The Effects of the Combined Use of a PDE5 Inhibitor and Medications for Hypertension, Lower Urinary Tract Symptoms and Dyslipidemia on Corporal Tissue Tone

Home , Mirodenafil, PDE5 inhibitor

ORIGINAL ARTICLE The effects of the combined use of a PDE5 inhibitor and medications for hypertension, lower urinary tract symptoms and dyslipidemia on corporal tissue tone

JH Lee1,2, MR Chae1,2, JK Park1,3, JH Jeon1,4 and SW Lee1,2

ED is closely associated with its comorbidities (hypertension, dyslipidemia and lower urinary tract symptoms (LUTS)). Therefore, several drugs have been prescribed simultaneously with PDE5 inhibitors. If a specific medication for ED comorbidities has enhancing effects on PDE5 inhibitors, it offers alternative combination therapy in nonresponders to monotherapy with PDE5 inhibitors and allows clinicians to treat ED and its comorbidities simultaneously. To establish theoretical basis of choosing an appropriate medication for ED and concomitant disease, we examined the effects combining a PDE5 inhibitor with representative drugs for hypertension, dyslipidemia and LUTS on relaxing the corpus cavernosum of rabbits using the organ- bath technique. The effect of mirodenafil on relaxing phenylephrine-induced cavernosal contractions was significantly enhanced À4 À6 À6 À7 À9 by the presence of 10 M losartan, 10 M nifedipine, 10 M amlodipine, 10 M doxazosin and 10 M tamsulosin (Po0.05). The maximum relaxation effects were 47.2±3.8%, 57.6±2.6%, 64.0±3.7%, 76.1±5.7% and 71.7±5.4%, respectively. Enalapril and simvastatin had no enhancing effects. The relaxation induced by sodium nitroprusside alone (39.0±4.0%) was significantly À4 enhanced in the presence of the 10 M losartan (66.0±6.0%, Po0.05). Tetraethylammonium (1 mM) significantly inhibited the enhancement effects of tamsulosin and doxazosin on mirodenafil-induced relaxation (doxazosin: 76.1±5.7% vs 45.3±2.3%; tamsulosin: 71.7±5.4% vs 48.1±3.5%). On the basis of these findings, losartan seemed to induce synergistic effects through an interaction with nitric oxide. In addition, K þ channel activation could be one of the mechanisms for the synergistic effect of combining mirodenafil with doxazosin or tamsulosin. We believe that the combination of a PDE5 inhibitor with losartan, nifedipine, amlodipine, doxazosin or tamsulosin could be a pharmacologic strategy for simultaneously treating ED and its comorbidities and increasing response rates to PDE5 inhibitors.

International Journal of Impotence Research (2012) 24, 221--227; doi:10.1038/ijir.2012.19; published online 5 July 2012 Keywords: comorbidity; ED; PDE5 inhibitor

INTRODUCTION Considering the aforementioned investigational and clinical ED is a common condition, with a reported prevalence of 52% in reports, we hypothesized that combining a PDE5 inhibitor with men aged 40--70 years in the United States1 and 49% in men aged antihypertensive drugs, alpha-adrenergic blockers or hypolipi- 50--80 years in Europe.2 It can affect patients’ lives in different demic agents might relax the corpus cavernosum synergistically. ways, including disrupting interpersonal relationships, interfering There is little information available in the literature concerning with sexual life, causing problems with partners and increasing these issues. Therefore, we examined the effects of combining stress, all of which make ED a major quality of life issue.3 a PDE5 inhibitor with antihypertensive drugs, alpha-adrenergic ED has multifactorial causes and is associated with several blockers and hypolipidemic agents on the in vitro relaxation of the chronic diseases, such as hypertension4--7 dyslipidemia,8 and lower rabbit corpus cavernosum. urinary tract symptoms (LUTS).2,9--13 Therefore, several drugs have been prescribed simultaneously with PDE5 inhibitors in ED patients. Several investigational and clinical reports have sug- MATERIALS AND METHODS gested that some types of medication, including antihypertensive Corporal tissue strip preparation drugs, alpha-adrenergic blockers and hypolipidemic agents, can All animal experiments were conducted with the approval of the Institute help treat ED. In vitro studies examining alpha-adrenergic for Animal Care and Use Committee of the Samsung Medical Center (Seoul, blockers,14,15 calcium channel blockers16,17 and angiotensin-con- South Korea). Sexually mature male New Zealand white rabbits (2.2± verting enzyme inhibitors18 have shown direct relaxation or induced 0.3 kg) were killed with an air embolism in the ear vein. Each entire penis increased relaxation in response to electrical ﬁeld stimulation was then surgically excised and cleaned by removing the corpus (EFS). Angiotensin receptor blockers have also been shown to spongiosum and urethra. The corporal tissue was subsequently carefully improve sexual function19--21 in clinical trials and have improved dissected from the surrounding tunica. Three to four corporal strips of endothelial function in hypercholesterolemic rats.22 approximately equal size (2 Â 2 Â 10 mm) were obtained from each penis

