JPET Assistant Professor of Medicine (Clinician-Educator)

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Vol. 292, Issue 3, 974-981, March 2000


Effects of Intracavernous Administration of Selective Antagonists of alpha 1-Adrenoceptor Subtypes on Erection in Anesthetized Rats and Dogs

Giorgio Sironi, Davide Colombo, Elena Poggesi, Amedeo Leonardi, Rodolfo Testa, Olivier Rampin1, Jacques Bernabé1 and Francois Giuliano2

Pharmaceutical R&D Division, Recordati S.p.A., Milan, Italy

    Abstract
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References

The proerectile properties of three novel alpha 1-adrenoceptor (alpha 1-ADR) antagonists with different profiles of selectivity for the alpha 1-ADR subtypes have been evaluated in anesthetized rats and dogs on intracavernous (IC) injection, in comparison with prazosin and phentolamine. In rats, the tested compounds decreased blood pressure (BP) and increased IC pressure (ICP), as well as the ratio ICP/BP. Rec 15/2841 (alpha 1a- plus alpha 1L-ADR-selective antagonist) and Rec 15/2615 (alpha 1b-ADR selective) were the most potent compounds. The ICP/BP ratios calculated after injection of Rec 15/3039 (alpha 1d-ADR selective) were not markedly different from those observed after vehicle injection. Prazosin and phentolamine proved poorly active, their main effect being hypotension. Approximate ED25 values (dose of compound in micrograms inducing 25% increase of ICP/BP ratio) were Rec 15/2615 (22 µg/kg) >= Rec 15/2841 (29 µg/kg) > prazosin (136 µg/kg) > phentolamine (1298 µg/kg) > Rec 15/3039 (9600 µg/kg). Submaximal stimulation of the cavernous nerve elicited an ICP rise whose amplitude was not altered by Rec compounds. In contrast, prazosin and phentolamine decreased this ICP rise. All compounds but 15/3039 induced significant increase of the ICP/BP ratio in dogs. Rec 15/2615 proved to be the most interesting compound, inducing significant increases of ICP/BP at doses practically devoid of effects on BP. The rank order of potency in dog in increasing the ICP/BP ratio was similar to that observed in rats. Only at the highest doses tested, all compounds, except Rec 15/3039, decreased the ICP rise elicited by submaximal stimulation of the cavernous nerve. Our data demonstrate that the alpha 1b- and alpha 1L-ADR subtypes are functionally relevant for the erectile function in these models, and that alpha 1b- and/or alpha 1L-ADR subtypes selective antagonists could represent a real advantage in erectile dysfunction therapy.

    Introduction
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References

During erection, blood fills the penis. This physiological response relies on arterial and intracavernous (IC) smooth muscle relaxation (Andersson and Wagner, 1995). It is controlled by the autonomic innervation to the penis. Parasympathetic pathways are considered the main proerectile pathways. Sympathetic pathways play a major role in flaccidity and detumescence. As recently pointed out (Melman and Gingell, 1999), a critical amount of relaxation of contracted arterial and trabecular smooth muscle is required to convert the flaccid penis to erect state. In the flaccid state, noradrenaline (NA), released by postganglionic sympathetic fibers and acting at postjunctional alpha 1-adrenoceptor (ADR) subtypes present on the smooth muscle fibers of the cavernous arteries and of the corpus cavernosum (CC), contributes to keep the penile smooth muscles contracted (Christ et al., 1990). IC injection of alpha 1-antagonists, e.g., phenoxybenzamine, phentolamine, or moxisylyte, produces tumescence and erection (for review, see Andersson, 1993). Furthermore, proerectile effects of transurethral delivery of the alpha 1-antagonist prazosin in humans have been recently reported (Peterson et al., 1998), as well as the relaxing effect of this compound on human and dog CC in vitro (Hedlund and Andersson, 1985; Christ et al., 1990; Holmquist et al., 1990; Hayashida et al., 1996).

Pharmacological, biochemical, and radioligand binding studies evidenced three different alpha 1-ADR subtypes with a high affinity for prazosin, namely, alpha 1A- (alpha 1a-), alpha 1B- (alpha 1b-), and alpha 1D- (alpha 1d-), with lowercase subscripts being used for recombinant receptors and uppercase subscripts for receptors in native tissues (Hieble et al., 1995). In functional studies alpha 1-ADRs with a low affinity for prazosin also have been identified (but not cloned yet) and termed alpha 1L-ADRs (Flavahan and Vanhoutte, 1986; Muramatsu et al., 1995).

