阅读说明：本技术 用于抑制平滑肌的非肾上腺素能收缩和前列腺细胞增殖的onvansertib (Onvantertib for inhibiting non-adrenergic contraction of smooth muscle and proliferation of prostate cells ) 是由 马克·埃兰德 玛雅·莱丁格 托马斯·H·亚当斯 于 2020-02-25 设计创作，主要内容包括：提供了一种治疗患者的良性前列腺增生(BPH)的方法。还提供了抑制平滑肌的非肾上腺素能收缩的方法。另外提供了一种抑制人类前列腺细胞的增殖的方法。(A method of treating Benign Prostatic Hyperplasia (BPH) in a patient is provided. Methods of inhibiting smooth muscle non-adrenergic contractions are also provided. Additionally provided is a method of inhibiting proliferation of human prostate cells.)

1. A method of treating Benign Prostatic Hyperplasia (BPH) in a patient, comprising treating the patient with (a) onvanisertib and (b) an α 1-blocker, a 5- α -reductase inhibitor, a phosphodiesterase-5 enzyme inhibitor, or a combination thereof.

2. The method of claim 1, wherein the patient is treated with onvansertib and an α 1-blocker.

3. The method of claim 1, wherein the method provides greater than 50% reduction in symptoms of BPH or improvement in urine flow (Qmax).

4. The method of claim 2, wherein the alpha 1-blocker is alfuzosin, doxazosin, tamsulosin, or silodosin.

5. A method of inhibiting non-adrenergic contraction of a smooth muscle, the method comprising treating the smooth muscle with onvansertib.

6. The method of claim 5, wherein the smooth muscle contraction is endothelin-1 induced or ATP induced (purinergic).

7. The method of claim 5, wherein the smooth muscle is prostate tissue.

8. The method of claim 7, wherein the prostate tissue is human prostate tissue.

9. The method of claim 7, wherein the prostate tissue is in a patient with Benign Prostatic Hyperplasia (BPH).

10. A method of inhibiting proliferation of prostate cells, the method comprising treating human prostate cells with onvansertib in a manner sufficient to inhibit proliferation of the prostate cells.

11. The method of claim 10, wherein the human prostate cell or tissue is in a patient with BPH.

(1) Field of the invention

The present application relates generally to the treatment of Benign Prostatic Hyperplasia (BPH). More specifically, treatment of BPH with a highly selective polo-like kinase 1(PLK1) inhibitor is provided.

(2) Description of the related Art

Benign Prostatic Hyperplasia (BPH) is commonly associated with Lower Urinary Tract Symptoms (LUTS), which are urethral compression due to prostatic hypertrophy and increased prostatic smooth muscle tone in BPH (Hennenberg et al, 2014). This symptomatic BPH condition, known as Bladder Outlet Obstruction (BOO), usually requires drug therapy (Oelke et al, 2013). However, this has limited effectiveness even in mild to moderate LUTS and is not applicable to severe LUTS (Oelke et al, 2013). In contrast to the limitation of improvement, the high number of cases, about 6 billion patients with obstructive symptoms worldwide in 2018 (Irwin et al, 2011). Due to age-dependence of prevalence and population shift, the importance of LUTS suggesting BPH will further increase, placing a high demand for new medical therapies with higher effectiveness.

Based on the contribution of prostate smooth muscle tone and prostate growth to LUTS suggesting BPH, medical treatment in BPH aims to inhibit 1) prostate smooth muscle contraction to rapidly ameliorate symptoms, and 2) prostate growth to prevent BPH progression and complications (Oelke et al, 2013). Medicine for treating prostatic diseasesSmooth muscle contraction is activated in part by norepinephrine released during adrenergic neurotransmission₁-adrenergic receptor induction (Hennenberg et al, 2014). Thus, α₁-adrenoceptor antagonists ('alpha')₁Blockers ") are the first line of choice to suggest treatment of LUTS for BPH, as they can alleviate BOO by relaxing the prostate smooth muscle (Caine et al, 1975, 1976; oelke et al, 2013). However, they reduce symptoms (international prostate symptom score, IPSS) and reduce urinary flow (Q)_{Maximum of}) Does not exceed 50%, while even placebo can cause up to 30% improvement (Hennenberg et al, 2014, 2017; oelke et al, 2013; strand et al, 2017). The phosphodiesterase-5 inhibitor tadalafil (tadalafil) has recently been approved for the treatment of obstructive LUTS, but its effectiveness is not higher than α₁Effectiveness of the blocking agent (Dahm et al, 2017). 5 α -reductase inhibitors (5-ARI) were applied to arrest prostate growth with the aim of reducing prostate size and preventing BPH progression (Oelke et al, 2013). However, the risk of symptom progression can be reduced by no more than 35-40% by monotherapy and 66% by combination therapy (Strand et al, 2017). Disappointing results of medical LUTS therapy have led to high drug withdrawal rates, with up to 70% of patients stopping within 12 months after the first prescription (Cindolo et al, 2015). The consequences are disease progression, hospitalization and surgery for BPH (Cindolo et al, 2015). Alpha is alpha₁The limitations of blockers may result from the contribution of non-adrenergic mediators to prostate smooth muscle tone (including endothelin-1), which reacts with alpha₁Adrenergic receptors concurrently induce prostate smooth muscle contraction (Hennenberg et al, 2014, 2017).

polo-like kinases (PLKs) are serine-threonine kinases and are primarily associated with promoting proliferation in different cell types, including airway smooth muscle cells, prostate cancer cells, and others (Jiang and Tang, 2015; Lin et al, 2019; Shao et al, 2015). Furthermore, recent findings indicate the function of PLK1 to promote airway, vascular and human prostate smooth muscle contraction (de Carcer et al, 2017; Hennenberg et al, 2018; Li et al, 20)16). However, PLK inhibitors and alpha₁The additive effects of blockers in prostate smooth muscle contraction, and their effects on prostate stromal cell growth are not known.

onvansertib

onvansertib (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) was the first oral route of administration of a PLK 1-specific ATP competitive inhibitor to enter clinical trials, demonstrating antitumor activity in different preclinical models. onvansertib has favourable pharmacological parameters and good oral bioavailability. onvansertib has several advantages over other PLK1 inhibitors, including very high selectivity for the PLK1 isoenzyme and very high oral bioavailability. onvansertib has an IC of 2nM for PLK1₅₀And for PLK2 and PLK3 are>10,000。

Summary of The Invention

A method of treating Benign Prostatic Hyperplasia (BPH) in a patient is provided. The method comprises treating the patient with (a) onvansertib and (b) an alpha 1-blocker, a 5-alpha-reductase inhibitor, a phosphodiesterase-5 enzyme inhibitor, or a combination thereof.

Methods of inhibiting smooth muscle non-adrenergic contractions are also provided. The method comprises treating smooth muscle with onvansertib.

Additionally provided is a method of inhibiting proliferation of human prostate cells. The method comprises treating human prostate cells with onvansertib in a manner sufficient to inhibit proliferation of the human prostate cells.

Brief description of several views of the drawings

Fig. 1A, 1B and 1C are graphs showing the effect of onvanterb on adrenergic contraction of human prostate bars (prostates). In organ baths, contraction of human prostate bars was induced by noradrenaline (fig. 1A), or by the alpha 1-adrenoceptor agonists phenylephrine and methoxamine (fig. 1B), after addition of onvanertib or DMSO at concentrations of 100nM or 1 μ M (control), or by noradrenaline (fig. 1C) after washing away DMSO and onvanertib (100nM) for 30 min. In each experiment, DMSO and onvansertib were applied to separate samples obtained from the same prostate. To eliminate heterogeneity due to individual differences, varying degrees of BPH, or other varying smooth muscle content (smooth muscle content), tone was expressed as a percentage of the contraction produced by high molar KCl, which was evaluated prior to application of onvansertib or DMSO. Data are mean ± s.e.m. from a series of tissues from n-5 (noradrenaline with 100nM onvanibertib, not washed), n-6 (noradrenaline with 1 μmonvanibertib), n-5 (phenylephrine), n-5 (methoxamine), and n-5 (noradrenaline after washing), where samples from each patient were assigned to both the control and inhibitor groups (# P <0.05 after multivariate analysis at indicated concentrations; P values for the whole group after two-way ANOVA are shown in the insert).

