NCX-911, a Novel Nitric Oxide-Releasing PDE5 Inhibitor Relaxes Rabbit Corpus Cavernosum in the Absence of Endogenous Nitric Oxide

NCX-911, a novel nitric oxide-releasing PDE5 inhibitor relaxes rabbit corpus cavernosum in the absence of endogenous nitric oxide

JS Kalsi1,2, PD Kell1, S Cellek2* and DJ Ralph1

1Institute of Urology and Nephrology, University College London, London, UK; and 2Wolfson Institute for Biomedical Research, University College London, London, UK

Phosphodiesterase type 5 (PDE5) inhibitors have reduced efficacy in treating erectile dysfunction (ED) in conditions where there is a lack of endogenous nitric oxide (NO). Therefore, NO-releasing PDE5 inhibitors have been developed. Here we report the effect of such a compound, NCX-911, on the tone and nitrergic relaxations of rabbit corpus cavernosum in the absence or presence of a NO G synthase inhibitor, N -nitro-L-arginine methyl ester (L-NAME; 500 lM). NCX-911 was found to be as 7 potent as sildenafil at inducing relaxation of rabbit cavernosum (EC50 values 997.8 195.7 and 1000.57140.8 nM, respectively). The potency of NCX-911 was not altered, but that of sildenafil decreased five-fold in the presence of L-NAME (EC50 values 1281.27268.3 and 4959.17882.1, nM respectively, Po0.001 for sildenafil). Both compounds potentiated nitrergic relaxations with similar potencies. These results suggest that NO-releasing PDE5 inhibitors could potentially be more useful than PDE5 inhibitors in the treatment of ED in conditions where there is a lack of endogenous NO. International Journal of Impotence Research (2004) 16, 195–200. doi:10.1038/sj.ijir.3901157 Published online 12 February 2004

Keywords: NCX-911; nitric oxide; corpus cavernosum; erectile dysfunction; PDE5 inhibitors

Introduction Two further PDE5 inhibitors (tadalafil Cialiss, vardenafil Levitras) have recently been launched. Clinical trials have demonstrated that both agents Male erectile dysfunction (MED) is a significant are also effective in a wide range of patient male health problem reported to affect the lives of subtypes;8,9 however, they too are less effective in 1 35–54% of men aged over 40 y. The management diabetic and postradical prostatectomy patients.10,11 of MED was revolutionized with the introduction of Therefore new therapeutic options, which do not the first oral phosphodiesterase type 5 (PDE5) require endogenous NO, are being developed. One s 2 inhibitor sildenafil (Viagra ) in 1998. Viagra has such approach is an NO-releasing derivative of been found to be highly effective in a wide range sildenafil (sildenafil nitrate; NCX-911) as a potential of patient subtypes; however, certain groups of medical therapy for MED. patients such as long-term diabetics and postradical NCX-911 and sildenafil have a similar selectivity prostatectomy patients do not gain the same and potency with respect to inhibiting PDE5.12 3–5 degree of benefit. This has been postulated to be However unlike sildenafil, NCX-911 has been due to a lack of endogenous nitric oxide (NO) shown to release NO spontaneously and increase 6,7 production. cGMP concentrations by activating soluble guanylate cyclase (sGC) and inhibiting PDE5.12,13 Further- more, NCX-911 has been shown to increase cGMP in a concentration-dependent manner in the absence of *Correspondence: Selim Cellek, MD, PhD, Wolfson In- endogenous NO, whereas sildenafil has little effect stitute for Biomedical Research, University College Lon- on cGMP concentrations under the same condi- don, Gower Street, Cruciform Building, London WC1E 12 6BT, UK. tions. In a hypercholesterolaemic rabbit model, E-mail: [email protected] NCX-911 was shown to be 5–10 times more potent Received 3 July 2003; revised 13 October 2003; accepted than sildenafil at reducing the phenylephrine- 27 October 2003 induced tone.12 Effect of NCX-911 on rabbit corpus cavernosum JS Kalsi et al 196 The aim of this study was to investigate the effect of NCX-911 on the tone and nitrergic responses of rabbit corpus cavernosum and compare the effects with that of sildenafil and an NO donor (spermine- NONOate) in the absence or presence of a NOS inhibitor.

