Potentiation of Penile Tumescence by T-1032, a New Potent and Specific Phosphodiesterase Type V Inhibitor, in Dogs

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1 /00/ $03.00/0 THE JOURNAL OF PHARMACOLOGY AND EXPERIMENTAL THERAPEUTICS Vol. 294, No. 3 Copyright 2000 by The American Society for Pharmacology and Experimental Therapeutics 2623/ JPET 294: , 2000 Printed in U.S.A. Potentiation of Penile Tumescence by T-1032, a New Potent and Specific Phosphodiesterase Type V Inhibitor, in Dogs TSUNEHISA NOTO, HIROTAKA INOUE, TOMIHIRO IKEO, and KOHEI KIKKAWA Discovery Research Laboratory, Tanabe Seiyaku Co., Ltd., Toda, Saitama, Japan Accepted for publication May 5, 2000 This paper is available online at Received for publication February 18, ABSTRACT We examined the mechanism underlying the potentiation of penile tumescence by methyl 2-(4-aminophenyl)-1,2- dihydro-1-oxo-7-(2-pyridinylmethoxy)-4-(3,4,5-trimethoxyphenyl)- 3-isoquinoline carboxylate sulfate (T-1032), a new potent and selective phosphodiesterase type V inhibitor. In vivo, pelvic nerve stimulation induced a penile tumescence together with increase of total nitric oxide metabolite levels within the corpus cavernosa of anesthetized dogs. Intravenous (1 100 g/kg) and intraduodenal (3, 30, 300 g/kg) treatment with T-1032 dose dependently potentiated the tumescence. The potency of T-1032 was equivalent to that of sildenafil. T-1032 did not influence the intracavernous pressure when the pelvic nerve stimulation was absent. The potentiation of tumescence was more pronounced by intracavernous than i.v. injection. Intracavernous N G -nitro-l-arginine, a nitric-oxide synthase inhibitor, but not N G -nitro-d-arginine diminished the effects of T-1032 on the tumescence. Furthermore, i.v. T-1032 augmented the tumescence induced by sodium nitroprusside (SNP) but not by vasoactive intestinal polypeptide (VIP). In vitro, in isolated preparations of canine corpus cavernosum precontracted with phenylephrine, SNP ( M) and VIP ( M) produced a dose-dependent relaxation accompanied by an increase in cgmp and camp levels, respectively. T-1032 augmented the relaxation induced by SNP but not by VIP. These data suggest that oral treatment with T-1032 has potential to improve erectile dysfunction through the inhibition of phosphodiesterase type V in the smooth muscles of corpus cavernosa. Penile erection is initiated by neuronal impulses in parasympathetic pelvic nerves that cause arteriolar vasodilatation and relaxation of smooth muscle elements in the corpus cavernosum, the erectile tissue of the penis. Inflow of blood in the corporal lacunae, together with relaxation of the smooth muscle surrounding the lacunae, and venous outflow obstruction, lead to turgor of the erectile tissue (Anderson, 1993; Andersson and Wagner, 1995). Over the years, accumulating data have supported that release of nitric oxide (NO) mediates the smooth muscle relaxant effect of pelvic nerve stimulation in corpus cavernosa (Ignarro et al., 1990; Rajfer et al., 1992; Trigo-Rocha et al., 1993b; Ayajiki et al., 1997). NO exerts its effect by stimulating soluble guanylate cyclase in the smooth muscle with a subsequent increase in the concentration of cgmp but not camp (Ignarro et al., 1990; Bush et al., 1992a; Dahiya et al., 1993; Martinez Pineiro et al., 1993; Sparwasser et al., 1994). After inducing the formation of cgmp, NO is rapidly degraded to nitrite and nitrate and the presence of these metabolites is one of the proof for the presence of NO (Bush et al., 1992b). Sildenafil, an inhibitor of the cgmp-specific phosphodiesterase type V (PDE V), is reported to be effective for the treatment of erectile dysfunction in humans (Boolell et al., 1996a,b; Goldstein et al., 1998). In animal studies, sildenafil facilitates tumescence in dogs and relaxes cavernous tissues of rabbits and humans in vitro (Carter et al., 1998; Chuang et al., 1998a; Stief et al., 1998). Because PDE V is a predominant isoenzyme hydrolyzing cgmp in the corpus cavernosum, it is thought that its inhibition and the subsequent accumulation of cgmp account