Figure 2 is a graph showing the effect of onvansertib on non-adrenergic contractions of human prostate bars. In organ baths, contraction of human prostate bars was induced by endothelin-1, ATP or thromboxane A2 analogue U46619 after addition of onvansertib or DMSO (control) at a concentration of 100 nM. In each experiment, DMSO and onvansertib were applied to separate samples obtained from the same prostate. To eliminate heterogeneity due to individual differences, varying degrees of BPH, or other varying smooth muscle content, tone was expressed as a percentage of the contraction produced by high molar KCl, which was evaluated prior to the application of onvansertib or DMSO. Data are mean ± s.e.m. from a series of tissues from n-6 (endothelin-1), n-5 (ATP), and n-5 (U46619) patients, where samples from each patient were assigned to both the control and inhibitor groups (P values for the whole group after two-way ANOVA are shown in the insert).

Figures 3A and 3B are graphs showing the effect of onvansertib on EFS-induced contraction of the human prostatic rod. In the organ bath, after the addition of onvansertib or DMSO (control) at a concentration of 100nM or 1 μ M (fig. 3A), and after washing away DMSO and onvansertib (100nM) for 30min (fig. 3B), the contraction of human prostate bars was induced by EFS. In each experiment, DMSO and onvansertib were applied to separate samples obtained from the same prostate. To eliminate heterogeneity due to individual differences, varying degrees of BPH, or other varying smooth muscle content, tone was expressed as a percentage of the contraction produced by high molar KCl, which was evaluated prior to the application of onvansertib or DMSO. Data are mean ± s.e.m. from a series of tissues from n-6 (DMSO vs 100nM onvansertib, without washing), n-7 (DMSO vs 1 μ Monvansertib) and n-5 (DMSO vs 100nM onvansertib, after washing) patients, where samples from each patient were assigned to both control and inhibitor groups (P values for the full group after two-way ANOVA are shown in the insert).

Fig. 4A and 4B are graphs showing the effect of an α 1-blocker on the contraction of a human prostate bar. In the organ bath, contraction of human prostate bars was induced by EFS (fig. 3A) or norepinephrine (fig. 4B) after addition of tamsulosin (300nM), silodosin (100nM) or an equal volume of distilled water containing 1% DMSO (control). In each experiment, solvent or α₁The blocking agent is applied to a separate sample obtained from the same prostate. To eliminate heterogeneity due to individual differences, varying degrees of BPH, or other varying smooth muscle content, tone is expressed as a percentage of the contraction produced by high molar KCl, which is applied at alpha₁Blocking agents or solvents were evaluated before. Data are a series of tissues from patients with n-5 (EFS/tamsulosin), n-5 (EFS/cilostacin), n-5 (noradrenaline/tamsulosin) and n-6 (noradrenaline/cilostacin)Wherein samples from each patient are assigned to both the control and inhibitor groups (# P after multivariate analysis on indicated concentrations)<0.05; p values for all groups after two-way ANOVA are shown in the insert).

FIGS. 5A and 5B are diagrams illustrating α alone₁Onvansertib compared to α as blocker₁Graph of the effect of the combination of blockers on EFS-induced contraction of the human prostatic bars. In an organ bath tamsulosin (300nM), silodosin (100nM), onvansertib (100nM) or a combination of onvansertib (100nM) with tamsulosin or silodosin, and equal amounts of solvent (DMSO as a sole absence of onvansertib to α)₁Control of blocking agent, or water/DMSO as control lacking α 1-blocking agent alone to onvanisertib), human prostatic bar contraction is induced by EFS. In each experiment, α₁The blocking agent or onvansertib and the combination are applied to separate samples obtained from the same prostate. Each figure represents a separate series of experiments using different prostate tissues (but samples from the same prostate were assigned to both groups in each figure). To eliminate heterogeneity due to individual differences, varying degrees of BPH, or other varying smooth muscle content, tone is expressed as a percentage of the contraction produced by high molar KCl, which is in application of onvansertib, α₁Blocking agents or DMSO prior to evaluation. The data are mean ± s.e.m. of a series of tissues from a patient of n-7 (tamsulosin versus tamsulosin and onvansertib), n-5 (cilostacin versus cilostacin and onvansertib), n-5 (onvansertib versus tamsulosin and onvansertib), and n-5 (onvansertib versus cilostacin and onvansertib), where samples from each patient were assigned to both control and inhibitor groups (# p.e.m. after multivariate analysis with indicated concentrations<0.05; p values for all groups after two-way ANOVA are shown in the insert).

Fig. 6A, 6B, 6C and 6D are photographs (fig. 6A) and graphs showing detection of PLK1 in human prostate tissue and WPMY-1 cells. Figure 6A shows human prostate tissue (n-4 patients) and WPMY-1 cells (from PLK1) using antibodies raised against PLK1n ═ 4 samples from independent experiments). Bands of the expected molecular weight (68kDa) size of PLK1 are shown. The position and size of the marker band is indicated on the right side of each blot (size in kDa). Fig. 6B shows the relative intensity of the putative PLK1 band. After quantification of the density of the 68kDa band in (fig. 6A), arbitrary units of all samples were normalized to the mean of the values obtained for prostate tissue. Values for all samples and median of the two groups are shown. Fig. 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). Ct value of RT-PCR was 2^-ΔCPAnd finally normalized to the mean obtained for prostate tissue. Values for all samples and median of the two groups are shown. FIG. 6D shows 2 from detection of PLK1 and PSA in human prostate tissue by RT-PCR^-ΔCPValues were plotted against each other and Spearman correlation analysis was performed.

Fig. 7A, 7B and 7C are graphs and micrographs showing the effect of onvansertib on viability and proliferation of WPMY-1 cells. Figure 7A is a graph showing the assessment of the viability of WPMY-1 cells by the CCK-8 assay after exposure to the indicated concentrations of onvansertib or solvent and for the indicated time periods. Data are mean ± s.e.m. (# P <0.05 vs. corresponding control) from 5 independent experiments per setup. Fig. 7B is a graph and micrograph showing proliferation assessed by a colony formation assay. WPMY-1 cells were exposed to the prescribed concentration of onvansertib. Representative micrographs are shown, as well as the mean ± s.e.m. (# P <0.05 control) from 5 independent experiments. Fig. 7C is a graph and micrograph showing proliferation assessed by EdU assay. WPMY-1 cells were exposed to the indicated concentration of onvansertib for 24 h. Nuclei of proliferating cells are shown in pink, while nuclei of non-proliferating cells are shown in blue. Representative pictures are shown, as well as mean ± s.e.m. from 5 independent experiments (# P <0.05 control).

Detailed Description

As used herein, the singular forms "a", "an" and "the" are intended to include the plural forms as well, unless the context clearly indicates otherwise. Moreover, use of "or" is intended to include "and/or" unless the context clearly indicates otherwise.

The present invention is based, in part, on the discovery that the highly selective polo-like kinase 1(PLK1) inhibitor onvansertib effectively inhibits both adrenergic and non-adrenergic contractions of smooth muscle, such as human prostate tissue. This is surprising because other PLK1 inhibitors inhibit adrenergic contraction of smooth muscle, but do not inhibit non-adrenergic contraction of smooth muscle (Hennenberg et al, 2018). Since onvansertib inhibits non-adrenergic contraction of smooth muscle, particularly endothelin-1-induced and ATP (purinergic) induced smooth muscle contraction (see examples below), onvansertib may be effective with inhibitors of adrenergic smooth muscle contraction such as alpha₁-a combination of blockers to treat Benign Prostatic Hyperplasia (BPH).

