Bimonthly publication annales d' urologie

109

VOLUME 27 N° 3 - 1993

LA REVUE D'UROLOGIE POUR LE PRATICIEN

contents

SPECIAL ISSUE DEVOTED

TO THE IMPOTENCE

Organized by Professor William von NIEDERHÄUSERN and by Doctor Jean-Marc WISARD

Curriculum vitae concerning Pr William von NIE-DERHÄUSERN ( G e n e v e , S u i s s e ) , and Dr Jean-Marc WISARD ( L a u s a n n e , S u i s s e ) 111 Introduction. W. von NIEDERHÄUSERN ( G e n e v e , S u i s s e ) , J .M. WISARD ( L a u s a n n e , S u i s s e ) 113 Flaccidity and erectipn of the human penis: morphological data. Giuseppe CONTI ( F r i b o u r g , S u i s s e ) 115-121 Investigating erectile dysfunction: value, interest and limitations of various examinations. M. WISARD, S . G A B E L L O N . D. FOURNIER, H J . LEISINGER ( L a u s a n n e , S u i s s e ) 122-130 Neurological and neurophysiological investigation of impotent men. M.C. S C H E I B E R - N O G U E I R A ( S t r a s b o u r g , F r a n c e ) 131-135 Single potential analysis of cavernous electrical activity (SPACE). Experiences, limitations and perspectives. M.H. DJAMILIAN, M.C. TRUSS, H.K. TAN, P. A N T O N , C h . G . STIEF, U. J O N A S ( H a n n o v e r , A l l e m a g n e ) 136-143

THERAPEUTIC METHODS

Revascularization of the penis in vascular impo-tence. D. HAURI (Zürich, S u i s s e ) 144-151 Prostaglandin E1 - The chemical implant. G. de B O C C A R D , Ch .H. R O C H A T ( G e n e v e , S u i s s e ) 152-155

Value and limitation of mechanical devices for assisting erection in the treatment of erectile dysfunction. G . G A B E L L O N , M. WISARD, H J . LEISINGER ( L a u s a n n e , S u i s s e ) 156-159 Erection sparing prostatovesiculectomy for Carci-noma of the prostate. D. HAURI (Zürich, S u i s s e ) . 160-165

PHYSIOLOGY

Intracavernous pressures increases and perineal muscles contractions in response to pressure stimulations of the glans penis. P. LAVOISIER, R. ALOUI ( L y o n , F r a n c e ) , M. SCHMIDT ( T o i e d o - O h i o , U S A ) , M. G A L L Y ( V i l i e u r b a n n e , F r a n c e ) 166-171

Evidence for a perineal vascular response induced by glans penis pressure Stimulation. P. LAVOISIER, R. ALOUI ( L y o n , F r a n c e ) , M. SCHMIDT ( T o i e d o - O h i o , U S A ) , M. G A L L Y ( V i l l e u r b a n n e , F r a n c e ) 172-175

Quantif ication of smooth muscle tissue using f uzzy logic. S. K A S A S ( F r i b o u r g , S u i s s e ) , M. WISARD ( L a u s a n n e , S u i s s e ) , K . S T O F F E L ( F r i b o u r g , S u i s s e ) . 176-178

Intracavernous application of SIN-I in rabbit and man: functional and toxicological results. M.F. MEYER, A .TAHER, H. K R A H ( H a n n o v e r , A l i e m a g n e ) , J . S T A U B E S A N D ( F r i b o u r g , A i l e m a g n e ) , A J . B E C K E R , M. KIRCHER ( H a n n o v e r , A l l e m a g n e ) , B . M A Y E R ( G r a z , A u t r i c h e ) , U . J O N A S , W . G . F O R S S M A N N , Ch .G . STIEF ( H a n n o v e r , A l l e m a g n e ) . 179-182

INSERTION OF PENILE IMPLANTS

Flexible penile implant in the restoration of erectile function. L. SUBRINI ( P a r i s , F r a n c e ) 183-191

Biomechanical study of flexible penile implants. L . S U B H M ( P a r i s , F r a n c e ) 192-196

INFORMATIONS 1 3 0 , 1 5 9 , 1 7 8

ANALYSIS 155, 171

SUBSCRIPTION RATES 135

Each article is accompagnied with an English summary

The contents page of «ANNALES D'UROLOGIE» appears regularly in «CURRENT CONTENTS», and is indexed in the bibliographical publications of the CNRS (Paris), in the « INDEX MEDICUS», and the Computerized Documentation System MEDLINE; in the publications of the Institute for Scientific Information of Philadelphia, and « BIOLOGICAL ABSTRACTS » of the Biosciences Information Service.

