ChronicSildenafilImprovesErectileFunctionand Endothelium-dependent Cavernosal Relaxations in Rats: Lack oftachyphylaxis $

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1 European Urology European Urology 47 (2005) ChronicSildenafilImprovesErectileFunctionand Endothelium-dependent Cavernosal Relaxations in Rats: Lack oftachyphylaxis $ Delphine Behr-Roussel a, Diane Gorny a, Katell Mevel a, Stéphanie Caisey a, Jacques Bernabé a, Gillian Burgess b, Chris Wayman b, Laurent Alexandre a, François Giuliano a,c, * a Pelvipharm, Domaine CNRS, 1 Avenue de la terrasse, Bâtiment 5, Gif sur Yvette, France b Pfizer Global Research and Development, Ramsgate Rd, Sandwich, Kent, CT13 9NJ, UK c Groupe de Recherche en Urologie, UPRES, EA 1602, University of Paris South, 63 rue du Général Leclerc, Le Kremlin Bicêtre, France Accepted 17 September 2004 Available online 7 October 2004 Abstract Objectives: Sildenafil is a widely-prescribed effective on-demand treatment of erectile dysfunction (ED). Chronic treatment with sildenafil could help patients with ED. Methods: The effects of an 8-week long treatment with sildenafil (60 mg/kg/d sc) in male Sprague Dawley rats were evaluated on electrically-elicited erectile responses in vivo before and after an acute injection of sildenafil (0.3 mg/ kg iv). In addition, endothelium-dependent and -independent relaxations of strips of corpus cavernosum in vitro were examined. All experiments were performed 36 hours after the last injection of sildenafil. Results: Endothelium-dependent relaxations of cavernosal strips to acetylcholine were enhanced after chronic treatment with sildenafil while relaxations to A23187 or sodium nitroprusside were unchanged. Frequencydependent erectile responses elicited by cavernous nerve stimulation were significantly improved. Moreover, the erectile responses to acute sildenafil were greater in chronically-treated rats with sildenafil. Conclusions: This is the first report providing experimental support for chronic dosing with sildenafil which could be of use for patients that are poor responders to on-demand treatment. Chronic sildenafil may regulate the transduction pathway leading to the activation of enos but has no effect on NO bioavailability or on the cgmp pathway, thereby eliminating a possible concern for tachyphylaxis. # 2004 Elsevier B.V. All rights reserved. Keywords: Erectile dysfunction; Sildenafil; PDE-5; Tachyphylaxis; Chronic administration; Corpus cavernosum; Endothelium 1.Introduction Upon sexual stimulation, penile erection, occurring in response to the activation of pro-erectile autonomic pathways, is greatly dependent on adequate inflow of blood to the erectile tissue and requires coordinated arterial endothelium-dependent vasodilatation and $ This work was supported by a grant from Pfizer and presented in abstract form at the last American Urological Association Annual Meeting (8 13 May 2004, San Fransisco, CA, USA). * Corresponding author. Tel ; fax: address: giuliano@cyber-sante.org (F. Giuliano). sinusoidal endothelium-dependent cavernosal smooth muscle relaxation [1]. NO is the principal peripheral pro-erectile neurotransmitter, released both by parasympathetic-nitrergic autonomic nerves and the sinusoidal endothelium to produce cgmp and relax cavernosal smooth muscle, and resulting ultimately in increased intracavernosal pressure (ICP) [2]. Sildenafil inhibits selectively type 5 cgmp-phosphodiesterase (PDE-5), resulting in an enhanced availability of cgmp in cavernosal tissues, relaxation of smooth muscle and increased blood flow into cavernosal spaces. Sildenafil was the first orally active drug to /$ see front matter # 2004 Elsevier B.V. All rights reserved. doi: /j.eururo

2 88 D. Behr-Roussel et al. / European Urology 47 (2005) treat patients with erectile dysfunction (ED) and has become the standard treatment for ED in more than 110 countries [3]. Although sildenafil has proven to be effective in treating ED, a report has suggested that its prolonged use may produce tachyphylaxis [4]. However, other clinical trials including more patients followed for longer periods of time have demonstrated only a small (less than 5%) patient dropout rate for lack of efficacy [5,6]. These low rates of withdrawal imply that men continued to be satisfied with sildenafil during longterm treatment. Furthermore, tachyphylaxis, in most pharmacological and human settings, does not occur with drugs that are used in an on-demand basis. Nonetheless, recent data has suggested that sildenafil could have additional and prolonged beneficial effects on endothelial function in diabetic patients if taken on a daily basis [7]. Furthermore, the use of chronic sildenafil could also improve nocturnal erectile activity, thus maintaining the morphodynamic integrity of smooth muscle cells within the corpora cavernosa [8]. Finally, despite broad clinical success of sildenafil in the treatment of ED, certain patients with severe ED i.e. Erectile Function Domain Scores between 1 and 10 [9] remain poor responders. Our hypothesis is that chronic treatment, far from inducing tachyphylaxis, could help salvage non-responders to sildenafil therapy. Thus, whether chronic treatment with sildenafil leads to tachyphylaxis is important to ascertain since it may have widespread implications, not only for sildenafil, but also for the other PDE-5 inhibitors, vardenafil and tadalafil if this is a class effect. The primary goal of this study was, therefore, to study the effects of chronic treatment with sildenafil on the erectile function in rats by (1) evaluating the effects of a chronic treatment with sildenafil on cavernosal tissue endothelial reactivity, and (2) studying their erectile responses elicited by electrical cavernous nerve stimulation under anaesthesia. Secondly, we investigated the sensitizing effect a chronic treatment with sildenafil on electrically-induced erectile responses before and after an acute administration of sildenafil in anaesthetized rats. 2.Methods 2.1. Animals Male Sprague-Dawley rats (Charles River, France, g, n = 44) were housed 7 days prior to the experiments with free access to food and water. All procedures were performed in accordance with the legislation on the Guide for the Care and Use of Laboratory Animals (NIH publication No , revised 1996) and Animal Care regulations in force in France as of Drugs and solutions Krebs bicarbonate solution of the following composition was used ((mmol l 1 ): NaCl 118.0; KCl 4.6; CaCl 2 2.5; KH 2 PO 4 1.2; MgSO 4 1.2; glucose 11.1; NaHCO ; HEPES, 20.85, ph ). The following drugs were used: phenylephrine, acetylcholine, A23187, sodium nitroprusside (Sigma, St Louis, USA), sildenafil mesylate (Pfizer, Sandwich, UK) Chronic treatment of rats Each experimental group received 20 mg/kg of sildenafil mesylate (or saline solution) via subcutaneous injection repeated three times a day (60 mg/kg/d in total) during 8 weeks. Body weight was recorded every day. Blood samples were collected before treatment, during the treatment period (bi-monthly, 6 8 h after a subcutaneous injection) and at the time of the in vivo experiments for the determination of free plasma concentrations of sildenafil (UK- 92,480) and its active metabolite (UK-103,320), as previously described [10]. All experiments were performed after a 36-h wash-out period Isometric tension studies in cavernosal strips Rats were terminally anaesthetized with sodium pentobarbital (50 mg/kg ip). The penis was carefully harvested, transferred into ice-cold Krebs bicarbonate solution and dissected. Cavernosal strips were placed in 5-ml organ baths filled with Krebs maintained at 37 8C, bubbled with 95% O 2-5%CO 2 and connected to a forcedisplacement transducer. Tissues were equilibrated for 60 min. Concentration-response curves to acetylcholine (ACh, 10 9 mol/l to 10 4 mol/l), A23187, a calcium ionophore (10 9 mol/l to mol/l) and sodium nitroprusside (SNP, 10 9 mol/l to 10 4 mol/l) were constructed on phenylephrine-induced pre-contracted tissues, as previously described [11] Erectile responses to cavernosal nerve stimulation in anaesthetized rats Erectile responses were elicited by electrical stimulation of the cavernous nerve in anaesthetized rats, as previously described [11]. Briefly, rats were anaesthetized with urethane (1.2 g/kg ip), tracheotomized and maintained at 37 8C. The carotid artery was catheterized to record arterial pressure and a 21-gauge needle was inserted into one of the corpus cavernosum of the penis to record ICP simultaneously via pressure transducers (Elcomatic 750, UK). The cavernous nerve was exposed at the lateral aspect of the prostate and mounted on a bipolar platinum electrode connected to an electrical stimulator (AMS 2100, Phymep, France). After stabilization, basal MAP was recorded. Electrical stimulations of the cavernous nerve (1 ms, 45 s, 6 V) at different frequencies (1, 2, 3, 4, 5 and 10 Hz) were performed in a randomized manner and repeated twice in order to establish frequency-response curves. Then, 4 minutes after an intravenous injection of sildenafil 0.3 mg/kg, electrical stimulations of the cavernous nerve at 1, 3 and 5 Hz were again performed before sacrifice of the rats with an overdose of urethane Calculations and statistical analysis Values are expressed as mean S.E. mean of n experiments. Tissue contractile responses were expressed as the absolute change in maximal developed tension (in g) normalized per gram tissue weight and relaxations as the percentage of change in phenylephrine-induced tone. Concentrations inducing 50% of the maximal effect were expressed as pd 2 values and calculated using GraphPad Prism. Erectile responses elicited by electrical stimulation were expressed as the ratio of DICP (mmhg)/map (mmhg) 100, with