for the effect of sildenafil (Boolell et al., 1996a). This is supported by in vitro findings that sildenafil specifically amplifies a relaxation of corpus cavernosum smooth muscle that depends on cgmp formation in rabbit (Chuang et al., 1998a). However, the mechanisms of sildenafil-induced augmentation of penile tumescence are not fully studied in vivo. T-1032 [methyl 2-(4-aminophenyl)-1,2-dihydro-1-oxo-7-(2- pyridinylmethoxy)-4-(3,4,5-trimethoxyphenyl)-3-isoquinoline carboxylate sulfate] (Fig. 1) is a selective PDE V inhibitor synthesized in our laboratory. This compound reversibly inhibits PDE V with a K i value of 1.2 nm (Kotera et al., 2000; Table 1). In this study, to know whether T-1032 is effective to improve erectile dysfunction, the effects of T-1032 on penile ABBREVIATIONS: NO, nitric oxide; PDE V, phosphodiesterase type V; T-1032, methyl 2-(4-aminophenyl)-1,2-dihydro-1-oxo-7-(2-pyridinylmethoxy)-4-(3,4,5-trimethoxyphenyl)-3-isoquinoline carboxylate sulfate; SNP, sodium nitroprusside; VIP, vasoactive intestinal polypeptide; L-NAME, N G -nitro-l-arginine; D-NAME, N G -nitro-d-arginine; i.d., intraduodenal. 870

2 2000 T-1032, a New Potent and Selective PDE V Inhibitor 871 Fig. 1. Chemical structure of T tumescence were examined in vivo with anesthetized dogs in comparison with those of sildenafil. Furthermore, the underlying mechanisms were studied both in vivo and in vitro. Materials and Methods Animal Preparation. This project was approved by the Ethical Committee at Tanabe Seiyaku Co., Ltd. Male mongrel dogs weighing between 13 and 23 kg were used. In Vivo Experiments. Dogs were anesthetized with pentobarbital sodium (30 mg/kg i.v. bolus injection, followed by 4.5 mg/kg/h i.v. infusion). An endotracheal tube was placed to ventilate (15 ml/kg/ stroke, 20 strokes/min) with room air. The femoral artery was cannulated for continuous blood pressure monitoring. The abdomen was opened through a midline abdominal incision. Polyethylene catheter was inserted into the duodenum and held in place by a ligature. The left pelvic nerve, located superior and lateral to the prostate was carefully isolated and placed on a bipolar electrode (IMT-1530; Inter Medical Co., Ltd., Nagoya, Japan) connected to an electronic stimulator (Nihon Kohden, Tokyo, Japan). Two 21-gauge venous needles were placed in the corpus cavernosum on the left side ( 1 cm apart): one was connected to the pressure transducer (TP-400T, Nihon Kohden) and linearecorder (WR3701; Graphtec Tokyo, Japan) for recording intracavernous pressure, and the other was used for drawing blood and for intracavernous injection of sodium nitroprusside (SNP), vasoactive intestinal polypeptide (VIP), N G -nitro-l-arginine methyl ester (L-NAME), and its enantiomer N G -nitro-d-arginine methyl ester (D-NAME). Blood (0.2 ml) was taken during the tumescence induced by the pelvic nerve stimulation and centrifuged at 4 C. The serum was stored at 80 C until the measurements of NO metabolites. Drugs were administered i.v. (0.1 ml/kg), intraduodenally (i.d., 0.25 ml/kg), or intracavernously (0.5 ml/head). At the i.v. treatment, T-1032 and sildenafil were applied 5 min before nerve stimulation because time to peak responses of both drugs was about 5 min in a preliminary study. L-NAME and D-NAME were treated intracavernously 15 min before the stimulation. The pelvic nerves TABLE 1 Inhibitory effects of T-1032 and sildenafil on six canine PDE isozymes PDE I, IV, and V were isolated from the lung and PDE III was from the heart. PDE II and VI were prepared from the adrenal gland and retinas, respectively. All data were cited from the previous study (Kotera et al., 2000). Compounds IC 50 PDE V PDE I PDE II PDE III PDE IV PDE VI M T Sildenafil Fig. 2. A, typical trace of the tumescence induced by the pelvic nerve stimulation (10 V, 200 s of pulse width for 40-s duration) in the anesthetized dog. B, tumescence induced by the pelvic nerve stimulation in anesthetized dogs (n 6). C, serum nitrite and nitrate levels from the corpus cavernosum during the tumescence induced by the pelvic nerve stimulation (n 6).

