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Effect of the Novel BKCa Channel Opener LDD175 on the Modulation of Corporal Smooth Muscle Tone Hyun Hwan Sung, MD, PhD,* Seol Ho Choo, MD,† Deok Hyun Han, MD,* Mee Ree Chae, MSc,* Su Jeong Kang, Ms,* Chul-Seung Park, PhD,‡ Insuk So, MD, PhD,§ Jong Kwan Park, MD, PhD,¶**†† and Sung Won Lee, MD, PhD* *The Department of Urology, Samsung Medical Center, Samsung Biomedical Research Institute, Sungkyunkwan University School of Medicine, Seoul, Korea; †Department of Urology, Ajou University School of Medicine, Suwon, Korea; ‡ School of Life Sciences and National Leading Research Laboratory, Gwangju Institute of Science and Technology, Gwangju, Korea; §Department of Physiology and Biophysics, Seoul National University College of Medicine, Seoul, Korea; ¶Department of Urology, Medical School, Jeonju, Korea; **Institute for Medical Sciences, Chonbuk National University, Jeonju, Korea; ††Research Institute and Clinical Trial Center of Medical Device of Chonbuk National University Hospital, Jeonju, Korea DOI: 10.1111/jsm.12744
ABSTRACT
Introduction. The BKCa channel has been reported to play an important role in erectile function. Recently, novel BKCa channel activator, LDD175, was introduced. Aim. This study aims to investigate whether LDD175 relaxes corporal smooth muscle (CSM) via BKCa channel activation. Methods. After isolation of CSM strip from a male rabbit model, contraction studies using organ bath was performed. Isolating human tissue and cell cultures, electrophysiological studies were done via whole-cell patch-clamp recording. Main Outcome Measures. Vasodilatory effects of LDD175 were evaluated by cumulative addition ranging from 10−7 to 10−4 M in corpus cavernosal strips after precontraction with 10−5 M phenylephrine via organ bath system. Using cultured human CSM cells, patch-clamp recording was performed. Erectile function was measured by in vivo rat cavernous nerve stimulation. Results. LDD175 caused an endothelium-independent relaxation of corporal tissues, and this effect was abolished by pretreatment with iberiotoxin. The relaxation effect of 10−4 M LDD175 was greater than that of 10−6 M udenafil (54.0 ± 3.1% vs. 34.5 ± 3.9%, P < 0.05); 10−5 M LDD175 with 10−6 M udenafil caused a greater relaxation effect on strips than 10−5 M LDD175 or 10−6 M udenafil alone (50.7%, 34.1%, vs. 20.7%, respectively, P < 0.001). In patch-clamp recordings, LDD175 increased K+ currents in a dose-dependent manner, and washout of LDD175 or the addition of iberiotoxin fully reversed the increase. Intravenous LDD175 improved erectile function measured by area under the curve (AUC) of the intracavernosal pressure (ICP)/arterial blood pressure (ABP) ratio (1,612.1 ± 135.6 vs. 1,093.7 ± 123.1, P < 0.05). There was no difference between 10 mg/kg LDD175 and 1 mg/kg udenafil regarding maximal ICP, maximal ICP/ABP ratio, and the AUC of the ICP/ABP ratio (P > 0.05). Conclusions. LDD175 leads to an endothelium-independent relaxation of erectile tissue, primarily through the opening of BKCa channels. The results suggest that LDD175 might be a new candidate treatment for erectile dysfunction. Sung HH, Choo SH, Han DH, Chae MR, Kang SJ, Park C-S, So I, Park JK, and Lee SW. Effect of the novel BKCa channel opener LDD175 on the modulation of corporal smooth muscle tone. J Sex Med **;**:**–**. Key Words. Erectile Dysfunction; BKCa Channel; Corpus Cavernosum; LDD175; PDE5
© 2014 International Society for Sexual Medicine
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2 Introduction
E
rectile dysfunction (ED) is a chronic disease defined as the consistent inability to initiate or maintain a sufficient erection for satisfactory sexual function [1,2]. Since the introduction of sildenafil, oral phosphodiesterase type 5 inhibitors (PDE5) have been the first-line treatment for patients with ED because they are noninvasive, effective, and well-tolerated therapeutic options [3]. However, the response rate is lower in subpopulations, including those with diabetes mellitus, patients with severe vasculogenic ED, and postradical prostatectomy patients [4,5]. Thus, the development of novel therapeutic options is imperative for patients who are nonresponsive to PDE5. The BKCa channel is a Ca2+-activated potassium channel, which is activated by an increase in the intracellular concentration of Ca2+ or by membrane depolarization with a large conductance (100–300 pS) [6]. BKCa channels may play an important role in the control of vascular smooth muscle tone under physiological conditions and in pathophysiological states, such as hypertension, stroke, atherosclerosis, diabetes, and ED [7]. BKCa channel openers stabilize the cell by increasing the efflux of potassium ions, leading to hyperpolarization and thus decreasing cell excitability and/or causing smooth muscle relaxation [8]. There are several types of BKCa channel openers such as NS004, NS1619, mefenamic, flufenamic acids, NS1608, and the quinolinone analog [9–13]. However, the major limitations of this class of compounds are weak potency and insufficient selectivity. Recently, Gormemis et al. optimized the pharmacophore groups of the benzofuroindole analogs [14]. They found that compound 22 (4-chloro-7-trifluoromethyl-10Hbenzo[4,5]furo[3,2-b]indole-1-carboxylic acid, LDD175) was the most potent and effective activator of BKCa channels out of benzofuroindole analogs [14]. This novel BKCa channel opener compound was already verified to have relaxant activities on the urinary bladder, ileum, and uterus of Sprague-Dawley rats in a dosedependent manner by activating the BKCa channels [15–17]. In addition, LDD175 has been reported to have no significant adverse cardiovascular effects in in vivo animal experiments using Wistar-Kyto rats [15]. Werner et al. verified that the BKCa channel plays an important role in erectile function and that loss of the BKCa channel
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Sung et al. leads to ED, as demonstrated by using a knockout mouse lacking the Slo gene (Slo-/-) [18]. Thus, the relaxant activity of LDD175 could be expected to occur in penile smooth muscle tissues. Aims
The aim of this study is to investigate the relaxant effect of LDD175 in the corporal smooth muscle (CSM) cells via BKCa channel activation and to evaluate its effect on erectile function. Methods
All studies were performed according to a protocol approved by the Internal Review Board of the Samsung Medical Center/Sungkyunkwan University School of Medicine. After acquiring informed consent from the patients, human tissue was obtained from the corpus cavernosum of patients with organic ED who were undergoing the implantation of penile prostheses. All of the rats utilized in these studies were treated according to the guidelines of the Institutional Animal Care and Use Committee of the Samsung Biomedical Research Institute.
Isolation of Corpus Cavernosal Strip from an Animal Model Sexually mature male New Zealand white rabbits (3.0 ± 0.3 kg) were killed with an air embolism in the ear vein. Each entire penis was then surgically excised and cleaned by removing the corpus spongiosum and urethra. The corporal tissue was subsequently carefully dissected from the surrounding tunica. Four corporal strips of approximately equal size (2 × 2 × 10 mm) were obtained from each penis. Contraction Studies Using Organ Bath Rabbit cavernosal strips were separately prepared for organ-bath studies. Organ-bath studies were performed as previously described method [19]. The vasodilatory effect of LDD175 was studied by the cumulative addition of the substance at concentrations ranging from 10−7 to 10−4 M to endothelium-intact and endothelium-denuded strips after precontraction with phenylephrine (PE) 10−5 M. The endothelial lining of the corpus cavernosum was removed by rubbing the strip between the thumb and index finger for 20 seconds and soaking in a 10% solution of CHAPS
LDD175 and Corporal Smooth Muscle Tone (3-[(3-cholamidopropyl)-dimethylammonio]-lpropanesulphonate) for 10 seconds. The success for removing endothelium was confirmed by the absence of relaxation to acetylcholine. The strips were preincubated with a specific inhibitor of BKCa channels, 100 nM iberiotoxin (IbTX), for 30 minutes before PE was added. After the addition of IbTX, LDD175 was added to the precontracted strips with PE. The relaxation effect for the PDE5 (udenafil, 10−6 and 10−5 M) was also separately studied in the same manner and compared with LDD175. In terms of the time of drug treatment, we have waited the relaxation plateau of the tension plot after the cumulative addition of drug treatment.