Several studies have demonstrated the presence of these alpha 1-ADR subtypes in rat and human CC (Traish et al., 1995, 1996; Dausse et al., 1998; Véronneau-Longueville et al., 1998). With in situ hybridization with specific oligonucleotide probes and RNase protection assays techniques, evidence was provided that both rat and human CC expressed the three cloned alpha 1-ADR subtypes. Functional studies on human CC in vitro, however, are controversial, either suggesting the involvement of all the three cloned alpha 1-ADR subtypes (Traish et al., 1995) or pointing out to the alpha 1L-ADR subtype as the main mediator of NA-induced contraction in this tissue (Davis et al., 1998), being this last assumption in contrast to the data of Christ et al. (1990), showing that 0.5 nM prazosin produced a substantial blockade of phenylephrine-induced contraction.

Pharmacological evidence for the univocal role of a well defined alpha 1-ADR subtype in the CC would represent a major advance in the field of male erectile dysfunction treatment, leading to the possible use of selective alpha 1-antagonists. Although the in vitro characterization of the expression of CC alpha 1-ADR subtypes represents a promising field for pharmacological research on male erectile dysfunction therapy, in vivo studies offer the advantage to analyze the effects of the compounds on intracavernous pressure (ICP) and blood pressure (BP).

Furthermore, in anesthetized animals, penile erection can be elicited by cavernous nerve (CN) or pelvic nerve (PN) stimulation. It elicits an increase of ICP whose amplitude is dependent on both the stimulation applied and BP (Carati et al., 1987; Giuliano et al., 1993). Because alpha 1-antagonists are known to lower BP, these compounds may alter indirectly ICP rise elicited by nerve stimulation.

For these reasons, we studied the effects of well characterized alpha 1-antagonists in in vivo models in rats and dogs. The radioligand-binding affinity of these compounds for the alpha 1-ADR subtypes, as well as their in vitro potency as antagonists of NA-induced contraction of rabbit aorta after chloroethylclonidine (CEC) alkylation (alpha 1L-ADR subtype), were used for profiling the tested antagonists.

Three novel alpha 1-antagonists were tested, namely, Rec 15/3039, Rec 15/2841, and Rec 15/2615. The last two compounds were reported to be selective for the alpha 1a/alpha 1L- and alpha 1b-ADR subtypes, respectively (Testa et al., 1997), whereas Rec 15/3039 has been found to be selective for the alpha 1d-ADR subtype. Prazosin was used as a nonsubtype-selective reference compound, and phentolamine was included in the study, being a nonselective alpha 1- and alpha 2-ADR blocking agent widely used for IC vasoactive injection therapy, alone or in combination with other active substances (Montorsi et al., 1995). Proerectile effects of the compounds were tested in anesthetized rats and dogs by measuring ICP and BP. Modulation of ICP increase elicited by CN or PN stimulation by the above-mentioned drugs also was investigated.

    Materials and Methods
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References

Male New Zealand White rabbits weighing 2.5 to 3.5 kg (Morini Allevamenti, Reggio Emilia, Italy), male Sprague-Dawley rats weighing 200 to 250 g (Charles River, Saint Aubin Les Elbeuf, France), and male beagle dogs weighing 10 to12 kg (Green Hill Allevamenti, Brescia, Italy) were used in this study. Animals were housed with free access to food and water and maintained on a forced 12-h light/dark cycle at 22-24°C for at least 1 week before the experiments were carried out. The animals were handled according to internationally accepted principles for care of laboratory animals (European Economic Community, Council Directive 86/609, OJL358, 1, December 12, 1987).

Binding Affinity for Recombinant alpha 1a-, alpha 1b-, and alpha 1d-ADR Subtypes. The affinity of Rec 15/3039 for the recombinant bovine alpha 1a-, hamster alpha 1b-, and rat alpha 1d-ADR subtypes in Chinese hamster ovary cells was evaluated as previously described with [3H]prazosin as labeled ligand (Testa et al., 1995). The affinity data of the other compounds tested were previously obtained with the same methods (Testa et al., 1997).

Functional Affinity for alpha 1L-ADR Subtype. The functional affinity of Rec 15/3039 for the alpha 1L-adrenoceptor subtype was evaluated as antagonism of NA-induced contraction of rabbit aorta pretreated with CEC with the same method previously reported for testing all other compounds used in the present study (Testa et al., 1997).

