Topical application of a Rho-kinase inhibitor in rats causes penile erection

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1 (2004) 16, & 2004 Nature Publishing Group All rights reserved /04 $ Topical application of a Rho-kinase inhibitor in rats causes penile erection Y Dai 1,2,3, K Chitaley 1, RC Webb 1, RW Lewis 2 and TM Mills 1,2 * 1 Department of Physiology, Medical College of Georgia, Augusta, Georgia, USA; 2 Department of Surgery (Urology), Medical College of Georgia, Augusta, Georgia, USA; and 3 Department of Urology, Nanjing Drum Tower Hospital, Nanjing, China Studies from this laboratory have demonstrated that RhoA/Rho-kinase signaling mediates vasoconstriction in the penile circulation of the rat and that erection results from inhibition of this activity with Y In prior animal studies, Y was administered to the rats by intracavernous injection. To determine if topical application of the Rho-kinase inhibitor is an effective mode of delivery, Y was applied to the surface of the tunica albuginea or to the glans penis and surrounding skin in intact or castrated rats. Both sites of drug administration resulted in a marked increase in the erectile response both with and without stimulation of the autonomic innervation of the penile vasculature. Although high doses of the drug were found to reduce systemic blood pressure, topical administration of the Rho-kinase inhibitor, in appropriate doses, may have clinical value for the treatment erectile dysfunction. (2004) 16, doi: /sj.ijir Published online 12 February 2004 Keywords: erectile response; rat; Rho-kinase; RhoA; topical drug application Introduction A variety of vasoactive agents have been utilized in the pharmacological treatment of erectile dysfunction (ED). 1,2 When injected into the cavernous sinuses, drugs such as prostaglandin E1 or papaverine cause smooth muscle relaxation leading to erection of the penis. 3 Transurethral PGE1 has also been introduced as a treatment of ED, but is not widely used. 4 Phosphodiesterase 5 inhibitors, which slow the degradation of cgmp and hence facilitate erection, are effective when given orally, are convenient to use and are well tolerated. 5,6 However, despite the variety of pharmacological treatment options available, each has shortcomings and contraindications so that the search for the safest and most effective treatment for ED must continue. 7,8 It has recently been demonstrated that a previously uninvestigated biochemical pathway may regulate *Correspondence: TM Mills, PhD, Department of Physiology, Medical College of Georgia, Augusta, GA , USA. tmills@mail.mcg.edu Received 4 March 2003; revised 5 March 2003; accepted 18 May 2003 smooth muscle contraction in the cavernosal circulation The results of these studies suggest that the RhoA/Rho-kinase calcium sensitization pathway is integral to the tonic contraction, which maintains the penis in the flaccid state, and that erection can result from the reduction in the activity of this pathway with the Rho-kinase inhibitor, Y This agent is a highly selective inhibitor of Rho-kinase activity. 14,15 Based on these reported findings, it was proposed that nitric oxide (NO) is a potent inhibitor of the RhoA/Rho-kinase pathway, thereby providing a mechanism by which erection can occur in the presence of strong basal vasoconstrictor activity. 16 Furthermore, inhibition of this pathway has been suggested as a potential treatment for ED. 17 In published studies in which Rho-kinase activity in the penis has been blocked with Y-27632, the drug was administered either by intracavernosal injection 10,16 or by addition to the bathing medium in in vitro experiments The present study was undertaken to determine if topical application of the Rho-kinase inhibitor, Y-27632, was also effective at enhancing the erectile response in laboratory rats. Included in these experiments were groups of normal intact rats in which Y was applied to the tunica albuginea. In addition, the drug was applied to the glans penis and surrounding skin of castrated rats in which the erectile response is sharply suppressed.

