of adrenergic contraction (induced by noradrenaline, phenylephrine, and methoxamine) for onvansertib appears to be greater than that reported for SBE 13 and cyclaporin 9 (Hennenberg et al., 2018). In addition to endothelin-1-induced contraction, onvansertib inhibited ATP-induced contraction. Notably, previous studies addressing ATP-induced contraction in the prostate were to our knowledge limited to animal models (Brandli et al., 2010; Buljubasich and Ventura, 2004; Hennenberg et al., 2017; White et al., 2015; Xu and Ventura, 2010), so our data may be the first evidence suggesting purinergic smooth muscle contraction in the human prostate. Consistent with other PLK inhibitors, U46619-induced contraction was again resistant to onvansertib. Non-adrenergic mediators, including endothelin-1, thromboxane A2, and possibly ATP, may contribute to prostatic smooth muscle tone in parallel with α1-adrenoceptors in BPH, and thus, they may maintain urethral obstruction despite treatment with α1-blockers, which may explain the limitations or large number of non-responders of α1-blockers (Hennenberg et al., 2014, 2017). Given its effects on endothelin-1- and ATP-induced contractions, the in vivo improvement of LUTS suggestive of BPH by onvancertive may exceed that of alpha-1 blockers.

[0042] Another important finding is that the inventors found that onvansertiv inhibited the proliferation of prostate stromal cells. Previous studies reporting the inhibition of proliferation by onvansertiv were performed in various types of tumor cells (Casolaro et al., 2013; Hartsink-Segers et al., 2013; Sero et al., 2014; Valsasina et al., 2012). In the prostate, other PLK inhibitors or knockdown of PLK1 inhibit tumor cell proliferation and tumor growth, enhancing the efficacy of antitumor treatment (Lin et al., 2019; Reagan-Shaw and Ahmad, 2005; Shao et al., 2015; Zhang et al., 2014). In general, PLK inhibitors, including onvansertiv, reduce proliferation by blocking the G2-M cell cycle (Valsasina et al., 2012). Prolonged blockade of mitosis by PLK inhibition can be followed by apoptosis and cell death (Valsasina et al., 2012). Both processes may explain the effects of onvansertib on the proliferation and viability of WPMY-1 cells in our study. The loss of viability in WPMY-1 cells occurred as early as 24 h, but was clearly time-dependent. For smooth muscle cells, a role for PLK1 in promoting proliferation was suggested for airway smooth muscle cells, where PLK1 knockdown inhibited growth factor-induced proliferation (Jiang and Tang, 2015). Taken together, our findings that proliferation and viability of WPMY-1 cells are affected by PLK1 inhibitors are consistent with previous studies performed in other cell types. WPMY-1 cells are derived from non-malignant human prostatic stroma and closely resemble prostatic smooth muscle cells and can be considered as such. (Wang et al., 2015). Smooth muscle cells are the major cell type in the prostatic stroma and may show increased proliferation in BPH (Strand et al., 2017). They contribute to prostatic hyperplasia in BPH (Strand et al., 2017). Comparison of expression levels by Western blot and RT-PCR suggested that WPMY-1 cells express PLK1 at a constant level comparable to the highest level in human prostate tissues with variable PLK1 content. It can be expected that onvancertive may prevent prostate growth or reduce prostate size in BPH, which is the main goal in 5-ARI treatment to prevent the progression of BPH and reduce the risk of complications and surgery (Oelke et al., 2013).

