Rationale for the Combination of PGE 1 and S-Nitrosoglutathione to Induce Relaxation of Human Penile Smooth Muscle 1

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1 /00/ $03.00/0 THE JOURNAL OF PHARMACOLOGY AND EXPERIMENTAL THERAPEUTICS Vol. 295, No. 2 Copyright 2000 by The American Society for Pharmacology and Experimental Therapeutics 2715/ JPET 295: , 2000 Printed in U.S.A. Rationale for the Combination of PGE 1 and S-Nitrosoglutathione to Induce Relaxation of Human Penile Smooth Muscle 1 JAVIER ANGULO, PEDRO CUEVAS, IGNACIO MONCADA, ANTONIO MARTÍN-MORALES, ANTONIO ALLONA, ARGENTINA FERNÁNDEZ, SONIA GABANCHO, PETER NEY, and IÑIGO SÁENZ DE TEJADA Fundación para la Investigación y el Desarrollo en Andrología, Madrid, Spain (J.A., I.M., A.M.-M., A.A. S.G., I.S.d.T.); Departmento de Investigación, Hospital Ramón y Cajal, Madrid, Spain (P.C., A.F.); and Corporate Development, Schwarz-Pharma, Monheim, Germany (P.N.) Accepted for publication June 22, 2000 This paper is available online at Intracavernosal prostaglandin E 1 (PGE 1 ) has been extensively used as a therapeutic agent for the treatment of erectile dysfunction (Buvat et al., 1996, 1998; Linet and Ogring, 1996). Although some success has been obtained, a considerable number of patients (about 30 40%) have not responded to treatment with PGE 1 (Porst, 1996). The reason for the lack of response to PGE 1 in refractory patients is not known, but could depend on many factors (number of receptors, transduction mechanisms, metabolism, tissue structure, etc.). Indeed, it has not been demonstrated that the efficacy in the clinical response to PGE 1 correlates with the relaxation of human penile smooth muscle (arterial and trabecular) to this drug. PGE 1 promotes relaxation of penile arterial smooth muscle via prostacyclin (IP) receptors and of trabecular smooth muscle through interaction with prostaglandin E (EP) receptors, with some preliminary data supporting a key role for the EP 2 Received for publication March 17, This study was partially supported by a grant from Schwarz Pharma. ABSTRACT Many men with erectile dysfunction have been successfully treated with intracavernosal injection of prostaglandin E 1 (PGE 1 ) but this treatment is ineffective in 30 to 40% of patients. The goals of this study were to characterize PGE 1 -induced relaxation of isolated human penile smooth muscle (penile arteries and trabecular strips), correlating this in vitro response with the clinical response to this drug, and to evaluate the effects of the combination of PGE 1 with S-nitrosoglutathione (SNO-Glu) on relaxation of isolated human penile smooth muscle. Large variability in the EC 50 and maximal relaxation induced by PGE 1 was observed between tissues of different patients. Patients with poor clinical response to intracavernosal alprostadil (PGE 1 ) had significantly larger EC 50 values and smaller maximal relaxation compared with patients with partial or complete clinical response to this drug. SNO-Glu consistently produced complete or near complete relaxation of human corpus cavernosum strips and penile arteries, even when the tissue responded poorly to PGE 1. In trabecular strips, the combination of PGE 1 and SNO-Glu in a 1:100 ratio demonstrated a synergistic relaxation effect. The combination of PGE 1 and SNO-Glu simultaneously increased the levels of both camp and cgmp in human corpus cavernosum tissue. Our results suggest that the clinical effectiveness of intracavernosal administration of PGE 1 is related to the variability of the relaxation responses of human trabecular tissue and penile arteries to this drug. The synergistic interaction of PGE 1 and SNO-Glu makes this combination an effective method to cause penile smooth muscle relaxation, a necessary step to initiate and maintain penile erection. receptor subtype (Andersson and Wagner, 1995; Sáenz de Tejada et al., 1998). IP and EP receptors, coupled to G- proteins, increase intracellular camp levels through adenylyl cyclase stimulation (Andersson and Wagner, 1995). Penile smooth muscle relaxation; however, is not only mediated by the camp pathway but also by the nitric oxide (NO)-cGMP pathway, which plays a fundamental role in the relaxation of human penile smooth muscle (Kim et al., 1991; Rajfer et al., 1992) and penile erection (Burnett et al., 1992; Trigo-Rocha et al., 1993). Activation of the cgmp pathway is brought about by the endogenous generation of NO from lacunar and vascular endothelia and nitrergic nerves. The existence of NO donors allows for pharmacological activation of the cgmp pathway and facilitates penile erection (Truss et al., 1994; Martínez-Piñeiro et al., 1998). S-Nitrosothiols are NO donor molecules, some of which are present in human plasma and tissues in physiological conditions and one of these compounds, S-nitrosoglutathione (SNO-Glu) has been reported to induce vascular (MacAllister et al., 1995) and penile tra- ABBREVIATIONS: PGE 1, prostaglandin E 1 ; NO, nitric oxide; SNO-Glu, S-nitrosoglutathione; SIN-1, linsidomine chlorohydrate; PSS, physiological salt solution. 586

2 2000 Relaxation of Human Penile Smooth Muscle to PGE 1 and SNO-Glu 587 becular (Gupta et al., 1995) smooth muscle relaxation. In addition, promising, although preliminary clinical data have shown that intracavernosal coadministration of PGE 1 and the NO donor linsidomine chlorhydrate (SIN-1) produces better erectile responses than those obtained with either compound individually (Tordjman, 1993). The mechanism behind such possible clinical benefit has not been determined. The aim of the present study was to analyze the relaxant responses to PGE 1 in human corpus cavernosum and penile resistance arteries from impotent patients and to determine whether the clinical response to PGE 1 would depend, at least in part, on the degree of penile smooth muscle relaxation induced by this prostanoid. We also sought to evaluate possible synergistic effects of combining PGE 1 and SNO-Glu to cause relaxation of penile smooth muscle. Experimental Procedures Human Corpus Cavernosum Tissues. Human corpus cavernosum specimens were obtained from impotent men at the time of penile prosthesis insertion. All experimental protocols were approved by the local ethics committee. Tissues were maintained at 4 6 C in M-400 solution (composition per 100 ml: 4.19 g of mannitol, g of KH 2 PO 4,0.97gofK 2 HPO 4 3H 2 O, g of KCl, g of NaHCO 3, ph 7.4) until used, which ranged between 2 and 16 h after extraction (Simonsen et al., 1997). Vascular Reactivity of Resistance Penile Arteries. Penile small arteries, helicine arteries (lumen diameter m), which are the terminal branches of deep penile arteries, were dissected by carefully removing the adhering trabecular tissue, and arterial ring segments (2 mm in length) were subsequently mounted on two 40- m wires on microvascular Halpern-Mulvany myographs (J.P. Trading, Aarhus, Denmark) for isometric tension recordings. The vessels were allowed to equilibrate for 30 min in physiological salt solution (PSS) of the following composition: 119 mm NaCl, 4.6 mm KCl, 1.5 mm CaCl 2, 1.2 mm MgCl 2, mm NaHCO 3,11mM glucose, 1.2 mm KH 2 PO 4, and mm EDTA at 37 C continuously bubbled with 95% O 2,5%CO 2 to maintain a ph of 7.4. Passive tension and internal circumference of vascular segments when relaxed in situ under a transmural pressure of 100 mm Hg (L 100 ), were determined. The arteries were then set to an internal circumference equivalent to 90% of L 100, at which the force development was close to maximal (Mulvany and Halpern, 1977). The preparations were then exposed to 125 mm K (equimolar substitution of NaCl for KCl in PSS) and the contractile response was measured. The arteries were contracted with 1 M norepinephrine and relaxation responses were evaluated by cumulative additions of compounds to the chambers. Relaxant responses are expressed as percentage of total relaxation (loss in tone) induced by the addition of 0.1 mm papaverine HCl to the chambers at the end of the experiment. All data are expressed as mean S.E. Organ Chamber Studies. Strips of corpus cavernosum tissue (3 3 7 mm) were immersed in 8-ml organ chambers containing PSS, maintained at 37 C, and aerated with 95% O 2,5%CO 2, ph 7.4. Each tissue strip was incrementally stretched to optimal