Patent Application
20090306093
The present invention relates to a theophylline-based compound capable of enhancing the production of cGMP, and more particularly to a compound of 7-[2-[4-(2-Chlorobenzene)piperazinyl]ethyl]-1,3-dimethyl xanthine capable of treating a patient having benign prostate hyperplasia (BPH).
BPH is a hormone- and age-dependent disease, resulting from a change in size, composition and function of the prostate gland, and leads to obstruction of the bladder and urethra and LUTS. The LUTS involves in an increase of prostatic cellular mass and smooth muscle tone. α1-adrenoceptor, the predominant receptor in the human prostate, bladder neck and urethra, crucially mediates the prostate smooth muscle contraction[1]. Since the best way to treating BPH is to decrease prostate size and smooth muscle tone, 5α-reductase inhibitors and selective α-androgenic blockers are thus clinically used[2]. However, the proportion of α1-adrenoceptor subtypes may be different, e.g., the ratio of α1A, α1B and α1D subtype mRNAs being 85:1:14 and 63:6:31 in BPH and non-BPH, respectively[3]. Therefore, α1A/α1D-adrenoceptor antagonist, tamsulosin, is preferred clinically to be useful in reducing cardiovascular side effects.
Cyclic adenosine monophosphate (cAMP) and cyclic guanosine monophosphate (cGMP) are synthesized by activation of adenylyl- and guanylyl-cyclases respectively and degraded by cyclic nucleotide phosphodiesterases (PDE)[4]. In general, nitric-oxide (NO) activates soluble guanylyl cyclase (sGC), which leads to an increase in intracellular cGMP, and subsequently activates cytosolic cGMP-dependent protein kinase (PKG). Studies on smooth muscle have shown that activation of PKG may lead to the opening of large conductance Ca2+ activated K+ (BKCa) channels[5] or ATP-sensitive potassium (KATP) channels[6].
Increasing evidences show that RhoA/Rho kinase (ROCK2) is involved in a variety of important physiological functions including smooth muscle contraction, cell proliferation, cell adhesion, cell migration, and various inflammatory responses[7]. Particularly, activities of ROCK2 play important roles in modulating smooth muscle contraction in various tissues, including cavernosum, prostate and bladder[8-10]. ROCK2 inhibitor, Y-27632, has been shown to relax rabbit and human cavernous smooth muscle and human prostate smooth muscle[10,11].
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From the above description, it is known whether KMUP-1 involves in treating BPH has become a major problem waited to be solved. In order to overcome the drawbacks in the prior art, another pharmaceutical activity of KMUP-1 is provided. The particular design in the present invention not only solves the problems described above, but also is easy to be implemented. Thus, the invention has the utility for the industry.
In the present invention, it is first shown that the relationship between expression of sGC/cGMP/PKG and inhibition of ROCK2 in rat prostate to certify the mentioned possibility. We investigated possible actions of KMUP-1 on prostate contraction by comparing its actions with those of PDE5A inhibitor (sildenafil), α-adrenergic blockers (doxazosin and tamsulosin) and PKG activator on rat prostate and on proliferation of human epithelial cell PZ-HPV-7 during cell growth to testify that KMUP-1-induced relaxation of the rat prostate is mediated by the activation of sGC/cGMP/PKG pathway, combined with α1A/α1D-adrenergic blocking activity, BKCa and KATP channels opening, and inhibition of ROCK2 and PDE.
In accordance with one aspect of the present invention, a method for treating a patient having a BPH is provided. The method comprises a step of administering to the patient a therapeutically effective amount of one selected from a group consisting of a treating compound of 7-[2-[4-(2-Chlorobenzene)piperazinyl]ethyl]-1,3-dimethyl xanthine, a salt of the treating compound, a solvate of the treating compound and a combination thereof, wherein the compound has an effect on treating the benign prostate hyperplasia via a mechanism selected from a group consisting of an, activation of PKG pathway, a blockage of an adrenoceptor, an opening of a potassium channel and an inhibition of PDE activity.
Preferably, the PKG pathway is sGC/cGMP/PKG pathway.