[0043] Prostate growth and smooth muscle contraction are targets of medical therapy in BPH, but have long been thought to be largely independent of each other. It is now becoming clear that both processes are intimately linked to each other, based on recent evidence showing that prostate smooth muscle contraction and stromal cell growth are sensitive to the same inhibitors (Hennenberg et al., 2014). Inhibitors of Rho kinase, Rac GTPase, or Src family kinases simultaneously inhibited agonist-induced contraction of human prostate tissue and growth of prostate stromal cells (Rees et al., 2003; Wang et al., 2015, 2016b). This is similar to the situation in other smooth muscle cells, e.g., vascular smooth muscle cells, where RhoA / Rho kinase mediates contraction and proliferation (Shimokawa et al., 2016). Notably, a similar role has been suggested for PLK1 in airway smooth muscle cells, where both proliferation and contraction depend on PLK1 (Jiang and Tang, 2015; Li et al., 2016). The same is apparently true for prostatic smooth muscle. 1In contrast to blockers or 5-ARIs, onvansertiv can simultaneously reduce the tone and growth of prostatic smooth muscle. Whether onvansertiv accesses the same or different intracellular signaling pathways to inhibit smooth muscle contraction and proliferation, or what other effectors participate in onvansertiv-sensitive signaling together with PLK1, cannot be assessed based on our data. The effects of PLK inhibitors on vascular smooth muscle contraction, which may cause cardiovascular side effects and limit in vivo applications, have not been reported to the best of our knowledge.

[0044] Taken together, it can be speculated that onvansertiv may be highly effective in vivo when it is applied to treat LUTS suggestive of BPH for three reasons. First, it can be hypothesized that the improvement of LUTS by adding onvansertiv to α1-blockers may be higher than that by α1-blockers alone, based on our displacement from combination experiments in EFS-induced contraction. Second, onvansertiv inhibited endothelin-1-induced contraction, which is not sensitive to α1-blockers but contributes to prostatic smooth muscle tone in BPH (Hennenberg et al., 2014, 2017), thus resulting in a high degree of symptom relief and Q10. maxThird, onvansertib strongly reduced the viability of stromal cells, which is expected to reduce prostate size in parallel with symptom relief. Since the number of smooth muscle cells can be reduced in vivo, the antiproliferative effect may reduce contractility even more strongly after chronic application in vivo than after short exposure in organ baths. In organ baths, 1-h exposure is usually not sufficient to reduce contractility by affecting viability (Yu et al., 2018, 2019a, 2019b), and therefore this anticontractile effect most likely resulted from a signaling mechanism mediated by PLK1 that promotes contraction. Combination therapy of α1-blockers and 5-ARIs is commonly applied to treat LUTS in men (Oelke et al., 2013). However, it is associated with side effects and has a high discontinuation rate (Fullhase et al., 2013). Single compounds such as onvancertive that can address adrenergic and non-adrenergic prostatic smooth muscle tone at once along with growth may be attractive for clinical trials as they may replace combination therapy with α1 blockers and 5-ARIs and may exceed the efficacy of monotherapy with α1 blockers. Onvancertive is orally available (Beria et al., 2011; Valsasina et al., 2012). Recently, pharmacokinetics and safety have been investigated in phase 1 trials, achieving plasma concentrations in the triple-digit nanomolar range in humans at the lowest dose tested (Weiss et al., 2018). As a result, it appears possible that concentrations similar to those in our organ bath experiments (100 nM) may be achieved in the prostate by oral administration, and thus clinical trials for LUTS indicative of BPH may be promising.

[0045] conclusion Onvancertive inhibits neurogenic and α1-adrenergic contraction of human prostatic smooth muscle and proliferation of prostatic stromal cells. Although α1-blockers reduce neurogenic contraction by at most 50%, the combination of α1-blockers and onvancertive provides additive and stronger inhibition of neurogenic prostatic contraction. It appears that onvancertive may simultaneously reduce prostatic smooth muscle tone and growth in BPH, and therefore onvancertive may be an attractive compound to be tested in vivo for LUTS indicative of BPH.

[0046] [References] TIFF0007621280000002.tif141170 TIFF0007621280000003.tif221170 TIFF0007621280000004.tif217170 TIFF0007621280000005.tif229170 TIFF0007621280000006.tif210170 TIFF0007621280000007.tif139170

[0047] In view of the above, it will be seen that the several objects of the invention are achieved and other advantageous results are attained. Because various changes can be made in the methods and compositions described above without departing from the scope of the invention, it is intended that all matter contained in the above specification and shown in the accompanying drawings should be interpreted in an illustrative and not limiting sense. All references cited herein are incorporated by reference as part of this specification. They are intended merely to summarize the assertions made by the authors and are not an admission that the references constitute prior art. Applicants reserve the right to challenge the accuracy and pertinence of the cited references.