Accordingly, in some embodiments, a method of treating Benign Prostatic Hyperplasia (BPH) in a patient is provided. The methods comprise treating the patient with (a) onvansertib and (b) an alpha 1-blocker, a 5-alpha-reductase inhibitor, a phosphodiesterase-5 enzyme inhibitor, or a combination thereof. Since onvansertib inhibits smooth muscle contraction by a mechanism different from that of an α 1-blocker, a 5- α -reductase inhibitor or a phosphodiesterase-5 enzyme inhibitor, combination therapy is superior to any inhibitor alone. Indeed, the combination therapy of onvansertib and tamsulosin or silodosin (both of which are α 1-blockers) inhibits prostate smooth muscle tissue to a greater extent than any of the individual compounds (see examples below).

In some embodiments, the methods reduce symptoms of BPH or reduce urinary flow (Q)_{Maximum of}) The improvement is greater than 30%. In other embodiments, the method is on the urine flow (Q)_{Maximum of}) The improvement is greater than 50%.

In various embodiments, onvansertib and α are used₁-treatment of the patient with a blocking agent. Alpha is alpha₁Non-limiting examples of blocking agents are cilostacin, tamsulosin, alfuzosin, doxazosin, prazosin and terazosin. Process for preparing 5-alpha-reductase inhibitorsNon-limiting examples are dutasteride (dutasteride) and finasteride (finasteride). Non-limiting examples of phosphodiesterase-5 inhibitors are avanafil (avanafil), lodenafil (lodenafil), milodenafil (mirodenafil), sildenafil (sildenafil), tadalafil, vardenafil (vardenafil), udenafil (udenafil), zaprinast (zaprinast), benzamidafil and dactadalafil (dasatafil).

Methods of inhibiting smooth muscle non-adrenergic contractions are also provided. The method comprises treating smooth muscle with onvansertib.

This method can be used to inhibit any non-adrenergic smooth muscle contraction. In some embodiments, smooth muscle contraction is endothelin-1 induced or ATP induced (purinergic).

In various embodiments of the 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 in any tissue of any mammal. In some embodiments, the prostate tissue is in a patient with Benign Prostatic Hyperplasia (BPH).

Additionally provided is a method of inhibiting proliferation of prostate cells. The method comprises treating prostate cells with onvansertib in a manner sufficient to inhibit the growth of human prostate cells. In various embodiments, the prostate cells are in a human patient with BPH, although the method should be effective in inhibiting prostate cell proliferation in vitro or in vivo in any mammal.

In some embodiments of these methods, the cells are also exposed to a second agent that inhibits proliferation of human prostate cells. The second agent can be any agent known to be at least partially effective in inhibiting the growth of human prostate cells. A non-limiting example of such a second drug is metformin (Wang et al, 2017).

In any of the methods provided herein, onvansertib may be administered by any means known in the art. In some embodiments, onvansertib is administered orally in a pharmaceutically acceptable excipient. Effective inhibition of any of these can be determined by methods known in the artThe dosage of onvansertib for smooth muscle tissue contraction or prostate cell proliferation in a particular patient without undue experimentation. In some embodiments, the dose of onvansertib administered is greater than 6mg/m²One day, 12mg/m²Daily or 24mg/m²The day is. In some of these embodiments, the dose of onvansertib is less than 50mg/m²A day, e.g. 24mg/m²Daily or 36mg/m²The day is.

In embodiments where onvansertib is administered with an additional drug, onvansertib may be administered before, simultaneously with, or after the additional drug. Further, the onvansertib and the second drug may be administered on different or the same schedules, e.g., daily administration, 5 days administration +2 days no administration, etc.

Preferred embodiments are described in the following examples. Other embodiments within the scope of the claims herein will be apparent to one skilled in the art from consideration of the specification or practice of the invention as disclosed herein. It is intended that the specification, together with the examples, be considered exemplary only, with the scope and spirit of the invention being indicated by the claims which follow the examples.

Example the Effect of onvansertib on prostate smooth muscle function in benign prostatic hyperplasia in humans

Summary of the embodiments

Prostate smooth muscle contraction and prostate hypertrophy contribute to lower urinary tract symptoms suggesting benign prostatic hyperplasia. Recent evidence has demonstrated that inhibitors of polo-like kinase (PLK) inhibit smooth muscle contraction of human prostate tissue. However, they are on α₁The additive effect of blockers and the effect on prostate growth are not known. Here we examined a novel and highly selective inhibitor onvansertib of PLK1 alone and with α₁-the effect of the combination of blockers on prostate smooth muscle contraction, and on the proliferation and viability of prostate stromal cells (WPMY-1). Prostate tissue was obtained from radical prostatectomy. Contraction was studied in an organ bath. Proliferation and viability were assessed by plate colony (plate colony), EdU and CCK-8 assays. Electric fieldStimulation (EFS) -induced contraction of human prostate tissue is inhibited by 100nM and 1 μ M onvansertib to 34% at 32Hz and by α₁Blockers tamsulosin and silodosin are inhibited to 48% and 47%. With alpha alone₁The combination of onvansertib with tamsulosin or silodosin further reduced EFS induced contraction compared to blocking agent (59% and 61%, respectively) and onvansertib alone (68% for both). Norepinephrine, phenylephrine, methoxamine, endothelin-1, and ATP-induced contraction are inhibited to a similar extent by onvanisertib (100 nM). The viability and proliferation of WPMY-1 cells decreased in a concentration and time dependent manner (24h-72h, 10nM-100 nM). onvansertib inhibits neurogenic, adrenergic, endothelin-1, and ATP-induced contraction of human prostate smooth muscle and proliferation of stromal cells. Alpha is alpha₁The blocking agent reduces shrinkage by no more than 50%. Alpha is alpha₁The combination of a blocking agent with onvansertib provides an additive inhibition of prostate constriction.

Materials and methods

Human prostate tissue

Human prostate tissue was obtained from patients undergoing radical prostatectomy for prostate cancer (n ═ 107). Patients who previously underwent transurethral prostatectomy (TURP) were excluded. This study was carried out according to the Declaration of Helsinki of the World Medical Association and has been approved by the ethical committee of the university of munich, mark vich-macmlinean-munich, germany (Ethikkommission bei der LMU munchen, approval text No. 19-737). Informed consent was obtained from all patients. Samples and data were collected and analyzed anonymously upon ethical approval, and therefore no data was stored regarding patient characteristics. Samples were taken immediately after prostatectomy, followed by macroscopic examination by a urologist. Considering that most prostate cancers occur in the peripheral zone (periurethral zone), all tissues were taken from the periurethral zone (praadirachheep et al, 2011; Shaikhibrahim et al, 2012). According to the pathological evaluation, only tissue samples showing no histological signs of tumor formation, cancer or inflammation were collected. BPH is present in 80% -83% of prostate cancer patients (Alcaraz et al, 2009; Orsted and Bojesen, 2013). Typically, such tissues show varying levels of Prostate Specific Antigen (PSA), as well as varying levels of smooth muscle and glandular epithelial cells, reflecting the presence of varying degrees of BPH (Wang et al, 2016a, 2016 c). For macroscopic examination and sampling, the prostate was opened by a single longitudinal incision from the sac to the urethra. Subsequently, any significant tumor infiltration of the two intersections (intersections) was examined macroscopically. Because tumors are usually located in the peripheral region, tumor infiltration in the periurethral region (where sampling is performed) is very rare (found in less than 1% of the prostate). The prostate showing tumors in the periurethral region in macroscopic examination was not sampled and was not included in the study. The organ bath studies were performed immediately after sampling, while samples for molecular analysis were snap frozen in liquid nitrogen and stored at-80 ℃.