Preferably, the adrenoceptor is an α1A/α1D-adrenergic receptor with a relatively high selectivity.
Preferably, the potassium channel is one selected from a group consisting of a BKCa channel, a KATP channel and a combination thereof.
Preferably, the inhibition of PDE is combined with an inhibition of ROCK2.
In accordance with another aspect of the present invention, a method for treating a patient having a lower urinary tract symptom is provided. The method comprises a step of administering to the patient a therapeutically effective amount of one selected from a group consisting of a treating compound of 7-[2-[4-(2-iodobenzene)piperazinyl]ethyl]-1,3-dimethyl xanthine, a salt of the treating compound, a solvate of the treating compound and a combination thereof, wherein the compound has an effect on treating the benign prostate hyperplasia via a mechanism selected from a group consisting of an activation of PKG pathway, a blockage of an adrenoceptor, an opening of a potassium channel and an inhibition of PDE activity.
Preferably, the PKG pathway is sGC/cGMP/PKG pathway.
Preferably, the adrenoceptor is an α1A/α1D-adrenergic receptor with a relatively high selectivity.
Preferably, the potassium channel is one selected from a group consisting of a BKCa channel, a KATP channel and a combination thereof.
Preferably, the inhibition of PDE is combined with an inhibition of ROCK2.
In accordance with a further aspect of the present invention, a method for treating a patient having an erectile dysfunction is provided. The method comprises a step of administering to the patient a therapeutically effective amount of one selected from a group consisting of a treating compound of 7-[2-[4-(2-fluorobenzene)piperazinyl]ethyl]-1,3-dimethyl xanthine, a salt of the treating compound, a solvate of the treating compound and a combination thereof, wherein the compound has an effect on treating the benign prostate hyperplasia via a mechanism selected from a group consisting of an activation of PKG pathway, a blockage of an adrenoceptor, an opening of a potassium channel and an inhibition of PDE activity.
Preferably, the PKG pathway is sGC/cGMP/PKG pathway.
Preferably, the adrenoceptor is an a1A/a1D-adrenergic receptor with a relatively high selectivity.
Preferably, the potassium channel is one selected from a group consisting of a BKCa channel, a KATP channel and a combination thereof.
Preferably, the inhibition of PDE is combined with an inhibition of ROCK2.
The above aspects and advantages of the present invention will become more readily apparent to those ordinarily skilled in the art after reviewing the following detailed descriptions and accompanying drawings, in which:
The present invention will now be described more specifically with reference to the following embodiments. It is to be noted that the following descriptions of preferred embodiments of this invention are presented herein for the purposes of illustration and description only; it is not intended to be exhaustive or to be limited to the precise form disclosed.
The present invention provides a chemical compound, KMUP-1, having a pharmaceutical activity on treating BPH, easing the lower urinary tract symptoms and treating the erectile dysfunction. The detailed description for the pharmaceutical experimental results of KMUP-1 is provided as below.
Pharmacological Experiments
The synthetic method of KMUP-1 of the present invention has been disclosed in U.S. Pat. No. 6,979,687, and is not described hereafter.
Drugs: 9-(tetrahydro-2-furanyl)-9H-purin-6-amine (SQ22536), Y-27632, doxazosin, zaprinast, 8-pCPT-cGMP, RP-8-pCPT-cGMP, charybdotoxin, apamin, 4-aminopyridine (4-AP), glibenclamide, phenylephrine, and 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one (ODQ) were obtained from Sigma Chemical Co. Sildenafil citrate was supplied by Cadila Healthcare Ltd (Maninagar, India). Tamsulosin was supplied by Standard Chem. and Pharm. CO., LTD (Tainan, Taiwan). KMUP-1, artificially synthesized, was dissolved in 10% absolute alcohol, 10% propylene glycol, 2% 1N HCl and 78% distilled water. Tamsulosin and Sildenafil citrate were dissolved in distilled water.