IC and BP Recording in Rats. The evaluation of the erectile properties of the different compounds tested in rats was performed according to a previously described method (Giuliano et al., 1993). Rats were anesthetized by an i.p. injection of urethane (l.5 g/kg in sterile saline) and placed on a homeothermic blanket. Their temperature was maintained at 37°C. Animals were tracheotomized to facilitate spontaneous breathing and to prevent aspiration of saliva. A catheter filled with heparinized saline (25 I.U./ml) was placed into the carotid artery to record arterial BP (millimeters Hg). The penis was desheathed and the CCs were exposed. A 25-gauge stainless steel needle was inserted into one CC to record ICP (millimeters Hg). The needle was attached to a catheter filled with heparinized saline (25 I.U./ml). Catheters were connected to pressure transducers (model 750, Elcomatic Ltd, Glasgow, UK). CN was exposed via a suprapubic midline incision and separated from surrounding connective tissue under dissecting microscope observation. The pelvic cavity was covered with mineral oil warmed to 37°C, and the CN was placed on bipolar platinum stimulating electrodes connected to an electrical stimulator (model 2100; A-M Systems, Phymep, Paris, France) delivering trains of square wave pulses (6 and 2V, 10Hz, l ms, 30s). ICP and BP signals were amplified (AZAP 90104; Bionic Instruments, Nozay, France), digitized, and recorded on a personal computer for further analysis.

Following a resting period of 10 min, the compounds solvent was IC injected (50 µl/injection). Then, increasing doses of one compound were injected every 10 min by the same route. Five injections (one solvent plus four cumulative doses) were performed in each rat, and five rats were used for the study of one compound. For each injection and for each compound, we measured BP averaged over the 10 min after the injection. The peak of ICP reached during the 10-min period following an injection also was recorded.

In a second set of experiments, the effects of the tested compounds were evaluated on the ICP increase elicited by CN stimulation. A stimulation of 6V, 10Hz, 1 ms was applied for 30 s to search for the maximal ICP increase, followed by a stimulation at 2V, 10Hz, 1 ms for 30 s, 10 min later, so as to elicit an incomplete ICP rise. IC injection of vehicle or compounds was performed, and a submaximal stimulation (2V) was delivered at the peak time effect of the compound. Five rats were used for the study and ICP and BP were measured as reported above.

IC and BP Recording in Dogs. The evaluation of the erectile properties in dogs was performed according to the method of Carati et al. (1987), with some modifications. Male beagle dogs were anesthetized with pentobarbital sodium (Nembutal, 35 mg/kg i.v. for induction and 4 mg/kg/h for maintenance) and intubated with an endotracheal cuffed tube to facilitate free ventilation. A collateral of the left femoral vein was cannulated with a PE catheter for infusion of the anesthetic. Systemic BP was monitored via a Mikro-tip 6F (Millar Instruments, Houston, TX) pressure transducer introduced into the aortic arch through the right common carotid artery. ICP was measured by means of a 20-gauge needle placed into the left or right CC and the same needle was used for IC injection of the compounds. The needle was attached to a catheter filled with heparinized saline (25 I.U./ml). Pressure signal was triggered by BM 614/2 carrier amplifiers (Biomedica Mangoni, Pisa, Italy) on a multichannel polygraph (Rectigraph 8K SAN-EI, Tokyo, Japan).

IC injections of the compounds were performed in a volume of 0.5 ml. Each injection was followed by a flush of 0.5 ml of saline. The vehicles for drug dissolution were tested before the first dose of each drug. The compounds were administered in a cumulative way, with 30-min interval between two doses. ICP (millimeters Hg) was measured at the peak effect after the administration of the compounds. Systolic BP (SBP) and diastolic BP (DBP) (millimeters Hg) were measured at the peak effect after the administration of drugs to evaluate the effects of the compounds on the BP independently from the effects on ICP. Moreover, SBP was measured at the time of maximal ICP value after IC injection.

To evaluate the effects of the tested compounds on electrically induced increase of ICP, the abdominal cavity was opened through a suprapubic midline incision, the right PN was cut laterally to the rectum, and the distal end placed on bipolar electrodes. Maximal electrical stimulation with trains of pulses of 10 V, 15 Hz, 8 ms for 25 s was delivered with a digital stimulator (BM ST6, Biomedica Mangoni) to analyze the effects of IC injections on ICP rises elicited by nerve stimulation. Electrical stimulation was delivered at the peak time effect of the compound, and ICP and SBP were measured.