Materials and methods

Rabbit corpus cavernosum Figure 1 Chemical structures of NCX-911 and sildenafil citrate.

Male New Zealand White rabbits (3.0–3.5 kg, Har- with 5% CO2 in O2 (pH 7.0–7.2). The inducible form lan, UK) were killed by an overdose of phentobarbi- of NOS (iNOS) can be induced by trace amounts of tone (Euthesate, Willows Francis Veterinary, UK) endotoxin in the buffer16 and cause loss of tone.17 injected into the marginal vein of the ear. The penis Dexamethasone was added to the modified Krebs’ was excised from pubic bone. The tunica albuginea solution to prevent induction of iNOS.16 The around the corpus cavernosum was cut open. The cyclooxygenase inhibitor indomethacin was added corpus cavernosum was excised and cut longitudin- to prevent synthesis of prostaglandins since these ally to obtain four identical strips (4 Â 10 mm). All can cause non-neurogenic relaxations.18 In our animal experiments were conducted according to experiments, we used spermine-NONOate which is the rules outlined by the Home Office, Animals a spontaneous NO donor, which does not require (Scientific Procedures) Act 1986. cofactors or catalysts to release NO in a physiologi- cal solution.19 Dexamethasone, guanethidine mono- G sulfate, indomethacin, N -nitro-L-arginine methyl Mounting the tissues and recording the mechanical ester (L-NAME) and scopolamine hydrochloride responses were obtained from Sigma, UK. Spermine-NONOate and tetrodotoxin were obtained from Calbiochem, UK. NCX-911 was provided by Nicox SA (Sophia Each preparation was cleaned of adherent tissues Antipolis, France). Sildenafil citrate was extracted and mounted horizontally between two ring electro- s from Viagra tablets as the free base, analysed by des (4 mm diameter) in superfusion chambers (371C) 1H-NMR and LC/MS to confirm identity and purity as described previously.14,15 The chambers were (497%) and then converted to its citrate salt by Dr perfused with modified Krebs’ solution at a constant D Madge at the Medicinal Chemistry Department, flow of 1.0 ml/min by means of peristaltic pumps Wolfson Institute for Biomedical Research, Univer- (Miniplus 2, Gilson). One end of the preparation sity College London. Chemical structures of NCX- was tied to a Grass FT 03C force–displacement 911 and sildenafil citrate are presented in Figure 1. transducer connected to a Linearcorder WR 3101 Drugs were applied directly to the medium (Graphtec, Tokyo, Japan) for registration of isometric reservoir. changes in tension. The preparations were given a tension (0.4–1 g) and allowed to equilibrate for 90 min. The preparations were stimulated electri- cally (electrical field stimulation; EFS) for 5 s with Statistical analysis and presentation of the results trains of rectangular pulses of 50 V, 0.3 ms pulse duration and at a range of frequency of 0.5–25 Hz, The effect of drugs on phenylephrine-induced tone delivered by Grass S88 stimulators. The recorder was expressed as the percentage of the height of was run at a rate of 25 mm/min to record the contraction of the same tissue under control condi- mechanical responses. The mechanical responses tions. The effect of drugs on EFS-induced relaxation were also recorded on a computer by a specialized responses was calculated as percentage of area data acquisition system (Axon Instruments, USA). under the relaxation curve (AUC) of the same tissue under control conditions. EC50 values were calcu- Chemicals and solutions lated using Microcal Origin software (Version 4.1). Results are expressed as mean7s.e. of the mean. Statistical analysis was performed using Student’s The composition of the modified Krebs’ solution unpaired two-tailed t-test. A probability value (P) was (mM): NaCl 136.9, KCl 2.7, CaCl2 1.8, MgSO4 less than 0.05 was considered to be statistically 0.6, NaHCO3 11.9, KH2PO4 0.5, glucose 11.5, significant. n denotes the number of animals used indomethacin 0.01, dexamethasone 0.01, and gassed for each set of experiments.

