Design and Evaluation of Nitrosylated -Adrenergic Receptor Antagonists as Potential Agents for the Treatment of Impotence 1

Transcription

1 /99/ $03.00/0 THE JOURNAL OF PHARMACOLOGY AND EXPERIMENTAL THERAPEUTICS Vol. 290, No. 1 Copyright 1999 by The American Society for Pharmacology and Experimental Therapeutics Printed in U.S.A. JPET 290: , 1999 Design and Evaluation of Nitrosylated -Adrenergic Receptor Antagonists as Potential Agents for the Treatment of Impotence 1 I. SÁENZ DE TEJADA, D. S. GARVEY, J. D. SCHROEDER, T. SHELEKHIN, L. G. LETTS, A. FERNÁNDEZ, B. CUEVAS, S. GABANCHO, V. MARTÍNEZ, J. ANGULO, M. TROCHA, P. MAREK, P. CUEVAS, and S. W. TAM Departamento de Investigación, Hospital Ramón y Cajal, Fundación para la Investigación y el Desarrollo en Andrología, Madrid, Spain (I.S. de T., A.F., B.C., S.G., V.M., J.A., P.C.); and NitroMed, Inc., Bedford, Massachusetts (D.S.G., J.D.S., T.S., L.G.L., M.T., P.M.) Accepted for publication March 2, 1999 This paper is available online at ABSTRACT We designed and evaluated a new class of molecules, nitrosylated -adrenergic receptor antagonists, as potential agents for the treatment of impotence. In in vitro studies with human and rabbit corpus cavernosum strips in organ chambers, the -adrenergic receptor antagonists ( -ARAs) moxisylyte and yohimbine and their corresponding nitrosylated compounds, SNOmoxisylyte (NMI-221) and SNO-yohimbine (NMI-187), concentration-dependently relaxed endothelin-induced contraction. The nitrosylated compounds were significantly more potent than the parent -ARA. In human tissues, the specific phosphodiesterase type 5 inhibitor zaprinast potentiated the relaxing effects of the nitrosylated compounds. Only nitrosylated compounds induced accumulation of cyclic GMP in rabbit corpus cavernosum strips. Yohimbine and NMI-187 demonstrated a potent 2 -blocking activity, with no significant differences in pa 2 values (8.9 versus 8.2, respectively). Moxisylyte and NMI- 221 showed moderate potency in antagonizing phenylephrine Penile erection requires the relaxation of arterial and trabecular smooth muscle (Sáenz de Tejada et al., 1991). The combination of the activation of sacral parasympathetic outflow and the inhibition of sympathetic input is believed to initiate and sustain penile smooth muscle relaxation and, therefore, erection (Sáenz de Tejada et al., 1989; Juneman, 1996). It has been demonstrated that a nitric oxide (NO)-like substance is a key mediator of penile smooth muscle relaxation (Kim et al., 1991; Azadzoi et al., 1992; Burnett et al., 1992). NO is released by nonadrenergic, noncholinergic nerves within the trabecular and penile arterial tissues as well as the endothelia that line the lacunar spaces and the intima of penile arteries (Kim et al., 1991). NO synthase-like immunoreactivity has been identified in nerves and endothelia in corpus cavernosum tissue (Burnett et al., 1993), and Received for publication October 26, This work was partially supported by a grant from NitroMed Inc. contraction, with comparable pa 2 values for both molecules (6.5 versus 6.6, respectively). -Adrenergic receptor-binding studies showed similar binding affinities for the -ARA and their corresponding nitrosylated compounds. In vivo, intracavernosal injection of nitrosylated molecules caused greater increases in intracavernosal pressure (NMI-221 versus moxisylyte) that were more long lasting than those of moxisylyte or yohimbine. There were no significant differences between nitrosylated and non-nitrosylated compounds in the magnitude of systemic mean arterial pressure decrease after intracavernosal injection. -ARA and the nitrosylated compounds showed no pain-inducing activity as evaluated with the paw-lick model in mice. In summary, nitrosylated -ARA have the dual functionalities of nitric oxide donors and -ARA. These drugs induced penile erection in animals, suggesting their possible