Tension measurement

Prostate strips (6 mm. times.3 mm) were mounted in 10ml air (95% O)₂And 5% CO₂) Tissue bath (Danish Myotechnology, Aarhus, Denmark) comprising a Krebs-Henseleit solution (37 ℃, pH 7.4) with the following composition: 118mM NaCl, 4.7mM KCl, 2.55mM CaCl₂，1.2mM KH₂PO₄，1.2mM MgSO₄，25mM NaHCO₃7.5mM glucose. In each single experiment, two of the four bars obtained from the same prostate were assigned to the control group (no inhibitor or antagonist), and the other two bars were assigned to the inhibitor/antagonist group (resulting in duplicate determinations for each group in each single experiment). All four samples of one experiment were examined in four chambers of the same organ bath. Thus, the control and inhibitor curves in each series/plot were obtained from the same prostate, but different prostates were examined for different series/plots. Due to different inhibitor concentrations (100nM, 1. mu.M) and onvansertib with alpha₁Combinations of blocking agents, the amount of solvent being different between series, which excludes any comparison between the level of shrinkage in different series. Thus, and is contemplated to be derived from a radical prostateResected prostate tissue may exhibit considerable heterogeneity, with statistical comparisons being made only between groups within the same series (i.e., containing tissue from the same prostate for the inhibitor and control groups), and not between series obtained from different prostates (i.e., organ bath experiments not spanning different series). Only one curve per sample was recorded, i.e. one agonist or Electric Field Stimulation (EFS) was recorded.

After installation in the organ bath chamber, the preparation was strained to 4.9mN and kept in equilibrium for 45 min. During the initial phase of the equilibration period, a spontaneous drop in tension is usually observed. Thus, the tension was adjusted three times during the equilibration period until a stable resting tension of 4.9mN was reached. After the equilibration period, the maximal contraction induced by 80mM KCl was assessed. Subsequently, the chamber was washed three times with Krebs-Henseleit solution for a total of 30 min. Cumulative concentration response curves for noradrenaline, phenylephrine, methoxamine, endothelin-1 and for U46619, or EFS induced frequency response curves were generated 30min after the addition of onvansertib, tamsulosin, silodosin and/or dimethyl sulfoxide (DMSO) as controls. The reversibility of the onvansertib action was evaluated in a separate series, where DMSO and onvansertib were washed off by replacing the Krebs-Henselit solution three times within 30min, starting 30min after the application of DMSO or onvansertib, followed by the construction of a concentration response curve for norepinephrine or a frequency response curve generated by EFS.

Application of EFS mimics action potentials, resulting in the release of endogenous neurotransmitters, including norepinephrine. Tetrodotoxin, an inhibitor of neurotransmitter release, has been previously used to demonstrate that it accounts for two thirds of EFS-induced contractions in the human prostate (Angulo et al, 2012). For EFS, a tissue strip was placed between two parallel platinum electrodes connected to a CS4 stimulator (Danish Myotechnology). Rectangular pulses of 1ms duration were applied with a voltage of 50V for a train duration (train duration) of 10s, and a delay of 1ms was used between the individual pulses. EFS-induced contractile responses were studied at frequencies of 2Hz, 4Hz, 8Hz, 16Hz, and 32Hz with 30s interval between stimuli.

Data analysis and calculation are based on the maximum peak height of the shrinkage. For calculation of agonist or EFS induced contraction, tone is expressed as% of KCl induced contraction as this can correct for different stromal/epithelial ratios, different smooth muscle content, different degrees of BPH or any other heterogeneity between prostate samples and patients.

Western blot analysis

Frozen prostate tissue using a composition containing matrix AThe system (MP Biomedicals, Illkirch, France) was homogenized in a buffer containing 25mM Tris/HCl, 10. mu.M phenylmethanesulfonyl fluoride, 1mM benzamidine and 10. mu.g/ml hemileupeptin sulfate. After centrifugation (20,000g, 4min), the protein concentration of the supernatant was determined using the Dc assay kit (Biorad, Munich, Germany) and boiled for 10min with Sodium Dodecyl Sulfate (SDS) sample buffer (Roth, Karlsruhe, Germany). Samples of cultured stromal cells were prepared as described below. The sample (20. mu.g/lane) was subjected to SDS-polyacrylamide gel electrophoresis, and the protein was blotted onNitrocellulose membrane (Schleicher)&Schuell, Dassel, Germany). The membranes were blocked overnight with Phosphate Buffered Saline (PBS) containing 5% milk powder (Roth, Karlsruhe, Germany) and incubated with rabbit anti-PLK 1(208G4) (#4513) (Cell Signaling Technology, Danvers, MA, USA) and mouse monoclonal anti- β -actin antibody (sc-47778) (Santa Cruz Biotechnology, Santa Cruz, CA, USA).

The primary antibody was diluted in PBS containing 0.1% Tween 20(PBS-T) and 5% milk powder. Detection was then continued using biotinylated secondary goat anti-rabbit or horse anti-mouse IgG (BA-1000, BA-2000) (Vector Laboratories, Burlingame, CA, USA) followed by incubation with avidin and biotinylated horseradish peroxidase (HRP) from the "Vector ABC kit" (Vector Laboratories, Burlingame, CA, USA), both diluted 1:200 in PBS. After incubation with primary or secondary antibodies or biotin-HRP, the membrane was washed with PBS-T. Finally, the blots were developed with Enhanced Chemiluminescence (ECL) using ECL Hyperfilm (GE Healthcare, Freiburg, Germany). Density quantification was performed on the intensity of the resulting bands of PLK1 using "Image J" (National Institutes of Health, Bethesda, Maryland, USA).

RT-PCR

RNA from frozen prostate tissue or cells was isolated using the RNeasy Mini kit (Qiagen, Hilden, Germany). For separation from tissue, using a matrix AThe system (MP Biomedicals, Illkirch, France) homogenized 30mg of tissue. The RNA concentration was measured spectrophotometrically. Reverse transcription into cDNA was carried out using the reverse transcription system (Promega, Madison, Wis., USA) with 1. mu.g of isolated RNA. RT-PCR for PLK1, PSA and glyceraldehyde 3-phosphate dehydrogenase (GAPDH) was performed with a Roche Light cycler (Roche, Basel, Switzerland) using primers supplied as a ready-to-use mixture by Qiagen (Hilden, Germany) based on Ref-Seq accession No. NM-005030 for PLK1, NM-001030047 for KLK3 (synonymous PSA) and NM-002046 for GAPDH. The PCR reaction was carried out in a volume of 25. mu.l, containing 5. mu.lFastStart DNA MasterPlus SYBR Green I (Roche, Basel, Switzerland), 1. mu.l template, 1. mu.l primer and 18. mu.l water. Denaturation was performed at 95 ℃ for 10min and amplification was performed with 45 cycles of: 15 seconds at 95 ℃ followed by 60 seconds at 60 ℃. The specificity of the primers and amplification was demonstrated by subsequent melting point (fusion point) analysis, which revealed a single peak for each target. The results are expressed using the Δ Δ CP method, where the cycle number (Ct) at which the fluorescence signal exceeds the defined threshold for GAPDH is subtracted from the Ct value of PLK (Ct)_Target-Ct_GAPDHΔ CP) and the value is calculated as 2^-ΔCPAnd come into mutualNormalized or normalized to the mean of prostate tissue.