Membrane Preparation and a1A-/a1B-/a1D-Adrenoceptor Binding
Wistar rats were obtained from National Laboratory Animal Breeding and Research Center (Taipei, Taiwan) and housed under a constant temperature and controlled illumination. The submaxillary glands, spleen, and aorta of rats were separately homogenized for 30 seconds with a Kinematica polytron in 20 volumes of ice-cold TE buffer (10 mM Tris HCl, 1 mM EDTA, 0.1 mM ascorbic acid, pH 7.4). The homogenate was filtered under pressure through muslin and the filtrate was centrifuged at 1,000 g for 10 minutes. The supernatant was centrifuged again at 10,000 g for 12 minutes at 4° C. The second supernatant was centrifuged at 30,000 g for 15 minutes at 4° C., and the final pellet was resuspended in the assay buffer (75 mM Tris HCl, 25 mM MgCl2, pH 7.4). Protein content was determined by Bradford's method.
[3H]-prazosin, KMUP-1 and membranes (200-300 μg) were incubated for 60 minutes at 25° C. with or without the addition of nonspecific binding agents, in a 75 mM Tris HCl buffer with 25 mM MgCl2, making a final volume of 500 μL. In this experiment, non-specific binding was defined by use of 10 μM phentolamine. In competitive-binding experiments, the competing agent was added directly to the incubation mixture. The incubation was terminated by adding 1 mL of ice cold assay buffer followed by immediate filtration through Whatman GF/C glass fiber filters supported on a 12-port filter manifold (Whatman Int. Ltd, Springfield Mill, Maidstone, UK). The filters were immediately washed 3 times with 5 mL of ice-cold assay buffer and dried in an oven at 60° C. for 2 hours before adding 5 mL of Triton-toluene based scintillation fluid. Membrane-bound radioligand trapped in the filters was counted in a Beckman LS6500 scintillation system (Fullerton, Calif.) with an efficiency of 45%. In each experiment, nonspecifically bound radioligand agents were measured by incubating membrane protein. Specific binding was then obtained by deducting this value from the total binding of radioligand agents for each sample.
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Receptor affinity and binding selectivity on a1-adrenergic receptor by KMUP-1 and previous doxazosin and tamsulosin[12] are shown in Table I.
a1-Adrenoceptor Antagonism in Isolated Rat Prostate
Smooth muscle contractility of isolated Wistar rat prostate was used to evaluate the possible involvement of a1-adrenoceptor antagonism by KMUP-1. KMUP-1 (0.01-1 μM) or vehicle was added to organ bath, containing the Tyrode solution, for 30 minutes after a concentration-response curve to phenylephrine (0.001-100 μM) was obtained.
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Isolated Prostate and Tension Measurement
The rat isolated prostate tissue was prepared as previously described with modifications[10]. Briefly, Wistar rats (300-400 g) were killed with pentobarbital sodium (30 mg/kg) and the ventral part of their prostates were quickly excised. Three or four strips (10×5 mm) were mounted under 0.2-0.5 gm tension and allowed to equilibrate for 90 minutes in Tyrode solution composed of 118 mM of NaCl; 25 NaHCO3, 4.7 KCl, 1.2 KH2PO4, 1.2 MgSO4, 11 glucose, and 2.5 CaCl2 (pH 7.4) maintained at 37° C. and aerated with 95%. O2 and 5% CO2.
Isometric tension was recorded with a force displacement transducer (UGO BASILE, Model 7004, Italy). After the equilibration period, the strips were primed twice by adding 10 μM of the agonist, phenylephrine, to reach 80-100% of maximum contraction. When stable constriction to phenylephrine was reached, KMUP-1 (10 μM), sildenaifil (10 μM), zaprinast (10 μM), doxazosin (10 μM), tamsulosin (10 μM) and 8-pCPT-cGMP (10 μM) were added to isolated prostate strips for 1 hour to examine whether PDE inhibition and a-adrenergic blockade could provide relaxation activity on rat prostate. The relaxation on rat prostate by KMUP-1 (0.01-100 μM) and tamsulosin (0.001 nM-100 μM) were also tested by their combination with sildenafil (0.1 μM), respectively. In isolated prostate strips, while stable constriction to phenylephrine was reached, sildenafil (0.1 μM) was pre-incubation for 30 minutes, before the cumulative addition of KMUP-1 (0.01-100 μM) or tamsulosin (0.001 nM-100 μM) to isolated prostate strips. In another experiment, to examine the possible mechanism of action of KMUP-1, rat prostates were pretreated with ODQ (1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one, 10 μM, sGC inhibitor), RP-8pCPT-cGMPs (10 μM, PKG inhibitor), glibenclamide (10 μM, KATP channel blocker), 4-AP (4-aminopyridine, 100 μM, voltage-dependent K+ channel blocker), apamin (1 μM, Ca2+-dependent K+ channel blocker) and charybdotoxin (ChTX) (0.1 μM, Ca2+-dependent K+ channel blocker) for 30 minutes before addition of KMUP-1 (100 μM).