1Genitourinary Disease Oriented Translational Research, Seoul, Korea; 2Department of Urology, Samsung Medical Center, Sungkyunkwan University School of Medicine, Seoul, Korea; 3Department of Urology, Jeonbuk National University of Medicine, Jeonju, Korea and 4Department of Physiology, Seoul National University School of Medicine, Seoul, Korea. Correspondence: SW Lee, Department of Urology, Samsung Medical Center, Sungkyunkwan University School of Medicine #50 Ilwon-dong, Kangnam-ku, Seoul 135-710, Korea. E-mail: [email protected] Received 19 August 2011; revised 21 February 2012; accepted 20 April 2012; published online 5 July 2012 Use of PDE5 inhibitor on corporal tissue tone JH Lee et al 222 À5 À6 À7 À7 À6 and were separately prepared for organ-bath studies. Each corporal strip 10 M), amlodipine (10 or 10 M), nifedipine (10 or 10 M), doxazosin À7 À6 À10 À9 À6 was tied with silk in one organ chamber, with one end fixed to a tissue (10 or 10 M), tamsulosin (10 or 10 M) or simvastatin (10 or À5 À9 À5 holder and the other secured to a force transducer. The latter was 10 M). The relaxation effects of mirodenafil (10 to 10 M) were then connected to an appropriately calibrated four-channel polygraph (Power- studied on PE-induced precontracted cavernosal strips. Lab; ADInstruments, Sydney, NSW, Australia) in which the transducer In the second series of experiments, to ascertain whether the synergetic output was recorded. The corporal strips were kept in the organ baths with relaxation effects of alpha-adrenergic blockers and mirodenafil were Krebs solution maintained at 37 1C by a thermoregulated water circuit and mediated through K þ channel activation, we investigated the response of À5 by continuously bubbling with a mixture of 95% O2 and 5% CO2. Each mirodenafil (10 M) on PE-induced tone after incubating the tissue with À9 À6 corporal strip was stretched to an optimal isometric tension of 1.0 g and tamsulosin (10 M) or doxazosin (10 M) for 20 min. After three washes at À5 was equilibrated for 90 min. During the equilibration period, the tissues 10 min intervals, the relaxation effects of mirodenafil (10 M) were studied were washed with fresh Krebs solution every 30 min, and the tension was on PE-induced precontracted cavernosal strips of the corpus tissue þ adjusted if necessary. exposed in the organ bath to 1 mM tetraethylammonium (TEA), a K channel inhibitor, for 40 min. We then incubated the tissue with tamsulosin À9 À6 Organ-bath studies in vitro (10 M) or doxazosin (10 M) for 20 min. Finally, we compared the relaxa- For antihypertensive drugs, we used losartan, an angiotensin II receptor tion magnitude of pre- and post-TEA incubation. Lastly, the following experiment was conducted to confirm the direct antagonist that blocks the activation of angiotensin II AT1 receptors, and enalapril, which inhibits the angiotensin-converting enzyme, a component effect of losartan on the corpus cavernosum. Control tissue strips were precontracted with PE, and cumulative response curves were constructed of the blood pressure-regulating renin--angiotensin system. We also used À9 À5 for sodium nitroprusside (SNP) (10 to 10 M). The tissues were then nifedipine and amlodipine, which block the calcium channels in cardiac À5 muscle and blood vessels. For LUTS, we used doxazosin and tamsulosin. washed three times, followed by the addition of losartan (10 M) to the Tamsulosin is a selective a receptor antagonist that has preferential organ bath for 20 min. The tissues were recontracted with PE, and cumu- 1 lative response curves were again constructed for SNP. We then compared selectivity for the a1A and a1D receptors. Doxazosin is a non-selective a1 receptor antagonist. Simvastatin was used as a class of drugs to lower the the relaxation magnitude of pre- and post-losartan incubation. cholesterol levels by inhibiting the enzyme HMG-CoA reductase, which has a central role in the production of cholesterol in the liver. Lastly, mirodenafil was used for PDE5 inhibitor and the receptor selectivity of Solution and drugs 23 --1 mirodenafil is summarized in Table 1. The Krebs solution had the following composition: NaCl, 118 mM l ; --1 --1 --1 --1 In the first series of experiments, the following experiment was con- NaHCO3,25mM l ; glucose, 5.6 mM l ; KCl, 4.7 mM l ;KH2PO4, 1.2 mM l ; --1 --1 ducted to ascertain whether amlodipine, nifedipine, enalapril, doxazosin, MgSO47H2O, 1.17 mM l ; and CaCl2 2H2O, 2.5 mM l . Mirodenafil and tamsulosin or simvastatin enhanced mirodenafil-induced relaxation in the enalapril were provided by SK (Seoul, South Korea), and tamsulosin and corpus cavernosum. The organ-bath concentrations have been decided doxazosin were provided by Astellas (Seoul, South Korea) and Pfizer (Seoul, based upon the human plasma level of each drug, and it was lower than South Korea), respectively. Simvastatin, amlodipine, losartan and nifedipine the maximal plasma concentration of each drug (Table 2).24--31 Initially, the were obtained from Hanmi (Seoul, South Korea). The stock solutions of the relaxation effect of mirodenafil was studied with a cumulative addition at drugs were prepared in dimethylsulfoxide, except for mirodenafil and À9 À5 À5 concentrations ranging from 10 to 10 M, with a plateau of a 10 M losartan, which were dissolved in distilled water. The final concentration of phenylephrine (PE)-induced contraction for the control group. After three dimethylsulfoxide was o0.1%, and this concentration did not affect the washes at 10 min intervals, the strips were incubated with one of the smooth muscle tone. All other chemicals were purchased from Sigma À5 À4 À6 following drugs for 20 min: losartan (10 or 10 M), enalapril (10 or Chemical (St Louis, MO, USA).