therapeutic value as agents for the local pharmacological treatment of impotence. the activity of this enzyme has been characterized in corpus cavernosum tissue homogenates (Kim et al., 1991). Inhibitors of NO synthase inhibit penile erection elicited by the stimulation of the pelvic nerves (Burnett et al., 1992; Holmquist et al., 1991). Furthermore, it has recently been shown that cyclic GMP (cgmp)-specific phosphodiesterase (PDE) inhibitors [PDE type 5 (PDE5) inhibitors] potentiate nonadrenergic, noncholinergic nerve-mediated relaxation of trabecular smooth muscle (Carter et al., 1998) and that these agents enhance penile erection (Goldstein et al., 1998). This would be expected from a physiological function that is dependent on the NO/cGMP pathway. Sympathetic pathways are inhibitory of penile erection and predominantly involve adrenergic nerves (Sáenz de Tejada et al., 1989). Neurogenic contraction of penile smooth muscle is mediated by adrenergic nerves by way of -adrenergic receptors. 1 -Adrenegic receptors are thought to be the ABBREVIATIONS: NO, nitric oxide; -ARA, -adrenergic receptor antagonist; -ARAs; PGE 1, prostaglandin E 1 ; DMSO, dimethyl sulfoxide; PDE, phosphodiesterase; PPP, platelet-poor plasma; DAM, desacetylmoxisylyte. 121

2 122 Sáenz de Tejada et al. Vol. 290 main mediators of the constrictor responses of penile smooth muscle (Sáenz de Tejada et al., 1989; Traish et al., 1995), although 2 postjunctional receptors have also been implicated as mediators of constrictor response in penile arteries and trabecular smooth muscle (Sáenz de Tejada et al., 1989; Simonsen et al., 1997a, b). -Adrenergic receptor antagonists ( -ARAs) have been shown to potentiate the erection-inducing activity of other vasodilator drugs [e.g., prostaglandin E 1 (PGE 1 ) or papaverine], and it is now common practice to treat patients with drug combinations that, on the one hand, induce direct smooth muscle relaxation and, on the other hand, inhibit adrenergic-mediated vasoconstriction. Such an approach has proved to be very successful in the pharmacological management of patients with erectile dysfunction (Govier et al., 1993). A shortcoming of vasoactive combinations that include PGE 1, which are the most effective combinations, is the ability of this molecule to induce pain, a common side effect in patients receiving intracavernosal pharmacotherapy with this agent (Buvat et al., 1996; Linet and Ogring, 1996). We designed a new class of molecules, nitrosylated -ARAs, that combine, in one molecule, the vasodilator activity of NO with -adrenergic receptor-blocking activity. The pharmacological characteristics of these molecules and their erection-inducing activity and nociceptive potential were investigated. Materials and Methods Animals. Studies were performed in accordance with the Declaration of Helsinki and with the Guide for the Care and Use of Laboratory Animals as adopted and promulgated by National Institutes of Health. Male New Zealand White rabbits (3 3.5 kg; Panlab, Navarra, Spain), male Sprague-Dawley rats ( g), and male CD-1 mice (22 25 g; Charles River, Wilmington, MA) were used in this study. Human and Rabbit Corpus Cavernosum Tissues. Human corpus cavernosum strips were obtained from impotent men at the time of penile prosthesis insertion. 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 ) until used, a time lapse of 2 to 16 h from extraction. Rabbits were euthanized with an overdose of pentobarbital (60 mg/ kg) and immediately exanguinated. The entire penis was then removed from the animal (Kim et al., 1991; Azadzoi et al., 1992). Rabbit corpus cavernosum tissue was dissected free from the surrounding tunica albuginea, cut into tissue strips (3 3 7 mm), and used for either organ chamber contractility studies or cgmp determinations. Drugs and Materials. NMI-187 and NMI-221 were synthesized at NitroMed, Inc. (Bedford, MA). The