3 872 Noto et al. Vol. 294 Fig. 3. A, representative curves showing potentiation of nerve-induced tumescence by T-1032 treatment. B and C, effects of i.v. treatment with T-1032 and sildenafil on the tumescence induced by the pelvic nerve stimulation, and on the blood pressure in anesthetized dogs (n 5 7). *P.05 versus vehicle. Fig. 4. A, intracavernous injection (0.5 ml) of T-1032 (100 M) exerts no effect on the intracavernous pressure without pelvic nerve stimulation. B and C, effects of intracavernous pretreatment with T-1032 on the tumescence induced by the pelvic nerve stimulation in anesthetized dogs (n 5). The dosages of 0.01, 0.1, 1, 10, and 100 M, 0.5 ml/head are equivalent to about , 0.002, 0.02, 0.2, and 2 g/kg, respectively. *P.05 versus vehicle. Fig. 5. Effects of i.d. treatment with T-1032 and sildenafil on the tumescence induced by the pelvic nerve stimulation in anesthetized dogs (n 5 7). *P.05, **P.01 versus vehicle. were stimulated by electrical square pulses (200- s pulse width) of 10 V at frequencies from 2.5 to 20 Hz for a period of 40 s at intervals of 20 min. All the experiments were started when the submaximal nerve stimulation evoked consistent responses. For quantitative determination of the tumescence, we measured the area under the curve and expressed it as millimeters of mercury multiplied by minute. Serum nitrite and nitrate levels were measured by a Griess reagent method with an automated HPLC system (ENO-20; Eicom, Kyoto, Japan) as described previously (Yamada and Nabeshima, 1997). In Vitro Experiments. Anesthetized dogs were sacrificed by bleeding from the carotid arteries. The penis was rapidly removed and the corpus cavernosa were isolated. The tunica albugina was carefully removed and strips of cavernous tissues (5 3 1 mm) were obtained. The specimens were mounted under 1.5-g resting tension on hooks in organ baths (10 ml) filled with the modified Ringer-Locke solution gassed with 95% O 2 and 5% CO 2 at 37 C. The tension was recorded with a isometric transducer (AP-601G; Nihon Kohden) on a six-channel multipen recorder (NC-6625; Graphtec). The tissues were contracted three times with potassium chloride (30 mm) after 60 min of equilibration. After that, strips were contracted submaximally by phenylephrine (5 M). L-NAME (100 M) was added 20 min before inducing contraction to inhibit the release of endogenous NO. Cumulative relaxation curves to SNP or VIP were obtained after the contracted tension was stabilized. Papaverine (100 M)-induced relaxation was taken as 100% at the end of experiment. T-1032 or its vehicle was pretreated 10 min before the contraction. T-1032 exerted no significant effects on the contraction induced by phenylephrine as did sildenafil (Ballard et al., 1998). The tissue content of cyclic nucleotides was measured as follows: when the relaxation by SNP or VIP reached a stable level, usually within 2 min, the strips were immediately frozen in liquid nitrogen

4 2000 T-1032, a New Potent and Selective PDE V Inhibitor 873 ANOVA except for the experiments in which drugs were administered i.d. with two-way repeated measure with Bonferroni s correction. Differences were considered significant when P.05. Fig. 6. Effects of N G -nitro-l-arginine (L-NAME, 3 mm, 0.5 ml intracavernous) and N G -nitro-d-arginine (D-NAME, 3 mm, 0.5 ml intracavernous) on T-1032-induced potentiation of the tumescence in the anesthetized dogs (n 5). *P.05 versus pretreatment (paired t test), P.05 (Dunnett s method). and then stored at 70 C until the assay. The tissues were homogenized by Polytron (Kinematica, Lucerne, Switzerland) in 10% trichloroacetic