Table 1. Selectivity of PDE5 inhibitors against human phosphodiesterase families

IC 50 (nM) /(selectivity ratio)

Sidenafil Vardenafil Tadalafil Mirodenafil

PDE1 281 (80) 70 (500) 430 000 (44450) 16 400 (48 520) PDE3 16 200 (4630) 41000 (47140) 4100 000 (414 800) 86 500 (255 917) PDE5 3.50 (1) 0.14 (1) 6.74 (1) 0.33 (1) PDE6 6 (11) 3.5 (25) 1260 (187) 10.2 (31) PDE11 2730 (780) 162 (1160) 37 (5) 3750 (11 360)

Selectivity ratio ¼ IC50 for other PDE/IC50 for PDE5.

Table 2. Comparison of the maximal plasma concentration after administration of clinical dose with the organ bath concentrations

Mirodenaﬁl Losartan Nifedipine Amlodipine Doxazosin Tamsulosin Enalapril Simvastatin

Clinical dose (mg) 100.0 100.0 10.0 10.0 2.0 0.4 10.0 40.0 Cmax (ng ml--1) 373.40 757.00 79.00 5.88 17.97 8.18 140.00 3.24 --1 À4 À3 À4 À5 À5 À5 À4 À6 Cmax (M l ) 7.0 Â 10 1.6 Â 10 2.3 Â 10 1.0 Â 10 3.3 Â 10 1.8 Â 10 2.8 Â 10 7.7 Â 10 Organ-bath concentration 10À9--10À5 10À5--10À4 10À7--10À6 10À6--10À7 10À7--10À6 10À10--10À9 10À6--10À5 10À6--10À5 --1 (M l ) Abbreviation: Cmax, maximal plasma concentration. The data are presented as the mean.

