Jae-Young Park¹, Hwancheol Son², Soo Woong Kim¹, Jae-Seung Paick^1
1Department of Urology, Seoul National University College of Medicine, Seoul, Korea
²Department of Urology, Seoul Municipal Boramae Hospital, Seoul, Korea

Sildenafil citrate is a potent, competitive cGMP-specific phosphodiesterase type 5 (PDE5) inhibitor that is the first line oral treatment for erectile dysfunction (ED) of various etiology [1, 2]. However, several studies showed that administering sildenafil could not resulted in rigidity sufficient for satisfactory sexual intercourse in  11 % - 22 % of patients with psychogenic and 36 % - 65 % with organic ED [1, 2].

Combination drug therapy is appealing to salvage patients in whom monotherapy with sildenafil failed. In an early empirical trial, the most popular approaches have been the use of sildenafil plus intraurethral or intracaver-nous PGE1 [3, 4]. However, previous study clearly demonstrated a patient preference for the least invasive forms of therapy (oral medication) despite the prior unavailability of effective oral agents [5]. Recently, the clinical efficacy and safety of sublingual apomorphine (APO), a dopamine receptor agonist, has been documented [6, 7]. Combining centrally acting drug (APO) with peripheral conditioner (sildenafil) might be an attractive approach. The aim of this study was to investigate a possible potentiating effect of APO on sildenafil-induced penile erection using the conscious rabbit model.

All studies were approved by the Institutional Animal Care and Use Committee at this medical center. Twelve male New Zealand White rabbits (3.5 - 4.0 kg) were used. Animals were housed individually in cages and food and drink were available ad libitum. Room lights were on from 7 am to 7 pm, the room temperature was 22 - 24 .

A 24 gauge angiocatheter filled with normal saline was inserted into a marginal ear vein and taped up in position. The animals became accustomed and were not disturbed by repeated handling. In a separate study to investigate effects of drugs on systemic blood pressure, a 24 gauge angiocatheter was placed into the central ear artery in a restrain cage and connected to a transducer to continuously monitor the systemic blood pressure (n=12). Sedation was maintained with an intravenous bolus injection of urethane (800 mg/kg) as required and 150 mL of normal saline was administered very slowly to maintain patency.

Sildenafil, an extract from Viagra (Pfizer Inc., Brooklyn, USA) was dissolved in normal saline (2.8 mg/mL) and APO (A4393, Sigma, USA) in dimethylsulfoxide (DMSO) and then diluted in distilled water (1 mg/mL).

The drug combinations injected were as follows: APO (0, 0.05, 0.1 and 0.4 mg/kg) and sildenafil (0.5, 1 and 5 mg/kg). As a result, 12 different combinations of drugs were each administered to 12 animals. APO was administered intravenously followed by sildenafil. Typically, the drugs were administered via an ear vein, followed by a small volume of saline and then the angiocatheter was removed. The time was noted and animals were gently removed from the cage and held by one individual. The length of uncovered penile mucosa was measured using a caliper as described previously [8]. Penile erection was evaluated at 5, 10, 15, 30, 45, 60, 90 and 120 min after agent administration and continued for 150 minutes in case of a persistent erection.

The penis was not handled at all to avoid direct stimulation. Between measurements, animals were returned to their cages and given access to water and food. Later, animals were randomized for administration of different doses. The minimal time between experiments for each animal was one week. Each dose was repeated six times in different animals randomly chosen. All animals tolerated this well.

Systemic blood pressure was recorded about 10 min after placing an angiocatheter into the artery and vein. Systemic blood pressure was continuously recorded before and after drug administration. The minimal interval between each experiment for each animal was three days. Each dose was repeated six times in different animals.

In the preliminary study, intravenous injection of drug vehicle (saline or DMSO) did not cause any change in penile length. Systemic administration of sildenafil produced a dose-dependent increase in the length of the exposed penile mucosa (Figure 1). Doses of 0.5, 1 and 5 mg/kg induced a maximal mucosa response of 2.3 2.1, 3.7 2.2 and 9.9 4.8 mm, respectively. The response to 5 mg/kg was significantly higher (P<0.01) compared with those to 0.5 and 1 mg/kg, while there was no significant difference between the responses to 0.5 and 1 mg/kg of sildenafil. Maximal responses were produced at about 5 min after sildenafil administration. Then, the response gradually waned, but sometimes there was a substantial and measurable erection after 2 h at the highest dose (5 mg/kg).

Figure 4. Change in length of uncovered penile mucosa measured after systemic administration of APO at indicated doses and 5 mg/kg sildenafil into ear vein of conscious rabbits. ^bP<0.05, compared with sildenafil alone.

As a result, the co-administration of 1 mg/kg sildenafil and 0.1 mg/kg APO produced the most effective penile erection among 12 different combinations. In addition, the synergistic effect caused by APO was more obvious at a sildenafil dose of 1 mg/kg than at 0.5 or 5 mg/kg. However, there was no increase in the duration of penile erection with the addition of APO.

The mean arterial blood pressure (MAP) before injection was 83.9 ?10.4 mmHg. Administration of sildenafil caused a transient, dose-dependent decrease in MAP (Figure 5), which returned to baseline values within 10 min of the injection. The administration of APO at 0.05 mg/kg and 0.1 mg/kg also caused a mild, transient decrease in MAP, while 0.4 mg/kg of APO increased MAP slightly (data not shown). Combined administration of sildenafil and APO did not significantly decrease the MAP compared with sildenafil alone (P>0.05) (Figure 5).