structure of these molecules is depicted in Fig. 1. Yohimbine HCl, moxisylyte HCl, phentolamine HCl, indomethacin, endothelin-1, phenylephrine, zaprinast, and PGE 1 were obtained from Sigma Chemical Co. (St. Louis, MO). UK-14,304 and 3-isobutyl-1-methylxanthine were obtained from Research Biochemicals Inc. (Natick, MA). [ 3 H]Prazosin (specific activity, 77.2 Ci/mmol) and [ 3 H]yohimbine (specific activity, 78.0 Ci/ mmol) were purchased from NEN Life Science Products (Boston, MA). NMI-187 and NMI-221 were dissolved in distilled water. Indomethacin was dissolved in one part Na 2 CO 3 and two parts NaH 2 PO 4 and zaprinast in dimethyl sulfoxide (DMSO). Organ Chamber Studies. Strips of corpus cavernosum tissue (3 3 7 mm) were immersed in 10-ml organ chambers containing physiological salt solution, maintained at 37 C, and aerated with 5% CO 2 /95% O 2, ph 7.4. Each tissue strip was incrementally stretched Fig. 1. Molecular structure of -adrenergic receptor antagonists yohimbine and moxisylyte and their respective nitrosylated analogs, NMI-187 and NMI-221. to optimal isometric tension, as determined by maximal contractile response to 1 M phenylephrine (Kim et al., 1991; Azadzoi et al., 1992). Tissues were contracted with 0.5 M phenylephrine or endothelin, and relaxation responses were evaluated by cumulative additions of compounds to the chambers. In some experiments, human strips were incubated with the inhibitor of PDE5 activity, zaprinast (1 M), 15 min before contraction with phenylephrine followed by the evaluation of relaxant responses to NMI-187 or NMI-221. 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 experiments designed to determine pa 2 of the different antagonists (parent or nitrosylated -ARAs), a full dose response to either phenylephrine (for moxisylyte or NMI-221) or UK- 14,304 (for yohimbine and NMI-187) were obtained in the presence or absence (control response) of various concentrations of the adrenergic receptor antagonist. pa 2 values, their S.E.M. values, and their 95% confidence limits were calculated according to the method of Tallarida and Murray (1987). Measurement of cgmp in Tissues. Corpus cavernosum strips were immersed in 10-ml organ chambers containing physiological salt solution, maintained at 37 C, and aerated with 5% CO 2 /95% O 2, ph 7.4. Each tissue strip was incrementally stretched to optimal isometric tension, as determined by maximal contractile response to 1 M phenylephrine. Then, to each tissue, we applied 0.5 M phenylephrine, 30 M zaprinast, and 100 M 3-isobutyl-1-methylxanthine and allowed the tissues to incubate for 15 min; after which the tissues were treated with the test drug (NMI-187 or NMI-221) or control drug (yohimbine or moxisylyte) at various concentrations or with vehicle. Tissues were allowed to incubate for an additional 5 min and then immediately frozen in liquid nitrogen and stored at 80 C until extraction for cyclic nucleotide assay. Tissues were extracted by homogenization in 6% trichloroacetic acid, followed by ether (H 2 O-saturated) extraction and lyophilization. cgmp was determined by enzyme-linked immunosorbent assay using a kit from Cayman Chemical Co. (Ann Arbor, MI). Membrane Preparation. Male Sprague-Dawley rats ( g) were euthanized with precharged CO 2, after which the brains were immediately removed and stored at 70 C until they were used. Cerebral cortex tissues were homogenized in 20 volumes of ice-cold buffer (50 mm Tris HCl, 0.5 mm EDTA, ph 7.4) using a Polytron homogenizer (Brinkmann Instruments, Westbury, NY) (19,000 rpm for 20 s). The homogenate was centrifuged at 41,000g for 20 min at 4 C. The resulting membrane pellet was resuspended in 40 volumes of buffer and centrifuged at 41,000g for 20 min. This washing procedure was repeated two more times, and the resultant pellet was resuspended in 10 volumes of buffer, aliquoted, and stored at 70 C to be used within 2 weeks.