Drugs and Chemicals. [3H]Prazosin (7-methoxy-3H), specific activity 76.2 Ci/mmol was from NEN Life Science Products (Milano, Italy). The compounds coded Rec 15/2615 (1-(4-amino-6,7-dimethoxy-2-guinazolinil)-4-[2-[2-metoxy-6-(1-methylethyl)phenoxy]acetyl]piperazine hydrochloride) and Rec 15/2841 (2-cyclohexyl-N-[3-4-(2-methoxyphenyl)-1-piperazinyl]propyl]-3-methyl-4-oxo-4H-1-benzopyran-8-carboxamide) (Testa et al., 1997), as well as Rec 15/3039 (8-[2-[4-(5-chloro-2-methoxyphenyl)-1-piperazinyl]ethyl]-8-azaspiro[4,5]decane-7,9-dione dihydrochloride) were synthesized in Recordati chemical laboratories. Prazosin and phentolamine were obtained from Sigma Chemical Co. (St. Louis, MO). All the other drugs or materials were obtained from commercial sources.

In binding studies, the compound was dissolved in absolute ethanol. For the isolated organ preparations (rabbit aorta), prazosin and phentolamine were dissolved in distilled water. Rec compounds were dissolved in distilled water containing 3% dimethylformamide and 3% Tween 80, and further diluted in deionized H2O.

In the rat experiments, all the compounds were dissolved and diluted in propylene glycol-Sorensen solvent. In the dog experiments, Rec 15/2615 and phentolamine were dissolved (1 or 3 mg/ml) in 10% (v/v) N,N-dimethylformamide and further diluted in deionized H2O. Rec 15/2841 and prazosin were dissolved in deionized H2O.

Statistical Analysis. The competition curves of Rec 15/3039 on the alpha 1-ADR subtypes were analyzed by nonlinear curve fitting of the logistic equation (De Lean et al., 1978). The IC50 values and pseudoHill slope coefficients were estimated by the program. The value for the inhibition constant, Ki, was calculated with the Cheng and Prusoff (1973) equation. Data were reported as pKi (-log10 Ki).

In the in vitro functional studies on rabbit aorta, the dissociation constant (KB) was estimated from a Schild plot. Because the slope of the plot was never significantly different from unity, it was constrained to 1, where the intercept represents the pKB (-log10 KB).

The in vivo data in rats and dogs (ICP and BP) were reported as means ± S.E. of the mean, or as percentage of variation (±S.E.) of the basal values. The significance of the difference between the basal (or vehicle) values and those observed after the IC injection of the tested compounds was assessed by ANOVA and Dunnett's test. Differences were considered statistically significant if P < .05 or P < .01.

The ratio (ICP/BP)·100, which corresponds to the percentage of BP reached by ICP, was differently calculated in rats and dogs. In the rat experiments, mean BP was calculated over the 10-min period after injection of the compounds. In the dog experiments, the SBP observed at the peak effect on ICP was used to evaluate the ICP/BP ratios.

Approximate ED25 values (dose of compound inducing 25% increase of ICP/BP ratio) also were evaluated by nonlinear curve fitting of the logistic equation (De Lean et al., 1978). The function used was as follows:
<UP>Response</UP>=E<SUB><UP>max</UP></SUB>+<FR><NU>(E<SUB><UP>min</UP></SUB>−E<SUB><UP>max</UP></SUB>)</NU><DE>1+X/(X<SUB>50</SUB>)<SUP>−<UP>s</UP></SUP></DE></FR>
where the response was the observed (ICP/BP) ·100; X was the dose giving the corresponding response; Emax and Emin were the maximum and minimum responses observable (always forced to 100 and 0%, respectively); X50 was the midpoint location parameter; and s was the slope of the curve.

    Results
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References

In Vitro Characterization of Antagonists. The binding affinity of the tested compounds for the animal recombinant alpha 1-ADR subtypes, as well as their functional affinity for the alpha 1L-ADR subtype are shown in Table 1. As previously reported (Testa et al., 1997), phentolamine is selective for the alpha 1a-ADR subtype, Rec 15/2841 shows the same affinity for the alpha 1a- and alpha 1L-ADR subtypes, Rec 15/2615 is selective for the alpha 1b-ADR subtype, and prazosin is a nonselective compound with low affinity for the alpha 1L-ADR subtype. Rec 15/3039 showed selectivity for the alpha 1d-ADR subtype versus all the other subtypes.