Cell culture

WPMY-1 cells are immortalized cell lines obtained from human prostate stroma that does not have prostate cancer (Webber et al, 1999). Cells were obtained from the American type culture Collection (ATCC; Manassas, Va., USA) and were cultured at 37 ℃ and 5% CO₂Maintained in RPMI 1640(Gibco, Carlsbad, CA, USA) supplemented with 10% Fetal Calf Serum (FCS) and 1% penicillin/streptomycin. Before the addition of onvansertib or DMSO, the medium was changed to medium without FCS. For western blot analysis, cells were lysed using radioimmunoprecipitation assay (RIPA) buffer (Sigma-Aldrich, st. louis, MO, USA) and removed from the flask after incubation for 15min on ice. Cell debris was removed by centrifugation (10,000g, 10min, 4 ℃) and different aliquots of the supernatant were subjected to protein determination or boiled with SDS sample buffer.

Cytotoxicity assays

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 24h, then the indicated concentrations of onvansertib or solvent were added. Subsequently, the cells were grown for different time periods (24h, 48h, 72 h). Separate controls were performed for each time period. At the end of this period, 10 μ l of [2- (2-methoxy-4-nitrophenyl) -3- (4-nitrophenyl) -5- (2, 4-disulfophenyl) -2H-tetrazolium monosodium salt (WST-8) from CCK-8 was added and the absorbance in each well was measured at 450nm after incubation for 2H at 37 ℃.

Colony formation assay

Approximately 100 cells were placed in each well of a 6-well plate and treated with onvansertib (final concentration 10nM, 50nM, 100nM) or solvent. Cells were incubated at 37 ℃ for 14 days, then washed twice with PBS and fixed by 2ml of 10% trichloroacetic acid overnight (4 ℃). Subsequently, all plates were washed five times with cold water and stained with 0.4% sulforhodamine B solution (diluted in 1% acetic acid) for 30min at room temperature. All plates were labeled and washed five times with 1% acetic acid before taking photographs. The number of colonies containing 50 or more cells was counted under a microscope.

EdU proliferation assay

WPMY-1 cells were plated at cell line specific density (50,000/well) on 16-well chambered coverslips (Thermo Scientific, Waltham, MA, USA). After 24h, cells were treated with onvansertib (final concentration 10nM, 50nM, 100nM) or solvent. After an additional 24h, the medium was changed to a 10mM solution of 5-ethynyl-2' -deoxyuridine (EdU) in FCS-free medium containing onvansertib or solvent. After 20h, cells were fixed with 3.7% formaldehyde. EdU incorporation was determined using the "EdU-Click 555" cell proliferation assay (Baseclick, Tutzing, Germany) according to the manufacturer's instructions. In this assay, incorporation of EdU into DNA was assessed by detection with a fluorescent 5-carboxytetramethylrhodamine (5-TAMRA). Counterstaining of all nuclei was performed with DAPI. Cells were analyzed by fluorescence microscopy (excitation: 546 nm; emission: 479 nm).

Medicine and nomenclature

onvansertib (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). Phenylepinephrine ((R) -3- [ -1-hydroxy-2- (methylamino) ethyl group]Phenol) and methoxamine (alpha- (1-aminoethyl) -2, 5-dimethoxybenzyl alcohol) is alpha₁-selective agonists of adrenergic receptors. U46619((Z) -7- [ (1S,4R,5R,6S) -5- [ (E,3S) -3-Hydroxyoct-1-enyl)]-3-oxabicyclo [2.2.1]Hept-6-yl]Hept-5-enoic acid) is an analog of thromboxane a2(TXA2), which specifically activates the thromboxane a2 receptor, and is frequently used as an agonist of the thromboxane a2 receptor (Malmsten, 1976; shen and Tai, 1998). Tamsulosin (5- [ (2R) -2- [2- (2-ethoxyphenoxy) ethylamino]Propyl radical]-2-methoxybenzenesulfonamide) and silodosin (1- (3-hydroxypropyl) -5- [ (2R) -2- [2- [2- (2,2, 2-trifluoroethoxy) phenoxy ] 2]Ethylamino group]Propyl radical]-2, 3-dihydroIndole-7-carboxamides) is alpha₁-adrenoceptor antagonist, p.alpha_1ASubtypes have high selectivity. Both are available and are routinely used to treat LUTS suggestive of BPH (oenke et al, 2013). Stock solutions of onvansertib (10mM) and appropriate dilutions were prepared in DMSO and stored at-20 ℃ until use. Aqueous stock solutions of noradrenaline, phenylephrine, methoxamine (each 10mM) and adenosine 5' -triphosphate (ATP) (500mM suspension) were freshly prepared prior to each experiment. Stock solutions of U46619(10mM) were prepared in ethanol and stored at-80 ℃ until use. An aqueous stock solution of endothelin-1 (0.4mM) was stored at-20 ℃ until use. Stock solutions of tamsulosin and silodosin (10mM) were prepared in DMSO and further diluted with distilled water (1:100 dilution to 100 μ M) and stored at-20 ℃ until use. onvansertib is supplied by Trovagene Inc (San Diego, CA, USA). Norepinephrine, phenylephrine, methoxamine and ATP are obtained from Sigma (Munich, Germany), U46619, tamsulosin and silodosin are obtained from Tocris (Bristol, UK), and endothelin-1 is obtained from Enzo Life Sciences (Hitachi, Inc.) (R) ((R))Germany).

Statistical analysis

Data are presented as mean ± standard error of mean (s.e.m.) of the indicated number of experiments (n). Multivariate analysis of variance (ANOVA) and two-way ANOVA were used for the unpaired observations andversion 20(IBM SPSS Statistics, IBM Corporation, Armonk, New York, USA). P value<0.05 was considered statistically significant. All groups included in the statistical analysis were based on five or more independent experiments and, in the case of experiments with human tissue, included tissues from five or more patients in each group. Thus, the smallest group size for statistical tests is n-5. Furthermore, all of them were compared with each other by statistical testsGroups show the same group size; thus, no statistical comparison between groups of different sample sizes or between groups containing tissue from different patients was performed. Spearman correlation analysis and calculation of pEC50 values by curve fitting were performed using GraphPadPrism6(Statcon, Witzenhausen, Germany).

Results

Effect of onvansertib on adrenergic contraction

Induction of human prostate tissue contraction in organ baths to evaluate onvansertib and α₁-the effect of the blocking agent on prostate smooth muscle contraction. The effect of onvansertib on norepinephrine-induced contraction of human prostate tissue was tested using two different concentrations of onvansertib. Both concentrations, 100nM and 1. mu.M, caused significant inhibition of noradrenaline-induced contraction (FIG. 1A). The degree of inhibition was similar for both concentrations, so increasing the onvansertib concentration from 100nM to 1. mu.M did not increase the degree of inhibition (100nM onvansertib produced 45. + -. 9.7% inhibition of 100. mu.M noradrenaline-induced contraction, 1. mu.M onvansertib produced 41. + -. 6.2% inhibition of 100. mu.M noradrenaline-induced contraction). In contrast to contractile force, onvansertib did not alter the pEC50 value for norepinephrine (5.9. + -. 0.2M after 100nM onvansertib and 5.5. + -. 0.2M in the corresponding control; 5.7. + -. 0.3M after 1. mu.M onvansertib and 6. + -. 0.3M in the corresponding control).

Using two kinds of alpha₁Adrenergic receptor agonists phenylephrine and methoxamine, confirming that onvansertib is alpha₁-the effect of adrenergic prostate smooth muscle contraction. onvansertib (100nM) caused significant inhibition of phenylephrine and methoxamine induced contraction (figure 1B). The degree of inhibition by both agonists was similar and similar to the inhibition by onvansertib of noradrenaline-induced contraction (100nM onvansertib produced 47 + -16.2% inhibition of 30. mu.M phenylephrine-induced contraction and 100nM onvansertib produced 43 + -19.4% inhibition of 100. mu.M methoxamine-induced contraction). In contrast to contractile force, onvansertib does not alter phenylephrine or formazanOxygen modified pEC₅₀Values (phenylephrine 7.8 + -1.3M after 100nM onvansertib and 6.7 + -0.8M in the corresponding control; methoxamine 5.2 + -0.2M after 100nM onvansertib and 5 + -0.1M in the corresponding control).