2 Materials and methods Animals Male, Sprague Dawley rats ( g) were obtained from Harlan and maintained on a 12 h light/dark cycle with rat chow and water available ad libitum according to guidelines at the Medical College of Georgia, Augusta, Georgia, USA. In some experiments, animals were surgically castrated 7 to 14 days prior to the measurements of erectile function. Experimental design In the first series of experiments, Y was applied topically to the tunica albuginea to determine if the Rho-kinase inhibitor would cross this barrier to cause vasodilation leading to erection. In these studies, the rats were anesthetized with ketamine (87 mg/kg body weight) plus xylazine (13 mg/kg) and then a cannula was inserted into the carotid artery to permit the continuous monitoring of mean arterial pressure (MAP). Next, the penis was partially degloved by retracting the skin and fascia overlying the tunica albuginea to allow insertion of a pressure transducer cannula into the right corpus cavernosum to permit continuous monitoring of intracavernosal pressure (ICP). 16,18 Care was taken in the location of the cavernosal cannula to avoid any chance of entry of the topically applied Y around the path created by the cannula. Once these cannulae were in place, the right major pelvic ganglion (MPG) was exposed through a mid-ventral incision. With the viscera retracted, stainless-steel bipolar electrodes were placed on the MPG and, with the stimulator set at 5 V, the position of the electrodes was adjusted until the maximal ICP was achieved. During the experiment, MAP and ICP were continuously recorded on a polygraph recorder and analyzed electronically (Polyview, Grass Instrument Company). Basal ICP and MAP measurements were collected before application of the Y and without ganglionic stimulation. MAP and ICP measurements were also carried out during stepwise stimulation of the MPG (1 5 V, 5 ms duration, 12 Hz frequency with 30 s at each voltage). 19 At 10 min after application of one of the three doses of Y (100, 200, 400 mg in2ml DMSO) to the tunica albuginea of the normal intact rats, the basal and stimulated erectile responses were measured, and these values compared to the basal and stimulated responses recorded before application of the drug. In addition, ICP and MAP measurements were carried out before and after application of the DMSO vehicle only. Once these experiments had established that the Rho-kinase inhibitor readily penetrates the tunica albuginea to cause erection, a second set of studies was completed to determine if the drug would penetrate penile skin as well. In this experiment, 1000 mg Y in 2 ml DMSO or 2 ml of the DMSO vehicle was applied in castrated rats with ICP and MAP measurements carried out 20 min after application. Drugs The selective Rho-kinase inhibitor, Y-27632, generously donated by Mitsubishi Pharma, Osaka Japan, was dissolved in DMSO in order to facilitate penetration into cavernous tissue. In the normal intact rats, 100, 200 or 400 mg Y-27632/2 ml Y was applied via a micropipette over the proximal dorsal tunica albuginea. In the castrated animals, 1000 mg Y-27632/2 ml DMSO was applied to the glans penis and surrounding skin. Expression of results and statistical analyses The erectile response was computed as the ratio of ICP to the concurrently measured MAP (ICP/MAP). ICP/MAP and MAP data were analyzed using oneway analysis of variance (ANOVA) or two-way ANOVA for repeated measures with post hoc analysis by Student s Newman Keul s test. Statistical significance was set at Po0.05. Results Direct application of the Rho-kinase inhibitor Y to the tunica albuginea resulted in an increase in ICP (Figure 1). This tracing shows that within a Figure 1 Tracing of the effect of topical application of 100 mg Y (arrow) on MAP and ICP. Note that within a few minutes of application of the Rho-kinase inhibitor in 2 ml DMSO, ICP rises reaching a maximum after about 10 min. Over the same time interval, there is a minor decline in MAP. 295

3 296 Figure 2 Effects of application of Y to the tunica albuginea on the erectile response (ICP/MAP) in the rat penis. Note that 10 min after application of the DMSO vehicle or 100 mg of Y-27632, the erectile response is not significantly changed as compared to the response BEFORE application (*). However, after application of 200 and 400 mg of the Rho-kinase inhibitor, the responses are significantly increased relative to before treatment. Each bar represents the mean7s.e.m. for measurements carried out in 4 6 animals. *Po0.05. few minutes of applying 100 mg of the Rho-kinase inhibitor in 2 ml DMSO, the ICP increased with the response reaching a maximum in min. Figure 1 also demonstrates that when this concentration of Y was applied to the tunica albuginea, there was a small change in MAP. In both normal intact and castrated rats, topical application of 2 ml of the DMSO vehicle had no effect on either the basal response or the response during stimulation of the MPG. Likewise, tunical application of the low dose of Y (100 mg) failed to increase significantly the basal response (Figure 2) or the responses to ganglionic stimulation at 1 5 V (Figure 3). However, application of higher doses of the Rho-kinase inhibitor (200 or 400 mg) in