International Journal of Impotence Research (2012), 221 -- 227 & 2012 Macmillan Publishers Limited Use of PDE5 inhibitor on corporal tissue tone JH Lee et al 223 Statistical analysis cavernous smooth muscle function because, in contrast to other All data are expressed as means±s.e. of the mean, where n represents the drugs, only a few have verified the direct effect of losartan on number of strips. For the analysis, a paired t-test and a repeated measures corporal smooth muscle. Cumulative response curves constructed analysis of variance were used, followed by the Bonferroni’s post hoc test. for SNP showed that tissue strip relaxation was significantly P-values of 0.05 or lower were considered statistically significant. The enhanced by losartan in six preparations (paired Student’s t-test, values of the relaxation responses were given as a percent ratio of the level Po0.05, Figure 1). The maximum responses to SNP (39.0±4.0%) of tension at the maximal relaxation to the level of maximum contraction were significantly enhanced by the presence of losartan (66.0± by PE. 6.0%, Po0.05). Consequently, it was confirmed that losartan enhanced NO-induced relaxation in rabbit corpus cavernous smooth muscle relaxation. This could be one mechanism, in enhancing mirodenafil-induced relaxation. RESULTS Angiotensin receptor blocker Losartan alone did not significantly affect the tone of the PE- Calcium channel blocker precontracted tissues during consecutive experiments. Mirodenafil Nifedipine, an L-type calcium channel blocker, and amlodipine, an À9 À5 from 10 to 10 M induced a concentration-dependent relaxation L- and N-type calcium channel blocker, did not significantly affect of the PE-induced contraction of the cavernosal strips. The effect of PE-induced contraction. Preincubation with nifedipine (10À7 and À6 mirodenafil on relaxing PE-induced cavernosal contractions was sig- 10 M) enhanced the relaxation of mirodenafil significantly (Po0.05, À4 nificantly enhanced by the presence of 10 M losartan in the organ Figure 2). The maximum effect was a 30.3±3.4% inhibition for the bath (Po0.05, Figure 1, Table 3). The maximum effect was a 32.8± PE-induced contraction of mirodenafil vs 41.9±1.7% for pre- À7 2.0% inhibition of PE-induced contraction for mirodenafil alone vs incubation with 10 M nifedipine (Po0.05) and 57.6±2.6% for À4 À6 47.2±3.8% for the preincubation of 10 M losartan (Po0.05). preincubation with 10 M nifedipine (Po0.05). Preincubation Other organ-bath studies of losartan were conducted to with amlodipine also increased the relaxation of mirodenafil on determine the losartan/nitric oxide (NO) interaction on corpus PE-precontracted strips (Po0.05, Figure 2, Table 3). The maximum

abLog [mirodenafil] : M Log [SNP] : M 9 8765 -9-8-7-6 0.0% 0.0%

20.0% 20.0%

* 40.0% 40.0% * * * 60.0% 60.0% Relaxation (%) Relaxation (%) *

80.0% Control 80.0% Control Losartan 10-5 M Losartan 10-5 M Losartan 10-4 M 100.0% 100.0% Figure 1. (a) The influence of losartan on the relaxation induced by mirodenafil on PE-induced cavernosal contractions. The concentration- À5 À4 response curves of mirodenafil were performed in the presence of 10 or 10 M losartan on rabbit cavernosal strips (n ¼ 6). (b) The influence of losartan on the relaxation induced by SNP on PE-induced cavernosal contractions. The concentration-response curves of SNP were À5 performed in the presence of 10 M losartan on rabbit cavernosal strips (n ¼ 6). The data are presented as the mean±s.e. of the mean. A repeated measures analysis of variance was followed by Bonferroni’s post hoc test (a); paired t-test (b); *o0.05 compared with control; SNP: sodium nitroprusside.

