BJUI. Study Type Diagnostic (case series) Level of Evidence 4 OBJECTIVE CONCLUSIONS

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1 2008 The Authors. Journal compilation 2008 BJU International Sexual Medicine PARK et al. BJUI BJU INTERNATIONAL Why a combined intracavernosal injection with trimix and oral sildenafil is reliable therapy in the ultrasonographic evaluation of erectile dysfunction Jong Kwan Park, Jung Sun Park, Sang Bong Jeon, Wan Shou Cui, Sung Zoo Kim*, Kyung Koo Kang and Sung Won Lee Departments of Urology and *Physiology, Medical School, and Institute for Medical Sciences, Chonbuk National University, Institute for Medical Sciences, and Research Institute of Clinical Medicine of Chonbuk National University Hospital Jeonju, Chonbuk, Research Laboratory, Dong A Pharmaceutical company, Yongin, and Department of Urology, Sungkyunkwan University, Seoul, Korea Accepted for publication 8 February 2008 Study Type Diagnostic (case series) Level of Evidence 4 OBJECTIVE To evaluate prospectively and compare the clinical response and the change in nucleotides correlating with haemodynamic changes in the cavernosal arteries after an intracavernosal injection (ICI) with vasoactive agents with or without oral sildenafil in men with erectile dysfunction (ED). PATIENTS AND METHODS In all, 80 patients with ED were prospectively evaluated by clinical assessments, measuring nucleotides in blood plasma and haemodynamics in cavernosal arteries. All patients had colour Doppler ultrasonography (CDU) twice with an interval of 5 h. First, each patient had CDU after ICI with trimix (0.25 ml) or prostaglandin E1 (PGE1, 5 µg), and the second CDU was after ICI trimix given 1 h after oral placebo (group I), sildenafil 25 mg (group II) or 100 mg (group III) and after ICI with PGE1 at 1 h after oral placebo (group IV) or 100 mg sildenafil (group V). Levels of cgmp and camp in peripheral venous and penile cavernosal blood plasma were measured at 15 min after ICI. RESULTS The mean peak systolic velocity (PSV) at 5, 10, 15 min, and resistive index at 10 min in the second CDU after ICI with trimix, were significantly increased in group III. The mean (SEM) levels of cavernosal cgmp were significantly increased in group III and V, from (313.5) to (580.4) and (214.2) to (915.0) fmol/ml, respectively. camp was significantly increased in group V, from (2068.4) to (3684.2) fmol/ml. CONCLUSIONS The haemodynamic changes and cgmp and camp production in the cavernosum were improved by trimix plus sildenafil more than with than PGE1 plus sildenafil or one ICI with trimix or PGE1. The results suggest that ICI with trimix and sildenafil is the best combination for a pharmacological erection test. KEYWORDS intracavernosal injection, colour Doppler ultrasonography, haemodynamic changes, sildenafil, erectile dysfunction INTRODUCTION Erectile dysfunction (ED) is defined as the persistent inability to attain and maintain an erection sufficient to permit satisfactory sexual performance. Since colour Doppler ultrasonography (CDU) was introduced by Lue et al. [1], a pharmacologically induced erection during CDU has become the most reliable test when evaluating the penile vascular system of patients with ED. During penile CDU with an intracavernosal injection (ICI) of vasoactive agents, there is sometimes inadequate smooth muscle relaxation in response to vasoactive agents because of a sympathetic discharge initiated by anxiety about the pain of injection, psychological inhibition by the unfamiliar procedure or an inadequate volume of the ICI [2,3]. To overcome this impaired response a booster injection of vasoactive medication, a high dose of a vasoactive agent, ICI with a mixture of the vasoactive agents, the combination of the ICI with audio-visual stimulation, oral sildenafil or genital stimulation have been used [4,5]. Phosphodiesterase type 5 (PDE5) inhibitors are recognized as first-line therapy for patients with ED; these agents maintain an erection by inhibiting the catabolism of cgmp, resulting in increased cgmp. So that sildenafil can become effective in facilitating erection after oral intake, the activation of the nitric oxide (NO)-cGMP pathway by sexual stimulation is required. Thus, in the absence of sexual stimulation in humans, sildenafil has little effect on cavernosal smooth muscle relaxation and does not cause a rigid penile erection [6]. Prostaglandin E1 (PGE1) is known to increase the intracellular camp concentration, inducing relaxation of the cavernosal smooth muscle and dilatation of cavernosal arteries [7]. Papaverine increases the production of camp and cgmp by an inhibitory action on PDE, although 2008 BJU INTERNATIONAL 102, doi: /j x x 993