isometric tension, as determined by the maximal contractile response to 1 M phenylephrine (Kim et al., 1991; Azadzoi et al., 1992). Relaxant responses were evaluated by adding increasing cumulative concentrations of compounds to strips contracted with 0.5 M phenylephrine. Relaxation responses are expressed as percentage of total relaxation (loss in tone) induced by the addition of 0.1 mm papaverine HCl to the chambers at the end of the experiment. All data are expressed as mean S.E. In studies using a combination of relaxant compounds, a molar proportion of 1:100, PGE 1 :SNO-Glu, was chosen based on the EC 50 value for penile smooth muscle relaxation for each compound individually. Measurement of Cyclic Nucleotides in Human Corpus Cavernosum Tissue. Corpus cavernosum strips were immersed in 8-ml organ chambers containing PSS, maintained at 37 C, and aerated with 95% O 2,5%CO 2, ph of 7.4. Each tissue strip was incrementally stretched to optimal isometric tension, as determined by the maximal contractile response to 1 M phenylephrine. Then each tissue was treated with the phosphodiesterase inhibitors zaprinast (30 M) and 3-isobutyl-1-methylxanthine (100 M) and allowed to incubate for 15 min, after which time tissues were treated with drug or vehicle. Tissues were allowed to incubate for another 30 min and then immediately frozen in liquid nitrogen and stored at 80 C until extraction for the cyclic nucleotide assay. Tissues were extracted by homogenization in 6% trichloroacetic acid followed by ether (H 2 O- saturated) extraction and lyophilization. Cyclic nucleotides were determined by enzyme-linked immunosorbent assay using a kit from Cayman Chemical Co. (Ann Arbor, MI). Protein Determinations. Protein were determined using the Bio-Rad Protein Assay kit microtiter plate assay procedure (Bio-Rad, Hercules, CA) with BSA as the standard. Drugs and Materials. Phenylephrine, norepinephrine (arterenol), and zaprinast were obtained from Sigma Chemical Co. (St. Louis, MO). 3-Isobuytl-1-methylxanthine was obtained from Research Biochemicals International (Natick, MA). PGE 1 - -cyclodextrine was kindly provided by Schwarz-Pharma (Monheim, Germany). SNO-Glu was kindly provided by Nitromed Inc. (Bedford, MA). Drugs were dissolved in distilled water at the time of the experiment. Statistical Evaluation. A total of 57 patients was included in the study of individual responses to PGE 1. We obtained penile resistance arteries from 22 of them and trabecular tissue from 39 (both arteries and cavernosal strips were collected from four patients). Individual EC 50 (concentration required to obtain the half-maximum response induced by the compound) and maximum relaxations to PGE 1 were calculated. Patients were divided into two groups according to the clinical erectile response (poor and partial or complete) to PGE 1, as assessed by the degree of penile tumescence after intracavernosal injection of alprostadil (PGE 1 ) determined by visual and physical examination of the penis by the attending physician. The comparison of EC 50, maximum relaxation, and clinical response to alprostadil between both groups was done by Student s t test analysis, which was evaluated using StatView software (Abacus Concepts, Inc., Cary, NC) for Apple computers. Evaluation of the effects of PGE 1 and SNO-Glu, alone or in combination, on penile smooth muscle relaxation and cyclic nucleotide accumulation was performed in tissues from another group of 10 patients (five for relaxation studies and five for cyclic nucleotide determinations). Complete concentration-response curves were obtained and compared by a two-factor ANOVA statistical analysis using StatView software for Apple computers. Statistical analysis of tissue cyclic nucleotide levels was performed by ANOVA followed by a Student-Newman-Keuls post hoc test using GraphPad (San Diego, CA) InStat software. Determination of synergism for the combination of PGE 1 and SNO-Glu was performed according to the isobole method (Berenbaum, 1989). This method applies the equation d a /D a d b /D b 1to define the additive effects between two agents, A and B, where d a and d b are the