To assess the reversibility of the effect of onvansertib on adrenergic contraction, noradrenaline-induced contraction was assessed after washing away DMSO and onvansertib (100nM) in a series of separate experiments. Under these conditions, the norepinephrine-induced contraction was not different, i.e. similar after washing away DMSO and onvanisertib (fig. 1C).

Effect of onvansertib on non-adrenergic contractions

The effect of onvansertib on the contraction of prostate smooth muscle induced by endothelin-1, ATP or the thromboxane a2 analogue U46619 was tested. onvansertib (100nM) caused significant inhibition of endothelin-1 induced contraction (100nM onvansertib produced 45 ± 6.6% inhibition of 3 μ M endothelin-1 induced contraction) and ATP-induced contraction (100nM onvansertib produced 51 ± 18.6% inhibition of 10mM ATP-induced contraction) (figure 2). onvansertib (100nM) did not alter the contraction induced by U46619 (figure 2).

Effect of onvansertib on EFS-induced shrinkage

The effect of onvansertib on neurogenic contraction of human prostate tissue induced by EFS was tested using two different concentrations of onvansertib. Both concentrations, 100nM and 1 μ M, caused significant inhibition of EFS-induced contraction (fig. 3A). The degree of inhibition was similar for both concentrations, so increasing the onvansertib concentration from 100nM to 1. mu.M did not increase the degree of inhibition (100nM onvansertib produced 34 + -11.3% inhibition of 32 Hz-induced contraction, 1. mu.M onvansertib produced 34 + -14% inhibition of 32 Hz-induced contraction).

To evaluate the reversibility of the effect of onvansertib on EFS-induced shrinkage, EFS-induced shrinkage was evaluated after washing away DMSO and onvansertib (100nM) in a series of separate experiments. Under these conditions, EFS-induced contraction was not different, i.e. similar after washing away DMSO and onvansertib (fig. 3B).

₁Effect of alpha-blockers on EFS-and norepinephrine-induced contraction

α₁The blockers tamsulosin and silodosin significantly inhibited EFS-induced contraction of human prostate tissue and to a similar extent (fig. 4A). The degree of inhibition was similar for both compounds (300nM tamsulosin produced 48 + -18.3% inhibition of 32Hz induced contraction and 100nM silodosin produced 47 + -16.4% inhibition of 32Hz induced contraction). In contrast to the incomplete inhibition of EFS-induced contraction, tamsulosin and cilostacin at the same concentrations almost completely inhibited norepinephrine-induced contraction (300nM tamsulosin produced 93 + -3.5% inhibition of 100. mu.M norepinephrine-induced contraction and 100nM cilostacin produced 84 + -4.99% inhibition of 100. mu.M norepinephrine-induced contraction) (FIG. 4B).

₁Effect of onvansertib in combination with alpha-blockers on EFS-induced shrinkage

In the addition of an alpha 1-blocker or alpha alone₁EFS-induced contraction between human prostate tissue was compared after combination of blocking agent and 100nM onvansertib (fig. 5 a). With addition of alpha alone₁Addition of alpha compared to EFS induced shrinkage after blocker₁The EFS-induced contraction after combination of the blocking agent with onvansertib was significantly lower (fig. 5 a). The effect of the addition of onvanestib was similar when tamsulosin and cilostacin were used (onvanestib produced 59 + -11.5% inhibition of 32Hz induced contraction compared to tamsulosin alone and onvanestib produced 61 + -9.8% inhibition of 32Hz induced contraction compared to cilostacin alone).

In two separate series of experiments, onvansertib and α were added₁The effect of the combination of blockers on EFS-induced contraction was compared to the effect of onvansertib alone (100nM) (fig. 5 b). Addition of onvansertib with α compared to EFS-induced shrinkage after addition of onvansertib alone₁The EFS-induced shrinkage after the combination of blockers was significantly lower (fig. 5 b). Use ofWhen tamsulosin and silodosin are used, alpha is added₁The effect of the blocking agent was similar (tamsulosin produced 68 ± 12.2% inhibition of 32Hz induced contraction compared to onvansertib alone, and silodosin produced 68 ± 12.7% inhibition of 32Hz induced contraction compared to onvansertib alone).

Detection of PLK1 in WPMY-1 cells

To compare the likely expression level of PLK1 in human prostate stromal cell line WPMY-1 and human prostate tissue, western blot analysis was performed using antibodies raised against PLK 1. Detection revealed a band of a size with the expected molecular weight of PLK1 (fig. 6). Consistent with previous findings (Hennenberg et al, 2018), the intensity of these bands varied widely between samples of prostate tissue from different patients (fig. 6A). In contrast, the intensities of four different samples of WPMY-1 cells were constant and the range at the highest expression level was observed in human prostate tissue (fig. 6B). A similar pattern was observed after detection of PLK1 mRNA by RT-PCR (FIG. 6C). Thus, PLK1 mRNA levels showed high changes in human prostate tissue (fig. 6C). In WPMY-1 cells, PLK1 mRNA levels were constant and ranged at levels similar to the highest levels observed in prostate tissue (fig. 6C). In human prostate tissue, no correlation between mRNA levels of PLK1 and PSA was observed (R ═ 0.543, P ═ 0.297) (fig. 6D).

Effect of onvansertib on the viability and proliferation of WPMY-1 cells

The effect of onvansertib on the viability of WPMY-1 cells was assessed by the CCK-8 assay. onvansertib induced a concentration (10nM, 50nM, 100nM) and time (24h-72h) dependent decrease in viability (figure 7A). The reduction range is between: after 24h, 15. + -. 8.8% at 10nM to 63. + -. 2.2% at 100nM, after 48h, 28. + -. 8.3% at 10nM to 86. + -. 1.9% at 100nM, and after 72h, 40. + -. 4.4% at 10nM to 89. + -. 1.4% at 100nM (FIG. 7A).

The effect of onvansertib on the proliferation of WPMY-1 cells was evaluated by two different readouts (readouts), where the results were confirmed to each other (fig. 7B and 7C). In the plate colony assay, onvansertib reduced colony formation in a concentration-dependent manner, with the following reductions in total: a 39 ± 4.5% reduction in 10nM, a 89 ± 1.9% reduction in 50nM, and a 97 ± 0.4% reduction in 100nM (fig. 7B). In the EdU assay, onvansertib reduced the percentage of proliferating cells in a concentration-dependent manner, with the following reductions in total after 24 h: 10nM was 18 + -0.1%, 50nM was 24 + -2.1%, and 100nM was 35 + -2.0% (FIG. 7C).

Discussion of the related Art

Our findings indicate that the PLK1 inhibitor onvansertib inhibits 1) neurogenic, α 1-adrenergic, and endothelin-induced contraction in the human prostate, and 2) proliferation of prostate stromal cells. Notably, the combination of onvansertib with α 1-blocker caused greater inhibition of neurogenic contraction than α 1-blocker alone. From a clinical point of view, the additive effects of onvansertib and α 1-blocker may be particularly interesting together with a simultaneous effect on growth. Both prostate smooth muscle contraction and prostate growth may contribute to BOO and LUTS in BPH (Hennenberg et al, 2014). Thus, both are targets for medical therapy, yet they still suffer from insufficient effectiveness, widespread use of combination therapy, and low compliance, ultimately leading to complications, hospitalization, and surgery (Hennenberg et al, 2014; Oelke et al, 2013). The identification of new targets is a prerequisite to overcome these limitations in the future. Based on our findings, onvansertib may be an attractive compound to be tested in vivo in the context of LUTS suggestive of BPH.