2 P ARK ET AL. papaverine acts at many levels, leading to a very complex mode of action in the smooth muscle [8 10]. To the best of our knowledge, no study has evaluated the clinical effectiveness of sildenafil during changes in intracavernosal nucleotides or correlations with cavernosal arterial haemodynamics during CDU using ICI with vasoactive agents. Thus the aim of the present study was to prospectively evaluate and compare the clinical response and changes in nucleotides correlating with haemodynamic changes in the cavernosal arteries after ICI with vasoactive agents, with or without oral sildenafil. PATIENTS AND METHODS The study was approved by the Institutional Review of Board of the Chonbuk National University Hospital, and written informed consent was obtained from every patient before starting the evaluation. The study comprised patients with ED who presented at our sexual dysfunction clinic, referred by other physicians or presenting themselves for further evaluation; in all, 80 consecutive patients with ED were evaluated. Inclusion criteria consisted of the patients with ED for 6 months. After taking a complete history, a physical examination and laboratory tests (serum testosterone, blood glucose, total cholesterol, homocysteine, high-sensitivity C-reactive protein, serum renal and liver profiles, and a complete blood count), all patients were instructed to complete self-administered questionnaires (a Korean version of the International Index of Erectile Function) to evaluate their baseline ED. Patients with ED had a clinical assessment and CDU; the peak systolic velocity (PSV), end-diastolic velocity (EDV), resistance index, RI ((PSV EDV)/PSV) and cavernosal artery diameters were measured (from the both cavernosal arteries) before and at 5, 10 and 15 min after ICI with trimix (5 mg papaverine, 5 µg PGE1, and 0.25 mg phentolamine) or PGE1. The levels of cgmp and camp in peripheral venous and penile cavernosal blood plasma were measured at 15 min after ICI with 0.25 ml trimix or 5 µg PGE1, with or without oral sildenafil. Patients were examined in two sessions, 5 h apart. All patients were assessed with the second CDU after complete detumescence from the first CDU; patients who did not detumesce after the first session or had a greater blood flow than in the first session were excluded. The patients were divided randomly into five groups; each group was exposed to a fixed dose of ICI with trimix or PGE1 and different doses of oral sildenafil before ICI in the second session of CDU. In the first three groups, patients had CDU before and after ICI with trimix; 5 h later each patient had CDU before and after ICI with trimix with no sildenafil (group I), 25 mg sildenafil (group II) or 100 mg sildenafil (group III); in group IV and V, each patient had CDU before and after ICI with PGE1, and 5 h later had CDU before and after ICI with PGE1 with no sildenafil (group IV) or 100 mg sildenafil (group V). All CDU and ICI of vasoactive agents were done by the same examiner in both the first and second sessions. For CDU a class 1 type-b (B&K Medical, Denmark) machine with a 7.0- MHz linear-array transducer was used for obtaining real-time images of the corpus cavernosum, visualization, and Doppler calculations of the blood flow in the cavernosal arteries before and after ICI; CDU was applied to the ventral surface at the penile base [4]. Initially, the penile PSV, EDV, RI and arterial diameter measurements were obtained from both proximal cavernosal arteries. The haemodynamic values were recorded before and at 5, 10 and 15 min after the ICI with PGE1 or