International Journal of Impotence Research Effect of NCX-911 on rabbit corpus cavernosum JS Kalsi et al 197

Figure 2 The effect of NCX-911 (0.01–30 mM) on phenylephrine- Figure 3 The effect of sildenafil (0.001–30 mM) on phenylephr- induced tone in the rabbit corpus cavernosum in the absence ine-induced tone in the rabbit corpus cavernosum in the absence (squares) or presence of L-NAME (500 mM; triangles). (squares) or presence of L-NAME (500 mM; triangles).

Table 1 EC50 values of NCX-911, sildenafil and spermine- NONOate for lowering the phenylephrine-induced tone in the rabbit corpus cavernosum

7 EC50 mean s.e.m. (nM) n

NCX-911 997.87195.7 8 þ L-NAME 500 mM 1281.27268.3 8

Sildenafil 1000.57140.8 10 þ L-NAME 500 mM 4959.17882.1* 8

Spermine-NONOate 843.3754.4 8 þ L-NAME 500 mM 698.4761.8 8 n denotes the number of animals used for each set of experiments. Figure 4 The effect of spermine-NONOate (0.001–30 mM) on *Po0.05 vs sildenafil in the absence of L-NAME. phenylephrine-induced tone in the rabbit corpus cavernosum in the absence (squares) or presence of L-NAME (500 mM; triangles). Results (EC50 ¼ 1000.57140.8 nM). The addition of L-NAME (500 mM) resulted in a significant rightward parallel Effect of NCX-911 on phenylephrine-induced tone shift in the concentration response curve to sildenafil. EC50 of sildenafil was 4959.17882.1 nM in the presence of 500 mM L-NAME (Po0.05 vs control) After treatment of the tissues with scopolamine (Figure 3 and Table 1). (10 mM) and guanethidine (10 mM) to inhibit choli- nergic and noradrenergic pathways, respectively, and elevation of tone with phenylephrine (3 mM, EC80), Effect of spermine-NONOate on phenylephrine- administration of NCX-911 into the superfusion fluid induced tone resulted in a complete relaxation of rabbit cavernosal strips in a concentration-dependent manner 7 (EC50 ¼ 997. 8 195.7 nM) (Figure 2 and Table 1). Spermine-NONOate elicited concentration-depen- The NO synthase inhibitor L-NAME (500 mM) dent relaxation of rabbit cavernosal strips 7 resulted in a slight but nonsignificant rightward (EC50 ¼ 843.3 54.4 nM). In the presence of L-NAME parallel shift in the concentration response curve to (500 mM), the potency of spermine-NONOate was 7 NCX-911; in the presence of 500 mM L-NAME, EC50 not changed (EC50 ¼ 698.42 61.86 nM; P ¼ 0.1002) of NCX-911 was 1281.27268.3 nM (P ¼ 0.41 vs (Figure 4 and Table 1). control) (Figure 2 and Table 1). Eliciting nitrergic relaxations

Effect of sildenafil on phenylephrine-induced tone EFS (50 V, 0.3 ms pulse duration, 0.5–25 Hz, for 5 s, every 120 s) elicited reproducible contractions of Sildenafil caused relaxation of rabbit cavernosal the tissues. After treatment of the tissues with strips in a concentration-dependent manner scopolamine (10 mM) and guanethidine (10 mM) and