3 D. Behr-Roussel et al. / European Urology 47 (2005) DICP, the difference between ICP in the flaccid state and ICP during the plateau phase of the erectile response and MAP, the mean arterial pressure during the plateau phase of the erectile response. This ratio accounts for the strong influence of the systemic blood pressure on the amplitude of ICP increase during the plateau phase [12]. Comparisons were performed using a twoway ANOVA statistical analysis followed by a Bonferroni s complementary analysis or Student s t-test where relevant. A p-value less than 0.05 was considered to be significant. 3.Results There was no statistical difference observed in body weight nor basal MAP between saline- or sildenafiltreated rats Functional in vitro studies Endothelial relaxation induced by ACh was significantly enhanced by 35% in rats treated chronically with sildenafil compared to untreated rats (n = 12, p < 0.01, two-way ANOVA) without a significant change in pd 2 ( vs , NS, Student s t-test). Conversely, relaxations elicited by A23187, a calcium ionophore, or by SNP were similar in tissues from chronically saline- or sildenafil-treated animals (Fig. 1) In vivo studies Mean trough plasma concentrations of free sildenafil (UK-92,480) and its active n-desmethyl metabolite, UK-103,320, during the 8-week long chronic treatment phase reached concentration above 30 nm throughout the study (Fig. 2). In untreated rats, there was no detectable levels of drug or metabolite. After a wash-out period of 36 h, concentrations of free sildenafil and its metabolite were still detected in the plasma (25 nm and 10 nm, respectively, Fig. 2). Chronic administration of sildenafil significantly enhanced frequency-dependent erectile responses in treated rats compared to untreated rats (i.e. maximal Fig. 1. Maximal relaxations induced by increasing concentrations of ACh (upper panel), A23187 (middle panel) or SNP (lower panel) in cavernosal strips from rats that have received saline (full circles) or sildenafil mesylate (20 mg/kg sc 3 /d, open circles) for 8 weeks. p < 0.01, Two-way ANOVA. Fig. 2. Mean plasma concentrations of free sildenafil (UK-92,480, upper panel) and its active n-desmethyl metabolite, UK-103,320 (lower panel) throughout the study.

4 90 D. Behr-Roussel et al. / European Urology 47 (2005) Fig. 3. Upper panel: Effect of a chronic treatment with saline (full circles) or sildenafil mesylate (20 mg/kg sc 3 /d, open circles) for 8 weeks on erectile responses elicited by cavernous nerve stimulation at increasing frequencies. p < 0.001, two-way ANOVA. Lower panel: Effect of an acute administration of sildenafil mesylate (0.3 mg/kg iv) on erectile responses elicited by cavernous nerve stimulation at increasing frequencies in rats that have received saline (hatched bars) or sildenafil mesylate (20 mg/ kg sc 3 /d, open bars) for 8 weeks. p < 0.01, two-way ANOVA; *p < 0.05, Bonferroni s complementary analysis. responses to 10 Hz-6Vof % vs %, p < 0.001, n = 10, two-way ANOVA, Fig. 3). The stimulation frequency required to produce 50% of the maximal erectile response following electrical stimulation was lower in the sildenafil-treated rats compared to saline rats ( versus Hz, respectively, p < 0.05, Student s t-test), thus indicating a shift to the left of the frequency-dependent erectile response curves. Most interestingly, rats treated with chronic sildenafil also gave a greater response (45% increase in the erectile response following electrical stimulation) toanacuteinjectionofsildenafilcomparedtoratsthathad received saline chronically (responses to 5 Hz-6V of % vs %, p < 0.05, n = 10, Bonferroni s complementary analysis, Fig. 3). 4.Discussion The main findings of these experiments are that a chronic treatment with sildenafil potentiates AChinduced endothelium-dependent cavernosal responses and enhances erectile responses in anaesthetized rats. A possible concern for tachyphylaxis with sildenafil has arisen with the suggestion that daily dosing with sildenafil may salvage poor responders to on-demand sildenafil therapy. Previous work with human cavernosal smooth muscle cells in culture had reported that tachyphylaxis could occur following repeated exposure to extremely high concentrations of sildenafil (25 mm) [13]. We addressed this issue in a different context using functional evaluation of endothelial and erectile responses after an exposure to sildenafil more closely related to those attained with treatment in humans [10] to investigate this concern. Overall rates of discontinuation from sildenafil treatment for ED have been reported to be very low in clinical trials [5,6]. Nonetheless, certain patients with severe ED remain poor responders. Indeed, the natural progression of the underlying