We tested the effect of onvansertib on EFS and norepinephrine-induced contraction using two different concentrations. Both concentrations of 100nM and 1. mu.M onvansertib inhibited contraction to a similar extent. Thus, together with the reported IC50 values, we concluded that inhibition of contraction is largely dependent on PLK1 inhibition, and that no other contraction-mediated kinases participate in this range up to 1 μ M. Indeed, it is reported that onvansertib is highly specific for PLK1, showing an IC50 value of 2nM for PLK1, a 10. mu.M IC50 value for PLK2, and a 10. mu.M IC50 value for PLK3 in biochemical assays (Beria et al),2011). In another screen by biochemical assay onvansertib was tested against 296 kinases and showed a decrease in activity of 11 kinases (including PLK1) when tested at 1 μ M: (<50%) and only inhibits PLK1 when tested at 100nM (valsnaina et al, 2012). Another series of biochemical assays in the same study with 63 kinases revealed that the IC of FLT3₅₀IC with value of 510nM, MELK₅₀IC with a value of 744nM and CK2₅₀The value was 826nM (Valnasina et al, 2012). Therefore, the concentration of 100nM used in most of our experiments is likely to be highly specific for PLK1, and most of the effect is likely due to PLK1 inhibition. Since a concentration of 1 μ M did not provide greater inhibition than that caused by 100nM, we hypothesized that 1)1 μ M did not cause off-target inhibition of the contraction-mediated kinase, and 2)100nM was sufficient to achieve the greatest, PLK 1-dependent effect. Consistent with previous studies (belia et al, 2011; valsnaina et al, 2012) that reported onvansertib to bind to the ATP pocket of PLK1 via hydrogen bonds and thus act as an ATP competitive inhibitor with reversible dissociation, in our studies the role of onvansertib was reversible. Therefore, after washing away onvansertib, the contraction returns to the level of the corresponding control.

Although the medical history of the included patients was not taken into account, inhibition of smooth muscle contraction was observed in our organ bath experiments. Thus, the tissue samples were anonymized and no patient data was collected, stored or analyzed for this study. To eliminate individual differences or heterogeneity caused by previous treatments, we referenced contraction with high molar KCl induced contraction and confirmed our findings by different settings, i.e., using different α 1-adrenoceptor agonists or different combination settings. However, the lack of reference to patient characteristics, particularly to previous drugs that may affect contractility, needs to be considered as a potential limitation. Neurogenic and alpha₁Inhibition of adrenergic prostate smooth muscle contraction is consistent with recent findings obtained with four other inhibitors with putative selectivity to PLK1 (Hennenberg et al, 2018). Similar to our current results, other PLK1 inhibitors were induced to EFSThe range of inhibition of contraction induced was about 50% (Hennenberg et al, 2018). Here, we tested two α' s₁Effect of blockers on EFS-induced contraction and reaction of onvansertib with α₁Combinations of blockers with alpha alone₁The blocking agent and onvansertib were compared. Two kinds of alpha₁The blockers tamsulosin and silodosin provided very similar results and inhibited EFS-induced shrinkage by less than 50%. Alpha is alpha₁This incomplete inhibition of the blocking agent is consistent with previous results, which report a₁Similar or even smaller effects of blockers in 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 excluded that the incomplete inhibition of EFS-induced contraction by tamsulosin and silodosin is due to alpha₁Too low a concentration of blocking agent. Indeed, the same concentrations used in the EFS experiments completely reduced the noradrenaline induced prostate contraction (at least over the range of noradrenaline concentrations used herein). We observed that alpha is different from alpha alone₁Comparison of the tension after the blocking agent or onvansertib, application of onvansertib and a₁The combination of blockers followed a significantly lower EFS-induced contraction, which can be considered as a key finding of our study. Presumably, in patients with BPH, α alone₁At a blocker to blocker ratio₁Addition of onvansertib or other PLK1 inhibitors on the basis of blocking agents may lead to higher LUTS improvement. Consistent with previous results obtained at the protein level, we confirmed here that PLK1 mRNA expression was not correlated with PSA expression, suggesting that it functions independently of the extent of BPH (Hennenberg et al, 2018).

In contrast to recent findings in which the PLK1 inhibitors SBE 13 and cyclapolin did not alter endothelin-1 induced contraction of human prostate tissue, we observed here inhibition of endothelin-1 induced contraction by onvansertib (Hennenberg et al, 2018). The reason for this difference is still presumed at the present stage, but may reflect a different pharmacological profile. However, it is worth noting thatEven the inhibition of adrenergic contraction (induced by norepinephrine, phenylephrine and methoxamine), the inhibition by onvansertib was shown to be greater than the reported inhibition of SBE 13 and cyclopolin 9 (Hennenberg et al, 2018). In addition to endothelin-1 induced contraction, onvansertib also inhibits ATP-induced contraction. Notably, our data may be the first evidence to suggest purinergic smooth muscle contraction in the human prostate, since, to our best of our knowledge, previous studies addressing ATP-induced contraction in the prostate were limited to animal models (Brandli et al, 2010; Buljubasich and Ventura, 2004; Hennenberg et al, 2017; White et al, 2015; Xu and Ventura, 2010). In line with other PLK inhibitors, the contraction induced by U46619 is here again resistant to onvansertib. Non-adrenergic mediators, including endothelin-1, thromboxane a2, and possibly ATP, may contribute to prostate smooth muscle tone in BPH in parallel with α 1-adrenergic receptors, so despite treatment with α 1-blockers they can maintain urethral obstruction, which may explain the limitations of α 1-blockers or the large number of non-responders (Hennenberg et al, 2014, 2017). Considering the effects on endothelin-1 and ATP-induced contraction, onvansertib may improve in vivo LUTS suggesting BPH more than α₁-a blocking agent.

As another key finding, we observed onvansertib to inhibit proliferation of prostate stromal cells. Previous studies reporting inhibition of proliferation by onvansertib were performed in different types of tumor cells (Casolaro et al, 2013; Hartsink-Segers et al, 2013; Sero et al, 2014; valsnaina et al, 2012). In the prostate, knockdown of other PLK inhibitors or PLK1 inhibited the proliferation of tumor cells and tumor growth, and enhanced the effectiveness of anti-tumor therapy (Lin et al, 2019; Reagan-Shaw and Ahmad, 2005; Shao et al, 2015; Zhang et al, 2014). Generally, PLK inhibitors, including onvansertib, reduce proliferation by G2-M cell cycle blockade (valsnaina et al, 2012). Prolonged mitotic block by PLK inhibition may be followed by apoptosis and cell death (valsnaina et al, 2012). These two processes probably explain the effect of onvansertib on the proliferation and viability of WPMY-1 cells in our study. Reduced viability of WPMY-1 cells occurred as early as 24h, but this was clearly time-dependent. With respect to smooth muscle cells, a proliferation-promoting effect of PLK1 has been proposed against airway smooth muscle cells, where PLK1 knockdown inhibits growth factor-induced proliferation (Jiang and Tang, 2015). In summary, our observations that proliferation and viability of WPMY-1 cells were affected by PLK1 inhibitors were consistent with previous studies in other cell types. WPMY-1 cells are derived from the non-malignant human prostate stroma and are very similar to prostate smooth muscle cells and can be considered as such prostate smooth muscle cells (Wang et al, 2015). Smooth muscle cells are the predominant cell type in the prostate stroma and may show increased proliferation in BPH (Strand et al, 2017). The latter contributes to prostatic hypertrophy in BPH (Strand et al, 2017). Comparison of expression levels by western blot and RT-PCR showed that WPMY-1 cells expressed PLK1 at a constant level, similar to the highest level in human prostate tissue, with varying levels of PLK1 in human prostate tissue. It is expected that onvansertib can prevent prostate growth in BPH or reduce prostate size (which is the primary target for treatment with 5-ARI) in order to prevent progression of BPH and reduce the risk of complications and surgery (Oelke et al, 2013).