concentrations of agents A and B in the combination that is needed to obtain a specified level of the effect (relaxation), and D a and D b are the single concentrations of the drugs required to achieve the same level of effect. Synergy is assumed when solving this equation gives a value less than unity. Applying this equation, we determined, in each patient, the theoretical concentrations of the PGE 1 and SNO-Glu combination (always in a 1:100 M ratio) needed to obtain several specified levels of relaxation of trabecular smooth muscle (10, 20, 40, 60, and 70%) when additive effects between the two agents are assumed. The following equation is the application of the isobole method described by Berenbaum (d a /D a d b /D b 1) to our model. We used

3 588 Angulo et al. Vol. 295 this equation to obtain the theoretical concentration of PGE 1 in the combination ([SNO-Glu] is 100-fold higher) needed to obtain a specified level of relaxation when only additive effects are present. [PGE 1 ] in the combination [PGE 1 alone 100 [PGE 1] in the combination 1 [SNO-Glu] alone Using these data we constructed a theoretical curve that was compared with the curve experimentally obtained with the combination of PGE 1 and SNO-Glu. Comparison of these curves was performed by a two-factor ANOVA using StatView software. Results Evaluation of Individual Responses to PGE 1 in Human Penile Smooth Muscle. Relaxations induced by PGE 1 were individually studied in strips of corpus cavernosum from 39 impotent patients and in penile resistance arteries from 22 patients (Fig. 1). Notable variability was observed Fig. 1. Individualized responses to PGE 1 (1 nm to 10 M) in human corpus cavernosum strips (top) contracted with phenylephrine (1 M) and human penile resistance arteries (bottom) contracted with norepinephrine (1 M) from 39 and 22 impotent patients, respectively. Data are expressed as the percentage of total relaxation induced by 0.1 mm papaverine. with respect to the relaxant responses produced by PGE 1 in trabecular smooth muscle from different patients, resulting in a range of several orders of magnitude in individual EC 50 values (mean M, standard deviation 0.418, range from 0.01 to 10 M) and variability in maximal relaxation (mean 83.5%, standard deviation 12.0, range from 60 to 100%). Similar results were obtained when PGE 1 -induced relaxations were evaluated in individual penile resistance arteries, showing variability in EC 50 values (mean M, standard deviation 1.097, range from 0.01 to 10 M), and more noteworthy in maximal relaxation (mean 73.3%, standard deviation 23.2, range from 50 to 100%). Analysis of the data showed a statistically significant correlation between the clinical effectiveness of PGE 1 and the magnitude of the relaxations obtained in vitro with PGE 1. Lower EC 50 values for PGE 1 and larger maximal relaxations were observed when corpus cavernosum strips or penile arteries originated from patients with better clinical response to intracavernosal injection of PGE 1 (Fig. 2). Relaxant Responses Induced by the Combination of SNO-Glu and PGE 1 on Human Penile Smooth Muscle. The nitrosothiol SNO-Glu produced consistent relaxations of human corpus cavernosum strips and penile resistance arteries in a concentration-dependent manner. Administration of SNO-Glu always elicited a near-to-full relaxation of both penile smooth muscle preparations even when tissue from the same patient responded poorly to PGE 1. Representative examples of this observation are shown in Fig. 3. The coadministration of PGE 1 and SNO-Glu in a 1:100 proportion (PGE 1 :SNO-Glu) caused concentration-dependent relaxation of both trabecular and arterial smooth muscle. Figure 4 shows relaxation to PGE 1 alone compared with relaxation caused by the combination of PGE 1 and SNO-Glu plotted as only PGE 1 concentrations. There was a significant shift to the left of the PGE 1 concentration-response curve with the coadministration of SNO-Glu. (In trabecular tissue, EC 50 for PGE 1 alone was M versus M for PGE 1 SNO-Glu, P.01; in penile arteries, EC 50 for PGE 1 alone was M versus M for PGE 1 SNO-Glu, P.01. Maximum responses