                              
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  		TABLE 1
Binding affinity of the tested compounds for the animal recombinant alpha 1-ADR subtypes, and their functional alpha 1-antagonistic activity against NA-induced contractions of rabbit aorta after CEC alkylation (alpha 1L-ADR subtype)

Data represent pKi values (recombinant receptors) or pKB values (functional). Standard deviations were always <10% and omitted. Data from Testa et al. (1997) with exception of Rec 15/3039.

Effects on IC and BP in Rats. In rats, cumulative IC injection of propylene glycol-Sorensen solvent transiently increased ICP (Table 2). Phentolamine, Rec 15/2841, and Rec 15/3039 elicited a significant ICP increase only at the highest dose injected, relative to vehicle-induced effect (Table 2). The ICP increase did not last more than 10 min (period of observation). The effects of prazosin and Rec 15/2615 were not different from vehicle at any dose tested, probably because of the consistent increase of ICP observed in matched vehicle-treated rats. All the compounds induced a significant and dose-related decrease in BP (Table 2).

                              
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  		TABLE 2
Effects of IC injection of vehicle and cumulative doses of the different compounds tested on ICP and BP in anesthetized rats

Data represent the mean ± S.E. (n = 5) values of ICP and mean BP in mm Hg.

When the effects on the ICP were corrected for the hypotensive effects, i.e., with the ICP/BP ratio, Rec 15/2841 and Rec 15/2615 were the most potent compounds. The ICP/BP ratio was dose-dependently increased and significantly different from vehicle effect after injection of 100 and 300 µg/kg Rec 15/2841 and 300 µg/kg Rec 15/2615 (Fig. 1). The ICP/BP ratios observed after injection of Rec 15/3039 were significantly different from those of vehicle-treated rats at the highest dose tested. These effects, however, were not markedly higher than those observed after vehicle injection and poorly dose-dependent (Fig. 1).


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  		Fig. 1.   Effects of vehicle and different doses of the compounds tested on the ICP/BP after IC injection in rats. Data represent mean + S.E. values of the ratio. Basal, first column; vehicle, second column; different tested doses, other columns (prazosin and phentolamine: 10, 30, 100, 300, and 1000 µg/kg; Rec 15/2841, 15/2615 and 15/3039: 10, 30, 100, and 300 µg/kg).a, P < .05 and b, P < .01 versus basal; c, P < .05 and d, P < .01 versus vehicle. The mean percentage of decreases (+S.E.) of BP evaluated versus the basal values also is shown. Statistical significance of the effects of different doses of each drug on BP was evaluated on absolute values (Table 2) and is not shown in the figure.

The increase of ICP/BP ratio induced by prazosin and phentolamine were poorly dose-dependent. Furthermore, these compounds induced a marked hypotensive effect also at the lowest tested doses (Fig. 1).

Approximate ED25 values (dose of compound in micrograms inducing 25% increase of ICP/BP ratio) were evaluated after subtraction of the vehicle effect, and gave the following rank order of potency: Rec 15/2615 (22 µg/kg) >= Rec 15/2841 (29 µg/kg) > prazosin (136 µg/kg) > phentolamine (1298 µg/kg) > Rec 15/3039 (9600 µg/kg). Rec compounds had no visible effects on the ICP rise induced by electrical stimulation (Table 3). In contrast, prazosin at any dose and 300 to 1000 µg/kg phentolamine reduced the amplitude of the ICP rise elicited by CN stimulation (Table 3). The ICP/BP ratios calculated during CN stimulation were not statistically different before and after IC injection of Rec compounds (Fig. 2). Conversely, IC injection of prazosin decreased the ICP/BP ratio. Phentolamine displayed biphasic effects on the ICP/BP ratio. It increased it at low doses and inhibited the response at the highest doses (Fig. 2), thereby confirming previously reported data (Giuliano et al., 1993).

                              
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  		TABLE 3
Effects of IC injection of vehicle and cumulative doses of the different compounds tested on ICP elicited by electrical stimulation (ES) in anesthetized rats

Data represent the mean ± S.E. (n = 5) values of ICP (mm Hg) elicited by submaximal (2V) electrical stimulation (ES) of CN.