3 1999 Nitrosylated -Adrenergic Receptor Antagonists in Impotence 123 Receptor Binding Assays. 1 -Adrenergic receptor-binding assay was performed according to a modified procedure of Buscher et al. (1996). 2 -Adrenergic receptor-binding assay was performed according to a modified procedure of Brown et al. (1990). Then, 25 l of drug solution was incubated with 25 l of either buffer (50 mm Tris HCl, 0.5 mm EDTA, ph 7.4) or human platelet-poor plasma (PPP) for 10 min at 25 C in a 96-well round-bottom microplate. A 25- l aliquot of a radioligand solution of either [ 3 H]prazosin (for 1 -adrenoceptor binding) or [ 3 H]yohimbine (for 2 -adrenergic receptor binding) was added. The binding assay was initiated by the addition of a 175- l aliquot of rat cerebral cortex membranes (80 g protein) and incubated at 25 C for 45 min for 1 -adrenergic receptor binding or 30 min for 2 -adrenergic receptor binding. The final radioligand concentration in a total volume of 250 l was 0.3 nm for [ 3 H]prazosin and 3 nm for [ 3 H]yohimbine. At the end of the incubation, samples were filtered rapidly through Whatman GF/B filters bonded to a 96-well microplate (UniFilter-96; Packard, Downers Grove, IL) and washed three times each with 350 l of ice-cold buffer. The filters were air dried, and 50 l of Microscint-20 liquid scintillator (Packard) was added to each filter and counted in a Packard Topcount microplate scintillation counter (Packard). Nonspecific binding was determined in the presence of 10 M phentolamine HCl for both binding assays. Protein Determinations. Proteins were determined using the Bio-Rad Protein Assay Kit microtiter plate assay procedure (Bio- Rad, Hercules, CA) with BSA as standard. Data Analyses. Binding data were analyzed by the LIGAND nonlinear model-fitting program (Munson and Rodbard, 1980). When complete concentration-response curves were obtained, a two-factor ANOVA test was performed to compare each curve with another (e.g., NMI-187 versus yohimbine, NMI-221 versus moxixylyte) using StatView software for Apple computers. Remainder data were analyzed by performing a ANOVA test followed by a Student-Newman- Keuls post hoc test with GraphPAD (San Diego, CA) InStat software for Apple computers. For in vitro studies, n represents the number of tissues, each from a different animal or patient, whereas in in vivo experiments, it represents the number of different animals studied. In Vivo Studies. Induction of anesthesia in the rabbits was accomplished with propofol (10 mg/kg) administered in a bolus, and anesthesia was maintained by i.v. pump infusion (syringe pump model 268; Harvard Apparatus) that delivered 0.5 to 1 mg of propofol/kg/min, as required. Systemic arterial blood pressure was measured in a femoral artery, dissected in the inguinal area, and catheterized with a 22-gauge angiocatheter connected to a pressure transducer (Hewlett-Parkard). After incision of the distal prepucial skin surrounding the penis, a 25-gauge butterfly needle was inserted into the cavernosal space and connected to a pressure transducer (Hewlett-Packard). Pressures were recorded using an MACLAB monitoring system. Drugs dissolved in distilled water were administered intracavernosally via a venocatheter (Venocath-16; Abbott Laboratories, North Chicago, IL) connected to a 25-gauge needle inserted into the corpora cavernosa, across the tunica albuginea. All doses of the drugs tested were dissolved so that the final volume administered intracavernosally was 160 l. Intracavernosal pressure was monitored continually until the effect of the drug tested disappeared and the intracavernosal pressure returned to baseline. At that point, a control vasodilator mixture of 20 g/ml PGE 1,30 mg/ml papaverine, and 1 mg/ml phentolamine was injected, which is known to consistently cause a full sustained maximum erectile response in the rabbit. Data on the peak intracavernosal pressure responses are expressed as a percentage of the maximum intracavernosal pressure response obtained with the control vasodilator mixture, which was considered to be 100%. Mouse Paw Lick Test. The procedure was similar to the formalin-induced paw lick test as described previously (Vaccarino et al., 1993). Male CD-1 mice were injected with 20 l saline, vehicle (DMSO), or drug solutions into the left hind paw. The time spent licking the injected paw was then monitored for 30 min at 5-min intervals. Results Effects of Moxisylyte, Yohimbine, NMI-221, and NMI- 187 on Corpus Cavernosum Strips Contracted with Endothelin. Human or rabbit corpus cavernosum contracted with endothelin (10 nm) relaxed when exposed to high concentrations ( 50 M) of either moxisylyte or yohimbine (Figs. 2 and 3). Nitrosylation of these molecules signif- Fig. 2. Concentration-response curves to moxisylyte and NMI-221 (A) and yohimbine and NMI-187 (B) on rabbit corpus cavernosum strips contracted with endothelin (10 nm). Data are expressed as mean S.E.M. of the percentage of total relaxation induced by 0.1 mm papaverine. n indicates number of strips used, with each strip from a different animal. Relaxations to NMI-221 and NMI-187 were greater than those to moxisylyte and yohimbine, respectively, compared with the use of ANOVA (P.005).