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Determination of cGMP/cAMP
Isolated prostate strips from Wistar rats were prepared to determine prostate levels of cGMP and cAMP. Briefly, prostate strips were respectively incubated with KMUP-1 (10-100 μM), sildenafil (100 μM), and zaprinast (100 μM) in 10 mL Tyrode solution aerated with 95% O2 and 5% CO2 for 1 hour. To examine the possible mechanisms of action of KMUP-1, the rat prostate was pretreated with ODQ (10 μM, sGC inhibitor) and SQ22536 (9-(tetrahydro-2-furanyl)-9H-purin-6-amine, 10 μM, adenylyl cyclase inhibitor) for 30 minutes before KMUP-1 was added. After the incubation, the prostate tissues were extracted with 1 mL of 0.1 N HCl. The extracts were then lyophilized for a further measurement of cyclic GMP or cyclic AMP of each sample, using commercially available radioimmunoassay kits (Amersham Pharmacia Biotech, Buckinghamshire, England).
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Moreover, the relaxation of prostate, pre-contracted with phenylephrine (10 μM) by KMUP-1 (100 μM), was attenuated by K+ channel blockers, such as glibenclamide (115.00±7.07%), 4-AP (122.00±3.74%) and apamin (126.00±9.09%) and charybdotoxin (138.00±7.01%), respectively.
Functional studies have shown the a1A-adrenoceptor subtype able to mediate prostate smooth muscle contraction in humans and animals. Both functional and radioligand binding studies have found terazosin and doxazosin to have potent cardiovascular effects and to have greater affinities for a1B-adrenoceptor than a1A-adrenoceptor[12]. Some studies have demonstrated that highly selective a1A-adrenoceptor antagonists lack cardiovascular side effects on LUTS[12]. a1D-adrenoceptor has also been found to be significantly up-regulated in rat obstructed bladder[13]. In addition, some studies have demonstrated that a1D-adrenoceptor selective antagonists ameliorate irritative symptoms[14]. Although the exact role of a1D-adrenoceptor in the prostate remains unclear, we cannot ignore their importance. Selectivity for the a1A and/or a1D subtypes over the a1B subtype is referred to as “uroselectivity”[12].
Table I shows that the subtype selection of KMUP-1 is a1A=a1D>a1B. Therefore, KMUP-1 might be able to serve as an uroselective antagonist, similar to tamsulosin, for the treatment of BPH.
In order to investigate whether KMUP-1 is involved in PKG, sGCa1 and PKA protein expressions and the relaxation activity of rat prostate, the following experiments are designed.
Protein Extraction and Western Blotting
Prostate strips were incubated with KMUP-1 (10-100 μM), sildenafil (100 μM) and zaprinast (100 μM) in 10 mL Tyrode solution aerated with 95% O2 and 5% CO2 for 1 hour. To examine the possible mechanisms of KMUP-1 action, the rat prostate were pretreated with ODQ (10 μM), SQ22536 (10 μM) and RP-8pCPT-cGMPs (10 μM) for 30 minutes prior to the addition of KMUP-1 (100 μM).