Preparation of the Corpus Cavernosal Strip from Human Tissue and Cell Cultures Homogeneous explant cell cultures of human corporal smooth muscle (CSM) cells were prepared as previously described methods [19,20]. The human CSM cultures were morphologically homogeneous. The cobblestone morphology characteristic of endothelial cells and the flattened and spread-out shapes characteristic of fibroblasts were not observed. Cell purity was more than 70%. Cellular homogeneity was further verified by observing smooth muscle-specific α-actin and myosin immunoreactivity. The cultures were maintained for no more than four passages; during this time, all measured pharmacological and molecular properties were evaluated in the intact tissues that were maintained in the cultures, for example, cyclic adenosine monophosphate formation [20], calcium mobilization [21], expression or function of the gap junction protein, connexin [22], and K+ channel activity [23]. Electrophysiological Studies To directly investigate the effect of LDD175 on the activity of the BKCa channel and compare the effect of LDD175 with NS1619, a typical selective BKCa channel opener, we performed whole-cell patch-clamp recordings in cultured human CSM cells. Electrical results were recorded 3 minutes after the addition of drug. The detailed description of patch-clamp study has been reported previously [19]. In Vivo Animal Experiments Using the Cavernous Nerve Stimulation Model The erectile response was measured using Sprague-Dawley rats (351.6 ± 8 g). The detailed methodology of measuring the erectile response
3 has been described previously [24]. Submaximal electrical field stimulation (EFS) was used with a 1 V, 2 Hz, 5 ms pulse width and duration of 60 seconds because it would be difficult for LDD175 to induce the additional effect of relaxation vs. control in the setting of maximal stimulation. Submaximal EFS was performed 10 minutes after a series of control (buffer solution alone), LDD175 (10 mg/kg), and udenafil (1 mg/kg) injections. At the end of the submaximal stimulation, the full response was measured using the EFS of a 6 V, 10 Hz, 5 ms pulse width with a duration of 60 seconds to compare it with submaximal stimulation in the presence of LDD175 and to ensure the cavernous nerve was intact. The buffer solution, LDD175, and udenafil were administered at intervals of 30 minutes for washout. The ratio of maximal intracavernosal pressure (ICP) to mean arterial BP at the peak of the erectile response was calculated to control for variations in arterial blood pressure (ABP). At the end of the experiment, the rats were euthanized with an air embolism.
Drugs and Solutions LDD175 was kindly provided by AnyGen Co., Ltd. (Gwangju, Korea), and udenafil was obtained from Dong-A Pharmaceutical (Seoul, Korea). All other chemical agents were purchased from Sigma Chemical (St. Louis, MO, USA). For the in vitro organ bath and electrophysiological studies, LDD175 was dissolved in dimethyl sulfoxide (DMSO), as described in previous studies [17]. Udenafil was dissolved in 0.05 mol/L citric acid at 30 mg/kg. NS1619 was dissolved in ethanol. The stock solution was diluted immediately before use. The final concentration of the solvents was less than 0.1%, and this bath concentration of solvents did not significantly affect smooth muscle tone or ion-channel activity. For the in vivo functional studies, LDD175 was dissolved in 10% Tween 20. All other drugs were dissolved in distilled water. Statistical Analysis All data are expressed as the mean ± standard error and were analyzed using IBM SPSS (Statistical Package for the Social Sciences) Statistics software version 21.0 (IBM, Armonk, NY, USA). The twotailed Student’s t-test, paired t-test, and one-way analysis of variance test with Tukey’s post hoc analysis were used, as appropriate, to compare group mean values, with differences being considered significant at P < 0.05. J Sex Med **;**:**–**
4 Results
Relaxation Effects of LDD175 on Rabbit Corporal Smooth Muscle via the BKCa Channel In the organ bath studies, precontracted rabbit CSM strips were relaxed by LDD175 in a dose-
Sung et al. dependent manner from 10−7 M to 10−4 M (n = 10, 10−7 M = 3.7 ± 1.4%, 10−6 M = 14.5 ± 2.6%, 10−5 M = 26.3 ± 2.0%, and 10−4 M = 54.0 ± 3.0%, Figure 1A). LDD175 induced endotheliumindependent relaxation of rabbit CSM strips (DMSO only, n = 5, 15.7 ± 2.9%; intact endothe-
Figure 1 (A) Concentration-response curve for LDD175 and the rabbit CSM strip (n = 10, 10−7 M = 3.7 ± 1.4%, 10−6 M = 14.5 ± 2.6%, 10−5 M = 26.3 ± 2.0%, and 10−4 M = 54.0 ± 3.0%, *P < 0.05 vs. control, Figure 1A). The relaxant effects are expressed as the % of the PE-induced contraction. (B) Effect of endothelium-denudation (n = 10) or IbTX (n = 7) on the relaxation response induced by LDD175. Removal of the endothelium did not significantly affect the relaxation potencies (DMSO only, n = 5, 15.7 ± 2.9%; intact endothelium, n = 7, 45.9 ± 6.2%, P < 0.05 vs. DMSO only; denuded endothelium, n = 10, 41.0 ± 4.9%, P > 0.05 vs. intact endothelium). The relaxation response induced by LDD175 was significantly inhibited after pretreatment of the strips with IbTX, a selective blocker of BKCa channels (LDD175, n = 7, 45.9 ± 6.2%; LDD175 plus IbTX, n = 7, 23.6 ± 3.3%, P < 0.05 vs. LDD175). (C) 10−4 M LDD175 (n = 10, 54.0 ± 3.1%) and udenafil (n = 6, 10−6 M, 34.5 ± 3.9%; 10−5 M, 67.1 ± 1.7%) induced relaxations in phenylephrine-contracted rabbit CSM strips. 10−4 M LDD175 had a greater relaxation effect than 10−6 M udenafil (P < 0.01). The combination of 10−5 M LDD175 and 10−6 M udenafil had additive effects on relaxation (n = 7, 10−5 M LDD, 32.7 ± 3.8%; 10−6 M udenafil, 21.6 ± 2.7%; 10−5 M LDD plus 10−6 M udenafil, 50.4 ± 4.5%, *P < 0.05). The results are expressed as the % relaxation of the phenylephrine-induced contraction. Values are expressed as the mean ± SE The meaning of n was the number of CSM strips of rabbit. Three or four strips were obtained from each rabbit. CSM = corporal smooth muscle; IbTX = iberiotoxin.
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5
LDD175 and Corporal Smooth Muscle Tone lium, n = 7, 45.9 ± 6.2%, P < 0.05 vs. DMSO only; denuded endothelium, n = 10, 41.0 ± 4.9%, P > 0.05 vs. intact endothelium, Figure 1B). However, pretreatment with IbTX significantly inhibited the relaxation response of 10−4 M LDD175 (LDD175, n = 7, 45.9 ± 6.2%; LDD175 plus IbTX, n = 7, 23.6 ± 3.3%, P < 0.05 vs. LDD175, Figure 1C).
Comparison with PDE5 and Additive Effect Udenafil also induced a dose-dependent relaxation of rabbit CSM strips (n = 6, 10−6 M, 34.5 ± 3.9%; 10−5 M, 67.1 ± 1.7%, Figure 2A). The 10−4 M LDD (n = 10, 54.0 ± 3.1%) had a greater relaxation effect than 10−6 M udenafil (P < 0.01, Figure 1C). The combination of 10−5 M LDD175
Figure 2 (A) Representative current traces recorded at a holding potential of −60 mV with the external solution containing 5 mM K+. (B) Current–voltage (I–V) relationship measured by a 500 ms ramp pulse from −100 to +80 mV. LDD175 activated outward-rectifying K+ currents by a maximum of 952.1% at +60 mV (n = 13, P < 0.05 vs. control). (C) LDD175-activated current was blocked completely by TEA (1 mM, n = 6, 78.2%, P < 0.05) or IbTX (100 nM, n = 4, 78.1%, P < 0.05). (D) LDD175 dose-dependently activated whole-cell K+ currents in CSM cells. (E) Summary of peak current densities at +60 mV (n = 10, control, 24.5 ± 5.8 pA/pF; 0.1 μM, 30.9 ± 6.5 pA/pF; 1 μM, 45.1 ± 8.2 pA/pF; 10 μM, 178.5 ± 30.0 pA/pF; 30 μM, 257.3 ± 48.3 pA/pF; 100 μM, 265.5 ± 53.4 pA/pF, at 60 mV, *P < 0.05 vs. control). Values are the mean ± SEM The meaning of n was the number of cultured human CSM cells used in the patch clamp. CSM = corporal smooth muscle; IbTX = iberiotoxin; TEA = tetraethylammonium; w/o = wash out.