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  		Fig. 2.   Effects of vehicle and different doses of the compounds tested on the ICP/BP ratio observed after IC injection followed by submaximal electrical stimulation (2V) of the CN in rats. Data represent the mean + S.E. values of the ratio. Legend for columns as in Fig. 1. c, P < .05 and d, P < .01 versus vehicle.

Effects on IC and BP in Dogs. The results of the effects on ICP and DBP of IC administration of drugs in anesthetized dogs are reported in Table 4. Vehicles used for drug dilution were tested before each dose of each compound and showed neither effects on ICP nor effects on systemic BP (data not shown).

                              
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  		TABLE 4
Effects of IC injection of vehicle and cumulative doses of the different compounds tested on ICP and BP in anesthetized dogs

Data represent the mean ± S.E. values of ICP and diastolic BP in mm Hg.

All Rec compounds induced a dose-dependent increase in ICP. The duration of the effect was very short after Rec 15/3039, and did not last >4 min at the highest dose tested. The effects of Rec 15/2841 were also transient, but lasted 12 min after injection of 1000 µg/kg. The increase of ICP induced by Rec 15/2615 was long-lasting and ranged from 15 min after the administration of 10 µg/kg to 62 min after the administration of the highest dose. The increase of ICP induced by prazosin lasted 23 min after injection of 1000 µg/kg, whereas the duration of phentolamine effects lasted 4 min after the highest dose (1000 µg/kg) was administered.

All drugs tested induced a dose-dependent decrease in BP, as shown by the decreases in DBP evaluated at the peak effect. The hypotensive effects were lower for Rec compounds and phentolamine relative to those induced by prazosin.

The ICP/BP ratios (where ICP is computed at the maximal effects after the injection of the compound and BP is the SBP computed at the same time) evaluated in dog experiments are shown in Fig. 3. All compounds, with the exception of Rec 15/3039, induced significant increase of ICP/BP. Taking into account the effects on BP, Rec 15/2615 was the most potent proerectile compound, inducing significant increases of ICP/BP at doses practically devoid of effects on BP. The rank order of potency in dogs, expressed as ED25 values, was similar to that observed in rats: Rec 15/2615 (5 µg/kg) > Rec 15/2841 (127 µg/kg) > prazosin (332 µg/kg) > phentolamine (627 µg/kg) >= Rec 15/3039 (674 µg/kg).


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  		Fig. 3.   Effects of different doses of the compounds tested on the ICP/BP after IC injection in dogs. Data represent the mean + S.E. values of the ratio. Basal, first column; different tested doses, other bars (prazosin: 30, 100, 300, and 1000 µg/kg; phentolamine: 10, 30, 100, 300, and 1000 µg/kg; Rec 15/2841: 10, 30, 100, 300, and 1000 µg/kg; Rec 15/2615 and 15/3039: 3, 10, 30, 100, 300, and 1000 µg/kg). a, P < .05 and b, P < .01 versus basal. The mean percentage of decreases (+S.E.) of DBP evaluated versus the basal values also is shown. Statistical significance of the effects of different doses of each drug on BP was evaluated on absolute values (Table 4), and is not shown in the figure.

In dogs, electrical stimulation of PN induced ICP rises whose amplitude was frequency-related (Fig. 4). Eight to 16 Hz elicited the greatest response. To evaluate the effects of the tested compounds on the increase of ICP induced by maximal electrical stimulation, we always used 15 Hz, and the results obtained are shown in Table 5. All compounds (but not Rec 15/3039) reduced the electrically induced increase of ICP only at the highest tested doses. The ICP/BP ratios were not different from the basal values with the exception of the highest doses tested (Fig. 5).


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  		Fig. 4.   Frequency-response curve of electrical stimulation-induced increase of ICP in dogs. Data represent the mean ± S.E. values of ICP increase (millimeters Hg) evaluated after stimulation at frequencies ranging from 0.25 to 16 Hz.

                              
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  		TABLE 5
Effects of IC injection of vehicle and cumulative doses of the different compounds tested on ICP elicited by electrical stimulation in anesthetized dogs

Data represent the mean ± S.E. values of ICP (mm Hg) elicited by maximal electrical stimulation (ES) of pelvic nerve.


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  		Fig. 5.   Effects of different doses of the compounds tested on the ICP/BP ratio observed after IC injection followed by maximal electrical stimulation (15 Hz) of the PN in dogs. Data represent the mean + S.E. values of the ratio. Legend for the columns as in Fig. 3. a, P < .05 and b, P < .01 versus basal.