In order to examine the expression of Rho kinase in prostate, the prostate strips were pre-incubated with phenylephrine (10 μM) for 30 minutes. After a pre-treatment of phenylephrine, KMUP-1 (10-100 μM) and Y-27632 (10 μM) were further incubated for 30 minutes. After the incubation time, the prostate were extracted with protein extraction reagent (Pierce Biotechnology, Inc., Rockford, Ill., USA.). The PZ-HPV-7 cells were incubated with and without KMUP-1 (0.1-10 μM) at 30 minutes, 8 and 24 hours. They were then washed rapidly with ice-cold PBS, incubated with protein extraction reagent, and scraped from the plates. The prostate tissues and cells were sonicated three times for 10 second each time and centrifuged at 13,000 rpm at 4° C. for 30 minutes. The protein concentrations of supernatants were determined, using bovine serum albumin as the standard. Tissue and cell extract protein were then boiled at a ratio of 4:1 with sample buffer (100 mM Tris, pH 6.8, 20% glycerol, 4% SDS, and 0.2% bromphenol blue). 40-60 μg protein was used in this experiment. Electrophoresis was performed using 10 or 12% SDS-polyacrylamide gel electrophoresis and then transferred to nitrocellulose membranes (Millipore Corporation, Billerica, Mass.). The membrane was blocked with Tris-buffered saline (20 mM Tris and 137 mM NaCl, pH 7.6) containing 0.1% Tween 20 (TTBS) and 5% non-fat milk at room temperature for 1 hour, washed with TTBS, and then incubated overnight at 4° C. in the appropriate primary antibody of PKG (Santa Cruz, 1:200), sGCa1 (Santa Cruz, 1:200), PKA (Abcam, 1:1000), ROCK2 (Upstate, 1:1000), and PDE5A (BD Biosciences, 1:1000), p21 (Upstate, 1:1000). The membranes were washed in TTBS before being incubated with horseradish peroxidase-conjugated antibody against mouse, goat, or rabbit IgG (Santa Cruz, 1:1000) for 1 hour. The membrane was then washed in TTBS and developed with the enhanced chemiluminescence (ECL) for the detection of the specific antigen. The intensity of the bands was measured by densitometry.
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Moreover, KMUP-1 (10, 100 μM) significantly increases cAMP levels (1.74±0.51, 2.82±0.42 pmol mg−1 protein) and which are inhibited by a pretreatment with SQ22536 (10 μM), respectively. The accumulation of cAMP by KMUP-1 (100 μM) alone and in the presence of SQ22536 (10 μM) are 2.82±0.42 and 2.16±0.79 pmol mg−1 protein, respectively.
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sGC, consisting of a1 and β1 subunits, is a target enzyme of endogenous NO, and it converts GTP to cGMP, resulting in smooth muscle relaxation[15]. Some studies have detected the expression of PDE4, PDE5 and PDE11 in human prostate[16, 17]. The current study has revealed that KMUP-1 strongly induces sGCa1 expression rather than sGCβ1. Please refer to
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In addition, it is found that a pretreatment of ODQ (10 μM) and RP-8-pCPT-cGMPs (10 μM) for 30 minutes significantly reduce the expression of sGCa1 and PKG in the presence of KMUP-1 (100 μM), respectively. As shown in
It is suggested that the activation of sGC by KMUP-1 plays an important role in relaxing rat prostate. This study further confirms that PDE5 inhibition, PKG activation and a-adrenergic blocking co-regulate or independently regulate the prostate smooth muscle tone in rats. The relaxation of prostate by KMUP-1 brings out not only sGC activation but also PDE inhibition (
In addition, previous studies have demonstrated that PKG inhibits the contractility through an activation of KATP channels in both human-cultured prostatic stromal cells and in prostate tissue[20, 21]. In human cultured prostatic stromal cells, the elevation of intracellular cAMP stimulates PKA to open BKCa channels. In this invention, it is also shown that KMUP-1 can potently induce the expression of PKG and PKA (
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In order to investigate whether KMUP-1 can protect against the growth of human prostate epithelial cells, the following experiments are designed.