trimix. There was no statistically significant difference between the right- and the left-side blood flow. Therefore, PSV, EDV, RI and diameter of the cavernosal arteries were evaluated using the mean values of the left and right cavernosal arteries. Blood samples were collected from antecubital vein before ICI, and then the antecubital vein and corpus cavernosum at 15 min after ICI in the first and second session, respectively. Blood samples were centrifuged and plasma samples maintained frozen at 82 C until analyses. cgmp was measured using an equilibrated radioimmunoassay (RIA), as described previously [11]. Briefly, standards or samples were incubated with diluted cgmp antiserum (Calbiochem-Novabiochem Co., San Diego, CA, USA) and iodinated cgmp ( cpm/ 100 µl) in a sodium acetate buffer (50 mm, ph 4.85) for 24 h at 4 C. The bound form was separated from the free form by charcoal absorption. The RIA for cgmp was done on the day of the experiments, and all samples in an experiment were analysed in one assay. Nonspecific binding was <2.5%; the 50% intercept was at 0.39 ± 0.03 pmol/tube (15 samples). The intra- and interassay coefficients of variations were 6.7 (12 samples) and 8.6% (nine samples), respectively. The mean values of the results were expressed as femtomoles of cgmp generated per milligram protein per millilitre. The production of camp was also measured by equilibrated RIA, as previously described [11]. Briefly, standards or samples were incubated with diluted camp antiserum (Calbiochem-Novabiochem) and 125 I-2-0-mono-succinyl-adenosine 3,5 -c monophosphate tyrosyl methyl ester, cpm/100 µl) in 50 mm sodium acetate buffer (ph 4.8) containing theophylline (8 mm), for 24 h at 4 C. For the acetylation reaction, 5 µl of a mixture of acetic anhydride and triethylamine (1:2) was added to the assay tube before adding antiserum and tracer. The bound form was separated from the free form by charcoal suspension. The RIA for camp was done on the day of the experiments, and all samples from one experiment were analysed in one assay. Non-specific binding was <2.0%; the 50% intercept was at ± 0.79 fmol/ tube (10 samples). The intra- and interassay coefficients of variation were 5.0 (10 samples) and 9.6% (10 samples), respectively. The results are expressed as the mean (SEM), with the statistical significance of differences calculated using one-way ANOVA followed by Bonferroni s multiple comparison test, and CDU with ICI of trimix or PGE1 before and after oral sildenafil compared using Student s paired-t-test; in all tests P < 0.05 was considered to indicate significance. RESULTS The PSV, EDV and RI before and at 5, 10 and 15 min after ICI with trimix in the first and second sessions are shown in Table 1. There were significant increases in the PSV at 5, 10 and 15 min, and in the RI at 10 min after ICI with trimix plus 100 mg sildenafil (group III). The EDV in the second session in the group III was slightly decreased at 5 and 10 min, but not statistically significantly BJU INTERNATIONAL

3 TABLE 1 Data obtained from the cavernosal artery during colour duplex Doppler evaluation in five groups 5 min 10 min 15 min Group Session PSV EDV RI PSV EDV RI PSV EDV RI I First 36.8 (2.52) 6.4 (1.22) 0.80 (0.38) 38.4 (2.71) 6.4 (1.08) 0.80 (0.35) 39 (2.64) 6.3 (0.99) 0.80 (0.03) Second 38.7 (2.70) 6.4 (1.12) 0.80 (0.34) 40.7 (3.02) 6.9 (1.09) 0.80 (0.33) 40.4 (3.33) 6.1 (0.9) 0.80 (0.03) II First 33.1 (1.89) 5.0 (0.64) 0.80 (0.02) 36.0 (2.5) 5.2 (0.67) 0.90 (0.14) 37.2 (2.2) 5.1 (0.54) 0.90 (0.14) Second 35.3 (1.75) 5.5 (0.79) 0.80 (0.02) 41.0 (2.23) 6.0 (0.83) 0.90 (0.17) 41.2 (2.29) 6.0 (0.8) 0.90 (0.18) III First 30.2 (1.54) 4.9 (0.34) 0.83 (0.01) 30.8 (1.4) 4.9 (0.44) 0.84 (0.01) 31.7 (1.4) 4.5 (0.40) 0.86 (0.01) Second 35.9 (1.65) 4.6 (0.45) 0.87 (0.01) 37.8 (1.5) 4.7 (0.38) 0.87 (0.01) 37.2 (1.4) 4.8 (0.35) 0.87 (0.01) P <0.016 <0.001 <0.041 <0.008 IV First 27.0 (2.2) 5.6 (1.32) 0.80 (0.03) 30.2 (2.7) 5.3 (1.07) 0.83 (0.02) 33.3 (2.7) 6.0 (1.25) 0.83 (0.03) Second 30.7 (2.2) 5.2 (0.72) 0.83 (0.01) 31.3 (2.6) 4.8 (0.53) 0.85 (0.01) 31.7 (2.6) 4.9 (0.69) 0.85 (0.10) V First 34.1 (2.6) 4.8 (0.40) 0.85 (0.01) 