    Discussion
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References

During the past 15 years, the field of erectile dysfunction treatment has grown tremendously, offering several options to impotent patients, such as penile prosthetic surgery, vacuum therapy, penile vascular surgery, IC injection or intraurethral delivery of vasoactive agents, and more recently oral pharmacotherapy. Besides the "revolution" created by the introduction of the phosphodiesterase V inhibitor sildenafil in this field (Goldstein et al., 1998), it is accepted opinion that there is still room for effective and safe drugs with different mechanisms of action and that are delivered through different routes.

Considering the use of IC injections of vasoactive agents, this approach not only shed light on the diagnosis and treatment of impotence but also enriched basic research studies. Postganglionic sympathetic fibers release NA in the penis, and NA is an important neurotransmitter in the control of flaccidity and detumescence (Andersson and Wagner, 1995). The latter is caused by adrenergic nerves constricting corporal smooth muscle through an effect of NA on alpha 1-ADRs (Lue and Tanagho, 1987, 1988). In vitro studies demonstrated that the responsiveness of human erectile tissue to alpha 1-ADR agonists was altered by a variety of factors, including aging, and that the efficiency of phenylephrine-induced contractions was significantly increased in corporal vascular tissue from impotent men (Christ et al., 1990, 1991).

The identification of different alpha 1-ADR subtypes in animal and human CC (Traish et al., 1995, 1996; Dausse et al., 1998; Véronneau-Longueville et al., 1998) suggests further investigations of the functional role of alpha 1-ADR subtypes in modulating CC tone. It is possible, in fact, that adrenergic imbalance toward vasoconstriction that prevents erection could be mediated by a specific alpha 1-ADR subtype. In this case, a subtype-selective antagonist could improve the therapeutic efficacy of these kinds of drugs over less selective ones, e.g., moxisylyte, although effects of the latter have been reported (Imagawa et al., 1989; Costa et al., 1993a,b; Bernabé et al., 1995; Buvat et al., 1996).

To this aim, we tested the effects of alpha 1-ADR antagonists with different selectivity for recombinant alpha 1-subtypes in anesthetized rat and dog models of penile erection. Because functional studies also have identified alpha 1-ADRs (not cloned yet) with a low affinity for prazosin and termed alpha 1L-ADRs (Flavahan and Vanhoutte, 1986; Muramatsu et al., 1995), the functional affinity for this subtype of the compounds also was evaluated. Among the compounds tested, Rec 15/2841 displayed selectivity for recombinant alpha 1a-ADR subtype, Rec 15/2615 was selective for the alpha 1b-ADR subtype (Testa et al., 1997), and Rec 15/3039 showed selectivity for the alpha 1d-ADR subtype (present data). On the alpha 1L-subtype, Rec 15/2841 was the most potent compound (pKB = 9.19), whereas Rec 15/3039 gave a low (<7.0) pKB value. The reference compounds prazosin (a nonselective antagonist) and phentolamine, selective for the alpha 1a-ADR subtype and showing high affinity for the alpha 2-ADR subtypes (Leonardi et al., 1997), also were used. The compounds have been injected IC and their effect on ICP and BP measured to search for their proerectile properties. Their effect on ICP increase elicited by CN or PN stimulation also has been assessed to evaluate their effects on an incomplete (rat) or complete (dog) erection.

IC injection of compound Rec 15/3039 did not consistently increase ICP in rats or in dogs. These findings clearly indicate that the alpha 1d-subtype is not relevant for penile erection, in agreement with the observations of Davis et al. (1998) showing that BMY7378 (a selective alpha 1d-antagonist) was inactive on human tissue. Compounds Rec 15/2841 and Rec 15/2615 displayed proerectile properties in the anesthetized rat and dog: they elicited a dose-dependent ICP increase in both species, whereas proerectile properties of prazosin and phentolamine were less evident.

It is noteworthy that in our experimental conditions, the ICP increase induced by 300 µg/kg Rec 15/2615 in dogs (Table 4) was similar to that of 100 µg/kg papaverine (ICP = 100 mm Hg; Recordati, data on file), and markedly higher than that of 1 µg/kg prostaglandin E1 (PGE1) (ICP = 35 mm Hg; Recordati, data on file).