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6 and 10−6 M udenafil had an additive effect on relaxation (Figure 1C).
Effect of LDD175 on BKCa Currents in Human CSM Cells To directly investigate the effect of LDD175 on the activity of the BKCa channel, we performed whole-cell patch-clamp recordings in cultured human corporal smooth muscle cells. At the −60 mV holding potential with the external solution containing 5 mM K+, an extracellular application of 30 μM LDD175 significantly increased outward currents at all membranes, and this effect was completely inhibited by treatment with 100 nM IbTX (Figure 2A). As shown in Figure 2B, the current–voltage (I–V) relationship showed that LDD175 activated outward-rectifying K+ currents by a maximum of 952.1% at +60 mV (n = 13, P < 0.05 vs. control); 1 mM tetraethylammonium (TEA) or 100 nM IbTX suppressed these currents by 78.2% (n = 6, P < 0.05) and 78.1% (n = 4, P < 0.05), respectively (Figure 2C). The effect of LDD175 on the outward current was dose depen-
Sung et al. dent, and the increased currents were readily reversed by drug washout (Figure 2D). Figure 2E summarizes the average values, showing that the current density was gradually increased by the addition of higher concentrations of LDD175.
Comparison of the Effects of NS1619 and LDD175 on BKCa Channels in Human Corporal Smooth Muscle Cells To compare the effect of LDD175 with other BKCa channel activators, the electrophysiological effect of NS1619, a typical selective BKCa channel opener, was examined. Extracellular application of 30 μM NS1619 also markedly increased the whole-cell currents at a holding potential of +60 mV by 880.0% (n = 7, P < 0.05 vs. control, Figure 3A), similar to activation by LDD175. This enhancement of outward currents by NS1619 was completely inhibited by the addition of 100 nM IbTX (Figure 3B). At −40 mV, which is near the resting membrane potential (or more physiological potentials) of the cell, 30 μM LDD175 still activated currents (n = 13, 23.9 ± 5.8 pA/pF,
Figure 3 (A) Typical current–voltage relationships obtained using a 500 ms ramp pulse from −80 to +80 mV. Membrane currents were recorded in the absence and presence of 30 μM NS1619 at a holding potential of +60 mV by 880.0% (n = 7, P < 0.05 vs. control ). (B) The NS1619-activated current was blocked completely by 100 nM IbTX. (C and D) Summary of peak current densities at a holding potential of +60 mV (C) or −40 mV (D). The relaxant capacities of 30 μM LDD175 were significantly higher than those of 30 μM NS1619, a selective BKCa channel opener (at −40 mV, NS1619: n = 7, 1.5 ± 0.6 pA/pF, 1.3fold vs. control, P = 0.6; LDD175: n = 13, 23.9 ± 5.8 pA/pF, 42.8-fold vs. control, P < 0.05). Values are the mean ± SEM, *P < 0.05 vs. con: control The meaning of n was the number of cultured human CSM cells used in the patch clamp. IbTX = iberiotoxin; w/o = wash out.
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LDD175 and Corporal Smooth Muscle Tone
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Figure 4 In vivo animal model of cavernous nerve stimulation to evaluate erectile function after intravenous administration of LDD175 and udenafil under submaximal EFS (2 Hz, 1 V, and 60 seconds) and maximal EFS (10 Hz, 6 V, and 60 seconds). (A) Maximal ICP (n = 8, 48.9 ± 5.4 [control] vs. 63.5 ± 5.8 [LDD175] vs. 66.2 ± 8.3 [udenafil], P > 0.05). (B) Maximal ICP/ABP ratio (n = 8, 41.0 ± 5.0 [control] vs. 50.2 ± 5.3 [LDD175] vs. 55.7 ± 6.9 [udenafil], P > 0.05). (C) AUC of the ICP/ABP ratio (n = 8, 1,093.7 ± 123.1 [control] vs. 1,612.1 ± 135.6 [LDD175] vs. 1,657.5 ± 122.3 [udenafil], P < 0.05). *P < 0.05 vs. control The meaning of n was the number of rats. ABP = arterial blood pressure; AUC = area under curve; EFS = electrical field stimulation; ICP = intracavernosal pressure.
P < 0.05), although 30 μM NS1619 did not change the currents (n = 7, 1.5 ± 0.6 pA/pF, P = 0.6, Figure 3D). These results demonstrated that LDD175 might be activated under physiological conditions as well, unlike NS1619.
In Vivo Nerve Stimulation Rat Model With submaximal EFS, intravenous administration of LDD175 improved the erectile function of rats in terms of area under the curve (AUC) of the ICP/ABP ratio (n = 8, 1,093.7 ± 123.1 [control] vs. 1,612.1 ± 135.6 [LDD175] vs. 1,657.5 ± 122.3 [udenafil], P = 0.01). There were trend in favor of LDD175 compared with control but no significant difference in the maximal ICP (n = 8, 48.9 ± 5.4 [control] vs. 63.5 ± 5.8 [LDD175], P = 0.08) and maximal ICP/ABP ratio (n = 8, 41.0 ± 5.0 [control] vs. 50.2 ± 5.3 [LDD175], P = 0.23). There was no difference between the effect of 10 mg/kg LDD175 and 1 mg/kg udenafil regarding all aspects of interest, including maximal ICP (63.5 ± 5.8 [LDD175] vs. 66.2 ± 8.3 [udenafil], P = 0.80), maximal ICP/ ABP ratio (50.2 ± 5.3 [LDD175] vs. 55.7 ± 6.9 [udenafil], P = 0.58), and the AUC of the ICP/ABP ratio (1,612.1 ± 135.6 [LDD175] vs. 1,657.5 ± 122.3 [udenafil], P = 0.85) (Figure 4). Discussion
The main results of the current study were that LDD175 relaxed penile CSM strips precontracted
by PE in organ bath studies, and this relaxation was completely blocked by IbTX, a BKCa channel blocker. Whole-cell patch-clamp recording was used to measure BKCa currents after activation with 30 μM LDD175 and inhibition with IbTX. Moreover, enhanced erectile responses shown with intravenous administration of LDD175 were demonstrated using an in vivo cavernous nerve stimulation study. BKCa channels are considered to be key players in maintaining normal vasomotor tone by regulating potassium efflux. Hyperpolarization plays an important role in vascular reactivity by providing negative feedback upon excessive constriction [8]. In addition, diverse ion channels have been related to the relaxation of smooth muscle. Potassium channels are abundantly expressed in the smooth muscles of diverse organs, including the penis, where they play an important role in the control mechanism of the contraction of smooth muscle cells [25]. Adenosine triphosphate sensitive potassium channel (KATP) openers, compounds first developed for overactive bladders, also activated KATP channels. However, this compound was activated in the cardiovascular systems and brought about unwanted hemodynamic side effects [26]. Compared with the KATP channels, BKCa channels are known to be less expressed in cardiovascular tissue [8]. Thus, fewer cardiovascular adverse events might be expected with the use of BKCa channel openers. J Sex Med **;**:**–**
8 Penile erection is mainly mediated by the cyclic guanosine monophosphate/nitric oxide (NO) pathway. The vasodilator effects of NO are partly mediated by the activation of BKCa channels [27– 29]. Kun et al. reported that the relaxant responses elicited by sildenafil involve the activation of BKCa channels in both the intracavernosal artery and human CSM strips [30]. A recent report also showed that the activation of BKCa improve vasodilatory capacity of PDE5 in the human penile vessels and enhance erectile responses in vivo. They concluded that therapeutic potential of BKCa channel activator might improve the efficacy of PDE5 in the management of ED [29]. We also verified that 10−5 M LDD175 plus 10−6 M udenafil had more relaxation effects than 10−5 M LDD175 or 10−6 M udenafil only. These results suggest that the mechanism of action of LDD175 could be augmented by PDE5 or vice versa and that LDD175 and PDE5 had additive effects on relaxation. Using cultured human CSM cells, we also investigated whole-cell K+ currents in the presence of 30 μM NS1619, a typical BKCa channel opener, in an attempt to compare it with 30 μM LDD175. At a holding potential of +60 mV, peak current densities were induced by NS1619 and inhibited by IbTX. At −40 mV, current densities did not form with NS1619, which is more similar to normal cell capacitance, although LDD175 induced a peak current. The activation threshold of BKCa channel currents was shifted by LDD175 to more negative voltages, which were close to the resting potential of the cells. This in vitro electrophysiological outcome showed that LDD175 activated BKCa channels under more physiological conditions (−40 mV) in contrast to NS1619. A more efficient effect of LDD175 on relaxing penile CSM would be expected compared with NS1619. In vivo cavernous nerve stimulations were conducted in a series of submaximal (control, in the presence of LDD175 and udenafil) and maximal stimulations in each experiment. In the current study, intravenous administration of LDD175 (10 mg/kg) significantly enhanced erectile response during electrical stimulation with regard to the AUC of the ICP/ABP ratio. These increases were also elicited with the administration of udenafil (1 mg/kg), and there was no difference between LDD175 and udenafil with respect to the ICP/ABP ratio and the AUC of the ICP/ABP ratio. Drug-related adverse reactions should be of concern in the field of new drug development. Some KATP channel openers (including croJ Sex Med **;**:**–**