Table 3. Effects of losartan, nifedipine, amlodipine, tamsulosin, doxazosin, enalapril and simvastatin on rabbit corpus cavernosum relaxations evoked by mirodenaﬁl

Relaxation (%)

Losartan (M) Nifedipine (M) Amlodipine (M) Tamsulosin (M) Doxazosin (M) Enalapril (M) Simvastatin (M) 10À5 10À4 10À7 10À6 10À7 10À6 10À10 10À9 10À8 10À7 10À5 10À5

Mirodenaﬁl (M) 10À9 5.9 8.6 10.6 17.9* 7.5 16.5 8.8 17.5* 11.4 24.7* 9.3 7.6 10À8 13.5 19.0 21.1* 33.1* 19.0 31.0* 18.8 36.5* 23.4 42.2* 16.8 19.3 10À7 20.0 28.3* 28.6* 41.6* 27.9 43.2* 27.5 50.3* 33.2 54.9* 23.0 23.5 10À6 26.8 39.1* 36.0* 51.2* 36.5 54.7* 34.2 61.0* 43.3 65.7* 30.2 28.8 10À5 36.6 47.2* 41.9* 57.6* 45.4 64.0* 45.7 71.7* 50.0 76.1* 34.3 34.6

Data were mean; *Po0.05 compared with the respective control.

& 2012 Macmillan Publishers Limited International Journal of Impotence Research (2012), 221 -- 227 Use of PDE5 inhibitor on corporal tissue tone JH Lee et al 224 a Log [mirodenafil] : M b Log [mirodenafil] : M -9 -8 -7 -6 -5 -9 -8 -7 -6 -5 0.0% 0.0%

* 20.0% 20.0% + * * 40.0% + * 40.0% * + # * * 60.0% +# 60.0%

Relaxation (%) + # Relaxation (%) * * 80.0% Control 80.0% Control -7 Nifedipine 10-7 M Amlodipine 10 M Nifedipine 10-6 M Amlodipine 10-6 M 100.0% 100.0% Figure 2. (a) The influence of nifedipine on the relaxation induced by mirodenafil on PE-induced cavernosal contractions. The concentration- À7 À6 response curves of mirodenafil were performed in the presence of 10 or 10 M nifedipine on rabbit cavernosal strips (n ¼ 6). (b) The influence of amlodipine on the relaxation induced by mirodenafil on PE-induced cavernosal contractions. The concentration-response curves À8 À7 of mirodenafil were performed in the presence of 10 or 10 M amlodipine on rabbit cavernosal strips (n ¼ 6). The data are presented as the mean±s.e. of the mean. A repeated measures analysis of variance was followed by Bonferroni’s post hoc test; *o0.05 compared with control; þ # À7 o0.05 compared with control; o0.05 compared with 10 M nifedipine.

a Log [mirodenafil] : Mb Log [mirodenafil] : M -9 -8 -7 -6 -5 -9 -8 -7 -6 -5 0.0% 0.0%

20.0% 20.0% * * 40.0% 40.0% * *

60.0% Relaxation (%) 60.0%

Relaxation (%) * * * Control Control * 80.0% * 80.0% Tamsulosin 10-10 M Doxazosin 10-8 M * Tamsulosin 10-9 M Doxazosin 10-7 M 100.0% 100.0% Figure 3. (a) The influence of tamsulosin on the relaxation induced by mirodenafil on PE-induced cavernosal contractions. The concentration- À10 À9 response curves of mirodenafil were performed in the presence of 10 or 10 M tamsulosin on rabbit cavernosal strips (n ¼ 6). (b) The influence of doxazosin on the relaxation induced by mirodenafil on PE-induced cavernosal contractions. The concentration-response curves of À8 À7 mirodenafil were performed in the presence of 10 or 10 M doxazosin on rabbit cavernosal strips (n ¼ 6). The data are presented as the mean±s.e. of the mean. A repeated measures analysis of variance was followed by Bonferroni’s post hoc test; *o0.05 compared with control.