179

INTRACAVERNOUS APPLICATION OF SIN-I IN RABBIT AND MAN: FUNCTIONAL AND TOXICOLOGICAL RESULTS

M . F . M E Y E R 1 2 , A . T A H E R 1 , H . K R A H 1 , J. S T A U B E S A N D 3 , A . J . B E C K E R 1 , M . K I R C H E R 1 , B. M A Y E R 4 , U . J O N A S 1 , W . G . F O R S S M A N N 2 , C h . G . STIEF 1

M E Y E R M.F. , T A H E R A , K R A H H. , STAUBESAND J., BECKER A . J , KIRCHER M , M A Y E R B , JONAS U., FORSSMANN W.G. , STIEF Ch.G. - Intracavernous application of SIN-I in rabbit and man: functional and toxicological results.

Ann Urol, 1993, 27, n° 3, 179-182.

S U M M A R Y : The mode of action of the active meta-bolite SIN-I of the vasodilator prodrug molsidomine was studied i n v i t r o and i n v i v o in corpus cavernosum of rabbit and man. SIN-I produces a dose-dependent relaxation of isolated human cavernous smooth muscle Strips. In the rabbit, the intracavernous appli-cation of SIN-I increased the intracavernous pressure to a füll erection (approximately 100 cm H 2 0 ) . This response wa shighly reproductible. SIN-I was also injected intracavernously 6 times in five rabbits over 2 weeks; no inflammatory or fibrotic reactions were found on histology. SIN-I may be a reliable drug for the treatment of impotence without side-effects.

KEY-WORDS : SIN-I. — Relaxation of cavernous smooth muscle Strips. — No inflammation and fibrosis.

M E Y E R M . F , T A H E R A , K R A H H. , STAUBESAND J , BECKER A.J., KIRCHER M , M A Y E R B , JONAS U., FORSSMANN W . G , STIEF Ch.G. - Injection intra-caverneuse de SIN-I chez le lapin et l'homme: resultats fonctionnels et toxico-logiques. ( E n A n g l a i s ) . Ann Urol, 1993, 27, n° 3, 179-182.

R E S U M E : L'action du metabolite actif SIN-I du pre-curseur du vasodilatateur molsidomine a ete etudiee in v i t r o et i n v i v o sur le corps caverneux du lapin et de l'homme. Le SIN-I cause une relaxation, dependante de la dose, de bandelettes de corps caverneux humain isolees. Chez le lapin, Pinjection intra-caverneuse de SIN-I augmente la pression intra-caverneuse jusqu'au niveau d'une erection complete (environ 100 cm H 2 0 ) . Cet effet est reproductible ä un fort pourcen-tage. D'autre part, on a procede ä des injections intra-caverneuses de SIN-I 6 fois en deux semaines chez le lapin ; aucune reaction inflammatoire ou fibreuse n'a ete constatee ä l'examen histologique. Le SIN-I pour-rait constituer un traitement de l'impuissance virile sans effets secondaires.

M O T S - C L E S : SIN-I. — Relaxation de fibres musculaires lisses caverneuses. — Absence d'inflammation et de fibröse.

I N T R O D U C T I O N

To induce an erection, the penile arteries and sinusoids have to dilatate, thereby decreasing the resistance to penile blood flow [1]. However, the mechanism of penile smooth muscle relaxation has not been fully elucidated yet. Nitric oxide (NO), which is believed to accound for the biological actions of endothelium-derived relaxing factor [2, 3] was recently suggested to be of importance in the regula-tion of penile smooth muscle tone, both in the flaccid State [4] and during erection [2, 4]. SIN-I causes its effects by « non-enzymatical liberation of N O » [5].

1. Urologische K l i n i k der Medizinischen Hochschule, H A N N O V E R (MHH) ( G e r m a n y ) . 2. Niedersächsisches I n s t i t u t für Peptidforschung (IPF), H A N N O V -E R ( G e r m a n y ) . 3. Anatomisches I n s t i t u t der Universität, F R E I B U R G ( G e r m a n y ) . 4. Pharmakologisches I n s t i t u t der Universität, G R A Z ( A u s t r i a ) .