Sung et al. makalim, ZD6169, and WAY-133537) caused significant decreases in arterial pressure at doses effective on the bladder and that these channel openers exhibited low bladder selectivity [26]. In contrast, BKCa channel openers seem to have fewer side effects in the cardiovascular system due to the lower expression of BKCa channels in heart tissue [8]. Pena et al. [15] reported an in vivo hemodynamic assessment of LDD175 using Wistar-Kyoto rats. In their experiments, the aortic rings of rats were not relaxed by LDD175. In addition, BP and heart rate changes following intravenous administration of the compound were evaluated, showing that 10 mg/kg LDD175 did not cause any significant change in BP or heart rate. The current study also demonstrated that BP fluctuation and hemodynamic instability during in vivo cavernous nerve stimulation were not observed. In the limitations, this study was to verify the effectiveness of LDD175 on the relaxation of cavernosal smooth muscle, not on the safety aspect. So, the current study lacked the safety assessment. Further experiments should be followed to measure the safety assessment of LDD175. In addition to that, the potential use of LDD175 will be applied in the preclinical and clinical setting. Second, the investigation about tissue selectivity of corpus cavernosum vs. vascular smooth muscle should be determined. Lastly, the evaluation of combined administration of LDD175 and udenafil in the in vivo setting will be valuable. In conclusion, present study provides evidence that LDD175, a novel opener of BKCa channels, leads to a concentration-dependent relaxation of erectile tissue in CSM strips precontracted by PE 10−5 M in vitro. This relaxation is endothelium independent and occurs primarily via BKCa channel opening. Moreover, enhanced erectile function of the in vivo animal model using cavernous nerve electrical stimulation tests was verified. These responses are comparable with those seen with udenafil. LDD175 may also have an additive effect with udenafil on the relaxation of smooth muscle. The results suggest that LDD175 might be a new candidate in ED treatment. Further investigation should be pursued to apply LDD175 to clinical settings. Acknowledgment
This study was supported by a grant of the Korean Health Technology R&D Project, Ministry of Health & Welfare, Republic of Korea (HI13C0104).
LDD175 and Corporal Smooth Muscle Tone Corresponding Author: Sung Won Lee, MD, PhD, Department of Urology, Samsung Medical Center, Samsung Biomedical Research Institute, Sungkyunkwan University School of Medicine, 81 Irwon-ro, Gangnamgu, Seoul 135-710, Korea. Tel: +82-2-3410-3559; Fax: +82-2-3410-3027; E-mail: [email protected] Conflict of Interest: The author(s) report no conflicts of interest.
Statement of Authorship
Category 1 (a) Conception and Design Hyun Hwan Sung; Sung Won Lee (b) Acquisition of Data Hyun Hwan Sung; Seol Ho Choo; Mee Ree Chae; Su Jeong Kang (c) Analysis and Interpretation of Data Hyun Hwan Sung; Seol Ho Choo; Deok Hyun Han; Mee Ree Chae; Su Jeong Kang; Sung Won Lee
Category 2 (a) Drafting the Article Hyun Hwan Sung (b) Revising It for Intellectual Content Chul-Seung Park; Insuk So; Jong Kwan Park; Sung Won Lee
Category 3 (a) Final Approval of the Completed Article Insuk So; Jong Kwan Park; Sung Won Lee
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9 7 Hill MA, Yang Y, Ella SR, Davis MJ, Braun AP. Large conductance, Ca2+-activated K+ channels (BKCa) and arteriolar myogenic signaling. FEBS Lett 2010;584:2033–42. 8 Ghatta S, Nimmagadda D, Xu X, O’Rourke ST. Largeconductance, calcium-activated potassium channels: Structural and functional implications. Pharmacol Ther 2006;110:103– 16. 9 Olesen SP, Munch E, Moldt P, Drejer J. Selective activation of Ca(2+)-dependent K+ channels by novel benzimidazolone. Eur J Pharmacol 1994;251:53–9. 10 Ottolia M, Toro L. Potentiation of large conductance KCa channels by niflumic, flufenamic, and mefenamic acids. Biophys J 1994;67:2272–9. 11 Siemer C, Bushfield M, Newgreen D, Grissmer S. Effects of NS1608 on MaxiK channels in smooth muscle cells from urinary bladder. J Membr Biol 2000;173:57–66. 12 Hewawasam P, Fan W, Ding M, Flint K, Cook D, Goggins GD, Myers RA, Gribkoff VK, Boissard CG, Dworetzky SI, Starrett JE Jr, Lodge NJ. 4-Aryl-3-(hydroxyalkyl)quinolin-2ones: Novel maxi-K channel opening relaxants of corporal smooth muscle targeted for erectile dysfunction. J Med Chem 2003;46:2819–22. 13 Butera JA, Antane SA, Hirth B, Lennox JR, Sheldon JH, Norton NW, Warga D, Argentieri TM. Synthesis and potassium channel opening activity of substituted 10Hbenzo[4,5]furo[3,2-b]indole-and 5,10-dihydro-indeno[1,2b]indole-1-carboxylic acids. Bioorg Med Chem Lett 2001;11: 2093–7. 14 Gormemis AE, Ha TS, Im I, Jung KY, Lee JY, Park CS, Kim YC. Benzofuroindole analogues as potent BK(Ca) channel openers. Chembiochem 2005;6:1745–8. 15 dela Pena IC, Yoon SY, Kim SM, Lee GS, Ryu JH, Park CS, Kim YC, Cheong JH. Bladder-relaxant properties of the novel benzofuroindole analogue LDD175. Pharmacology 2009; 83:367–78. 16 Dela Pena IC, Yoon SY, Kim SM, Lee GS, Park CS, Kim YC, Cheong JH. Inhibition of intestinal motility by the putative BK(Ca) channel opener LDD175. Arch Pharm Res 2009; 32:413–20. 17 Ahn HS, dela Pena I, Kim YC, Cheong JH. 4-chloro-7trifluoromethyl-10H-benzo[4,5]furo[3,2-b]indole-1carboxylic acid (TBIC), a putative BK(Ca) channel opener with uterine relaxant activities. Pharmacology 2011;87:331–40. 18 Werner ME, Zvara P, Meredith AL, Aldrich RW, Nelson MT. Erectile dysfunction in mice lacking the large-conductance calcium-activated potassium (BK) channel. J Physiol 2005; 567:545–56. 19 Sung HH, Chae MR, So I, Jeon JH, Park JK, Lee SW. Effects of ginsenoside on large-conductance K(Ca) channels in human corporal smooth muscle cells. Int J Impot Res 2011;23:193–9. 20 Palmer LS, Valcic M, Melman A, Giraldi A, Wagner G, Christ GJ. Characterization of cyclic AMP accumulation in cultured human corpus cavernosum smooth muscle cells. J Urol 1994;152:1308–14. 21 Zhao W, Christ GJ. Endothelin-1 as a putative modulator of erectile dysfunction. II. Calcium mobilization in cultured human corporal smooth muscle cells. J Urol 1995;154:1571–9. 22 Brink PR, Ramanan SV, Christ GJ. Human connexin 43 gap junction channel gating: Evidence for mode shifts and/or heterogeneity. Am J Physiol 1996;271:C321–31. 23 Lee SW, Wang HZ, Zhao W, Ney P, Brink PR, Christ GJ. Prostaglandin E1 activates the large-conductance KCa channel in human corporal smooth muscle cells. Int J Impot Res 1999;11:189–99. 24 Rehman J, Chenven E, Brink P, Peterson B, Walcott B, Wen YP, Melman A, Christ G. Diminished neurogenic but not pharmacological erections in the 2- to 3-month experimentally diabetic F-344 rat. Am J Physiol 1997;272:H1960–71.