[0048] As used herein, the singular forms "a," "an," and "the" are intended to include the plural forms as well, unless the context clearly dictates otherwise. Additionally, the use of "or" is intended to include "and / or" unless the context clearly dictates otherwise. As used herein, particularly in the embodiments, the term "about" or "approximately" when preceding a numerical value indicates a value plus or minus a range of 10%. When a range of values ​​is given, it is understood that each intervening value between the upper and lower limits of the range, to one tenth of the unit of the lower limit, and any other stated range or intervening value within that stated range, is encompassed in the disclosure unless the context clearly indicates otherwise. It is also encompassed within the disclosure that the upper and lower limits of these smaller ranges may be independently included within the smaller range (subject to any limit specifically excluded in the stated range). When a stated range includes one or both limits, ranges excluding one or both of the included limits are also included in the disclosure.

[0049] As used herein and in the embodiments, the phrase "and / or" should be understood to mean "one or both" of the elements thus conjoined, i.e., elements that are in some cases conjunctive and in other cases disjoint. Multiple elements listed with "and / or" should be construed in the same manner, i.e., "one or more" of the elements thus conjoined. In some cases, other elements may be present other than the elements specifically identified by the "and / or" clause, whether related or unrelated to the elements specifically identified. Thus, as a non-limiting example, when used with open-ended terms such as "comprising," a reference to "A and / or B" may refer in one embodiment to only A (optionally including elements other than B); in another embodiment to only B (optionally including elements other than A); in yet another embodiment to both A and B (optionally including other elements), etc.

[0050] Here, as used herein and in the embodiments, "or" should be understood to have the same meaning as "and / or" as defined above. For example, when inserted between items in a list, "or" or "and / or" should be interpreted as inclusive, i.e., the inclusion of at least one (including more than one) of a plurality of elements or a list of elements, and optionally additional items not listed. The term "only" as used in contrast, such as "only one of" or "exactly one of," or "consisting of," when used in the embodiments, refers to the inclusion of exactly one element of a plurality of elements or a list of elements. In general, the term "or" as used herein should only be interpreted as an exclusive alternative (i.e., "one or the other but not both") when preceded by a term of exclusion, such as "any of," "one of," "only one of," or "exactly one of." When used in the embodiments, "consisting essentially of" shall have its ordinary meaning as used in the field of patent law.

[0051] As used herein and in the embodiments, "at least one" in reference to a list of one or more elements should be understood to mean at least one element selected from any one or more of the elements in the list of elements, and not necessarily including at least one of each and every element specifically listed in the list of elements, and not excluding any combination of elements in the list of elements. This definition also allows that in some cases elements other than those specifically identified in the list of elements may be presented, whether related or unrelated to the specifically identified elements, to which the phrase "at least one" refers. Thus, as a non-limiting example, "at least one of A and B" (or, equivalently, "at least one of A or B" or, equivalently, "at least one of A and / or B") can refer, in one embodiment, to at least one A, optionally including more than one, with no B present (and optionally including elements other than B); in another embodiment, to at least one B, optionally including more than one, with no A present (and optionally including elements other than A); in yet another embodiment, to at least one A, optionally including more than one, and at least one B, optionally including more than one, with other elements present (and optionally including other elements), etc.

Claims

1. A pharmaceutical composition comprising onvancertive for use in a method of treating benign prostatic hyperplasia (BPH) accompanied by action potential-induced smooth muscle contraction with (a) onvancertive and (b) silodosin or tamsulosin.

2. 2. The pharmaceutical composition of claim 1, wherein the method of treating relieves symptoms of BPH or improves urinary flow (Qmax) by more than 50%.

3. 1. A pharmaceutical composition comprising onvancertive for use in a method for inhibiting action potential-induced contraction of smooth muscle, the method comprising treating smooth muscle with (a) onvancertive and (b) silodosin or tamsulosin.

4. The pharmaceutical composition of claim 3 , wherein the smooth muscle is prostate tissue.

5. The pharmaceutical composition of claim 4 , wherein the prostate tissue is human prostate tissue.

6. 5. The pharmaceutical composition of claim 4, wherein the prostate tissue is from a patient with benign prostatic hyperplasia (BPH).

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