4 124 Sáenz de Tejada et al. Vol. 290 Fig. 3. Concentration-response curves to moxisylyte and NMI-221 (A) and yohimbine and NMI-187 (B) on human corpus cavernosum strips contracted with endothelin (10 nm). Effects of treatment with the PDE5 inhibitor zaprinast on relaxations induced by NMI-221 (A) and NMI-187 (B). Data are expressed as mean S.E.M. of the percentage of total relaxation induced by 0.1 mm papaverine. n indicates number of strips used, with each strip from a different patient. Relaxations to NMI-221 and NMI-187 were greater than those to moxisylyte and yohimbine, respectively, compared with the use of ANOVA (P.005). This test yielded a significant enhancement of responses to NMI-221 and NMI-187 in the presence of zaprinast (P.005). icantly enhanced their ability to inhibit tone generated by endothelin (Figs. 2 and 3). A significant shift to the left on the concentration-response curve was observed with the nitrosylated compounds when comparing the effects of moxisylyte with those of NMI-221 (Fig. 2A) or of yohimbine with those of NMI-187 (Fig. 2B). Effects of PDE5 Inhibitor Zaprinast on Concentration-Response Curve to NMI-187 and NMI-221. Treatment of human corpus cavernosum tissues with zaprinast (1 M) did not modify basal tone or phenylephrine-induced contractions (data not shown) but caused a significant shift to the left on the concentration relaxation response curve to NMI-221 (Fig. 3A) and NMI-187 (Fig. 3B). Effects of Yohimbine, Moxisylyte, NMI-187, and NMI- 221 on cgmp Tissue Content. cgmp tissue content in rabbit corpus cavernosum tissue after a 5-min exposure to either moxisylyte (30 M) or yohimbine (300 M) was comparable to the levels measured under basal control conditions (Fig. 4). In contrast, the nitrosylated compounds induced a concentration-dependent significant increase in cgmp accumulated in rabbit cavernosal tissues (Fig. 4). Effects of Moxisylyte and NMI-221 on Contraction Induced by Phenylephrine and Effects of Yohimbine and NMI-187 on Contraction Induced by UK-14,304. Moxisylyte and NMI-221 had moderate potency in antagonizing the constrictor response induced by the selective 1 - adrenergic receptor agonist phenylephrine. There was no significant difference between the pa 2 values of moxisylyte and NMI-221, suggesting similar affinity for 1 -adrenergic Fig. 4. cgmp tissue content of rabbit corpus cavernosum after exposure to moxisylyte or NMI-221 (A) and yohimbine or NMI-187 (B). Data are expressed as mean S.E.M. of pmol cgmp/mg of tissue protein content. n indicates number of strips used for each treatment, with each strip from a different animal. *P.05, **P.01, ***P.005 versus control. P.05, P.01 versus 300 M (Student-Newman-Keuls post hoc test).