International Journal of Impotence Research Effect of NCX-911 on rabbit corpus cavernosum JS Kalsi et al 198 elevation of tone with phenylephrine (3 mM, EC80), Effect of sildenafil on nitrergic relaxations EFS elicited reproducible frequency-dependent relaxation responses with an optimum frequency of 5 Hz (not shown). These relaxations were comple- At subthreshold concentrations of sildenafil (10 and tely inhibited with an inhibitor of sGC, ODQ 30 nM), the duration and magnitude of the nitrergic (30 mM), an inhibitor of NO synthase, L-NAME relaxation responses were potentiated (Figures 5b (500 mM) or tetrodotoxin (1 mM) indicating that they and 6; 10 nM not shown). Sildenafil citrate and were nitrergic in nature and neurogenic in origin NCX-911 had similar potency to enhance nitrergic (not shown). relaxations at all frequencies (Figures 5 and 6).

Effect of sildenafil and NCX-911 on blocked Effect of NCX-911 on nitrergic relaxations nitrergic responses

Since above 100 nM NCX-911 was able to reduce the After the nitrergic relaxations were blocked by L- phenylephrine-induced tone and it is not possible to NAME (500 mM), administration of sildenafil and elucidate the nitrergic relaxation responses at NCX-911 decreased the phenylephrine-induced reduced tone, two concentrations (10 and 30 nM) tone as mentioned above; however, nitrergic resp- were chosen to study the effect of the drug on onses were not observed to appear again (Figure 7). nitrergic responses. At those subthreshold concentrations, NCX-911 potentiated both the duration and magnitude of the relaxation responses (Figures 5a and 6; 10 nM not shown). Discussion

Relaxation of penile cavernosal and vascular smooth muscle is the most important step in penile erection.20 NO is now accepted to have a fundamental role in this process. It has been shown to induce an increased production of the second messenger cGMP via stimulation of the enzyme sGC in the penile smooth muscle. In turn, cGMP has been shown to activate a number of downstream systems, ultimately resulting in relaxation of the cavernosal smooth muscle.21

Figure 5 The effect of NCX-911 (a) or sildenafil (b), both at 30 nM, on the nitrergic responses of the rabbit corpus cavernosum. Each dot represents EFS (50 V, 0.3 ms pulse duration, for 5 s, every 120 s) at indicated frequencies.

Figure 7 Two representative tracings showing the effect of NCX- 911 (upper panel) or sildenafil (lower panel) on the tone and blocked nitrergic responses of the rabbit corpus cavernosum. EFS (indicated by dots; 50 V, 0.3 ms pulse duration, 5 Hz, for 5 s, every Figure 6 The effect of NCX-911 (solid circles) or sildenafil (open 120 s) elicited nitrergic responses that were completely inhibited circles), both at 30 nM, on the nitrergic relaxation responses of the by L-NAME (500 mM). NCX-911 or sildenafil lowered the tone of rabbit corpus cavernosum. *Po0.05, significantly different from the tissue with different potencies in the presence of L-NAME; control. The difference between NCX-911- and sildenafil-induced however, they failed to reverse the inhibition of the nitrergic potentiation was insignificant. responses.