Rec 15/2841 and Rec 15/2615 had no effect on the incomplete erection elicited by submaximal cavernous nerve stimulation in rats (when expressed as ICP/BP ratio), and decreased ICP/BP in dogs only at the highest doses. The duration of the effect (increase of ICP) in dogs exerted by Rec 15/2615 was markedly longer than that of all other compounds tested.

Rat CC express the three cloned alpha 1-ADR subtypes (alpha 1a, alpha 1b and alpha 1 day), whereas no comparable data are available concerning the alpha 1-subtypes present in dog tissues (Véronneau-Longueville et al., 1998). Our data, on the whole, unmask the role of alpha 1b-and alpha 1L-ADR subtypes in rat and dog penile erection. The most active compounds, in fact, were Rec 15/2841 (alpha 1a, and alpha 1L selective), and Rec 15/2615 (alpha 1b selective). Phentolamine, although displaying selectivity for the alpha 1a-ADR subtype (but lower than Rec 15/2841), was one of the less potent compounds at the alpha 1L-ADR. Prazosin showed approximately the same affinity for the alpha 1a and alpha 1L-ADR subtypes as Rec 15/2615. It is possible, however, that its proerectile effect is blunted by the marked hypotensive effects.

For the last fifteen years, intracavernosal pharmacotherapy has been the cornerstone for the medical treatment of erectile dysfunction, both as monotherapy and using drug combinations (Fallon, 1995; Porst, 1996). PGE1 is recognized today as the most potent proerectile drug, and success rate, depending on the studies, ranges from 70 to 80%. However some patients complain of pain and clinicians report rare priapism (Buvat et al., 1996; Porst, 1996). Another vasoactive agent officially approved for the IC monotherapy treatment of impotence in several countries is moxisylyte, a nonselective alpha 1-ADR antagonist showing a lower incidence of penile pain and prolonged erections/priapism than PGE1. Moxisylyte is efficient in vitro (Imagawa et al. 1989; Costa et al., 1993b) and in humans (Costa et al., 1993a; Buvat et al., 1998) but displays less proerectile effects than PGE1 in patients with erectile dysfunction (Buvat et al., 1996).

Therefore, more research is required to search for a more efficient IC treatment that would lead to a lower rate of side effects. In this view, selective alpha 1-ADR antagonists alone or in combination with other drugs deserve further studies in humans (Dinsmore and Alderdice, 1998).

A question that remains to be answered is the potential use of oral delivery of selective alpha 1-ADR antagonists. Testing the effects of single oral treatment with the alpha 1-ADR antagonist phentolamine was recently performed (Becker et al., 1998). The authors concluded that oral phentolamine could benefit patients with recent onset erectile dysfunction. The addition of an oral alpha 1-ADR antagonist with IC injections of PGE1 had beneficial effects, relative to IC of PGE1 alone (Kaplan et al., 1998). Given their depressant effect on BP, one should keep in mind their possible side effects, altering the mechanisms of erection. Nevertheless, the evidence from large hypertension trials on different antihypertensive drugs suggests that patients on doxazosin had a lower incidence of erectile dysfuncion than placebo group (Grimm et al., 1997). In summary, there is a potential role for treatment with selective alpha 1-ADR antagonists alone or in combination with other agents to be used in the treatment of erectile dysfunction.

    Acknowledgements

We thank L. Greto for his skilfull technical assistance.

    Footnotes

Accepted for publication November 9, 1999.

Received for publication June 17, 1999.

1 Current address: Laboratoire de Neurobiologie des Fonctions Végétatives, Institut National de la Recherche Agronomique, 78352 Jouy-en-Josas, Cedex, France.

2 Current address: Service d'Urologie, C.H.U. de Bicêtre, Assitance Publique-Hôpitaux de Paris 78 rue du Général Leclerc 94270 Le Kremlin-Bicêtre, France.

Send reprint requests to: Giorgio Sironi, Pharmaceutical R&D Division, RECORDATI S.p.A., Via Civitali 1, 20148 Milano, Italy. E-mail: sironi.g{at}recordati.it

    Abbreviations

IC, intracavernous; NA, noradrenaline; ADR, adrenoceptor; CC, corpus cavernosum; ICP, intracavernous pressure; BP, blood pressure; CN, cavernous nerve; PN, pelvic nerve; CEC, chloroethylclonidine; SBP, systolic blood pressure; DBP, diastolic blood pressure; PGE1, prostaglandin E1.

    References
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Abstract
Introduction
Materials and Methods
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Discussion
References

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