Supported by a grand from the Deutsche Forschungsgemeinschaft D F G Sti 96/2-2. Correspondance address: Pr C G . Stief, Urologische Klinik der Medizinischen Hochschule Hannover, Postfach 610180, D-3000 H A N N O V E R 61. Manuscrit recu ä la Redactlon le 24 avril 1993. Accepte par les Comltes de Lecture et de Redactlon le 3 mal 1993.

e Sociele d'Edition de {'Association d'Enseignement M e d i c a f des Höpitaux de Paris

180 I N T R A C A V E R N O U S A P P L I C A T I O N O F S I N - I I N R A B B I T A N D M A N A N N A L E S D ' U R O L O G I E

In view of the observations that N O is involved in mediating penile erection and that certain nitrovaso-dilators have been employed clinically to induce erection in impotent patients, the present study was initiated to assess the dose-dependent relation between the intracavernously applied SIN-I dose and the evoked penile erection in the rabbit model. As it was previously described the chronic application of the non-specific phosphodiesterase-inhibitor papa-verine causes fibrosis and inflammation [6]. Therefore the present study was designed to evaluate whether the chronic short-term intracavernous appli-cation of 0,1 mg SIN-I (6x in 14 days) in the corpus cavernosum of the rabbit causes undesired side efifects of any degeneration of penile tissue. Further-more, / v i t r o and in v i v o studies of the effect of SIN-I on cavernous smooth muscle relaxation were done.

M A T E R I A L S A N D M E T H O D S

Organ bath experiments

Corpus cavernosum excised from human penis during penectomy for penile Carcinoma or penile prosthesis implantations is dissected into small strips of 0,3 x 0,7 cm. The specimen are mounted under 0.5 gm, tension in organ Chambers containing Krebs — bicarbonate Solution at 37 °C gassed with 95 % 0 2 5 % C 0 2 . Isometric contractions are measured using Sta-tham transducers connected to Linseis polygraphs. The preparations are allowed to equilibrate for 60-90 min. After this period, norepinephrine or endothe-lin 10"6 are added to contract the strips ; after a stable tension is reached, SIN-1 is added in doses between 10-9-10~4 M .

Intracavernous application of SIN 1

To evaluate the effect of SIN-1 in v i v o , the rabbit model was chosen ; we did not want to present human data since a patients response to any intracavernously applied drug is modified by his etiology of the erectile dysfunction he is suffering from.

After sedation with i.m. ketamine (10 mg), 9 ani-mals (3.5 to 5 kg BW) were anaesthetized with i.v. pentobarbital (15 mg kg"1) through a needle placed in an ear vein. The rabbits (New Zeeland white rabbits) are placed in a supine position on a thermoregulated operating table (model I I A , Hugo Sachs Elektronik, Germany. Additional heat is provided with a heating lamp. The penile skin is removed by blunt dissection and a 21-gauge needle is inserted into the left corpus cavernosum for pressure recording. The needle is connected to a fluid line via a threeway stopcock which allows for the intracavernosal application of SIN 1. To prevent clotting, 50I .U. heparin is given through this mute every 2-3 hours. This dose of hepa-rin is well below the dosis needed to induce changes in penile hemodynamics [7]. After the pressure returns to baseline, 1 ml saline is given intracaverno-sally in order to flush the drug away and to avoid clotting.

To study the effects of SIN-I the drug is adminis-tered intracavernously at dosages of 0,01, 0,02, 0,05 and 0,1 mg through the 21-gauge needle until in every animal a dose - response - curve is achieved.

Histopathological study

SIN-I of 0,1 mg is injected intracavernously 6 times in 5 rabbits in between 14 days. Then the rabbits are sacrified. A long seghment (3 cm) of the penis includ-ing the injected site is collected for light-microscopic examination, fixed in 10% neutral formalin Solution and stained with H E and Masson - trichrome. For immunohistofluorescence the penile tissue is fixed by immersion in 4% formaldehyde at 4°C overnight. Five minutes sections are cut on a Reichert-Jung microtome and incubated with antisera diluted 1 : 250 (NO-synthetase-antibody against neuronal NO-syn-thase, no staining of endothelial NO-synthase ; raised in rabbit). Bound antiserum is visualized with fluores-cin-conjugated pig anti-rabbit IgG diluted 1 :40.

Drugs

The following drugs are used : ketamine, pentobar-bital, heparin, SIN-I, a gift from Dr Heenning and Dr Grewe, Cassella, Germany) NO-synthetase antise-rum (Dr Meyer, Universität Graz), fluorescin conju-gated pig anti-rabbit IgG.