5 1999 Nitrosylated -Adrenergic Receptor Antagonists in Impotence 125 TABLE 1 pa 2 values of parent and nitrosylated -ARA Isolated rabbit corpus cavernosum tissues were induced to contract with either UK-14,304 or phenylephrine in the presence or absence of antagonists in tissue baths. Dose response of UK-14,304 was determined in the absence and presence of three concentrations of yohimbine and four concentrations of NMI-187 ranging from 10 to 100 nm. Dose response of phenylephrine was determined in the absence or presence of three concentrations of either moxisylyte or NMI-221 ranging from 1 to 10 M. Values represent mean S.E.M. with 95% confidence limits in parentheses. n represents number of experiments performed. Compound pa 2 n Against UK 14,304 Yohimbine ( ) 4 NMI ( ) 5 Against phenylephrine Moxisylyte ( ) 4 NMI ( ) 4 receptors (Table 1). UK-14,304 caused a concentration-dependent contraction of rabbit corpus cavernosum strips. Yohimbine and NMI-187 demonstrated potent antagonist activity against the contraction induced by the selective 2 - adrenoceptor agonist UK-14,304,. There was no significant difference between the pa 2 values of yohimbine and NMI- 187, suggesting similar affinity for 2 -adrenergic receptors (Table 1). -Adrenergic Receptor Binding. The K d values of [ 3 H]yohimbine and [ 3 H]prazosin and the K i values of yohimbine in the 1 - and 2 -receptor-binding assays were similar to published values (Brown et al., 1990; Buscher et al., 1996). Yohimbine and NMI-187 were selective for 2 -adrenergic receptors. Both compounds had a similarly high affinity for 2 -adrenergic receptors and had moderate affinity for 1 - adrenergic receptors in rat cerebral cortex membranes (Table 2). Preincubation with human PPP had no effect on the binding of yohimbine or NMI-187 to 1 - or 2 -adrenergic receptors. NMI-187 had about 3-fold higher affinity for 1 - adrenergic receptors than yohimbine with and without preincubation in PPP and thus was slightly less 2 selective than yohimbine. Both moxisylyte and NMI-221 were selective for 1 -adrenergic receptors (Table 2). NMI-221 had low affinity for 1 -adrenergic receptors compared with moxisylyte, which was 49 times more potent. However, the affinity of NMI-221 for 1 -adrenergic receptors increased by more than 65 times after incubation with PPP. Incubation of moxisylyte with PPP increased its affinity for the 1 -adrenergic receptor by about 4-fold. Incubation with PPP did not significantly affect the binding of moxisylyte or NMI-221 to 2 - adrenergic receptors. Effects of Intracavernosal Administration of Moxisylyte, NMI-221, Yohimbine, or NMI-187 on Intracavernosal Pressure in Anesthetized Rabbit. Intracavernosal administration of moxisylyte (1 and 2 mg) caused modest and short-lasting increases in intracavernosal pressure (Fig. 5A). NMI-221 caused a significantly larger increase than moxisylyte in peak intracavernosal pressure, a response comparable to that measured after the standard control vasodilator mixture (PGE 1, papaverine, and phentolamine) (Fig. 5A). The duration of the response was also significantly enhanced with 2 mg of the nitrosylated compound (Fig. 5B). Intracavernosal administration of yohimbine (0.5 or 1 mg) caused a transient increase in peak intracavernosal pressure approaching the response observed after the administration of the control standard vasodilator combination (Fig. 6A). NMI- 187, at the same doses, provoked a similar increase in intracavernosal pressure (Fig. 6A). However, the response after 1 mg of drug was significantly longer lasting with the nitrosylated compound than with the parent molecule (Fig. 6B). Effects of Intracavernosal Administration of Moxisylyte, NMI-221, Yohimbine, or NMI-187 on Systemic Mean Arterial Pressure in Anesthetized Rabbit. As shown in Fig. 7A, moxisylyte and NMI-221 caused a similar transient decrease in systemic mean arterial pressure that typically recovered within a few minutes. Yohimbine (0.5 or 1 mg) caused a modest transient decrease followed by a sustained increase in blood pressure (Fig. 7B). NMI-187 provoked a slight decrease in blood pressure that recovered promptly. The increase in blood pressure observed after yohimbine treatment was