effect was 31.4±1.7% inhibition for the PE-induced contraction described for alpha-adrenergic blockers32 and PDE5 inhibitors.33 À6 þ of mirodenafil vs 64.0±3.7% for preincubation with 10 M As shown in Figure 4, the nonspecific K channel blocker tetrae- nifedipine. thylammonium (1 mM, n ¼ 6, Po0.05) significantly inhibited the enhancement effect of tamsulosin and doxazosin on mirodenafil- þ Alpha-adrenergic blocker induced relaxation. We suggest that K channel activation with alpha-adrenergic blockers and mirodenafil is one of the action Tamsulosin, a selective a and a receptor antagonist, and 1A 1D mechanisms in this enhancing effect. doxazosin, a nonselective alpha-receptor antagonist, did not significantly affect the tone of the PE-precontracted tissues during the consecutive experiment compared with controls. Preincuba- Angiotensin-converting enzyme inhibitor À9 tion with tamsulosin (10 M) enhanced the relaxation effects of Enalapril did not significantly affect the tone of the PE- mirodenafil significantly (Po0.05, Figure 3, Table 3). The maximum precontracted tissues. Cumulative doses of mirodenafil induced effect was 32.7±7.6% inhibition of the PE-induced contraction of concentration-dependent relaxation in contracted corporal tissue À9 À9 À8 mirodenafil vs 71.7±5.4% for pre-incubation with 10 M tamsu- as follows: 9.0±1.3% at 10 M, 16.7±2.2% at 10 M, 23.1±2.8% À7 À7 À6 À5 losin (Po0.05). Preincubation with doxazosin (10 M) also enhanced at 10 M, 28.7±2.9% at 10 M and 34.6±3.2% at 10 M (n ¼ 6). À5 the relaxation effects of mirodenafil significantly (Po0.05, Figure 3). Preincubation with enalapril (10 M)didnotenhancethe The maximum effect was 32.1±7.2% inhibition of the PE-induced relaxation effects of mirodenafil, as shown in the following À9 À8 contraction with mirodenafil vs 76.1±5.7% for preincubation with percentages: 9.3±1.3% at 10 M, 16.8±1.8% at 10 M, À7 À7 À6 10 M doxazosin. 23.0±2.0% at 10 M, 30.2±2.1% at 10 M and 34.3±2.3% at þ À5 À6 We investigated the role of K channel opening with respect 10 M (n ¼ 6, P40.05, Table 3). Preincubation with 10 M to the synergistic effects of mirodenafil and alpha-adrenergic enalapril did not significantly suppress the relaxation effects blockers because the K þ channel-opening effect has been of mirodenafil.

International Journal of Impotence Research (2012), 221 -- 227 & 2012 Macmillan Publishers Limited Use of PDE5 inhibitor on corporal tissue tone JH Lee et al 225 -9 -9 a Doxazosin 10-7 M TEA+Doxazosin 10-7 M b Tamsulosin 10 M TEA+Tamsulosin 10 M 0.0% 0.0%

20.0% 20.0%

40.0% 40.0% * 60.0% 60.0% *

80.0% 80.0%

100.0% 100.0% À5 À7 Figure 4. (a) The influence of TEA (1 mM) on the relaxation induced by 10 M mirodenafil and preincubated with 10 M doxazosin on PE- À5 induced cavernosal contractions. (n ¼ 6). (b) The influence of TEA (1 mM) on the relaxation induced by 10 M mirodenafil and preincubated À9 with 10 M tamsulosin on PE-induced cavernosal contractions (n ¼ 6). The data are presented as the mean±s.e. of the mean; paired t-test; *a significant difference at Po0.05. TEA, tetraethylammonium.