37.1 (2.6) 4.9 (0.53) 0.87 (0.01) 37.3 (2.3) 5.0 (0.43) 0.86 (0.01) Second 38.0 (2.5) 5.3 (0.60) 0.85 (0.02) 38.0 (2.4) 5.9 (0.79) 0.85 (0.02) 41.6 (2.65) 5.8 (0.62) 0.86 (0.01) TABLE 2 cgmp and camp levels in the cavernosal and peripheral venous blood in the first and second sessions Cavernosal, fmol/ml Venous, fmol/ml Group 1st session 2nd session 1st session 2nd session cgmp I (491.4) (242.0) (514.1) (315.2) II (401.0) (935.4) (652.5) (317.4) III (313.5) (580.4) (545.8) (172.7) IV (370.5) (215.1) (450.1) (128.8) V (214.2) (915.0) (684.4) (403.0)* camp I (3271.1) (2208.8) (2582.6) (1147.1) II (3313.4) (3577.7) (1854.6) (295.7) III (2068.4) (3684.2)* (1732.7) (3110.7) IV (2116.8) (1866.8) (2156.9) (1838.7) V (3908.0) (3420.0) (3662.6) (896.1) *P < 0.05; P < The PSV, EDV and RI before and at 5, 10 and 15 min after ICI with PGE1 in the first and second sessions are also shown in Table 1; in group IV there were increases in PSV at 5 and 10 min and in RI at 5, 10 and 15 min after ICI in the second session. The EDV at 5, 10 and 15 min after ICI with PGE1 in the second session were lower, but not statistically significantly. In group V there were increases in the PSV at 5, 10 and 15 min, and in RI at 5 and 15 min, but not significantly in the second session (Table 1). The level of cgmp in the cavernosal blood plasma in the second session was increased in groups I, II, III and V but not group IV (Table 2). There were statistically significant differences in group III and V. The level of cgmp obtained from venous blood plasma was increased in the second session in all groups, but not statistically significantly. The level of camp in cavernosal blood plasma in the second session was significantly increased in group III (P < 0.05) but decreased in the group IV (P < 0.02; Table 2). DISCUSSION The results of the present study suggest that combined oral sildenafil and ICI with trimix leads to a significant clinical response, by increasing cyclic nucleotides and blood flow in the cavernosal arteries, and more than one ICI with trimix. CDU provides highly reliable and detailed information on penile haemodynamics and vascular anatomy, and is used as an objective test to evaluate penile vascular function in patients with ED [12]. However, the presence of a high state of anxiety can be involved in the failure to reach a complete erectile response during the pharmacological erection test using maximal and supra-maximal doses of trimix or PGE1. As a rigid, good-quality erection might not be achieved in all patients during the test, repeated injections, higher doses of the vasoactive medications, and the combination of the ICI with sexual stimulation and oral sildenafil have been used to overcome the problem and improve erectile quality [4,5,12,13]. The present results showed that the PSV obtained after combined ICI with trimix and oral sildenafil was significantly higher than the PSV after one ICI with trimix. Interestingly, more patients responded better clinically to the combination of ICI with trimix plus oral sildenafil than to the combination of ICI with PGE1 and oral sildenafil. These results suggest that the NO-cGMP pathway is a key modulator of penile erection, although camp is also important in inducing an erection [14,15]. The concentration of camp and cgmp are regulated by different physiological mechanisms; camp is a second messenger, which induces relaxation of penile cavernosal smooth muscle, and PGE1 also induces penile erection by increasing the production of camp [7]. The camp-specific PDE isoenzymes 3 and 4 have been isolated from human cavernosal artery, and PGE1 and forskolin induce relaxation of cavernosal smooth muscle [16]. The use of combined therapy with several agents increases the production 2008 BJU INTERNATIONAL 995