R E S U L T S

Organ bath study

Concentration response relationships of the effect of SIN-I on isolated human cavernous tissue are depicted in figure 1. The dose-response-curves for the relaxing effect of SIN-I on isolated human cavernous tissue precontracted with norepinephrine and as well as the effect of papaverin, a non-specific phosphodies-terase-inhibitor show that concentrations of 10~9 and 10"7 M SIN-I causes dose-dependent relaxations which are close to their maximum at 10~6 M . Papaver-ine showed the most potent effect at concentrations of 10"5 M and 10~4 M in a very small therapeutical width, shown by the step increase of the dose effect curve.

Intracavernous application of SIN-I

The intracavernous application of SIN-I induces a dosage - dependent erectile response. A injection of 0,02 mg SIN-I evoked a füll penile erection for 10 minutes. 0,05 mg induced a füll erection for 16 minutes whereas the intracavernos application of 0,1 mg leads to a long-lasting response of füll penile erection and persisted for about 84 minutes with severe side — effects concerning the systemic blood pressure (the blood pressure drops for about 45-50 cm H 2 0 in the first 4 minutes).

V O L U M E 27 N ° 3 - 1993 I N T R A C A V E R N O U S A P P L I C A T I O N O F S I N - I I N R A B B I T A N D M A N 181

Fig. 1. - Cavernous tissue of the SIN-I treated rabbits. A u. B) Massen-Goldner (x400, x100); FITC-method with NO-synthase-antibody in man.

Fig . 1. — T issu c a v e r n e u x d e s lap ins t ra i tes pa r SINH A -x 4 0 0 ; B x 100.

120-

100-

80-c o cu 60-X 35 Ö 40 -

SS 20 -

0 -

-1

n = 5-7

• sin-1 • sin-1

• papaverine

•9 - 8 - 7 - 6 - 5

concentration (log. molar)

Histopathological study

It was shown by H E and Masson - Goldner stain that the caernous tissue from the rabbits consists of loose sinusoid Spaces separated by connective tissue trabecula containing bundles of smooth muscle cells, nerve fibers and arterioles. In the corpus cavernosum of the rabbits treated by repeated injections of SIN-I, nor local fibrosis neither any sign of inflammation was seen. The intracavernous tissue does not exhibit any hyperplasia including fibrous tissue and smooth muscle mass (fig. 2 a et b).

Immunofluorescence

The staining of the NO-synthetase-containing axons supplying penile smooth muscle is substantia-ted by immunocytochemistry as seen in figure 2c.

D I S C U S S I O N

The present study shows that the NO-donor SIN-I induces a relaxation of the cavernous smooth muscle in v i t r o and i n v i v o . Previous studies showed that N O which is involved in the mechanism of penil smooth

Fig. 2. - Relaxation of cavernosal smooth muscle to SIN-I (a, b) and papaverine (c). Tone was induced by norepi-nephrine. Data represent mean responses to each con-centration of SIN-I. Vertical bars represent Standard error of mean. Concentrations of the drug are indicated as logarithm of molarity (log M).

Fig. 2. — Re laxa t i on du m u s c l e l isse c a v e r n e u x trai te par SIN-I (a, b) et papave r i ne (c). C o n t r a c t i o n indui te par no re -p inephr ine . C e s t r a c e s rep resen ten t les r e p o n s e s m o y e n n e s ä c h a q u e c o n c e n t r a t i o n d e SIN-I. Bar re ver t i -c a l e : e r reur S tandard m o y e n n e . L e s concen t ra t i ons d e s agen t s c h i m i q u e s sont i nd i quees par le logar i thme de s a molar i te ( log M) .

182 I N T R A C A V E R N O U S A P P L I C A T I O N O F S I N - I I N R A B B I T A N D M A N A N N A L E S D ' U R O L O G I E

Fig. 3. - Tracing showing the increase in intracavernous pressure induced by intracavernous injection of 0.1 mg SIN-I. Then rise in pressure was described in terms of öP, which was defined as the maximal pressure obtained by SIN-l-injection minus the basal pressure before the injec-tion. The low pressure increase directly after the SIN'I injection as well as the steady pressure below the final maximum in the f irst 3-4 minutes is due to the drop in sys-temic blood pressure.

Fig . 3. — T r a c e mont rant l 'augmenta t ion de la p r e s s i o n i n t r a - cave rneuse indui te par in ject ion i n t r a - c a v e r n e u s e de 0,1 m g de SIN-I. L 'e levat ion est e x p r i m e e en t e r m e de de l ta P ; c 'es t la p ress ion max ima le o b t e n u e par in jec t ion de SIN-I d i m i n u e e de la p ress ion d e b a s e avant in jec t ion . L a p ress ion b a s s e s 'e leve ap res in jec t ion de SIN-I. L a b a i s s e d e p r e s s i o n en fin d ' e x p e r i e n c e est d u e ä la b a i s s e d e p r e s s i o n sys tem ique .

muscle tone is directly released from nerves. Further-more it was shown that inhibition of NO-synthesis in v i v o inhibited nerve-induced penile erection [8]. This is supported by the finding that we could stain N O -synthetase containing axons supplying penile smooth muscle cells. This also shows that the relaxing effect of SIN-I is specific and physiological.