ED causing reduction in neurotransmitter availability (i.e. endothelial dysfunction) with or without further reduction of vascular supply is nowadays considered as the main factor in any loss of efficacy of the treatment. Thus, in this study, we used normal rats to eliminate possible interference from a cardiovascular or metabolic-associated disease process on ED. In the present study, a chronic treatment with sildenafil potentiates ACh-induced endothelium-dependent cavernosal responses while A23187-induced and SNPinduced relaxant responses are not enhanced. This could indicate that either the muscarinic receptors or the transduction mechanisms leading to the activation of endothelial NOS are upregulated by a chronic treatment with sildenafil. Indeed, the activation of the PI3-kinase/Akt/eNOS phosphorylation cascade could be implicated [14,15], thus causing a more sustained NO release and relaxation [2]. The resulting increase in NO production could be still strictly-speaking calcium-dependent although the enzyme could be in an activated state at resting calcium levels [14,15]. This is of particular relevance since shear-stress induced NO-dependent relaxation, a more physiological stimulus of cavernosal smooth muscle endothelium-dependent relaxation, also relies on this signaling cascade to activate enos. In any case, it rules out an effect of chronic treatment with sildenafil on NO bioavailability, or on the subsequent guanylate cyclase pathway and smooth muscle responsiveness to NO, thereby eliminating concern about tachyphylaxis. Furthermore, erectile responses to electrical stimulation of the cavernous nerve are significantly enhanced following chronic exposure to sildenafil. Moreover, chronic treatment may actually sensitize the rats to an acute administration of sildenafil. It is to be noted that mean plasma concentrations of free

5 D. Behr-Roussel et al. / European Urology 47 (2005) sildenafil (UK-92,480) and its active n-desmethyl metabolite, UK-103,320 reached steady-state concentrations above 30 nm in rats during the treatment phase (free concentrations of 20 nm are known to give efficacy in man and 30 nm is equivalent to free plasma concentrations observed following a 100 mg dose of sildenafil in man [16]). However, the wash-out period that was selected (36 h), although sensible considering the half-life of sildenafil in rats (18 min) [10], was not sufficient to insure total elimination of sildenafil from the plasma of the chronically-treated rats. Although it is not possible to conclude that the enhancement of erectile responses induced by cavernous nerve stimulation was solely imputable to the chronic treatment in the in vivo experiments, the in vitro experiments support the concept of profound tissue modifications following chronic treatment with sildenafil. Indeed, in these experiments, tissue samples underwent extensive washings during the stabilization period, thus removing residual compound from the tissue. In spite of these extensive washings, cavernosal endotheliumdependent ACh-induced relaxations were significantly enhanced in tissue samples from chronically-treated rats. Thus, the modifications induced by a chronic treatment with sildenafil were still present although the compound had been most likely washed away. In conclusion, chronic treatment with sildenafil may actually exert beneficial effects on erectile function that go beyond those obtained from acute administration. If confirmed in humans, this could help salvage poor responders to sildenafil therapy. Furthermore, because of its effect on the potentiation of endothelium-dependent cavernosal relaxations, chronic treatment with sildenafil could be of particular benefit in patients with cardiovascular disease-related ED where cavernosal endothelial dysfunction occurs. It will thus be of great interest to test this concept in pathophysiological experimental animal models known to display this abnormality [11,17,18]. Moreover, further investigations are needed to determine whether this beneficial effect is specific to sildenafil or is a class effect associated with inhibition of PDE-5. Acknowledgements We thank Sophie Goyer for skilled help in data management. References [1] Andersson KE, Wagner G. Physiology of penile erection. Physiol Rev 1995;75: [2] Hurt KJ, Musicki B, Palese MA, Crone JK, Becker RE, Moriarity JL, et al. Akt-dependent phosphorylation of endothelial nitric-oxide synthase mediates penile erection. Proc Natl Acad Sci USA 2002;99: [3] Goldstein I, Lue TF, Padma-Nathan H, Rosen RC, Steers WD, Wicker PA. Oral sildenafil in the treatment of erectile dysfunction. Sildenafil Study Group. N Engl J Med 1998;338: [4] El Galley R, Rutland H, Talic R, Keane T, Clark H. Long-term efficacy of sildenafil and tachyphylaxis effect. 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