Prostate growth and smooth muscle contraction are targets for drug therapy in BPH, but have long been mostly considered separately from each other. Based on recent evidence that prostate smooth muscle contraction and stromal cell growth are susceptible to the same inhibitors, it is now increasingly clear that these two processes are closely related to each other (Hennenberg et al, 2014). Inhibitors of Rho kinase, Rac GTP enzyme or Src family kinases simultaneously inhibit agonist-induced contraction of human prostate tissue and growth of prostate stromal cells (Rees et al, 2003; Wang et al, 2015, 2016 b). This may be similar to that in other smooth muscle cells, for example vascular smooth muscle cells, where RhoA/Rho kinase mediates contraction and proliferation (Shimokawa et al, 2016). Notably, a similar effect of PLK1 in airway smooth muscle cells was suggested, where it was increasedBoth colonization and contraction are dependent on PLK1(Jiang and Tang, 2015; Li et al, 2016). This obviously also applies to prostate smooth muscle. Thus, with α₁Blocking agent or 5-ARI in contrast, onvansertib may immediately reduce prostate smooth muscle tone and growth. onvansertib addresses (addresses) the same or different intracellular signaling pathways to inhibit smooth muscle contraction and proliferation, and which other effectors participate in onvansertib-sensitive signaling with PLK1, cannot be estimated based on our data. To our knowledge, the effect of PLK inhibitors on vascular smooth muscle contraction (which may cause cardiovascular side effects and may limit in vivo applications) has not been reported to date.

In summary, it can be speculated that onvansertib may be very effective in vivo if it is applied to treat LUTS suggestive of BPH for three reasons. First, it can be assumed that the improvement in LUTS by adding onvansertib on the basis of α 1-blocker is likely to be higher than by α 1-blocker alone, based on our findings from a combination experiment of EFS-induced contraction. Second, a high degree of symptom relief and Q can be expected_{Maximum of}Because onvansertib inhibits endothelin-1 induced contraction, which is on alpha₁Blockade insensitivity, but contributes to prostate smooth muscle tone in BPH (Hennenberg et al, 2014, 2017). Third, it might be expected that the reduction in prostate size parallels the reduction in symptoms, as onvansertib strongly reduces the viability of stromal cells. After long-term use in vivo, the antiproliferative effect may even decrease contractility more strongly than short-time exposure in organ baths, since the number of smooth muscle cells in vivo may decrease. In organ baths, exposure 1h is usually not sufficient to reduce contractility by affecting vitality (Yu et al, 2018, 2019a, 2019b), so anti-contractile effects most likely occur due to PLK1 mediated signaling mechanisms that promote contraction. Alpha is alpha₁Combination therapy of-blockers and 5-ARI is commonly applied for the treatment of male LUTS (oenke et al, 2013). However, side effects are additive and the drug withdrawal rate is high (Fullhase et al, 2013). Single compounds that can simultaneously address both adrenergic and non-adrenergic prostate smooth muscle tone and growthSubstances, such as onvansertib, may be attractive for clinical trials because they may replace α₁Combination therapy with a blocking agent and 5-ARI, and possibly with more than alpha₁-effectiveness of monotherapy with blockers. onvansertib is orally available (bearlyavailable) (belia et al, 2011; valsnaina et al, 2012). Pharmacokinetics and safety have recently been examined in phase I studies in which plasma concentrations in humans reach the three-digit nanomolar range, even with the lowest dose tested (Weiss et al, 2018). Thus, it appears likely that concentrations similar to those in our organ bath experiments (100nM) can be obtained in the prostate by oral administration, making clinical studies in the context of LUTS suggestive of BPH likely promising.

Conclusion

onvansertib inhibits neurogenic and alpha of human prostate smooth muscle₁-adrenergic contraction and proliferation of prostate stromal cells. Alpha is alpha₁-the blocking agent reduces neurogenic contraction by no more than 50%, but α₁The combination of a blocking agent with onvansertib provides an additive and stronger inhibition of neurogenic prostate constriction. It seems likely that onvansertib can simultaneously reduce prostate smooth muscle tone and growth in BPH, so onvansertib can be an attractive compound to be tested in vivo against the LUTS background suggestive of BPH.

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In view of the above, it will be seen that the several objects of the invention are achieved and other advantages attained.

As various changes could be made in the above methods and compositions without departing from the scope of the invention, it is intended that all matter contained in the above description and shown in the accompanying drawings shall be interpreted as illustrative and not in a limiting sense.

All references cited in this specification are hereby incorporated by reference. The discussion of the references herein is intended merely to summarize the assertions made by the authors and no admission is made that any reference constitutes prior art. Applicants reserve the right to challenge the accuracy and pertinency of the cited references.

As used herein, the singular forms "a", "an" and "the" are intended to include the plural forms as well, unless the context clearly indicates otherwise. Moreover, use of "or" is intended to include "and/or" unless the context clearly indicates otherwise.

As used herein, in particular embodiments, the terms "about" or "approximately" when preceding a value indicate a range of plus or minus 10% of the value. Where a range of values is provided, it is understood that each intervening value, to the tenth of the unit of the lower limit unless the context clearly dictates otherwise, between the upper and lower limit of that range and any other stated or intervening value in that stated range is encompassed within the disclosure. The upper and lower limits of these smaller ranges may independently be included in the smaller ranges, and are also included in the disclosure, subject to any specifically excluded limit in the stated range. Where a stated range includes one or both of an upper limit and a lower limit, ranges excluding either or both of those included limits are also included in the disclosure.

The term "and/or" as used in the specification and embodiments is understood to mean "one or two" of the elements so combined, i.e., elements that are present in combination in some cases and elements that are present in isolation in other cases. Multiple elements listed with "and/or" should be interpreted in the same manner, i.e., "one or more" of the elements so combined. In addition to the elements specifically identified by the "and/or" clause, other elements may optionally be present, whether related or unrelated to those specifically identified elements. Thus, as a non-limiting example, when used in conjunction with an open language such as "comprising," reference to "a and/or B" may refer in one embodiment to a alone (optionally including elements other than B); in another embodiment, to B only (optionally including elements other than a); in yet another embodiment refers to both a and B (optionally including other elements); and so on.

As used in the specification and embodiments, "or" should be understood to have the same meaning as "and/or" as defined above. For example, when items in a list are separated, "or" and/or "should be interpreted as being inclusive, i.e., including at least one of the plurality of elements or list of elements, but also including more than one, and optionally including additional unlisted items. It is expressly intended that only terms such as "only one of. In general, when the foregoing is a term of exclusivity such as "any," one of. When used in an embodiment, "consisting essentially of shall have its ordinary meaning as used in the art of patent law.

As used in the specification and embodiments, referring to a list of one or more elements, the term "at least one" should be understood to mean at least one element selected from any one or more of the elements in the list of elements, but not necessarily including at least one of each element specifically listed in the list of elements, and not excluding any combination of elements in the list of elements. The definition also allows that elements other than the elements specifically identified in the list of elements to which the term "at least one" refers may optionally be present, whether related or unrelated to those specifically identified elements. 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, optionally including more than one, a, with no B present (and optionally including elements other than B); in another embodiment refers to at least one, optionally including more than one, B, with no a present (and optionally including elements other than a); in yet another embodiment means at least one, optionally including more than one, a, and at least one, optionally including more than one, B (and optionally including other elements); and so on.