The dose-response-curve of SIN-I is in agreement with a reliable control using the drug in therapeutical dose ränge. Due to the fask kinetics of decomposition [9] in the cavernous tissue SIN-I should not provoke a prolonged erection. In contrast to these findings, the non-specific phosphodiesterase-inhibitor papaverin has a very narrow therapeutical with and the drug also implies the risk of a priapism [10-11].

Our in v i t r o findings are supported by the in v i v o experiments. The intracavernous injection sinduces a dose-dependent erectile response, but even high doses of SIN-I regarding the penile volume of the rabbit compared to the human, dot not induce a prolonged erection.

After repetitive intracavernous injections of SIN-I in the rabbit no signs of fibrosis and/or inflammation are detected. These results may attribute to the anti-inflammatory effects as well as to the good tissue tolerability of the nitric oxide donor SIN-I. So when only a relatively low number of papaverine injections (n = 6) are performed, the low dose injections induc a marked cavernous inflammation and fibrosis [12]. So, intracavernous injection of SIN-I seems, at least, to be much better tolerated than papaverine. Further studies with more injections and different species have to be carried out before a final conclusion may be drawn.

Finally, we may emphasize that SIN-I seems to be an adequate therapeutical agent in the treatment of erectile dysfunction in terms of its therapeutical with and the nontoxic short-term effects concerning local degenerations after repeated applications. Future long-term studies in various species are necessary for the judgement of local toxicity.

REFERENCES

1. ANDERSSON K.E. , HOLMQUIST F. - Mechanisms for contraction and relaxation of human penile smooth muscles. I n t J Impotence Res, 1990; 2: 209-225.

2. IGNARRO L.J. - Nitric oxide. A novel Signal transduction mechanism for transcellular communication. Hypertension, 1990; 76 : 477-483.

3. MARIN J., SANCHEZ-FERRER C.F. - Role of endothelium-formed nitric oxide on vascular responses. Gen Pharmacol, 1990 ; 21 :575-587.

4. HOLMQUIST F., H E D L U N D H., ANDERSSON K.E. - Characteriza-tion of inhibitory neurotransmission in the isolated corpus cavernosum from rabbit and man. Submitted, 1991.

5. RAND M.J. - Nitrergic transmission : nitric oxide as a mediator of non-adrenergic, non-cholinergic neuroeffector transmission. C l i n i Experim Pharmacol Physiol, 1992 ; 19: 147-169.

6. HWANG T.I., Y A N G C , HO W.L. , C H U H. - Histopathological change of corpora cavernosa after long-term intracavernous injection. E u r U r o l , 1991 ; 20: 301-306.

7. KIRKEBY J.H., LUNDBECH P.E., FORMAN A., ANDERSSON K.E. , DJURHUUS J.C. — Penile venous occlusion — an experimental model with autologue extracorporal penile blood perfusion and possibility for aortic occlusion. I n t J Impotence Res, 1990; 2: 181-191.

8. HOLMQUIST F., STIEF C G . , JONAS U., ANDERSSON K.E. -Effects of the nitric oxide synthase inhibitor NG-nitro-L-arginine on the erectile response to cavernous nerve Stimulation in the rabbit. Acta Physiol Scand, 1991 ; 143: 299.

9. REDEN J. - Molsidomine. Blood Vessels, 1990; 27: 282. 10. ABOSEIF S.R., BREZA J., DIEDERICHS W., BOSCH R., BENARD

F., STIEF C G . , LUE T.F. , T A N A G H O E.A. - Effect of chronic intra-cavernous injection of papaverine and Prostaglandin El on erectile tissue in monkeys. J U r o l , 1988; 139 : 257A.

11. J Ü N E M A N N K.P., A L K E N P. - Pharmacotherapy of erectile dysfunc-tion. I n t J Impotence Research, 1989 ; / : 71.

12. S T A C K L W., STIEF C G . , BENARD F., BOSCH G. , L O U P A L G. , L U E T.F . , T A N A G H O E T . - Intracavernous injections of Solutions with different osmolarity and pH in the rabbit. I n t J Impotence Res, 1989; / : 197-200.