intact rats significantly elevated both the basal response (Figure 2) and the response during ganglionic stimulation (Figure 3). The single exception to this was the failure of the 400 mg dose of Y to enhance the erectile response during 5 V stimulation (Figure 3). To investigate any hypotensive effect of tunical application of Y-27632, systemic blood pressure was monitored following drug application. The results in Figure 4 show that MAP declined significantly with the topical application of 200 and 400 mg ofy , although there was no significant effect after application of the DMSO vehicle or 100 mg. At both the high doses of the Rho-kinase inhibitor, MAP remained reduced for more than 30 min after topical application (not shown). Having established that Y readily crosses the tunica albuginea to dilate the cavernosal vasculature, the Rho-kinase inhibitor was applied next to Figure 3 Effects of application of Y to the tunica albuginea on the electrically stimulated (1 5 V) erectile response (ICP/MAP). At 10 min AFTER application of 100 mg Y-27632, the erectile response at all levels of electrical stimulation is not significantly different from the response at each voltage measured before application. However, in the 200 and 400 mg treatment groups, the response after drug application is significantly greater at all voltages (*) as compared to the response before application with the exception of 5 V in the 400 mg treated animals. Each bar represents the mean7s.e.m. for measurements carried out in 4 6 animals. *Po0.05. the glans penis and surrounding skin in hypogonadal animals that show severe ED with measurements of ICP and MAP 20 min after application. In the absence of ganglionic stimulation, ICP rose significantly from 771 mmhg before application to 2172 mmhg after application (Po0.05). Simultaneous measurement of MAP revealed values of mmhg declining to 8177 mmhg 20 min after application (Po0.05). Figure 5 shows that the basal erectile response and the responses at all levels of ganglionic stimulation (1 5 V) were significantly enhanced after drug application when compared to the response before the drug was applied. Discussion The results of the present study demonstrate that, when applied topically to the tunica albuginea or to the glans penis and surrounding skin, an inhibitor of Rho-kinase (Y-27632) caused penile erection. This observation extends the prior findings that intracavernous injection of Y will both initiate erection and enhance erection resulting from electrical stimulation of the MPG or administration of a NO donor drug. 10,16,20 As would be expected, the rate at which Y increased intracavernous blood pressure was greater when the drug was given by intracavernous route (maximum ICP within 5 min of injection) compared to min after topical application to the tunica albuginea or glans and

297 Figure 4 MAP responses to application of Y-27632 to the tunica albuginea.

4 297 Figure 4 MAP responses to application of Y to the tunica albuginea. Comparison of MAP measurements carried out before and 10 min after application of the Rho-kinase inhibitor show no effect in the DMSO vehicle and 100 mg treated rats. However, MAP is significantly decreased in the 200 and 400 mg treated animals. Each bar represents the mean7s.e.m. for measurements carried out in 4 6 animals. *Po0.05. adjacent skin. In all instances, ICP remained elevated for more than 30 min after topical administration. Furthermore, when compared to the doses required to induce erection when Y was injected, it is clear that a greater amount of drug is required to achieve a similar erectile response when it was administered topically. Figure 1 shows that application of 100 mg Y to the tunica albuginea caused a marked rise in ICP. However, this figure also shows that after application there was a small decline in MAP. The results in Figure 4 show that at higher doses Y (200 or 400 mg) applied to the tunica albuginea, MAP was significantly suppressed, while application of the DMSO vehicle or 100 mg of the Rho-kinase inhibitor failed to reduce MAP significantly. We have previously reported that this same compound lowers blood pressure when high doses are given by intracavernous injection to normal, intact rats 10 or to severely hypertensive rats. 21 Other investigators have reported that oral administration of Y lowers blood pressure in hypertensive rats but not in normotensive animals. 14 The present study utilizes the castrated rat with 1000 mg Y applied to the glans penis and surrounding skin. As shown in Figure 5, this treatment strongly enhanced the erectile response in the castrated animals. Castration leads to a reduced erectile response based on diminished production of NO and increased sensitivity to alpha-adrenergic-mediated vasoconstriction. 25 This model was selected for these studies because the erectile response is sharply reduced compared to the response in intact animals. Furthermore, it has recently been reported that the Rho-kinase pathway is upregulated in castrated rats and that Figure 5 Comparison of the erectile response (ICP/AMP) before and 20 min after application of Y (1000 mg) to the glans penis and surrounding skin of castrated rats shows a significant stimulation (*) at all levels of ganglionic stimulation (1 5 V). Each bar represents the mean7s.e.m. for measurements carried out in four animals. *Po0.05. intracavernous injection of Y fully restores the erectile response. 26 Taken together, the results presented in this report and in prior publications support the possibility that inhibition of the RhoA/Rho-kinase signaling pathway may represent a new method for the treatment of ED. Despite the finding that some doses