In this study, we used the conscious rabbit model, which was originally proposed by Naganuma et al [9]. To investigate the effects of drugs on penile erection, the anesthetized animals have been commonly used, in which the intracavernosal pressure is recorded during electro-stimulation of the peripheral nerves to the penis. Compared with this conventional method the conscious animal model is simpler, less invasive and can exclude the potential influence of anesthesia on the activity of drugs. One prerequisite of this method is that the agents should be able to induce erection by themselves without sexual or direct penile stimulation.

Usually the rabbit penis is not visible as it is covered by fur. However, the length of the penis and the duration of the erection are easily measured during penile erection [8]. We produced penile erection in the conscious rabbit by intravenous administration of sildenafil and demonstrated the potentiation effect of APO on sildenafil-induced penile erection for the first time. As for the conscious rat, Andersson et al [10] reported that APO elicits a rise in intracavernosal pressure, which can be prolonged by sildenafil.

The doses of sildenafil and APO used in this study were determined based on previous reports [8, 11] and our pilot study. The bioavailability of sildenafil and APO in humans are about 40 % [12] and 17 % [13], respectively, thus the doses of both drugs used in this experiment may be relatively higher than those prescribed in men [14]. The conscious rabbits are not sexually stimulated and higher doses seem to be needed to induce penile erection.

Sildenafil effect is dose-dependent. However, intravenous administration of APO at high concentrations could not induce penile erection. This finding is consistent with that of the previous study using the conscious rabbit model [9]. In contrast, subcutaneously and centrally administered APO has been shown to induce penile erection in rats [10, 15]. Thus, the mode of action of APO may be species-specific.

Interestingly, APO, which did not induce penile erection by itself, significantly enhanced the penile erection produced by sildenafil. The present results showed that the administration of sildenafil at doses of 5 mg/kg or 0.5 mg/kg was less effective than at 1 mg/kg when combined with APO. This reduced effectiveness may be due to the fact that sildenafil at 5 mg/kg might have already triggered the maximal response and sildenafil at 0.5 mg/kg might not have induced sufficient response to be affected by APO co-administration.

Previous studies in rats showed that the maximum effect on penile erection was obtained with APO at 0.1 mg/kg and that the response decreased on increasing APO doses from 0.1 to 0.5 mg/kg [10, 16]. Tarcan et al [11] also reported that APO at 0.1 and 0.2 mg/kg most effectively increases nerve-stimulated peak clitoral intracavernous and vaginal wall blood flows in the rabbits and that at more than 0.2 mg/kg it is less effective and produces adverse effects. Based on these results, the most effective dose of APO for inducing penile erection in the rabbits or rats is 0.1 - 0.2 mg/kg and doses greater than 0.2 mg/kg may not be as effective due to activation of the D1 dopaminergic receptor.

The site of action of APO stimulation is considered to be in the central nervous system. The fact that the effects of APO were abolished after NO synthase inhibition suggests that the action of APO is dependent on release of NO [17]. If minimal amount of nitric oxide (NO) is present at the nerve terminals even in the absence of sexual stimulation, lower than usual doses of PDE5 inhibitor could induce penile erection. Furthermore the efficacy of vardenafil, another PDE5 inhibitor, was also strengthened by the presence of NO donor in the same model [8].

APO activates the central dopaminergic receptor and then induces penile erection via NO secretion [17]. Nitrates, precursors of NO, enhance the production of cyclic GMP and in the presence of PDE 5 inhibitors, this can lead to severe hypotension [18]. If the combination of APO and PDE 5 inhibitors induces hypotension, the combination therapy should not be used. Vardi et al [19] reported that sildenafil (100 mg) caused small, clinically insignificant reductions in ambulatory blood pressure in active and resting normotensive and hypertensive men. Moreira et al [20] reported that the side-effect profile of sildenafil was dose related and mild. On the other hand, Nakayama et al [21] studied the cardiovascular function in anesthetized dogs given APO at the standard emetic dose (0.05 mg/kg) or at 10 times this dose; no significant changes were seen at 0.05 mg/kg and the reduction in systemic vascular resistance and mean systemic arterial pressure were minimal at 0.5 mg/kg. Fagan et al [22] showed that in patients receiving common antihypertensive agents and nitrates, sublingual APO at higher than recommended doses produced no clinically significant changes in heart rate or blood pressure as with APO alone.

The results of the above studies are consistent with our results that sildenafil induced a dose-dependent decrease in the mean blood pressure, but that additional APO caused no further significant decrease. Recently, it was indicated that the combined administration of sildenafil and APO with maximal doses (100 mg and 3 mg, respectively) did not lead to any increase in the rate of the cardiovascular side effects compared with monotherapy group in healthy volunteers [23]. Due to different NO release sites, the hypotension caused by the nitrate/sildenafil combination might not be duplicated by APO/sildenafil combination [24]. According to the present and other study, the cardiovascular effects of APO and sildenafil/APO combination seems to be insignificant. Moreover, the smaller effective dose of APO and sildenafil may further reduce these effects.