Cell Proliferation
PZ-HPV-7, a human prostate epithelial cell line derived from normal peripheral zone tissue and transfected with human papillomavirus type 18, was obtained from the American Type Culture Collection (ATCC). Cells were grown in K-SFM medium supplemented with 100 U/mL penicillin, 100 gm/L, streptomycin 5 ng/ml, EGF human recombinant and 50 g/mL BPE in 75-cm2 flasks at 37° C. in a humidified atmosphere of O2/5% and CO2/95% air. The culture medium was renewed every 2 days and cells were sub-cultured every 6 days. For the experiments, cells were seeded in 24-well plates at a density of 1×104 cells/well. After 24 hours and 48 hours, when cells were 80% confluent, KMUP-1 (1-100 μM) was added to the 24-well plates. Cell proliferation was measured using MTT assay (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl-tetrazolium bromide, Sigma). The trypan blue (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl-tetrazoliumbromide) exclusion assay was performed to determine the proportion of living and dead cells in 24 hours and 48 hours after treatment. After 24 hours and 48 hours of treatment with KMUP-1 (1-100 μM), attached cells were collected by trypsinization. Cells were counted in a hemocytometer using equal volume of cell suspension and trypan blue solution (0.2% in PBS).
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BPH is believed to be secondary to an increased cell proliferation of both stromal and epithelial prostate cells[22]. Some studies have shown that vadenafil, tadalafil, NO donors and sildenafil display anti-proliferation activity in human prostate smooth muscle cells isolated from BPH patients[23, 24]. As shown in
Cell Cycle Distribution
PZ-HPV-7 cells were incubated for 2 days with or without KMUP-1 (0.1-10 μM), after trypsinized for 10 minutes with 0.5% trypsin supplemented with 5.3 mM EDTA in HBBS. The cell suspension was then centrifuged at 200 g for 5 minutes at 4° C., resuspended in 1 mL ice-cold HBSS and fixed by the drop-wise addition of 2 mL of ice-cold methanol followed by continuously shaking. The processed cells were kept on ice for 30 minutes and then cells were collected by centrifugation at 90 g for 5 min and stained with propidium iodide (50 μg/mL, Sigma). RNase A (Promega, Madison, Wis.) was then added to a final concentration of 100 U/mL. Samples were stored in the dark for at least 30 minutes before an analysis on a Coulter Epics XL-MCL flow cytometer. Flow cytometry analysis was performed on a Coulter Epics XL-MCL.
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RNA Isolation and cDNA Synthesis
PZ-HPV-7 cells were incubated for 2 days with or without KMUP-1 (1-100 μM). After the incubation, total RNA is isolated by processing the PZ-HPV-7 cells with TRI REAGEANT (Molecular Research Center, INC., Cincinnati, Ohio, USA) and 1-bromo-3chloropropane (BCP). Isopropanol was used to precipitate the isolated RNA; ethanol (75%) was used to wash the unwanted solution. The samples were then air-dried for 15 minutes at the room temperature. The pellet was re-suspended in 20 μL of DEPC-water (Ambion). After the RNA isolation, total RNA (2 μg) was used for the reverse transcription to cDNA by High-Capacity cDNA Reverse Transcription Kits (Applied Biosystems).
Quantitative Reverse Transcription-PCR
Quantitation of Rho kinase and an internal reference gene (GAPDH) were done by fluorescence-based real-time detection (ABI PRISM 7900HT Sequence Detection System (Taqman); Applied Biosystems, Foster City, Calif.). The probe sequences used are listed in Table II. The PCR reaction mixture consisted of 1× TaqMan Master Mix, 1× Probe/primers Assay Mix, 100 ng cDNA, and water containing a reference dye, to a final volume of 20 μL (all reagents from Applied Biosystems). Cycling conditions were 50° C. for 2 minutes, 95° C. for 10 minutes, followed by 40 cycles at 95° C. for 15 second and 60° C. for 1 minute. Gene expression values (relative mRNA levels) are expressed as ratios (differences between the Ct values) between the genes of interest and an internal reference gene (GAPDH) providing a normalization factor for the amount of RNA isolated from cells. This analysis was performed by 7900 HT SDS 2.2.1 software.
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Cyclin/cyclin-dependent kinase (CDK) protein complexes function at different cell cycle stages. p21 protein is a CDK inhibitor known to bind to cyclin-CDK complexes following the anti-mitogenic signals to induce cell cycle arrest at G0/G1 phase. p21 protein may be potentially used as a therapeutic target to reduce cell proliferation. The present results suggest the ability of KMUP-1 to inhibit the proliferation of prostate epithelial cells coming through the increase of p21 protein and arrested the cell cycle at G0/G1 phase.
Besides, Y-27632, a ROCK2 inhibitor, is able to inhibit the expression of ROCK2, which is usually increased, resulting from various stimulations, including by x-adrenergic agonist phenylephrine. Y-27632 has been described to have anti-proliferation and anti-adrenergic contraction activity in prostatic smooth muscle cells and tissues[10]. In this study, the expression of ROCK2 in rat prostate is certainly induced by phenylephrine. In contrast to Y-27632, KMUP-1 not only inhibits phenylephrine-induced expression of ROCK2, which is a downstream signaling of cGMP/PKG pathway in vascular system[25], but also inhibits the α1A/α1D-adrenoceptor binding ability in rat prostate smooth muscle. Theoretically, it is reasonable to suggest that KMUP-1 also inhibits cGMP-regulated expression of ROCK2 in human prostatic epithelial cells. Obviously, KMUP-1 can inhibit the growth of human prostate epithelial cells by Rho kinase inhibition, evidenced by expression of p21 protein in epithelial PZ-HPV-7 cells in
Statistical Analysis
All data are expressed as the mean±SEM. Statistical differences were analyzed by two-tailed unpaired Student's t test respectively. Whenever a control group was compared with more than one treatment group, one-way analysis of variance was used. When the analysis of variance found a statistical difference, results were further analyzed with Dunnett's test. A P value less than 0.05 were considered to significant in all experiments. Analysis of the data and plotting of the figures were performed using statistical software (SigmaPlot version 8.0 and SigmaStat Version 2.03, Chicago, Ill.) run on an IBM-compatible computer.
Clinical use of 5α-reductase inhibitors and α-adrenergic blockers can result in sexual dysfunction or ejaculatory disorders for the treatment of BPH and LUTS. Although PDE5 inhibitors are commonly used to treat erectile dysfunction, PDE inhibitors are also suggested to regulate bladder and prostate smooth muscle tone. Recent researches suggest that the combination of α1A and/or α1D-adrenergic blocker and PDE5 inhibitor may be useful in treating patients having BPH and LUTS-associated erectile dysfunction[26, 27]. In the mentioned studies, it has shown that low concentration of sildenafil (0.1 mM) can sensitize tamsulosin-induced concentration-response relaxation and shift the response curve to the left side. Accordingly, it is strongly suggested that the combination of PDE inhibition with α1A and/or α1D-adrenergic blockade by KMUP-1 might have positive impact in the treatment of patients having BPH/LUTS and associated male erectile dysfunction.
This invention first demonstrates that KMUP-1 has the selective α1A/α1D-adrenergic blocking activity on prostate smooth muscle tone. KMUP-1 multiply possesses PDE inhibition, sGC activation and selective α1A/α1D-adrenergic blocking activity. This is the reason why KMUP-1 can display more extinguish maximal efficacy than tamsulosin in relaxing rat prostate smooth muscle (
As mentioned in the above, the present invention indicates that KMUP-1-induced relaxation of the rat prostate is mediated by a mechanism selected from a group consisting of the activation of sGC/cGMP/PKG pathway, a blockage of α1A/α1D-adrenoreceptors, BKCa and KATP channels opening, and inhibition of ROCK2 and PDE. Moreover, KMUP-1 not only reduces prostate smooth muscle tone but also inhibits prostatic epithelial cell growth through up-regulating the expression of p21. It is suggested that KMUP-1 can be used by means of altering the prostate contractility, often associated with BPH, LUTS and erectile dysfunction. Accordingly, the present invention firstly shows that a cGMP-enhancer KMUP-1 might be used in the treatment of BPH, and thus it fits the demand of the industry and is industrially valuable.
While the invention has been described in terms of what is presently considered to be the most practical and preferred embodiments, it is to be understood that the invention needs not be limited to the disclosed embodiments. On the contrary, it is intended to cover various modifications and similar arrangements included within the spirit and scope of the appended claims which are to be accorded with the broadest interpretation so as to encompass all such modifications and similar structures.
