International Journal of Impotence Research (2000) 12, Suppl 1, S37±S40 ß 2000 Macmillan Publishers Ltd All rights reserved 0955-9930/00 $15.00 www.nature.com/ijir
Abanoquil, a new alpha-1 adrenoceptor antagonist. In vitro and in vivo effect on erectile tissue A Giraldi1*, M Wyllie2 and G Wagner1 1
Department of Medical Physiology, University of Copenhagen, Rigshospitalet, 7121, Blegdamsvej 9, DK-2100, Copenhagen, Denmark; 2Urodoc, Kent, UK Using an organ bath model with porcine cavernosal tissue strips and an in vivo monkey model we demonstrated that abanoquil, a novel alpha adrenoceptor antagonist, is able to relax contracted tissue strips and induce erectile response when injected intracorporally. The erectile response in the monkeys was not dose-related and compared to the effect of papaverine injections, abanoquil induced a lower level of tumescence and rigidity. Hence, abanoquil might be useful as a facilitator of erection in the pharmacological treatment of erectile dysfunction. International Journal of Impotence Research (2000) 12, Suppl 1, S37±S40 Keywords: abanoquil; alpha-1 adrenoceptor antagonist; erectile dysfunction
Introduction It is generally accepted that ¯accidity of the penis is a consequence of contraction of the smooth muscle cells of corpus cavernosum (cc) and the penile arteries, caused in part, by sympathetic nervous activity stimulating alpha adrenoceptors.1 The dominating alpha adrenoceptor in cc is alpha-1 subtype, whereas it is alpha-2 in the cavernosal arteries.2±4 Relaxation of the trabecular smooth muscle cells of cc is crucial to develop an erection.5,6 In the ongoing search for agents that have the capacity of relaxing cc smooth muscle cells and thereby have a possible role in the pharmacological treatment of erectile dysfunction, multiple, in vivo and in vitro studies on cc smooth muscle have been carried out. These studies have demonstrated relaxatory response of cc smooth muscle to naturally occuring compounds such as prostaglandin,7 vasoactive intestinal polypeptide8 and pharmacological agents such as papaverine,9 phosphodiesterase inhibitors,10 calcium channel blocking compounds,11 potassium channel openers12,13 and alpha adrenoceptor blocking agents.14,15 Abanoquil, UK-52,046 (4-amino-6,7-dimethoxy2(1,2,3,4-tetrahydro-6,7-dimethoxyisoquinol-2yl) quinoline methanesulphonate) (Figure 1) is a novel alpha adrenoceptor antagonist with a high selectivity to the alpha-1 subtype.16 The aim of the present study was to evaluate the effect of abanoquil *Correspondence: Dr A Giraldi, Department of Medical Physiology, University of Copenhagen, Rigshospitalet, Dept. 7121, Blegdamsvej 9, DK-2100, Copenhagen, Denmark. E-mail:
[email protected]
on corpus cavernosum smooth muscle in vitro and in vivo.
Material and methods In vitro studies Porcine corpus cavernosum was obtained from boars immediately after slaughtering. Tissue strips (~3 6 3 6 8 mm) were prepared and mounted between two L-shaped metal hooks immersed in standard model organ baths for recording of the isometric tension. All tissue strips that were used for experiments showed spontaneous activity and responded to addition of norepinephrine (NE) by contraction. Pharmacological experiments The effect of abanoquil (UK-52,046 from P®zer, UK) on spontaneous contractions was studied by addition of increasing concentrations to the organ bath (10ÿ8±10ÿ5 M, n 11). Dose±response curves to NE were constructed by cumulative addition of NE (10ÿ7±10ÿ4 M) to the organ bath followed by a complete washout when steady-state was achieved after addition of the highest concentration. Thereafter, abanoquil (10ÿ10±10ÿ7 M) was added to the organ baths and after 30 min incubation new NE, dose±response curves were constructed. To study the effect of abanoquil on precontracted tissue-strips, the strips were precontracted with NE (10ÿ6±10ÿ4 M). When maximal contraction was
Effect of abanoquil on erectile tissue A Giraldi et al S38
Figure 1 Chemical structure of abanoquil UK52,046.
monkeys had in previous experiments responded to intracavernosal (ic) injections of papaverine by increasing tumescence and rigidity of the penis. The monkeys were anaesthetized with intramuscular apozepam (0.1 mg=kg) and ketamine-chloride (20 mg=kg) and placed in a supine position with the penis stretched out. A rubber band was placed around the root of the penis as a tourniquet. The agents of use were dissolved in 0.3 ml saline and injected into one of the copora cavernosa using a 27 G needle; the tourniquet was kept in place for 3 min after the injection. Five, 10, 15, 20, 30, 60 and 120 min after the injection, tumescence (increase in volume) and rigidity of the penis were estimated visually and by palpation by the same experienced investigator. The estimations were graded on a ®ve point scale (0-4), 0 representing no change in tumescence=rigidity and 4 full tumescence=rigidity. Before and during the experiments the brachial arterial systolic pressure and pulse were measured using a neonatal cuff and an 8 MHz Doppler to localize the artery.