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10 25 Edwards G, Henshaw M, Miller M, Weston AH. Comparison of the effects of several potassium-channel openers on rat bladder and rat portal vein in vitro. Br J Pharmacol 1991; 102:679–86. 26 Fabiyi AC, Gopalakrishnan M, Lynch JJ III, Brioni JD, Coghlan MJ, Brune ME. In vivo evaluation of the potency and bladder-vascular selectivity of the ATP-sensitive potassium channel openers (-)-cromakalim, ZD6169 and WAY-133537 in rats. BJU Int 2003;91:284–90. 27 Fukami Y, Toki Y, Numaguchi Y, Nakashima Y, Mukawa H, Matsui H, Okumura K, Ito T. Nitroglycerin-induced aortic relaxation mediated by calcium-activated potassium channel is markedly diminished in hypertensive rats. Life Sci 1998;63: 1047–55.
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Sung et al. 28 Lee SW, Kang TM. Effects of nitric oxide on the Ca2+activated potassium channels in smooth muscle cells of the human corpus cavernosum. Urol Res 2001;29:359–65. 29 Gonzalez-Corrochano R, La Fuente J, Cuevas P, Fernandez A, Chen M, Saenz de Tejada I, Angulo J. Ca2+ -activated K+ channel (KCa) stimulation improves relaxant capacity of PDE5 inhibitors in human penile arteries and recovers the reduced efficacy of PDE5 inhibition in diabetic erectile dysfunction. Br J Pharmacol 2013;169:449–61. 30 Kun A, Matchkov VV, Stankevicius E, Nardi A, Hughes AD, Kirkeby HJ, Demnitz J, Simonsen U. NS11021, a novel opener of large-conductance Ca(2+)-activated K(+) channels, enhances erectile responses in rats. Br J Pharmacol 2009; 158:1465–76.
Effect of the Novel BKCa Channel Opener LDD175 on the Modulation of Corporal Smooth Muscle Tone Hyun Hwan Sung, MD, PhD,* Seol Ho Choo, MD,† Deok Hyun Han, MD,* Mee Ree Chae, MSc,* Su Jeong Kang, Ms,* Chul-Seung Park, PhD,‡ Insuk So, MD, PhD,§ Jong Kwan Park, MD, PhD,¶**†† and Sung Won Lee, MD, PhD* *The Department of Urology, Samsung Medical Center, Samsung Biomedical Research Institute, Sungkyunkwan University School of Medicine, Seoul, Korea; †Department of Urology, Ajou University School of Medicine, Suwon, Korea; ‡ School of Life Sciences and National Leading Research Laboratory, Gwangju Institute of Science and Technology, Gwangju, Korea; §Department of Physiology and Biophysics, Seoul National University College of Medicine, Seoul, Korea; ¶Department of Urology, Medical School, Jeonju, Korea; **Institute for Medical Sciences, Chonbuk National University, Jeonju, Korea; ††Research Institute and Clinical Trial Center of Medical Device of Chonbuk National University Hospital, Jeonju, Korea DOI: 10.1111/jsm.12744
ABSTRACT
Introduction. The BKCa channel has been reported to play an important role in erectile function. Recently, novel BKCa channel activator, LDD175, was introduced. Aim. This study aims to investigate whether LDD175 relaxes corporal smooth muscle (CSM) via BKCa channel activation. Methods. After isolation of CSM strip from a male rabbit model, contraction studies using organ bath was performed. Isolating human tissue and cell cultures, electrophysiological studies were done via whole-cell patch-clamp recording. Main Outcome Measures. Vasodilatory effects of LDD175 were evaluated by cumulative addition ranging from 10−7 to 10−4 M in corpus cavernosal strips after precontraction with 10−5 M phenylephrine via organ bath system. Using cultured human CSM cells, patch-clamp recording was performed. Erectile function was measured by in vivo rat cavernous nerve stimulation. Results. LDD175 caused an endothelium-independent relaxation of corporal tissues, and this effect was abolished by pretreatment with iberiotoxin. The relaxation effect of 10−4 M LDD175 was greater than that of 10−6 M udenafil (54.0 ± 3.1% vs. 34.5 ± 3.9%, P < 0.05); 10−5 M LDD175 with 10−6 M udenafil caused a greater relaxation effect on strips than 10−5 M LDD175 or 10−6 M udenafil alone (50.7%, 34.1%, vs. 20.7%, respectively, P < 0.001). In patch-clamp recordings, LDD175 increased K+ currents in a dose-dependent manner, and washout of LDD175 or the addition of iberiotoxin fully reversed the increase. Intravenous LDD175 improved erectile function measured by area under the curve (AUC) of the intracavernosal pressure (ICP)/arterial blood pressure (ABP) ratio (1,612.1 ± 135.6 vs. 1,093.7 ± 123.1, P < 0.05). There was no difference between 10 mg/kg LDD175 and 1 mg/kg udenafil regarding maximal ICP, maximal ICP/ABP ratio, and the AUC of the ICP/ABP ratio (P > 0.05). Conclusions. LDD175 leads to an endothelium-independent relaxation of erectile tissue, primarily through the opening of BKCa channels. The results suggest that LDD175 might be a new candidate treatment for erectile dysfunction. Sung HH, Choo SH, Han DH, Chae MR, Kang SJ, Park C-S, So I, Park JK, and Lee SW. Effect of the novel BKCa channel opener LDD175 on the modulation of corporal smooth muscle tone. J Sex Med **;**:**–**. Key Words. Erectile Dysfunction; BKCa Channel; Corpus Cavernosum; LDD175; PDE5
© 2014 International Society for Sexual Medicine
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2 Introduction
E
rectile dysfunction (ED) is a chronic disease defined as the consistent inability to initiate or maintain a sufficient erection for satisfactory sexual function [1,2]. Since the introduction of sildenafil, oral phosphodiesterase type 5 inhibitors (PDE5) have been the first-line treatment for patients with ED because they are noninvasive, effective, and well-tolerated therapeutic options [3]. However, the response rate is lower in subpopulations, including those with diabetes mellitus, patients with severe vasculogenic ED, and postradical prostatectomy patients [4,5]. Thus, the development of novel therapeutic options is imperative for patients who are nonresponsive to PDE5. The BKCa channel is a Ca2+-activated potassium channel, which is activated by an increase in the intracellular concentration of Ca2+ or by membrane depolarization with a large conductance (100–300 pS) [6]. BKCa channels may play an important role in the control of vascular smooth muscle tone under physiological conditions and in pathophysiological states, such as hypertension, stroke, atherosclerosis, diabetes, and ED [7]. BKCa channel openers stabilize the cell by increasing the efflux of potassium ions, leading to hyperpolarization and thus decreasing cell excitability and/or causing smooth muscle relaxation [8]. There are several types of BKCa channel openers such as NS004, NS1619, mefenamic, flufenamic acids, NS1608, and the quinolinone analog [9–13]. However, the major limitations of this class of compounds are weak potency and insufficient selectivity. Recently, Gormemis et al. optimized the pharmacophore groups of the benzofuroindole analogs [14]. They found that compound 22 (4-chloro-7-trifluoromethyl-10Hbenzo[4,5]furo[3,2-b]indole-1-carboxylic acid, LDD175) was the most potent and effective activator of BKCa channels out of benzofuroindole analogs [14]. This novel BKCa channel opener compound was already verified to have relaxant activities on the urinary bladder, ileum, and uterus of Sprague-Dawley rats in a dosedependent manner by activating the BKCa channels [15–17]. In addition, LDD175 has been reported to have no significant adverse cardiovascular effects in in vivo animal experiments using Wistar-Kyto rats [15]. Werner et al. verified that the BKCa channel plays an important role in erectile function and that loss of the BKCa channel
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Sung et al. leads to ED, as demonstrated by using a knockout mouse lacking the Slo gene (Slo-/-) [18]. Thus, the relaxant activity of LDD175 could be expected to occur in penile smooth muscle tissues. Aims
The aim of this study is to investigate the relaxant effect of LDD175 in the corporal smooth muscle (CSM) cells via BKCa channel activation and to evaluate its effect on erectile function. Methods
All studies were performed according to a protocol approved by the Internal Review Board of the Samsung Medical Center/Sungkyunkwan University School of Medicine. After acquiring informed consent from the patients, human tissue was obtained from the corpus cavernosum of patients with organic ED who were undergoing the implantation of penile prostheses. All of the rats utilized in these studies were treated according to the guidelines of the Institutional Animal Care and Use Committee of the Samsung Biomedical Research Institute.