acid (1 ml). After centrifugation (2000g, 15 min), the supernatants were washed with water-saturated diethyl ether three times. The liquid phase were lyophilized and assayed for cgmp and camp contents with cgmp enzyme immunoassay system and camp enzyme immunoassay system (Amersham Corp., Amersham, UK), respectively. Protein contents of pellets were quantificated as described and the tissue contents of the cyclic nucleotides were expressed as picomoles per gram protein (Bradford, 1976). The composition of the modified Ringer-Locke solution was as follows: 120 mm NaCl, 5.4 mm KCl, 2.2 mm CaCl 2, 1.0 mm MgCl 2, 25.0 mm NaHCO 3, and 5.6 mm glucose. Drugs. T-1032 (synthesized at Tanabe Seiyaku Co., Ltd., Toda, Japan) and sildenafil citrate (synthesized at Tanabe Seiyaku Co., Ltd.) were dissolved in N HCl for i.d. administration or N HCl in saline for i.v. and intracavernous injections. VIP (Peptide Institute, Osaka, Japan) and SNP (Nacalai Tesque, Kyoto, Japan) were dissolved in saline (in vivo) or the modified Ringer-Locke solution (in vitro). L-NAME and D-NAME (Sigma, St. Louis, MO) were dissolved in saline. All other chemicals used were of reagent grade. Data Analysis. The results are expressed as mean S.E. Statistical analyses were made with the Dunnett s method after one-way Results In Vivo Experiments. Pelvic nerve stimulation (5 20 Hz) increased the intracavernous pressure accompanied by the elevation of nitrite and nitrate levels in the blood drawn from the corpus cavernosum in a frequency-dependent manner (Fig. 2). Intravenous treatment with T-1032 and sildenafil dose dependently potentiated the penile tumescence induced by submaximal pelvic nerve stimulation (Fig. 3, A and B). Both compounds caused a slight but significant decrease in arterial blood pressure (Fig. 3C). The effect of T-1032 on the tumescence was more potent by the intracavernous than i.v. treatment (Fig. 4). T-1032 did not influence the intracavernous pressure without the pelvic nerve stimulation (Fig. 4A). Intraduodenal treatment with T-1032 and sildenafil induced a dose-dependent potentiation of tumescence (Fig. 5). Although the time to reach the maximal increase was the same, the duration of the response was much longer by T-1032 than sildenafil (Fig. 5). Intracavernous pretreatment with L-NAME (3 mm, 0.5 ml) but not D-NAME (3 mm, 0.5 ml) diminished the effects of T-1032 on the tumescence (Fig. 6), without any effects on the blood pressure (data not shown). Intracavernous injection of SNP (100 or 300 M, 0.5 ml) and VIP (3 M, 0.5 ml) caused the tumescence to the same extent. T-1032 (30 g/kg i.v. followed by 1 g/kg/min i.v. infusion) potentiated the tumescence induced by intracavernous SNP but not VIP (Fig. 7). In Vitro Experiments. In isolated preparations of the canine corpus cavernosa precontracted with phenylephrine, SNP ( M) and VIP ( M) produced a concentration-dependent relaxation that was accompanied by the increase in tissue cgmp and camp levels, respectively. T-1032 ( M) augmented the relaxation induced by SNP but not by VIP (Figs. 8 and 9). Discussion Penile erection involves parasympathetic nerve-mediated relaxation of the blood vessels and the trabecular meshwork Fig. 7. A, typical trace of the effects of T-1032 (30 g/kg i.v. bolus followed by 1 g/kg/min i.v. infusion) on the tumescence induced by intracavernous VIP (3 M, 0.5 ml) or SNP (SNP, 100 M, 0.5 ml) treatments in the anesthetized dog. B, effects of T-1032 (30 g/kg i.v. bolus followed by 1 g/kg/min i.v. infusion) on the tumescence induced by intracavernous VIP or SNP in anesthetized dogs (n 6). *P.05 versus pretreatment.