International Journal of Impotence Research Effect of NCX-911 on rabbit corpus cavernosum JS Kalsi et al 199 PDE5 inhibitors such as sildenafil, tadalafil However, it is uncertain whether NCX-911 will and vardenafil have been very successful in the have the same potency in vivo to potentiate penile management of erectile dysfunction. They improve erection and/or the same capacity to donate NO as it erectile function by reducing the breakdown has in vitro. Further in vivo studies are required to of the active cGMP and thereby potentiating its address these issues. action. However, diabetic patients5,10,11 and patients In the presence of an inhibitor of NO synthase, the with cavernosal nerve injury following radical nitrergic relaxations were completely inhibited in prostatectomy4 have had less success with PDE5 the rabbit corpus cavernosum. Increasing concentra- inhibitors than other patient groups. It has been tions of NCX-911 or sildenafil were not able to proposed that this is the result of a lack of reverse the loss of nitrergic relaxations. This is in endogenous NO production.6,7,22 It has been contrast to the effect of an NO-independent sGC postulated that in order for a PDE5 to be activator, BAY41-2272.15 This agent has been re- successful, a certain degree of residual NO must be ported to be able reverse the loss of nitrergic present.23 relaxation at concentrations greater than 300 nM.15 In order to address this problem, novel agents that BAY-41 2272 has been shown to bind to an allosteric are combinations of a PDE5 inhibitor and NO- site on the sGC enzyme different to the binding site releasing compounds have been produced. NCX- for NO.24 Binding at the allosteric site may lead 911 (sildenafil nitrate) is one such agent. to a conformational change such that there is an In this study, we have shown that NCX-911 lowers increased affinity for NO. The conformational the phenylephrine-induced tone in rabbit corpus change may be responsible for the reversal of cavernosum with an EC50 of approximately 990 nM. inhibition of nitrergic relaxations. In contrast, there This is in agreement with a previous study where are no reported conformational changes associated the EC50 of the compound to relax human corpus with the addition of either a PDE5 inhibitor or an cavernosum was in the range of 1–10 mM.13 In our NO donor. study, the potency of NCX-911 on lowering pheny- In conclusion, our results suggest that NCX-911 is lephrine-induced tone was similar to both sildenafil a potent relaxant of rabbit cavernosal smooth muscle and the NO donor in control tissues. However, after with a similar potency to NO. It is equally addition of the NOS inhibitor the potencies of both efficacious with respect to relaxation of rabbit NCX-911 and NO donor remained unchanged, corpus cavernosum independent of NO availability. whereas that of sildenafil was decreased five-fold. Like PDE5 inhibitors, NCX-911 potentiates both These results suggest that NCX-911 and NO donors the duration and the magnitude of the nitrergic do not require endogenous NO to relax cavernosal relaxation. Therefore, NCX-911 may be a novel way smooth muscle. Sildenafil, however, seems to be of treating MED particularly in patients where relatively dependent on endogenous NO to elicit its endogenous NO production is dysfunctional. relaxant action. These results are in accordance with a previous study where endogenous NO production was reduced in a hypercholesterolaemic rabbit model and Acknowledgements NCX-911 was 5–10 times more potent than sildenafil citrate in reducing phenylephrine-induced tension.12 Furthermore, in anaesthetized rats, the This work and JS Kalsi are supported by the cavernous nerve-stimulation-induced increase in St Peter’s Andrology Research Fund. JS Kalsi is a intracavernous pressure was enhanced by intrave- recipient of Young Investigator’s Award from the nous administration of both sildenafil and NCX-911; International Society for Sexual and Impotence however, at submaximal stimulation NCX-911 was Research (2002). S Cellek is a fellow of the Juvenile more potent than sildenafil.12 Similarly in strepto- Diabetes Research Foundation. We thank D Madge zotocin-induced diabetic rats, NCX-911 has been for extracting sildenafil citrate. found to be more potent than sildenafil citrate (JS Kalsi, PD Kell, DJ Ralph and S Cellek, unpublished observations). Both NCX-911 and sildenafil increased the mag- References nitude and the duration of nitrergic relaxations with similar potencies in the rabbit corpus cavernosum. 1 Kubin M, Wagner G, Fugl-Meyer AR. Epidemiology of erectile These results suggest that both compounds have dysfunction. Int J Impot Res 2003; 15: 63–71. similar mechanisms of action and properties under 2 Goldstein I et al. Oral sildenafil in the treatment of erectile control conditions despite the differences in chemi- dysfunction. Sildenafil Study Group. N Engl J Med 1998; 338: cal structure. Release of the NO from the NCX-911 1397–1404. 3 Rendell MS, Rajfer J, Wicker PA, Smith MD. Sildenafil for the compound does not seem to further potentiate treatment of erectile dysfunction in men with diabetes: a the nitrergic relaxations in normal rabbit corpus randomized controlled trial. Sildenafil Diabetes Study Group. cavernosum. JAMA 1999; 281: 421–426.

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