Pharmacological experiments
Figure 2 Concentration response curve showing norepinephrine-induced contraction of porcine corpus cavernosum after incubation with abanoquil (10ÿ10±10ÿ7 M). Calculated as percent contraction of NE-induced contraction before abanoquil incubation. Control tissues were not incubated with abanoquil.
obtained, abanoquil was added to the organ bath in increasing concentrations (10ÿ9±10ÿ7 M). Statistical analysis The in vitro experiments were analysed using the paired Student's t-test, P 0.05 was considered as signi®cant. In vivo monkey studies In accordance with local ethical guidelines, each monkey participated in only one experiment a day with an interval of at least two weeks between each experiment. In these series of experiments, 12 male monkeys were used (Maccaca Fascicularis, weight 4±6 kg). All International Journal of Impotence Research
In order to investigate the effect of abanoquil on corporal smooth muscle the monkeys were injected with 0.7 mg (n 12), 1.8 mg (n 8) or 3.5 mg (n 11) abanoquil. Six monkeys were injected with papaverine (9 mg, SAD, DK) in order to compare the effect of papaverine to the effect of abanoquil. A further six monkeys served as controls and were injected with saline 0.3 ml. In order to elucidate the receptor speci®city and reversibility of the effect of abanoquil, seven monkeys were injected with 10 mg abanoquil and tumescence and rigidity were evaluated. Thereafter, the monkeys were injected with NE (10 mg, n 3) or phenylephrine (PE, 10 mg, n 4, Sigma, USA). Ensuing tumescence and rigidity were evaluated 5 and 10 min after the NE=PE injection. Using the same procedure, six monkeys were injected with 25 mg abanoquil followed by 50 mg clonidine. Statistical analysis The data in each series of experiments were analysed using the non-paired-parametric Friedmann test and the Page test for trend. To analyse differences between the maximal obtained tumescence and rigidity with each agent the non-paired, non-parametrical Kruskal-Wallis test was used. In order to compare the values obtained with abanoquil to those obtained with papaverine, the WilcoxonPratt test was used. All these statistical calculations were performed with the MEDSTAT program.17
Effect of abanoquil on erectile tissue A Giraldi et al
Blood pressure and pulse were expressed as mean SE and analysed using the paired Student's t-test. P < 0.05 was considered as signi®cant.
Results In vitro experiments Abanoquil, in concentrations ranging from 10ÿ7 to 10ÿ4 M, did not affect the spontaneous activity of the smooth muscle tissue strips. Addition of abanoquil (10ÿ10±10ÿ7 M) prior to NE-induced contraction inhibited the NE-induced contraction in a dose dependent manner as illustrated by the rightward shift of the NE-dose±response curve shown in Figure 2. Abanoquil 10ÿ10 M had no signi®cant effect compared to control tissue, whilst abanoquil 10ÿ7 M almost completely inhibited contraction of the tissue strips. Addition of abanoquil to NE-precontracted porcine tissue strips resulted in a dose-related relaxation of the tissue with a minimal relaxation using abanoquil 10ÿ9 M and an almost complete relaxation when the dose of abanoquil is increased to 10ÿ7 M (data not shown). In vivo experiments Injections of 0.7, 1.8 and 3.5 mg abanoquil resulted in a signi®cant change in tumescence and rigidity of the penis in the monkeys (P < 0.001 for all three concentrations). Judged by the rank sums (Friedmann test) the responses for all three concentrations peaked 15±20 min after the intracavernosal injections. The responses then levelled off, but were still present in 27 of the 31 trials after 120 min. On this basis we applied the Page-test for trend on the period of 0±20 min and 20±120 min. This showed that a steady increase in tumescence and rigidity occurred in all three groups from 0 to 20 min after the injection (P < 0.001), whereas there were no signi®cant changes from 20 to 120 min. The monkeys were controlled 4 h after the injections and none of them developed long lasting erections. Furthermore there was no signi®cant difference in the obtained maximum values for tumescence or rigidity when comparing the effect of three different abanoquil concentrations. Arterial blood pressure and pulse were unaffected during the experiments. Papaverine injections resulted in a signi®cant increase in both penile tumescence and rigidity from the time of intracavernosal injection to time of peak erectile response (15 min) whereafter the response levelled off with a signi®cant decrease in tumescense but not in rigidity after 120 min. Four h after the experiment, no lasting erections were observed. Comparison of the attained values at peak
time showed no statistically signi®cant difference between the responses induced by abanoquil and papaverine using the Wilcoxon-Pratt test (P 0.063). If the responses were compared through the whole time period (0±120 min) signi®cant higher erectile responses (both tumescence and rigidity) were observed after papaverine injections compared to abanoquil. Saline injections had no effect on penile tumescence and rigidity. Injection of NE or PE subsequent to abanoquil injection resulted in a signi®cant decrease in tumescence and rigidity during the following 10 min after the injection. In contrast, clonidine did not reverse the effect of abanoquil, since no signi®cant decrease in tumescence and rigidity was observed after the injection.