4 P ARK ET AL. of cgmp and camp in the cavernosum, resulting in the most effective treatment for ED. In the present study the combination of oral sildenafil and ICI with trimix significantly increased the levels of cgmp and camp in the cavernosal but not the peripheral blood plasma. These results suggest that cross-talk between PDE5 and PDE3 might be activated during ICI with trimix plus oral sildenafil in the corpus cavernosum, but not in peripheral blood. Sudden increases in intracavernosal cgmp by ICI with trimix and oral sildenafil might have effectively enhanced intracellular camp accumulation in isolated human cavernosal tissues, because increases in cgmp inhibit PDE3 and lead to the accumulation of camp, which also enhances the relaxation of cavernosal smooth muscle induced by cgmp [17 19]. There is cross-talk between cgmp and camp-dependent signal transduction pathways in human cavernosal and cardiac muscle [18]. However, in the present study the level of camp was significantly increased from (2068.4) to (3684.2) fmol/ml (P < 0.05) in cavernosal blood plasma by treatment with trimix plus sildenafil, but not in peripheral blood plasma. High levels of cgmp or camp, which cause cavernosal arterial dilatation resulting in penile erection, is facilitated by NO release that is a direct result of sexual stimulation. In the absence of sexual stimulation, there is minimal NO released from the parasympathetic nonadrenergic noncholinergic (NANC) nerves and the cavernosal endothelium. Signals from the sympathetic nervous system dominate the signal from the parasympathetic NANC nervous system, resulting in a flaccid penis. Therefore, therapy with an oral PDE5 inhibitor plus ICI with trimix induces a powerful signal for erection, which is a response of the NOcGMP and camp pathway. Taken together, we recommend the combination of oral PDE5 inhibitor plus ICI with trimix to evaluate patients with ED when using ICI. Also, this combination might be effective in the patients with ED who do not respond to PDE5 inhibitors or ICI. In conclusion, the combination of oral sildenafil and ICI with trimix significantly improves cavernosal haemodynamics and increases the production of the cgmp and camp in the cavernosal plasma. In addition, the combination might also activate interactions between the cgmp- and campmediated signalling pathways in the cavernosum. The combination of ICI with trimix plus oral PDE5 inhibitor is an excellent method for the pharmacologically induced erection test and possibly an effective therapy in the patients with ED who do not respond to PDE5 inhibitors or ICI. ACKNOWLEDGEMENTS This study was supported by grants from Clinical Trial Center of Chonbuk National University Hospital (2007) and Ministry of Health and Welfare, Republic of Korea (A060520). CONFLICT OF INTEREST None declared. REFERENCES 1 Lue TF, Hricak H, Marich KW, Tanagho EA. Vasculogenic impotence evaluated by high-resolution ultrasonography and pulsed Doppler spectrum analysis. Radiology 1985; 155: Chen J, Greenstein A, Matzkin H. Is a second injection of vasoactive medication necessary during color duplex doppler evaluation of young patients with venoocclusive erectile dysfunction? Urology 2000; 55: Lane BR, Ausmundson SJ, Butler RS, Zippe CD, Jones JS, Lakin MM. Use of a visual analog scale to assess pain of injection with intracavernous injection therapy. J Sex Med 2005; 2: Park K, Kwon DD, Oh BR, Ryu SB, Park YI. Efficacy of virtual glasses in audiovisual sexual stimulation during penile color duplex Doppler ultrasonography. 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5 involved in the regulation of penile smooth muscle tone. World J Urol 2004; 22: Stief CG, Uckert S, Becker AJ et al. Effects of sildenafil on cgmp and camp levels in isolated human cavernous and cardiac tissue. Urology 2000; 55: Waldkirch E, Uckert S, Yildirim H et al. Cyclic AMP-specific and cyclic-gmp specific phosphodiesterase isoenzymes in human cavernous arteriesimmunohistochemical distribution and functional significance. World J Urol 2005; 23: Correspondence: Jong Kwan Park, Department of Urology, Medical School, Chonbuk National University, Jeonju, Korea. rain@chonbuk.ac.kr Abbreviations: ED, erectile dysfunction; CDU, colour Doppler ultrasonography; ICI, intracavernosal injection; PSV, peak systolic velocity; EDV, end-diastolic velocity; RI, resistance index; PDE, phosphodiesterase; NO, nitric oxide; PGE1, prostaglandin E1; RIA, radioimmunoassay; NANC, nonadrenergic noncholinergic BJU INTERNATIONAL 997