À8 À7 --1 À10 À9 --1 HMG-Co A reductase inhibitor doxazosin (10 ,10 M l ), tamsulosin (10 ,10 M l ), À6 À5 --1 À6 À5 --1 Simvastatin did not signicantly affect the tone of the PE- enalapril (10 ,10 M l ) and simvastatin (10 ,10 M l ) were precontracted tissues. The cumulative dosing of mirodenafil- not effective in relaxing PE-induced contractions in a rabbit corpus induced concentration-dependent relaxation in contracted cor- cavernosum if applied alone. However, losartan, nifedipine, À9 À8 poral tissue was 6.9±3.1% at 10 M, 17.6±4.5% at 10 M, amlodipine, tamsulosin and doxazosin, when applied in combination À7 À6 22.6±5.3% at 10 M, 29.8±5.0% at 10 M and 33.5±4.2% at with mirodenafil, did potentiate mirodenafil-induced relaxation. À5 À5 10 M (n ¼ 6). Preincubation with 10 M simvastatin did not In clinical settings, this result would suggest that angiotensin À9 enhance the relaxation effects of mirodenafil: 7.6±2.1% at 10 M, receptor blockers or calcium channel blockers could be better À8 À7 À6 19.3±1.0% at 10 M, 23.5±1.6% at 10 M, 28.8 ±2.0% at 10 M options than other types of antihypertensive drugs if hypertensive À5 and 34.6±2.5% at 10 M (n ¼ 6, P40.05, Table 3). Preincubation patients need a PDE5 inhibitor for intercourse. Because there is a À6 with 10 M of simvastatin also did not significantly suppress the considerable population of patients with ED who do not respond relaxation effects of mirodenafil. to PDE5 inhibitors and because there is an increased prevalence of ED in patients with hypertension, the combination of a PDE5 inhibitor with an angiotensin receptor blocker or a calcium channel blocker could be a pharmacologic strategy to increase DISCUSSION the response rates of ED patients with hypertension to PDE5 Several reports have suggested that medication for ED comorbid- inhibitors. ities (hypertension, LUTS and dyslipidemia) can help treat ED. In The enhancing mechanism of angiotensin receptor blockers can the acute application setting of an organ bath, alpha-adrenergic be explained as follows. Recent data suggest that angiotensin II is blockers and calcium channel blockers showed relaxation effects produced and secreted by the human corpus cavernosum via on the corpus cavernous. Contraction induced by 5-hydroxytryp- adrenergic stimulation. Although the production of angiotensin II tamine was significantly impacted by doxazosin.14 Nifedipine from angiotensin I is catalyzed by angiotensin-converting enzyme, and verapamil, which are L-type calcium channel blockers, were other conversion pathways have been identied.38 The degradation effective in relaxing corpus cavernous smooth muscle induced by of angiotensin II by angiotensinase results in a peptide with a half- PE in vitro.16--17 In the animal disease model, calcium channel life of only a few seconds,39 giving credence to the suggestion blockers, angiotensin-converting enzyme inhibitors and angioten- that the human corpus cavernosum is a local angiotensin II sin-receptor blockers showed a re-establishment of ED. The producing paracrine system.40 This is supported by the ndings spontaneous treatment of hypertensive rats with amlodipine or that human corpus cavernosum produces and secretes physiolo- imidapril for 4 weeks induced a relaxation increase in the response gical amounts of angiotensin II, as much as 200fold greater than to EFS of the corpus cavernosum.18 Treating hypercholesterolemic that in plasma,41 and healthy men produce angiotensin II corpus rats with irbesartan also improved endothelial function.22 In clinical cavernous blood levels that are higher during penile detumes- trials, preliminary data from several randomized and open studies cence than tumescence.39 Immunohistochemical staining of have suggested that angiotensin type 1 receptor antagonists may dog and rat corpus cavernous tissue revealed that angiotensin be associated with improvement in sexual function.19--21 II-containing cells are distributed in endothelium and smooth Although the effects of the aforementioned drugs on the muscle.41 In addition, angiotensin II is secreted upon adrenergic corpus cavernosum have been addressed, only a few studies have stimulation by a variety of paracrine cells such as vascular focused on the possible enhancement interaction with PDE5 endothelial cells, vascular smooth muscle cells, cardiocytes and inhibitors.34,35 However, considering that approximately 30--40% granular cells of the juxtaglomerular apparatus.42--44 As the human of all ED patients do not respond to PDE5 inhibitors36 and that corpus cavernosum is considered a specialized vascular tissue,39,41 many ED patients may take several drugs37 for their comorbidities, a similar stimulatory mechanism likely exists in the human penis.40 this issue is important. The enhancing interaction with PDE5 EFS-induced relaxation of corpus cavernous strips was increased inhibitors offers alternative combination therapy in nonrespon- by 29.4% after exposure to guanethidine (inhibitors of the ders to monotherapy with PDE5 inhibitors and allows clinicians to adrenergic pathway), atropine (inhibitors of the cholinergic treat ED and its comorbidities simultaneously. pathway) and indomethacin (inhibitors of prostaglandin path- Therefore, we evaluated the effects of the combined use of a ways) when values before and after losartan were compared.40 PDE5 inhibitor and medication for ED comorbidities (for example, The difference between EFS-induced relaxation of corpus caver- À5 À4 --1 hypertension, LUTS and dyslipidemia). Losartan (10 ,10 M l ), nous strips before and after losartan administration was greater À7 À6 --1 À7 À6 --1 nifedipine (10 ,10 M l ), amlodipine (10 ,10 M l ), (140%) when guanethidine was omitted from the inhibitor