Isolation of Corpus Cavernosal Strip from an Animal Model Sexually mature male New Zealand white rabbits (3.0 ± 0.3 kg) were killed with an air embolism in the ear vein. Each entire penis was then surgically excised and cleaned by removing the corpus spongiosum and urethra. The corporal tissue was subsequently carefully dissected from the surrounding tunica. Four corporal strips of approximately equal size (2 × 2 × 10 mm) were obtained from each penis. Contraction Studies Using Organ Bath Rabbit cavernosal strips were separately prepared for organ-bath studies. Organ-bath studies were performed as previously described method [19]. The vasodilatory effect of LDD175 was studied by the cumulative addition of the substance at concentrations ranging from 10−7 to 10−4 M to endothelium-intact and endothelium-denuded strips after precontraction with phenylephrine (PE) 10−5 M. The endothelial lining of the corpus cavernosum was removed by rubbing the strip between the thumb and index finger for 20 seconds and soaking in a 10% solution of CHAPS
LDD175 and Corporal Smooth Muscle Tone (3-[(3-cholamidopropyl)-dimethylammonio]-lpropanesulphonate) for 10 seconds. The success for removing endothelium was confirmed by the absence of relaxation to acetylcholine. The strips were preincubated with a specific inhibitor of BKCa channels, 100 nM iberiotoxin (IbTX), for 30 minutes before PE was added. After the addition of IbTX, LDD175 was added to the precontracted strips with PE. The relaxation effect for the PDE5 (udenafil, 10−6 and 10−5 M) was also separately studied in the same manner and compared with LDD175. In terms of the time of drug treatment, we have waited the relaxation plateau of the tension plot after the cumulative addition of drug treatment.
Preparation of the Corpus Cavernosal Strip from Human Tissue and Cell Cultures Homogeneous explant cell cultures of human corporal smooth muscle (CSM) cells were prepared as previously described methods [19,20]. The human CSM cultures were morphologically homogeneous. The cobblestone morphology characteristic of endothelial cells and the flattened and spread-out shapes characteristic of fibroblasts were not observed. Cell purity was more than 70%. Cellular homogeneity was further verified by observing smooth muscle-specific α-actin and myosin immunoreactivity. The cultures were maintained for no more than four passages; during this time, all measured pharmacological and molecular properties were evaluated in the intact tissues that were maintained in the cultures, for example, cyclic adenosine monophosphate formation [20], calcium mobilization [21], expression or function of the gap junction protein, connexin [22], and K+ channel activity [23]. Electrophysiological Studies To directly investigate the effect of LDD175 on the activity of the BKCa channel and compare the effect of LDD175 with NS1619, a typical selective BKCa channel opener, we performed whole-cell patch-clamp recordings in cultured human CSM cells. Electrical results were recorded 3 minutes after the addition of drug. The detailed description of patch-clamp study has been reported previously [19]. In Vivo Animal Experiments Using the Cavernous Nerve Stimulation Model The erectile response was measured using Sprague-Dawley rats (351.6 ± 8 g). The detailed methodology of measuring the erectile response
3 has been described previously [24]. Submaximal electrical field stimulation (EFS) was used with a 1 V, 2 Hz, 5 ms pulse width and duration of 60 seconds because it would be difficult for LDD175 to induce the additional effect of relaxation vs. control in the setting of maximal stimulation. Submaximal EFS was performed 10 minutes after a series of control (buffer solution alone), LDD175 (10 mg/kg), and udenafil (1 mg/kg) injections. At the end of the submaximal stimulation, the full response was measured using the EFS of a 6 V, 10 Hz, 5 ms pulse width with a duration of 60 seconds to compare it with submaximal stimulation in the presence of LDD175 and to ensure the cavernous nerve was intact. The buffer solution, LDD175, and udenafil were administered at intervals of 30 minutes for washout. The ratio of maximal intracavernosal pressure (ICP) to mean arterial BP at the peak of the erectile response was calculated to control for variations in arterial blood pressure (ABP). At the end of the experiment, the rats were euthanized with an air embolism.
Drugs and Solutions LDD175 was kindly provided by AnyGen Co., Ltd. (Gwangju, Korea), and udenafil was obtained from Dong-A Pharmaceutical (Seoul, Korea). All other chemical agents were purchased from Sigma Chemical (St. Louis, MO, USA). For the in vitro organ bath and electrophysiological studies, LDD175 was dissolved in dimethyl sulfoxide (DMSO), as described in previous studies [17]. Udenafil was dissolved in 0.05 mol/L citric acid at 30 mg/kg. NS1619 was dissolved in ethanol. The stock solution was diluted immediately before use. The final concentration of the solvents was less than 0.1%, and this bath concentration of solvents did not significantly affect smooth muscle tone or ion-channel activity. For the in vivo functional studies, LDD175 was dissolved in 10% Tween 20. All other drugs were dissolved in distilled water. Statistical Analysis All data are expressed as the mean ± standard error and were analyzed using IBM SPSS (Statistical Package for the Social Sciences) Statistics software version 21.0 (IBM, Armonk, NY, USA). The twotailed Student’s t-test, paired t-test, and one-way analysis of variance test with Tukey’s post hoc analysis were used, as appropriate, to compare group mean values, with differences being considered significant at P < 0.05. J Sex Med **;**:**–**
4 Results
Relaxation Effects of LDD175 on Rabbit Corporal Smooth Muscle via the BKCa Channel In the organ bath studies, precontracted rabbit CSM strips were relaxed by LDD175 in a dose-
Sung et al. dependent manner from 10−7 M to 10−4 M (n = 10, 10−7 M = 3.7 ± 1.4%, 10−6 M = 14.5 ± 2.6%, 10−5 M = 26.3 ± 2.0%, and 10−4 M = 54.0 ± 3.0%, Figure 1A). LDD175 induced endotheliumindependent relaxation of rabbit CSM strips (DMSO only, n = 5, 15.7 ± 2.9%; intact endothe-
Figure 1 (A) Concentration-response curve for LDD175 and the rabbit CSM strip (n = 10, 10−7 M = 3.7 ± 1.4%, 10−6 M = 14.5 ± 2.6%, 10−5 M = 26.3 ± 2.0%, and 10−4 M = 54.0 ± 3.0%, *P < 0.05 vs. control, Figure 1A). The relaxant effects are expressed as the % of the PE-induced contraction. (B) Effect of endothelium-denudation (n = 10) or IbTX (n = 7) on the relaxation response induced by LDD175. Removal of the endothelium did not significantly affect the relaxation potencies (DMSO only, n = 5, 15.7 ± 2.9%; intact endothelium, n = 7, 45.9 ± 6.2%, P < 0.05 vs. DMSO only; denuded endothelium, n = 10, 41.0 ± 4.9%, P > 0.05 vs. intact endothelium). The relaxation response induced by LDD175 was significantly inhibited after pretreatment of the strips with IbTX, a selective blocker of BKCa channels (LDD175, n = 7, 45.9 ± 6.2%; LDD175 plus IbTX, n = 7, 23.6 ± 3.3%, P < 0.05 vs. LDD175). (C) 10−4 M LDD175 (n = 10, 54.0 ± 3.1%) and udenafil (n = 6, 10−6 M, 34.5 ± 3.9%; 10−5 M, 67.1 ± 1.7%) induced relaxations in phenylephrine-contracted rabbit CSM strips. 10−4 M LDD175 had a greater relaxation effect than 10−6 M udenafil (P < 0.01). The combination of 10−5 M LDD175 and 10−6 M udenafil had additive effects on relaxation (n = 7, 10−5 M LDD, 32.7 ± 3.8%; 10−6 M udenafil, 21.6 ± 2.7%; 10−5 M LDD plus 10−6 M udenafil, 50.4 ± 4.5%, *P < 0.05). The results are expressed as the % relaxation of the phenylephrine-induced contraction. Values are expressed as the mean ± SE The meaning of n was the number of CSM strips of rabbit. Three or four strips were obtained from each rabbit. CSM = corporal smooth muscle; IbTX = iberiotoxin.