not observed with NMI-187, whereas the initial drop in blood pressure was similar to that of yohimbine (Fig. 7B). Effects of PGE 1, NMI-187, and NMI-221 on Mouse Paw Lick. Injection of saline into the hind paw of mice did not produce a significant response. Injection of vehicle (DMSO) into the hind paw produced a significant paw lick response compared with saline (Fig. 8). PGE 1 produced a dose-related increase in the time spent on paw licking. Neither NMI-187 at 66 and 333 g nor NMI-221 at 66 to 660 g increased the time spent on paw licking (Fig. 8). Discussion Our study shows the feasibility of nitrosylation of -ARAs through the use of an ester linker. Such nitrosothiol molecules release nitrosonium ions, acting as NO donors, and once the ester linker group has been cleaved, they exert their full activity to antagonize -adrenergic receptors. The pharmacologically induced relaxation of penile smooth muscle by NO together with the inhibition of the tone generated by endogenous catecholamines mimics, at least in part, the physiological events that initiate and sustain penile erection (Sáenz de Tejada et al., 1989; Juneman, 1996). This was the rationale for combining NO donor with -ARA activities in the design of these molecules for the treatment of erectile dysfunction. The ability of NMI-187 and NMI-221, more so than the parent -ARAs, to reverse tone induced by endothelin in corpus cavernosum strips was indicative of their NO donor activity; an effect that was significantly enhanced by an inhibitor of the cgmp-specific PDE5 zaprinast (Shahid et al., 1991; Taher et al., 1997). Furthermore, the concentrationdependent accumulation of cgmp induced by NMI-187 and NMI-221, but not yohimbine or moxisylyte, further demonstrates the NO donor capacity of these nitrosylated compounds. The -ARA activity of the molecules was investigated in contractility experiments with corpus cavernosum tissues in organ chambers and in receptor binding studies with rat cerebral cortex membranes. The ester linker in the 17-hydroxy position of the yohimbine molecule does not seem to affect its -adrenergic-binding activity because yohimbine and NMI-187 showed similar 2 -adrenergic receptor antagonist potencies, as indicated by similar pa 2 values in the contractility studies and similar K i values in the receptorbinding studies. In contrast, moxisylyte and NMI-221 showed similar 1 -adrenergic receptor antagonist activity in the isolated tissue bioassay even though the binding affinity

6 126 Sáenz de Tejada et al. Vol. 290 TABLE 2 -Adrenergic receptor binding to rat cerebral cortex membranes with and without preincubation with human PPP [ 3 H]Yohimbine K d 5.3 nm, 3 nm used for binding (30 min at room temperature). [ 3 H]Prazosin K d 184 pm, 0.3 nm used (45 min at room temperature). K i 1 2 Control PPP Control PPP nm Yohimbine NMI a Moxisylyte NMI b c 24, a 15, a Data represent the mean S.E.M. of three or four experiments in duplicate or triplicate. a P.05 and b P.01 compared with its respective parent molecule and c P.01 compared with no platelet-poor plasma control of the same molecule, as determined by Student-Neuman-Keuls post hoc test. Fig. 5. Effects of intracavernosal administration of moxisylyte (MOX) or NMI-221 on intracavernosal pressure (ICP) (A) and the duration of response (B) in the anesthetized rabbit. Data are expressed as mean S.E.M. of the percentage of the response obtained with the control standard vasodilator combination (PGE 1, papaverine, and phentolamine). n indicates number of animals studied. *P.05 (Student-Newman-Keuls post hoc test). of moxisylyte for 1 -adrenergic receptor is 49 times higher than that of NMI-221. This significant difference in binding affinity disappeared when NMI-221 was preincubated with PPP. Preincubation with PPP increased the 1 -adrenergic Fig. 6. Effects of intracavernosal administration of yohimbine (YOH) or NMI-187 on intracavernosal pressure (ICP) (A) and the duration of response (B) in the anesthetized rabbit. Data are expressed as mean S.E.M. of the percentage of the response obtained with the control standard vasodilator combination (PGE 1, papaverine, and phentolamine). n indicates number of animals studied. *P.05 (Student-Newman-Keuls post hoc test). receptor-binding affinity of moxisylyte and NMI-221 by 4.7 and 65 times, respectively. This increase in binding affinity is consistent with the hypothesis that the ester bond of both moxisylyte and NMI-221 was hydrolyzed by esterases to