5 874 Noto et al. Vol. 294 Fig. 8. Effects of SNP and VIP on the levels of cgmp (f) and camp ( ) in the canine corpus cavernosum precontracted by 5 M phenylephrine (n 4 6). *P.05 versus vehicle. of smooth muscles that constitute the corpora cavernosa (Anderson, 1993; Andersson and Wagner, 1995). Several in vitro studies have demonstrated that NO and its second messenger cgmp are responsible for the nerve-induced relaxation of rabbit, dog, and human corpus cavernosum smooth muscle strips (Kim et al., 1991; Pickard et al., 1991; Holmquist et al., 1992; Hedlund et al., 1995b). In in vivo studies in rats, dogs, cats, and rabbits, prevention of nerveinduced erection by the inhibition of the NO synthesis demonstrated that NO is essential for the physiological process of erection (Holmquist et al., 1991; Burnett et al., 1992; Finberg et al., 1993; Trigo-Rocha et al., 1993a; Wang et al., 1994). Although NO levels in the rat penis were increased by the electrical stimulation of cavernosal nerves, nitrite and nitrate levels in human cavernosal blood do not change during the erection (Moriel et al., 1993; Escrig et al., 1999). There have been no reports about the change of these levels in the canine cavernosum during the tumescence. The increase of nitrite and nitrate levels during the tumescence in this study again confirms the crucial role of NO in the erection. T-1032 dose dependently enhanced the tumescence by the i.d. treatment with the same potency as sildenafil in this study. Because oral treatment with sildenafil improves erectile dysfunction in humans (Boolell et al., 1996b; Goldstein et al., 1998), T-1032 is supposed to have the same potentials. The potentiation of tumescence was more pronounced by intracavernous than i.v. administration. This is consistent with the hypothesis that systemically (i.d. or i.v.) applied T-1032 locally acts in the corpus cavernosa. Although PDE V was inhibited by T-1032 with a K i value of 1.2 nm in vitro (Kotera et al., 2000), in vivo effect was evoked only at dosages of 10 M. We suspect that the injected T-1032 was immediately diluted in the blood stream in the corpus cavernosum. N G -substituted analogs of L-arginine, such as L-NAME and L-N G -amino-l-arginine, inhibit NO synthase by an enantiomer-specific manner (Rees et al., 1990). Intracavernous pretreatment with L-NAME but not D-NAME, an enantiomer, diminished the potentiating effect of T It has been reported that intracavernous L-NAME blocks pelvic nervestimulated tumescence with the partial reversal by the NO precursor L-arginine (Trigo-Rocha et al., 1993a). Together with the previous findings, it is concluded that nerve-stimulated tumescence as well as T-1032-induced potentiation Fig. 9. Effects of T-1032 on the relaxation induced by SNP (A) or VIP (B) in the isolated canine corpus cavernosa (n 5)., vehicle; Œ, 0.01 M T-1032;, 0.1 M T-1032;, 1 M T needs NO synthase activity. This is in accord with the hypothesis that T-1032 augments the effect of NO by inhibiting PDE V activity. SNP generates NO and relaxes isolated rabbit and human corpus cavernosa with the elevation of the tissue contents of cgmp (Bush et al., 1992b; Holmquist et al., 1993; Chuang et al., 1998b). Although VIP is unlikely to be involved in the relaxation of the corpus cavernosa induced by physiological pelvic nerve stimulation, exogenously injected VIP relaxes isolated human corpus cavernosa via adenylate cyclase activation with the increase of the tissue contents of camp (Azadzoi et al., 1992; Hedlund et al., 1995a; Hempelmann et al., 1995; Okamura et al., 1999). In this study, both SNP and VIP concentration dependently relaxed isolated canine corpus cavernosa in association with increases in the tissue contents of cgmp and camp, respectively. Selective enhancement of the relaxation induced by SNP but not by VIP suggests that the effect of T-1032 is mediated by the inhibition of the cgmp-specific pathway, presumably PDE V. The potency of T-1032 to augment the relaxation is lower than that expected from the inhibition of the isolated enzyme. This may be due to a low permeability to the cell membrane.

6 2000 T-1032, a New Potent and Selective PDE V Inhibitor 875 Intracavernous injection of SNP and VIP induced the tumescence in anesthetized dogs. Although the relaxant effects of both drugs were similar in the isolated cavernosa, VIP was more potent on tumescence than SNP in in vivo studies. We suspect that the VIP-induced relaxation is potentiated by some unidentified mechanism in vivo, which is lost in the isolated muscles in vitro. Systemically applied T-1032 selectively enhanced the tumescence induced by SNP, like isolated muscles. This clearly shows that the downstream signal of the elevation of cgmp is enhanced by T-1032 in the cavernous smooth muscles in vivo. Together with the in vitro studies, T-1032 seems to elicit the effect through the inhibition of PDE V within the cavernous smooth muscles. In summary, oral treatment with T-1032 may improve erectile dysfunction as does sildenafil through inhibition of PDE V in the smooth muscles of corpus cavernosa. 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