for PGE 1 in trabecular tissue were % versus % with the PGE 1 SNO-Glu combination, P.01; and % versus % for PGE 1 versus PGE 1 SNO-Glu, respectively, P.01 in penile arteries.) The relaxation response of the PGE 1 and SNO-Glu combination was also compared with the relaxation-response curve induced by SNO-Glu alone, but this time plotted as only the concentration of SNO-Glu in the combination, as shown in Fig. 5. There was a significant shift to the left of the SNO-Glu concentration-response curve with the coadministration of PGE 1 in trabecular tissue, but not in penile arteries. The EC 50 for SNO-Glu alone was M versus M for SNO-Glu PGE 1 (P.05) in trabecular tissue. Maximum responses were % for SNO-Glu versus % for SNO-Glu PGE 1 and were not significantly different from each other. The possible existence of synergism in the relaxation of penile smooth muscle between PGE 1 and SNO-Glu was analyzed from the responses obtained with the drug combination in human penile trabecular and arterial smooth muscle. The actual experimental curve with the concentrations of the PGE 1 and SNO-Glu combination needed to obtain several

4 2000 Relaxation of Human Penile Smooth Muscle to PGE 1 and SNO-Glu 589 Fig. 2. EC 50 values for PGE 1 (top) and maximum relaxation responses to PGE 1 (bottom) obtained in human corpus cavernosum strips contracted with phenylephrine (1 M) (left) and human resistance penile arteries contracted with norepinephrine (1 M) (right). The tissues were divided into two groups depending on the patients clinical erectile response (poor, n 20 and n 14; partial or complete, n 19 and n 8 for trabecular tissue strips and penile arteries, respectively) to intracavernosal injection of alprostadil (PGE 1 ). EC 50 is defined as the concentration of PGE 1 (in M) required to obtain half of the maximum response to this drug. Maximum relaxation data are expressed as the percentage of total relaxation induced by 0.1 mm papaverine. *P.05 and **P.01 between groups by two-tailed Student s t test. specified levels of relaxation (10, 20, 40, 60, and 70%) was compared with the calculated theoretical curve assuming additive effects of both drugs (under Experimental Procedures). Significantly lower concentrations of the PGE 1 and SNO-Glu combination were required than those that would be expected if only additive effects occurred between both compounds. This suggests a synergistic interaction between PGE 1 and SNO-Glu in the relaxation of trabecular smooth muscle (Fig. 6, top). However, PGE 1 and SNO-Glu did not exert synergistic effects in causing relaxation of penile arterial smooth muscle (Fig. 6, bottom). Effect of the Combination of PGE 1 and SNO-Glu on Cyclic Nucleotide Accumulation in Corpus Cavernosum Tissue. Tissue levels of cgmp were not significantly altered by incubation with PGE 1 (1 M), but a remarkable increase was observed when treating the tissues with SNO- Glu (100 M). Incubation of trabecular tissues with the combination of PGE 1 and SNO-Glu induced a significant increase in cgmp content compared with control (Fig. 7, top). camp content was increased by incubation of tissues with PGE 1 (1 M). Incubation of the strips with SNO-Glu (100 M) did not modify tissue camp levels and the combination of SNO-Glu PGE 1 produced an increase in camp content that was similar to that obtained with PGE 1 alone (Fig. 7, bottom). Discussion In clinical trials, response rates in the range of 40 to 70% to intracavernosal injection of PGE 1 have been reported in patients suffering from erectile dysfunction (Buvat et al., 1996, 1998; Linet and Ogring, 1996; Porst, 1996). This observation, supported by extensive clinical experience, presents intracavernous injection of PGE 1 as an efficacious treatment for impotence. However, a considerable number of patients do not respond to such treatment for unknown reasons. Furthermore, the dose of PGE 1 required to achieve satisfactory erections is markedly variable, ranging from 0.5 to 20 g (Linet and Ogring, 1996), with some patients needing doses as high as 40 g to obtain responses (Porst, 1996). Analysis of in vitro responses to PGE 1 in penile arterial and