7 1999 Nitrosylated -Adrenergic Receptor Antagonists in Impotence 127 Fig. 7. Effects of intracavernosal administration of moxisylyte and NMI- 221 (A) and yohimbine (YOH) and NMI-187 (B) on systemic mean arterial pressure (MAP) in the anesthetized rabbit. Data are expressed as mean S.E.M. of the percentage of change of MAP from the baseline. n indicates number of animals studied. Both doses of yohimbine induced a significant increase of MAP compared with the respective doses of NMI-187 by ANOVA (P.05). Fig. 8. Effects of PGE 1, NMI-187, and NMI-221 on the paw lick response in mice. Data represent the mean S.E.M. of 8 to 11 mice per group. *P.05 (Student-Newman-Keuls post hoc test) versus DMSO. yield a more potent compound. In vivo, moxisylyte is immediately metabolized to desacetylmoxisylyte (DAM), conjugated DAM, and conjugates of desmethylated DAM after intracavernosal administration in humans (Costa et al., 1992). Thus, it is possible that incubation of moxisylyte and NMI-221 with human PPP may result in the formation of DAM, which has a higher affinity for 1 -adrenergic receptors than the parent molecule. Moxisylyte and yohimbine are currently used for the pharmacological treatment of erectile dysfunction. Intracavernosal injection of moxisylyte has been shown to facilitate erection compared with placebo. The use of this drug was associated with a low incidence of pain, although it was less effective than PGE 1 in terms of erection-inducing activity (Buvat et al., 1996). Yohimbine has been used for decades as an oral agent for the treatment of erectile dysfunction. Although its effects on facilitating erection in animal models is well demonstrated (Clark et al., 1984), its therapeutic value for the treatment of impotence in humans has been questioned (Kunelius et al., 1997). This drug was found to be superior to placebo in the treatment of psychogenic impotence (Reid et al., 1987) but ineffective in the treatment of impotence with an organic basis (Teloken et al., 1998). The in vivo experiments demonstrate that nitrosylation of the -ARAs significantly enhances their erection-inducing activity. The responses, particularly those to NMI-221, were comparable to the erectile response provoked by the administration of the triple mixture of PGE 1, papaverine, and phentolamine, which represents the most effective vasoactive combination that is currently used for the treatment of erectile dysfunction (Govier et al., 1993). Within the doses used in this study, the nitrosylation of the -ARAs did not significantly enhance the systemic effects of these molecules on systemic blood pressure. A rapid local transfer of NO in the cavernosal space may explain why the nitrosylated compounds, despite being more potent vasoactive agents than the parent -ARAs, did not significantly enhance systemic hypotension. The use of intracavernosal PGE 1, alone or in combination with other agents, has encountered pain as a significant side effect in more than 30% of patients (Gerber and Levine, 1991; Linet and Ogring, 1996). For this reason, the nitrosylated -ARAs were compared with PGE 1 in the mouse paw lick test. PGE 1 produced nociception in the mouse paw lick test. In a previous study, PGE 1 was found to be nociceptive in the abdominal constriction (Gyires and Knoll, 1975). NMI-187 and NMI-221, on the other hand, did not produce nociception in the mouse paw lick test; thus, these compounds have the advantage of not inducing pain compared with PGE 1. In summary, we synthesized and characterized the pharmacological and biological activity of two nitrosylated -ARAs that have the dual functionalities of donating NO and antagonizing -adrenergic receptors. These drugs induce penile erection in animal models, suggesting that they may be useful therapeutic agents for the local (intracavernosal, transglansdular, or transurethral), pharmacological treatment of impotence.

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