& 2012 Macmillan Publishers Limited International Journal of Impotence Research (2012), 221 -- 227 Use of PDE5 inhibitor on corporal tissue tone JH Lee et al 226 cocktail.40 Guanethidine-induced chemical sympathectomy made citrate is capable of producing cavernosal smooth-muscle relaxa- losartan less effective. This nding suggested angiotensin II released tion by an additional mechanism that may involve a-receptors via adrenergic stimulation had been blocked by losartan. and K þ channel openings.33 In addition, the K þ channel opening In a previous study, angiotensin II caused a dose-dependent effect of phentolamine in the corpus cavernosum has been contraction of cavernous tissue through the activation of angi- previously described.32 In our study, TEA significantly inhibited the otensin type 1 receptors, and this contraction was blocked by the enhancement effect of tamsulosin and doxazosin on mirodenafil- angiotensin type 1 receptor antagonist losartan.40 Another recent induced relaxation. Thus, it may be hypothesized that the K þ study indicated that angiotensin II was a potent stimulator of the channel-opening effect of mirodenafil and alpha-adrenergic smooth-muscle enzyme NAD(P)H oxidase, which stimulates the blockers is one of the possible action mechanisms for enhancing production of reactive oxygen species, such as superoxide.45 the relaxation effect along with alpha-receptor blockage by The interaction of superoxide with NO decreases NO bioavail- mirodenafil and alpha-adrenergic blockers. In addition, effect of ability by promoting superoxide and generating the potent adrenergic blocker in increasing the efficacy of cyclic nucle- oxidative peroxynitrite radical, which is thought to have an otide-dependent vasodilation could be another enhancing important role in vascular pathophysiology.46,47 In this study, the mechanism.34 relaxation of the rabbit corpus cavernosum PE-induced tone by In the present study, we also investigated the effects of an HMG SNP was significantly enhanced in the presence of losartan. A Co-A reductase inhibitor and an angiotensin-converting enzyme similar result has also been reported in recent studies using the inhibitor on mirodenafil-induced relaxation, both of which are human corpus cavernosum.40 Therefore, it may be postulated among the most common drugs simultaneously prescribed with that angiotensin II produced via adrenergic stimulation was PDE5 inhibitors. The role of HMG Co-A reductase inhibitors in blocked by losartan, inhibiting the decreased NO bioavailability relation to RhoA prenylation in the pathophysiology of the effects of angiotensin II. Consequently, the effect of losartan on spontaneously tonic smooth muscle has been investigated.57 NO bioavaiability and its direct relaxing ability by blocking In a previous study, captopril directly enhanced endothelium- angiotensin II may be possible mechanisms for losartan-enhanced dependent relaxation in the rat aorta.58 However, no enhance- mirodenafil-induced relaxation. ment or significant detrimental effects with mirodenafil were Regarding the enhancing mechanism of calcium channel observed in our study. blockers, it has been reported that there are L-type voltage-gated In conclusion, the present study confirms that losartan, calcium channels in the smooth muscle of the corpus cavernosum, nifedipine, amlodipine, tamsulosin and doxazosin enhance and the contraction of this muscle and detumescence of the penis mirodenafil-induced relaxation on PE-contracted corpus caverno- are highly dependent on an increase in the cytosolic concentra- sum of rabbits. We believe that these experimental data could be 2 þ 48,49 À5 À4 tion of Ca . It has also been reported that 10 and 10 M useful for choosing medications for chronic diseases in patients nifedipine, an L-type calcium channel blocker, were effective in using PDE5 inhibitors. relaxing norepinephrine-induced contractions in the rabbit corpus cavernosum.16 A recent study also reported that vardenafil and sildenafil possess direct muscle relaxant potential, possibly via CONFLICT OF INTEREST inhibiting Ca2 þ influx through both receptor-operated and The authors declare no conflict of interest. voltage-dependent Ca2 þ channels.50 Therefore, we assumed that the collaborating effect of mirodenafil and nifedipine on Ca2 þ modulation caused this enhancing effect. Amlodipine, which is an ACKNOWLEDGEMENTS inhibitor of L- and N-type calcium channels, also had enhancing This study was supported by a grant from the Korean Health Technology R & D effects on mirodenafil in the present study. 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