trabecular tissues from impotent patients shows a great degree of variability in the relaxation of human penile smooth muscle to this prostanoid. The variability of PGE 1 - induced relaxations was observed in human penile resistance arteries as well as corpus cavernosum strips and it affected both the sensitivity to PGE 1, obtaining EC 50 values separated by several orders of magnitude, and the maximum relaxation, which varied, especially in penile arteries, from less than 50% to complete relaxation (100%). It is interesting to note that the in vitro response to PGE 1 in human penile tissues correlated with the clinical response to PGE 1 in the patients from whom the tissue was obtained. Our results show that poor responses to intracavernosal alprostadil are associated with diminished maximum response and increased EC 50 of the relaxations to PGE 1 in vitro. These data suggest that a lack of clinical response to PGE 1 may be, in many cases, due to the inability of this prostanoid to relax penile arterial and/or trabecular smooth muscle sufficiently.

5 590 Angulo et al. Vol. 295 Fig. 3. Tracings of concentration-response curves to PGE 1 (0.1 nm to 10 M) and SNO-Glu (10 nm to 1 mm) in human corpus cavernosum strips contracted with 1 M phenylephrine (PE) (top) and penile resistance arteries contracted with 1 M norepinephrine (NE) (bottom). Note the poor response to PGE 1 and full relaxation to SNO-Glu. Concentrations are expressed as the log of molarity. Concentration-response curves to PGE 1 and SNO-Glu were performed in parallel tissues from the same patient. The mechanisms underlying reduced penile smooth muscle relaxation to PGE 1 are currently unknown. The rationale for the use of a combination of PGE 1 with other drugs, for the treatment of erectile dysfunction, is to increase the number of responding patients and to diminish the dose of PGE 1 required to achieve adequate responses because this prostanoid may cause penile pain. To date, the most widely used combination therapy includes PGE 1,papaverine, and phentolamine, which has been shown to improve response rates (Padma-Nathan, 1990; McMahon, 1991; von Heyden et al., 1993). In the present study, we have investigated the possibility of combining the administration of PGE 1, which activates the camp pathway, with the activation of the NO/cGMP pathway, which plays a prominent role in the physiological mechanism of penile erection (Kim et al., 1991; Burnett et al., 1992; Rajfer et al., 1992; Trigo-Rocha et al., 1993). Pharmacological stimulation of cgmp production in penile smooth muscle can be achieved with NO donors, which are compounds that release NO but are more stable, allowing for their use as therapeutic drugs. These NO donors have been reported to relax penile smooth muscle (Sáenz de Tejada et al., 1989; Bush et al., 1992) and have been evaluated for the treatment of erectile dysfunction as a potential alternative treatment to PGE 1 (Truss et al., 1994; Martínez-Piñeiro et al., 1998). Our results show that SNO-Glu, a nitrosothiol present in plasma, is an effective relaxant of human penile resistance arteries and human trabecular smooth muscle. When the tissues, primarily arteries but also trabecular smooth muscle, relaxed poorly to PGE 1, SNO-Glu alone was able to induce a strong response (near full relaxation) in those tissues. The combination with SNO-Glu improved the relaxations to PGE 1 in human penile arteries and in trabecular smooth muscle, causing a shift to the left of the PGE 1 concentration-response curve and a larger maximum relaxation compared with PGE 1 alone. Although in penile arteries the combination of PGE 1 and SNO-Glu did not improve the relaxation response to SNO-Glu alone, this combination did relax human trabecular smooth muscle more efficiently than SNO-Glu or PGE 1 alone. Furthermore, a synergistic interaction between the two compounds was observed when given in combination to relax trabecular smooth muscle, as shown in the analysis of synergism using the isobole method (Berenbaum, 1989). The constructed theoretical curve for the existence of additive effects between PGE 1 and SNO-Glu predicted significantly higher concentrations of both drugs than those experimentally determined to produce the same level of relaxation, confirming the existence of synergism. Based on these results, we propose that the combination of PGE 1 and SNO-Glu has significant advantages for the relaxation of penile trabecular smooth muscle compared with the separate administration of these drugs. Improved relaxant properties of the combination of PGE 1 and SNO-Glu are probably due to the ability of this treatment to trigger the activity of the two essential pathways responsible of penile smooth muscle relaxation (i.e., camp and cgmp pathways). The activation of both pathways was demonstrated in our experiments by the accumulation of both cyclic nucleotides in human cavernosal tissue after

6 2000 Relaxation of Human Penile Smooth Muscle to PGE 1 and SNO-Glu 591 Fig. 4. Concentration-response curves to PGE 1 (0.1 nm to 10 M) and to the combination of PGE 1 (0.1 nm to 10 M) and SNO-Glu (PGE 1 /SNO- Glu, in 1:100 M ratio) in human corpus cavernosum strips contracted with phenylephrine (1 M) (top) and human penile resistance arteries contracted with norepinephrine (1 M) (bottom). The concentration-response curve obtained with the combination of PGE 1 and SNO-Glu is plotted only as PGE 1 concentrations. Data are expressed as mean S.E. of the percentage of total relaxation induced by 0.1 mm papaverine. n indicates the number of patients from whom the tissues were collected for the experiments. Concentration-response curves to PGE 1 and to the combination of PGE 1 and SNO-Glu were carried out in parallel tissues from the same patients., two-factor ANOVA indicates significant differences (P.01) between the concentration-response curve obtained with the combination of PGE 1 and SNO-Glu and that elicited by PGE 1 alone. treatment with the combination. Potentiation of the NO/ cgmp pathway with oral sildenafil together with local PGE 1 is used on occasions in the clinical management of patients who are partial responders to PGE 1. The existence of synergism between NO and camp is not well recognized in human penile smooth muscle. Furthermore, other investigators have tested the effect of combining two other molecules, vasoactive intestinal polypeptide and SIN-1, stimulants of camp and cgmp, respectively, in human corpus cavernosum and cavernous artery, reporting a lack of synergistic interaction (Hempelmann et al., 1995). The difference between the results of Hempelmann et al. (1995) and those of our study may be due to the specificity of Fig. 5. Concentration-response curves to SNO-Glu (10 nm to 1 mm) and to the combination of PGE 1 and SNO-Glu (10 nm to 1 mm; PGE 1 /SNO- Glu, in 1:100 M ratio) in human corpus cavernosum strips contracted with phenylephrine (1 M) (top) and human penile resistance arteries contracted with norepinephrine (1 M) (bottom). The concentration-response curve obtained with the combination of PGE 1 and SNO-Glu is plotted only as SNO-Glu concentrations. Data are expressed as mean S.E. of the percentage of total relaxation induced by 0.1 mm papaverine. n indicates the number of patients from whom the tissues were collected for the experiments. Concentration-response curves to SNO-Glu and to the combination of PGE 1 and SNO-Glu were carried out in parallel tissues from the same patients., two-factor ANOVA indicates significant differences (P.01) between the concentration-response curve obtained with the combination of PGE 1 and SNO-Glu and that elicited by SNO-Glu alone in human corpus cavernosum strips. stimulating different receptor pathways. The combination of PGE 1 with SIN-1 has been shown to be more effective to produce erections than PGE 1 or SIN-1 individually, when evaluated in a clinical trial involving 50 impotent patients (Tordjman, 1993). Our study provides new information that may explain the mechanism underlying this clinical finding. The synergistic interaction of PGE 1 and SNO-Glu was specific to trabecular smooth muscle because it was not observed in penile arteries, despite the fact that these two tissues are anatomically related, and functionally key to the erectile process.

7 592 Angulo et al. Vol. 295 Fig. 6. Analysis of synergism of the relaxations elicited by the combination of PGE 1 and SNO-Glu in human corpus cavernosum strips (top) and penile resistance arteries (bottom). Starting from the responses obtained with the two compounds individually, the theoretical concentrations of the drugs in the combination required to achieve several specified levels of relaxation, when additive effects are assumed, were calculated and compared with those experimentally determined. Data are expressed as mean S.E. of the concentrations of PGE 1 (in nm) present in the combination (concentration of SNO-Glu is 100-fold higher). n indicates the number of patients from whom the tissues were collected for the determinations. Synergistic effects were observed in human trabecular smooth muscle (*P.05 versus theoretical additive effects by two-factor ANOVA) but not in penile resistance arteries. In summary, the present study characterizes the responses to PGE 1 in human trabecular smooth muscle and penile resistance arteries, which both show large variability in response to PGE 1. We found a correlation of the in vitro response with the clinical erectile response that had not been previously reported. These results may explain why some patients respond and others do not to intracavernosal PGE 1. This study also demonstrates that SNO-Glu consistently relaxes penile smooth muscle whether it relaxes well or not to PGE 1. This suggests that the clinical response to PGE 1 may be limited in some patients by the specific lack of response of penile smooth muscle to this prostanoid, while maintaining the ability to relax in response to agents that activate alternative relaxant pathways. We also found that a combination of PGE 1 and SNO-Glu has a synergistic interaction to relax Fig. 7. cgmp (top) and camp (bottom) tissue content of human corpus cavernosum after exposure to PGE 1 (1 M), SNO-Glu (100 M), or both. Data are expressed as mean S.E. of picomoles of cgmp or camp per milligram of tissue protein content. n indicates the number of patients from whom the tissues were collected for the determinations. *P.05, **P.01 versus control. (Student-Newman-Keuls post hoc test). penile trabecular smooth muscle. Such a combination may have significant therapeutic advantages in the treatment of male erectile dysfunction. Acknowledgments We thank Dr. Gordon Letts (Nitromed Inc.) for kindly providing SNO-Glu. We also thank Fundación Futuro for administrative management. References Andersson K-E and Wagner G (1995) Physiology of penile erection. Physiol Rev 75: Azadzoi KM, Kim N, Brown ML, Goldstein I, Cohen RA and Saenz de Tejada I (1992) Endothelium-derived nitric oxide and cyclooxygenase products modulate corpus cavernosum smooth muscle tone. J Urol 147: Berenbaum MC (1989) What is synergy? Pharmacol Rev 41: Burnett AL, Lowenstein CJ, Bredt DS, Chang TS and Snyder SH (1992) Nitric oxide: A physiologic mediator of penile erection. Science (Wash DC) 257: Bush PA, Aronson WJ, Buga GM, Rajfer J and Ignarro LJ (1992) Nitric oxide is a potent relaxant of human and rabbit corpus cavernosum. J Urol 147: Buvat J, Costa P, Morlier D, Lecocq B, Stegmann B and Albrecht D (1998) Double blind multicenter study comparing alprostadil -cyclodextrin with moxisylyte chlorhydrate in patients with chronic erectile dysfunction. J Urol 159: Buvat J, Lemaire A and Herbaut-Buvat M (1996) Intracavernous pharmacotherapy: Comparison of moxisylyte and prostaglandin E1. Int J Impot Res 8: Gupta S, Moreland RB, Munarriz R, Daley J, Goldstein I and Sáenz de Tejada I (1995) Possible role of Na -K -ATPase in the regulation of human corpus caver-

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