S39
Discussion In the present study it was shown that abanoquil induced an erectile response when injected intracorporally in monkeys. Abanoquil induced relaxation of pre-contracted porcine corporal smooth muscle strips in vitro, but had no effect on the spontaneous activity. The penile response to intracavernosal injection achieved in monkeys was characterized by a greater effect on tumescence than on rigidity. Most of the monkeys developed an erection after abanoquil injection, however, most of the erections were scored lower than the attainable maximum (4=4). When compared to the effect of intracavernosally administered papaverine a signi®cantly lower attainable erectile response was observed with abanoquil, even though there was no difference when comparing the attained level at peaking time, probably due to the small numbers of monkeys used. The achieved erectile responses were not doserelated. This may be attributed to the high doses utilized or because the responses were induced by a non-speci®c reaction caused by abanoquil. The latter does not seem to be the case, since the alpha-1 agonists NE and PE were able to reverse the effect of high concentrations of abanoquil in vivo and the alpha-2 agonist, clonidine, had no effect on the response. These ®ndings indicate that abanoquil primarily exerts its effect by alpha-1 antagonism. This is in agreement with the ®ndings that alpha-1 receptors are the predominant alpha-adrenoceptor type in the corpus cavernosum. In the search for an erectogenic agent, it is crucial that the agent does not cause priapism. In the present in vivo studies, no long lasting erections were observed in the monkeys. It should, however, be emphasized that all studies were done under anaesthesia and the number of experiments were limited. Previous studies of alpha-adrenoceptor antagonists have shown that not all compounds are useful in the treatment of erectile dysfunction. International Journal of Impotence Research
Effect of abanoquil on erectile tissue A Giraldi et al S40
Phentolamine, given intracavernosally itself does not elicit a satisfactory erectile response and is usually used in combination with other agents such as papaverine and prostaglandin E1. The present study suggests that intracavernosally administered abanoquil, similar to phentolamine, might be useful as a facilitator of erection in the pharmacological treatment of erectile dysfunction rather than an agent inducing a full erection as intracavernosal prostaglandin E1 or papaverine.
References 1 Stief CG et al. Cavernous smooth muscle changes during penile erection and sympathetic stimulation. Int J Imp Res 1991; 3: 15±20. 2 Andersson KE. Pharmacology of lower urinary tract smooth muscles and penile erectile tissues. Pharmacol Rev 1993; 45: 253±308. 3 Wagner G, Wei MQ. Alpha-a-adrenoceptor subtypes A and B exist in penile smooth muscle tissue. Int J Imp Res 1992; 4(Suppl 2): 42. 4 Traish AM, et al. Identi®cation of alpha-1-adrenergic receptor subtypes in human corpus cavernosum tissue and in cultured trabecular smooth muscle cells. Receptor 1993; 5: 145±157. 5 Wagner G, Saenz de Tejada I. Update on male erectile dysfunction. BMJ 1998; 316: 678±682.
International Journal of Impotence Research
6 Andersson KE, Wagner G. Physiology of penile erection. Physiol Rev 1995; 75: 191±236. 7 Adaikan PG. Pharmacology of the human penis. MSc. Thesis, University of Singapore, Singapore 1979, PP 1±213. 8 Willis E, et al. Vasoactive Intestinal Polypeptide (VIP) as a possible neurotransmitter involved in penile erection. Acta Physiol Scand 1981; 113: 545±547. 9 Virag R. Intracavernous injection of papaverine for erectile failure. Lancet 1982; II: 938. 10 Boolell M et al. Sildena®l: an orally active type 5 cyclic GMPspeci®c phosphodiesterase inhibitor for the treatment of penile erectile dysfunction. Int J Imp Res 1996; 8: 47±52. 11 Fovaeus M, Andersson KE, Hedlund H. Effects of some calcium channel blockers on isolated human penile erectile tissue. J Urol 1987; 138: 1267±1272. 12 Giraldi A, Wagner G. Effects of pinacidil upon erectile tissue, in vitro and in vivo. Pharmacol Toxicol 1990; 67: 235±238. 13 Holmquist F, Andersson KE, Hedlund H. Effects of pinacidil on isolated human corpus cavernosum penis. Acta Physiol Scand 1990; 138(4): 463±469. 14 Brindley GS. Cavernosal alpha blockade: a new technique for investigating and treating impotence. Br J Psychiat 1983; 143: 332±337. 15 Blum MD, Bahnson RR, Porter TN, Carter MF. Effect of local alpha-adrenergic blockade on human penile erection. J Urol 1985; 134: 479±481. 16 Greengrass PM, Russel MJ, Wyllie MG. Abanoquil: a novel alpha1-adrenoceptor antagonist. Br J Pharmacol 1991; 104: 321P. 17 Wulff HR, Schlichting P. Manual for MEDSTAT, Herlev University Hospital, November 1988.