5 298 of Y exert a hypotensive action when injected 10,16 or applied topically (Figure 3), further research may reveal tissue-specific isoforms of RhoA or Rho-kinase. If these isoforms are found and one proves to be unique to the urogenital system, then it is possible that selective drugs could be designed to inhibit selectively the enzyme in the cavernous circulation and not in other vascular beds. We have demonstrated that gene transfer of dominant-negative RhoA into the penis enhances erectile function. 27 This observation provides background for gene therapy in ED with respect to this cell signaling cascade. Alternatively, combining the Rho-kinase inhibitor with other ED treatment drugs could be of value. For example, a combination treatment with low doses of a PDE 5 inhibitor (such as sildenafil) with a low dose of Y may result in an enhanced erectile response while minimizing the hypotensive actions of the Rho-kinase inhibitor or adverse effects of the PDE 5 inhibitor. References 1 Pryor JL, Redmon B. New therapies and delivery mechanisms for treatment of erectile dysfunction. Int J Impot Res 2000; 12: S158 S Morales A. Developmental status of topical therapies for erectile and ejaculatory dysfunction. Int J Impot Res 2000; 12: S80 S85. 3 Bechara A et al. Comparative study of papaverine plus phentolamine versus prostaglandin E1 in erectile dysfunction. J Urol 1997; 157: Fulgham PF et al. Disappointing initial results with transurethral alprostadil for erectile dysfunction in a urology practice setting. J Urol 1998; 160: Carson CC, Burnett AL, Levine LA, Nehra A. The efficacy of sildenafil citrate (Viagra) in clinical populations: an update. Urology 2002; 60(Suppl 2): Steers W et al. Assessment of the efficacy and safety of Viagra (sildenafil citrate) in men with erectile dysfunction during long-term treatment. Int J Impot Res 2001; 13: Meuleman EJ. Prevalence of erectile dysfunction: need for treatment? Int J Impot Res 2002; 14: S22 S28. 8 Carson CC. Erectile dysfunction in the 21st century: whom we can treat, whom we cannot treat and patient education. Int J Impot Res 2002; 14: S29 S34. 9 Chitaley K, Webb RC, Mills TM. The ups and downs of Rho-kinase and penile erection: upstream regulators and downstream substrates of Rho-kinase and their potential role in the erectile response. Int J Impot Res 2003; 15: Chitaley K et al. Antagonism of Rho-kinase stimulates rat penile erection via a nitric oxide-independent pathway. Nat Med 2001; 7: Rees RW et al. Human and rabbit cavernosal smooth muscle cells express Rho-kinase. Int J Impot Res 2002; 14: Rees RW et al. Y-27632, an inhibitor of Rho-kinase, antagonizes noradrenergic contractions in the rabbit and human penile corpus cavernosum. Br J Pharmacol 2001; 133: Wang H et al. RhoA-mediated Ca 2 þ sensitization in erectile function. J Biol Chem 2003; 277: Uehata M et al. Calcium sensitization of smooth muscle mediated by a Rho-associated protein kinase in hypertension. Nature 1997; 389: Ishizaki T et al. Pharmacological properties of Y-27632, a specific inhibitor of rho-associated kinases. Mol Pharmacol 2000; 57: Mills TM et al. Effect of Rho-kinase inhibition on vasoconstriction in the penile circulation. J Appl Physiol 2001; 91: Mills TM, Chitaley K, Lewis R. Vasoconstrictors in erectile physiology. Int J Impot Res 2001; 13: S29 S Mills T et al. Endothelin-1 induced vasoconstriction is inhibited during erection in rats. Am J Physiol 2001; 281: R476 R Somlyo AP, Somlyo AV. Signal transduction by G-proteins, rho-kinase and protein phosphatase to smooth muscle and non-muscle myosin II. J Physiol 2000; 522: Mills TM, Chitaley K, Lewis RW, Webb RC. Nitric oxide inhibits RhoA/Rho-kinase signaling to cause erection. Eur J Pharmacol 2002; 439: Chitaley K, Webb RC, Dorrance AM, Mills TM. Decreased penile erection in DOCA-salt and stroke prone-spontaneously hypertensive rats. Int J Impot Res 2001; 13: S16 S Shabsigh R. The effects of testosterone on the cavernous tissue and erectile function. World J Urol 1997; 15: Marin R, Escrig A, Abreu P, Mas M. Androgen-dependent nitric oxide release in rat penis correlates with levels of constitutive nitric oxide synthase isoenzymes. Biol Reprod 1999; 61: Reilly CM, Zamorano P, Stopper VS, Mills TM. Androgenic regulation of NO availability in rat penile erection. J Androl 1997; 18: Reilly CM, Stopper VS, Mills TM. Androgens modulate the alpha-adrenergic responsiveness of vascular smooth muscle in the corpus cavernosum. J Androl 1997; 18: Wingard C, Johnson J, Holmes A, Prikosh A. Improved erectile function following Rho-kinase inhibition in a rat castrate model of erectile dysfunction. Am J Physiol 2003; 284: R1572 R Chitaley K et al. Adeno-associated viral gene transfer of dominant negative RhoA enhances erectile function in rats. Biochem Biophys Res Commun 2002; 298: