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INDUCTION OF PENILE ERECTION BY INTRACAVERNOSAL AND TRANSURETHRAL ADMINISTRATION OF NOVEL NITRIC OXIDE DONORS IN THE CAT
0022-534719911616-201310 T H E J O r R N t U . OF UROLOGY Copyright 0 1999 by AMERICANUROLOCICAL ASSOCIATION, INC.
Vol. 161,2013-2019, June 1999 Printed in U S A
INDUCTION OF PENILE ERECTION BY INTRACAVERNOSAL AND TRANSURETHRAL ADMINISTRATION OF NOVEL NITRIC OXIDE DONORS IN THE CAT HUNTER C. CHAMPION, TRINITY J . BIVALACQUA, RUN WANG, PHILIP J. KADOWITZ, LARRY K. KEEFER, JOSEPH E. SAAVEDRA, JOSEPH A. HRABIE, PAUL C. DOHERTY AND WAYNE J . G. HELLSTROM* From the Departments of Urology and Pharmacology, Tulane University School of Medicine, New Orleans, Louisiana, the Labomtory of Comparative Carcinogenesis and SAlC Frederick, National Cancer Institute, Frederick Cancer Research and Development Center, Frederick, Maryland, and VTWS, Inc., Mountain View, California
ABSTRACT
Purpose: The effects of novel nitric oxide (NO) donors administered intracavernosally and transurethrally on erectile function in the anesthetized cat were evaluated. Materials and Methods: In pentobarbital-anesthetized cats, increases in intracavernosal pressure, penile length, and duration of erectile response were determined after intracavernosal and transurethral injections of novel NO donors (MAHMA/NO, PAPANO, DEA/NO, PIPERAZVNO and PROLINO). All parameters were measured after administration of NO donors intracavernosally via a 30-gauge needle and urethrally via a Jelco i.v. catheter in a volume of 200 p1. Systemic arterial pressure was also assessed in these experiments. All NO donors were compared with a triple-drug control combination comprised of papaverine (1.65 mg.), prostaglandin E, (0.5 pg.), and phentolamine (25 pg.). Results: MAHMA/NO, PAPANO, DEA/NO, PIPERAZVNO and PROLUNO induced dose dependent increases in intracavernosal pressure and penile length (p <0.05) when administered intracavernosally. The increases in cavernosal pressure and penile length were comparable to those observed with the triple-drug control combination. The maximum increase in cavernosal pressure in response to PROLVNO and PAPANO was associated with no significant change in systemic arterial pressure. Transurethral administration of PROLINO and PIPERAZVNO induced dose-dependent increases in cavernosal pressure and penile length (p <0.05). The response was similar to that of the triple-drug control combination, except that transurethral PROLVNO and PIPERAZIINO had no significant effect on systemic blood pressure. Conclusions: NO donors caused dose-dependent increases in cavernosal pressure when administered intracavernosally and transurethrally. These data suggest further exploration of the use of NO donors for the treatment of erectile dysfunction. KEYWORDS:penile erection, novel nitric oxide donors, diazeniumdiolates, transurethral, intracavernosal
An estimated 20 to 30 million American men suffer from peripheral nervous systems, as well as in local vascular conerectile dysfunction, and 50 to 60% of these men have a t r 0 1 . ~ - ~ Although NO solution can produce an erectile response, it vascular component to their condition.'.' Penile erection is caused by increased arterial inflow and restricted venous is not clinically efficacious in the management of impotence outflow, coordinated with the relaxation of blood vessels and because of its extremely short half-life in aerobic the trabecular smooth muscle that constitutes the corpora Recently, vasodilators of the diazeniumdiolate class have c a ~ e r n o s a .Although ~ parasympathetic innervation is in- been developed that allow for the controlled pharmacological volved in erectile dysfunction, the erectile response cannot be delivery of NO. Half-lives of ions in this series range from 2 completely blocked by atropine, suggesting that a nonadren- seconds to 20 hours, depending on the structure, allowing for ergic, noncholinergic (NANC) mechanism is involved in pe- the study of dosage rate as a determinant of pharmacological nile e r e ~ t i o n Nitric .~ oxide (NO) is a free radical molecule response.**' which serves as a chemical messenger in both the central and The use of intracavernosal pharmacotherapy has become peripheral nervous systems. NO is a powerful vasodilator increasingly widespread for the local treatment of erectile invoking an integral role in the physiological and pathophys- dysfunction. Until recently, the only method of introducing iological regulation of blood flow and blood pressure. More vasoactive agents into the corpus cavernosum was direct recently, the role of NO in the mediation of penile erection injection. Although this method is successful for most pahas been revealed with actions defined a t the central and tients, long-term compliance is low, and there are a number of acknowledged side effects, including pain, penile fibrosis, Accepted for publication February 1, 1999. hematoma formation* and priapism." a number of Department of Urology SL42,Tulane Uni* Requests for versity School of Medicine, 1430 Tulane Avenue, New Orleans, LA studies have documented the medicated urethral system of delivery (MUSE) for the transurethral administration of 70112. Supported by a fellowship from the American Foundation for UrO- prostaglandin El (alprostadil) as an effective and lesslogical Disease, Inc. (TJB),an educational grant from VTWS, Inc., invasive method for introducing vasoactive agents for the and National Institutes of Health Contract NO. ~ 0 1 - ~ 0 - 5 6 0 0to 0 purpose of treating erectile dy~fUnction."-'~ Although the SAIC Frederick. 2013
2014
NITRIC OXIDE DONORS AND ERECTION IN THE CAT
transurethral administration of alprostadil has proven efficacious in double-blind studies, it is not without side effects (for example, dizziness, hypotension, and penile pain). Moreover, long-term efficacy and compliance in the general population remain to be established. It has been postulated that these untoward consequences may be due to both local and systemic effects of the prostaglandin E l and/or local effects of the solvent used in the formulation. Increases in efficacy and a reduction of side effects are the two key issues in the development of new agents for the treatment of erectile dysfunction. Efficacy has been improved in intracavernosal therapies by the addition of other agents. However, since alprostadil appears to sensitize nerve fibers to noxious stimuli through interactions with specific prostaglandin receptors, l4 some degree of pain can be expected with administration of this agent regardless of the route of delivery. Ideally, agents with short half-lives that could be administered locally would reduce the potential for systemic side effects. Such agents should activate pathways that induce the erectile response without the sensitizing nerve conduction pathways. Nitric oxide donors would prove useful in this regard, however, attempts to use currently available NO NO donors have met with limited S U C C ~ S SThe .~~ - ~generat~ ing diazeniumdiolate complexes may have a n advantage in this regard since they spontaneously and predictably release NO. Although these compounds have been shown to cause smooth muscle relaxation and increased blood flow in a number of organ systems," little is known about their effects on penile erection. This study explores the effects of intracavernosal injection of these NO donors on erectile function in the cat and investigates their potential to induce penile erection via the transurethral route of administration. MATERIALS A N D METHODS
Adult male cats weighing 2.8 to 4.8 kg. were sedated with ketamine hydrochloride (10 to 15 mg./kg. i.mJ and anesthetized with pentobarbital sodium (30 mg./kg. i.v.). Supplemental doses of pentobarbital were administered as needed to maintain a uniform level of anesthesia. A vertical, circumcision-like incision was made to expose the two ventral corpora cavernosa and the dorsal corpus spongiosum. A 30gauge needle was placed into the right corpus cavernosum to permit administration of drugs into the penis. A 25-gauge needle was placed midway into the left corpus cavernosum for the measurement of intracavernosal pressure (mm. Hg). Systemic and intracavernosal pressures (mm. Hg) were measured with Statham P23 transducers connected to a Grass model-7 polygraph, and mean pressures were obtained by electronic averaging. Penile length (mm.)was measured with a ruler. These procedures have been previously described and were approved by the Tulane University Animal Care and Use Committee.22z23 In experiments in which the agonists were administered intracavernosally, injections were made via a 30-gauge needle inserted into the corpus cavernosum. In experiments in which the transurethral administration of NO donors was employed, drugs were introduced into the urethra via a Jelco i.v. catheter in a volume of 200 p1. In all experiments, injections of drugs were made when the cavernosal pressure was at baseline value. The effect of a single injection of a random-
ized dose of a drug on cavernosal pressure and penile length was measured until cavernosal pressure had returned t o pre-injection level. Each injection was made aRer a period of at least 15 to 25 minutes from the completion of the preceding response to ensure a stable baseline. Injection of 200 pl. of a saline vehicle had no significant effect on cavernosal pressure or penile length. In the first series of experiments, the effects of intracavernosal injection of NO donors on cavernosal pressure and penile length were studied. In each animal, a given NO donor in a volume of 200 p1. was injected intracavernosally in random order in doses of 1to 100 pg. Based on pilot studies, an additional series of experiments was conducted to evaluate the ability of two of the NO donor drugs to induce penile erection via transurethral administration. In this series of experiments, the agents dissolved in a volume of 200 pL were administered transurethrally in random order in doses of 100 t o 2000 Fg. After dose-response curves for the NO donors were attained, the standard control combination of papaverine (1.65 mg.), prostaglandin E l (PGE,) (0.5 pg.), and phentolamine (25 pg.) was administered intracavernosally for comparative purposes. Erectile responses to each of the NO donors were determined in separate animals. Drugs of the diazeniumdiolate class used in this study Their full were synthesized as separately described.', chemical names, abbreviations, and half-lives for NO release in pH 7.4 phosphate buffer at 37C are given in table 1.Each agent was dissolved in 10 mM NaOH; this alkaline stock solution was then diluted with phosphate buffered saline (PBS) (pH 7.41, and the dosing solution was administered immediately after preparation. The drugs were administered intracavernosally in small volumes (200 pl.) and control experiments with injection of the drug vehicles had no effect on cavernosal or systemic arterial pressure. The control tripledrug combination of papaverine, PGE and phentolamine (Sigma Chemical CO., St. Louis, MO) was prepared and injected as previously d e ~ c r i b e d .The ~ ~ .triple-drug ~~ combination was used as a standard for comparison for all erectile agents studied. The data were expressed as mean 2 SEM and analyzed by one-way analysis of variance (ANOVA) for multiple-group comparisons and by Student's t test for individual group comparison. The value of p c0.05 was established as the criterion for statistical significance. RESULTS
Erectile response to intracavernosal injection of NO donors. The effects of intracavernosal injection of NO donors on cavernosal pressure and penile length were investigated in the cat, and the results of these experiments are shown in fig. 1. Intracavernosal injections of MAHMA/NO (1 to 30 pg.), PAPMNO (1 to 30 pg.), DEA/NO (1 to 30 pg.), PROLI/NO (3 to 100 pg.) and P I P E M U N O (1 to 30 pg.) induced dosedependent increases in cavernosal pressure and penile length (fig. 1). Intracavernosal injection of MAHMA/NO, PAPMNO, DEA/NO, PROLI/NO and PIPERAZVNO induced increases in cavernosal pressure and penile length that were comparable to the triple-drug combination (C) (fig. 1). When the increases in cavernosal pressure in response to the NO donors were compared on a molar basis to take
TABLE1. Xdentities and half-liues of diazeniumdiolate NO donors used in this study Chemical Name
Abbrev .
C and pHat7.4 Half-Life 37
Reference
Disodium 1-~2-carboxylatopyrrolidin-l-yl~diazen-l-ium-l,2-diolate, methanol solvate l-fN-Methyl-N-IB-( N-methylammoniohexyl)aminolIdiazen-l-ium-l,2-diolate Sodium l-(N,N-diethyIamino)diazen-l-ium-1,2-diolate Sodium l-(piperazin-l-yl)diazen-l-ium-l,2-diolate
PROLUNO MAHMAMO DEMO PIPEMINO PAPAIN0
2 sec.
17 16 8, 19 20 21
1-IN-( 3-Ammoniopropyl~-N-~n-propyl~aminoldiazen-l-ium-l,2-diolate
1 min. 2 min. 5 min. 15 min.
NITRIC OXIDE DONORS AND ERECTION IN THE CAT
2015
h
OD
$
150
-1 E
2a
n=5-6
100
2 a
3 1030C
V 1 3 1030 C
V 1 3 1030 C
PAPA/NO
DEA/NO
MA iMA/NO 30
-E
s
**
n=5-6
h
n=6-8
** ** **
** T
T T
V 3 10301OOC
PROLI/NO
1
n=6
2s
V 1 3 1030C
PIPERAZVNO
1
n=5-7
** T
OD
8 a
-.aJ
20
E
a" 1s
I
I
I
I
I
I
V 1 3 1030 C
V 1 3 1030 C
V 1 3 1030 C
MAHMA/NO
PAPNNO
DEA/NO
V 3 1030100C
PROLIWO
v
1 3 1030
c
PIPERAZVNO
FIG. 1. Bar graphs showing cavernosal pressure and penile len h In res onse to intracavernosal injections of NO donors, M A H W O (pg.), PAPA/NO (pg.), DEA/NO (pg.), PROLI/NO (pg.), and PIPkkAZrIN8 (pg.).'C" denotes triple-drug control combination ( 1.65 mg. papaverine, 25 pg. phentolamine and 0.5 pg. PGE,) administered at end of experiment; n = no. of experiments. One asterisk indicates that
response is significantly different from baseline with p value less than 0.05 *ip <0.05);two asterisks indicate that p value was less than 0.01 **ip <0.01).
The duration of the erectile response, as determined by the duration of the peak response and the total duration of erectile response, was compared for each of the NO donors, and these data are shown in fig. 3 . The doses selected in fig. 3 represent the duration of the dose of drug that produced the greatest increase in cavernosal pressure obtained. The peak durations of the erectile response induced by PIPERAZVNO, MAHMA/NO and DEAN0 were similar (fig. 3). The peak 125 duration of the erectile responses induced by PAF'MO was n=5-8 slightly less than that of PIPERAZUNO, MAHhWNO, and PROLIMO D E M O , although the difference was not statistically signifPIPERAZIMO icant ( p >0.05) (fig. 3). The duration of the peak erectile response to PROLVNO was significantly less (p c0.05) than IS PAPA/NO that obtained by PIPERAZLNO, MAHMA/NO, and D E M O (fig. 3). When the peak durations of the erectile response to MAHh4"O 50 the NO donors were compared with the peak duration of the E response elicited by the triple-drug control combination, the DEA/NO > duration of the response to the triple-drug control combina8 25 tion was significantly longer ( p c0.05) (fig. 3 ) . The total durations of the erectile responses induced by the NO donors are compared in fig. 3 . The durations of the 1 erectile response induced by intracavernosal PIPERAZVNO and MAHMA/NO were not significantly different (p >0.05) Dose (nmol) from the duration of the response to the triple-drug control FIG. 2. Dose-response curves comparing increases in intracavern- combination (fig. 3 ) . The total duration of the erectile reosal ressure in response to M A H M O (pg.), PAPAIN0 ( w ) , sponse to PROLIMO, P A P M O , and D E M O was signifiDE&O (pg.), PROLI/NO (pg.), and PIPERAZI/NO (pg.).Doses of NO donors are expressed on nmol. basis to take molecular weight cantly shorter in duration (p c0.05) than the response to the triple-drug control combination (fig. 3). into account; n = no. of experiments.
molecular weight into account, the dose-response curves for PIPERAZLNO, PAPA/NO, MAHMA/NO, and D E M O were similar (fig. 2). The dose-response curve for PROLVNO was 1 to 2 log units to the right of the dose-response curves for the other NO donors (fig. 2).
h
2
NITRIC OXIDE DONORS AND ERECTION IN THE CAT
60.
40 n=5-8
50.
T
n=5-8
30
40
*
20
*
*
30
*
20
10
10
0
0
FIG.3. Bar graphs comparing peak duration of erectile response and total duration of erectile response to intracavernosal injections of MAHMA/NO (10 pg.), PAPA/NO (10pg.), D E M O (30 pg.), PROLI/NO (100 pg.), and PIPERAZUNO (10 pg.). Control denotes response to triple-drug control combination (1.65 mg. papaverine, 25 pg. phentolamine and 0.5 pg. PGE,) administered at end of experiment; n = no. of experiments. One asterisk indicates that response is significantly different from that of triple-drug control combination with p value less than 0.05 *(p <0.05).
The effects of intracavernosal injection of PROLVNO (100 pg.), PAPA/NO (10 p g . ) , PIPERAZI/NO (30 pg.), MAHhWNO (10 pg.) and DEADTO (30 pg.) on systemic arterial pressure are shown in table 2. Intracavernosal injections of PAFANO, PIPERAZVNO, MAHMA/NO, DENNO, and the triple-drug control combination induced significant decreases in systemic arterial pressure when compared with pre-injection baseline values (table 2). However, the injection of PROLUNO, in a dose that produced a maximum increase in cavernosal pressure, did not significantly alter systemic arterial pressure in the cat (table 2). The decreases in systemic arterial pressure in response to intracavernosal injection of PROLI/NO, PAFANO, PIPERAZVNO, MAHMA/NO, but not DEANO, were significantly less (p <0.05) than the decrease in systemic arterial pressure induced by the tripledrug control combination (table 2 ) . Erectile response to transurethral injection of NO donors. Given the favorable erectile profile of PROLI/NO and PIPERAZUNO, these NO donors were administered transurethrally in the cat, and the effects on cavernosal pressure and penile length are shown in fig. 4. When administered transurethrally, PROLUNO (100 to 400 p g . ) and PIPERAZI/NO (200 to 2000 pg.) induced dose-related increases in cavernosal pressure and penile length (fig. 4). The durations of the erectile response, as determined by
the duration of the peak response and the total duration of erectile response induced by the transurethral injection of PROLI/NO (400 pg.) and PIPERAZVNO (2000 fig.), are shown in fig. 5 . The peak durations of the erectile response induced by PROLUNO and PIPERAZVNO are very similar (fig. 5 ) . When the peak durations of the erectile response t o the NO donors were compared with the peak duration of the response t o the triple-drug control combination, the peak duration exhibited by the triple-drug control combination was significantly longer (p <0.05). The total durations of the erectile responses induced by the transurethral administration of the NO donors are compared in fig. 5 . The durations of the erectile responses induced by the transurethral injection of PIPERAZVNO and PROLVNO were not significantly different (p B0.05) (fig. 5). However, the total duration of erectile response to PIPERAZI/NO and PROLVNO was significantly shorter in duration (p c0.05) than the response to the triple-drug control combination (fig. 5). The effects of transurethral injection of PROLLNO (400 pg.) and PIPERAZUNO (2000 fig.) on systemic arterial pressure are shown in table 3 . Intracavernosal injections of PROLVNO and PIPERAZVNO did not significantly alter systemic arterial pressure (p >0.05). The triple-drug control combination induced significant decreases (p <0.05) in sys-
TABLE2. Change in mean systemic arterial pressure (mm.Hg) NO Donors Before Use
After Use
.____
.
~~
-
Control Triple-Drug Difference
Before Use
ARer Use
DiKerence
PROLUNO 156.0 2 5.8 150.0 i 4.2 6.0 2 2.4 157.0 t 3.4 80.0 2 9.4 76.0 2 9.3* 98.3 i 9.4 60.6 2 8.4" PAPA/NO 165.6 2 9.0 153.3 -t 9.4 12.2 2 1.9 158.9 2 10.0 PIPERAZL/NO 168.3 i 10.1 150.0 i 7.6 18.3 -C 6.0* 168.3 % 8.8 101.7 2 13.3 66.7 i 8 8' MAHMO 178.6 ? 5.9 110.0 2 10.4 65.0 2 10.4% 152.9 2 6.7 25.0 2 4.8* 175.0 2 5.6 76.2 2 14.6% DEA/NO 167.5 i 16.4 121.2 2 13.4 46.2 i 16.0' 161.2 5 18.6 ____ 82.5 2 11.1 Change in mean systemic arterial pressure (mm.Hg) in response to intracavernosal injections of PROLVNO (100 pg.), P A p m O (10 pg..),PIPERAZLNO (30 pg.L MAHMA/NO (10 fig.) and D E M O (30 fig.) and control triple-drug combination. N = 5-8. *p c0.05. response is significantly different from baseline,
NITRIC OXIDE DONORS AND ERECTION IN THE CAT
2017
Transurethral Injection
50 -
*
T
200
400
* T
v
100
c
v
PROLVNO
200
400 1oO02oO0
c
PIPERAZVNO 30
n=9-10
n=9- 10
*
2ou rir II *
25
25
*
*
T
20
15
-
v
100
200
400
c
PROLVNO
15
v
200
400 1o0o2oO0
c
PIPERAZVNO
FIG. 4. Bar graphs showing cavernosal pressure and penile length in response to transurethral administration of NO donors PROLI/NO (pg.) and PIPERAZI/NO (pg.)."C" denotes triple-drug control combination (1.65 mg. papaverine, 25 pg. phentolamine and 0.5 gg. PGE ) administered at end of experiment; n = no. of experiments. One asterisk indicates that response is significantly different from baseline with p value less than 0.05 *(p <0.05); two asterisks indicate that p value was less than 0.01 **(p <0.01).
temic arterial pressure when compared with the pre-injection baseline values (table 3). DISCUSSION
Three neuroeffector pathways - adrenergic, cholinergic, and nonadrenergic, noncholinergic (NANC) - work in unison to coordinate smooth muscle tone in the corpus cavernosum. Evidence suggests that NO is the NANC neurotransmitter responsible for activation of soluble guanylyl cyclase resulting in production of intracellular c G M P . Other ~ ~ studies suggest that release of acetylcholine stimulates the muscarinic receptors on endothelial cells and induces the release of NO, creating a common pathway for cavernosal relaxation and the induction of penile e r e ~ t i o n In . ~ addition, studies have shown that a decrease in NO synthase in rats is correlated with a decrease in erectile function.25 Based on these findings, it has been postulated that the administration of exog-
enous NO may be useful in the management of male erectile dysfunction. However, NO itself is very labile, with a short physiological half-life. Intracavernosal injection of sodium nitroprusside in patients who suffer from erectile dysfunction was not feasible because of the severe hypotension that occurred after admini~tration.'~Other studies with linsidomine chlorohydrate (SIN-l), another NO donor, have demonstrated relatively good efficacy in inducing penile erection in humans with fewer sideeffects when compared with intracavernosal injection of prostaglandin E, and/or papaverine."j However, this has not been universally ~onfirmed.'~ The use of NO donors for the treatment of erectile dysfunction has been hampered by the lack of a controlled release of NO. Recently, a new class of NO-generating diazeniumdiolate complexes, which spontaneously and predictively release NO through a nonenzymatic reaction, has been developed. The results of the present study demonstrate the ability of
2018
NITRIC OXIDE DONORS AND ERECTION IN THE CAT
Transurethral Injection
60
.!
50
n=9- 10
40 x
30
30
ll
PROLI/NO PIPERAZVNO
T
20 10
0
7
PROLVNO PIPERAZVNO
Control
7
Control
Flc;. 5. Bar graphs comparing ak duration of erectile response and total duration of erectile response to transurethral administration of PROLL/NO ( 4 0 0 p g . ) and P I P E b L / N O (2000 pg.1. Control denotes response to triple-drug control combination (1.65 mg. papaverine, 25 pg. phentolamine and 0.5 p PGE,, administered at end of experiment; n = no. of experiments. One asterisk indicates that response is significantly different from tkat of triple-drug control combination with p value less than 0.05 *(p <0.05). TM1.k: 3. Change in mean s-ystemic arterial blood pressure (mm.Hg) ...
~
~
~- ~.
~~~~
K O Donors .
Hetiire LJse
ARer Use ~~
I'ROI,l/M) 1'II'EWlM)
Control Triple-Drug -
~~
Difference ~
~
~~~
Before Use
After Use
Difference
.~
6.2 146.0 * 6.7 3.0 z 5.4 149.4 -C 5.1 76.2 2 7.0 72.3 t 8.4* 9.3 154.0 I 7.9 2.1 5 5.0 159.3 2 6.7 80.1 2 9.4 78.7 t 7.3* ('hange in mean sy?itemicartenal pressure tmm.Hg) in response to transurethral administration of PROLIMO (400 pg.) and PIPERAZUNO (2000 pg.) and intracavern~isel injection ( 1 1 the triple-drug control combination. N = 9-10, 'p < 0.05. response is significantly different from baseline value. 149 0 156.6
* '
~
MAHMA/NO, PAPNNO, DENNO, PIPERAZVNO and PROLVNO, all novel NO donors, to induce dose dependent increases in cavernosal pressure and penile length when administered intracavernosally in the cat. The increases in cavernosal pressure and penile length in response to intracavernosal injection of MAHMA/NO, PAPA/NO, DEA/NO, PIPERAZVNO, and PROLVNO were comparable to the increases induced by the triple-drug control combination, suggesting that these agents possess the ability to induce penile erection in this animal model. Electrical stimulation of the cavernosal nerve was not conducted in this study because the control triple-drug combination served as a control comparison, as described in previous papers from this laboratory, to gauge the potency of all NO donors tested.".'" When compared on a nmol. basis. taking molecular weight into account, the dose-responses for MAHMA/NO, PAPADTO, DEA/NO, and PIPERAZVNO were similar and were 3 to 10-fold more potent than PROLVNO. Moreover, the durations of the maximum increases in cavernosal pressure in response to NO donors were similar to each other, but were significantly shorter in duration when compared with the triple-drug control combination. However. the decrease in systemic arterial pressure following injection of the NO donors was also significantly less than that observed with the triple-drug control combination. In fact. intracavernosal injections of PROLVNO and PAPA/NO resulted in slight but not significant decreases in systemic arterial pressurr a t all doscs studied. The reason for the diffrrenct. in the c4fcct on systemic arterial pressure is not known, but thcsc data suggrst that PROLI/NO. at the d o w ~nwdt.d to induct, penile ert.ction. dow not alter sys-
temic arterial pressure, perhaps because of its more rapid release of NO into the cavernosal tissue with less systemic absorption. The relative lack of systemic absorption may be augmented by the onset of the veno-occlusive response. The triple-drug combination induced severe hypotension, which may be attributed to the doses used of papavenne, phentolamine, and PGE, employed in this combination. The results of this study show that these novel NO donors can induce a dose-dependent increase in cavernosal pressure and penile length when injected intracavernosally in the cat. Transurethral administration of PROLUNO and PAPANO in doses of 100 to 400 pg. and 200 to 2000 pg., respectively, induced dose-dependent increases in cavernosal pressure and penile length to values comparable to those observed with the triple-drug control combination. These data provide evidence that, like prostaglandin E,, these NO donors can induce an erectile response when administered transurethrally. This may be a significant advantage over other vasodilator agents with a shorter duration of action and over those which alter systemic arterial pressure. Moreover, the use of local drug application is appealing for treating erectile dysfunction because of reduced side effects and greater clinical acceptability.2fi The finding that PROLUNO generally gave a shorter lasting and less potent erectile response than the other test agents presumably stems from its short half-life ( 2 seconds a t pH 7.4 and 37C).'' While it was stable a s a stock solution in 10 mM sodium hydroxide, we felt it was inadvisable to administer the compound to the animals in a n alkaline vehicle. Accordingly. w e neutralized the stock solution with PBS but
NITRIC OXIDE DONORS AND ERECTION IN THE CAT
made every effort to inject the neutral dosing solution as soon as possible thereafter, usually within seconds. However, this procedure invariably took enough time that premature dissociation of PROLI/NO and subsequent loss of NO must have occurred to a significant extent. This was not a problem with the other drugs, whose half-lives are long compared with the time that elapsed between neutralization of their stock solutions and administration to the animals. We view it as likely that PROLVNOs potency would be much greater in a more accommodating vehicle. When administered intracavernosally and transurethrally, the NO-releasing diazeniumdiolates evaluated in this study did not substantially lower systemic arterial pressure. This observation can be attributed to the fact that these novel NO donors are ionic or zwitterionicand are not easily absorbed as such into the systemic circulation. Inactivation of this highly diffusible species (NO) through its extremely rapid destruction by oxyheme species, superoxide and other reactive biomolecules prohibits extension of its vasodilatory effects much beyond the immediate vicinity of the corpus cavernosum thus causing less of a systemic and erectile response than that of the control triple-drug combination. The ability of these spontaneous NOreleasers to concentrate their vasodilatory effect solely in the penis when administered locally, without hemodynamic side effects is a major advantage and provides a strong rationale for further investigation of this class of agents in the clinical management of erectile dysfunction. CONCLUSIONS
The resultsof the present study provide evidence that MAHMA/ NO, PAPADTO, DENNO, PIPERAZI/NO, and P R O W 0 can induce dose-dependent increases in cavernosal pressure and penile length in the cat when administered intxacavemosdy. The increases in cavernosal pressure and penile length were comparable to those observed with the triple-drug control combination. Although the magnitudes of the increases in cavemosalpressure and penile length were similar,the duration of the maximum erectile response induced by the NO donors was sigmfmmtly less than that observed by the triple-drug control combmation. The maximum increase in cavernosal pressure in response to PROLVNO and PAPADTO was assOciated with no sgdicant change in systemic arterial pressure. The decreases in systemic arterial pressure in response to intracavernosal injection of -0, D M O , and PIPERAZINO were sigm6cantly less than those observed with the tripledrug control combination. Moreover, the results of the present study demonstrate that transmthral administration of PROWNO and PIPERAzIlNO can induce dosedependent increases in cavernosal pressure and penile length, similar to those observed with the triple-drug control combmation but without the decrease in systemicarterial pressure. These data support further clinical investigationsof diazeniumdiolatesfor the pharmacologic management of erectile dysfunction. Acknowledgments. The authors would like to thank Melanie Cross for her assistance in the preparation of this manuscript. REFERENCES
1. Feldman, H. A., Goldstein, I., Hatzichristou, D. G., Krane, R. J.
and McKinlay, J. B.: Impotence and its medical and psychosocial correlates: results of the Massachusetts male aging study. J. Urol., 151: 54, 1994. 2. NIH Consensus Conference. Impotence. NIH Consensus Development Panel on Impotence. JAMA, 270 83, 1993. 3. Andersson, K-E. and Wagner, G.: Physiology of penile erection. Phvsiol Rev.. 75: 191, 1995. 4. Anderson, K-E.: Pharmacology of lower urinary tract smooth muscles and penile erectile tissues. Pharmacol. Rev , 45: 253, 1993. 5. Burnett, A. L., Lowenstein, C. J., Bredt, D. S., Chang, T. S. K. and Snyder, S. H.: Nitric oxide: a physiologic mediator of penile erection. Science, 257: 401, 1992. 6. Melis, M. R. and Argiolas, A,: Nitric oxide donors induce penile
2019
erection and yawning when injected in the central nervous system of male rats. Eur. J. Pharmacol., 294: 1, 1995. 7. Burnett, A. L.: Nitric oxide in the penis: physiology and pathology. J . Urol., 157: 320, 1997. 8. Maragos, C. M., Morley, D., Wink, D. A,, Dunams, T. M., Saavedra, J. E., Hoffman, A., Bove, A. A,, Isaac, L., Hrabie, J. A. and Keefer, L. R: Complexes of NO with nucleophiles as agents for the controlled biological release of nitric oxide. Vasorelaxant effects. J. Med. Chem., 34:3242, 1991. 9. Morley, D. and Keefer, L. R: Nitric oxiddnucleophile complexes: a unique class of nitric oxide-based vasodilators. J. Cardiovasc. Pharmacol., 22(Suppl. 7): S3, 1993. 10. Lakin, M. M., Montague, D. K., Medendorp, S. V., Tesar, L. and Schover, L. R.: Intracavernous injection therapy: analysis of results and complications. J. Urol., 1 4 3 1138, 1990. 11. Hellstrom, W. J . G., Bennett, A. H., Gesundheit, N., Kaiser, F. E., Lue, T. F., Padma-Nathan, H., Peterson, C. A., Tam, P. Y., Todd, L. K., Varady, J. C. and Place, V. A.: A double blind, placebo-controlled evaluation of the erectile response to transurethral alprostadil. Urology, 48:851, 1996. 12. Padma-Nathan, H., Hellstrom, W. J. G., Kaiser, F. E., Labasky, R. F., Lue, T. F., Nolten, W. E., Norwood, P. C., Peterson, C. A,, Shabsigh, R., Tam, P. Y., Place, V. A. and Gesundheit, N.: Treatment of men with erectile dysfunction with transurethral Alprostadil. N. Engl. J. Med., 336 1, 1997. 13. Williams, G., Abbou, C. C., b a r , E. T., Desvaw, P., Flam, T. A,, Lycklama, A,, Nijeholt, G. A., Lynch, S. F., Morgan, R. J., Muller, S. C., Porst, H., Pryor, J. P., Ryan, P., Witzch, U. K., Hall, M. M., Place, V. A,, Spivack, A. P. and Gesundheit, N.: Efficacy and safety of transurethral alprostadil therapy in men with erectile d y s h c t i o n . MUSE Study Group. Br. J. Urol., 81: 889, 1998. 14. Lue, T. F. and Levine, J. D.: Impotence (editorial). J. Urol., 154: 85, 1995. 15. Brock, G., Breza, J . and Lue. T. F.: Intracavernous sodium nitroprusside: inappropriate impotence treatment. J . Urol., 150 864, 1993. 16. Stief, C. G., Holmquist, F., Djamilian, M., Krah, H., Andersson, K-E. and Jonas, U.: Preliminary results with nitric oxide donor linsidomine chlorhydate in the treatment of human erectile dysfunction. J . Urol., 148: 1437, 1992. 17. Porst, H.: Prostaglandin E, and the nitric oxide donor linsidomine for erectile failure: a diagnostic comparative study of 40 patients. J. Urol., 1 4 9 1280, 1993. 18. Saavedra, J. E., Southan, G . J., Davies, K. M., Lundell, A., Markou, C., Hanson, S. R., Adrie, C., Hurford, W. E., Zapol, W. M. and Keefer, L. K.: Localizing antithrombotic and vasodilatory activity with a novel, ultrafast nitric oxide donor. J. Med. Chem., 39 4361, 1996. 19. Hrabie, J. A., Klose, J . R., Wink, D. A. and Keefer, L. K.: New nitric oxide-releasing zwitterions derived from polyamines. J. Org. Chem., 5 8 1472, 1993. 20. Hrabie, J. A., Saavedra, J. E., Davies, K. M. and Keefer, L. K.: Adducts of piperazine with nitric oxide. Org. Prep. Proc. Intl., 1999. 21. Maragos, C. M., Wang, J. M., Hrabie, J. A., Oppenheim, J. J. and Keefer, L. K.: Nitric oxiddnucleophile complexes inhibit the in vitro proliferation of A375 melanoma cells via nitric oxide release. Cancer Res., 53: 564, 1993. 22. Wang, R., Domer, F. R.. Sikka, S. C., Kadowitz, P. J. and Hellstrom, W. J . G.: Nitnc oxide mediates penile erection in cats. J . Urol., 151: 234, 1994. 23. Champion, H. C., Wang, R., Hellstrorn, W. J. G. and Kadowitz, P. J.: Nociceptin, a novel endogenous ligand for the ORL, receptor, has potent erectile activity in the cat. Am. J. Physiol.. 273 E214, 1997. 24. Kerwin Jr., J. F., Lancaster Jr., J. R. and Feldman, P. L.: Nitric oxide: a new paradigm for second messengers. J . Med. Chem., 3 8 4343, 1995. 25. Garban, H., Vernet, D., Freedman, A., Rajfer, J. and Gonzalez-Cadavid, N.: Effect of aging on nitric oxide-mediated oenile erection in rats. Am. J. Phvsiol.. 268: H467. 1995. 26. Doherty, P. C.: Oral, transdermal, i n d transurethral therapies for erectile dysfunction. In: Male Infertility and Sexual Dysfunction. Edited by Wayne J. G. Hellstrom. New York Springer Verlag, chapter 33, pp. 452-467, 1997.
Vol. 161,2013-2019, June 1999 Printed in U S A
INDUCTION OF PENILE ERECTION BY INTRACAVERNOSAL AND TRANSURETHRAL ADMINISTRATION OF NOVEL NITRIC OXIDE DONORS IN THE CAT HUNTER C. CHAMPION, TRINITY J . BIVALACQUA, RUN WANG, PHILIP J. KADOWITZ, LARRY K. KEEFER, JOSEPH E. SAAVEDRA, JOSEPH A. HRABIE, PAUL C. DOHERTY AND WAYNE J . G. HELLSTROM* From the Departments of Urology and Pharmacology, Tulane University School of Medicine, New Orleans, Louisiana, the Labomtory of Comparative Carcinogenesis and SAlC Frederick, National Cancer Institute, Frederick Cancer Research and Development Center, Frederick, Maryland, and VTWS, Inc., Mountain View, California
ABSTRACT
Purpose: The effects of novel nitric oxide (NO) donors administered intracavernosally and transurethrally on erectile function in the anesthetized cat were evaluated. Materials and Methods: In pentobarbital-anesthetized cats, increases in intracavernosal pressure, penile length, and duration of erectile response were determined after intracavernosal and transurethral injections of novel NO donors (MAHMA/NO, PAPANO, DEA/NO, PIPERAZVNO and PROLINO). All parameters were measured after administration of NO donors intracavernosally via a 30-gauge needle and urethrally via a Jelco i.v. catheter in a volume of 200 p1. Systemic arterial pressure was also assessed in these experiments. All NO donors were compared with a triple-drug control combination comprised of papaverine (1.65 mg.), prostaglandin E, (0.5 pg.), and phentolamine (25 pg.). Results: MAHMA/NO, PAPANO, DEA/NO, PIPERAZVNO and PROLUNO induced dose dependent increases in intracavernosal pressure and penile length (p <0.05) when administered intracavernosally. The increases in cavernosal pressure and penile length were comparable to those observed with the triple-drug control combination. The maximum increase in cavernosal pressure in response to PROLVNO and PAPANO was associated with no significant change in systemic arterial pressure. Transurethral administration of PROLINO and PIPERAZVNO induced dose-dependent increases in cavernosal pressure and penile length (p <0.05). The response was similar to that of the triple-drug control combination, except that transurethral PROLVNO and PIPERAZIINO had no significant effect on systemic blood pressure. Conclusions: NO donors caused dose-dependent increases in cavernosal pressure when administered intracavernosally and transurethrally. These data suggest further exploration of the use of NO donors for the treatment of erectile dysfunction. KEYWORDS:penile erection, novel nitric oxide donors, diazeniumdiolates, transurethral, intracavernosal
An estimated 20 to 30 million American men suffer from peripheral nervous systems, as well as in local vascular conerectile dysfunction, and 50 to 60% of these men have a t r 0 1 . ~ - ~ Although NO solution can produce an erectile response, it vascular component to their condition.'.' Penile erection is caused by increased arterial inflow and restricted venous is not clinically efficacious in the management of impotence outflow, coordinated with the relaxation of blood vessels and because of its extremely short half-life in aerobic the trabecular smooth muscle that constitutes the corpora Recently, vasodilators of the diazeniumdiolate class have c a ~ e r n o s a .Although ~ parasympathetic innervation is in- been developed that allow for the controlled pharmacological volved in erectile dysfunction, the erectile response cannot be delivery of NO. Half-lives of ions in this series range from 2 completely blocked by atropine, suggesting that a nonadren- seconds to 20 hours, depending on the structure, allowing for ergic, noncholinergic (NANC) mechanism is involved in pe- the study of dosage rate as a determinant of pharmacological nile e r e ~ t i o n Nitric .~ oxide (NO) is a free radical molecule response.**' which serves as a chemical messenger in both the central and The use of intracavernosal pharmacotherapy has become peripheral nervous systems. NO is a powerful vasodilator increasingly widespread for the local treatment of erectile invoking an integral role in the physiological and pathophys- dysfunction. Until recently, the only method of introducing iological regulation of blood flow and blood pressure. More vasoactive agents into the corpus cavernosum was direct recently, the role of NO in the mediation of penile erection injection. Although this method is successful for most pahas been revealed with actions defined a t the central and tients, long-term compliance is low, and there are a number of acknowledged side effects, including pain, penile fibrosis, Accepted for publication February 1, 1999. hematoma formation* and priapism." a number of Department of Urology SL42,Tulane Uni* Requests for versity School of Medicine, 1430 Tulane Avenue, New Orleans, LA studies have documented the medicated urethral system of delivery (MUSE) for the transurethral administration of 70112. Supported by a fellowship from the American Foundation for UrO- prostaglandin El (alprostadil) as an effective and lesslogical Disease, Inc. (TJB),an educational grant from VTWS, Inc., invasive method for introducing vasoactive agents for the and National Institutes of Health Contract NO. ~ 0 1 - ~ 0 - 5 6 0 0to 0 purpose of treating erectile dy~fUnction."-'~ Although the SAIC Frederick. 2013
2014
NITRIC OXIDE DONORS AND ERECTION IN THE CAT
transurethral administration of alprostadil has proven efficacious in double-blind studies, it is not without side effects (for example, dizziness, hypotension, and penile pain). Moreover, long-term efficacy and compliance in the general population remain to be established. It has been postulated that these untoward consequences may be due to both local and systemic effects of the prostaglandin E l and/or local effects of the solvent used in the formulation. Increases in efficacy and a reduction of side effects are the two key issues in the development of new agents for the treatment of erectile dysfunction. Efficacy has been improved in intracavernosal therapies by the addition of other agents. However, since alprostadil appears to sensitize nerve fibers to noxious stimuli through interactions with specific prostaglandin receptors, l4 some degree of pain can be expected with administration of this agent regardless of the route of delivery. Ideally, agents with short half-lives that could be administered locally would reduce the potential for systemic side effects. Such agents should activate pathways that induce the erectile response without the sensitizing nerve conduction pathways. Nitric oxide donors would prove useful in this regard, however, attempts to use currently available NO NO donors have met with limited S U C C ~ S SThe .~~ - ~generat~ ing diazeniumdiolate complexes may have a n advantage in this regard since they spontaneously and predictably release NO. Although these compounds have been shown to cause smooth muscle relaxation and increased blood flow in a number of organ systems," little is known about their effects on penile erection. This study explores the effects of intracavernosal injection of these NO donors on erectile function in the cat and investigates their potential to induce penile erection via the transurethral route of administration. MATERIALS A N D METHODS
Adult male cats weighing 2.8 to 4.8 kg. were sedated with ketamine hydrochloride (10 to 15 mg./kg. i.mJ and anesthetized with pentobarbital sodium (30 mg./kg. i.v.). Supplemental doses of pentobarbital were administered as needed to maintain a uniform level of anesthesia. A vertical, circumcision-like incision was made to expose the two ventral corpora cavernosa and the dorsal corpus spongiosum. A 30gauge needle was placed into the right corpus cavernosum to permit administration of drugs into the penis. A 25-gauge needle was placed midway into the left corpus cavernosum for the measurement of intracavernosal pressure (mm. Hg). Systemic and intracavernosal pressures (mm. Hg) were measured with Statham P23 transducers connected to a Grass model-7 polygraph, and mean pressures were obtained by electronic averaging. Penile length (mm.)was measured with a ruler. These procedures have been previously described and were approved by the Tulane University Animal Care and Use Committee.22z23 In experiments in which the agonists were administered intracavernosally, injections were made via a 30-gauge needle inserted into the corpus cavernosum. In experiments in which the transurethral administration of NO donors was employed, drugs were introduced into the urethra via a Jelco i.v. catheter in a volume of 200 p1. In all experiments, injections of drugs were made when the cavernosal pressure was at baseline value. The effect of a single injection of a random-
ized dose of a drug on cavernosal pressure and penile length was measured until cavernosal pressure had returned t o pre-injection level. Each injection was made aRer a period of at least 15 to 25 minutes from the completion of the preceding response to ensure a stable baseline. Injection of 200 pl. of a saline vehicle had no significant effect on cavernosal pressure or penile length. In the first series of experiments, the effects of intracavernosal injection of NO donors on cavernosal pressure and penile length were studied. In each animal, a given NO donor in a volume of 200 p1. was injected intracavernosally in random order in doses of 1to 100 pg. Based on pilot studies, an additional series of experiments was conducted to evaluate the ability of two of the NO donor drugs to induce penile erection via transurethral administration. In this series of experiments, the agents dissolved in a volume of 200 pL were administered transurethrally in random order in doses of 100 t o 2000 Fg. After dose-response curves for the NO donors were attained, the standard control combination of papaverine (1.65 mg.), prostaglandin E l (PGE,) (0.5 pg.), and phentolamine (25 pg.) was administered intracavernosally for comparative purposes. Erectile responses to each of the NO donors were determined in separate animals. Drugs of the diazeniumdiolate class used in this study Their full were synthesized as separately described.', chemical names, abbreviations, and half-lives for NO release in pH 7.4 phosphate buffer at 37C are given in table 1.Each agent was dissolved in 10 mM NaOH; this alkaline stock solution was then diluted with phosphate buffered saline (PBS) (pH 7.41, and the dosing solution was administered immediately after preparation. The drugs were administered intracavernosally in small volumes (200 pl.) and control experiments with injection of the drug vehicles had no effect on cavernosal or systemic arterial pressure. The control tripledrug combination of papaverine, PGE and phentolamine (Sigma Chemical CO., St. Louis, MO) was prepared and injected as previously d e ~ c r i b e d .The ~ ~ .triple-drug ~~ combination was used as a standard for comparison for all erectile agents studied. The data were expressed as mean 2 SEM and analyzed by one-way analysis of variance (ANOVA) for multiple-group comparisons and by Student's t test for individual group comparison. The value of p c0.05 was established as the criterion for statistical significance. RESULTS
Erectile response to intracavernosal injection of NO donors. The effects of intracavernosal injection of NO donors on cavernosal pressure and penile length were investigated in the cat, and the results of these experiments are shown in fig. 1. Intracavernosal injections of MAHMA/NO (1 to 30 pg.), PAPMNO (1 to 30 pg.), DEA/NO (1 to 30 pg.), PROLI/NO (3 to 100 pg.) and P I P E M U N O (1 to 30 pg.) induced dosedependent increases in cavernosal pressure and penile length (fig. 1). Intracavernosal injection of MAHMA/NO, PAPMNO, DEA/NO, PROLI/NO and PIPERAZVNO induced increases in cavernosal pressure and penile length that were comparable to the triple-drug combination (C) (fig. 1). When the increases in cavernosal pressure in response to the NO donors were compared on a molar basis to take
TABLE1. Xdentities and half-liues of diazeniumdiolate NO donors used in this study Chemical Name
Abbrev .
C and pHat7.4 Half-Life 37
Reference
Disodium 1-~2-carboxylatopyrrolidin-l-yl~diazen-l-ium-l,2-diolate, methanol solvate l-fN-Methyl-N-IB-( N-methylammoniohexyl)aminolIdiazen-l-ium-l,2-diolate Sodium l-(N,N-diethyIamino)diazen-l-ium-1,2-diolate Sodium l-(piperazin-l-yl)diazen-l-ium-l,2-diolate
PROLUNO MAHMAMO DEMO PIPEMINO PAPAIN0
2 sec.
17 16 8, 19 20 21
1-IN-( 3-Ammoniopropyl~-N-~n-propyl~aminoldiazen-l-ium-l,2-diolate
1 min. 2 min. 5 min. 15 min.
NITRIC OXIDE DONORS AND ERECTION IN THE CAT
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FIG. 1. Bar graphs showing cavernosal pressure and penile len h In res onse to intracavernosal injections of NO donors, M A H W O (pg.), PAPA/NO (pg.), DEA/NO (pg.), PROLI/NO (pg.), and PIPkkAZrIN8 (pg.).'C" denotes triple-drug control combination ( 1.65 mg. papaverine, 25 pg. phentolamine and 0.5 pg. PGE,) administered at end of experiment; n = no. of experiments. One asterisk indicates that
response is significantly different from baseline with p value less than 0.05 *ip <0.05);two asterisks indicate that p value was less than 0.01 **ip <0.01).
The duration of the erectile response, as determined by the duration of the peak response and the total duration of erectile response, was compared for each of the NO donors, and these data are shown in fig. 3 . The doses selected in fig. 3 represent the duration of the dose of drug that produced the greatest increase in cavernosal pressure obtained. The peak durations of the erectile response induced by PIPERAZVNO, MAHMA/NO and DEAN0 were similar (fig. 3). The peak 125 duration of the erectile responses induced by PAF'MO was n=5-8 slightly less than that of PIPERAZUNO, MAHhWNO, and PROLIMO D E M O , although the difference was not statistically signifPIPERAZIMO icant ( p >0.05) (fig. 3). The duration of the peak erectile response to PROLVNO was significantly less (p c0.05) than IS PAPA/NO that obtained by PIPERAZLNO, MAHMA/NO, and D E M O (fig. 3). When the peak durations of the erectile response to MAHh4"O 50 the NO donors were compared with the peak duration of the E response elicited by the triple-drug control combination, the DEA/NO > duration of the response to the triple-drug control combina8 25 tion was significantly longer ( p c0.05) (fig. 3 ) . The total durations of the erectile responses induced by the NO donors are compared in fig. 3 . The durations of the 1 erectile response induced by intracavernosal PIPERAZVNO and MAHMA/NO were not significantly different (p >0.05) Dose (nmol) from the duration of the response to the triple-drug control FIG. 2. Dose-response curves comparing increases in intracavern- combination (fig. 3 ) . The total duration of the erectile reosal ressure in response to M A H M O (pg.), PAPAIN0 ( w ) , sponse to PROLIMO, P A P M O , and D E M O was signifiDE&O (pg.), PROLI/NO (pg.), and PIPERAZI/NO (pg.).Doses of NO donors are expressed on nmol. basis to take molecular weight cantly shorter in duration (p c0.05) than the response to the triple-drug control combination (fig. 3). into account; n = no. of experiments.
molecular weight into account, the dose-response curves for PIPERAZLNO, PAPA/NO, MAHMA/NO, and D E M O were similar (fig. 2). The dose-response curve for PROLVNO was 1 to 2 log units to the right of the dose-response curves for the other NO donors (fig. 2).
h
2
NITRIC OXIDE DONORS AND ERECTION IN THE CAT
60.
40 n=5-8
50.
T
n=5-8
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40
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20
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FIG.3. Bar graphs comparing peak duration of erectile response and total duration of erectile response to intracavernosal injections of MAHMA/NO (10 pg.), PAPA/NO (10pg.), D E M O (30 pg.), PROLI/NO (100 pg.), and PIPERAZUNO (10 pg.). Control denotes response to triple-drug control combination (1.65 mg. papaverine, 25 pg. phentolamine and 0.5 pg. PGE,) administered at end of experiment; n = no. of experiments. One asterisk indicates that response is significantly different from that of triple-drug control combination with p value less than 0.05 *(p <0.05).
The effects of intracavernosal injection of PROLVNO (100 pg.), PAPA/NO (10 p g . ) , PIPERAZI/NO (30 pg.), MAHhWNO (10 pg.) and DEADTO (30 pg.) on systemic arterial pressure are shown in table 2. Intracavernosal injections of PAFANO, PIPERAZVNO, MAHMA/NO, DENNO, and the triple-drug control combination induced significant decreases in systemic arterial pressure when compared with pre-injection baseline values (table 2). However, the injection of PROLUNO, in a dose that produced a maximum increase in cavernosal pressure, did not significantly alter systemic arterial pressure in the cat (table 2). The decreases in systemic arterial pressure in response to intracavernosal injection of PROLI/NO, PAFANO, PIPERAZVNO, MAHMA/NO, but not DEANO, were significantly less (p <0.05) than the decrease in systemic arterial pressure induced by the tripledrug control combination (table 2 ) . Erectile response to transurethral injection of NO donors. Given the favorable erectile profile of PROLI/NO and PIPERAZUNO, these NO donors were administered transurethrally in the cat, and the effects on cavernosal pressure and penile length are shown in fig. 4. When administered transurethrally, PROLUNO (100 to 400 p g . ) and PIPERAZI/NO (200 to 2000 pg.) induced dose-related increases in cavernosal pressure and penile length (fig. 4). The durations of the erectile response, as determined by
the duration of the peak response and the total duration of erectile response induced by the transurethral injection of PROLI/NO (400 pg.) and PIPERAZVNO (2000 fig.), are shown in fig. 5 . The peak durations of the erectile response induced by PROLUNO and PIPERAZVNO are very similar (fig. 5 ) . When the peak durations of the erectile response t o the NO donors were compared with the peak duration of the response t o the triple-drug control combination, the peak duration exhibited by the triple-drug control combination was significantly longer (p <0.05). The total durations of the erectile responses induced by the transurethral administration of the NO donors are compared in fig. 5 . The durations of the erectile responses induced by the transurethral injection of PIPERAZVNO and PROLVNO were not significantly different (p B0.05) (fig. 5). However, the total duration of erectile response to PIPERAZI/NO and PROLVNO was significantly shorter in duration (p c0.05) than the response to the triple-drug control combination (fig. 5). The effects of transurethral injection of PROLLNO (400 pg.) and PIPERAZUNO (2000 fig.) on systemic arterial pressure are shown in table 3 . Intracavernosal injections of PROLVNO and PIPERAZVNO did not significantly alter systemic arterial pressure (p >0.05). The triple-drug control combination induced significant decreases (p <0.05) in sys-
TABLE2. Change in mean systemic arterial pressure (mm.Hg) NO Donors Before Use
After Use
.____
.
~~
-
Control Triple-Drug Difference
Before Use
ARer Use
DiKerence
PROLUNO 156.0 2 5.8 150.0 i 4.2 6.0 2 2.4 157.0 t 3.4 80.0 2 9.4 76.0 2 9.3* 98.3 i 9.4 60.6 2 8.4" PAPA/NO 165.6 2 9.0 153.3 -t 9.4 12.2 2 1.9 158.9 2 10.0 PIPERAZL/NO 168.3 i 10.1 150.0 i 7.6 18.3 -C 6.0* 168.3 % 8.8 101.7 2 13.3 66.7 i 8 8' MAHMO 178.6 ? 5.9 110.0 2 10.4 65.0 2 10.4% 152.9 2 6.7 25.0 2 4.8* 175.0 2 5.6 76.2 2 14.6% DEA/NO 167.5 i 16.4 121.2 2 13.4 46.2 i 16.0' 161.2 5 18.6 ____ 82.5 2 11.1 Change in mean systemic arterial pressure (mm.Hg) in response to intracavernosal injections of PROLVNO (100 pg.), P A p m O (10 pg..),PIPERAZLNO (30 pg.L MAHMA/NO (10 fig.) and D E M O (30 fig.) and control triple-drug combination. N = 5-8. *p c0.05. response is significantly different from baseline,
NITRIC OXIDE DONORS AND ERECTION IN THE CAT
2017
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15
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FIG. 4. Bar graphs showing cavernosal pressure and penile length in response to transurethral administration of NO donors PROLI/NO (pg.) and PIPERAZI/NO (pg.)."C" denotes triple-drug control combination (1.65 mg. papaverine, 25 pg. phentolamine and 0.5 gg. PGE ) administered at end of experiment; n = no. of experiments. One asterisk indicates that response is significantly different from baseline with p value less than 0.05 *(p <0.05); two asterisks indicate that p value was less than 0.01 **(p <0.01).
temic arterial pressure when compared with the pre-injection baseline values (table 3). DISCUSSION
Three neuroeffector pathways - adrenergic, cholinergic, and nonadrenergic, noncholinergic (NANC) - work in unison to coordinate smooth muscle tone in the corpus cavernosum. Evidence suggests that NO is the NANC neurotransmitter responsible for activation of soluble guanylyl cyclase resulting in production of intracellular c G M P . Other ~ ~ studies suggest that release of acetylcholine stimulates the muscarinic receptors on endothelial cells and induces the release of NO, creating a common pathway for cavernosal relaxation and the induction of penile e r e ~ t i o n In . ~ addition, studies have shown that a decrease in NO synthase in rats is correlated with a decrease in erectile function.25 Based on these findings, it has been postulated that the administration of exog-
enous NO may be useful in the management of male erectile dysfunction. However, NO itself is very labile, with a short physiological half-life. Intracavernosal injection of sodium nitroprusside in patients who suffer from erectile dysfunction was not feasible because of the severe hypotension that occurred after admini~tration.'~Other studies with linsidomine chlorohydrate (SIN-l), another NO donor, have demonstrated relatively good efficacy in inducing penile erection in humans with fewer sideeffects when compared with intracavernosal injection of prostaglandin E, and/or papaverine."j However, this has not been universally ~onfirmed.'~ The use of NO donors for the treatment of erectile dysfunction has been hampered by the lack of a controlled release of NO. Recently, a new class of NO-generating diazeniumdiolate complexes, which spontaneously and predictively release NO through a nonenzymatic reaction, has been developed. The results of the present study demonstrate the ability of
2018
NITRIC OXIDE DONORS AND ERECTION IN THE CAT
Transurethral Injection
60
.!
50
n=9- 10
40 x
30
30
ll
PROLI/NO PIPERAZVNO
T
20 10
0
7
PROLVNO PIPERAZVNO
Control
7
Control
Flc;. 5. Bar graphs comparing ak duration of erectile response and total duration of erectile response to transurethral administration of PROLL/NO ( 4 0 0 p g . ) and P I P E b L / N O (2000 pg.1. Control denotes response to triple-drug control combination (1.65 mg. papaverine, 25 pg. phentolamine and 0.5 p PGE,, administered at end of experiment; n = no. of experiments. One asterisk indicates that response is significantly different from tkat of triple-drug control combination with p value less than 0.05 *(p <0.05). TM1.k: 3. Change in mean s-ystemic arterial blood pressure (mm.Hg) ...
~
~
~- ~.
~~~~
K O Donors .
Hetiire LJse
ARer Use ~~
I'ROI,l/M) 1'II'EWlM)
Control Triple-Drug -
~~
Difference ~
~
~~~
Before Use
After Use
Difference
.~
6.2 146.0 * 6.7 3.0 z 5.4 149.4 -C 5.1 76.2 2 7.0 72.3 t 8.4* 9.3 154.0 I 7.9 2.1 5 5.0 159.3 2 6.7 80.1 2 9.4 78.7 t 7.3* ('hange in mean sy?itemicartenal pressure tmm.Hg) in response to transurethral administration of PROLIMO (400 pg.) and PIPERAZUNO (2000 pg.) and intracavern~isel injection ( 1 1 the triple-drug control combination. N = 9-10, 'p < 0.05. response is significantly different from baseline value. 149 0 156.6
* '
~
MAHMA/NO, PAPNNO, DENNO, PIPERAZVNO and PROLVNO, all novel NO donors, to induce dose dependent increases in cavernosal pressure and penile length when administered intracavernosally in the cat. The increases in cavernosal pressure and penile length in response to intracavernosal injection of MAHMA/NO, PAPA/NO, DEA/NO, PIPERAZVNO, and PROLVNO were comparable to the increases induced by the triple-drug control combination, suggesting that these agents possess the ability to induce penile erection in this animal model. Electrical stimulation of the cavernosal nerve was not conducted in this study because the control triple-drug combination served as a control comparison, as described in previous papers from this laboratory, to gauge the potency of all NO donors tested.".'" When compared on a nmol. basis. taking molecular weight into account, the dose-responses for MAHMA/NO, PAPADTO, DEA/NO, and PIPERAZVNO were similar and were 3 to 10-fold more potent than PROLVNO. Moreover, the durations of the maximum increases in cavernosal pressure in response to NO donors were similar to each other, but were significantly shorter in duration when compared with the triple-drug control combination. However. the decrease in systemic arterial pressure following injection of the NO donors was also significantly less than that observed with the triple-drug control combination. In fact. intracavernosal injections of PROLVNO and PAPA/NO resulted in slight but not significant decreases in systemic arterial pressurr a t all doscs studied. The reason for the diffrrenct. in the c4fcct on systemic arterial pressure is not known, but thcsc data suggrst that PROLI/NO. at the d o w ~nwdt.d to induct, penile ert.ction. dow not alter sys-
temic arterial pressure, perhaps because of its more rapid release of NO into the cavernosal tissue with less systemic absorption. The relative lack of systemic absorption may be augmented by the onset of the veno-occlusive response. The triple-drug combination induced severe hypotension, which may be attributed to the doses used of papavenne, phentolamine, and PGE, employed in this combination. The results of this study show that these novel NO donors can induce a dose-dependent increase in cavernosal pressure and penile length when injected intracavernosally in the cat. Transurethral administration of PROLUNO and PAPANO in doses of 100 to 400 pg. and 200 to 2000 pg., respectively, induced dose-dependent increases in cavernosal pressure and penile length to values comparable to those observed with the triple-drug control combination. These data provide evidence that, like prostaglandin E,, these NO donors can induce an erectile response when administered transurethrally. This may be a significant advantage over other vasodilator agents with a shorter duration of action and over those which alter systemic arterial pressure. Moreover, the use of local drug application is appealing for treating erectile dysfunction because of reduced side effects and greater clinical acceptability.2fi The finding that PROLUNO generally gave a shorter lasting and less potent erectile response than the other test agents presumably stems from its short half-life ( 2 seconds a t pH 7.4 and 37C).'' While it was stable a s a stock solution in 10 mM sodium hydroxide, we felt it was inadvisable to administer the compound to the animals in a n alkaline vehicle. Accordingly. w e neutralized the stock solution with PBS but
NITRIC OXIDE DONORS AND ERECTION IN THE CAT
made every effort to inject the neutral dosing solution as soon as possible thereafter, usually within seconds. However, this procedure invariably took enough time that premature dissociation of PROLI/NO and subsequent loss of NO must have occurred to a significant extent. This was not a problem with the other drugs, whose half-lives are long compared with the time that elapsed between neutralization of their stock solutions and administration to the animals. We view it as likely that PROLVNOs potency would be much greater in a more accommodating vehicle. When administered intracavernosally and transurethrally, the NO-releasing diazeniumdiolates evaluated in this study did not substantially lower systemic arterial pressure. This observation can be attributed to the fact that these novel NO donors are ionic or zwitterionicand are not easily absorbed as such into the systemic circulation. Inactivation of this highly diffusible species (NO) through its extremely rapid destruction by oxyheme species, superoxide and other reactive biomolecules prohibits extension of its vasodilatory effects much beyond the immediate vicinity of the corpus cavernosum thus causing less of a systemic and erectile response than that of the control triple-drug combination. The ability of these spontaneous NOreleasers to concentrate their vasodilatory effect solely in the penis when administered locally, without hemodynamic side effects is a major advantage and provides a strong rationale for further investigation of this class of agents in the clinical management of erectile dysfunction. CONCLUSIONS
The resultsof the present study provide evidence that MAHMA/ NO, PAPADTO, DENNO, PIPERAZI/NO, and P R O W 0 can induce dose-dependent increases in cavernosal pressure and penile length in the cat when administered intxacavemosdy. The increases in cavernosal pressure and penile length were comparable to those observed with the triple-drug control combination. Although the magnitudes of the increases in cavemosalpressure and penile length were similar,the duration of the maximum erectile response induced by the NO donors was sigmfmmtly less than that observed by the triple-drug control combmation. The maximum increase in cavernosal pressure in response to PROLVNO and PAPADTO was assOciated with no sgdicant change in systemic arterial pressure. The decreases in systemic arterial pressure in response to intracavernosal injection of -0, D M O , and PIPERAZINO were sigm6cantly less than those observed with the tripledrug control combination. Moreover, the results of the present study demonstrate that transmthral administration of PROWNO and PIPERAzIlNO can induce dosedependent increases in cavernosal pressure and penile length, similar to those observed with the triple-drug control combmation but without the decrease in systemicarterial pressure. These data support further clinical investigationsof diazeniumdiolatesfor the pharmacologic management of erectile dysfunction. Acknowledgments. The authors would like to thank Melanie Cross for her assistance in the preparation of this manuscript. REFERENCES
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