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5
LDD175 and Corporal Smooth Muscle Tone lium, n = 7, 45.9 ± 6.2%, P < 0.05 vs. DMSO only; denuded endothelium, n = 10, 41.0 ± 4.9%, P > 0.05 vs. intact endothelium, Figure 1B). However, pretreatment with IbTX significantly inhibited the relaxation response of 10−4 M LDD175 (LDD175, n = 7, 45.9 ± 6.2%; LDD175 plus IbTX, n = 7, 23.6 ± 3.3%, P < 0.05 vs. LDD175, Figure 1C).
Comparison with PDE5 and Additive Effect Udenafil also induced a dose-dependent relaxation of rabbit CSM strips (n = 6, 10−6 M, 34.5 ± 3.9%; 10−5 M, 67.1 ± 1.7%, Figure 2A). The 10−4 M LDD (n = 10, 54.0 ± 3.1%) had a greater relaxation effect than 10−6 M udenafil (P < 0.01, Figure 1C). The combination of 10−5 M LDD175
Figure 2 (A) Representative current traces recorded at a holding potential of −60 mV with the external solution containing 5 mM K+. (B) Current–voltage (I–V) relationship measured by a 500 ms ramp pulse from −100 to +80 mV. LDD175 activated outward-rectifying K+ currents by a maximum of 952.1% at +60 mV (n = 13, P < 0.05 vs. control). (C) LDD175-activated current was blocked completely by TEA (1 mM, n = 6, 78.2%, P < 0.05) or IbTX (100 nM, n = 4, 78.1%, P < 0.05). (D) LDD175 dose-dependently activated whole-cell K+ currents in CSM cells. (E) Summary of peak current densities at +60 mV (n = 10, control, 24.5 ± 5.8 pA/pF; 0.1 μM, 30.9 ± 6.5 pA/pF; 1 μM, 45.1 ± 8.2 pA/pF; 10 μM, 178.5 ± 30.0 pA/pF; 30 μM, 257.3 ± 48.3 pA/pF; 100 μM, 265.5 ± 53.4 pA/pF, at 60 mV, *P < 0.05 vs. control). Values are the mean ± SEM The meaning of n was the number of cultured human CSM cells used in the patch clamp. CSM = corporal smooth muscle; IbTX = iberiotoxin; TEA = tetraethylammonium; w/o = wash out.
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6 and 10−6 M udenafil had an additive effect on relaxation (Figure 1C).
Effect of LDD175 on BKCa Currents in Human CSM Cells To directly investigate the effect of LDD175 on the activity of the BKCa channel, we performed whole-cell patch-clamp recordings in cultured human corporal smooth muscle cells. At the −60 mV holding potential with the external solution containing 5 mM K+, an extracellular application of 30 μM LDD175 significantly increased outward currents at all membranes, and this effect was completely inhibited by treatment with 100 nM IbTX (Figure 2A). As shown in Figure 2B, the current–voltage (I–V) relationship showed that LDD175 activated outward-rectifying K+ currents by a maximum of 952.1% at +60 mV (n = 13, P < 0.05 vs. control); 1 mM tetraethylammonium (TEA) or 100 nM IbTX suppressed these currents by 78.2% (n = 6, P < 0.05) and 78.1% (n = 4, P < 0.05), respectively (Figure 2C). The effect of LDD175 on the outward current was dose depen-
Sung et al. dent, and the increased currents were readily reversed by drug washout (Figure 2D). Figure 2E summarizes the average values, showing that the current density was gradually increased by the addition of higher concentrations of LDD175.
Comparison of the Effects of NS1619 and LDD175 on BKCa Channels in Human Corporal Smooth Muscle Cells To compare the effect of LDD175 with other BKCa channel activators, the electrophysiological effect of NS1619, a typical selective BKCa channel opener, was examined. Extracellular application of 30 μM NS1619 also markedly increased the whole-cell currents at a holding potential of +60 mV by 880.0% (n = 7, P < 0.05 vs. control, Figure 3A), similar to activation by LDD175. This enhancement of outward currents by NS1619 was completely inhibited by the addition of 100 nM IbTX (Figure 3B). At −40 mV, which is near the resting membrane potential (or more physiological potentials) of the cell, 30 μM LDD175 still activated currents (n = 13, 23.9 ± 5.8 pA/pF,
Figure 3 (A) Typical current–voltage relationships obtained using a 500 ms ramp pulse from −80 to +80 mV. Membrane currents were recorded in the absence and presence of 30 μM NS1619 at a holding potential of +60 mV by 880.0% (n = 7, P < 0.05 vs. control ). (B) The NS1619-activated current was blocked completely by 100 nM IbTX. (C and D) Summary of peak current densities at a holding potential of +60 mV (C) or −40 mV (D). The relaxant capacities of 30 μM LDD175 were significantly higher than those of 30 μM NS1619, a selective BKCa channel opener (at −40 mV, NS1619: n = 7, 1.5 ± 0.6 pA/pF, 1.3fold vs. control, P = 0.6; LDD175: n = 13, 23.9 ± 5.8 pA/pF, 42.8-fold vs. control, P < 0.05). Values are the mean ± SEM, *P < 0.05 vs. con: control The meaning of n was the number of cultured human CSM cells used in the patch clamp. IbTX = iberiotoxin; w/o = wash out.
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LDD175 and Corporal Smooth Muscle Tone
7
Figure 4 In vivo animal model of cavernous nerve stimulation to evaluate erectile function after intravenous administration of LDD175 and udenafil under submaximal EFS (2 Hz, 1 V, and 60 seconds) and maximal EFS (10 Hz, 6 V, and 60 seconds). (A) Maximal ICP (n = 8, 48.9 ± 5.4 [control] vs. 63.5 ± 5.8 [LDD175] vs. 66.2 ± 8.3 [udenafil], P > 0.05). (B) Maximal ICP/ABP ratio (n = 8, 41.0 ± 5.0 [control] vs. 50.2 ± 5.3 [LDD175] vs. 55.7 ± 6.9 [udenafil], P > 0.05). (C) AUC of the ICP/ABP ratio (n = 8, 1,093.7 ± 123.1 [control] vs. 1,612.1 ± 135.6 [LDD175] vs. 1,657.5 ± 122.3 [udenafil], P < 0.05). *P < 0.05 vs. control The meaning of n was the number of rats. ABP = arterial blood pressure; AUC = area under curve; EFS = electrical field stimulation; ICP = intracavernosal pressure.
P < 0.05), although 30 μM NS1619 did not change the currents (n = 7, 1.5 ± 0.6 pA/pF, P = 0.6, Figure 3D). These results demonstrated that LDD175 might be activated under physiological conditions as well, unlike NS1619.
In Vivo Nerve Stimulation Rat Model With submaximal EFS, intravenous administration of LDD175 improved the erectile function of rats in terms of area under the curve (AUC) of the ICP/ABP ratio (n = 8, 1,093.7 ± 123.1 [control] vs. 1,612.1 ± 135.6 [LDD175] vs. 1,657.5 ± 122.3 [udenafil], P = 0.01). There were trend in favor of LDD175 compared with control but no significant difference in the maximal ICP (n = 8, 48.9 ± 5.4 [control] vs. 63.5 ± 5.8 [LDD175], P = 0.08) and maximal ICP/ABP ratio (n = 8, 41.0 ± 5.0 [control] vs. 50.2 ± 5.3 [LDD175], P = 0.23). There was no difference between the effect of 10 mg/kg LDD175 and 1 mg/kg udenafil regarding all aspects of interest, including maximal ICP (63.5 ± 5.8 [LDD175] vs. 66.2 ± 8.3 [udenafil], P = 0.80), maximal ICP/ ABP ratio (50.2 ± 5.3 [LDD175] vs. 55.7 ± 6.9 [udenafil], P = 0.58), and the AUC of the ICP/ABP ratio (1,612.1 ± 135.6 [LDD175] vs. 1,657.5 ± 122.3 [udenafil], P = 0.85) (Figure 4). Discussion
The main results of the current study were that LDD175 relaxed penile CSM strips precontracted
by PE in organ bath studies, and this relaxation was completely blocked by IbTX, a BKCa channel blocker. Whole-cell patch-clamp recording was used to measure BKCa currents after activation with 30 μM LDD175 and inhibition with IbTX. Moreover, enhanced erectile responses shown with intravenous administration of LDD175 were demonstrated using an in vivo cavernous nerve stimulation study. BKCa channels are considered to be key players in maintaining normal vasomotor tone by regulating potassium efflux. Hyperpolarization plays an important role in vascular reactivity by providing negative feedback upon excessive constriction [8]. In addition, diverse ion channels have been related to the relaxation of smooth muscle. Potassium channels are abundantly expressed in the smooth muscles of diverse organs, including the penis, where they play an important role in the control mechanism of the contraction of smooth muscle cells [25]. Adenosine triphosphate sensitive potassium channel (KATP) openers, compounds first developed for overactive bladders, also activated KATP channels. However, this compound was activated in the cardiovascular systems and brought about unwanted hemodynamic side effects [26]. Compared with the KATP channels, BKCa channels are known to be less expressed in cardiovascular tissue [8]. Thus, fewer cardiovascular adverse events might be expected with the use of BKCa channel openers. J Sex Med **;**:**–**
8 Penile erection is mainly mediated by the cyclic guanosine monophosphate/nitric oxide (NO) pathway. The vasodilator effects of NO are partly mediated by the activation of BKCa channels [27– 29]. Kun et al. reported that the relaxant responses elicited by sildenafil involve the activation of BKCa channels in both the intracavernosal artery and human CSM strips [30]. A recent report also showed that the activation of BKCa improve vasodilatory capacity of PDE5 in the human penile vessels and enhance erectile responses in vivo. They concluded that therapeutic potential of BKCa channel activator might improve the efficacy of PDE5 in the management of ED [29]. We also verified that 10−5 M LDD175 plus 10−6 M udenafil had more relaxation effects than 10−5 M LDD175 or 10−6 M udenafil only. These results suggest that the mechanism of action of LDD175 could be augmented by PDE5 or vice versa and that LDD175 and PDE5 had additive effects on relaxation. Using cultured human CSM cells, we also investigated whole-cell K+ currents in the presence of 30 μM NS1619, a typical BKCa channel opener, in an attempt to compare it with 30 μM LDD175. At a holding potential of +60 mV, peak current densities were induced by NS1619 and inhibited by IbTX. At −40 mV, current densities did not form with NS1619, which is more similar to normal cell capacitance, although LDD175 induced a peak current. The activation threshold of BKCa channel currents was shifted by LDD175 to more negative voltages, which were close to the resting potential of the cells. This in vitro electrophysiological outcome showed that LDD175 activated BKCa channels under more physiological conditions (−40 mV) in contrast to NS1619. A more efficient effect of LDD175 on relaxing penile CSM would be expected compared with NS1619. In vivo cavernous nerve stimulations were conducted in a series of submaximal (control, in the presence of LDD175 and udenafil) and maximal stimulations in each experiment. In the current study, intravenous administration of LDD175 (10 mg/kg) significantly enhanced erectile response during electrical stimulation with regard to the AUC of the ICP/ABP ratio. These increases were also elicited with the administration of udenafil (1 mg/kg), and there was no difference between LDD175 and udenafil with respect to the ICP/ABP ratio and the AUC of the ICP/ABP ratio. Drug-related adverse reactions should be of concern in the field of new drug development. Some KATP channel openers (including croJ Sex Med **;**:**–**
Sung et al. makalim, ZD6169, and WAY-133537) caused significant decreases in arterial pressure at doses effective on the bladder and that these channel openers exhibited low bladder selectivity [26]. In contrast, BKCa channel openers seem to have fewer side effects in the cardiovascular system due to the lower expression of BKCa channels in heart tissue [8]. Pena et al. [15] reported an in vivo hemodynamic assessment of LDD175 using Wistar-Kyoto rats. In their experiments, the aortic rings of rats were not relaxed by LDD175. In addition, BP and heart rate changes following intravenous administration of the compound were evaluated, showing that 10 mg/kg LDD175 did not cause any significant change in BP or heart rate. The current study also demonstrated that BP fluctuation and hemodynamic instability during in vivo cavernous nerve stimulation were not observed. In the limitations, this study was to verify the effectiveness of LDD175 on the relaxation of cavernosal smooth muscle, not on the safety aspect. So, the current study lacked the safety assessment. Further experiments should be followed to measure the safety assessment of LDD175. In addition to that, the potential use of LDD175 will be applied in the preclinical and clinical setting. Second, the investigation about tissue selectivity of corpus cavernosum vs. vascular smooth muscle should be determined. Lastly, the evaluation of combined administration of LDD175 and udenafil in the in vivo setting will be valuable. In conclusion, present study provides evidence that LDD175, a novel opener of BKCa channels, leads to a concentration-dependent relaxation of erectile tissue in CSM strips precontracted by PE 10−5 M in vitro. This relaxation is endothelium independent and occurs primarily via BKCa channel opening. Moreover, enhanced erectile function of the in vivo animal model using cavernous nerve electrical stimulation tests was verified. These responses are comparable with those seen with udenafil. LDD175 may also have an additive effect with udenafil on the relaxation of smooth muscle. The results suggest that LDD175 might be a new candidate in ED treatment. Further investigation should be pursued to apply LDD175 to clinical settings. Acknowledgment
This study was supported by a grant of the Korean Health Technology R&D Project, Ministry of Health & Welfare, Republic of Korea (HI13C0104).
LDD175 and Corporal Smooth Muscle Tone Corresponding Author: Sung Won Lee, MD, PhD, Department of Urology, Samsung Medical Center, Samsung Biomedical Research Institute, Sungkyunkwan University School of Medicine, 81 Irwon-ro, Gangnamgu, Seoul 135-710, Korea. Tel: +82-2-3410-3559; Fax: +82-2-3410-3027; E-mail: [email protected] Conflict of Interest: The author(s) report no conflicts of interest.
Statement of Authorship
Category 1 (a) Conception and Design Hyun Hwan Sung; Sung Won Lee (b) Acquisition of Data Hyun Hwan Sung; Seol Ho Choo; Mee Ree Chae; Su Jeong Kang (c) Analysis and Interpretation of Data Hyun Hwan Sung; Seol Ho Choo; Deok Hyun Han; Mee Ree Chae; Su Jeong Kang; Sung Won Lee
Category 2 (a) Drafting the Article Hyun Hwan Sung (b) Revising It for Intellectual Content Chul-Seung Park; Insuk So; Jong Kwan Park; Sung Won Lee
Category 3 (a) Final Approval of the Completed Article Insuk So; Jong Kwan Park; Sung Won Lee
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